Health Environment Aquaculture I To 12
Health Environment Aquaculture I To 12
Health Environment Aquaculture I To 12
C
and 34 ups of salinity (Ontiveros-Garca, 2008). Its growing and feeding phase occurs on the
skin and gills of fish, followed by a detachment of the trophont (with stalk withdrawal),
which then produces a reproductive cyst (tomont) that settles in the tank walls. The cyst
performs several asexual divisions, releasing motile forms (dinospores of 12 to 15 m) after
the last division, which is then capable of infecting new hosts. Outbreaks of A. ocellatum
have been frequently observed on bullseye puffer fish, spotted rose snapper, Pacific red
snapper, Pacific cubera snapper, yellow snapper and mullet snapper causing gill lesions,
impaired oxygen exchange and even 100% of fish mortality (Fajer-vila et al., 2011; Prez-
Urbiola et al., 2008a). The rearing conditions of juvenile leopard groupers were favorable for
the proliferation of this dinoflagellate, resulting in a cumulative mortality rate of 89.5% at 7
days post-infection, where the highest virulence was observed at 48 h (Reyes-Becerril et al.,
Parasitic Diseases in Cultured Marine Fish in Northwest Mexico
67
2008). The infection is characterized by initial signs of appetite loss, followed by opacity of
the skin with whitish areas and erosion of the caudal fin. Fish start swimming sideways and
rubbing against the tanks bottom. Severe infection cases have been observed on the spotted
rose snapper (Ontiveros-Garca, 2008), with hundreds of parasites per fish. A notorious fish
response was an increment on erythrocyte size, which could be considered as respiratory
exchange compensation due to the effect of parasites on the gills. A positive correspondence
between the level of infections of A. ocellatum and the total leucocyte number was also
observed, indicating that this is a protective response to parasitic stress (Das et al., 2006).
High infection levels of A. ocellatum were further associated to a severe proliferative
epithelial response on gills, high number of mucous cells, infiltrating inflammatory cells and
lamellar fusion (Ontiveros-Garca, 2008).
3. Infections by monogeneans
Monogeneans are flatworm ectoparasites with a direct life cycle, frequently found in
mariculture systems. The main characteristic of the monogenean group is presence of an
opisthaptor, an adhesive apparatus equipped by sclerotized structures located in the
posterior region of the worm. Most of monogeneans infecting marine fish are ovoviviparous
(Buchmann & Bresciani, 2006). They are considered important pathogens due to their
velocity of propagation among fish in culture systems (Thoney & Hargis, 1991). Mass
infections by monogeneans are result of the innate susceptibility that stressful
environmental conditions could elicit on fish (Buchman & Bresciani, 2006).
3.1 Capsalids
The capsalid family has a characteristic flattened, leaf-like body. The haptor is also
remarkably conservative. The basic arrangement comprises a saucer-shaped attachment
organ armed with three pairs of median sclerites that are usually large, 14 small hooklets at
the periphery of the haptor proper and a thin, membranous marginal valve around the edge
(Whittington, 2004). The capsalid monogeneans are considered important pathogens in fish
culture facilities (Ogawa et al., 1995; Whittington et al., 2001a). They normally inhabit on the
fish skin and under the scales, although some can be found on the gills and nostrils
(Buchmann & Bresciani, 2006). These parasites feed on epithelial cells and mucus and are
relatively large and flat, with prominent muscular disc haptors at the posterior end (Leong
& Colorni, 2002).
Some species have been linked to epizootics of wild fishes (Paperna & Overstreet, 1981),
while other species have shown low host specificity in marine fish cultured in floating net
cages, such as the ones of the genera Benedenia, Neobenedenia and Megalocotyloides (Leong &
Colorni, 2002). Benedenia spp., B. epinepheli, B. lutjani, and Neobenedia sp. were reported to the
snappers L. johni and L. argentimaculatus, while N. girellae was reported to L. johni (Leong &
Colorni, 2002) and T. rubripes (Ogawa et al., 1995). B. epinepheli was reported in 25 fish
species, where tetraodontid fish was considered the most susceptible for severe infections
(Leong & Colorni, 2002). Five Neobenedenia species were found in the Pacific coast and
Cortez Sea, Mexico: N. girellae, morphologically synonymized as N. melleni by Whittington &
Horton (1996) molecularly confirmed by Li et al. (2005), and N. adenea, on the groupers M.
pardalis and M. rosacea in Baja California Sur, Mexico (Kohn et al., 2006) and N. isabellae on
gills of M. olfax in Nayarit; N. longiprostata on the serranid family in Baja California Norte
Health and Environment in Aquaculture
68
and N. pacifica on the flathead mullet (Mugil cephalus) in Baja California Norte (Kohn et al.,
2006). Wild bullseye puffer fish were found infested by N. melleni at low levels (1-3 parasite
per fish) (Fajer-vila et al., 2004), while in tanks the infection levels reached values of 28-60
parasites per fish, causing skin lesions, anorexia and mortality on juveniles (Fajer-vila et
al., 2008). A potential new species of Neobenedenia (Fig. 4a) was found in spotted rose
snapper causing hemorrhage of the caudal fin (Fig. 4b), emaciation and mortality in
juveniles reared in tanks (unpublished results). Additional reports of Neobenedenia spp. on
adults of L. peru, L. aratus, L. novemfasciatus and L. argentiventris reared in tanks indicates that
fish show opaque eyes, mucus proliferation, coloration changes and anorexia, while this
parasite was not found in juveniles of the same fish species (Prez-Urbiola et al., 2008a). As
pathogen, Neobenedenia is particularly important as there are reports of the introduction of
this parasite into fish stocks due to unregulated fish transference across international
borders (Ogawa et al., 1995).
Fig. 4. Neobenedenia sp. (Scale bar = 1mm at 40 X; Staining Van Cleaves hematoxilin) (a)
causing hemorrhage on the caudal fin in spotted rose snapper (b).
Benedenia seriolae is the most common capsalid species in the intensive culture of yellowtail
(Seriola quinqueradiata), where approximately 20% of the total production costs is dedicated
to control this parasite (Whittington et al., 2001b). In Bahia Magdalena, BCS, Mexico, 5%
mortality of S. lalandi cultured in floating cages has been attributed to mismanagement and
the presence of opportunistic parasites, such as Benedenia sp. (Avils-Quevedo & Castell-
Orvay, 2004). Benedenia sp. is commonly found in wild fish such as croakers, flounders, and
mackerels (Avils-Quevedo & Castell-Orvay, 2004). In Mexico most of the capture-based
tuna aquaculture is located along the Pacific Coast of Baja California. The farms normally
rear the pacific bluefin tuna a species recognized as resistant to diseases, so although several
parasites have been identified, no serious problems have been reported. The capsalids
reported in pacific bluefin tuna are B. seriolae, Capsala paucispinosa and Capsala sp. (Munday
et al., 2003). No reports of monogeneans were found for the culture of the California halibut
a common fish along the Pacific coast of North America. However, there are reports of
capsalids parasitizing this fish species such as Entobdella squamula and E. hippoglossi
(Castillo-Snchez et al., 1998; Kalman, 2006).
3.2 Dactylogyrids
Dactylogyrids are common gill parasites from teleost fish distributed throughout warm seas
(Wu et al., 2006). The monogeneans of the Dactylogyridae family presents two pairs of large
hooks or hamuli that oppose each other and allow the parasite to attach to the secondary
a b
Parasitic Diseases in Cultured Marine Fish in Northwest Mexico
69
lamellae of the gill filament. The hamuli break the gill tissue and cause epithelial
hyperplasia, oedema and haemorrhages (Whittington, 2005). Heavily affected fish may die
due to asphyxia as a result of gill pathology and interference with the exchange of
respiratory gases and ions (Stephens et al., 2003). Dactylogyrids could have an impact on
cultured fish as they can easily multiply and disperse as result of their direct life cycle,
sometimes reaching very high densities.
Some Haliotrema species are pathogens of marine fish. H. johni and H. abaddon were reported
infecting gills of snapper L. johni, in floating cages in Penang, Malaysia (Leong & Wong,
1987), and the cultured Australian dhufish (Glaucosoma hebraicum) (Pironet & Jones, 2000).
Dactylogyrid monogeneans (Fig. 5) such as Haliotrematoides spp. and Euryhaliotrema sp. are
commonly found on the gills of wild and cultured spotted rose snapper (Fajer-vila et al.,
2007).
Fig. 5. Dactylogyrid monogeneans on a gill filament of spotted rose snapper.
Scale bar = 50 m (40 x).
Three dactylogyrid genera, Euryhaliotrema, Haliotrematoides and Tetrancistrum, include
species that have been found on the gills of snappers (Kritsky et al., 2009). Fifteen species of
Euryhaliotrema are currently known from snappers worldwide (Fuentes-Zambrano & Silva
Rojas, 2006; Garca-Vargas et al., 2008; Kritsky & Boeger, 2002, Li, 2005, 2006; Li et al., 2005;
Pan & Zhang, 2006) and 22 of Haliotrematoides (Kritsky et al., 2009). In Mexico four species of
Haliotrematoides have been reported from lutjanids from the Atlantic (Kritsky et al., 2009;
Zhukov, 1976). E. perezponcei and H. guttati (Garca-Vargas et al., 2008), H. spinatus, H.
plectridium and Euryhaliotrematoides sp. were found on the spotted rose snapper in
northwestern coast of Mexico (Soler-Jimnez & Fajer-vila, in press) Perez-Urbiola et al.
(2008a) reported high levels of dactylogyrids (Haliotrema spp.) associated to mortalities of
snappers (L. aratus, L. argentiventris and L. novemfasciatus) confined at high densities in little
ponds with low water exchange.
Experimental studies with L. guttatus subjected to different levels of dactylogyrid infections
showed that the attachment of dactylogyrids increased the erythrocyte sedimentation rate in
fish with a low level of infection (mean intensity of 22 parasites per fish). Glucose was high
for all infection levels. An increase in the infection load was positively correlated with the
total leucocyte count (68,000 to 152,000 leucocytes), representing an increase of more than
Health and Environment in Aquaculture
70
200 percent in fish with the infection level higher respect to control group. The infection load
was also positively correlated with the thrombocyte percentage ratio and the percentage of
granulocytes, whereas increased infection level was negatively correlated with the leucocyte
percentage ratio and the number of lymphocytes. A high infection level (100 parasites per
fish) led to severe proliferative epithelial response in gills, as well as high number of mucous
cells, a moderate increase in chloride cells, infiltrating inflammatory cells and lamellar
fusion (Del Ro-Zaragoza et al., 2010).
3.3 Diclidophorids
Diclidophoridae monogeneans have an elongated body, which usually have an
opisthohaptor with several suckers or clamps in two mostly symmetrical rows. The number
of clamps never exceeds four on one side and they have also a cirrus and or genital atrium
armed or unarmed (Yamaguti, 1961). The genus Heterobothrium is a very common
diclidophorid parasite of tetraodontid fish, with four pair of sessile clamps and a ring of
claw-shaped spines at the terminal end of the male copulatory organ. Heterobothrium has a
direct cycle, invading fish hosts through free-swimming larvae, oncomiracidia. Outbreaks of
diclidophorids have been reported in several Asian countries, such as H. okamotoi infecting
gills and the branchial cavity of the tiger puffer fish, while Neoheterobothrium hirame infects
the gills and the buccal cavity of the Japanese flounder (P. olivaceus), the latter parasite being
considered the causative agent of anaemia among Japanese fisheries since the late 1990s
(Ogawa, 2002).
In the Pacific coast of Mexico, H. ecuadori has been reported in the bullseye puffer fish from
Oaxaca (Lamothe-Argumedo, 1967) and Sinaloa (Fajer-vila et al., 2004). In the Gulf of
Mexico, H. lamothei from the gills of checkered puffer (S. testudineus) was described. Reports
regarding the occurrence of the platyhelminth parasites on wild bullseye puffers fish from
two localities in Sinaloa revealed that H. ecuadori was the most prevalent species, likely to be
present at water temperature of 23-24.5
g
g
-
1
Time (days)
A B
Fig. 1. Muscle oxytetracycline levels -time profiles in the shrimp Litopenaeus vannamei after
an oral OTC dosage through a medicated feed. The vertical bars represent the Standard
Error (n=3). A: treatment period, B: withdrawal period.
The most frequent administration route for antibiotics in shrimp is oral, in which the
antibiotic is incorporated in the feed with subsequent exposure to the extremely aggressive
aquatic environment. For this reason, it is important that the antibiotic be contained within a
pellet to maintain its stability and protect it from factors such as leaching and binding to
trivalent and divalent cations (Cabello, 2004).
It must be certain that the shrimp will eat the food when the antibiotic therapy is applied,
because the disease will otherwise not be treated, the environment will be contaminated,
and the emergence of bacterial resistant strains will be favored. The consumption of food by
the farmed organisms may decrease during the molting period, due to environmental
factors, or factors related to the infections, reducing the quantity of the antibiotic ingested
(Cuzon et al., 2004).
The water temperature of the farm ponds is a critical point to consider, because parameters
such as the maximum concentration, distribution volume, and rate of elimination of the
The Use of Antibiotics in Shrimp Farming
207
0
50
100
150
200
250
300
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
g
g
-
1
Time (days)
A B
Fig. 2. Hepatopancreas oxytetracycline levels time profiles in the shrimp Litopenaeus
vannamei after an oral OTC dosage through a medicated feed. The vertical bars represent the
Standard Error (n=3). A: treatment period, B: withdrawal period.
antibiotic may be affected. The pH, oxygenation, salinity, stage of disease, climatic changes,
and presence of natural food in the ponds are other factors that affect antibiotic therapies
among aquatic organisms (Chvez & Montoya, 2004; Montoya, 2002).
The use of pharmacological agents, antibiotics, and other chemical agents should be
considered as methods of last resort in shrimp farming and aquaculture in general. None of
the antibiotics is approved for use in shrimp in the United States. The medicated feed is
used in an extralabel manner only for treatment of minor species as defined in the Code of
Federal Regulations (21 CFR 514.1(d)(1)(ii). In an aquatic species, the extralabel use of
medications added to feed in limited to products approved for use in other aquatic species
(FDA, 2001).
6. The development of bacterial resistance
Drug resistance in when a formerly effective drug dose is no longer effective. This can be a
natural resistance or an acquired resistance. Resistance arises mainly by natural selection,
Health and Environment in Aquaculture
208
the replication of a naturally resistant strain after the drugs has killed all of the susceptible
strains. Since mutagenic drugs generally are not used, resistance by drug-induced mutation
seldom occurs. Drug resistance also can develop from gene transfer or gene amplification
(Albert, 1985).
The most worrying effect of the use of antibiotics in aquaculture production and its
relationship with human health is the generation of resistant bacteria strains and the transfer
of this resistance from the aquatic environment to land, where strains that are highly
immune to antibiotics may originate that are capable of causing disease among humans. The
transfer of resistance may occur through mechanisms as simple as the consumption of
seafood products that contain bacteria that are resistant to various antibiotics (Grslund &
Bengtsson, 2001).
It has been demonstrated that the use and abuse of antibiotics has given rise to multiple
resistance among microbial populations associated with shrimp production. Various studies
have shown that antibiotics persist in the sediment and aquatic environment for several
months after their administration (Matyar et al., 2008), and that these may affect native
bacterial community in detrimental to the ecosystem since this community plays key roles in
biogeochemical processes. Some antimicrobials can inhibit important microbial processes as
denitrification or primary production by cyanobacteria (Garcia-Armisen et al., 2011).
The capacity of the microorganisms to reduce sulfates may also be reduced (Pez-Osuna et
al., 2003), affecting the quality of the sediment and the environment (Ma et al., 2006), thereby
promoting the proliferation of resistant bacterial strains or pathogens (Capone et al., 1996;
Hektoen et al., 1995; Tendencia & De la Pea, 2002;), which may place the viability of shrimp
crops at risk.
It is estimated that between 15 and 40% of the administered medicated diet is not ingested by
the organisms and remains in the substrates. Another part of the medication is not absorbed
during its passage through the intestinal tract of the organism and returns to the environment
in fecal matter. The amount of antibiotic transferred to the environment varies from 1%
(chloramphenicol) to 90% (oxytetracycline) (Capone et al., 1996). Hektoen et al. (1995) reported
that approximately 70-90% of the antibiotic used in the therapy of farmed organisms ends up
in the environment and sediment, and a high percentage exhibits antibacterial activity. It has
been reported that residues of oxolinic acid and oxytetracycline are very persistent under
certain conditions, with half-lives exceeding 100 days (Samuelsen et al., 1992).
Three mechanisms of resistance to tetracycline have been described: (1) decreased
intracellular accumulation due to either impaired influx or increased efflux by an active
transport protein pump; (2) ribosome protection due to production of protein that interfere
with tetracycline binding to the ribosome; and (3) enzymatic inactivation of tetracyclines.
The most important of these is production of an efflux pump. The pump protein is encoded
on a plasmid and may be transmitted by transduction or by conjugation. Because these
plasmids commonly encode resistance genes for other drugs, eg, aminoglycosides,
sulfonamides and chloramphenicol, tetracycline resistance is marker for resistance to
multiple drugs (Tenover, 2006).
Some studies have demonstrated that the concentrations of oxytetracycline in the sediment
after therapy may range from 0.4 to 495 g g
-1
. Therapeutic dosages of oxytetracycline in fish
The Use of Antibiotics in Shrimp Farming
209
may cause sub-lethal effects, including alteration of the levels of immunoglobulin in the
serum and suppression of the phagocytic response and macrophages (Uyaguari et al., 2009).
International regulations regarding the use of antibiotics in aquaculture have established a
list of prohibited products (Stolker & Brinkman, 2005). Shrimp with traces of these products
are subject to measures against their importation. The strongest restrictions are on the use of
chloramphenicol, dimetridazole, furazolidone, nitrofurazone, other nitrofurans, and
fluoroquinolones, and these antibiotics should not be used at any stage of the production
process (Defoirdt, et al., 2007; Tittlemier et al., 2007).
Epidemiological and molecular assays have indicated that genes mediating resistance might
be transmitted from aquatic bacteria to bacteria capable of producing infections among
humans and terrestrial animals. This demonstrates that the aquatic and terrestrial
compartments lack borders with respect to the flow of resistance genes and that the
resistance phenomenon is global, because the use of antibiotics in an environment will have,
over time, repercussions in other, apparently distant, ecosystems (Cabello, 2002; Rhodes et
al., 2000). To decrease the contamination of the environment and bacterial resistance,
appropriate aquaculture production practices must be carried out, and biosecurity measures
must be applied to reduce outbreaks of disease and the propagation of pathogenic agents
(Kemper, 2008).
Global efforts are needed to promote more judicious use of antibiotics in aquaculture and
the new strategies to control phatogenic bacteria are needed to make the industry more
sustainable. However, it is not always economically feasible to culture the organism in the
most optimal conditions, so there will always be a risk to infection and a need for effective
biocontrol techniques (Defoirdt, et al., 2007).
It is important to highlight that the application of highly sensitive analytical methodologies
is indispensably in measuring the concentrations of antibiotics and their metabolites with
certainty in the distinct tissues of aquaculture products. This would help in establishing
regulations that protect the environment, generating products that are safe for human
consumption, and allowing the growth of aquaculture.
A practical use of the pharmacokinetic data is the possibility to design dosage regimens in
which levels of a specific drug can be maintained above the Minimum Inhibitory
Concentration and below toxic effects by means of repeated dosages. However, this method
requires information on Minimal Inhibitory Concentration established for bacterial
pathogens of interest. Although there are reports on available Minimal Inhibitory
Concentration for bacterial strains potentially pathogens to shrimp species, they reveal a
wide range of values. Takahashi et al., (1985) reported that Minimal Inhibitory
Concentration of oxytetracycline against 49 strains of Vibrio sp., range from 0.1 to 12.5 g
mL
-1
. Monhey et al., (1992) found the Minimal Inhibitory Concentration to be in the range of
2.0 g mL
-1
or less for Vibrio isolated mainly from American shrimp. Furthermore, Roque et
al., (2001) in their study of 144 isolated of Vibrio reported a Minimal Inhibitory
Concentration of 304.0 g mL
-1
for oxytetracycline with a range from 0.26 to 1064 g mL
-1
.
Given the wide range of Minimal Inhibitory Concentration it is recommended to isolate
local bacterial strains and evaluate their Minimal Inhibitory Concentration. Additionally, it
still requires performing such studies in natural farming conditions (Gmez-Jimenez et al.,
2008).
Health and Environment in Aquaculture
210
7. Conclusion
Scientific studies have been conclusive with respect to the health risk that the massive and
unlimited use of antibiotics in aquaculture represents. When health certifications are
implemented for aquaculture products demanded by domestic and foreign markets, the
control over the use of these compounds needs to be increased, together with other aspects
of primary importance such as food safety, protection of the environment, and the health of
farmed organisms. These aspects should be considered and resolved through the
implementation of Best Management Practices.
8. Acknowledgment
The authors wish to express their gratitude to PhD. Evelia Acedo Flix and Q.B. Rosalva
Prez Morales of the Molecular Microbiology Laboratory, for their technical support and
contribution to help facilitate the completion of the project. Furthermore, we acknowledge
the kind donation of the shrimp from the farm La Borbolla, specialty engineers Roberto
Federico Aguayo Valenzuela and Csar Patio Patio, and the technical assistance of Biol.
Adolfo Prez lvarez and Tech. Juan Carlos Gastlum Domnguez.
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8
Probiotics in Aquaculture Benefits to the
Health, Technological Applications and Safety
Xuxia Zhou
1
and Yanbo Wang
2,*
1
College of Biological and Environmental Engineering,
Zhejiang University of Technology, Hangzhou
2
Food Quality and Safety Department, Zhejiang
Gongshang University, Hangzhou
China
1. Introduction
Aquaculture is the fastest growing food-producing sector in the world at an average rate of
8.9% per year since 1970, compared with only 1.2% for capture fisheries and 2.8% for
terrestrial farmed meat production systems over the same period (Subasinghe, 2005).
Although aquatic food production through aquaculture is the fastest growing sector and
vaccines are being developed and marketed in aquaculture, the disease is still a major
problem in the aquaculture farming industry (Bondad-Reantaso et al., 2005). During the last
decades, chemical additives and veterinary medicines, especially antimicrobial agents, to
prevent and control disease have been also applied in aquaculture (Wang and Xu, 2004;
Cabello, 2006; Lupin, 2009). However, the risks associated with the transmission of resistant
bacteria from aquaculture environments to humans, and the introduction in the human
environment of nonpathogenic bacteria, containing antimicrobial resistance genes, and the
subsequent transfer of such genes to human pathogens existed according to FAO (2005).
Previous studies also show the aquatic bacteria can develop resistance genes as a
consequence of exposure to antimicrobial agents (Smith et al., 1994; Kim et al., 2004; Srum,
2006). Therefore, the need for alternative techniques is increasing and the contribution of
probiotics may be considerable.
The use of probiotics in aquaculture is now widely accepted with an increasing demand for
environment friendly aquaculture (Ring and Gatesoupe, 1998; Gatesoupe, 1999; Sharma
and Bhukhar, 2000; Irianto and Austin, 2002; Wang and Xu, 2006; Vine et al., 2006; Wang,
2007; Denev et al., 2009; Qi et al., 2009). Nowadays, a number of preparations of probiotics
are commercially available and have been introduced to fish, shellfish and molluscan
farming as feed additives, or are incorporated in pond water (Moriarty, 1998; Wang et al.,
2005; Prado et al., 2010). According to the claims of the producers, these products are
effective in supporting the health of aquatic animals and are also safe. However, there are
doubts with regard to the general concept of probiotics and to these claims on the other
hand. Indeed, the current explanations and principles are still not enough to describe what
*
Corresponding Author
Health and Environment in Aquaculture
216
probiotics actually are, where they come from, and what they can do (Wang et al., 2008).
Thus, there is clearly a need in increasing our knowledge of aquacultural animals and of
effective preparation, technological applications and safety evaluation of probiotics. This
chapter provided a summary of the status and challenges of probiotics application in
aquaculture. In this chapter, the benefits to the health, technological application and safety
evaluation were discussed. In addition, the probiotics information in aquaculture obtained
from authentic and highly regarded sources was contained and listed.
2. Probiotics and gut microbiota
Three general modes of probiotics actions have been classified and presented by Oelschlaeger
(2010) as follow: (1) Probiotics might be able to modulate the hosts gut defences including the
innate as well as the acquired immune system and this mode of action is most likely important
for the prevention and therapy of infectious diseases but also for the treatment of
inflammation of the digestive tract or parts thereof. (2) Probiotics can also have a direct effect
on other microorganisms, commensal and/or pathogenic ones and this principle is in many
cases of importance for the prevention and therapy of infections and restoration of the
microbial equilibrium in the gut. (3) Finally, probiotic effects may be based on actions affecting
microbial products, host products and food ingredients and such actions may result in
inactivation of toxins and detoxification of host and food components in the gut. According to
above summary, all three modes of probiotics actions are all likelihood associated with gut
and/or gut microbiota. Therefore, it has become apparent that we are in fact dealing with
another organ, the so called microbiotic canal with the increased knowledge of the specific
activity of the gut microbiota (Wolf, 2006). In general, the gut microbiota remain relatively
stable throughout life once established although they can be influenced by several factors such
as mode of delivery, hygiene and the use of antibiotics.
The gut microbiota with the epithelium and mucosal immune system orchestrate a network
of immunological and nonimmunological defenses, providing both protection against
pathogens and tolerance to commensal bacteria and harmless antigens (Sanz and Palma,
2009). The important role of commensal bacteria in development of optimally functioning
mucosal immune system was demonstrated in germ-free animals (Tlaskalov-Hogenov,
2004). Therefore, the imbalance of gut microbiota has been linked to several diseases
including inflammatory bowel diseases, periodontal disease, rheumatoid arthritis,
atherosclerosis and allergy. So probiotics, that is, microbial strains that have beneficial
effects on the host, are thought to benefit this intestinal ecosystem (Julio and Marie-Jos,
2011). In addition, some probiotics strains also induce the secretion of multiple antimicrobial
materials by intestinal Paneth cells through cell-autonomous MyD88-dependent toll-like
receptor activation (Vaishnava et al., 2008) and regulate the alterations of permeability
related with infections, stress, and inflammatory conditions (Lutgendorff et al., 2008). There
is evidence that probiotics produce a protective effect on the gut microbiota and the
beneficial effects of probiotics on several microbial disorders have been well reviewed
(Gismondo et al., 1999).
As for the aquatic animals such as fish and shrimp, the colonization of the gastrointestinal
tract starts immediately after hatching and is completed within a few hours to modulate
expression of genes in the digestive tract, thus creating a favorable habitat for them and
preventing invasion by other bacteria introduced later into the ecosystem (Balczar et al.,
Probiotics in Aquaculture Benefits to the Health, Technological Applications and Safety
217
2006). This is attributed to competitive exclusion mechanisms and improved immune
system development and maturation. Intake of probiotics has been demonstrated to modify
the composition of the microbiota, and therefore assist in returning a disturbed microbiota
(by antibiotics or other risk factors) to its normal beneficial composition (Gmez and
Balczar, 2008). As for the mechanisms during this physiological process, the production of
antimicrobial substances, competition for nutrients or adhesion receptors, inhibition of
virulence gene expression and enhancement of the immune response are all included
(Irianto and Austin, 2002; Nikoskelainen, et al., 2003; Vine et al., 2004; Kim and Austin, 2006;
Balczar, et al., 2007). However, the exact mechanism by which these probiotics do this is not
known. Advances in the understanding of the mechanisms between gut microbiota and
probiotics and how the immune system of aquatic animals generally responds to gut
microbiota would be of great help to identify the molecular targets of probiotics and the
biomarkers of their effects, and to provide sounder evidences on their benefits on
physiologic conditions and immune-mediated disorders.
3. Probiotics effects in aquaculture: Benefits to the health
When looking at probiotics intended for an aquatic usage it is important to consider certain
influencing factors that are fundamentally different from terrestrial based probiotics
(Kesarcodi-Watson et al., 2008). Indeed, aquatic animals are quite different from the land
animals and a consequence of the specificity of aquatic microbiota is that the most efficient
probiotics for aquaculture may be different from those of terrestrial species (Gatesoupe,
1999). A fairly constant habitat of resident microbiota in the gastrointestinal tract of
terrestrial livestock is important, whereas most microbiota is transient in aquatic animals
(Moriarty, 1990). Shift in intestinal microflora of Atlantic halibut (Hippoglossus hippoglossus)
larvae during first feeding was studied and the results showed the transition from a
prevailing Flavobacterium spp. intestinal flora to an Aeromonas spp./Vibrio spp. dominant
flora occurred when first feeding commenced (Bergh et al., 1994). It indicated that the gut
microbiota of aquatic animals may change rapidly with the intrusion of microflora from
water, live food and artificial diet. In addition, aquatic animal and microorganisms share the
same ecosystem in the aquatic environment and it suggested that the interaction between
the microbiota, including probiotics, and the host is not limited to the intestinal tract.
Therefore, the definition of a probiotic for aquatic environments needs to be modified,
which allows a broader application of the term probiotic. A probiotic is then defined by
Verschuere et al. (2000) as a live microbial adjunct which has a beneficial effect on the host
by modifying the host-associated or ambient microbial community, by ensuring improved
use of the feed or enhancing its nutritional value, by enhancing the host response towards
disease, or by improving the quality of its ambient environment.
Most probiotics used in aquaculture belong to the lactic acid bacteria, of the genus Bacillus,
to the photosynthetic bacteria or to the yeast, although other genera or species have also
been mentioned (Fig. 1). Many studies have reported promising results using a single
beneficial bacterial strain as probiotic in the culture of many aquatic species (Gatesoupe,
1991; Noh et al., 1994; Bogut et al., 1998; Carnevali, et al., 2006; Daz-Rosales et al., 2009; Li et
al., 2009; Zhou et al., 2009; Tovar-Ramrez et al., 2010; Wang and Gu, 2010; Zhou et al., 2010;
Wang, 2011). It is important to consider the possibility of using different species, as
suggested by Noh et al. (1994) and Bogut et al. (1998). The effect of probiotics, photosynthetic
Health and Environment in Aquaculture
218
(a) (b)
(c) (d)
Fig. 1. The configurations of putative probiotics strains isolated and stored in our laboratory
using scanning electron microscope (Philips XL30ESEM, Netherlands). a, Lactococcus lactis;
b, Bacillus coagulans; c, Rhodopseudomonas palustris; d, Saccaromyces cerevisiae.
bacteria (Rhodobacter sphaeroides) and Bacillus sp. (B. coagulans), on growth performance and
digestive enzyme activity of the shrimp, Penaeus vannamei, was investigated and the results
showed that the effects were related with supplementation concentrations of probiotics and
thus use of a 10 g/kg (wet weight) supplement of probiotics in shrimp diet was
recommended to stimulate productive performance (Wang, 2007). A mixture of Bacillus
probiotic bacteria (Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus) was also
evaluated in the gilthead sea bream (Sparus aurata) larviculture focusing on their effects on
survival, growth and general welfare (Avella et al., 2010). The data generated in this study
show the benefit of the administration of Bacillus probiotic mixture in terms of stress
response and growth and provide scientific and technical support for the implementation of
sustainable development of sea bream aquaculture. Similar results were also observed in
olive flounder supplemented with Lactobacillus plantarum, Lactobacillus acidophilus,
Lactobacillus sakei, Bacillus subtilis, and Saccharomyces cerevisiae as individual and mixed
enriched diet (Harikrishnan et al., 2011a). Lactobacil probiotics individually or mixed with
Sporolac enriched diet were used to enhance the immune status, thereby improving the
disease resistance in lymphocystis disease virus infected olive flounder (Paralichthys
olivaceus) and the results showed that the better innate immune response and disease
resistance were found in groups supplemented with mixed probiotics (Harikrishnan et al.,
Probiotics in Aquaculture Benefits to the Health, Technological Applications and Safety
219
2010). However, feeding experiments conducted on 600 O. niloticus using the diets
containing single or mixed isolated probiotic bacteria show the different results in survival
rates and the highest with fish fed diets supplemented with B. pumilus was observed,
followed by a mixture of probiotics (B. firmus, B. pumilus and C. freundii in equal numbers),
and then C. freundii (Aly et al., 2008). It indicates that the beneficial effects of probiotics fed
aquatic animals are associated with probiotic strains, isolation species, culture animals and
water quality. Altogether, the data reported above may well explain the current trend to
prefer alternative probiotics for the application in aquaculture.
Additionally, a large number of studies have combined probiotics with prebiotics, a
selectively fermented ingredient that allows specific changes both in the composition
and/or activity in the gastrointestinal microflora that confers benefits upon host well-being
and health (Gibson et al., 2004). Thus the synbiotics, as a combination of probiotics and
prebiotics, have been studied to expect the synergistic effects. Nowadays, there are several
recognized functional prebiotic oligosaccharides such as fructooligosaccharides (FOS),
mannan oligosaccharides (MOS), insulin, -glucan, and xylo-oligosaccharides (XOS) in use
around the world. The effect of dietary application of a commercial probiotic (Bacillus spp.)
and MOS, used singularly and combined, on the survival, growth performance and feed
cost-benefit of European lobster (Homarus gammarus) larval was assessed and the results in
this study strongly suggest that the dietary combination of Bacillus spp. And MOS is cost
effective when used to promote survival and provides the added benefits of improved
growth performance, compared to their individual supplementation (Daniels et al., 2010).
Similar results have been reported on shrimp, Litopenaeus vannamei, and the disease
resistance was also improve by enhancing immunity, as well as presumably modulating
microflora in the shrimp's gut (Li et al., 2009). It suggested that the combined application of
probiotics and prebiotics is an interesting prospect for replacement of growth-promoting
chemotherapeutics in the aquaculture industry and could be a useful tool in the rearing of
certain aquatic animals. Recently, herbs and probiotics are combined in diet and treated as
one of the promising alternative tools to supplement and supplant antibiotics, chemicals or
vaccines (Sahu et al., 2008; Nayak, 2010). According to Harikrishnan et al. (2011b),
administration of probiotics (Lactobacillus sakei BK19) and herb (Scutellaria baicalensis) can
effectively minimize the mortality and restore the altered heamatological parameters and
enhancing the innate immunity in O. fasciatus against Edwardsiella tarda, which indicate a
promising role to prevent diseases and disease outbreaks in aquaculture. Similar results
were also determined in olive flounder, Paralichthys olivaceus, against Streptococcus parauberis
and the enhanced growth, blood biochemical constituents, and nonspecific immunity were
observed in the groups treated with probiotics and herbals mixture supplementation diet
(Harikrishnan et al., 2011c). Further investigations on the interaction between probiotics and
other functional additives at molecular level are warranted in aquaculture.
4. Manufacture and safety evaluation of probiotics
The continuing expansion of interest in probiotic bacteria has led to an increase in
manufactured functional foods and feeds containing these bacteria. Given the natural
and/or intestinal origin of these microorganisms, the challenges these putative probiotics
face in order to be in a highly viable state throughout processing, manufacture, and storage
are enormous. Environmental stresses such as temperature, acid, exposure and osmotic
Health and Environment in Aquaculture
220
pressure, oxygen have important effects on probiotics survival and activity both in product
and animal gut. However, like all bacteria, probiotic bacteria retain a broad arsenal of
molecular mechanisms to combat the often lethal environmental stresses encountered
during processing and following ingestion and therefore the comprehensive appreciation of
these mechanisms should inevitably lead to the design and manufacture of probiotic
cultures, which retain greater viability through to the target site in the intestine (Corcoran et
al., 2008). Environmental stress responses in Lactobacillus, which have been investigated
mainly by proteomics approaches, are reviewed by De Angelis and Gobbetti (2004) and the
physiological and molecular mechanisms of responses to heat, cold, acid, osmotic, oxygen,
high pressure and starvation stresses are described. As for the proteomics approaches, the
technique primarily bases on two-dimensional gel electrophoresis (2-DE) (Kellner, 2000).
The intensity of an individual spot indicates how much the cell has produced of that actual
protein and thus it has facilitated the rapid characterization of thousands of proteins in a
single polyacrylamide gel for the molecular mechanism studies of probiotics. Such studies
associated with the cellular processes and metabolism mechanisms available to probiotic
bacteria to facilitate survival in various stressful conditions can lead to production of
designer probiotic strains with enhanced viability in feed systems and efficacy following
ingestion for aquatic animals. Additionally, several other factors including the physiologic
state of the probiotics, the chemical composition of the product and possible interactions of
the probiotics with the starter cultures must be considered to ensure the abilities of
probiotics in aquaculture.
Although the probiotic species such as Lactobacillus acidophilus have been safely used for a
long time, the safety aspects have always to be considered and possible adverse effects
should continuously be evaluated as illustrated by literature (Salminen et al., 1998).
However, a growing number of diseases that appeared with the worldwide development
of aquaculture may be assigned to distinct bacteria belonging to the genera Streptococcus,
Lactococcus, Vagococcus and Carnobacterium, but, in most cases, the clear mechanisms have
not been found (Ring and Gatesoupe, 1998). In addition, safety considerations regarding
antimicrobial resistance neglected for a long time are now taken into account for the
development and marketing of probiotics (Courvalin, 2006). The question whether genetic
exchange may occur between probiotics and gut microflora or pathogens is raised because
the genes can be transferred between microorganisms. As a result, the antibiotic multi-
resistance existent of probiotics shows the possible insecurity caused by the possibility of
resistance genes transfer from probiotic strains to bacterial pathogens or from aquatic
commensals to probiotics. According to O'Brien et al. (1999), it is important to
differentiate between intrinsic resistance and that mediated by special genetic elements
when evaluating the antibiotic resistance profiles among different species and strains.
Indeed, safety is the state of being certain that adverse effects will not be caused by an
agent under defined conditions. Therefore, feeding of novel probiotics to healthy aquatic
animals is not only concerned with efficacy but safety even though lactobacilli and
bifidobacteria are generally regarded as safe. With the development of molecular biology
and other advanced modern techniques, the critical, tailored approaches such as cell
culture to safety evaluation of probiotics can ensure that healthy benefits are accessible to
aquatic animals. The epithelial cells of tilapia (Oreochromis nilotica) were isolated and
primarily cultured as the cells model to evaluate the probiotic, Rhodopseudomonas palustris,
through the morphologic characters, cells viability, livability and permeability (Wang and
Probiotics in Aquaculture Benefits to the Health, Technological Applications and Safety
221
Xu, 2007). This study shows cell culture is one of the promising approaches to safety
evaluation of probiotic in the future.
5. Future probiotics for aquaculture
The important role of the gut flora in the maintenance of health and in the prevention of
disease is well recognized (Holzapfel and Schillinger, 2002). Use of probiotics is likely to be
the most natural and safe means for improving gut flora balance to prevent bacterial
pathogens by competing for essential nutrients or attachment sites (Chukeatirote, 2003). As
for aquatic animals gut flora, the continuous interaction with the environment, the body
system and intrinsic microorganisms is very complex. Although the explosion in recent
years of publications dealing with probiotic organisms has been increased, central and vital
information is still needed and therefore more advanced methods should be developed to
assess the changes in the composition of the gut flora and their mutual interaction with the
metabolism of aquatic animals. Currently, probiotics may serve to partially replace the
presently reduced or even prohibited application of nutritive antibiotics or
chemotherapeutics in animal nutrition and in fulfillment of health claims in man and
animals (Reuter, 2001). According to Kesarcodi-Watson et al. (2008), a probiotic for the new,
effective and safe products in aquaculture must possess certain properties as follow: (1) the
probiotic should not be harmful to the host it is desired for, (2) it should be accepted by the
host, e.g. through ingestion and potential colonization and replication within the host, (3) it
should reach the location where the effect is required to take place, (4) it should actually
work in vivo as opposed to in vitro findings, and (5) it should preferably not contain
virulence resistance genes or antibiotic resistance genes. These properties should be
considered during the manufacture process and safety evaluation of novel probiotics. Then
the future will provide targeted probiotic bacteria accord with above properties for specific
use with carefully controlled studies on clearly defined selected strains. In addition, an
increasing demand for alternative to antibiotics products applied in aquaculture indicates a
bright future for probiotics and a number of better commercial probiotics will be available,
particular directed at larval culture.
6. Acknowledgments
This study is supported by the National Natural Science Foundation of China (No. 30901044
and 31072221) and Zhejiang Provincial Natural Science Foundation of China (No.
R3110345). Special appreciation goes to Mr. Junhui Wen, Mrs. Yaqing Zhu and Mr. Gentu
Wu for their works which lead to success of this review.
7. References
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9
Probiotics in Aquaculture of Kuwait
Current State and Prospect
Ahmed Al-marzouk and Azad I. Saheb
Mariculture and Fisheries Department (MFD)
Kuwait Institute for Scientific Research (KISR), Salmiya
Kuwait
1. Introduction
The blue-fin porgy, Sparidentex hasta, known as sobaity in Kuwait, is a commercially
valuable food fish greatly preferred in Kuwait and other Arabian Gulf countries. This
species has been cultured at the Kuwait Institute for Scientific Research (KISR) since 1979
(Hussain et al., 1981). From 1982 to1986, research efforts were carried out with the intension
of developing a commercially valid culture technology for sobaity (Teng et al., 1984). The
research efforts were completed, and the results were assessed to formulate a culture
technology recommended for commercial application (Teng et al., 1999). However, a
number of bacterial diseases have been reported that cause severe losses in sobaity larvae.
Outbreaks of vibriosis caused by Gram-negative bacteria Vibrio spp., is the most serious
bacterial disease of both grouper and sobaity (Rasheed, 1989a). They were identified as V.
anguillarum, V. ordalli and V. carchariae and V. damsela. In addition, V. harveyi was associated
with mortalities in hamoor and sobaity (Saeed, 1995). So far, conventional approaches, such
as the use of antimicrobial drugs, have had limited success in the prevention or cure of
aquatic animal disease. Furthermore, there is a growing concern about the use and,
particularly, the abuse of antimicrobial drugs in aquaculture. The practice of using
antibiotics indiscriminately for the treatment of diseases in aquaculture could result in the
accumulation of residues and the development of resistant strains of bacteria (Uma, 1999).
This leads to the search for new, more effective antibiotics thus increasing the consumption
of antibiotics in aquaculture. Vaccination can not prevent the development of the disease in
young and small fish (Ellis, 1999; Magnadottir et al. 2006). Conventional vaccination is,
accordingly, not of value before this time, and the larvae are wholly reliant on the non-
specific immune parameters. Thus, an alternative methods are to be evolved to maintain a
healthy microbial environment in fish rearing tanks. One such method that is gaining recent
acceptance within the aquaculture industry is the use of probiotics bacteria to control
potential bacterial pathogens (Wang et al., 2008; Decamp et al., 2010). Thus, the application
of probiotics may provide a potential alternative method to protect fish from infectious
diseases and improve the survival of cultured marine fish (Irianto and Austin, 2003). The
aim of the present study was to evaluate the effect of three autochthonous probiotics, coded
as SHBP, 4SQ and 5L8 and a standard isolate Lactobacillus divergens (ATCC, 35677) on
growth performance of rotifers and the effect of dietary administration of SHBP, L. divergens
and a combination (SHBP& L. divergens) on the survival rate of sobaity larvae.
Health and Environment in Aquaculture
228
2. Screening, isolation and in vitro antagonism test of autochthonous
probiotics
Performing an in vitro antagonism test is considered to be an important step in screening
potential probiotics, in which pathogenic bacteria are exposed to
the selective probiotics in
liquid
(Gildberg, et al., 1995, 1997) or solid (Austin et al., 1992; Dopazo, et al., 1988;
Westerdahl et al., 1990) medium. The preselection of candidate probiotics based on these
in vitro antagonism tests has usually led to the finding of effective
probiotics (Verschuere
et al., 2000). Bacterial isolates were obtained from the mid and hind gut of cultured
yellow-fin porgy Acanthopagrus latus (shaem), wild silver pomfret Pampus argenteus
(zobaidy), wild orange-spotted grouper Epinephelus coioides (hamoor), wild tigertooth
croaker, Otolithes argenteus (newaiby), cultured blue- fin porgy Sparidentex hasta (sobaity),
Lactobacillus sp., (Alken-Clear FIO-1006, Alken-Murray Corp, USA) and a Lactobacillus
divergens (ATCC, 35677). Autochthonous probiotic, Gram-positive bacteria isolated from
cultured shaem was coded as SHPB, form wild zobaidy, coded as 4SQI, from cultured
sobaity coded as S24, form wild newaiby coded as 5L82 and from wild hamoor coded as
5M99b. Probiotic bacteria were cultured in brain heart infusion broth (BHIB, Oxoid,
Basingstoke, UK) with 2% NaCl. After cultivation, bacteria were harvested by
centrifugation (2000 rpm for 10 min), washed twice and resuspended in phosphate-
buffered saline (PBS). The in vitro antagonism of the isolated autochthonous probiotics
against Vibrio alginolyticus (locally isolated from diseased cultured sobaity), V. anguillarum
(ATCC 43310), V. harveyi (locally isolated from diseased cultured mullet Liza klunzingeri),
V. parahaemolyticus (ATCC, 27159), V. vulnificus (ATCC, 33149) and Streptococcus agalactiae
(locally isolated from diseased zobaidy) was investigated. The inhibitory activity was
assessed by three antagonism tests, the first test is the well-diffusion test (Perez et al.,
1990) and the second test is the double-layer method (Dopazo et al., 1988). The third test
was a modification to the double-layer method. The new modified technique applied was
named as filter paper disc method, which aimed to obtain precise and accurate
inhibition zones created by the probiotics bacteria against the pathogenic bacteria. During
the current research the third method was selected. Five sterile filter paper disks were
placed on the BHIA, and a drop of the probiotic culture (2 L) was placed on the sterile
filter papers, and incubated overnight at 30C. The overnight cultures of the pathogenic
bacteria were prepared at 1:10 dilution using sterile phosphate buffered saline. All the
probiotic cells were killed by exposing the plate to each diluted pathogenic bacteria were
overlayed. The cultures were incubated overnight at 30 C and the zone of inhibition was
measured. The results showed that all the methods were suitable to assess the effect of
probiotic bacteria on pathogenic bacteria. However, the modified overlay method seems
to be more effective for the selection of probiotics. This method showed consistent results
on the zone of inhibition compared to the other two methods that some times produced
doubtful false negative results mainly due to the swarming nature of growth in the case of
V. alginolyticus. In addition, the administration of suitable concentration of probiotics and
allowing growth and production of antimicrobial compounds before the addition of Vibrio
spp produced a reliable inhibition results. The probiotics coded as SHPB and 4SQI
showed a significant zone of inhibition against all the pathogenic bacteria (Plate1). The
other probiotics showed some effect, but they were unable to inhibit the growth of all the
pathogenic bacteria.
Probiotics in Aquaculture of Kuwait Current State and Prospect
229
1: Lactobacillus sp; 2: Lactobacillus divergens; 3: 4SQI; 4: SHPB; 5: 5L82; 6: M99b; 7:S24.
Val: V. alginolyticus; VP: V. parahaemolyticus; Vh: V. harveyi; Strep: Streptococcus agalactiae; VV: V.
vulnificus; Van: V. anguillarum.
Plate 1. Antagonism test of seven putative probiotics against six pathogenic bacteria by the
modified double-layer method.
3. Competitive exclusion of vibrio co-cultured with autochthonous probiotics
(SHPB)
Competitive exclusion of potential pathogenic bacteria effectively reduces or eliminates the
need for antibiotic prophylaxis in intensive larviculture systems (Garriques & Arevalo,
1995). It has been reported that bacterial strains associated with intestinal and skin mucus of
adult marine turbot Scophthalmus maximus and dab Limanda limanda, suppressed the growth
of the fish pathogen V. anguillarum (Olsson et al., 1992). Thus, the manipulation of microbial
constitutes is a viable tool to reduce or eliminate the incidence of opportunist pathogens
(Balcazar et al., 2006). In this study, co-culture of Vibrio sp. and SHPB was plated on a BHI
agar plate. A 24 h BHI broth culture of Vibrio spp., and SHPB was used. The suspensions of
individual bacteria after harvesting and adjusting the cell density to 10
4
/ mL were used.
Both the suspensions were spread (100 L each) on a BHI agar plate and incubated for
observing the colony formation. The colonies were observed, under the microscope, at 0, 3
and 6 h of incubation at 30
o
C for competitive exclusion or invasive growth. The SHPB
showed a distinctive competitive exclusion against Vibrio spp., (plate 2).
Health and Environment in Aquaculture
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Plate 2. Competitive exclusion of SHPB (P) against one pathogenic Vibrio (V) After 6 h
(1a &1b) and after 3 h (1c & 1d). 20 x.
4. Effect of autochthonous probiotics on rotifer proliferation
Several studies related to marine fish larviculture have attempted to find suitable probiotics
that has a positive effect on the live food and improves their dietary value (Benetti et al.,
2004; Robertson et al., 2000; Gomez-Gil et al., 2000; Ringo and Birkbeck, 1999; Harzevilli et
al., 1998; Gatesoupe, 1991a). Douillet (2000) reported that rotifers cultured with an
Altermonas strain or blend of strains resulted in a consistent enhancement of the rotifers
culture. In this study, rotifers Brachionus plicatilis were enriched for 24 h with a high quality
mixture of algae (Nanochloropsis, Tetraselmis and Isochrysis) along with the available
commercial enrichments (Super-Selco and DHA protein selco). The rotifer density was 22
cell/ mL. A one week experiment was carried out to evaluate the proliferation of the rotifers
treated with probiotics SHPB, 4SQI, 5L82 and L. divergens. Daily 1 mL of the probiotic and 1
mL of the algal suspension was added to each flask, which corresponds to 10
10
CFU/ mL.
The control flasks received 2 mL algae daily. The rotifer count was determined for seven
days to assess the effect of the probiotics. An increase in rotifers proliferation and
reproducibility within different treatments was obtained, as described by the differences in
the coefficient of determination for four probiotics treatments and the control (Fig. 1). The
probiotic SHPB and L.divergens showed better enhancement on the cell population with time,
while 4SQI and 5L8 showed lower cell proliferation response compared to the control. The
SHPB gave the best compared to the control and other probiotics, mainly during the first
three days. All the probiotics tested showed better effect on the rotifer counts compared to
the control. The Vibrio load in the rotifer population without probiotic treatment was
significantly higher than that of the probiotic treated sets during the first three days.
However, the vibrio population was significantly lower in SHPB treated rotifer all through
the week. The sobaity larvae usually start feeding on day two or three post hatch. Thus, the
Probiotics in Aquaculture of Kuwait Current State and Prospect
231
Fig. 1. The effect of SHPB (B. halotolerance), LD (L. divergens), 4SQI, 5L82 on the growth
performance of rotifers compared with the control (Con).
rotifers are initially added on these days with a starting density of 5 rotifer/ mL. The
probiotics were added daily, this could explain the significant effect mainly for the SHPB on
rotifers proliferation all through out the experiment. Repetitive addition of the probiotics
can significantly enhance the rotifers performance and survival. The result of this study is in
agreement with the results obtained by Planas et al., (2006). They reported that continuous
additions of probiotic (Rosebacter strain) are necessary to maintain a minimum level of it in
the rotifer and the culture water. In addition, exposing the larvae to sufficient probiotic
concentrations will increase the chance of the probiotics being ingested by the larvae. Thus,
this could lead to the advantage of improving the survival rate of the fish larvae.
5. Effect of SHPB and L. divergens used alone and in combination (SHPB &
L. divergens) on the survival of sobaity larvae
Owing to the problem of antibiotic resistance and subsequent reluctance of using antibiotics,
the use of probiotics in larviculture is becoming increasingly popular. During the early
stages of development, manipulation of the larval digestive system seems possible through
the addition of probiotics, either through the culture water, or via the live food (Vine et al.,
2006). Geovanny et al. (2007) reported that the use of probiotics can increase the survival
rate and control the high incidences of larval diseases. Thus, by shifting the bacterial flora in
live feed organisms to probiotic species, this can assist the fish larvae to minimize the
pathogenic bacteria from the feed, and the fry will benefit from the probiotic bacteria.
Several bacterial probiotics were used in the larval culture of aquatic organisms. Kozasa
(1986) reported that the spores of Bacillus toyoi increased the growth rate of yellowtail and
reduced the mortality of Japanese eel that were infected by Edwardsiella sp. The Gram-
negative Vibrio pelagius decreased the mortality of the turbot larvae Scophthalmus maximus
challenged with Aeromonas caviae (Ringo and Vadstein, 1998). Gatesoupe (1991b) showed
that Bacillus toyoi and Bacillus sp spores increased the growth rate of larval turbot introduced
via the rotifer Brachionus plicatilis. Pirarat et al., (2006), reported that supplementation of
Health and Environment in Aquaculture
232
L. rhamnosus significantly reduced cumulative mortality due to E. tarda, confirming the
protective effect of a probiotic bacterium against this pathogenic bacterium. Suzer et al.,
2008 showed significant increase in the survival rate of Sparus auratus larvae fed with
Lactobacillus spp. via live food and water. In this study, the effect of single and combined
administration of SHPB and L. divergens on the survival rate of sobaity larvae was evaluated.
The larvae were reared in 1m
3
circular fiberglass tanks with stocking density of 60 larvae/ L.
The enriched rotifers were added to the larval tanks and the rotifer density was maintained
at 5/ mL. The survival rate of the larvae was determined at 28 days post hatch. The results
showed that all the treatment with probiotics significantly enhanced the survival rate
compared to the control. The survival in L. divergens fed larvae was the highest (11.7%)
whereas it was 9.2% in the mixed probiotics, 8.9% in SHPB and 6.3% in the control (Fig 2).
Balcazar et al., 2007, demonstrated that the administration of a mixture of bacterial strains
(Bacillus sp. and Vibrio sp.) positively influenced the growth and survival of white shrimp
juvenile. Salinas et al., 2008, showed that the combined probiotics, L. delbruekii and B. subtilis
enhanced the cellular innate immune system of gilthead seabream. Suzer et al., 2008,
showed that Sparus aurata larvae fed with commercial Lactobacillus spp via live food
increases survival rate and specific growth rate. To our knowledge there have been no
studies on any probiotic on the blue- finned Sparidentex hasta (sobaity). So, in our study,
since all the tested probiotics showed significant survival rate compared to the control, the
dietary administration of both singly SHPB and L.divergens and in combination (SHPB and
L. divergens) were used and they showed significant increased in the survival rate of sobaity
larvae and seems to be a promising probiotic candidate. However, feeding sobaity larvae
with a combination of them needs further dose adjustment to achieve the best survival rate
and possible beneficial interaction between both bacteria in sobaity gut microenvironment,
which may make the use of a mixture of different bacterial strains more interesting than
using a single bacterium.
Fig. 2. The survival rate of sobaity larvae fed with SHPB (B. halotolerance), LD (L. divergens)
and their combination (Mix) compared with a control (Con) group fed with un-
supplemented diet.
Probiotics in Aquaculture of Kuwait Current State and Prospect
233
6. Pathogen challenge test for probiotic-fed sobaity larvae
Increased resistance to the pathogen by probiotics has been extensively reported. Growth
inhibition against pathogens by Carnobacterium was reported (Joborn et al., 1997). The
tolerance of rainbow trout Oncorhynchus mykiss to furunculosis was enhanced when fed with a
diet including the probiotic L. rhamnosus (Nikoskelainen et al., 2001). In Atlantic cod Gadus
morhua, tolerance to V. anguillarum increased by feeding with lactic acid bacteria
Carnobacterium divergens supplemented in the diet (Gildberg et al., 1998). Robertson et al ., 2000)
reported that Atlantic salmon Salmo salar and rainbow trout O. mykiss, fed with Carnobacterium
spp. supplemented in the diet were more tolerant to disease. Chiu et al., 2010, showed that S.
cerevisiae colonized the intestines of the grouper E. coioides fed S. cerevisiae-supplemented diets
improved and increased the resistance to challenge by Streptococcus sp. and a grouper
iridovirus. In our study, after feeding the larvae with probiotic-enriched rotifers (SHBP/ L.
divergens / mixture of SHBP and L. divergens) for 28 days, the larvae challenged through
immersion against virulent Vibrio harveyi. The bath suspension contained a cell density of 10
7
cells m
-1
and the duration of challenge was 30 min. Fish larvae were transferred to 50 L
aquarium tanks (50 fish/ tank) after the challenge, mortalities were observed and recorded for
one week and the squash preparations of washed freshly dead larvae were plated on TCBS
agar to record specific mortalities. The results showed that fish larvae fed with SHPB and
combination probiotics showed clear disease resistance as indicated by distinctive 85.3% and
58% survival rate for SHPB and mixed treatments respectively compared to L.divergens-fed fish
54% and control 50%. Pieters et al., 2008, reported that challenge with A. bestiarum, the
probiotics GC2 and BA211 led to 76% and 88% survival, respectively, in contrast to 22%
survival for the controls. In the current study, it is apparent that SHPB and the mixed
probiotics (SHPB & L. divergens) fulfilled the major requirements of being an effective
probiotics by enhancing the survival rate of sobaity larvae after challenge with virulent V.
harveyi. However, the survival of larvae fed a combination of probiotics was not as good as
that of SHPB and here, probably the dose structuring needs to be optimized for furthering the
effects of a combination of probiotics (Fig 3).
Fig. 3. The survival rate of sobaity larvae challenged against V. harveyi after feeding with
SHPB (B. halotolerance), LD (L. divergens) and their combination (Mix) compared with a
control (Con) group fed with un-supplemented diet.
Health and Environment in Aquaculture
234
7. Molecular characterization of SHPB probiotic and bacteriocin-like
compound
The SHPB was characterized using the PCR and 16s rDNA gene amplification (Al-Marzouk
et al., 2009). The identification of SHBP probiotic confirmed as Bacillus halotolerance. The
modes of action of probiotics include the inhibition of a pathogen through the production of
bacteriocin-like compounds, competition for attachment sites, competition for nutrients
(particularly iron in marine microbes), alteration of enzymatic activity of pathogens,
immunostimulatory functions, and nutritional benefits such as improving feed digestibility
and feed utilization (Kesarcodi-Watson et al., 2008; Fuller, 1989). Thus, an understanding of
the mechanisms probiotics use to compete with pathogens is important when designing a
protocol for their selection. Bacteriocins are antibacterial proteins produced by bacteria to
kill or inhibit the growth of other bacteria (Cleveland et al., 2001). They are ribosomally
synthesized unlike antibiotics, which are synthesized by other mechanisms (Brock &
Madigan, 1997). In this study, B. halotolerance (SHBP) cultures of different age (12h, 24h, 36h
and 48h) were used for detecting the possible role of bacteriocin in the antibacterial activity.
The mode of action of B. halotolerance was confirmed through its ability to produce
bacteriocin-like compound, which is considered as a significant criterion of the defense
system displayed by it. It produced an amplicon of approximately 1500 bp and for the
bacteriocin gene a 1000 bp amplicon (Plate 3). Cultures of different age, however, showed
interesting amplification pattern with clear amplification of the bacteriocin-like gene in 24-h
culture and a very mild amplification in 12-h culture. This state was tested by treating the
compound (probiotic bacteria free BHI broth) with different pH and temperatures. Later, the
treated broth was used in antibacterial assay. The persistence of antibacterial activity of the
Plate 3. Polymerase Chain Reaction (PCR) amplification of the bacteria specific 16s rDNA
(approximately 1500 bp, brighter band) and bacteriocin gene (approximately 1000 bp).
Lanes: 1: 100 bp DNA ladder; 2: Negative sample; 3: Positive amplicon (kit); 4 to 7 probiotic
bacterial cultures (12h, 24h, 36 h and 48h) of B. halotolerance.
Probiotics in Aquaculture of Kuwait Current State and Prospect
235
treated broth confirmed that it a bacteriocin- like compound of the probiotic that was
responsible for the antagonism. One of the most well known bacteriocins is nisin, which is a
ribosomally synthesized antimicrobial peptide produced by certain strains of Lactococcus
lactis which has been proved to act against human multidrug resistant pathogens such
Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis
and others (Gatesoupe, 2008). Further research will be required to specify the exact type of
bacteriocin produced by the probiotic B. halotolerance.
8. Persistence of probiotics in the fish gut
Adhesion and colonization are important for selection and use of probiotic strains (Bussarin
& Rakshit, 2006; Vine et al., 2004; Olsson et al., 1992) and because it is considered a pre-
requisite for colonization (Beachey, 1981). Adhesion of probiotic bacteria to the intestinal
mucosa has been shown to enhance their antagonistic activity against pathogens (Coconnier
et al., 2003). In this study, the ability of SHPB, L. divergens and their combination to attach to
fish intestinal mucus were examined. Colonization of probiotics fed through diets was
monitored at 15 and 30 d during the probiotic feeding and at one month after the probiotic-
enriched feed was withdrawn. Colonization of the SHPB was evident even at 15 d (Plate 4).
The intense localization on the brush boarders of the hindgut intestinal epithelium noticed
at 15 and 30 d (Plates 4 & 5) during feeding, suggests that the SHPB is more likely to
Plate 4. Sobaity gut fed with SHPB, L. divergens & mixed (SHPB & L. divergens) and control
diets. Arrows: IMP: Immunopositive; GV: Gut vacuole; BB: brush boarder. Left (40 x) and
right. 10 x.
Health and Environment in Aquaculture
236
Arrows: HE: hindgut epithelium; LP= lamina propria of the hindgut.
Plate 5. Gut of sobaity sampled after 30 days of continuous feeding with SHPB and L.
divergens-(top right, 20 x), mixed probiotics (bottom left, 40x) and control (bottom right,
10x) diets. Note immunopositive SHPB colonization on the hindgut epithelium (top left). No
immunopositive bluish-purple reaction in the gut of control fish (bottom right, 10x).
colonize in the hind gut of sobaity than the L. divergens, which was processed as an antigen
and did not remain in the gut long enough (almost no localization at one month post
withdrawal). Clear and significant immunopositive localization (Plate 4) was noticed in gut
of both probiotic SHPB and mixed (SHPB and L. divergens) probiotic-fed sobaity even after
withdrawal of probiotic feeding. This indicates the higher persistence of the SHPB than that
of L. divergens. The process of colonization is characterized by attraction of bacteria to the
mucosal surface, followed by association within the mucous gel or attachment to epithelial
cells. Adhesion and colonization of the mucosal surfaces are possible protective mechanisms
against pathogens through competition for binding sites and nutrients, or immune
modulation (Balcazar et al., 2006). However it needs to be accepted that the efficiency of a
selected probiotic in vitro may significantly change when administered to the host because it
is influenced by more complex factors such as the selective ingestion and the death in the
intestinal tract (Vine et al., 2006) caused by the failure of the probiotic to maintain its in vitro
physiology under circumstances of a more complex microbial interactions and/or
nutritional environment. In general, there is a sense of the lack of correlation between in
vitro and in vivo experiments in the latest reviews on probiotic use in aquaculture (Balcazar
et al., 2006; Vine et al., 2006). The main claimed mechanisms are: competitive exclusion,
digestion enhancement, immune response enhancement, water quality improvement and
antiviral effects.
Probiotics in Aquaculture of Kuwait Current State and Prospect
237
No immunopositive bluish-purple reaction in the gut of control fish (bottom right).
Arrows: IPV: immunopositive vacuoles.
Plate 6. After 30 days of withdrawal of feeding with SHPB-(top left, 40x), L.divergens-(top
right, 40x), mixed probiotics (bottom left 40x)) and control (bottom right (40 x)) diets.
9. Bacterial count in gastrointestinal tract in probiotics-fed sobaity
No Vibrio sp., was detected in the vibrio selective medium (TCBS), from the fish gut fed
from SHPB and mixed probiotics (SHPB and L. divergens). The highest bacterial count was
counted in the control and L.divergens treatment (Table 1). The main bacterial colonies that
were detected in the brain heart infusion agar (BHIA) from fish fed with SHBP.
Treatment Media 45d 60d 75d 105d
SHPB BHIA 6.30 10
4
1.00 10
3
0.30 10
2
0.80 x 10
3
TCBS - - - -
LD BHIA 3.20 10
3
4.00 10
2
1.70 10
3
0.90 10
4
TCBS - - 1.00 10
2
15.00 0
4
Mix BHIA 3.00 10
3
6.60 10
3
6.20 10
3
0.30 10
4
TCBS - - - -
Con BHIA 1.30 10
4
6.50 10
2
4.10 10
3
2.10 10
3
TCBS - - 0.30 10
3
1.00 10
3
Table 1. Bacterial Count from the Gut of Sobaity Fed with SHPB (B. halotolerance), LD (L.
divergens) and their Combination (Mix) Compared with Control (Con).
Health and Environment in Aquaculture
238
10. Pathogen challenge test for probiotic-fed sobaity fry
Vendrell et al., 2008 showed that Probiotic supplementation of Leuconostoc mesenteroides and
Lactobacillus plantarum reduced fish mortality significantly from 78% in the control group to
46-54% in the probiotic groups after challenged with Lactococcus garvieae. In this study,
sobaity fry fed with SHPB and mixed probiotics showed clear disease resistance as indicated
by distinctive 100% survival rate compared to L.divergens fed fish and control (Table 2).
Based on the ability of the SHPB and mixed probiotics to attach to fish gut, the growth of the
pathogen in the digestive tract might be suppressed by the candidate probiotics presence.
Treatments Survival Rate (%)
SHBP 100
LD 16.6
Mix 100
Con 66
Table 2. Survival Rate of Control (Con) and Probiotic-Fed (SHPB, LD and Mix) Sobaity Fry
after Challenge with V. anguillarum
11. Immunological assays
The non-specific immune system can be stimulated by probiotics. Taoka et al., 2006
indicated that probiotics supplied in the rearing water and the diet of fish enhanced the
stress tolerance and the non-specific immune system of Japanese flounder, providing them a
higher resistance against stress conditions and pathogens. It has been demonstrated that
oral administration of Clostridium butyricum bacteria to rainbow trout enhanced the
resistance of fish to vibriosis, by increasing the phagocytic activity of leucocytes (Sakai et al.,
1995). Balcazar (2003) demonstrated that the administration of a mixture of bacterial strains
(Bacillus sp. & Vibrio sp.) positively influenced the growth and survival of juveniles of white
shrimp and presented a protective effect against the pathogens Vibrio harveyi and white spot
syndrome virus. This protection was due to a stimulation of the immune system, by
increasing phagocytosis and antibacterial activity. In this study, custom-production and
characterization of rabbit polyvalent antibodies against B. halotolerance and L. divergens were
carried out using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
and Western blot analysis, and in addition, to evaluate both the humoral and cellular innate
immunity responses of sobaity fed for 75 d with the above mentioned probiotics. These
included the phagocytic activity; lysozyme activity, serum immunoglobulin and alternative
complement (ACH50). Different immunological parameters, mainly serum and mucus
lysozyme, phagocyte activity and complement were enhanced in sobaity fry fed with B.
halotolerance and the mixed probiotics for 75 and 105 days.
11.1 Lysozyme activity
Lysozyme has an important role in non-specific immune defense system and it contained in
the mucus on the fish body surface, and in plasma and liver. Lysozyme has an antibiotic
ability and is released by leukocytes. It can damage bacterial cell walls, especially of Gram-
positive and some Gram-negative bacteria (Grinde, 1989). Lysozyme activity varies between
species of fish, genetic strains and different pathogens. Several reports showed the effect of
Probiotics in Aquaculture of Kuwait Current State and Prospect
239
probiotics on lysozyme activity. A significant increase in lysozyme activity was observed in
Nile tilapia, O. niloticus, fed diets containing S. cerevisiae for 21 days (El-Boshy et al., 2010).
Gatesoupe (2008) reported that feeding with Gram-positive and Gram negative potential
probiotics caused increase in the cellular parameters such as macrophages and enhanced
lysozyme activity. Kim & Austin (2006) recorded high gut mucosal lysozyme activity in fish
fed with Caronbacterium divergens B 33 and Carnobacterium maltaromaticum B26. Taoka et al.,
2006, also reported an enhanced lysozyme level in tilapia fed with live and dead probiotics
and recorded a high survival rate when challenged with Edwardsiella tarda. Panigrahi et al.,
2004, showed significantly higher lysozyme activity in rainbow trout fed with Lactobacilllus
rhamnosus. Apart from serum lysozyme content, probiotics can also enhance the lysozyme
level in skin mucosa of fish (Song et al., 2006). Lysozyme in fish can be measured either by
the turbidimetric method or the agarose plate assay. Each method was developed based on
the amount of lysis of the gram positive bacteria Micrococcus lysodeikticus. In this study,
lysozyme activity in serum was determined according to the method of Demers and Bayne
(1997) based on the lysis of the lysozyme- sensitive Gram positive bacterium, Micrococcus
lysodeikticus (Sigma, St. Louis). With the help of a computer application software, Delta Soft
3 (Biometalics Inc., New Jersey, USA) the equivalent unit of activity of the sample as
compared to the standard were determined and expressed as g ml
-1
serum. The
turbidimetric assay results can be reported by two methods; relative lysozyme activity
measured in Units/min, and by comparison to a standard curve generated using purified.
Our turbidimetric assay results were reported by relative lysozyme activity which measured
in Units/min, and by comparison to a standard curve generated using purified hen egg
white lysozyme. The results showed that sobaity fry fed with three types of probiotics has
significant higher lysozyme activity (Table 3).
Treatments
ACH50 (log
2
) titers
Lysozyme (units/min)
Serum Mucus
75d 105d 75d 105d 75d 105d
SHPB 6.67 0.58 5.67 0.58 18.63 4.50 17.00 6.61 21.25 4.53 26.38 7.37
LD 3.67 0.58 4.67 0.58 15.50 4.34 15.38 5.66 18.63 5.15 20.63 4.60
Mix 4.67 0.58 6.67 0.58 20.13 4.26 17.13 7.24 22.38 4.69 20.38 2.26
Con 2.33 0.58 3.67 0.58 11.63 3.81 11.75 2.38 14.13 3.98 12.38 3.50
ACH50 Tukeys HSD=1.35 for treatments; for time HSD= 1.0. ; serum lysozyme HSD= 5.74 for
treatments; for time HSD= 4.344; mucus lysozyme HSD= 4.56 for treatments; for time HSD= 3.45.
Values less than HSD values considered to be not significant
Table 3. Alternative Complement (ACH50) Titers (log
2
), Serum and Mucus Lysozyme
Content of Probiotic-Fed Sobaity (75 d) = After Probiotic feeding & (105 d) = After 30 d of
Withdrawal of Probiotic feed
11.2 Alternative complement activity (ACH50)
The alternative pathway of complement activity has emerged as a powerful nonspecific
defense mechanism for protecting fish against a wide range of potentially invasive
organisms, such as bacteria, fungi, viruses and parasites (Muller-Eberhard, 1988). Chiu et
al., 2010, showed that Epinephelus coioides fed S. cerevisiae-supplemented diets exhibited
Health and Environment in Aquaculture
240
significant increases in both serum lysozyme and alternative complement pathway
activities (ACH50). The same result was also observed in hybrid tilapia fed diets containing
DVAQUA
alloantigen-specific
cytotoxic cells presumed to be oT cells (Zhou et al., 2001). In the model using rainbow
trout PBLs, the presence and function of CTLs has been documented thanks to the use of
clonal trout effectors and MHC I-matching RTG-2 cell line targets, both sharing the same
allele (Dijkstra et al., 2003). Sensitized-rainbow trout showed that only sorted IgM
negative (sIgM
-
) PBLs were able to kill the targets in a specific manner (Fischer et al., 2003,
2006). These data suggested the involvement of trout CTLs that was further evidenced by
the up-regulation of TCRo and CD8o genes in these sIgM
-
cells
after allogeneic cell
immunization. The generation of monoclonal antibodies for rainbow trout CD8o has
allowed further characterization of this population (Takizawa et al., 2011). Sorted trout
CD8o
+
lymphocytes showed great expression of perforin or NK-lysin genes (related to the
cytotoxic activity, either specific or not), as well as their up-regulation upon stimulation
with the T-lymphocyte-mitogen PHA-L. However, further studies are still needed to
clearly identify them as the trout specific cytotoxic cells or effectors since tissue
distribution and gene expression pattern in CD8o
+
cells show some contradictory results
and deserve deeper analysis. In the last model, the use of clonal ginbuna crucian carp has
been very productive. They firstly proved the existence of specific cytotoxic response
against syngeneic virus-infected cells (Somamoto et al., 2000, 2002, 2006). Afterwards,
they have purified CD8o
+
, CD4
+
, IgM
+
and CD8o
-
CD4
-
IgM
-
leucocytes by means of
house-produced antibodies and found that only the CD8o
+
population was able to kill the
allotargets in a specific manner, what definitely demonstrates the specific cytotoxic
activity of fish CTLs (Toda et al., 2009). Moreover, they have also showed that these CTLs
mediate the target cell killing by the perforin-mediated pathway since perforin and
granzyme B inhibitors abolished almost completely the cytotoxic activity (Toda et al.,
2011a, 2011b).
Further studies in other fish species have documented the presence of TCR and CD8 genes
indicating presence of CTLs, but functional characterization of the CTL-mediated CMC
activity is still lacking. Thus, CD8 genes, alpha or beta chains, have also been sequenced in
fugu (Takifugu rubripes) (Suetake et al., 2007), Atlantic salmon (Salmo salar) (Moore et al.,
2005), European sea bass (Buonocore et al., 2006), gilthead seabream (Randelli et al., 2006),
Atlantic halibut (Hippoglossus hippoglossus) (Patel et al., 2008), common carp (Sun et al., 2007)
or orange-spotted grouper (Xu et al., 2011). Unfortunately, CD8o gene might not be the
definite CTL marker. In mammals, CTLs are characterized by the presence of the CD8o|
while the expression of the homodimer CD8oo is detected in NK cells, oT cells and
intestinal intraepithelial lymphocytes (Bonneville & Lang, 2002). Thus, unexpected data
obtained in the functional characterization of CD8o
+
-purified lymphocytes could reside in
the potential purification of other cells different to CTLs with non-specific activity.
However, further studies are needed to clearly ascertain the CTL presence, distribution and
role in these fish species, some of them with aquaculture interest.
Fighting Virus and Parasites with Fish Cytotoxic Cells
283
3. Cytotoxic response against fish tumors
Fish tumors are quite rare in the wild. However, aquaculture management, intensive culture
conditions and environmental contamination may increase the incidence of fish tumors.
Although some aspects, such as tumour structure and nature, metastasis or lethal effects
have been studied, little information exists concerning the involvement of the immune
system in protection against tumours (Campbell et al., 2001; McKinney & Schmale, 1994a,
1994b, 1997; Romano & Marozzi, 2004; Schmale et al., 1994, 2004; Thompson & Kostiala,
1990; Vicha & Schmale, 1994). Thus, most of the information regarding fish cytotoxic activity
comes from the use of hapten-modified autologous cells or xenogeneic/allogeneic cell lines
(Evans et al., 1984a-d, 1987; Fischer et al., 2006; Graves et al., 1984; Manning & Nakanishi,
1996; Nakanishi et al., 2002; Shen et al., 2002; Verlhac et al., 1990). So far, fish immune
response against tumors has been slightly evaluated. In the bicolour damselfish naturally
suffering of neurofibromatosis (DNF) (caused by a retrovirus), study of the immune
response has provided information with respect to CMC activity, morphology and
distribution, degranulation of eosinophilic granular cells (EGCs) and lymphocyte
proliferation (Vicha & Schmale, 1994; McKinney and Schmale, 1994a, 1994b; Campbell et al.
2001; Schmale et al. 2004). Most of the cytotoxic activity of damselfish leucocytes against
DNF-derived target cell lines resided in the spleen whilst in the head-kidney it was quite
low. Interestingly, specificity suggested that this activity was likely carried out by CTLs in
the spleen and by NCCs in the pronephros (McKinney & Schmale, 1994a). Later, they
demonstrated that the 5C6
-
lymphocytes showed all the cytotoxic activity against the
retrovirus-infected DNF tumor cell lines, suggesting the presence and role of damselfish
CTLs, whilst the 5C6
+
leucocytes were only able to kill xenogeneic erythrocytes (McKinney
& Schmale, 1997). Unfortunately, deeper characterization of this CMC model has been
abandoned.
The use of zebrafish as a model for human cancer would also help to understand the fish
immune response against tumors, and concretely the role played by cytotoxic cells. As
mentioned above, zebrafish showed NCCs in the peritoneal cavity that were positive for the
5C6 antibody and exerted cytotoxic activity against xenogeneic tumor cells (Moss et al.,
2009). Moreover, the complete genome sequence allow to identify major molecules involved
in the cytotoxic activity such as NCCRP-1, TCR, CD8, perforin, granzymes, Fas/FasL, etc.
The easy generation of transgenic zebrafish and mutants would also be a very valuable tool
to study the fish CMC activity against tumors. Further studies should focus on the leucocyte
infiltration to the tumor site and identification of the potential molecules involved in the
activity of the cytotoxic cells.
4. Cytotoxic response against parasites
Fish parasites represent a serious problem in the aquaculture since there are no available
vaccines or effective treatments. Whilst some aspects of the fish immune response against
parasites have been studied very little is known about the role of the cell-mediated cytotoxic
activity (Buchmann et al., 2001; Jones, 2001). First study evaluated the NCC activity in
catfish parasitized with Ichthyophthirius multifiliis (Graves et al., 1985a). They found that
moribund Ichthyophthirius multifiliis-infected fish showed decreased NCC activity in the
head-kidney against xenogeneic cells when compared to control specimens. Strikingly, this
activity was increased in the PBLs of the same fish as consequence of an activation of the
Health and Environment in Aquaculture
284
NCC killing capacity and affinity (Graves et a., 1985a). A second study determined that
catfish NCC were able to bind and kill 50-60% of Tetrahymena pyriformis after 10 h of co-
incubation (Graves et al., 1985b). Furthermore, NCC binding to xenogeneic tumor cells and
Ichthyophthirius multifiliis or Tetrahymena pyriformis parasites shared the same antigen, that in
the case of parasites, consist on a 46 kDa (Evans et al., 1998a, 1996; Graves et al., 1985a; Jaso-
Friedmann et al., 1997b; Lester et al., 1994). In another study, gilthead seabream specimens
were parasitized with the enteric Enteromyxum leei parasite (Cuesta et al., 2006). This
parasitation increased head-kidney NCC activity against tumor cells indicating that
parasitized fish posses enhanced cytotoxic cells activity. Moreover, parasite-exposed fish
either parasitized or not, showed increased NCC activity. However, no other study has
evaluated the role of the cell-mediated cytotoxicity against fish parasites and deserves
further evaluation due to the interest for aquaculture industry.
5. Cytotoxic response against viral infections
Viral diseases are responsible for most of the economic losses suffered in modern
aquaculture since they produce high levels of mortality and no effective antiviral
treatments are available. Moreover, fish farming practices such as growth under very high
densities, introduction of species in new areas, continuous transport between hatcheries,
nurseries and growing plants are increasing the spread of pathogens and the number of
susceptible and reservoir species. However, while most available information focuses on
the mechanisms involved in pathogen susceptible fish immune responses, further
knowledge is also important in pathogen-reservoir fish systems. Among the major
immune mechanisms to kill virus, the interferon (IFN) and the CMC are the most
important, but most efforts have only focused on the IFN pathway (Ellis, 2001; Robertsen,
2006). Regarding the CMC activity against virus, this can be mediated by innate or
specific cytotoxic cells (Table 2). Regarding the innate CMC activity against virus-infected
cells, first studies demonstrated that salmonid kidney, spleen and PBL leucocytes were
able to kill infectious pancreatic necrosis virus (IPNV)-infected cells much more than to
non-infected cells (Moody et al., 1985; Yoshinaga et al., 1994), and similarly in catfish
against channel catfish virus (CCV)-infected cells (Hogan et al., 1996), demonstrating the
antiviral activity of fish NCC and NK-like cells, respectively. In the orange-spotted
grouper, CD8
+
PBLs also showed non-specific cytotoxic activity against nodavirus
(nervous necrosis virus or NNV)-infected cells suggesting a role for NK-like or oT cells
(Chang et al., 2011). Fish exposure to virus also increases the fish innate cytotoxic activity.
Thus, gilthead seabream injected with viral hemorrhagic septicemia virus (VHSV), which
did not replicate at the assayed conditions, increased the NCC activity, demonstrating the
importance of studying the antiviral immune response in reservoir fish species (Esteban et
al., 2008). Moreover, NNV-infection increased the NCC activity of head-kidney leucocytes
from 1 to 15 days post-injection in both gilthead seabream and European sea bass
(unpublished data). Recently, we have also demonstrated that trout RTS11 (monocyte-
macrophage cell line) cells exposed to VHSV increased their cytotoxic activity against
xenogeneic tumor cells and up-regulated the NKEF (natural killer enhancing factor),
granzyme and perforin gene expression whilst trout head-kidney leucocyte infection with
the VHSV increased the innate cytotoxic activity but failed to significantly change the
expression of these genes (Ords et al., 2011).
Fighting Virus and Parasites with Fish Cytotoxic Cells
285
Fish CMC activity References
Channel catfish NK-like activity against CCV-infected cells Hogan et al., 1996
Atlantic salmon CMC activity against IPNV-infected cells Moody et al., 1985
Rainbow trout CMC activity against IPNV-infected cells Moody et al., 1985
Yoshinaga et al., 1994
VHSV infection induced innate CMC activity, up-
regulated NKEF, CD8o, perforin and granzyme
genes
Cuesta & Tafalla, 2009
Utke et al., 2007
Unpublished data
VHSV infection elicited specific CMC activity, up-
regulated CD8 o gene
Fischer et al., 2006
Utke et al., 2007
VHSV DNA vaccine elicited specific CMC activity Utke et al., 2008
VHSV and IPNV DNA vaccines up-regulated
NKEF, perforin and granzyme genes
Cuesta & Tafalla, 2009
Cuesta et al., 2010
Unpublished data
VHSV infection of RTS11 cells increased the CMC
activity, up-regulated NKEF, granzyme and
perforin genes
Ords et al., 2011
Ginbuna crucian
carp
IPNV or EVA infection elicited specific CMC
activity
Somamoto et al., 2000
CHNV infection elicited specific CMC activity, up-
regulated TCR| and CD8o genes
Somamoto et al., 2002
Somamoto et al., 2006
Generation of in vitro virus-specific CTLs and up-
regulation of TCR| and CD8 o genes
Somamoto et al., 2009
Anal immunization with CHNV-infected cells
elicited specific CMC activity
Sato & Okamoto, 2010
Common carp SVCV infection up-regulated, granzyme A/K or
CD8o genes
Forlenza et al., 2008
Huang et al., 2010
Gilthead
seabream
NCC activity induced by VHSV injection Esteban et al., 2008
NCC activity induced by NNV infection Unpublished data
Sea bass NNV infection no affected TCR| and CD8o genes Scapigliati et al., 2010
Atlantic halibut NNV infection no affected CD8o and CD8| genes Patel et al., 2008
Orange-spotted
grouper
CMC activity against NNV- or RSIV-infected cells Chang et al., 2011
NNV infection elicited specific CMC activity,
increased CD8o
+
cells and CD8o gene
Chang et al., 2011
Japanese
flounder
VHSV infection up-regulated CD8 gene Byon et al., 2005
VHSV DNA vaccine up-regulated CD8 gene Byon et al., 2006
CMC, cell-mediated cytotoxicity; CCV, channel catfish virus; IPNV, infectious pancreatic necrosis virus;
VHSV, viral hemorrhagic septicaemia virus; EVA, eel virus from America; CHNV, crucian carp
haematopoietic virus; RSIV, red seabream iridovirus; SVCV, spring viremia carp virus; NNV, nervous
necrosis virus; CTL, cytotoxic T lymphocytes; TCR, T cell receptor; NKEF, natural killer enhancing factor.
Table 2. Major studies evaluating the fish CMC activity against virus.
Health and Environment in Aquaculture
286
Viral infections also elicited the specific immune response by inducing antibody production
and CTL activity (Table 2) (Nakanishi et al., in press). First studies demonstrated that
isogeneic ginbuna crucian carp elicited CTL activity against virus. Thus, ginbuna crucian
carps immunized with hematopoietic necrosis virus (CHNV) specifically killed CHNV-
infected syngeneic cells in a viral antigen and MHC I-restricted manner (Somamoto et al.,
2000, 2002), increased the TCR| and CD8o gene expression (Somamoto et al., 2006) and
helped to establish virus-dependent CTL clones in vitro (Somamoto et al., 2009). In rainbow
trout, infection with VHSV greatly elicited specific- and MHC I-matched cytotoxic cells but a
non-specific and MHC I-mismatched cytotoxic activity was also found (Fischer et al., 2006;
Utke et al., 2007). Surprisingly, they found that specific CMC activity mediated by CTLs was
produced much earlier than the innate activity, in sharp contrast to all the information at
this respect. Strikingly, the NKEF gene expression followed the same time-profile than the
CTL activity but in the case of CD8o was opposite, adding more controversy to these data
(Utke et al., 2007). Furthermore, trout vaccination with VHSV DNA vaccines also elicited
CMC activity against MHC I-matched infected cells, suggesting a role for CTLs (Utke et al.,
2008). However, they also found a bit lower CMC activity against non-matching-infected
cells or cells infected with a different virus, suggesting a role for NCCs or even the ADCC
activity since these fish showed high antibody levels, but this has not been confirmed. In
other studies, VHSV infection increased the trout NKEF and CD8o gene in vivo but failed to
modulate the NKEF, perforin and granzyme genes in vitro (Cuesta & Tafalla, 2009; Ords et
al., 2011). VHSV and IPNV DNA vaccination also up-regulated the trout CD8o, perforin and
granzyme gene expression (Cuesta et al., 2010; unpublished data), giving more consistency
to the involvement of CMC activity against viral infections and its activation by DNA
vaccines. Moreover, oral vaccination with inactivated CHNV elicited specific CMC activity
that resulted viral antigen-specific and restricted to the MHC I (Sato & Okamoto, 2010). In
the orange-spotted grouper, nodavirus infection also elicited a CTL response when viral
antigens were properly presented by MHC I receptors, as well as increased the CD8o
expression at gene and CTL surfaces (Chang et al., 2011). This study represents the first one
demonstrating the CTL role against viral infection in marine fish species with great interest
for aquaculture industry. Further studies would help to understand the CMC activity
against viral infections and to design and probe viral vaccines.
6. Modulation of the fish cytotoxic activity
Fish CMC activity regulation has been widely evaluated and mostly focused on NCC
modulation. Fish NCC activity has been shown to be modulated by several chemicals,
cytokines, environmental contaminants, stress factors, immunostimulants, etc. First studies
dealt with the NCC inhibition by blocking the binding to target in order to characterize the
role of NCCRP-1, or inhibiting the killing mechanisms in order to evaluate the perforin- or
Fas/FasL-mediated lytic pathway (Bishop et al., 2002; Carlson et al., 1985; Evans et al., 1988,
2000; Hogan et al., 1999; Iwanowicz et al., 2004; Jaso-Friedmann et al., 1988, 2001; Kaur et al.,
2004; Shen et al., 2002). Further studies demonstrated that catfish NCC activity is increased
by leucocyte treatment with ionophore A23187, A23187 plus phorbol myristate acetate
(PMA) or vanadate but no with PMA alone or poly I:C (a mimic for viral infections) (Evans
et al., 1984b, 1990, 1998b). Moreover, serum from stressed fish contained cytokine-like
factors able to increase the tilapia NCC activity suggesting a role for FasL (Jaso-Friedmann
Fighting Virus and Parasites with Fish Cytotoxic Cells
287
et al., 2000; Ruiz et al., 2001). Fish NCC activity is also increased by bacterial infections:
Edwardsiella ictaluri in channel catfish (Evans et al., 1998b), Aeromonas salmonicida in brook
trout (Salvelinus fontinalis) (Dautremepuits et al., 2006) or Streptococcus iniae in tilapia (Taylor
et al., 2001). In our lab, we have been investigating the immunostimulatory role of many
substances and conditions in the gilthead seabream, one of the most important farmed
species in the marine aquaculture. This has allowed us to get a lot of information about the
regulation of the seabream immune response, and concretely the NCC activity. Thus, we
have shown in vitro and/or in vivo modulation of seabream NCC activity by vitamins C
(Cuesta et al., 2002b), E (Cuesta et al., 2001), A (Cuesta et al., 2003b) and D3 (Cerezuela et al.,
2009), chitin (Cuesta et al., 2003c; Esteban et al., 2000, 2001), levamisole (Cuesta et al., 2002c),
lactoferrin (Esteban et al., 2005), melatonin (Cuesta et al., 2008a), propolis (Cuesta et al.,
2005b), inulin (Cerezuela et al., 2008), unmethylated oligodeoxynucleotides (ODNs)
containing cytosine-phosphodiester-guanosine (CpG) motifs (Cuesta et al., 2008b, 2008c),
probiotic bacteria (Daz-Rosales et al., 2006; Salinas et al., 2005, 2006, 2008), yeast (Cuesta et
al., 2007; Ortuo et al., 2002; Reyes-Becerril et al., 2008; Rodrguez et al., 2003), fungi
(Rodrguez et al., 2004), virus (Esteban et al., 2008), environmental contaminants (p,p-DDE
and lindane) (Cuesta et al., 2008d) or stress factors (air exposure, crowding and anaesthetics)
(Cuesta et al., 2003d). In general, we have demonstrated great NCC increments after these
treatments. Moreover, we have also observed that NCC activity reached the greatest
activation, compared to other innate cellular immune responses such as phagocytosis or
respiratory burst activity, and did at shorter treatment times and lower dosages.
Unfortunately, most of this information has been obtained evaluating the NCC activity
against xenogeneic tumor cells and whether this is correlated to the in vivo activity against
viral infections deserves further investigation. In this sense, few recent studies have
correlated the stimulatory role of immunostimulants with an increased viral disease
resistance. Thus, probiotic-supplemented diets resulted in reduced mortality of Japanese
flounder specimens exposed to lymphocystis disease virus (LCDV) (Harikrishnan et al.,
2010) whilst feeding of shrimp with immunostimulant herbs reduced their mortality upon
viral disease (Citarasu et al., 2006). Further characterization of the beneficial
immunostimulants against viral diseases is needed to control the virus spreading and lethal
effects.
Apart from the direct activation of fish cytotoxic activity, the expression of some CMC-
related genes (NCCRP-1, CD8, perforin, granzyme, etc.) is also modulated (Table 2),
suggesting an increase in the CMC activity. First, the NCCRP-1 gene expression was altered
after bacterial infection (Reyes-Becerril et al., 2011; Sakata et al., 2005), administration of
immunostimulants (Cuesta et al., 2008b, 2008d; Lazado et al., ; Reyes-Becerril et al., 2008),
exposure to contaminants (Cuesta et al., 2008d) or bacterial vaccination (Caipang et al.,
2008), depending on the fish species, tissue, time and dose of exposure, and suggests a
parallel effect of fish NCC activity. Perforin gene expression is usually up-regulated after
immunization of ginbuna crucian carp with tumor cells (Toda et al., 2011a), after PHA-L
(Phaseolus vulgaris leucoagglutinin) stimulation of trout CD8o
+
cells (Takizawa et al., 2011),
after VHSV infection of RTS11 cell line (Ords et al., 2011) and after viral infection or DNA
vaccination in rainbow trout (unpublished data), whilst down-regulated after cadmium
exposure (Auslander et al., 2008). In a similar fashion, granzyme genes are up-regulated by
bacterial vaccination (Caipang et al., 2008), viral infections (VHSV in RTS11 cell line and
SVCV in carp) (Huang et al., 2010; Ords et al., 2011) and viral infection or DNA vaccination
Health and Environment in Aquaculture
288
(unpublished data). The transcript level of CD8 gene is related to the CTL presence,
abundance and activity. Thus, fish CD8 transcripts are up-regulated by viral and bacterial
infections, viral DNA vaccines, scale grafts, poly I:C or mitogens (Byon et al., 2005 2006;
Cuesta et al., 2010; Cuesta & Tafalla, 2009; Forlenza et al., 2008; Overturf & LaPatra, 2006;
Somamoto et al., 2005, 2006; Utke et al., 2007; Xu et al., 2011). In some studies, these CD8
gene levels have been correlated with increased CTL activity. Finally, other genes related to
the cytotoxic activity have received less attention. In this category, the natural killer
enhancing factor (NKEF), which increase the cytotoxic activity in humans but its role is
unknown in fish, is up-regulated by viral infections and DNA vaccines (Cuesta & Tafalla,
2009; Ords et al., 2011; Utke et al., 2007) while granulysin, which is secreted together to
granzymes and lyses target cells, gene is up-regulated in CD8
+
lymphocytes by mitogen
stimulation (Takizawa et al., 2011). Further studies are needed to clearly state the gene
expression with either innate or specific cytotoxic activity in fish. Future development of
more molecular tools will help to elucidate this fascinating and complex immune response.
7. Future directions
As summarized above, fish posses a wide range of cytotoxic cells with killing activity
against tumor cells, virus-infected cells and parasites. Further studies in the future should
identify, describe and characterize the cytotoxic cells and mechanisms in the most cultured
fish species and those susceptible to be farmed in the future. Another issue is the generation
of molecular tools to evaluate the fish CMC and clearly identify the function of NCCs, NK-
like and CTLs as well as assay models such as clonal fish, cytotoxic cell clones or MHC I-
paired effector and targets (virally infected or not). These tools will also help to design
powerful and safe vaccines against problematic virus and parasites for fish aquaculture.
Finally, these studies have also to be applied to marine fish, which culture is continuously
increasing because of the human demand and high economic value.
8. Glossary
ADCC Antibody-dependent cytotoxic cells
ALAK Lymphokine-activated killer cells
CCV Channel catfish virus
CD4+ T helper lymphocyte
CD8+ T cytotoxic lymphocyte or CTL
CHNV Crucian carp haematopoietic virus
CMC Cell-mediated cytotoxicity
CpG Cytosine-phosphodiester-guanosine
CTLs Cytotoxic T lymphocytes
DNA Deoxyribonucleic acid
DNF Damselfish neurofibromatosis
DTH Delayed hypersensitivity reaction
EGCs Eosinophilic granular cells
EVA Eel virus from America
HK Head-kidney
IgM Immunoglobulin M
IPNV Infectious pancreatic necrosis virus
Fighting Virus and Parasites with Fish Cytotoxic Cells
289
ITAM Activating intracellular motifs
ITIM Inhibitory intracellular motifs
Jak Janus kinase
KIR Killer immunoglobulin
LAK Lymphokine-activated killer cells
LCDV Lymphocystis disease virus
LFA-1 Leucocyte-function-associated antigen-1
MHC Major histocompatibility complex
MLR Mixed leucocyte reaction
NCC Non-specific cytotoxic cells
NCCRP-1 non-specific cytotoxic cell receptor protein-1
NITR Novel immune-type receptor
NK Natural killer
NKEF Natural killer enhancing factor
NKG2/CD94 C-type lectin membrane receptors
NNV Nervous necrosis virus
ODNs Unmethylated oligodeoxynucleotides
PBL Peripheral blood leucocytes
PE Peritoneal exudate
PHA-L Phaseolus vulgaris leucoagglutinin
PMA Phorbol myristate acetate
RSIV Red seabream iridovirus
RTG-2 Rainbow trout gonad cell line
Sp Spleen
STAT Signal Transducer and Activator of Transcription
SVCV Spring viremia carp virus
TCR T cell receptor
Th Thymus
VHSV Viral hemorrhagic septicaemia virus
9. Acknowledgments
This work has been funded by national (AGL2008-05119-C02-01 and AGL2010-20801-C02-
02) and regional projects (04538/GERM/06). A. Cuesta thanks to the Ministerio de Ciencia e
Innovacin for the Ramn y Cajal contract.
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12
Bacteriocins of Aquatic
Microorganisms and Their Potential
Applications in the Seafood Industry
Suphan Bakkal, Sandra M. Robinson and Margaret A. Riley
University of Massachusetts Amherst
USA
1. Introduction
Bacteriocins are potent antimicrobial polypeptides and proteins produced by most lineages
of Bacteria and, perhaps, by all members of Archaea (O'Connor & Shand, 2002; Riley &
Wertz, 2002a, 2002b; Tagg et al., 1976). Although initially the focus of numerous
biochemical, evolutionary, and ecological studies, more recently, their potential to serve in
human and animal health applications has taken center stage (Gillor et al., 2008). The use of
bacteriocins in probiotic applications, as preservatives, and, (most excitingly) as alternatives
to classical antibiotics is being broadly explored (Abee et al., 1995; Einarsson & Lauzon,
1995; Gillor & Ghazaryan, 2007; Gillor et al., 2007).
Most bacterial species produce one or more bacteriocins (Cascales et al., 2007). One of the
most prolific bacteriocin-producing species is Pseudomonas aeruginosa, of which 90% or more
of the strains tested produce their own version of bacteriocins, known as pyocins (Govan &
Harris, 1985). In contrast, only 15-50% of Escherichia coli produce their brand of bacteriocins,
known as colicins (Riley & Gordon, 1992). The colicins are exceedingly well characterized
proteins, and have been the subject of numerous detailed biochemical, molecular,
evolutionary, and ecological analyses (Cascales et al., 2007; Riley et al., 2003; Riley &
Gordon, 1999; Riley & Wertz, 2002a, 2002b). Some species of bacteria produce toxins that
may exhibit numerous bacteriocin-like features, but have not yet been fully characterized;
these toxins are referred to as bacteriocin-like inhibitory substances, or BLIS (Messi et al.,
2003; Moro et al., 1997).
In this chapter, we will explore the bacteriocins of aquatic bacteria, particularly those of
potential interest in the seafood industry. A short primer of bacteriocin biology is followed
by a detailed review of the diversity of bacteriocins described from marine microorganisms.
These toxins have received far less attention than bacteriocins produced by terrestrial or
human-commensal bacteria, yet they have equivalent potential as antibiotics and even
greater promise for use in the creation of probiotic strains for the seafood industry.
2. Bacteriocin basics
Bacteriocins are proteins or short polypeptides, which are generally only toxic to bacteria that
are closely related to the producing strain. A typical bacteriocin contains a toxin (bacteriocin)
Health and Environment in Aquaculture
304
gene, an immunity gene (which confers resistance to the aforementioned toxin), and a lysis
gene, which encodes a protein that aids in toxin release from the producing cell (Chavan &
Riley, 2007). Bacteriocins work by binding to and killing only cells with surface receptors that
are recognized by that specific bacteriocin (Cascales et al., 2007; Chavan & Riley, 2007). In a
microbial community, cells can either be bacteriocinogenic (produce bacteriocin), sensitive, or
resistant to each bacteriocin. When all three cell-types are present and are competing for
limiting resources, the strain interactions mimic the childrens game rock-paper-scissors
(Kerr et al., 2002). The premise of this game is that paper covers rock, scissors cut paper, and
rock breaks scissors, creating a cycle of wins and losses with no one matter dominating as long
as all three states are present. The same interaction is observed in microbial communities that
employ bacteriocins (Table 1). Only a small percentage of bacteriocinogenic cells will be
induced to produce and release bacteriocin. Some sensitive cells are immediately killed by the
bacteriocin, while others harbor mutations that confer resistance. These resistant cells rapidly
displace the producer cells, due to the cost of bacteriocin production. However, the resistant
cells grow more slowly than their sensitive counterparts, because resistance mutations often
have a negative effect on fitness (Kerr et al., 2002).
Strain More Fit Than Less Fit Than
Bacteriocin-producer Sensitive Resistant
Sensitive Resistant Bacteriocin-producer
Resistant Bacteriocin-producer Sensitive
Table 1. Competition for resources results in a rock-paper-scissors-like interaction of
microorganisms (adapted from Kerr et al., 2002).
In contrast to traditional antibiotics, which are used in human health applications precisely
because of their ability to kill a diversity of bacterial pathogens, bacteriocins generally target
only members of their producing species and its closest relatives (although numerous
exceptions abound)(Riley et al., 2003; Tagg et al., 1976). Riley et al. (2003) mapped the killing
spectrum of bacteriocins from seven enteric species (Escherichia coli, Klebsiella pneumoniae,
Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Hafnia alvei, and Serratia plymuthica)
onto the molecular phylogeny of the same species (Fig. 1). This study showed that
bacteriocin producers tend to kill strains belonging to their same species. However, there are
some exceptions, such as bacteriocins of E. coli that inhibit distantly related H. alvei (Fig. 1).
2.1 Bacteriocin naming
Bacteriocins were originally named based on the producer species such as colicins produced
by Escherichia coli, pyocins of Pseudomonas aeruginosa (formerly named pyocyania), cloacins of
Enterobacter cloacae, cerecins of Bacillus cereus, and pesticins of Yersinia pestis (Reeves, 1965).
Fredericq (1957) created the first classification, and thus nomenclature, of bacteriocins focusing
on the colicins of E. coli (Fredericq, 1957). Fredericq grouped colicins into 17 different types
(colicins A, B, C, D, E, F, G, H, I, J, K, V, S1, S2, S3, S4, and S5) based on their receptor
specificity. These colicins were then further subtyped (colicin E1, E2, and E3, etc.) based on
their immunity patterns. In this scheme, all subtypes were recognized by the same receptor,
but they possessed different immunity phenotypes (Fredericq, 1957). Later, the addition of the
producer strains name provided further differentiation of bacteriocins produced by strains of
the same species (Daw & Falkiner, 1996). This scheme is still used today.
Bacteriocins of Aquatic Microorganisms and
Their Potential Applications in the Seafood Industry
305
Fig. 1. The breadth of bacteriocin killing in enteric bacteria (adapted from Riley et al., 2003).
The bacteriocin killing phenotype of six enteric bacterial species were mapped onto their
molecular phylogeny constructed with concatenated sequences of five housekeeping genes
and 16s RNA. Vibrio cholerae (VC) was used as an outgroup to root the phylogenetic tree. EC,
Escherichia coli; KP, Klebsiella pneumoniae; KO, Klebsiella oxytoca; EB, Enterobacter cloacae; CF,
Citrobacter freundii; HA, Hafnia alvei; SP, Serratia plymuthica.
2.2 Bacteriocin classes
In general, bacteriocins are produced by Bacteria and studied based on the gram designation
of their producing species (Gram-negative versus Gram-positive). Additionally, a relatively
small number of bacteriocins from Archaeal species have also been characterized. A
comprehensive review of bacteriocins from Bacteria and Archaea can be found elsewhere
(O'Connor & Shand, 2002; Reeves, 1965; Riley & Gordon, 1999; Riley & Wertz, 2002a, 2002b;
Tagg et al., 1976). Below are short descriptions of the bacteriocin classes of Bacteria and
Archaea and examples of bacteriocins belonging to each class (Table 2).
Bacteriocins Bacteriocin Types /Class Size (kDa) Examples References
G
r
a
m
-
n
e
g
a
t
i
v
e
B
a
c
t
e
r
i
a
Colicins
Pore Formers
Nucleases
20-80
Colicins A, B
Colicins E2, E3
Cascales et al., 2007
Michel-Briand & Baysse, 2002
Gillor et al., 2004
Reeves, 1965
Colicin-like NA 20-80
S-pyocins
Klebicins
Phage-tail like
NA
> 80 R and F pyocins
Microcins
Post-translationally modified
Unmodified
< 10
Microcin C7
Microcin B17
Colicin V
G
r
a
m
-
p
o
s
i
t
i
v
e
B
a
c
t
e
r
i
a
Class I
Type A-positively charged and linear
Type B-uncharged or negatively charged
globular
Type C-synergistic
< 5
Nisin
Mersacidin
Lacticin 3147
Heng et al., 2007
Drider et al., 2006
Field et al., 2007
Maqueda et al., 2004
Class II
Class IIa-antilisterial
Class IIb-synergistic
< 10
Pediocin PA1
Carnobacteriocin B2
Class III
Type IIIa-Bacteriolytic enzymes
Type IIIb-Nonlytic peptides
> 10
Lysostaphin
Helveticin
Class IV Cyclic peptides < 10 Enterocin AS-48
A
r
c
h
a
e
a
Halocins
Microhalocins
Protein halocins
< 10
> 10
Halocin A4, C8, G1
Halocin H1, H4
Shand et al., 2007
OConnor & Shand, 2002
Ellen et al., 2011
Sun et al., 2005
Sulfolobicin
NA
~20
Sulfolobicin
Table 2. Bacteriocins of Bacteria and Archaea
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306
2.2.1 Bacteriocins of Gram-negative bacteria
Bacteriocins of Gram-negative bacteria are categorized into four main classes: colicins, colicin-
like bacteriocins, phage-tail like bacteriocins, and microcins (Table 2) (Chavan & Riley, 2007).
The colicins, produced by E. coli, are the most studied bacteriocins (Cascales et al., 2007).
Indeed, they have been used as a model system to study bacteriocin structure, function, and
evolution (Cascales et al., 2007; Riley & Gordon, 1999; Riley & Wertz, 2002a, 2002b). In general,
colicins are protease sensitive, thermosensitive proteins that vary in size from 25 to 90 kDa
(Pugsley & Oudega, 1987). There are two major colicin types based on their mode of killing;
pore former and nuclease colicins. Pore former colicins (colicins A, B, E1, Ia, Ib, K, E1, 5) kill
sensitive strains by forming pores in the cell membrane. Nuclease colicins (Colicins E2, E3, E4,
E5, E6, E7, E8, E9) kill by acting as DNases, RNases, or tRNAses (Gillor et al., 2004).
Proteinaceous bacteriocins produced by other Gram-negative species are classified as colicin-
like due to the presence of similar structural and functional characteristics (Table 2). Like
colicins, they can be nucleases (pyocins S1, S2) and pore formers (pyocin S5) (Michel-Briand &
Baysse, 2002). Klebicins of Klebsiella species, S-pyocins of Pseudomonas aeruginosa, and alveicins
of Hafnia alvei are among the most studied colicin-like bacteriocins.
Phage-tail like bacteriocins are larger structures that resemble the tails of bacteriophages.
Some even argue that they are defective phage particles (Bradley, 1967). R and F pyocins of
P. aeruginosa are some of the most thoroughly studied phage-tail like bacteriocins (Michel-
Briand & Baysse, 2002; Nakayama et al., 2000). They are encoded in a large gene cluster,
which spans a DNA region greater than 40 kb (Nakayama et al., 2000). There are 44 open
reading frames associated with the R2/F2 phenotypes, which include regulatory, lysis, and
toxin genes. The R2 and F2 pyocins show sequence similarity to the tail fiber genes of P2 and
lambda phages, respectively (Nakayama et al., 2000). Finally, Gram-negative bacteria
produce much smaller (<10 kDa) peptide bacteriocins called microcins. Microcins can be
divided into two classes: post-translationally modified (microcins B17, C7, J25, and D93) and
unmodified microcins (microcins E492, V, L, H47, and 24) (Table 2). Microcins are
chromosomally encoded (Gillor et al., 2004).
2.2.2 Bacteriocins of Gram-positive bacteria
Bacteriocins of Gram-positive bacteria are generally divided into four classes based on size,
morphology, physical, and chemical properties (Lee & Kim, 2011). Class I bacteriocins are
lantibiotics, which are small peptides (<5 kDa)(Field et al., 2007). They are post-
translationally modified, incorporating non-traditional amino acids such as dehydroalanine,
dehydrobutyrine, methyl-lanthione, and lantionine (Cleveland et al., 2001). This class is
subdivided into Type A, B, and C with the distinction being that members of Type A are
positively charged, linear peptides whereas those in Type B are either neutrally or
negatively charged rigid globular peptides. Members of Type C require synergistic activity
of two peptides to be active. This class includes the well-studied bacteriocins nisin and
lacticin (McAuliffe et al., 2001).
Class II bacteriocins are small (<10 kDa), heat-stable peptides that are not post-translationally
modified (Heng et al., 2007). Class II is also subdivided into two subgroups. Class IIa are
pediocin-like or Listeria-active peptides, which contain a conserved N-terminal sequences
(YGNGVxCxxxxCxV). Class IIb bacteriocins require the synergistic activity of two peptides to
be fully active (Nissen-Meyer et al., 1992). Class III bacteriocins are generally large (>10 kDa),
Bacteriocins of Aquatic Microorganisms and
Their Potential Applications in the Seafood Industry
307
heat-labile peptides. They are subdivided into two subtypes. Type IIIa are bacteriolysins,
which are bacteriolytic enzymes. Lysostaphin is the most studied bacteriocin in this subtype
(Schindler & Schuhardt, 1964). Type IIIb are non-lytic bacteriocins. Helveticin J (37 kDa)
produced by Lactobacillus helveticus belongs to this type (Joerger & Klaenhammer, 1986).
Finally, Class IV bacteriocins have unique structural characteristics. The first and last amino
acids of these bacteriocins are covalently bonded, thus they have cyclic structures. Enterocin
AS-48 produced by Enterococcus faecalis subsp. liquefaciens S-48 was the first characterized
bacteriocin belonging to this class (Maqueda et al., 2004).
2.2.3 Bacteriocins of Archaea
The Archaea also produce unique types of bacteriocin-like antimicrobial compounds, called
archaeocins (Shand & Leyva, 2007). They have been much less studied than the bacteriocins
of Bacteria. Thus far, two major types of archaeocins have been identified: halocins of
halobacteria and sulfolobicins of Sulfolobus species. Halocins can be peptides (< 10 kDa)
and/or proteins (>10 kDa) (Shand & Leyva, 2007). According to Torreblanca and Meseguer
(1994), halocin production is a universal feature of halobacteria. Halocins are located on
megaplasmids (or minichromosomes). Halocins H4 and S8 are located on ~300 kbp and
~200 kbp plasmids, respectively (Cheung et al., 1997; Price & Shand, 2000). Their activity is
usually detected at the late exponential to early stationary growth phase (Cheung et al.,
1997; Price & Shand, 2000). There is not much known about sulfolobicins. Prangishvili et al.
(2000) screened sulfolobicin production from Sulfolobus islandicus isolated from volcanic
vents throughout Iceland. This study predicted that sulfolobicin activity is membrane-
associated and is not released from the cell. Sulfolobicins are also associated with
membranous vesicles ranging in size from 90 to 180 nm in diameter (Prangishvili et al.,
2000). Like many bacteriocins, they are thermostable and sensitive to protease treatment.
Their mode of action is still unknown (Ellen et al., 2011).
2.3 Bacteriocin genetics
Bacteriocins can be encoded on chromosomes, plasmids, and other transposable elements.
For example, the colicins of E. coli and halocin H4 are plasmid-encoded while the pyocins of
P. aeruginosa are chromosomal (Chavan & Riley, 2007; Cheung et al., 1997; Michel-Briand &
Baysse, 2002). Lacticin 481 has been shown to reside on the transposon Tn5721 (Dufour et
al., 2000) and some bacterial species such as Serratia marcescens possess both plasmid and
chromosomally encoded bacteriocins (Riley & Wertz, 2002b). Just as we see differences in
the function of Gram-negative, Gram-positive, and Archaeal bacteriocins, we can also trace
these distinctions to the genetic level.
In general, the full function of bacteriocins produced by Gram-negative bacteria is encoded
via three tightly linked genes, the toxin, immunity, and lysis genes (Fig. 2A). However, there
are significant differences in the genetics of colicins, colicin-like bacteriocins, phage-tail-like
bacteriocins, and microcins (Fig. 2). For example, the colicin gene cluster consists of the three
bacteriocin-related genes in close proximity, whereas colicin-like pyocin S3 does not possess
a lysis gene (Fig. 2A-B). Two representative phage-tail like bacteriocins, R and F-type
pyocins, R2 and F2, are encoded in a large gene cluster that spans more than 40 kb
(Nakayama et al., 2000) and includes 44 open reading frames (Nakayama et al., 2000). The
open reading frames include regulatory, lysis, R, and F pyocin genes (Fig. 2C).
Health and Environment in Aquaculture
308
P
SOS
P
imm
caa cai cal
pyoS3A pyo S3I
P-box
prtN prtR
PRF9 PRF10-23 PRF24-26
PRF28-43
A- Colicin A
B-Pyocin S3
C- R2F2 Pyocins
D- Nisin A
nisA nisB nisT nisC nisI nisP nisR nisK nisF nisE nisG
PnisA PnisRK PnisF
E- Sulfolobicin
sulA sulB
PRF5-8
P-box
Fig. 2. Genetic organization of bacteriocins
(Horizontal arrows represent genes; not to scale)
A- Genetic organization of colicin gene cluster. PSOS and Pimm represent promoter regions of the SOS
and immunity genes. Gene caa encodes colicin A; cai encodes the immunity gene; and cal encodes the
lysis gene (adapted from Cascales et al., 2007).
B-Genetic organization of colicin-like gene cluster. P-box refers to the binding site for transcriptional
regulator (PrtN); pyoS3A gene encodes pyocin S3; pyoS3I encodes the immunity gene (adapted from
Duport et al., 1995).
C- Genetic organization of phage-tail like bacteriocin gene cluster. prtN and prtR encode
transcriptional activator (PrtN) and repressor (PrtR), respectively. PRF 9 and PRF24-26 encodes the lysis
genes; PRF10-23 and PRF28-43 encode the R2 and F2 pyocin structural genes, respectively (adapted
from Nakayama et al., 2000).
D-Genetic organization of lantibiotic gene cluster. P
nisA
, P
nisRK
, and P
nisF
encode promoter genes for nis
A, nisRK, and nisFEG, respectively; nisA encodes nisin A precursor; nisR and nisK encode proteins for
nisin biosynthesis; nisB, nisC, nisT, and nisP encode proteins for nisin processing and translocation; nisI,
nisF, nisE, and nisG encode immunity proteins (adapted from Kuipers et al., 1993; Mierau &
Kleerebezem, 2005).
E-Genetic organization of archaeocin gene cluster. Sulfolobicin is composed of SulA and SulB proteins,
encoded by sulA and sulB genes (adapted from Ellen et al., 2011).
The Gram-positive bacteriocins are more complicated genetically, with genes that encode
post-translational modification of the toxin. The genetic organization varies between the
Gram-positive bacteriocin classes as well such as the requirement of two peptides for the
full activation of Class II bacteriocins. An example is provided by the nisin gene cluster (Fig.
2D), which includes 11 genes (nisABTCIPRKFEG) encoding functions such as synthesis of
the nisin precursor (nisA), regulation of nisin biosynthesis (nisRK), the processing and
translocation of nisin (nisBCTP), and immunity (nisIFEG) (Kuipers et al., 1993; Mierau &
Kleerebezem, 2005).
The genetic organization of archaeocins is relatively unknown in comparison to other
bacteriocins. A recent study showed that sulfolobicin of Sulfolobus acidocaldarius is comprised
Bacteriocins of Aquatic Microorganisms and
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309
of two proteins, SulA and SulB, which are encoded by sulA and sulB genes, respectively (Fig.
2E). These two proteins are associated with membrane vesicles in the extracellular medium
(Ellen et al., 2011). The gene organization of archaeocins is also unique. Sun et al. (2005)
showed that the gene halC8 encoded both halocin C8 and its immunity protein HalI (Sun et
al., 2005). It differs from other bacteriocins in that typically separate genes encode
bacteriocin toxin and immunity proteins.
2.4 Bacteriocin biosynthesis
Bacteriocins are often produced under stress conditions, such as nutrient limitation and
overpopulation (Riley & Gordon, 1999). The biosynthesis of Gram-negative bacteriocins is
regulated by the host; often involving the SOS system. The SOS system is comprised of
RecA and LexA proteins. LexA is a transcriptional repressor, which binds to the bacteriocin
promoter and prevents its transcription. In stress conditions such as DNA damage and UV
exposure expression of the recA gene is induced. RecA binds to LexA and therefore prevents
the repression of bacteriocin expression (Cascales et al., 2007). The expression of colicin-like
S-pyocins and phage-tail like RF pyocins also depends on RecA, except these genes possess
a P-box in their promoter region instead of an SOS box (Nakayama et al., 2000; Sano et al.,
1993). Further, there is no LexA-dependent repression of bacteriocin expression. Activated
RecA cleaves the PtrR transcriptional repressor protein, which leads to expression of the
transcriptional activator ptrN gene. PtrN binds to the P-box and induces the expression of
the pyocin genes (Matsui et al., 1993; Michel-Briand & Baysse, 2002). Alternatively, Gram-
positive bacteria possess bacteriocin-specific biosynthesis pathways. For example, nisin
regulates its own biosynthesis in cell-density dependent conditions (Eijsink et al., 2002).
Finally, there is much less known about archaeocins. Regulation of their biosynthesis is still
under investigation.
3. Bacteriocins of marine bacteria
Bacteriocins produced by marine bacteria have generated a great deal of excitement due
primarily to their potential to serve as probiotics and antibiotics in the seafood industry
(Galvez et al., 2008; Garca et al., 2010; Pilet & Leroi, 2011). A recent antimicrobial screen of
258 bacterial strains isolated from water and sediment in the Yucatan peninsula revealed
that 46 strains belonging to the genera Aeromonas, Bacillus, Burkholderia, Photobacterium,
Pseudomonas, Serratia, and Stenotrophomonas possessed antimicrobial activity. Approximately
fifty percent of this antimicrobial activity was due to bacteriocins or BLIS (De la Rosa-Garcia
et al., 2007). Further, Wilson et al. (2000) investigated surface-attached bacteria isolated from
Sydney Harbor, Australia. He showed that approximately 10% of surface-attached marine
bacteria possess antibacterial activity. Proteinase K treatment showed that this inhibitory
activity was associated with proteinaceous substances such as bacteriocins or BLIS (Wilson
et al., 2010). Given the fact that bacteriocins and BLIS have been characterized in most
culturable bacterial species, it is tempting to speculate about the diversity of new substances
the marine environment will reveal.
The first bacteriocin isolated from marine microorganisms was detected in Vibrio harveyi
(formerly Beneckea harveyi). McCall and Sizemore (1979) screened a total of 795 Vibrio spp.
strains isolated from Galveston Island, Texas for bacteriocin production (McCall &
Sizemore, 1979). This study revealed that approximately 5% of the Vibrio spp. possessed a
Health and Environment in Aquaculture
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high molecular weight bacteriocin-like killing agent. Further investigation revealed that the
source of the bacteriocin-like killing was a plasmid. It was also determined that the killing
range was limited to strains of B. harveyi. This bacteriocin was named harveyicin (McCall &
Sizemore, 1979).
In 1982, Hoyt and Sizemore investigated the chemical nature of harveyicin. They showed that it
is a proteinaceous substance, sensitive to protease, trypsin, and papain treatment, and resistant
to pepsin and lipase treatment. Furthermore, it is stable at room temperature and -20 C for
several weeks and several months, respectively. However, the protein did lose killing activity
after heat treatment at 55 C for 4 hours (Hoyt & Sizemore, 1982). Hoyt and Sizemore (1982)
also investigated the role of harveyicin in bacterial competition in enteric and planktonic
environments at various temperatures (4-39C), pHs (5-9.5), and salt concentrations (1.75%
and 3.5%). They also performed a competition assay on nonluminous bacteriocinogenic
(SYLum-) and luminous bacteriocin sensitive strains (SYcured) in equal concentrations (10
6
cells/ml). The ratio of harveyicin producer strain (SYLum-) to the harveyicin sensitive strain
(SYcured) peaked in an enteric environment (25 C, high salinity (3%), and alkaline pH (pH
9.5)). The harveyicin producer strain also outcompeted the harveyicin sensitive strain at an
acidic pH (pH 5.0) (Hoyt and Sizemore, 1982). Given the fact that the natural habitat of these
species is in the guts of fish (an acidic environment), bacteriocin production may serve as a
competitive advantage to the bacteriocin producer strain in this environment.
The identification of harveyicin led to numerous bacteriocin-screening studies in marine
bacteria, which focused primarily on biochemical characterization of bacteriocins and
bacteriocin-like inhibitory substances (Bagenda et al., 2008; Bhugaloo-Vial et al., 1996;
Carraturo et al., 2006; Hosseini et al., 2009; Hoyt & Sizemore, 1982; Longeon et al., 2004; McCall
& Sizemore, 1979; Messi et al., 2003; Metivier et al., 1998; Moro et al., 1997; Nilsson et al., 2002;
Pinto et al., 2009; Pirzada et al., 2004; Prasad et al., 2005; Rajaram et al., 2010; Selvin et al., 2004;
Shehane & Sizemore, 2002; Stoffels et al., 1992; Sugita et al., 1997; Suzuki et al., 2005; Tahiri et
al., 2004; Valenzuela et al., 2010; Yamazaki et al., 2005; Zai et al., 2009). The majority of these
studies focused on the killing breadth of the producing strains due to their potential for use as
antimicrobials and probiotics. However, the authors generally did not further characterize the
identified bacteriocins/BLIS, although see Table 3 for rough classifications (Table 3).
The relatively few characterized marine bacteriocins are primarily isolated from
Carnobacterium species, which are ubiquitous, Gram-positive lactic acid bacteria isolated from
marine organisms (such as fish and marine sponges), from cold and temperate environments,
as well as from terrestrial environments including Canadian winter soil, permafrost ice,
composite piles, and horse manure (Leisner et al., 2007). C. divergens and C. maltaromaticum
(formerly known as C. piscicola) are the most-studied species from this genus.
Carnobacterium species can produce bacteriocins at low temperatures and high salt
concentrations (Buchanan & Bagi, 1997). Further, the bacteria survive in fish products and
have low acidifying capacity (Tahiri et al., 2009). Thus, these species have been the focus of
intense research due to their potential as probiotics (Leisner et al., 2007; Rihakova et al.,
2009). Piscicocin V1a, V1b, divercin V41, piscicocin CS526, divergicin M35, carnocin U149,
and carnobacteriocin B2 are some of the bacteriocins isolated from marine Carnobacterium
species (Table 3) (Bhugaloo-Vial et al., 1996; Duffes et al., 1999; Metivier et al., 1998; Stoffels
et al., 1992; Suzuki et al., 2005; Tahiri et al., 2004; Yamazaki et al., 2005). These bacteriocins
share similar characteristics with the Class II bacteriocins of Gram-positive bacteria.
Bacteriocins of Aquatic Microorganisms and
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Table 3. Bacteriocin and BLIS activity characterized from marine bacteria (NA: Not available)
Health and Environment in Aquaculture
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Table 3. Continued
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BLIS from the marine environment are a relatively uncharacterized group of inhibitory
substances. While they may not be well characterized, they are abundant. Indeed, numerous
BLIS have been identified from marine species belonging to Vibrio, Aeromonas,
Carnobacterium, Lactococcus, Streptomyces, Pseudoalteromonas, Enterococcus, and Pediococcus
genera (Table 3) (Bagenda et al., 2008; Carraturo et al., 2006; Longeon et al., 2004; Messi et
al., 2003; Moro et al., 1997; Pinto et al., 2009; Pirzada et al., 2004; Prasad et al., 2005; Rajaram
et al., 2010; Satish Kumar & Arul, 2009; Selvin et al., 2004; Shehane & Sizemore, 2002; Sugita
et al., 1997; Valenzuela et al., 2010; Zai et al., 2009). These species are important in the
seafood industry and in human health.
The bacteriocins and BLIS isolated from marine microorganisms are diverse. However, they
do share common characteristics with bacteriocins from Bacteria and Archaea (Fig. 3). They
can be small peptides (5-10 kDa) like microcins of Gram-negative bacteria, microhalocins of
halobacteria, and class I and II bacteriocins of Gram-positive bacteria. They can also be
larger in size (10-90 kDa) like colicins and colicin-like bacteriocins of Gram-negative
bacteria. The majority of marine BLIS have been tested against a number of proteases
including trypsin, proteinase K, and pronase A, which are commonly used to identify
bacteriocin activity. Some have unique characteristics. For example, BLIS-IW1, BLIS-BC1,
and BC2 from Vibrio species possess a carbohydrate moiety while BLIS VIB 571 from
V. harveyi has a lipid moiety (Shehane & Sizemore, 2002). It is not clear if these moieties are
Fig. 3. Shared and unique characteristics of bacteriocins and BLIS from marine Bacteria with
bacteriocins of terrestrial Bacteria and Archaea
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involved in BLIS activity. The mode of actions of these BLIS is unknown and their killing
ranges are variable; either narrow - like bacteriocins of Gram-negative bacteria - or quite
broad - like that observed in bacteriocins of Gram-positive bacteria (Bhugaloo-Vial et al.,
1996; Moro et al., 1997; Sugita et al., 1997; Suzuki et al., 2005; Yamazaki et al., 2005).
Further, the stability of BLIS activity varies. BLIS IW1 is inactive after cold storage at -70C;
like phage-tail-like bacteriocins. Others, such as BLIS-BC1, BLIS-BC2, and BLIS-400, are still
active after cold treatment, as is observed in colicin and colicin-like bacteriocins. Some BLIS
are also resistant to treatment with organic solvents. For example, BLIS-400 is resistant to
organic chemicals including ethanol, methanol, acetone, and chloroform (Carraturo et al.,
2006). This feature is similar to some bacteriocins isolated from Gram-positive bacteria and
Archaea (Carraturo et al., 2006). Finally, most BLIS from marine bacteria are produced
during the stationary phase of growth, similar to bacteriocins from Gram-positive bacteria
(Pinto et al., 2009; Tahiri et al., 2004).
4. Applications and implications of marine bacteriocins
The international seafood industry is one of the worlds most profitable commodities, worth
more than $75 billion per year (Food and Agriculture Organization of the United Nations
[FAO], 2006). Fish and seafood are major proteins in some areas of the world. In 2006, of the
143 million tons of total fishery production (including fish, crustacean, and mollusks), 110
million tons was for direct human consumption (Pilet & Leroi, 2011). To meet this demand, we
have seen a marked rise in aquaculture (the farming of aquatic plants and animals) in the last
decade (FAO, 2006). Recently, there have also been dramatic changes in the seafood industry
due to technological advances, consumer habits, and globalization of the food market (Galvez
et al., 2008). In particular, there has been an increase in consumer preference for foods that are
minimally processed or preserved (especially those that claim health-promoting benefits).
Consumers are also demanding that these foods be fresh tasting and ready-to-eat (Galvez et
al., 2008). The demands on the industry to provide fresh, minimally preserved products in the
ever-growing globalized food market is requiring a longer and more complex food-chain and
increasing the risk of microbial contamination and spoilage (Garca et al., 2010).
4.1 Challenges in the seafood Industry: Spoilage and disease
With the expansion of the seafood industry and the rise in seafood consumption, spoilage
and disease in fish are the main challenges the industry faces (Gram & Dalgaard, 2002;
Toranzo et al., 2005). Microorganisms are the major cause of spoilage and diseases in the
seafood industry. It is estimated that nearly 25% of all the seafood produced is lost to
microbial spoilage (Baird-Parker, 2000). Microbes cause changes in the sensory properties of
the seafood (smell, taste, color), which make it less appealing and often dangerous to eat
(Gram & Dalgaard, 2002). Disease severely affects the production and trade of farmed
seafood, creating high economic impacts for many countries (Bondad-Reantaso et al., 2005).
4.2 Common microbial diseases in aquaculture
Fear of disease, as well as climate change, are acting as deterrents to aquaculture farming
(Bondad-Reantaso et al., 2005). It has been shown that bacteria exhibit greater pathogenesis
at higher temperatures, leading to greater and more virulent disease in aquaculture (Desriac
Bacteriocins of Aquatic Microorganisms and
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et al., 2010). The bacterial microflora of fish can become pathogenic under stress conditions
such as sudden temperature changes, overcrowding, and poor water quality conditions and
thus can cause diseases in fish. Furunculosis, vibriosis, columnaris disease, streptococcosis,
pasteurellosis, fish tuberculosis, and enteric septicemia are common microbial diseases
observed in economically important aquaculture fish species. These diseases are
predominantly caused by Aeromonas, Vibrio, Cytophaga, Streptococcus, Pasteurella,
Mycobacterium, and Edwardsiella genera (Table 4).
Microorganism Hosts Diseases Antibiotic Treatment References
Aeromonas
Salmonids, catfish,
carp, tilapia, trout,
perch, goby
Frunculosis
MAS
Erythrodermatitis,
Ulcer
Florfenicol
Sulfadimethoxine and Ormetoprim
Oxytetracycline dihydrate
Noga, 2010
Vibrio
Salmonids, cod,
ayu, Japanese eels
Vibriosis
Cold water vibriosis,
Hemorrhagic septicmia
Skin ulcers
Oxytetracycline
Potentiated sulfonomides
Oxolinic acid
Noga, 2010
Toranzo et al., 2005
Cytophaga
Salmonids, catfish,
tilapia, stripped
bass
Columnaris disease
Oxytetracycline dihydrate
Florfenicol
Noga, 2010
Streptococcus
Stripped bass,
tilapia, turbot,
barramundi,
Atlantic Salmon
Streptococcosis
Oxytetracycline
Amoxicillin
Erythromycin
Noga, 2010
Toranzo et al., 2005
Pasteurella Yellow tail, seabass Pasteurellosis Oxytetracycline
Noga, 2010
Toranzo et al., 2005
Mycobacterium
Striped bass,
seabass, Atlantic
salmon
Fish tuberculosis
Ampicillin
Erythromycin thiocyanate
Noga, 2010
Jacobs et al., 2009
Edwardsiella Channel catfish
Enteric septicemia
Fish gangrene
Florfenicol
Sulfadimethoxine and Ormetoprim
Noga, 2010
Mohanty & Shaoo, 2007
Table 4. Fish pathogens, corresponding diseases, and antibiotic treatment regimens
Furunculosis (skin infection) and motile Aeromonas septicemia (MAS) are two major fish
diseases caused by Aeromonas species (Noga, 2010). Nonmotile, psychrophilic Aeromonas
salmonicida is the causative agent of furunculosis in salmonids (O'Brien et al., 1994). Besides
furunculosis in salmonids, it causes erythrodermatitis in carp and ulcers in trout (Noga,
2010). Furthermore, motile mesophilic Aeromonas hydrophila and Aeromonas veronii are the
causative agents of MAS in carp, tilapia, perch, catfish, and salmon. They also cause ulcer
disease in catfish, cod, carp, and goby (Noga, 2010).
Vibrio species cause systemic infections in fish (Vibriosis). The common symptoms are loss of
appetite and skin ulcers, which are associated with septicemia. Vibrio anguillarum and Vibrio
salmonicida are the causative agents of vibriosis and cold water vibriosis in salmon and cod,
respectively (Colwell & Grimes, 1984). Further, Vibrio vulnificus causes hemorrhagic septicemia
in Japanese eels and V. damsela causes skin ulcers (Toranzo et al., 2005).
Columnaris disease affects the skin and gills of freshwater fish including salmonids, catfish,
striped bass, and tilapia (Noga, 2010). Infection causes degradation of the cartilage tissue,
the major cause of death. Cytophaga columnaris as well as Bacillus columnaris, Flexibacter
columnaris, and Flavobacterium columnare are the causative agents of columnaris disease
(Noga, 2010).
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316
Streptococcosis is a fish disease caused by Streptococcus species. Some of the clinical
symptoms of streptococcosis are anorexia, loss of orientation, erratic swimming, lethargy,
and hemorrhaging (Noga, 2010). Streptococcus iniae is the causative agent of streptococcosis
in at least 27 species of farmed finfish. The worldwide impact of streptococcosis was
estimated globally to be around $100 million (USD) (Agnew &Barnes, 2007).
Pasteurellosis (pseudotuberculosis) is one of the most important diseases in Japanese
aquaculture affecting commercial yellowtail, ayu, black sea bream, red sea bream, and
hybrid striped seabass (Noga, 2010). Pasteurella piscidium is the causative agent of this
disease (Romalde, 2002). Hemorrhaging around the gills and lesions in the skin, liver, and
kidneys are common symptoms in acute and chronic forms of this disease (Noga, 2010).
Mycobacteriosis (or fish tuberculosis) can be observed in nearly 200 species including
salmonids, seabass, turbo, cod, and halibut (Noga, 2010). Mycobacterium marinum is the most
common Mycobacterium species causing tuberculosis. Greyish-white nodules in the spleen,
liver, and kidney, and hemorrhagic lesions are among the symptoms of this disease (Jacobs
et al., 2009).
Edwardsiellosis (emphysematous putrefactive disease) is commonly observed in carp,
tilapia, eel, catfish, salmon, and trout (Noga, 2010). Edwardsiella tarda and Edwardsiella
ictaluri are the main species causing Edwardsiellosis. Gas-filled lesions in the skeletal
musculature are the major clinical sign of this disease (Mohanty & Sahoo, 2007). Further,
Edwardsiella ictaluri is associated with enteric septicemia in channel catfish (Mohanty &
Sahoo, 2007)
4.3 Current practices in the seafood industry to combat spoilage and disease
The seafood industry employs numerous techniques to eliminate microorganisms from their
products. The oldest and still widely used form of seafood preservation is drying/salting.
Besides keeping the seafood chilled in cold-water or on ice, this is the most low-tech
preservation technique. There are many variations to this method such as wet-salting or
additional acidification, but most achieve the same results. In this method, the fish is dried
(with or without salt), which creates an environment devoid of vast nutrients (and possibly
with high salinity). This prevents most bacterial growth, but spoilage can still occur due to
filamentous fungi growth or insect infestation. Yeast is also able to grow in heavily wet-
salted fish (Gram & Dalgaard, 2002).
Other preservation methods include washing with disinfectants, including chlorinated
water, iodophores, salts, organic compounds, aldehydes, hydrogen peroxide, quaternary
ammonium compounds, and antiseptic dyes (Calo-Mata et al., 2007; Shao, 2001).
Disinfectants are mostly used to kill fungi and parasites. However, they may also select for
antibiotic resistance in bacteria (Murray et al., 1984). Seafood can also be marinated in an
acidic solution to prevent bacterial growth. Vacuum-packing and preservatives such as
sorbate and benzoate have also been employed to prevent microbial growth (Einarsson &
Lauzon, 1995; Gram & Dalgaard, 2002). Recently, complex solutions such as carbon-dioxide
packing, spray-drying of antimicrobials, and radiofrequency heating have been applied to
fight these ever-present problems of spoilage and contamination (Calo-Mata et al., 2007;
Galvez et al., 2007; Gram & Dalgaard, 2002).
Bacteriocins of Aquatic Microorganisms and
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The use of vaccines and antibiotics in aquaculture is aimed at preventing the initial
colonization by microorganisms. The means of administration of these prophylactics is
simple, they are either added to the water or feed, or given by injection (Shao, 2001). A large
body of research in the 1970s resulted in vaccines against numerous seafood pathogens,
primarily species of Vibrio (Shao, 2001). Although vaccines are effective (and cost-effective),
there are still no vaccines against shrimp and mollusc pathogens (Subasinghe, 2009).
An alternative to vaccination is the use of antibiotics to prevent bacterial infections.
Tetracycline has become one of the most popular antibiotics in aquaculture due to its low-
cost, low-toxicity, and high efficacy. Further, florfenicol, sulfadimethoxine/ormetoprim,
oxytetracycline, and sulfonamides, are used to treat common bacterial infections (Table 4).
However, the rampant use of antimicrobials in this industry has created massive selective
pressure for bacteria to develop resistance (World Health Organization [WHO], Fact sheet
194). While these drugs are effective in killing bacteria, they also play a much more
nefarious role in aquaculture. Antibiotics have varying half-lives, meaning they degrade at
different rates. Some antibiotics degrade slowly and thus proliferate in the aquatic
environment (Cabello, 2006). Worse still, the drugs flow into open waterways, sewage
systems, sediments, and can even remain in the flesh of the farmed seafood (Benbrook, 2002;
Cabello, 2006). These antibiotics continue to impose selective pressures leading to resistance
until they are eventually degraded. This resistance is not only seen in the bacteria that
inhabit the seafood produced in antibiotic-using aquaculture facilities, but in the animals
neighboring the facilities as well (Benbrook, 2002). It has also been shown that these
resistant bacteria are able to horizontally transfer their resistance-conferring genes to other
human pathogens (Benbrook, 2002). For these reasons, governing agencies, such as the US
Food and Drug Administration (FDA) and Environmental Protection Agency (EPA), strictly
regulate the use of antibiotics in aquaculture.
Due to the problems associated with antibiotic use, the seafood industry is exploring the use
of probiotics to promote the growth of healthy microflora in the seafood that can combat
infecting pathogens. Probiotics are live microbial feed supplements that beneficially affect
the host animal by improving its intestinal health (Fuller, 1989). While this definition needs
a bit of tweaking to fit the seafood industry, it does still apply.
One area of active research in seafood aquaculture is the utilization of bacteriocins as
antimicrobials. Bacteriocins have a long history of use in dairy or meat applications and
there is an increasing number of studies on the effect of bacteriocins as antimicrobials in the
seafood industry (Table 5) (Aasen et al., 2003; Al-Holy et al., 2004; Budu-Amoako et al., 1999;
Einarsson & Lauzon, 1995; Elotmani & Assobhei, 2004; Luders et al., 2003; Neetoo et al.,
2008; Nilsson et al., 1997; Nykanen et al., 2000; Szabo &Cahill, 1999; Tahiri et al., 2009;
Tsironi & Taoukis, 2010; Zuckerman & Ben Avraham, 2002). These studies have focused
largely on the effects of nisin, a Gram-positive bacteriocin that has been generally
recognized as safe (GRAS) by the FDA. Early studies of nisin indicated that it delayed
growth of L. monocytogenes in cold-smoked salmon. Later research revealed that the addition
of CO
2
atmospheric packing significantly increased the effectiveness of nisin against Listeria
(Nilsson et al., 1997).
There has also been encouraging research into nisin-coated packaging. Neetoo et al. (2008)
investigated the effect of nisin-coated plastic films on the survival of L. monocytogenes on
vacuum-packed cold smoked salmon. This study showed that nisin-coated plastic films
Health and Environment in Aquaculture
318
Bacteriocin Target Seafood product Reference
Bavaricin A Extended shelf-life Shrimp Einarsson et al., 1995
Carnocin U149 Extended shelf-life Shrimp Einarsson et al., 1995
Divergicin M35 L. moncytogenes Salmon Tahiri et al., 2009
Nisin L. moncytogenes Salmon Nilsson et al., 1997
Nisin L. moncytogenes Salmon Nilsson et al., 1997
Nisin L. moncytogenes Salmon Szabo and Cahill, 1999
Nisin L. moncytogenes Salmon Neetoo et al., 2008
Nisin L. moncytogenes Salmon Zuckerman and Ben Avraham, 2002
Nisin L. moncytogenes Trout Nykanen et al., 2000
Nisin L. moncytogenes Lobster Budu-Amoako et al., 1999
Nisin L. innocua Caviar and ikura Al-Holy et al., 2004
Nisin Aerobic bacteria Salmon Zuckerman and Ben Avraham, 2002
Nisin Bacterial flora Sardines Elotmani et al., 2004
Nisin Extended shelf-life Fish Tsironi and Taoukis, 2010
Nisin Z Extended shelf-life Shrimp Einarsson et al., 1995
Pediocin L. moncytogenes Salmon Szabo and Cahill, 1999
Sakacin P L. moncytogenes Salmon Aasen et al., 2003
Sakacin P E. coli Salmon Luders et al., 2003
Table 5. Examples of bacteriocin trials in seafood products (Adapted from Galvez et al.
2008).
reduced the number of L. monocytogenes by 3.9 log CFU/cm
2
at 4
o
C and 10
o
C after 56 and
49 days of incubation, respectively. Further, this study also showed that nisin-coated plastic
films suppressed the growth of other aerobic and anaerobic spoilage microorganisms in a
concentration-dependent manner (Neetoo et al., 2008).
The combination of nisin with heat has also been shown as an effective method to prevent L.
monocytogenes contamination. Budu-Amoako et al. (1999) applied nisin along with moderate
heating on cold-packed lobster and showed a reduction of L. monocytogenes by 3-5 logs in
comparison to nisin and/or heat treatment alone (Budu-Amoako et al., 1999). Further, Al-
Holy et al. (2004) used a radio-frequency heating method to apply heat treatment in
conjunction with nisin. The combination of nisin and radio-frequency heating caused
reduction of L. innocua by 100% (Al-Holy et al., 2004). With an industry moving away from
traditional preservation techniques, bacteriocins (such as nisin) offer a promising alternative
as antimicrobials in the seafood industry.
4.4 Bacteriocin potential in the seafood industry
There are a number of factors that play a significant role in the potential to use bacteriocins
as probiotics and/or bio-preservatives in the seafood industry. The natural microbiota of the
seafood needs to continue to be surveyed for its sensitivity to bacteriocins. This information
should be incorporated into the guidelines for bacteriocin use in order to use these proteins
prudently against relevant pathogens. The environmental conditions, such as pH and
temperature, during seafood growth and processing could also affect the activity of applied
bacteriocins and requires further investigation (Galvez et al., 2007).
Bacteriocins of Aquatic Microorganisms and
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319
Despite these factors, research on aquatic microorganisms has shown that bacteriocin
production and diversity in aquatic environment is abundant (Bagenda et al., 2008;
Bhugaloo-Vial et al., 1996; Carraturo et al., 2006; Hosseini et al., 2009; Hoyt & Sizemore,
1982; Longeon et al., 2004; McCall & Sizemore, 1979; Messi et al., 2003; Metivier et al., 1998;
Moro et al., 1997; Nilsson et al., 2002; Pinto et al., 2009; Pirzada et al., 2004; Prasad et al.,
2005; Rajaram et al., 2010; Selvin et al., 2004; Shehane & Sizemore, 2002; Stoffels et al., 1992;
Sugita et al., 1997; Suzuki et al., 2005; Tahiri et al., 2004; Valenzuela et al., 2010; Yamazaki et
al., 2005; Zai et al., 2009). Bacteriocins have numerous qualities that make them attractive as
alternatives to antibiotics. They have been shown to be non-toxic to eukaryotic cells and are
GRAS, making them a safe alternative to traditional antimicrobials (Galvez et al., 2008). It
has also been shown that purified bacteriocins do not effect the sensory qualities of seafood
and that they are stable up to a salinity concentration of 10%. Additionally, the relatively
narrow killing spectrum of bacteriocins compared to traditional antibiotics limits the
selective pressure for bacteria to evolve resistance to these antimicrobials and thus reduces
the incidence of drug-resistant pathogens.
Because of the above stated reasons, some have suggested that bacteriocins should be
applied to foods by spray-drying as either dried bacteriocins or probiotic bacteriocinogenic
strains (Calo-Mata et al., 2007; Galvez et al., 2007). For example, Brillet et al. (2005) has
shown that bacteriocin producer Carnobacterium divergens V41 can be used as a
biopreservative to inhibit the growth of Listeria monocytogenes in cold smoked salmon (Brillet
et al., 2005). This study showed that spray application of C. divergens V41 on commercial
smoked salmon did not affect the sensory qualities of the salmon (Brillet et al., 2005).
Additionally, Schobitz et al. (1999) directly applied a BLIS from Carnobacterium piscicola into
vacuum-packed meat, which inhibited the growth of L. monocytogenes in the vacuum-packed
meat after 14 days of storage at 4
o
C (Schobitz et al., 1999). These studies aid in the argument
that bacteriocins should be used as a biopreservation technique in the seafood industry. This
technology has already emerged in the terrestrial food industry as we see with nisin (an
FDA approved food additive) and Microgard
TM
(a milk-based BLIS).
It has also been suggested that bacteriocins could be combined with current methods of
antimicrobial treatment and preservation to produce synergistic effects, such as
incorporating bacteriocins into bio-active packaging (Calo-Mata et al., 2007; Galvez et al.,
2007; Pilet & Leroi, 2011). For example, bacteriocins can be impregnated into gel coatings
and/or polyethylene films and can be applied to seafood during packaging (Neetoo et al.,
2008). The application of bacteriocins on packaged seafood is steadily being seen as a very
promising biopreservation method (Aasen et al., 2003; Al-Holy et al., 2004; Budu-Amoako et
al., 1999; Einarsson & Lauzon, 1995; Elotmani & Assobhei, 2004; Luders et al., 2003; Neetoo
et al., 2008; Nilsson et al., 1997; Nykanen et al., 2000; Szabo &Cahill, 1999; Tahiri et al., 2009;
Tsironi & Taoukis, 2010; Zuckerman & Ben Avraham, 2002). In fact, immobilization of
bacteriocins on coating materials for biopreservation may actually reduce the cost of
packaging due to the reduced amount and cost of the antibacterial needed to attach to the
film (Galvez et al., 2008). Creating combinations of bacteriocins and current methods used in
the seafood industry has the potential to increase the guarantee of freshness by assuring the
inhibition of spoilage causing microorganisms.
One trouble that the industry was having was the scale-up of these bacteriocins to levels that
were high enough for use in pilot studies and/or on the industrial scale. However, there is
Health and Environment in Aquaculture
320
technology in the pipeline that will make this an issue of the past (Galvez et al., 2008). As the
exploration of the aquatic environments of our planet increases, we are sure to find new and
exciting bacteriocins, which could play a vital role in antimicrobial effects and
biopreservation in the seafood industry.
5. Conclusions and future research
Bacteriocins have been the focus of an extensive number of studies for the past sixty years
due to their important role in nature and more recently, their potential for use as
therapeutics and probiotics. Most studies of bacteriocins initially focused on phenotypic and
molecular characterization of these toxins. However, due to their high potency and
relatively narrow killing spectrum, they were quickly recognized as a natural alternative to
antibiotics.
Knowledge regarding bacteriocins and their potential applications from terrestrial bacteria
is vast. Gram-negative bacteriocins such as colicins and microcins of enteric bacteria and
pyocins of P. aeruginosa have great promise in human and veterinary medicine. In addition,
Gram-positive bacteriocins such as nisin, pediocin, and lacticin have been developed for use
as food preservatives.
The focus of this chapter was to explore the bacteriocin and BLIS activity characterized from
marine microorganisms and assess their potential applications in aquaculture. The
bacteriocins and BLIS from marine microorganisms are under-studied relative to their
terrestrial counterparts. Thus far, most studies are limited to the identification of BLIS
activity and characterization of its killing breadth. Some studies have further characterized
these proteinecous killing agents and classified them based on similarities to known
bacteriocins. However, the abundance and diversity of bacteriocins in marine
microorganisms remains to be fully explored. We predict that a wealth of interesting
bacteriocin proteins can be easily identified as the screening efforts proceed.
Further, there is increased interest in the use of bacteriocins as alternatives to classical
antibiotics in aquaculture. Bacteriocins are highly potent against marine pathogens and
environmentally safe, due to the fact that they do not create intensive selection pressures for
antibiotic resistance. Clearly, bacteriocins could prove extremely beneficial to the seafood
industry and more research should be dedicated to exploring their potential applications as
probiotics and therapeutics. Given the fact that all species of bacteria have the potential to
produce bacteriocins, and only a handful have thus far been identified from marine
microorganisms, we are confident that existing studies have exposed only the tip of the
iceberg, in terms of bacteriocin diversity and potential use in the seafood industry.
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13
The Atlantic Salmon (Salmo salar) Vertebra
and Cellular Pathways to Vertebral Deformities
Elisabeth Ytteborg
1
, Jacob Torgersen
1
,
Grete Baeverfjord
1
and Harald Takle
1,2
1
Nofima AS, Norwegian Institute of Food,
Fisheries and Aquaculture Research
2
AVS Chile S.A.
1
Norway
2
Chile
1. Introduction
Stagnating fish stocks and a growing population demanding for aquatic food products have
been the major driving forces behind the rapid increase in aquaculture production.
Federation of European Aquaculture Producers (FEAP) estimates that 650 000 tons of fish
are farmed in the EU annually (compared to 60 000 tons in 1970). Within Europe as a whole,
the total production is more than 1.6 million tons (FEAP, 2009). Norway is a major
contributor to Europe's aquaculture sector with over 860 000 tons of Atlantic salmon (Salmo
salar) and trout produced each year, a production that has been more than doubled the last
ten years (Directorates of fisheries, Norway 2009). Forecasts predict that production will
need to increase for decades to come if demands are to be met (Brugere & Ridler 2004). To
keep up with the growing demand, the aquaculture industry is constantly searching for new
strategies to improve the rearing conditions and reduce production time and cost. However,
as a relatively new industry, and as a consequence of intensified production regimes, the
aquaculture sector faces growth constraints.
Farmed salmon is bred for rapid growth, and the industry aim at obtaining the optimal
growth rate by optimizing both diets and environmental factors accordingly. However,
intensive rearing conditions are linked to increased occurrence of production related
diseases and malformations. Elevated temperature during the fresh water period was
commonly used in the 90`ies to speed up developmental rate. An increasing number of fish
developing manufacturing defects, such as skeletal abnormalities (figure 1), heart failure
and jaw deformities was observed. Recommendations limiting temperatures to safe levels,
8C during egg rearing and 12C after fist feeding, led to substantial reductions in skeletal
malformations (Baeverfjord et al., 1999). However, in the last few years, the start feeding
temperature has been increased again, due to the stakeholders demand for reduced
production time. Further, the growing need of replacing fish meal in commercial fish feeds
have come into focus and deformities related to feed ingredient replacements, malnutrition
and mineral deficiency are investigated.
Health and Environment in Aquaculture
330
Fig. 1. Deformed (top) and non-deformed Atlantic salmon and corresponding radiographic
pictures. Photo: Grete Baeverford, Nofima.
In the present situation, fast growth in combination with unpredictable and potentially low
bioavailability of nutrients is considered the main challenge for adequate skeletal
development. Suboptimal supply of minerals (phosphorous, magnesium, zinc) and
nutritional imbalances of fatty acids, vitamins (A, C and D) and amino acids are considered
the main challenges in regard to skeletal malformations. The challenges related to
bioavailability are further amplified with the introduction of vegetable meals, some of
which are rich in antinutrients (e.g. phytic acid) that may further impair absorption. It is
therefore important to completely understand the molecular and cellular events in bone
development in salmon in order to deal with upcoming questions
Most of the knowledge currently available on cellular mechanisms for bone development is
adopted from studies using mammalian species. However, information from teleosts, like
zebrafish (Danio rerio), sea bass (Dicentrarchus labrax), sea bream (Sparus aurata) and Atlantic
salmon and related in vitro studies, is emerging. Molecular tools like in situ hybridization,
microarray, real time quantitative PCR, immunohistochemistry and cell culture systems
have allowed researchers working with teleosts to do more comparative and functional
studies. Also, transgenic model organisms, such as zebrafish, and in vitro transfection with
reporter gene constructs are now being more common and will provide valuable
information on processes involved in bone metabolism. Relevant examples include studies
on gene expression of several bone and cartilage associated marker genes, such as bone
morphogenetic protein 2 (BMP-2) (Rafael et al., 2006), osteocalcin (Wargelius et al., 2009;
Ytteborg et al.,2010b), osteopontin (Fonseca et al., 2007), vitamin D receptor (Lock et al.,
2009), parahyroid related hormone (Flanagan et al., 2000) and proteoglycans (Pedersen et al
2010;Conceio et al., 2008). The Sea bream vertebrae cell lines described by Braga et al.
(2006) as well as two reports on zebrafish (DeLaurier et al., 2010; Kimmel et al., 2010) show
the possibilities with fluorescent reporter gene constructs in bone research. The work
developed during the last few years has provided clear evidence that fish can be adequate
supplementary model systems to study bone and cartilage biology. Teleosts have been
successfully used to analyze molecular and cellular mechanisms involved in different
developmental pathways and revealed that the key genetic factors regulating lineage
determination and differentiation of stem cells are conserved among vertebrates at the
molecular level in both sequence and expression pattern (Kikuta et al., 2007; Shafizadeh et
al., 2004; Nakashima et al., 2003; Aubin, 1998; Pinto et al., 2001; Renn et al., 2006; Wise et al.,
2006; Ytteborg et al., 2010a). Due to the similar physiologic pathways and genetic
The Atlantic Salmon (Salmo salar) Vertebra
and Cellular Pathways to Vertebral Deformities
331
background of fish and mammals, this alternative system is also an interesting model to
unveil some of the molecular determinants of human bone related diseases and
malformations, like osteogenesis imperfecta, degenerated disc disease, persistent
notochordal canal and scoliosis (Gorman and Breden, 2007;Nissen et al., 2006;Fisher et al.,
2003). A number of animal models have been used to explore the pathology of spinal
deformities and revealed that vertebral pathology presents a complex but comparable cross
species etiology. With regard to complex disorders in humans, multiple models are critical
for the investigation and manipulation of etiological factors.
Fish systems could be of benefit to vertebral research because they exhibit a diverse range of
deformities, are free from skeletal appendicles and substantial genomic resources have been
developed for several species. Skeletal deformities in commercial salmon production have
been recognized as a problem of obvious relevance to economy as well as animal welfare.
Much effort has been put into understanding malformed development of Atlantic salmon
vertebrae during the years due to the importance of this organism to the aquaculture
industry. As a consequence, Atlantic salmon is emerging as an excellent model to study
vertebral deformities and other relevant vertebral pathological states. In this review the
current knowledge on the cellular and molecular mechanisms for skeletal homeostasis in the
mature Atlantic salmon vertebrae is discussed. Further, the cellular mechanisms for
differentiation and activation of osteoblasts and chondrocytes are described in relation to
pathways for pathological development and discussed in the light of related pathological
conditions in mammalian species.
2. Cellular and molecular mechanisms controlling bone formation
Bone formation basically occur via two mechanisms in both mammals and teleosts:
mesenchymal stem cells (MSC) either differentiate directly into bone producing osteoblasts
(intramembranous ossification) or by first forming a cartilaginous template secreted by
chondrocytes which is later replaced by bone (endochondral ossification) (Erlebacher et al.,
1995). However, similarities and differences in tissue structure between teleost and
mammalian bone have been described (Witten et al., 2009; Huysseune et al., 2000). In
general, fish possess rather few long bones with growth plate-like arrangements exhibiting
typical endochondral bone formation as seen in mammals. In the Atlantic salmon vertebrae,
compact bone of the amphicoel and trabeculae is formed directly through intramembranous
ossification, whereas the arch centra are modelled through endochondral ossification. Both
mechanisms lead to the formation of mineralized extracellular matrix (ECM), consisting of
fibers, mainly collagen embedded in a matrix of proteoglycans (PGs) and proteins. An
overview of the two different processes in the Atlantic salmon vertebra is shown in figure 2a
and b.
2.1 Linage determination and cellular differentiation
The cellular lineage determination and differentiation of osteoblasts and chondrocytes from
the MSC lineage are determined by a number of transcription factors, regulatory
mechanisms, environmental conditions and mineral availability. The pathways are
interconnected during vertebral formation and must be coordinated. In particular, the
transcription factors Runx2, Osterix, Sox9, Twist and Mef2c have distinct functions both in
the establishment of the vertebral bodies and later in the differentiation and maturation of
Health and Environment in Aquaculture
332
Fig. 2a. Overview of the intramembraneous and endochondral ossification in the Atlantic
salmon vertebra. See the text below for details.
Fig. 2b. Bone and cartilage in the Atlantic salmon vertebra. Both endochondral and
intramembraneous ossification leads to mineralized bone formation (red Alizarin red S
staining of bone in the centra and arches; blue Toluidine blue staining of cartilage in the
arches). See text below for further details on bone and cartilage formation. Vertebrae from
15g fish, scale bar= 200 m
specific skeletal cell types (Karsenty et al., 2009). Similarly, signaling molecules like bone
morphogenetic proteins (Bmp2 and Bmp4) and hedgehog proteins (Ihh and Shh) play
different roles both during cell differentiation and skeletal tissue ontogeny (Karp et al., 2000;
Hogan et al., 1996; Spinella-Jaegle et al., 2001). Important signalling pathways that induce
transcription of matrix producing and mineralizing genes in osteoblasts and chondrocytes
include the downstream targets of Bmps; Runt-related transcription factor 2 (Runx2) the
The Atlantic Salmon (Salmo salar) Vertebra
and Cellular Pathways to Vertebral Deformities
333
zinc finger containing transcription factor Osterix and Sex determining region Y box 9
(Sox9). Whereas Runx2 and Osterix activates genes in the osteoblastic lineage (Karsenty et
al., 1999; Otto et al., 1997; Nakashima et al., 2002), Sox9 regulates transcription of
chondrocytic genes (Bell et al., 1997).
The differentiation of MSC into mature osteoblasts involves several phases, which may be
divided into three subsequent stages; commitment, extracellular matrix production and
mineralization. Estrogen and 1,25-dihydroxy vitamin D
3
are among the hormones shown to
increase osteogenic differentiation via up-regulation of osteogenic growth factors, such as
BMP2. Among the many transcription factors expressed early in osteogenesis, runx2 is
noteworthy because it is required for bone formation and is an important early indicator of
osteogenic capacity of cells. Downstream targets of Runx2 and Osterix include genes
encoding both collagenous (e.g. Collagen 1 and 1) and non-collagenous (e.g. Osteopontin,
Osteocalcin, Osteonectin, Bone sialoprotein and Alp) proteins, which make osteoblasts
capable of producing and mineralizing bone matrix (osteoid). In both teleosts and
mammalian MSCs, alkaline phosphatase (Alp), col1a and osteopontin serve as useful
markers of early osteogenesis and the expression of these genes usually increases
throughout maturation. Col1 is the major structural component of bone, whereas the non-
collagenous proteins binds inorganic minerals and are involved in the mineralization
process (Cowles et al., 1998; Ikeda et al., 1992; Bolander et al., 1988; Termine et al., 1981).
Upon maturation, osteoblasts start secreting osteoid and mineralizing components, leading
to direct formation of bone via the intramembraneous ossification pathway. The key
markers involved in osteogenesis are shown in Figure 3.
Fig. 3. Osteoblast differentiation, maturation and key factors involved. After commitment to
the osteoblast lineage, matrix deposition starts. Mature osteoblasts are responsible for both
osteoid production and mineralization. See text for details.
The chondrocytes undergo a more synchronized process of proliferation, differentiation and
maturation so that three pronounced zones can be identified in the growing cartilage:
resting, proliferating and hypertrophic zones (Hunziker et al., 1994). Chondrocytes in the
resting zone are irregularly scattered in cartilage matrix, whereas chondrocytes in the
proliferating and hypertrophic zones are arranged in columns. The chondrocytes in the
resting zone serves as stem-like cells in the growth plate, stimulated by e.g. growth hormone
(GH) and insulin like growth factor (IGF). The proliferating zone is the region for active cell
Health and Environment in Aquaculture
334
replication and chondrocytes in this zone are mostly devoted to cell cycle processes.
Chondrocyte hypertrophy is the final step of chondrocyte maturation, regulated by the
transcription factors Myocyte enhancer factor 2c (Mef2c) and Runx2 (Arnold et al., 2007;
Kim et al., 1999). Parathyroid hormone related protein (PTHrP) and Ihh appear to play
important roles in proliferating chondrocytes by maintaining cells in a proliferative
condition, hence preventing chondrocyte hypertrophy. After commitment to the
hypertrophic state, chondrocytes start expressing Col10 (Ytteborg et al., 2010b; Arnold et al.,
2007), a unique component of the matrix produced by hypertrophic cells and extensively
used as a marker for chondrocyte hypertrophy (Iyama et al., 1991). Once hypertrophy is
reached, endochondral ossification can be initiated (Mackie et al., 2008). Hypertrophic
chondrocytes induce angiogenesis by secreting angiogenetic factors, such as the Matrix
metalloproteinases (Mmps) and Vascular endothelia growth factor (VEGF) so that
osteoblasts and osteoclasts may enter via newly formed blood vessels (Blavier et al., 1995).
The key markers involved in chondrogenesis are shown in Figure 4.
Fig. 4. Chondrocytic differentiation, maturation and key factors involved. Resting,
proliferating and hypertrophic chondrocytes are clearly visible as zones in the growth plate.
See text for details.
Osteoclasts are cells involved in removing damaged bone, repair mechanisms, mineral
homeostasis and replacement of cartilage with bone, both in mammals (review in Boyle et
al., 2003) and teleosts (reviewed in Witten et al., 2009). Osteoclasts provide an acidic
environment where mineralized matrix may be dissolved through secretion of cathepsins,
mmps and tartrate resistante acid phosphatase (TRAP) (Delaisse et al., 2003; Motyckova et
al., 2001; Ortega et al., 2003; Engsig et al., 2000). As in mammals, osteoclasts in Atlantic
salmon are multinucleated and the mechanisms involved in activation and differentiation of
osteoclasts are conserved (review in Witten et al., 2009). Mononuclear cells respond to
macrophage
colony stimulating factor (M-CSF) produced by nearby stromal
cells and
osteoblasts, through activation of c-fms, the receptor for M-CSF (Wiktorjedrzejczak et al.,
1990; Yoshida et al., 1990). The other signaling system essential
for osteoclast differentiation
is triggered when receptor
activator of nuclear factor kappa () B ligand (RANKL), a
member of the tumor necrosis factor (TNF) family, activates its receptor RANK (reviewed in
Collin-Osdoy et al., 2004). Among the downstream genes of RANKL are genes directly
ivolved osteoclast function (e.g. TRAP and Cathepsin K). The key markers involved in
The Atlantic Salmon (Salmo salar) Vertebra
and Cellular Pathways to Vertebral Deformities
335
osteoclastogenesis are shown in Figure 5. In addition, mononucleated osteoclasts are also
found in both mammals and teleosts and are considered to participate in minor, fine tuning
bone resorption (Witten et al., 2009). However, since teleost lack haemapoietic tissue in bone
marrow, the question of the origin of these cells remains unknown. In the vertebrae of
Atlantic salmon, multinucleated osteoclasts have been identified in the arch centra and
trabeculae but not in the compact bone of the amphicoel (Witten et al., 2009).
Fig. 5. Osteoclast differentiation, maturation and key factors involved. Fully mature
osteoclasts are able to dissolve bone. See text for details.
2.2 Matrix mineralization
Skeletal formation and growth occurs as a result of mineralization of ECM. A time lag where
collagen synthesis decreases and mineralization increase appears to be required for allowing
modifications of the osteoid so that it is able to support mineralization and hydroxyapatite
(Ca
10
[PO
4
]
6
[OH]
2
) formation (Hernandez et al., 2000). Mineralization of both bone and
cartilage occurs by deposition of inorganic hydroxyapatite crystals inthe ECM. This process
has not yet been described in teleosts. In mammals, the initiating step of hydroxyapatite
formation occurs in ECM vesicles secreted from mature osteoblasts (Anderson et al., 2005,
1996, 1995). These vesicles create an environment where deposition of minerals (mainly Ca
2+
and P
i
) occurs and hydroxyapatite is produced, a process involving proteins like Annexins
and Alp (Balcerzak et al., 2003; Kirsch et al., 2005). The attachment of the vesicles to bone is
not well understood, but Alp and Annexin in the vesicle membrane are reported to anchor
to collagen fibrils (Wu et al., 1991). Vesicle formation is followed by the linking of
hydroxyapatite crystals to ECM components (Balcerzak et al., 2003) using the Ca
2+
and
hydroxyapatite binding properties of Osteonectin, Osteopontin, Osteocalcin and Bone
sialoprotein (Hoffmann et al., 1996; Pinto et al., 2001; Furie et al., 1991). Hypertrophic
chondrocytes are also capable of initiating calcification processes by releasing similar matrix
vesicles as osteoblasts and it has been suggested that hypertrophic chondrocytes may
participate actively in bone formation (Anderson et al., 1975; Kirsch et al., 1997). Moreover,
hypertrophic chondrocytes from both mammals and teleosts express genes like osteocalcin,
osteonectin and alp (Ytteborg et al., 2010b; Ishizeki et al., 1996; Lian et al., 1993). Cancedda et
al. (1992) showed that hypertrophic chondrocytes from chicken can be induced to obtain a
strictly osteoblastic phenotype in vitro. These findings are supported by Yasui et al. (1997)
Health and Environment in Aquaculture
336
who suggested that hypertrophic chondrocytes are able to trans-differentiate into
osteoblasts and produce bone through a process called trans-chondroid ossification. More
than 10 different forms of cartilage and several other tissues with histological characteristics
between bone and cartilage have so far been identified in fish (Huysseune et al., 1986;
Huysseune et al., 1990). This makes bone studies in Atlantic salmon more complicated, as
strict lines between cell types and distinct borders between tissue structures are difficult to
define. However, intermediate tissue is instructive due to the many molecular pathways
and cellular adaptations during pathological development and normal growth.
3. Pipeline for studying vertebral development
Bone deformities in Atlantic salmon are a complex problem, which may have diverse causes,
acting either one by one or in combination, hence, a number of different tools are important
to establish in order to cover different mechanisms involved in their development. The
pipeline for studying bone development in teleosts is shown in figure 6. In vertebrates, both
bone and cartilaginous structures coexist during development of the vertebral column and
both tissues are built up mainly of the organic ECM. Cartilage and bone cellular activity
largely depends on the interaction with ECM components. ECM components regulate cell
growth and differentiation by interacting with growth factors and enzymes, provide the
tissue with mechanical strength and resilience and constitute the template for mineralization
during development of the vertebral column. The composition and structure of molecules in
the ECM are shown to play pivotal roles in bone formation and changes therein may result
in deformities in the spine of both mammals and teleosts (Pedersen et al., 2010).
Radiography, or the use of X-rays for analysis, is the preferred method for fish skeletal
deformity diagnostics. X-rays have enough energy to penetrate soft tissues, but not bone
and other hard substances. Moreover radiography thus allows the creation of a negative
image of the skeletal structures of the fish, which allows the evaluation of calcification level
and for identification of pathology in the bones, without cutting into or even killing the fish.
However, fish radiography has its limits and it is difficult to diagnose fish before the
deformity has developed. More sensitive techniques are therefore necessary. So far in vivo
trials with Atlantic salmon using different temperatures and light regimes, water speed for
studying the effect of training and feeding trials using custom-made feeds for studying
mineral and vitamin components has been applied for deformity studies. In addition to
radiography and measuring rate of development and growth, essential minerals have been
followed from uptake and secretion in the intestines using quantitative real time PCR, to
incorporation in the bone matrix using mineral analysis and Fourier Transform InfraRed
(FT-IR), histological staining techniques and screening techniques such as microarray.
Important pathways for cellular differentiation of bone and cartilage have been followed
using gene expressional tools, like quantitative real time PCR, in situ hybridization and
immunohistochemistry. In a recent puplication, (Ytteborg et al., 2010b), it was shown by
using molecular markers and gene transcription techniques, that fish susceptible for
developing vertebral fusions could be detected already at 2g size. Atlantic salmon in vitro
based systems are also developed, where cellular differentiation and lineage determination
can be studied in more controlled environments (Ytteborg et al, 2010a). Combining
radiography, histological staining techniques and molecular tools has led to a more
complete understanding of how normal and pathological bone formation in Atlantic
salmon progress and opens up for prospective advanced functional studies in
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Fig. 6. Pipeline for studying vertebral development.
commercial teleosts species. Importantly, management control of deformities and health in
general demands precise tools and knowledge to depict any problem as early as possible in
the production line. The reliable correlation between defined skeletal markers and the risk of
developing vertebral deformities has indicated that these genes can be developed as
prognostic markers and further be used to investigate how the progression of skeletogenesis
is modulated in response to other stimuli.
4. The teleost vertebra
The vertebral column is the defining feature of all vertebrates, composed of an alternating
pattern of vertebral bodies (centra) and intervertebral regions. While centra give support
and strength to the organism, intervertebral regions provide flexibility. The segmented
pattern of the spine is established during embryogenesis when the precursors of the
vertebrae, the somites, are formed (review in Brand-Saberi et al., 2000). The mature Atlantic
salmon vertebra consists of approximately 58 vertebral bodies with neural and heamal
arches protruding from the top and bottom of the centrum, respectively (Kacem et al., 1998).
Grotmol and co-workers (Grotmol et al., 2006, 2005, 2003) have previously described the
early development of the Atlantic salmon vertebrae in details. However, few studies have
defined the nutritional needs or described the functions needed to keep continuous growth,
remodelling and homeostasis in the mature vertebrae. An overview of the Atlantic salmon
vertebra features is shown in figure 7.
4.1 The intervertebral regions
The notochord is found in embryos of all chordates, being well conserved between species
as the forerunner of the spinal column. However, whereas only remnants of the notochord
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Fig. 7. Overview of the Atlantic salmon vertebra.
exist in the mammalian intervertebral disc (IVD) between adjacent vertebrae (Walmsley et
al., 2009), the notochord persists throughout all life stages and throughout the entire length
of the fully developed vertebral column in many teleosts, including Atlantic salmon. The
morphology and function of the notochord of the mature vertebrae has not been thoroughly
described. However, the layers and cell types found at early larval stages persist throughout
all life stages in the salmonides. Hence, the mature notochord of Atlantic salmon consists of
a core of chordocytes, a layer of chordoblasts, an acellular fibrous sheath and an outer elastic
membrane (Grotmol et al., 2006). The chordoblasts continue to divide throughout life in
accordance with sustained notochordal growth (Grotmol et al., 2006) and maturate into
chordocytes, containing large fluid filled vacuoles (Adams et al., 1990; Glickman et al., 2003;
Nordvik et al., 2005). The chordoblasts also produce the basal membrane and ECM
components of the notochordal sheath, which in both mammals and teleosts like Atlantic
salmon, has been shown to consist of mainly Col2 fibrils (Domowicz et al., 1995;Linsenma et
al., 1973; Sandell et al., 1994). In mammals, the remnants of the notochord, the chordoblasts
and their subsequent matrix, develop into the intervertebral discs (IVDs), which separate the
vertebral bodies. The annulus fibrosus (AF) surrounding the nucleus pulposus (NP) of
mammalian discs consists of overlapping collagen and elastin fibrils, forming transversing
bands crossing the joint in opposite directions, hence, stabilizing and supporting the
intervertebral regions. The NP consists of a fluid filled matrix which distributes the
hydraulic pressure in all directions within each disc under compressive loads. Similarly in
teleosts, the helical geometry shift between adjacent collagen lamella in the acellular
notochordal sheath restricts expansion of the vacuolated chordocytes (Grotmol et al., 2005;
Grotmol et al., 2006; Koehl et al., 2000). The elastic membrane surrounding the notochordal
sheath has a thickened structure in the intervertebral regions, further contributing to
increased strength in these regions. At more mature stages, the notochordal sheath consists
of folded structures (Ytteborg et al., 2010d), which may be the consequence of compressions
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of the notochordal sheath upon formation and mineralization of the centra. As the cross-
helical architecture of parallel Col2 fibrils probably is important for flexural stiffness of the
larval body during development (Grotmol et al., 2006) the folded pattern may contribute to
increased flexibility and normal functioning of the mature spinal column.
In addition to its structural role, the notochord secretes factors to surrounding tissues and
contributes to vertebral patterning during embryogenesis (Cleaver et al., 2001; Fleming et
al., 2004). The role of the notochord in patterning of the somites is known from several
studies from chicken, mouse and zebrafish, in which secretion of Sonic hedgehog (Shh) from
the notochord appears to be essential both for somite survival during the early
somitogenesis and for induction of the sclerotome during later somitogenesis (review in
Monsoro-Burq et al., 2005). In vertebrate species with limited growth, such as humans, the
notochord ceases its regulating role for vertebral development as part of the normal
ontogeny, followed by the transformation of notochordal tissue into cartilage (Hunter et al.,
2003; Oegema et al., 2002). In Atlantic salmon, however, the notochord should fulfil its
regulating role for vertebral body differentiation throughout life, since salmon and other
fish species do not stop growing. Immunohistochemistry with the proteoglycan component
Perlecan has revealed that this protein is abundantly present in the notochordal sheath of
Atlantic salmon (Ytteborg et al., 2010d). Perlecan has structural roles in mammalian cartilage
and IVD (Sivan et al., 2006) and is important for proper establishment of basement
membranes in different vertebrates including teleosts (Parsons et al., 2002; Aviezer et al.,
1994). An interesting aspect of Perlecan is its link to nutritional transportation over the
notochordal sheath. Parsons et al. (2002) have previous suggested similarities between the
structural role of the teleost notochordal sheath and the mammalian glomerular kidney
membrane (GBM). GBM is an important part of the filtration machinery in the kidneys and
involved in hydrostatic pressure maintenance (Timpl et al., 1996). The heparan sulfate
chains of perlecan have further been shown to play important roles in glomerular filtration
(Morita et al., 2005) and to be involved in diffusion of nutrients during tooth development in
mice (Ida-Yonemochi et al., 2005). The mammalian IVD basically relies on diffusion for
nutrient supplies and removal of waste products. As no evidences for vascularization of the
Atlantic salmon notochord exists today, it seems likely that a similar transportation system
must apply for the vacuolated chordocytes in the notochord core.
4.2 The centra
The Atlantic salmon spinal column is formed directly in bone, in contrasts to the formation
of the vertebrae of avian and mammalian species, which are first formed in cartilage (Arratia
et al., 2001; Smith et al., 2009). At early stages, the precursors for the osteoblasts are situated
on the external elastic membrane only interrupted by the neural and haemal arch cartilages.
The segmentation process leading to formation of vertebral and intervertebral regions starts
with the formation of the chordacentra, where matrix in the outer half of the notochordal
sheath becomes mineralized (Fleming et al., 2004; Arratia et al., 2001; Laerm et al., 1976;
Grotmol et al., 2003). Osteoblast at the vertebral growth zones and osteoblasts lining the
trabeculae are involved in intramembraneous ossification. Denser osteoblast populations are
located along the cranial and caudal rims of each vertebral body, leading to the biconid
hour-glass shaped vertebra. In situ hybridization has confirmed transcription of osteogenic
marker genes like runx2, col1a, osteocalcin and osteonectin in these populations at mature
stages in Atlantic salmon ontogeny, confirming their actively involvement in osteoid
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production throughout life (Krossy et al., 2009; Ytteborg et al., 2010b and c), shown in
figure 8. In the arch centra of Atlantic salmon, in situ hybridization have identified sub-
populations of chondrocytes corresponding to the resting, proliferating and hypertrophic
chondrocytes described in mammals (Ytteborg et al., 2010b; Hunziker et al., 1994).
Chondrogenic marker genes, like col2a, col10a, sox9 and mef2c, are characteristic for specific
maturation zones and have been used to characterize the maturation process in the arches of
Atlantic salmon (Ytteborg et al., 2010b). TRAP secreting osteoclasts has further been
identified at the ossifying borders of the salmon arch centra, marking the ossification front
during endochondral ossification (Witten et al., 2009, Helland et al., 2006; Ytteborg et al.,
2010c). In the vertebrae of Atlantic salmon, multinucleated osteoclasts have also been
identified in the trabeculae but not in the compact bone of the amphicoel (Witten et al., 2009).
As the vertebra grow through the activity of osteoblasts located along the distal ridges, the
trabeculae becomes more branched and filled with adipose tissue. After finishing shaping
the scaffold for the vertebral bodies, the Atlantic salmon vertebrae continue to grow
throughout life (Nordvik et al., 2005). Compared to mammals, where bone is constantly
remodeled, the shape and constant growth of the salmon vertebrae have indicated that the
need for bone remodeling is scarce. During stressful or unfavorable conditions or during
periods of rapid growth, the mammalian skeleton is used as a mineral reservoir, where
minerals are released through the activity from the osteoclasts. In Atlantic salmon however,
such reservoirs are mostly found in the scales. Experiments have shown that long-term
Fig. 8. Overview of histological, immunohistochemical and molecular findings in non-
deformed vertebrae. Vertebral endbones (top): Toluidine, PCNA, Caspase 3, col1a, runx2,
col2a Elastin in elastic membrane (left), Perlecan in notochordal sheath (left). Osteocalcin in
trabeculae (right). Aggrecan in chordocytes (right) Arch centra (bottom): Alizarin
red/Toluidine blue, TRAP, PCNA, col2a, col10a, mef2c. Trabeculae, tb; Notochordal sheath,
ns; Notochord, nc; End bone, eb; Arch centra, ac. Scale bare = 100 m.
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stressful conditions rather manifests in salmon as overall improper bone formation and
cellular disturbances rather than increased bone resorption. This has been shown through x-
ray visualization of lower radiodensity (e.g. in ghost and hyperdence vertebrae),
development of soft bone phenotype, transcriptional analysis (e.g. reduced transcription
of genes involved in production and mineralization of ECM) and immunohistochemistry
showing disturbed cell cycling (e.g. using PCNA antibodies) in vertebrae not yet possessing
skeletal malformations. However, these disturbances might further develop into vertebral
deformities at later stages.
5. Vertebral deformities
Deformities in the spinal column have been observed in a diverse array of vertebrates and a
number of causatives have been suggested. Spinal disorders are a major concern for human
health and often related to painful conditions (Freemont et al., 2009). Spinal lesions observed
in wild animals, such as brown bear, sandtiger shark and smallmouth bass are occasionally
found and often reflect environmental problems (Preziosi et al., 2006; Bengtsson et al., 1979;
Vandenavyle et al., 1989; Wagner et al., 2005). Deformities in domesticated animals like
chicken, broilers, pigs and farmed fish are recognized as a reoccurring problem in intensive
production system and represent both ethical and economical challenges (Berg et al., 2006;
Hammond et al., 2007; Julian et al., 1998; Reiland et al., 1978; Sullivan et al., 2007). Fish with
spinal deformities, such as salmon, trout, cod, halibut, sea bass and sea bream, do not swim
efficiently, are less capable of acquiring food, are at a greater risk of predation and are more
susceptible to physiological imbalance, in addition to being down-graded at slaughter
(Silverstone et al., 2002). Most deformity studies in teleosts have been largely descriptive
and primarily performed to reveal factors contributing to increased occurrence of skeletal
deformities, e.g. genetics, infections, fast growth, light regimes, vaccination, water current
and quality, pollution, malnutrition and elevated temperatures (Berg et al., 2006; Berntssen
et al., 2003; Cahu et al., 2003; Divanach et al., 1997; Gjerde et al., 2005; Koumoundouros et al.,
2001; Lall et al., 2007; Madsen et al., 2000; Roy et al., 2002; Vagsholm et al., 1998). Spinal
deformities in Atlantic salmon have been intensively studied during the past years due to
the importance of this specie to the aquaculture industry. Bone deformities in Atlantic
salmon are a complex problem, which may have diverse causes that may act alone or in
combination. Among these causes of bone deformities, the effect of temperature stress
during the early developmental stages is best documented (Ytteborg et al., 2010a,b;
Wargelius et al,. 2005). Malformations later in life are often related to abnormal nutritional
preferences, malnutrition or fast growth. Until recently, the molecular development of
spinal deformities in fish has received relatively little attention and few deformities have
been explored beyond the level of association with particular causative factors. However,
accumulated studies on intensive production regimes and incidence of deformities have
been followed by more and more advanced studies on vertebral development and bone
biology. Below is the current state of knowledge on cellular mechanisms for pathological
bone development. In figure 9, major causatives, radiography and histological staining of
normal and deformed salmon is shown.
5.1 Cellular mechanisms behind weakened bone structures and development
Conditions that accompany fast growth in farmed animals, e.g. light and feeding regimes,
elevated temperatures and breeding, are linked to increased numbers of spinal deformities
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Fig. 9. Normal (left) and deformed (right) Atlantic salmon. From the top: photography of the
fish, radiographic image, enlarged radiography, Alizarine red S and Tolouidine blue double
staining.
(Julian et al., 1998; Reiland et al., 1978; Wargelius et al., 2009). Fast growing Atlantic salmon
has been shown to develop soft, low mineralized, bone compared to fish with lower growth
rates (Fjelldal et al., 2006) and to have an increased risk of developing vertebral deformities
(Fjelldal et al., 2007; Fjelldal et al., 2005). In fast growing Atlantic salmon, elevated muscle
mass exercise pressure on under-calcified bone that increases the mechanical pressure,
which might trigger formation of intermediate tissues and malformations (Witten et al.,
2005). Comparative studies have been performed in commercially farmed chicken, which
are the product of long-term selective breeding for high growth rates (Leterrier et al., 1992).
Fast growing chicken have weaker bone structures and increased rates of skeletal
abnormalities than slower growing broilers, which reduces the bone's ability to adapt to the
higher loads induced by the increasing body weight (Rawlinson et al., 2009). In Atlantic
salmon, however, high genetic growth rates have not been correlated to increased rates of
deformities (Gjerde et al., 2005). To fulfil the requirements for bone mineralization, fast
growing animals needs to assimilate a higher proportion of the mineral intake (Hernandez
et al., 2000). However, knowledge concerning mineral uptake and transportation in the fish
intestines are lacking and needs to be studied further. The current change in fish feed
production, switching to a vegetable based lipid diet, may further change the intestinal
uptake of minerals, vitamins and amino acids (Jutfelt et al., 2007). Achieving predictable
production of high-quality fish that perform well later in life therefore requires a high level
of control of various factors influencing normal development and growth during early
phases of life. Understanding the interactions between dietary mineral levels, n6/n3 fatty
acid ratios, bioavailability, growth rate, temperature and intestinal uptake is imperative to
be able to balance diet composition and use available feed ingredients adequately.
At the cellular level, a general trade-off between proliferation and differentiation has been
suggested as a cause for delayed skeletal development in fast growing species of birds
(Arendt et al., 2000; Rawlinson et al., 2009). It has further been suggested that during rapid
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growth the time required for bone matrix to be produced and mineralized may be reduced
to a critical level (Hernandez et al, 2000); hence development of a soft bone phenotype. This
causative relation has been suggested for fast growing under-yearling Atlantic salmon smolt
that has a higher incidence of vertebral deformities than slower growing yearling smolt
(Fjelldal et al., 2006). Temperature and light regimes are factors shown to speed up
developmental rate in Atlantic salmon, but also to delay production of osteoid. It therefore
seems that bone remodeling in Atlantic salmon is generally sensitive to elevated growth
rates (Ytteborg et al., 2010a). Osteoblasts and chondrocytes are cell types producing large
quantities of ECM and may therefore be particularly sensitive to stressful conditions, due to
reduced normal protein synthesis (Tsang et al., 2007; Haynes et al., 2004). Quantification of
mRNA in vertebrae from fast growing Atlantic salmon has revealed a reduced transcription
of important genes encoding structural proteins taking part in the bone matrix and
mineralization, e.g. col1a1, osteocalcin and osteonectin (Ytteborg et al., 2010b). Furthermore,
generally weaker in situ hybridization signals were detected for probes targeting these ECM
transcripts in areas where intramembranous ossification takes place. These findings further
correlated to an impaired mineralization and supported the assumption that disturbances in
bone formation constitute an important part of the mechanisms involved in soft bone
formation. These observations are further consistent with an Atlantic salmon osteoblast in
vitro experiment, where long-term 16C heat exposed cells showed a decreased transcription
of alp, col1a1 and osteocalcin. Based on in vitro and in vivo results it seems that Atlantic
salmon osteoblasts may be particularly sensitive to elevated temperatures during the early
stages of differentiation.
In mammals and teleosts like Atlantic salmon, elevated temperatures and fast growth may
also interrupt the normal chondrocytic differentiation pattern and delay endochondral bone
formation, further weakening the bony structures (Tsang et al., 2007). A number of studies
have linked skeletal malformations to disturbances in chondrocytic maturation (Kieswetter
et al., 1997; Farquharson et al., 2000; Julian et al., 1998). Recent results have suggested that
fast growth caused by elevated temperatures leads to an arrest prior to the final maturation
of chondrocytes in the Atlantic salmon vertebral arch centra (Ytteborg et al., 2010c).
Morphological studies of the arch centra of juvenile Atlantic salmon reared under intensive
temperatures have identified chondrocytes with a distorted maturation pattern and an
increased zone of hypertrophic chondrocytes (Ytteborg et al., 2010c). In this study, an
increased zone of hypertrophic chondrocytes correlated with increased transcription of
hypertrophic marker genes such as col10a1 and mef2c. Fast growing chickens are also
characterized by disturbed chondrocytic maturation where cartilage do not mature enough
to ossify (Julian et al., 2005; Farquharson et al., 2000) and increased mechanical load is
associated with an increased hypertrophic zone in the growth plate of rat ulnae along with a
suppressed mineralization rate (Robling et al., 2001; Ohashi et al., 2002). Furthermore,
mammalian osteoclasts are temperature sensitive and hypothermic conditions may
stimulate their activity (Patel et al., 2009). Similar observations have been described in
Atlantic salmon where no TRAP activity was observed in the arch centra of fish reared at
intensive temperatures. Also transcription of osteoclast associated marker genes, like Mmps
and Cathepsin K was reduced (Ytteborg et al., 2010c). Absence of Mmps may cause delays in
endochondral ossification and runx2 deficiency may inhibit mmp expression and lead to
mild disturbances of chondrocyte differentiation (Inada et al., 1999; Kirsch et al., 1997;
Pratap, 2005). Disturbances in chondrocytic maturation and endochondral ossification will
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overall weaken the vertebrae, and may be an explanation for wrinkled and shortened ribs
observed in Atlantic salmon suffering from P deficiency (reviewed in Sugiura et al 2005).
Overall, both bone and cartilage formation seems disturbed during fast growth and may
equally contribute to weakened skeletal structures. In Atlantic salmon, experiments have
indicated that during rapid growth, both endochondral and intramembraneous ossification
is affected. Moreover, fast growth leading to weakened bone and cartilage structures at
juvenile stages increases the risk of developing severe deformities later in ontogeny. This
might be a result of local cellular compensation and an effort to restore and strengthen a
weakened area in the vertebrae, as described in the next chapter.
5.2 Cellular mechanisms behind vertebral deformities
Witten et al. (2009) recently published a survey on commonly observed vertebral
malformations in Atlantic salmon which included different grades and combinations of
platyspondyly (compressions), ankylosis (fusions), lordosis (-shaped vertebral column),
kyphosis (-shaped vertebral column) and scoliosis (S-shaped vertebral column).
Histological characterization of compressions and fusions have described shape alterations
of vertebral body endplates, reduced intervertebral space, transformation of intervertebral
notochord tissue into cartilage, mineralization of the intervertebral cartilage and
replacement of intervertebral cartilage by bone (Witten et al., 2005; Kvellestad et al., 2000;
Witten et al., 2006), independent of the factor inducing the malformation. Changes in
transcriptional processes in osteoblasts and chondrocytes from both mammals and teleosts
are involved in pathological vertebral formation (Hammond et al., 2007; Breen et al., 1999;
Wargelius et al., 2005). The development of vertebral fusions is a dynamic process but recent
publications have shown that the underlying cellular and molecular mechanisms may be
summarized as four key events (Ytteborg et al., 2010a, b and c). These events are illustrated
in figure 10 and described in the text below.
I: Disorganization
The initiation of the fusion process includes disorganization and proliferation of osteoblasts
and chordoblasts. Osteoblasts at the growth zones of the vertebral body endplates have a
markedly increased cell proliferation rate and the growth zones extend spatially along the
rims of fusing vertebral bodies. As the intervertebral space narrows, proliferating
chordoblasts and denser packet chordocytes appear. With a progressing pathology,
proliferating chordoblasts occupy most of the intervertebral space and vacuolated
chordocytes disappear.
II: Metaplastic shift
Proliferating cells at the border between the osteoblast growth zones and the arch centra
show a transcriptional shift, where co-transcription of osteogenic (col1a, runx2, osteocalcin
and osteonectin) and chondrogenic (col2a, mef2c and col10a) marker genes are prominent. The
marked border between the osteoblast growth zones and the chondrocytic areas connected
to the arches becomes less distinct, as proliferating cells and chondrocytes blend through an
intermediate zone. A similar shift is found in the notochord where co-transcription of genes
such as col2a, sox9, col1a and runx2 increase with proliferation of chordoblasts. In the
central notochord of developing fusions, hyperdense regions of denser packet chordocytes
lacking vacuoles appear as the number of proliferating cell increase.
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Fig. 10. Major findings during the development of vertebral fusions in Atlantic salmon. I
Disorganization, II Metaplsatic shift, III Loss of notochordal sheath integrity, and IV Ectopic
bone formation. See text for details. Scale bar = 100 m (a-l), 50 m (m-r), 200 m (s-u).
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III: Loss of notochordal sheath integrity
The elastic membrane surrounding the notochord becomes fragmented and the notochordal
sheath loose its integrity. Verhoeff`s hematoxylin staining has visualized a thinner elastic
membrane surrounding the notochordal sheath of developing vertebral fusions. In the most
severe cases, the elastic membrane is fragmented. Furthermore, the highly folded structures
in the notochordal sheath are lost during development of spinal fusions.
IV: Ectopic bone formation
Ectopic bone formation in the affected areas gives the vertebral bodies a squared
morphology as the arch centra fuse and ossify. Ectopic mineralization of intervertebral
regions and arch centra is formed, indicating that the proliferating and metaplastic cells not
only differentiates towards osteoblast-like cells, but also complete the differentiation to cells
that are capable of producing mineralized matrix. The intervertebral space narrows
completely down and the notochord mineralizes.
The overall structural and molecular features of bone and cartilage development in vertebral
fusions in Atlantic salmon have shown resemblance with similar pathological spinal
conditions in mammals (Ytteborg et al., 2010c and d; Gorman et al., 2007; Witten et al., 2006).
For example several mammalian studies have suggest that changes in the balance between cell
death and cell proliferation is involved in bone and cartilage defects which may lead to
malformations (Cockroft et al., 1978; Miura et al., 2004; Breen et al., 1999; Farquharson et al.,
2000). Spinal fusions in Atlantic salmon are characterized by changes in ECM components and
mineralization of the intervertebral regions (Ytteborg et al., 2010c; Witten et al., 2006). Similarly,
intervertebral disc degeneration (IDD) in mammals involves breakdown of ECM components
in the AF and calcification of the NP (Takaishi et al., 1997; Kanemoto et al., 1996; Antoniou et
al., 1996). Fusion, compression and chondrogenic transformation of skeletal tissue have also
been reported from lordosis and kyphosis in sea bass. Histological examinations of both
lordosis and hyperdense vertebrae have further indicated cellular plasticity (like metaplastic
shifts and trans-differentiation) and development of intermediate tissues as pathological
events (Ytteborg et al., 2010c; Helland et al., 2006; Kranenbarg et al., 2006; Witten et al., 2006;
Witten et al., 2005). It has previously been suggested that a metaplastic shift is involved in the
development of spinal fusions, leading to the formation of chondroid bone which at later
stages in the fusion process is replaced by bone. As previously discussed, chondrocytes
associated with calcifying cartilage can acquire properties of osteoblasts (Cancedda et al., 1992)
and are able to change their phenotype from a primarily cartilage synthesizing cell type to a
bone synthesizing cell type (Lian et al., 1993). Co-transcription of chondrogenic and
osteogenic marker genes in the arch centra and notochord supports the suggestion of an
adaptation through metaplastic shifts during development of vertebral fusions, which may be
induced to produce more robust cells that are able to withstand increased mechanical load. A
pathway to bone formation through chondrocytes might be possible during development of
vertebral fusions and fast growth, which could be similar to trans-chondroid ossification, as
described by Yasui et al. (1997). Trans-differentiation and ectopic calcification has also been
suggested as pathological pathways in lordotic sea bass where deformations stimulate ectopic
bone formation in the intervertebral regions between two affected vertebral bodies and
along the rims of the vertebral body endplates (Kranenbarg et al., 2006). Similarly, a
shift in the mammalian IVD NP cell population coincides with spinal disorders
like intervertebral disc degeneration and changes in the synthesis of matrix molecules differ
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with the degree of degeneration (Handa et al., 1997). The mammalian AF is further
strengthened through cartilage formation upon elevated mechanical load (Lotz et al., 2002;
Prescher et al., 1998). Moreover, breakdown of PG components, like Aggrecan and Perlecan,
may lead to reduced hydrostatic pressure, invasion of nerves and blood vessels and loss of
transportation of nutrients and waste products in degenerating IVD (Kauppila, 1995; Urban et
al., 2003 and 2004; Melrose et al., 2002; Yasuma et al., 1993). Loss of Aggrecan resulting in
tissue dehydration, reduces the ability of mammalian IVD to transmit and absorb compressive
load (Kanemoto et al., 1996; Urban et al., 1985). Loss of Aggrecan and Perlecan has also been
observed in the notochord of Atlantic salmon during development of vertebral fusions
(Ytteborg et al. 2010d), which may possibly reduce the hydrostatic pressure and hence the
transportation of nutrients and alteration of pH values. Another comparative pathological
process to teleost vertebral fusions is the mammalian Bamboo spine, describing a condition
where vertebral bodies have fused and reshaped through ectopic bone formation (Bakay et al.,
1970; Resnick et al., 1983). Witten et al.(2005) have described similar processes in Atlantic
salmon. Fusing vertebral bodies may either stabilize as on large vertebral body or continue to
develop through neighbouring vertebrae. What kind of cellular actions leading to a stabilized
or aggravating fusion remains to be answered. However, it seems that different types of
deformities have similar pathways of cellular pathological development, processes involving
proliferation, metaplastic shifts, cellular instability and trans-differentiation.
6. Conclusion
During the last decade, fish have emerged as suitable animal models for studying bone and
cartilage biology and have shown to be a suitable supplement to mammalian systems
aiming to uncover the corresponding fundamental cellular and molecular mechanisms of
action. In the light of metaplastic shifts during skeletal deformities in Atlantic salmon, a cell
culture based system allowing for cellular differentiation and lineage determination studies
have been developed. In this particular system, precursor cells are stimulated to myogenic,
adipogenic and osteogenic differentiation, and opens up for studies where these cells can be
manipulated upon different stimuli to undergo metaplastic shifts. Hence, functional studies
can be performed to better characterize the pathology, define particular requirements and
minimize the occurrence of bone disorders. Advanced methods and defined molecular
markers should enable us to detect the risk of developing deformities early in ontogeny.
Similar diagnostics and medications as those existing in the human medicine will not be
applicable for farmed animals. However, treatments and diets have shown to be well suited
also for teleosts. Exercise and addition of minerals in the feed have already shown positive
effects in regards of bone quality and should be further addressed in future research.
7. Acknowledgments
The authors would like to thank Johanne Halvorsen (www.forestry.no) for help with
graphic illustrations.
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Part 5
Ecological Impacts of Fish Farming
14
Ecological Features of Large
Neotropical Reservoirs and Its
Relation to Health of Cage Reared Fish
Edmir Daniel Carvalho, Reinaldo Jos da Silva, Igor Paiva Ramos,
Jaciara Vanessa Krger Paes, Augusto Seawright Zanatta,
Heleno Brando, rica de Oliveira Penha Zica, Andr Batista Nobile,
Aline Angelina Acosta and Gianmarco Silva David
So Paulo State University (UNESP)
Brazil
1. Introduction
Environmental Brazilian legislation allow the implementation of cage culture fish farm
enterprises in large public reservoirs (Ayroza et. al., 2006), aiming a qualitative and
quantitative increase of inland aquaculture, regarding concerns with environmental,
economic and social sustainability (Costa-Pierce, 2002; Valente, 2000). Fish farming is an
important activity for animal protein production, and if well planned, is benefic to the
economic development of the country. However, it is necessary continuous assistance of
appropriate expertise and scientific support in order to organize and improve fishery and
aquaculture (Agostinho et al., 2007). Due to low productivity of inland native fish stocks,
Brazilian government aquaculture programs initially focused on change artisanal fisher to
fish farmers, assuming that this could improve their economic situation. This is a
mistaken philosophy, since the extrativist way of life contradicts the planned life of a
modern aquaculturist. This is especially applicable for Brazilian Southeastern region,
where this activity is basically maintained by intensive culture of tilapia (Furlaneto et al.,
2006) by capitalized stakeholders. Food and Agriculture Organization of the United
Nations [FAO] (2010) and Rojas & Wadsworth (2007) highlight that aquaculture annual
growth rate surpasses other zootechnical activities. Currently, annual fish yield in Brazil,
by means of aquaculture and extractive fishing, has accomplished a 1,240,000 metric
tons/year baseline, of which approximately 10% are related to tilapias (Ministrio da Pesca
[MPA], 2010), predominantly Oreochromis niloticus and derived hibrids Lovshin, 1982).
Within this context, Brazil has been showing vigorous growth (over 25% per year) in this
decade, however this growth is still modest considering the prodigious potential of water
resources and suitable weather that Brazil offers (Godinho, 2007; MPA, 2010). It is evident
that Brazilian aquaculture production is behind its potential comparing to Chinese fish
yield, that produces approximately 47.5 millions of metric tons/year (FAO, 2010).
Fitzsimmons (2006) highlights Brazil as a prominent country that could compete with
China as biggest fish producer in the world. The Paran River is the second largest
catchment in South America, with 3,780 km of extension, and is the main River of La Plata
Health and Environment in Aquaculture
362
River basin originating at the confluence of Paranaba and Grande Rivers and has a
watershed area of 2,800,000 km
2
, which consists chiefly of sedimentary and volcanic
rocks. The Paran River stretches are divided into an upper course, from its source to
Itaipu reservoir; a middle course along Paraguay-Argentina border; and a lower course
from Paraguay River confluence to La Plata River estuary. The Upper Paran River basin,
with an extension of 809 km and area 820,000 km
2
, has about 250 km without
impoundments, resulting in a deeply altered hydrological and limnological regime
(Stevaux, 1994). Currently, Brazilian inland net cage aquaculture is integrated to these
large reservoirs. Within this context, in the last five decades it is noticed that Brazilian
large Rivers have been impounded to build dams and power-plants, aiming
hidroelectricity as a priority (Tundisi, 1993; Zocchi, 2002), to meet the increasing demand
for energy in the country. This way of producing hidroelectricity energy represents 14.8%
of all Brazilian energy matrix (Ministrio das Minas e Energia [MME], 2006), with So
Paulo state responsible for over 22% of this type of energy. These impoundments were
built as a cascade system in large rivers (Grande, Tiet, Paranapanema and Paran Rivers)
(Agncia Nacional de Energia Eltrica [ANEEL], 2009; Agostinho et al., 2007). Under an
ecological perspective and environmental legislation, a good water quality and aquatic
ecosystem integrity are fundamental to allocate the multiple uses of these large reservoirs,
especially to effective organization by policy makers for aquaculture and fishing activities.
In limnological terms, the determination of trophic state index (TSI) sensu Carlson (1977),
based upon phosphorus and a chlorophyll contents to a specific water body, is a
satisfactory and practical tool as environmental indicator, considering different human
interventions that induce artificial eutrophication process. Various studies in
hydrographic sub-basins of Tiet and Paranapanema Rivers show that this index usually
varies. As an example, the index varies between oligotrophic to mesotrophic state for
upper and middle Paranapanema stretches (Nogueira et al. 2006), and also between
oligotrophic to eutrophic for Tiet River (Barbosa et al. 1999; Moretto et al., 2008; Tundisi
& Strakraba, 1999). These variations are mainly due to anthropogenic actions, such as
occupation of lands for agriculture, livestock, increasing urbanization due to growth of
human population, and emissions of organic wastes (Tundisi, 2005). Brazilian native
ichthyofauna of large rivers has been subjected to negative impacts, such as these
impoundments (Agostinho et al., 2007), introduction of non-native species (Brando et al.,
2009; Latini & Petrere, 2004; Orsi & Agostinho, 1999; Santos & Formagio, 2000; Souto et
al., 2011), environmental contamination, loss of riparian vegetation, sedimentation, and
erosion (Agostinho et al., 2007). Currently, a new form of impact in Brazil is the increasing
development of fish farming in floating cages (Ramos et al., 2008). In cage systems, the
input of organic matter and nutrients is done by artificial feeds, and output is
done through the removal of fish produced, similar to what occurs in fish ponds
(Beveridge, 2004). However, Beveridge (2004); Munday et al. (1992); Persson (1988); and
Pillay (2004) report that in fish cage farming systems up to 30% of feed is lost into the
aquatic environment, in the form of unconsumed feed and wastes. These feed losses can
cause problems related to eutrophication (Beveridge, 1984) and/or be used as a food
resource by local biota (Beveridge, 2004; Hkanson, 2005; Ramos et al., 2008; Vita et al.,
2004), resulting in ecological changes around these systems (Beveridge, 2004; Hkanson,
2005; Ramos et al., 2008). Besides these impacts, several authors as Agostinho et al. (2007);
Beveridge (1984, 1996 e 2004); Dempster et al. (2002); Hkanson (2005); Karakassis et al.
Ecological Features of Large Neotropical
Reservoirs and Its Relation to Health of Cage Reared Fish
363
(2000, 2002 e 2005); Machias et al. (2004, 2005 e 2006); Pitta et al. (2005); Ramos et al.
(2008); Yucel-Gier et al. (2007); and Zanatta et al. (2010) discuss the problems of this
activity in coastal and inland waters. These authors cite impacts upon water quality and
sediment which have implications on the structure of benthic communities, plankton and
fish, and furthermore, the inherent scapes of caged fish. Thus, it is evident the necessity of
developing new technologies aiming the enhancement of fish yield, associated to decrease
of environmental impacts caused by this zootechnical activity. This is a big challenge for
Brazilian aquaculture that needs to guarantee its economic and social sustainability with
preservation of water resources and multiple uses of public reservoirs.
2. Situation of the aquaculture in cage farms in Brazilian Southeastern
reservoirs: An overview
The effects of cage aquaculture enterprises in Brazilian inland waters upon the biota and
water quality have not been satisfactory elucidated yet, thus these effects still require studies
aiming a full comprehension to better ordinate these activities. However, despite divergent
opinions of some sectors of Brazilian society, the Brazilian government has been sponsoring
studies focusing on taking advantage of the potential for aquaculture of large public
reservoirs (Ayroza et al., 2006). Thus, Brazilian government has been selecting, delimitating,
and controlling areas to install Aquaculture Parks in different reservoirs, based upon
premises that promote sustainable development, enhancement of Brazilian fish yield, social
inclusion, and food safety. Effective success of production in aquaculture founds in
limnological studies and environmental carrying capacity, regarding concerns with water
quality and maintenance of water resources (Costa-Pierce, 2002; Tundisi, 2005), as well as its
interference upon aquatic biota, through escapes of non-native species, and pathogen
dispersion (Agostinho et al., 2007; Orsi & Agostinho, 1999). According to Dillon & Rigler
(1975) and Beveridge (2004), the modeling of environmental carrying capacity is done based
upon two fundamental equations, which define phosphorus emission to aquatic ecosystem
without inducing eutrophication, and how much P is emitted per product unit. The lack of
suitable information to define application values of this modeling has been repeatedly
emphasized as the highest difficulty to appraise environmental impacts caused by
aquaculture (Pillay, 2004). In Brazil, cage fish farming systems has gained impulse in the
mid-90s, especially in Brazilian Southeastern (Medeiros, 2002; Ono, 1998; Rojas &
Wadsworth, 2007). Nowadays, this activity is in accelerated expansion in Brazilian
reservoirs, and at least 40 freshwater fish species are used in Brazil, considering all kinds of
pisciculture (Godinho, 2007). However, despite this rich fish diversity, the most used species
for inland aquaculture in Brazil is the non-native Nile tilapia (O. niloticus) due to its
favorable zootechnical features (Castagnolli et al., 2000; David et al, 2006; Rojas &
Wadsworth, 2007). The Middle Paranapanema River area has about 800 net-cages
designated to Tilapia culture along its reservoirs and ponds. It is estimated in 200 kg of
fish/m
3
/cycle in small net-cages (up to 6m
3
); and 100 kg of fish/m
3
/cycle in net-cages of
great capacity (over 10m
3
) (Furlaneto et al., 2006). In this sense, three issues are eminent
within this approach: 1) artificial eutrophication, which is interconnected with
environmental carrying capacity; 2) dispersion of parasites and pathogens and; 3) escapes,
relating to depletion of fish biodiversity lato sensu by ecological processes of competition
and predation. Below, it is followed the results of our main researches aiming to identify
and quantify the ecological interferences of tilapia farming.
Health and Environment in Aquaculture
364
A
50
o
00W 45
o
00W
25
o
00S
20
o
00S
South America
Brazil
So Paulo State
Studied sites
Nova
Avanhandava
Chavantes
Jurumirim
Fig. 1. Reservoirs studied in the Upper Paran River Basin, So Paulo State, Brazil.
2.1 Limnological conditions and trophic state index
A comparative approach was used to evaluate the impact of three cage farms upon
limnological conditions of the stretches were the cages are installed. These farms are located
in three different reservoirs in the upper Paran River Basin, So Paulo state, Brazil (fig. 1).
The farms are small and medium-sized enterprises (ranging from 30 to 200 cages with 6 m
3
each), mainly for the culture of Nile tilapia (O. niloticus) using an intensive model, with high
densities and fed with pelleted compound feed. An example of such farms is shown in fig. 2.
Monthly surveys were performed in the farms in different years from 2003 to 2009, sampling
limnological data and water samples inside the Farm Sites (referred as FS) and in similar
Control areas sited upstream (CT). Differences among farm sites and control areas were
regarded as effect of the nutrient loads from the farms. Limnological sampling included
Secchi depth (Z
DS
), water temperature, pH, dissolved oxygen (DO), electric conductivity
(K
25
) measured in situ using a water quality multiprobe Horiba model U-22; water samples
were collected for nutrients (total Nitrogen - TN and total Phosphorus TP following
Valderrama, 1981; Strickland & Parsons, 1968), total suspended solids (Teixeira et al., 1978)
and chlorophyll a (CHL, Golterman et al., 1978) analyses. TSI was determined based on CHL
and TP by formulae 1 and 2, combined using formulae 3 (Carlson, 1996, as cited in Lin,
2001). The first reservoir studied by our research group was Nova Avanhandava, which is
the fifth of a cascade of reservoirs on the Middle Tiet River, sampled from 2003 to 2004
(Paes, 2006). It is a run-of-river plant, that has been operated since 1982, located at 358 m
above sea level, with surface area of 210 km
2
, total water volume of 2,720 x 10
6
m
3
, mean
discharge rate of 688 m
3
/s, maximum depth 30 m, level oscillation of less than 1 m
throughout the year, and water residence time of 46 days (Torloni et al., 1993; Rodgher et al.,
2002). The second reservoir was Jurumirim, where the surveys were done from 2004 to 2005;
it is a storage plant at Upper Paranapanema River, operated since 1962, located at 568 m
above sea level, total area 484 km
2
, total volume of 7,900 x 10
6
m
3
mean discharge rate of 210
Ecological Features of Large Neotropical
Reservoirs and Its Relation to Health of Cage Reared Fish
365
m
3
s
-1
, mean depth 12.9 m, residence time 334 days (Henry et al., 2006) and significant level
oscillation of 2.2 m along the year.
TSI (CHL) = 9.81*ln(CHL)+30.60
(1)
TSI (TP) = 14.42*ln(TP)+4.15 (2)
TSI = (TSI (CHL)+ TSI (TP))/2 (3)
Fig. 2. Cage farm at Chavantes reservoir, Middle Paranapanema River Basin, So Paulo
State, Brazil.
Lastly, it was studied Chavantes reservoir, that is also a storage plant in Paranapanema
River, operating since 1970 in its middle stretch at 474 m above sea level. This reservoir area
is 400 km
2
, total volume of 8,800 x 10
6
m
3
, maximum depth 80 m, residence time 281 days,
mean outflow 322 m
3
s
-1
, and seasonal water level oscillation is more than 3 m (Nogueira et
al., 2006). The three reservoirs are in a region with annual precipitation above 1,500 mm, and
with a rainy season from September to February and dry season from March to August.
Limnological characterization of the three sites studied are shown in Table 1.
Limnological features of Tiet River basin are quite different from Paranapanema River
basin (table 1). Ph values are higher in Tiet, reaching 9.6, while Paranapanema values were
close to neutrality. Conductivity was three fold higher in Tiet, and CHL were almost ten
fold higher. TP values were slightly higher at Tiet, and TN was two times higher in Nova
Avanhandava than in Jurumirim, and five fold higher than Chavantes. OD mean values
Health and Environment in Aquaculture
366
Reservoirs Nova Avanhandava
1
Jurumirim
2
Chavantes
3
Parameters/Sites FS CT FS CT FS CT
Temperature (C)
20.3-31.3
(26.33.3)
20.0-31.2
(26.23.5)
18.9-27.4
(23.63.0)
18.4-28.2
(23.23.0)
19.3-27.4
(23.42.5)
19.6-28.7
(23.82.5)
pH
5.8-8.9
(8.00.7)
7.2-9.6
(8.20.8)
6.0-8.3
(6.80.6)
6.0-8.2
(6.70.8)
6.0-7.6
(7.00.4)
6.6-7.8
(7.10.3)
Dissolved Oxygen
(mg/L)
6.4-9.9
(8.51.1)
7.0-10.0
(8.71.0))
6.5-8.9
(8.00.8)
6.8-9.0
(8.00.7)
6.5-11.4
(8.11.3)
2.2-11.5
(8.21.7)
Conductivity
(S/cm)
143.7-208.0
(184.818.5)
148.0-207.0
(183.919.0)
50.0-83.0
(59.39.4)
50.0-86.0
(59.89.1)
36.8-60.0
(40.56.0)
36.0-60.0
(40.36.2)
Secchi depth (m)
0.7-2.7
(1.40.6)
0.8-2.5
(1.30.5)
0.6-1.5
(0.90.2)
0.4-1.5
(1.00.3)
1.5-5.0
(3.10.7)
1.5-5.0
(3.10.8)
Chlorophyll a
(g/L)
2.2-30.5
(17.79.3)
5.1-30.1
(18.08.7)
0.5-8.0
(3.32.8)
0.9-3.8
(2.40.8)
0.9-2.3
(1.50.5)
0.7-5.4
(1.91.3)
Total dissolved
solids (mg/L)
0.0-11.1
(3.82.8)
0.5-4.0
(2.71.1)
0.1-5.0
(2.01.5)
0.1-4.4
(1.31.4)
- -
Total P
(g/L)
7.7-23.7
(14.13,7)
5.9-21.3
(12.74.3)
3.4-9.8
(7.02.0)
4.9-15.0
(8.23.6)
4.9-23.5
(11.15.2)
3.1-23.1
(9.74.1)
Total N
(g/L)
763.7-1950.2
(1282.6
283.9)
871.0-1973.4
(1213.2
342.5)
600.0-825.0
(669.472.0)
520.0-780.0
(621.383.5)
108.2-404.6
(244.974.0)
120.0-376.9
(236.078.7)
Table 1. Limnological characterization of surface waters of the three reservoirs studied in the
Paranapanema and Tiet Rivers, Upper Paran Watershed. Modified from
1
Paes (2006),
2
Zanatta (2007) and
3
David et al. (2011).
were above 6 mg/L in all sites, considered adequate for tilapia culture, and average Z
DS
reached 3.1 m at Chavantes, while in the other sites were about 1 m. A concerning issue
about the improvement of cage aquaculture in Upper and Middle Paranapanema reservoirs
is that winter temperatures were far below 23
o
C (fig. 3) and in a less degree for Nova
Avanhandava, assumed as a lower limit for efficient tilapia grow-out commercial systems
(Shelton & Popma, 2006; Suresh, 2002). This fact can hinder aquaculture sustainability and
profitability in this watershed, and suitable native species would be desirable. Paes (2006)
studied a medium-sized farm, with 80 fish cages of six m
3
volume, in Nova Avanhandava
reservoir and concluded that the limnological variables measured showed no statistically
significant differences between FS and CT (Table 1). Some of the variables were considered
high, especially electrical conductivity and total nitrogen, indicating risks of water quality
deterioration, a typical feature of Tiet River Watershed (Barbosa et al., 1999). The Trophic
state index (TSI) was mesotrophic for both areas (fig. 4A). These conditions can be seriously
aggravated by nutrient loads from cage farming activities, which can induce autopollution
and loss of water quality for aquaculture purposes. Alves & Baccarin (2006) found similar
values for these limnological variables, in a cage farm that uses more than 2 tons of feed per
day to produce 1,500 to 1,800 kg of fish per cage per cycle. Water quality depletion is object
of concerns in this basin because of widespread blooms of cyanobacteria (Fraccio et al.,
2002, Tundisi, 2005). In Jurumirim reservoir, Zanatta (2007) studied a small farm with only
30 cages of six m
3
volume, and no significant differences between FS and CT was found for
all limnological parameters measured. The very limited scale of farming operations
probably prevented impacts on water quality in this large reservoir. Thus, only natural
Ecological Features of Large Neotropical
Reservoirs and Its Relation to Health of Cage Reared Fish
367
17
20
23
26
29
32
35
Jan Feb Mar Apr May June July Aug Sept Oct Nov Dec
W
a
t
e
r
t
e
m
p
e
r
a
t
u
r
e
(
C
)
Nova Avanhandava Jurumirim Chavantes
Fig. 3. Seasonal variation of water surface temperature in the studied sites.
and seasonal differences were reported. Jurumirim reservoir was oligotrophic in both areas
throughout the year, except for FS (mesotrophic in August), (fig. 4B).
David et al. (2011) using data from a cage farm in Chavantes reservoir with 200 cages, found
significant seasonal variation for all limnological parameters measured, but no significant
differences among areas at monthly scale. In this same study, comparison of mean values
(pooled data per year) of phosphorus in the water between FS and CT indicates that fish
farming may be related to hipernutrification of FS in the euphotic layer (TP=1.65mg/m
3
,
p<0.05), although no significant increase in chlorophyll a was detected. However, in the
euphotic layer other limnological variables measured showed no significant differences
between areas, even though depletion of dissolved oxygen in the bottom layer in FS was
verified. Most of the year, oligotrophic conditions were also found in Chavantes, but
switched to mesotrophic in January for CT, and in December/January for FS (fig. 4C).
Simulations on the carrying capacity, due to availability of more detailed data on
hydrodynamics and field farming practices in Chavantes reservoir, was also performed by
18 David et al. (2011). Data used for carrying capacity modelling were: Feed Conversion
Rate (FCR): 1.5; feed total phosphorus content: 1.5%; whole fish phosphorus content: 0.9%
(Dantas & Athayde, 2007), resulting on of 13.5 kg P/ton of fish produced. It is worth
emphasizing that phosphorus loads can be greatly influenced by FCR (fig. 5), which itself is
related to the welfare of fish and their physiological condition. In addition to unprofitability,
the cultivation of tilapia at temperatures below the optimal nutrient emissions increases,
which brings higher risks of eutrophication. Hydrological data of FS used in modelling
were: original total phosphorus water content of 10.85 mg/m
3
, maximum allowable TP
water content of 30.0 mg/m
3
, and sedimentation rate of 0.083 (Larsen & Mercier, 1976).
Retention time used was 1 day for precautionary purposes, according to estimates of
Persson et al. (1994) and direct ADCP measurements was less than 1 day, with measured
water current velocity from 0.1 to 0.3 m/s.
Maximum allowable fish production in the area was estimated to be 1666 tons/year, while
the theoretical fish production that would result on the observed increase of TP was 144
tons/year while total tilapia production of approximately 55 tons/year. These results
Health and Environment in Aquaculture
368
25
30
35
40
45
50
55
60
65
Oct/03 Dec/03 Feb/04 Apr/04 June/04 Aug/04
T
r
o
p
h
i
c
S
t
a
t
e
I
n
d
e
x
FS CT
mesotrophic
eutrophic
oligotrophic
25
30
35
40
45
50
55
60
65
Feb/05 May/05 Aug/05 Dec/05
T
r
o
p
h
i
c
S
t
a
t
e
I
n
d
e
x
FS CT
mesotrophic
eutrophic
oligotrophic
25
30
35
40
45
50
55
60
65
May/08 July/08 Sept/08 Nov/08 Jan/09 Mar/09
T
r
o
p
h
i
c
S
t
a
t
e
I
n
d
e
x
FS CT
mesotrophic
eutrophic
oligotrophic
A B C
Fig. 4. Seasonal variation of Trophic State Index (TSI) for FS and CT for Nova Avanhandava
(A), Jurumirim (B) and Chavantes (C) reservoirs.
1
.
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8
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.
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2
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P load
kg/ton fish
produced
FCR
TP feed
kg/ton
Fig. 5. Effect of FCR and feed composition on Phosphorus loads by fish cage culture
indicate that fish production at FS is compatible with the local carrying capacity for the
assimilation and recycling of nutrients derived from aquaculture.
Regarding water quality, conditions may be considered adequate, with no signals of
surpassing eutrophication thresholds. Dillon & Rigler mass balance model overestimated
the fish production needed to cause the observed FS increase, indicating that specific models
are needed for the management of aquaculture in the ecosystems studied here.
2.2 Main fish parasites in cage farms
The interest in fish parasites has increased in recent decades due to economic implications,
particularly for intensive fish farms. The high rates of parasitic infestations or infections can
Ecological Features of Large Neotropical
Reservoirs and Its Relation to Health of Cage Reared Fish
369
cause considerable mortality in several species of farmed fish and its treatment in some
cases is very difficult, while in others, there are no currently effective treatments. The
economic losses caused by parasites can often be verified in an indirect way, either by
reducing the rates of assimilation and growth of infected animals, or by decreasing the value
of marketable final product (Eiras, 1993). Many organisms have been associated with fish
diseases. Although parasites can occur in natural environments, these organisms become
more abundant in conditions of intensive cultivation and depending on the conditions of
culture, they can have a deleterious effect to fishes. In eutrophic environments this situation
could worsen due to the presence of many intermediate host species, favouring the life cycle
of many parasites (Pillay, 2004). Our studies performed with O. niloticus in cage farms have
shown that the main parasites affecting fish health in intensive cage systems are
monogenoids, Trichodina spp., Ichthyophthirius multifiliis and Henneguya sp. Monogeneans
(fig. 6A) are ectoparasites responsible for the most important parasitic disease of fish
farming in Brazil (Martins, 1998). They are characterized by the presence of a fixation
structure generally located in the back of the body, called haptor, which contain hooks, bars
and anchors, in different numbers and sizes according to the species and its function is the
fixation of parasite in the hosts (Gerasev, 1990). The adult parasites are elongated, oval or
round and measure one to 75 millimeters. The most important monogeneans in fish farms
belong to the families Gyrodactylidae and Dactylogyridae. Gyrodactilids are viviparous
species and they are mostly parasites of the body surface of fish. Dactylogirids are oviparous
and are found in the gills, but can also become placed in the nasal cavities and, more rarely,
other parts of the body (Kubtiza & Kubtiza, 1999). The studies performed with O. niloticus
from fish farms from Chavantes reservoir showed that the dactylogyrids Cichlidogyrus halli
(fig. 6B-C) and Scutogyrus longicornis were the most abundant monogeneans found in the
gills of this fish species. Dactylogyrid monogeneans are hermaphrodites and have direct life
cycle, which facilitates the parasitic re-infestation. Generally in monogeneans the eggs
(dactylogyrids) or larvae (gyrodactylids) leave the uterus by the genital pore, attach to the
host and develops in the same host (Cheng, 1986). According to Eiras (1993), pathogenesis
caused by monogeneans varies with the species and the fixation site. Monogeneans which
parasitize the gills frequently cause cell hyperplasia and mucus hypersecretion. The lesions
are much more serious as the parasites are most abundant, which can reach high densities.
When attached to the tegument, necrosis of cells, destruction of scales and mucus
hypersecretion can be observed. In cases of high intensity of infection, monogeneans can
cause mortalities especially in small fish (Noga, 1996), which has been recorded for
numerous fish species (Cone et al., 1983; Ergens, 1983; Lester & Adams, 1974; Mackenzie,
1970). Considering the seriousness of this disease and also the difficulty of eradicating this
disease in fish after installed, it is suggested that all new fish bought undergo to quarantine
and prophylactic baths using commercial formalin diluted 1:4,000 for an hour or sodium
chloride in 1 to 3% from 30 minutes to 3 hours (Pavanelli et al., 1999). Another important
group of organisms that can affect fish health, especially those in breeding system, includes
the protozoans of the phylum Ciliophora, highlighting, Trichodina spp. (fig. 6D) and I.
multifiliis (fig. 6E). Apparently, these ciliates live as ectocomensal in the tegument and gills
of the fish without causing major damage, except in cases of heavy infestations, which is
particularly evident in species that multiply rapidly by successive binary divisions,
especially in environments with excess of organic material and low amounts of dissolved
oxygen in water (Eiras, 1993). The rearing of O. niloticus in cage farms in Chavantes
reservoir have high infection rates by Trichodina spp. (fig. 6D), in some cases and periods,
Health and Environment in Aquaculture
370
the prevalence is 100%. Trichodinids are ciliated protozoa commonly found in both
freshwater and saltwater, and show no host specificity, which favors their widespread
distribution. Their morphology is characteristic, with a circular shape and the presence of an
adhesive disc with a series of denticles that help fix the parasites in the host. They are
usually considered ectoparasites of skin and gills of the host and can rapidly proliferate in
the presence of decaying material (Heckmann, 1996). The life cycle of Trichodina spp. occurs
by binary fission, in which the parasite divides and fixes in the host's skin (Cheng, 1986). Its
pathogenesis is related to the rotatory movements of these ciliates on the gills and tegument
of the host, leading to an abrasive action of the skeletal structures and denticles present in
the adhesive disk, which damage the epithelial cells. Trichodinidiasis signs include loss of
appetite, lethargy, excessive mucus production in the gill epithelium and skin, erythema,
and sometimes bleeding skin (Heckmann, 1996). This is more evident in cases of intense
parasitism, which is observed when environmental conditions favour the reproduction of
the parasite and weakens the host, which happens when there is a decline in water quality.
These parasites are easily transmitted through infected fish, water, plants or utensils used
on fish farms. The treatment can be accomplished by bath with malachite green 2-3 g/10 m
3
,
when the fish are not used for consumption, or a bath for two hours in commercial formalin
1:4.000-6.000 (Pavanelli et al., 1999). Ichthyophthirius multifiliis (fig. 6E) however, has not been
frequently found in O. niloticus from Chavantes reservoir. It causes a disease commonly
known as freshwater white spot disease. This protozoa is a ciliate parasite, ovoid, measuring
100-1000 m, the cytoplasm is granular and contains numerous vacuoles and contractile
structures. These protozoans are characterized by the presence of a horseshoe-shaped
macronucleus and a micronucleus barely visible (Cheng, 1986). Although often cited as
ectoparasites, it is located under the epidermis, presenting the appearance of small white
spots on skin and gills of fish (Eiras, 1993). The life cycle of I. multifiliis is completed in 3-4
days at 25.5 C, but can also occur in up to 5 weeks at 18 C and in lower temperatures the
parasite will remain dormant. The adult parasite called trophont reach the maturity, leaves
the host as tomont forms and fixes in the substrate of fish cage forming a cyst. Inside it,
multiple cell divisions form many tomites which are the infective forms to new fishes when
the cyst breaks (Ewing & Kocani, 1987). Probably this protozoan is responsible for major
economic losses in fish farms in the world. Young fish are usually more susceptible, and
high infestations are usually associated with sudden drops in temperature in the water fish
farm (Eiras, 1993). In Brazil, I. multifiliis was already reported infecting Colossoma
macropomum; Prochilodus cearensis, Cichla ocellaris, Piaractus mesopotamicus; Leporinus
macrocephalus; tambacu hybrid (P. mesopotamicus x C. macropomum); Tilapia rendalli;
O.niloticus and Cyprinus carpio (Bksi, 2002; Tavares-Dias et al., 2001).
The most appropriate way to avoid freshwater white spot disease is maintaining good water
quality, adequate food, and avoiding stress, caused mainly by changes in temperature. In
this case, fish are more sensitive to attack by parasites. It should be considered that
freshwater white spot disease is difficult to treat, especially in large cages. Treatment should
be done in special cages for therapeutic baths. One product that can be used is sodium
chloride 0.3%, where the fish stay sunk for about 24 hours. More concentrated doses, 5% for
example, may be used in severe cases, with the fish remaining in the solution for 30 minutes
(Pavanelli et al., 1999). Myxozoa includes parasites frequently found in marine fish and
freshwater. Currently the phylum Myxozoa includes the classes Myxosporea and
Malacosporea, and almost all fish parasites belong to Myxosporea (Eiras et al., 2006). The life
Ecological Features of Large Neotropical
Reservoirs and Its Relation to Health of Cage Reared Fish
371
Fig. 6. A) Monogenoid in the gill of Oreochromis niloticus (10x); B) Cichlidogyrus halli
specimen (10x); C) detail of the haptor and sclerotized structures of Cichlidogyrus halli (40x);
D) Ichthyophthirius multifiliis specimen (40x); E) Trichodina sp. specimen (40x); F) Henneguya
sp. spores in fish tegument (100x).
cycle of mixosporeans occurs in two hosts: a vertebrate (fish) and invertebrates (oligochaete).
The spore is released when infected fish dies and decomposes, or through contaminated
faeces of a predator fish that have eaten infected fish. The oligochaetes are infected when they
ingest the sporoplasms that fix to the intestinal wall using polar filaments; these proliferate
and remain incubated for 3 months. After this period the Actinospore are released to infect
Health and Environment in Aquaculture
372
the fish in the water column and deposit the sporoplasm within the epidermis which will
migrate to the organs and gills (Stevens et al., 2001). The main genera of Myxozoa infecting
fishes are Myxobolus and Henneguya (fig. 6F). In Brazil there are only reports on the
occurrence of Henneguya sp. infecting O. niloticus (Ranzani-Paiva et al., 2005).
Histopathological analysis of the gills of fish infected Henneguya sp. reveal the presence of
severe bleeding and inflammatory foci in the gill epithelium, where the cysts are located.
Lesions such as compression of capillaries causing edema in superficial lamellae more
frequently in primary and secondary lamellae occasionally can be observed. In later stages,
cysts dilate the respiratory lamellae decreasing respiratory efficiency of infected fish (Martins
et al., 1999). The treatment of this disease has been carried out using 10 ml of formalin/m
3
,
which has been quite effective (Martins et al., 1999). Other studies have been conducted
evaluating the oral administration of chemotherapeutics (quinine, salinomycin) that had
significant effect on the treatment of infections in the gills of fish mixosporeans (Dohle et al.,
2002). However, studies on the treatment of infections Henneguya spp. are still scarce. Our
studies with O. niloticus in Chavantes reservoir have also demonstrated that the occurrence of
parasites in cage farms is associated with environmental variables. Therefore, we have
observed a higher prevalence, abundance and intensity of infection of parasites in the
summer. No difference was noted in the parasitism levels among different fish tanks,
considering the groups established by the zootechnical methods in fish farm. Depending on
the parasite species, fish size also influences the parasitism. For example, a negative
correlation was observed between monogenoids and fish size and a positive correlation
between Trichodina sp. and fish size. This is important because demonstrates that some
parasites are more important in the initial breeding phase while others are problems in the
final stages. In conclusion, we can note that despite the wide distribution and incidence of
parasites in fish rearing, there are few studies on the prevalence, pathogenesis, and potential
biological cycle of transmission of these parasites in Brazilian cage fish farms. It is important
to consider that in cage farming there must be a balance between the health of the host, the
proliferation of pathogens and conditions of the aquatic environment. Thus, poor water
quality, reduction of dissolved oxygen, changes in temperature, high fish density, inadequate
management or unbalanced nutrition are factors able to induce stress to the animals,
predisposing them to various types of infections, including parasites. Water offers an
extremely favorable environment for the proliferation of these agents and the parasites are
responsible for major losses in fish farms worldwide, with more relevance in the neotropics,
due to the climatic characteristics of these regions (Martins, 1998; Thatcher, 1994).
2.3 Impacts on the resident ichthyofauna and fish feeding ecology
Brazilian freshwater fish fauna has been managing to adjust to continuous environmental
impacts, such as damming and deforestation, due to its great diversity of species, with
different tactics of life cycle (Agostinho et al., 2007). Currently, a new expanding form of
impact is fish farming in floating cages. However, knowledge of the impacts from this
activity on the ichthyofauna is still precarious. This activity provides food resources and
shelter to resident fish fauna, attracting a large number of fish (Brando, 2010; Nobile, 2010;
Paes, 2006; Zanatta, 2007, 2011), which is also observed for marine fish farms (Boyra et al.,
2004; Machias et al., 2004, 2005, 2006). This attraction is due to availability of food resources,
such as feed losses, fish scales, and fish faeces. According to Beveridge (2004) and Pillay
(2004), these losses can reach 30% of all feed used in aquaculture enterprises. Thus, the
Ecological Features of Large Neotropical
Reservoirs and Its Relation to Health of Cage Reared Fish
373
contribution of allochthonous energy in aquatic ecosystems can cause changes in the food
chain, especially upon plankton community, benthic community, and fish fauna, interfering
with the dynamics of the aquatic ecosystem. Our studies in the Paran River basin (Middle
Tiet River and Upper/Middle Paranapanema River) reported impacts on the ichthyofauna
(Nobile, 2010; Paes, 2006; Zanatta, 2007, 2011) from this activity, such as changes in
population structure of fish fauna (Ramos et al., 2008), changes in diet (Brando, 2010;
Ramos et al., 2008, 2009), and changes in bromatological composition of some fish species
(Queiroz, 2010). A research made by Paes (2006) in 2003 and 2004 at Nova Avanhandava
reservoir, evaluated some impacts of fish farms upon ichthyofauna. While in FS was
recorded 18 species, a greater number of fish species (N=20) was recorded in CT. However,
a greater number of individuals was captured in FS (n=684) than in CT (n=518). For species,
the Shannon-Wiener diversity index H' (Krebs, 1999) showed no significant differences
between these two sites (p> 0.05). For Simpson dominance 1/D (Krebs, 1999), similar values
were recorded for FS (1/D = 5701) and CT (1/D = 5555). Even though this difference was
small, the dominant species in the FS was Metynnis maculatus, an omnivorous species with
tendency to herbivory, while in CT the dominant species was Plagioscion squamosissimus, a
strict carnivore species. A study conducted by Ramos et al. (2008) aimed to compare the diet
of the species M. maculatus, Astyanax altiparanae and P. squamosissimus between FS and CT.
The results show that the omnivorous species M. maculatus changed its diet in FS. This
species used a new alimentary source with high availability and low energy cost, and its diet
consists almost exclusively of feed from fish farming (85%). Such a change in diet led to a
change in population structure of this species, showing higher values of weight and length
in FS (p <0.05). Similar results were found at Jurumirim by Zanatta (2007). A greater number
of species (N=24) was captured in CT than in FS (N=21), and a greater number of
individuals were captured in CT (n=1,601) than in FS (n=1,470). There was no significant
differences for H ' (p> 0.05) between the two sites, and Pielou evenness E (Krebs, 1999)
values were similar for FS (E = 0.73) and CT (E = 0.76). At Chavantes reservoir, Nobile (2010)
showed differences only between the abundance of individuals in both sites, recording the
78% of the capture (n=3096) in FS. Furthermore, it was recorded introduction of the non-
native fish Ictalurus punctatus by aquaculture activities, whose impacts upon native fish
fauna of Brazilian reservoirs are still unknown (Zanatta et al., 2010). There was also the
capture of O. niloticus juveniles, which we believe that they are recruited from reproductive
processes occurring in the reservoir rather than escaped, due to its great abundance, and
size smaller than juveniles stocked in the cages (fig. 7). If these recruits are born from adult
scaped fish from fish farms, than efficiency of the sexual reversion process in use is not
enough to avoid breeding in this aquatic ecosystem, which may lead to disruptions of
ecological interactions with native fish assemblages. In Chavantes reservoir, Brando (2010)
and Ramos (2009) observed that the most abundant species showed differences in diet
composition between the FS and CT. Remains of feed was major component of the diet of
Pimelodus maculatus (98%) and A. altiparanae (99%) in FS. For CT, the diet of P. maculatus was
composed of detritus (27%), aquatic insects (22%), vegetables (21%) and molluscs (21%); and
A. altiparanae by terrestrial insects (61%) and vegetables (30%). Another species analyzed
was Apareiodon affinis, which diet was composed of detritus (80%) and feed remains (20%) in
FS, and only by detritus in CT. Moreover, two carnivorous species were analyzed
(Galeocharax knerii and P. squamosissimus), and there was no differences in their diet between
the sites studied. Comparative analysis of weight and length show that omnivorous species
grows faster at FS due to use of remains of feed as main food source. In addition,
Health and Environment in Aquaculture
374
10 cm
Fig. 7. Oreochromis niloticus juveniles captured around FS at Chavantes reservoir.
Queiroz (2010) observed change in bromatological composition of muscle tissue of P.
maculatus.
Significant differences for total lipids and gross protein were observed between fish caught
around FS and CT (p <0.05). It is evident that this type of fish farm attracts native
ichthyofauna in these reservoirs. Attractiveness is directly related to availability of food
resources, especially remains of feed. These are similar to the findings for marine
aquaculture in coastal ecosystems, which also found the effect of attractiveness of these
systems (Boyra et al., 2004; Dempster et al., 2002; Hkanson, 2005). The results presented
allow the conclusion that remains of feed are used by omnivorous species with large feeding
plasticity, which certainly justifies its dominance in sites used for cage aquaculture.
Furthermore, the changes in the diet of these species have direct effects on their population
structure, and the effects upon fish fauna still need to be clarified. Hkanson (2005) and
Ramos et al. (2008) report that such changes in the diet of abundant species in aquatic
ecosystems may affect the food chain in long term, and the effects upon ichthyofauna are
still unknown. We conclude that where fish farms in cages are deployed, there is a change
upon the population structure of fish communities, due to attractiveness and changes in the
diet of omnivorous species in nearby sites, which may interfere in the local ecological
dynamics, especially in the food web, altering ecological relationships between the
components of local biota.
3. Conclusion
The results showed that cage aquaculture fish production in oligotrophic ecosystems, as
Jurumirim and Chavantes reservoirs (Paranapanema River) is compatible with
environmental carrying capacity, and wild fishes play a relevant role recycling nutrients
derived from aquaculture. The main constraint to cultivation of tilapia in these reservoirs is
the low temperatures during the winter months, with recent records of mass mortality of
fish due to parasite infestation in this season. These aquaculture enterprises are an
important source of dispersion of non-native fish species, as O. niloticus and I. punctatus. In
Tiet river (Nova Avanhandava reservoir), the situation is more complex because of low
Ecological Features of Large Neotropical
Reservoirs and Its Relation to Health of Cage Reared Fish
375
water quality due to high nutrient loadings of sewage and agriculture runoff, related to the
presence of cyanobacteria blooms, causing severe consequences for the environmental and
economic sustainability of aquaculture. After some years operating cage farms in middle
and lower Tiet River, events of fish mass mortality associated with eutrophication
motivated fish farmers to move to sites with better water quality. It is also pertinent to note
that fish farming in cages still need the development of scientific and technological
knowledge, which besides basic technical support, could also allow the improvement of this
activity. The lack of expertise and the inappropriate implementation of best management
practices can lead to failure of these enterprises, making them economically unviable in the
short and medium term. Measures for the effective planning for farming public waters
require further discussion and guidance at governmental levels, in order to reach a truly
sustainable aquaculture, in all its socioeconomic and environmental interfaces.
4. Acknowledgments
The authors are grateful to the Conselho Nacional de Desenvolvimento Cientfico e
Tecnolgico CNPq, Financiadora de Estudos e Projetos FINEP, Fundao de Amparo
Pesquisa do Estado de So Paulo FAPESP, Fundao para o Desenvolvimento da Unesp -
FUNDUNESP for grants and financial support. Authors also thank Botucatu Bioscience
Institute (IBB) and Aquaculture Center So Paulo State University (CAUNESP) for the use
of laboratory facilities and logistics. This work complies with the current Brazilian laws.
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Part 6
Work-Related Hazards
Prevention and Mitigation
15
Aquacultural Safety and Health
Melvin L. Myers
1
and Robert M. Durborow
2
1
University of Kentucky, Lexington, Kentucky
2
Kentucky State University, Frankfort, Kentucky
USA
1. Introduction
Worldwide, 11,289,000 people worked in aquaculture in 2004up nearly three-fold from
3,832,000 workers in 1990 (Watterson et al., 2008). Aquaculture, including mariculture, is a
fast growing sector of worldwide agriculture but has unaddressed occupational safety and
health issues. Many fish farming tasks are dangerous; working around water poses a
particular danger, and Working at night and alone compounds the danger. A safety or
health hazard is any work design or property (physiological, physical, chemical, biological,
or psychological) that may cause harm to workers or bystanders.
As the cultivation of aquatic organisms, aquaculture may include the complete value chain
of production including feed production and fish processing, but for our purposes, we
delimit the scope of our writing to aquatic organism production that includes the hatchery,
nursery, and grow-out phases of production. Feeding, controlling predators, applying
chemicals, harvesting, and refurbishing or constructing structures or ponds are examples of
typical fish farm operations. While onshore aquaculture is associated with many of the same
hazards that are present in agriculture generally, offshore aquaculture is more closely akin
to a combination of hazards associated with shallow water commercial fishing and offshore
drilling.
Mariculture has many hazards. SINTEF, an independent research organization
headquartered in Norway, has presented data for Norwegian aquaculture showing that the
fatality rate (9.13 deaths/100,000 work yearsIs "work years" the correct unit here?) is 17
times the average rate for other industries (0.53 deaths/100,000 work years) and equivalent
to that of its fishing fleet (Clausen, 2000). In another study with more detail, 16 fatalities
occurred in the Norwegian salmon farming sector between 1980 and 1999. Ten of the deaths
were associated with using a boat: five in small boats, three occurring: in one type of
incident, boats capsized with overloading and shifting loads along with bad weather. Three
deaths occurred in workboats when two workers were either stuck by a crane or loads from
a crane, and another incident on a well boat, a worker was struck by an anchor that was
propelled into his face from a hang-up under the boat by the recoil of the anchor line. Three
additional workers died while diving, all of whom lacked a professional diving certificate
(Norwegian Labor Inspection Authority, 2001).
Figure 1 shows the percentage and type of non-fatal mariculture-related injuries. Most of
these injuries were associated with machinery followed by slips trips. Knife cuts and fish
Health and Environment in Aquaculture 386
Fig. 1. Percentage of nonfatal occupational injuries associated with aquaculture in Norway,
1980-1999. Source: Norwegian Labor Inspection Authority, 2001
bile in the eye were likely processing-related injuries, but boat-related injuries and needle
sticks were associated with fish production, as most probably were the climbing-related
injuries.
Figure 2 shows the frequency of mariculture-related illnesses in Norway during the 1980-
1999 period. The highest number of reported cases was for musculoskeletal disorders
followed by skin allergies and hearing loss.
Fig. 2. Number and type of aquaculture-related occupational illness cases reported in
Norway, 1980-1999. Source: Norwegian Labor Inspection Authority, 2001
In the United States, the non-fatal occupational injury rate in 2006 for onshore aquaculture
was 6.8 injuries per 100 full-time employees according to the U.S. Bureau of Labor Statistics.
20%
18%
5%
8%
4%
8%
7%
6%
24%
0%
5%
10%
15%
20%
25%
30%
8
5
4
1 1 1
0
1
2
3
4
5
6
7
8
9
Aquacultural Safety and Health 387
In comparison, rates were 5.3 and 7.8 injuries per 100 full-time employees for terrestrial crop
and animal production, respectively, and 4.6 injuries per 100 full-time employees across all
occupational sectors (Myers & Durborow, 2011; Cole et al., 2009).
Potential occupational hazards in aquaculture have been associated with fatalities that
include drownings, electrocutions, crushing-related injuries, hydrogen sulfide poisonings,
and fatal head injuries. Non-fatal injuries have been associated with slips, trips, falls,
machine operation and repair, strains and sprains, chemicals, and fires. Risk factors include
cranes (tip over and power line contact), aerators (entanglement and trauma), tractors and
sprayer-equipped all-terrain vehicles (overturn), heavy loads (lifting), boat propellers, high
pressure sprayers, slippery surfaces, rotting waste (hydrogen sulfide production), eroding
levees (overturn hazard), storm-related rushing water, diving conditions (bends and
drowning), night-time conditions, working alone, lack of training, no personal flotation
devices (PFD), and all-terrain vehicle use (ATV, also known as a quad-bike). Other hazards
include punctures or cuts from fish teeth or spines, needle-sticks, exposure to low
temperatures, and bacterial and parasitic infections (Myers, 2010).A fatality of an
aquaculture farm manager occurred in Kentucky when he was entangled in a tractor power-
take-off during fence post installation, and a diver drowned offshore in Hawaii while
working with submerged fish cages (Shikina, 2011).
In this chapter we follow an order that has been developed in industrial hygiene for
protection against hazards: identify, evaluate, and control the hazards. In the following
sections, we describe approaches for recognizing hazards, including descriptions of the
known occupational hazards involved with aquacultural work, and for evaluation, a job
hazard analysis approach is described. A risk matrix is used for priority setting so as to deal
with the worst hazards first and risk assessment is described. We describe precedence
modelsa safety hierarchyfor valuing the effectiveness of hazard controls, and finally, we
provide an outline that can be used for developing a safety manual for the individual
enterprise.
2. Identifying occupational hazards in aquaculture
This section describes the known hazards of aquacultural work and identifies and discusses
the hazards associated with different species and rearing technologies. This information will
aid fish farmers in recognizing hazards associated with their operations. Recognized
hazards involves employees and includes observed close calls and occupational injury or
illness history. The endpoint for the recognition of hazards is an inventory that lists the
hazards associated with all tasks, equipment, and substances:
Injury and illness information and data regarding the industry and related industries,
e.g., farming or fishing.
Information from past incidents and workplace injuries.
Information from your workers as well as family members and neighbors.
Product literature and information from suppliers.
Best industry practices.
Examine areas or activities where children or visitors may be present.
To identify and better understand hazards before product use, employers need to obtain
and read the manuals and safety sheets that are provided by equipment, machinery, and
Health and Environment in Aquaculture 388
chemical manufacturers. Employers should also develop and implement communication
and emergency plans to allow for a timely response in the event of an incident.
Five categories of hazards are 1) physiological (work design), 2) physical, 3) chemical, 4)
biological, and 5) psychological (Moreau & Neis, 2009). These categories are described in
Table 1. Onshore and offshore exposures to hazards differ greatly, and most species raised
offshore involve onshore tasks for the hatchery and nursery phases.
Categories Exposures Potential Consequences
Physiological (work
design)
Heavy lifting, prolonged standing,
awkward postures, repetitive
motion, overexertion, lack of
visibility
Low back pain, neck and
shoulder pain, bursitis,
tendonitis, tenosynovitis, carpal
tunnel syndrome
Physical Slips and trips, falls from height, falls
overboard, transport and trucking,
machinery, electricity, fire, heat and
cold, diving, noise, vibration,
confined spaces, entanglement,
underwater entrapment, solar
radiation
Injuries, cuts, burns, broken
bones, amputation,
hypothermia, hyperthermia,
drowning, electrocution, injury-
related death, asphyxiation,
decompression illness, sprains
and strains
Chemical (toxic,
flammable,
corrosive,
explosive)
Disinfectants, parasiticides,
piscicides, fungicides, antifoulants,
anesthetics, antibiotics, radon gas
from water sources, hydrogen
sulfide, carbon monoxide, sulfites,
dusts, fumes, styrene, needlesticks,
flammabilities, battery explosion
Respiratory illness, burns,
cancer, central nervous system
effects, birth defects,
reproductive effects, poisoning,
hematopoietic effects, and lung,
eye, or skin irritations
Biological Sharp teeth, spines, aerosolized
proteins, bacteria, parasites, skin
contact with shellfish and finfish
tissues and fluids, enzymes, airborne
proteins and endotoxins, fish feed
dust
Bites, cuts, punctures and
related infections; allergy,
asthma, eczema, urticaria
(hives), chapped skin, itching.
Psychological High demand and low control
situations, remote locations away
from family, potential for large fish
kills, abusive social environment
Work-related stress
Sources: Moreau & Neis, 2009; Myers, 2010; Durborow 1997; Erondu & Anyanwu, 2005.
Table 1. Occupational Hazards Associated with Aquaculture
Table 2 provides a summary of common occupational hazards and associated consequences
that have been related with work in aquaculture, focusing on the hazards particular to
rearing different fish and plant species. Hazards by species vary, but occupational hazards
also vary by the phase of production. While hazards associated with the nursery and grow-
Aquacultural Safety and Health 389
Species Potential Fatal Incident Potential Non-fatal Incident
Catfish Injuries from traffic collision,
unused seatbelt, or tractor
overturns and electrocution
from electrical contact.
Disease or illness from chemical exposure: formalin,
potassium permanganate, hydrogen peroxide,
fertilizers, lime, oxidants, disinfectants, algaecides,
herbicides; burns from exposure to sulfuric acid or
fires; injury related to fatigue or slips and falls; as
well as hand infections, back injury, spine
envenomation, and venomous injuries.
Trout Injury related to falls from
raceways or live tank trucks,
roadway collisions or, crane
overturn; drowning in
raceways.
Injury related to high pressure water jet penetration,
no guard rails, falls, and slips and trips; hearing loss
from noise exposure; leptospirosis infection; toxic
exposure to formaldehyde.
Shellfish Drowning when scuba diving. Electrical shock from broken electrical conduits;
injuries related to loose hand rails, missing safety
latches on electric hoists, and working on dredges;
shrimp meal allergy; crab scrape-related infections.
Crawfish, snail
(rice field
flooding)
Crushing injury from tractor
overturns.
Injury from boat propeller entanglement;
wrist/hand and back strains.
Tilapia Electrical shock from extension cords in water; injury
related to poor flooring, missing hand rails, working
alone, forklift, algae growing on walking surfaces,
trip hazards, or no fall protection; toxic effects or
burns related to chlorine, hydrogen peroxide, or
hydrochloric acid exposure; musculoskeletal
disorders; and spine prick infection (finger
amputation).
Ornamental fish Salt and coral dermatitis, fish tank granuloma, skin
infection, cercarial dermatitis, skin granulomas after
abrasions, Mycobacterium marinum infection.
Salmon Respiratory failure from H2S
exposure; drowning related to
fall overboard, boat capsizing,
or diving; injury when struck
by crane or crane loads or gear
entanglement
Decompression illness; toxic effects from styrene and
acetone exposure during fiberglass tank
construction; anaphylactic shock or infection from
self-injection of vaccine; and pesticide poisonings.
Tuna Diving-related drowning. Decompression illness.
Sturgeon Toxic effects or burns from exposure to hydrogen
peroxide, lime, or muriatic acid; fork lift-related
injuries; fire danger from oxygen.
Plant production
(taro, cress,
water spinach,
mimosa,
dropwort, water
lilies)
Drowning related to fall
overboard or no PFD.
Dermatitis, wastewater-related dermatitis,
leptospirosis.
Source: Myers 2010.
Table 2. Recognized Hazards Associated with Specific Species or Phases of Production
Health and Environment in Aquaculture 390
out phases may differ between species and rearing technology, hatchery operations have
much in common between species production. Deaths have occurred in hatcheries
associated with hydrogen sulfide exposure and slips and falls. Other hazards associated
with hatcheries include exposures to aerators, pumps, heaters, and other types of
machinery; fuels, solvents, hypochlorite, formaldehyde, formalin, confined spaces, water
jets, unguarded saws, ozone, and hair entanglement in hatching trough paddles.
Erondu & Anyanwu (2005) identified hazards in African aquaculture including noise, cuts,
sprains, fractures, asthma, rhinitis, snake and fish bites, bronchitis, chemical burns,
pesticides and disinfectant poisoning, parasites, and pathogens. In another review, Conway
and RaLonde (1998) identified hazards associated with worldwide aquaculture including
machine entanglements, hearing loss, slips and falls, drowning, lacerations, infections,
electric shock, hypothermia, repetitive strains, sleep deprivation, decompression illness,
organophosphate poisoning, respiratory illness, sunburn, keratotic injury, leptospirosis, and
dermatitis. In another two reviews, Durborow (1997; 1999) identified several hazards
associated with aquaculture including exposures to bacterial infection, hydrogen sulfide,
sodium metasulfide, sodium bisulfite, anesthetics, antibiotics, vaccines, tractor overturns,
electricity, overhead power lines, power-take-off or aerator entanglements, falls, muscle
strains, drowning, hypothermia, and decompression illness.
Tables 1 and 2 can be helpful in listing known or potential hazards into a hazard inventory.
This inventory should include the following (Myers, 2011):
Process descriptions,
Controls related to the hazards,
Location organizationally and physically of each process,
Supervisor name and contact information,
Number of employees who work in the process,
Medical information related to the hazards, and
Historical information about the process and related hazards
3. Job hazard analysis
The evaluation step aids in decision-making about the nature and control of hazards given
the circumstances of the work and exposure to the hazards (Myers, 2011). Priorities for
focusing on the worst first are effected by using a risk matrix. The matrix maps the
likelihood of an incident occurring versus the severity of the consequence, which informs
the decision maker of the risk and the urgency for taking preventive action. A risk
assessment tool summarizes and evaluates the workers exposure, job redesign, and actual
or potential control measures by the processes of the enterprise.
The job hazard analysis, which is currently used at some fish farms, identifies a job such as
climbing a ladder and lists the steps involved in the job and the associated hazards with
possible countermeasures to reduce exposure to the hazard. The job hazard analysis tool is
presented in a table format in Table 3.
A likely hazardous exposure to workers or bystanders must be corrected immediately.
When a hazard has been identified, the risk can be assessed by examining the likelihood
of the hazard resulting in injury to workers or other persons (is it likely or unlikely to
Aquacultural Safety and Health 391
Job:Lifting and Carrying Feed Bags Date: 02.11.2003
Title of Person doing the job:
Analysis by:
Organization: Location: Reviewed by:
Required or recommended personal protective equipment:
Approved by:
Sequence of job steps Potential hazards Recommended control
1. Planning ahead. 1. Holding the bag may
cause strain, or it may be
dropped.
1. Know where the bag is to go;
Have a place to put the bag.
Consider lighter loads with
more trips.
2. Lifting bag. 2. Back injury. 2. Use arm and leg muscles,
not your back; When in doubt
make it a 2-person task.
3. Carrying the bag. 3. The bag may slip and fall
resulting in strain from
overexertion.
3. Grasp bag firmly and secure
your footing; Keep your back
straight and the bag close to
your body. Avoid twisting
your back while carrying the
load; the back muscles may be
strained or the foot could stick
to a high traction surface while
the rest of the body is twisting,
causing knee sprain.
4. Seeing where you are
walking.
4. Carrying the load in front
of you can cause injury to
you or another person.
4. Make sure the load does not
block your view while
walking; Use a hand truck or
move bags stacked on pallets
with a forklift.
5. Setting the load down or
dumping the load.
5. Back injury. 5. Use your arm and leg
muscles. Avoid bending back.
Table 3. Sample Job Hazard Analysis Form
occur) and if the incident occurs, what is its potential severity, i.e., death, serious injury, or
minor injury?
Ask questions like: How many people come in contact with the hazard? How often? How
seriously could someone be harmed? How quickly could a dangerous situation occur if
something goes wrong (PEI, 2005)? Other factors to consider include abilities of the
individual, the weather and terrain, and how equipment is used.
The UN Food and Agriculture Organization provides a typical risk matrix as shown in Table
4 that can be used to set priorities for controlling hazards. In this table catastrophic refers to
death or a lethal disease, critical relates to severe injury or occupational disease, serious
relates to injury or disease requiring medical care but is not critical or catastrophic, minor
Health and Environment in Aquaculture 392
Likelihood
Consequence
Insignificant Minor Serious Critical Catastrophic
Rare N L L M M
Very low N L M H H
Low N L H H E
Moderate N M H E E
High N M E E E
Risk level denoted by: N = negligible, L = low, M = moderate, H = high, E = extreme
Source: Arthur et al. 2009.
Table 4. A Risk Matrix
refers to a minor injury or disease, and insignificant refers to property damage only
(Myers, 2011).
In Norway, job hazard analyses are summarized by job location. This summary is a risk
assessment that is made easy by asking three questions (Norwegian Labor Inspection
Authority, 2001):
1. What can go wrong (hazard)?
2. What can we do to prevent this (recommended control)?
3. What can we do to reduce the consequences if something occurs (recommended control)?
The emphasis should be aimed at the greatest risk. The format for this assessment is to place
answers to these questions as shown in Table 5. The examples developed in Norway suggest
different tables for each location in salmon farming: (1) fish hatchery, (2) dock, (3) fish fry
boat, (4) feed boat/work boat, (5) plant base, floating or on land, (6) plastic net pens, (7) steel
installation, and (8) feeding station. One example is shown in Table 5, which is consistent
with the Job Hazard Analysis and uses recommended control information from the US
Occupational Safety and Health Administration and the US National Institute for
Occupational Safety and Health.
4. Hierarchy of controls
This section introduces the reader to the hierarchy of controls as an extension of the
identified countermeasures listed in the job hazard analysis. It distinguishes passive from
active controls. Passive controls involve no human action for protection and include the
elimination of the hazard at the top of the hierarchy, followed by substitution of a less
hazardous technology or an engineered guard against the hazard. Active controls include
awareness through warnings or training and the use of personal protective equipment.
Farm operators are encouraged to adopt or develop inherently safety technologies by first
(eliminating), then (guarding against), and finally (warning about) the hazard. Warnings
are not always reliable in preventing contact with hazards. Examples of this hierarchy,
which has evolved for safety engineering, are presented in Table 6 (Wogalter, 2006).
The hierarchy of controls is an approach for evaluating the inherently safer technologies
with an emphasis on moving from active to passive controls. This simple two-step hierarchy
was used in highway safety with the highest precedence based upon (1) passive control that
Aquacultural Safety and Health 393
Work task
What can go wrong?
(Potential hazards)
What can we do?
(Recommended control)
Walking on
the dock..
Slipping or tripping on
dock.
Eliminate, to the extent possible, conditions
causing slippery working and walking surfaces
in immediate work areas, e.g., brush poured
cement before it dries to provide traction to the
walking surface. Active work areas shall be kept
free of equipment and materials not in use, and
clear of debris and other objects not necessary
for the work in progress. Cargo and material
shall not obstruct access to or egress from boats,
cranes, vehicles, or buildings. PFDs must be
available.
Climbing
aboard and
ashore from
boats.
Falling into sea. An adequate gangway must be provided. When
a gangway is not practical, then a ladder or
floating bridge must be used. The gangway must
have clear access, have hand rails, and be
properly trimmed and illuminated. When
boarding, leaving, or working from small boats
or floats, workers shall be protected by personal
flotation devices.
Loading or
unloading
with cranes.
Crushed by load or crane
contact.
Cranes are to be operated only by qualified and
trained personnel. Inspect all rigging prior to
use. Do not exceed the load chart capacity while
making lifts. Suspended loads must not pass
over the gangway or above workers. Shut down
the operation during extreme wind or during
storms.
Forklift
loading or
unloading.
Crushed by runover. Do not operate a forklift unless you are trained
and licensed. Do not handle loads that are
heavier than the weight capacity of the forklift.
Operate the forklift at a speed that will permit
it to be stopped safely.
Source: Norwegian Labor Inspection Authority, 2001. Adapted to recommendations by US agencies: the
Occupational Safety and Health Administration and the National Institute for Occupational Safety and
Health.
Table 5. An Example of a Job Hazard Analysis Related to a Work Location: The Dock
requires no human intervention at the work interface, whereas the less safe approach was
(2) the active control that depends upon human behavior at the work interface (Haddon,
1974). The passive control emphasized roadway and vehicle design features while active
controls focused on the driver.
Health and Environment in Aquaculture 394
PASSIVE CONTROLSprotection does not depend upon the workers actions (Haddon,
1974).
1. Eliminate hazards posed by equipment, animals, and the environment if at all possible
or substitute something safer by using a different machine, material or work practice
that poses less risk to perform the same task. For example, replace a faulty machine or
use a safer chemical instead of a more dangerous chemical.
2. Guard against the hazard when it is not possible to eliminate hazards. Engineered
controls include machinery guards and PTO shields. Design controls, such as locked
fences, isolate the worker from the hazard.
ACTIVE CONTROLSprotection depends upon the workers actions (Haddon, 1974).
3. Warn against the hazard if other controls are inadequate. Protect workers through
training, supervision, and personal protective equipment (PPE). For example, supervise
new workers until they are competent to deal with hazardous situations.Use and
provide proper clothes and respirator protection for handling dangerous chemicals or
biohazards.
With more than 50 fish farm visits, investigators were able to identify hazards on fish farms
as well as different levels of hazard control on different farms (Durborow, et al., 2011;
Ogunsanya et al.,2011). Farmers were generally aware of the hazards but were less aware of
controls that different farmers had used to prevent injury from the hazards. Twelve hazards
and a range of controls for each hazard are summarized below in Table 6, which classifies
the interventions against the precedence hierarchy of controls (Myers & Durborow, 2011;
Myers & Cole, 2009).
5. Model safety manual
The Global Aquaculture Alliance for Best Aquaculture Practices (BAP) is a standards-based
certification system that combines site inspections and records review to help program
participants meet the global demands for wholesome seafood produced in an
environmentally and socially responsible manner. BAP has developed standards to certify
shrimp hatcheries and shrimp, tilapia, channel catfish, pangasius, and salmon farms. One
section of 13 sections in the certifications process includes Worker Safety and Employee
Relations (BAPa, 2011). Other sections have an effect on worker safety and health: Storage
and Disposal of Farm Supplies, Drug and Chemical Management, Microbial Sanitation, and
Harvest and Transport.
In the certification process, the following standards regard worker safety: Living quarters
provided by the employer shall be well ventilated and have an adequate shower and toilet
and potable water; and wholesome meals should be available for workers. National labor
laws or criteria in the International Labor Organization Conventions for minimum age
and child labor shall be followed. Minimum standards for occupational safety and health
include (1) medical care access, (2) an emergency response plan regarding serious
illnesses or injuries, (3) workers trained in first aid and the emergency response plan, (4)
available first aid kits, and (5) personal protective devices and clothing should be
Aquacultural Safety and Health 395
Hazard Warning Guarding Elimination
Falling lid on live tanks
Post sign urging
caution when
working near raised
lid
Place wooden wedge
under open lid
Install locking or
pneumatic hinges
Impalement on electric
fence rods (to deter
otters)
Be careful and dont
fall
Use top insulators as
impalement caps
Place rods
horizontally on
raceway walls
Fall from feed bin roof
Hang on tight to
ladder
Install a ladder guard
or use a harness
attached to a cable
Install hatch handles
at ground level
Needle stick while
vaccinating fish
Keep fingers away
from injection site.
Use corrugated table
top to immobilize the
fish in the corrugated
groove
Install automatic fish
vaccination machine
Overhead electric
power line contact
Flag areas under
power lines
Raise power lines,
e.g., 30 to 45
Bury power lines
Lifting fish with a dip
net
Keep good posture
while lifting
Lift smaller loads of
fish making more
frequent trips
Install a pulley to
raise fish nets from
tank and a track to
slide loaded net to the
weighing point
Tractor overturn
Stay off of slopes
that could cause
rollover
Install rollover
protection and use the
seatbelt
Net entanglement &
drowning while diving
Dont panic
Place regulator
shrouds on O2 tanks
Traffic collision hazard Dont drive sleepy
Maintain distance
from other drivers
Aerator PTO
entanglement
Keep away from
rotating PTO shafts
Place guards on
power-take offs
Use electric-powered
aerators
Hatchery paddle
entanglement
Use panic wire to
stop shaft when hair
entangles
Replace bolted metal
paddles with plastic
paddles that slip upon
contact
Mount motor that
drives the paddles on
a movable platform
that completely
disengages the drive
belts if disturbed
Solar radiation
Dont expose bare
skin
Wear sun block Work in covered areas
Table 6. Examples of Prevention Effectiveness Related to the Hierarchy of Controls
Health and Environment in Aquaculture 396
provided as needed. Best Available Practices are established for the following categories of
fish farms and hatcheries:
Tilapia Farms (BAPb, 2011):
The above standards apply.
Shrimp Farms (BAPc, 2011):
The above standards apply.
Shrimp Hatcheries (BAPd, 2011):
The above standards apply, and
Train and assure appropriate licensing of machinery operators, drivers, and repair
personnel in machine safety.
Channel Catfish Farms (BAPe, 2011):
The above standards apply.
Electrical pumps and aerators must be wired according to standard and safe procedures.
Pangasius Farms (BAPf, 2011):
The above standards apply, and
Comply with laws that govern diving on fish farms.
Dive safety plans that include diver training, maintenance of diving logs, and
equipment maintenance.
Written procedures and trained staff to handle diving emergencies, and regular audit of
records and procedures.
Salmon Farms (BAPg, 2011):
The above standards apply, and
Initial training of workers for their assigned tasks and safety procedures and use of
boats and related equipment.
Familiarize workers with emergency response plans and train them in first aid, one of
whom shall be present among untrained personnel.
The employer and diving contractors shall comply with laws that govern diving on fish
farms or implement a dive safety plan requiring diver training and certification.
Minimize the frequency of ascents during the diving day.
Maintain dive logs that document procedures and safety-related incidents.
Require records on equipment maintenance.
Dive safety equipment shall include the availability of bottled oxygen.
Written safety policies for contractors.
A possible safety manual is provided by the Prince Edward Island Workers Compensation
Board in its Aquaculture Safety Code of Practice. It can be accessed online at
http://www.wcb.pe.ca/DocumentManagement/Document/pub_aquaculturesafetycodeof
practice.pdf. An outline for this manual is shown in Table 7. It does not address confined
spaces, but a US Occupational Safety and Health Administration publication can be used to
develop policies regarding confined spaces (OSHA, 2004).
Aquacultural Safety and Health 397
Responsibilities Under the Law
Aquaculture Safety Planning
Boat, Deck & Navigational Safety
Chainsaw Safety
Chemical, Fuel & Lubricant Safety
Diving Safety
Electrical Safety
Equipment & Machinery Safety
Ergonomics
Finfish Safety
Fire Prevention
First Aid and Emergencies
Confined Spaces*
Hand & Power Tools
Hoisting and Conveyor Systems
Hydraulic Safety
New & Young Workers
Personal Protective Equipment
Rescue Procedures
Sharp Objects Safety
Slip, Trip and Fall Prevention
Transportation Safety
Weather Hazards
Welding, Cutting or Soldering Safety
Winter Harvesting Safety
Workplace Housekeeping
Sources: PEI, 2005.
*OSHA 2004.
Table 7. Possible Sections That Can be Chosen for an Aquacultural Safety Manual
6. Conclusion
This chapter aims to provide information for establishing programs for protecting
aquaculturalists from occupational hazards. It presents many occupational hazards
associated with aquaculture with some regarding specific species and rearing technologies.
These recognized hazards can help the aquaculture production enterprise identify potential
hazards in its operation. Next, approaches are described for evaluating these hazards
including the job hazard analysis and risk assessment approaches with a description of the
risk matrix that can aid in setting priorities for controlling hazards. Section 4 addresses the
use of the hierarchy of controls to implement the most effective protection by emphasizing
passive controls (protection independent of the worker) over active controls (protection
dependent on the actions of the worker). Finally, a possible table of contents for developing
a safety manual for an operation is presented (Section 5).
We began with the model developed by industrial hygienists to protect against occupational
hazards: the identification, evaluation, and control of hazards. More recently, industrial
hygienists have added another purpose for their profession, the anticipation of hazards
(Myers, 2005). The anticipation of hazards is of high importance to aquaculture, which is
developing rapidly worldwide. One approach is to use known hazards and controls from
other sectors. Procedures in the fish processing sector can be expanded into the fish
production sector, and the unique procedures of the fishing sector can be adapted to
offshore aquaculture. In addition, the traditional regulatory sector regimes for onshore and
offshore operations need to come together to protect aquacultural workers (Claussen, 2000),
many of whom work both onshore and offshore.
Other sources may aid in more specific approaches. As an example, regarding channel
catfish, the Catfish Farmers of America, USDA Southern Regional Aquaculture Center, and
National Aquaculture Association developed Safety for Fish Farm Workers program
guidelines, which can be accessed at http://www.cdc.gov/nasd/docs/d001701-
d001800/d001756/d001756.html. A manual for aquaculture safety in cold waters Spawn,
Health and Environment in Aquaculture 398
Spat, and Sprains is available at http://seagrant.uaf.edu/lib/an/17/AN-17.pdf, which deals
not only with safety and ergonomics, but also with survival in the event of a vessel
capsizing. A Guide to Drug, Vaccine, and Pesticide Use in Aquaculture produced by the Federal
Joint Subcommittee on Aquaculture revised in 2007 can be accessed at
http://www.aquanic.org/jsa/wgqaap/drugguide/drugguide.htm.
7. Acknowledgments
The project that informed this chapter was supported by the Centers for Disease Control
and Prevention (NIOSH) Cooperative Agreement number 2 U50 OH007547-07, and human
subjects review was provided by the University of Kentucky Office of Research Integrity
under IRB protocol number 06-0564-P2G. We are grateful to Teresa Donovan of the
University of Kentucky College of Public Health for her help in editing this paper.
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Part 7
Spread of Pathogens from Marine Cage
16
Spread of Pathogens from Marine Cage
Aquaculture A Potential Threat for Wild Fish
Assemblages Under Protection Regimes?
Antonio Terlizzi
*
, Perla Tedesco and Pierpaolo Patarnello
Department of Biological and Environmental Sciences and Technologies,
University of Salento, CoNISMa, Lecce
Italy
1. Introduction
The spread and the control of pathogens represent a serious problem in aquaculture,
implying important environmental and economic issues (Huntington, 2006; Sapkota et al.,
2008).
With the rapid development of aquaculture in recent decades (Food and Agriculture
Organization [FAO], 2008), an increasing number of pathogens involved in fish disease, and
the range of susceptible fish species have been identified and described. The increasing use
of molecular tools has helped improve our knowledge in this area.
Few studies have been performed to evaluate the interactions host/pathogen in the natural
environment (Hedrick, 1998), or to investigate the mechanisms of infection and interaction
between farmed and wild specimens, nor the ecological role assumed by infected wild hosts
has been explored yet.
In view of the current decline of fish stocks, which has recently been attributed not only to
habitat loss and overfishing, but also even to the spread of pathogens (Daszak, 2000; Gozlan
et al., 2005), understanding these mechanisms is essential for a proper conservation and
management of marine and fisheries resources.
The effect of parasites and other pathogens on fish populations under protection regimes
has received virtually no attention (McCallum et al., 2005). Marine Protected Areas (MPAs)
can provide unique protection for critical areas and spatial escape for overexploited species
and are regarded as essential tools for the conservation of marine environments (Lubchenco
et al., 2003; Pauly, 2005; Roberts et al., 2005). However, the effectiveness of MPAs is greatly
limited by several anthropogenic and natural impacts acting at scales much larger than that
encompassing by the reserve boundaries. Examples of such impacts toward which MPAs
cannot offer any direct protection are represented by climate change (Graham et al., 2008;
McClanahan, 2000), with all the problems related to it (Keller et al., 2009), the spread of
pollutants (Jarman et al., 1992; Loganathan & Kannan, 1994; Terlizzi et al., 2004), invasive
*
Corresponding Author
Health and Environment in Aquaculture
404
species (Carlton et al., 1990; Katsanevakis et al. 2010; Trowbridge, 1995), and pathogens
(Lessios, 1988; Littler & Littler, 1995; Rasmussen, 1977; Steinbeck et al., 1992).
In this chapter we briefly review the potential threats to wild population represented by the
spread of pathogens from marine cage fish aquaculture. As an emblematic example we
focus particularly on the effects of a serious disease, namely the Viral Nervous Necrosis
(VNN), affecting several reared fish species worldwide.
In order to warn about how the spread of pathogens could represent a risk for natural
populations, even under protection regimes, we report on the results of a preliminary
survey for the detection of viral particles responsible for VNN in the gonads and brain
homogenates in some wild specimens collected in two no take-no access Marine Protected
Areas in the Southern Italy (South Adriatic and North Ionian Sea).
2. The spread of pathogens from marine cage aquaculture
There are several ways by which new pathogens can attain natural populations.
For example, a benign organism can undergo a genetic change that may increase its degree
of pathogenicity (Bull, 1994; Cunningham, 2002). A pathogen may also be introduced
through the expansion of the range of wild species in new regions as a result of global
warming, the removal of barriers, or the transport of animals for commercial and
recreational purposes (Gozlan et al., 2006).
It is now known that the activities of aquaculture farms, particularly those of offshore floating
cages, are a source of various types of environmental (Pusceddu et al., 2007) and biological
(Terlizzi et al., 2010) impact, including the facilitation of the spread of potential pathogens
(Krkoek et al., 2006). The high density to which organisms are reared and the unnatural and
stressful conditions to which they are subjected may in fact facilitate the emergence and spread
of diseases, which can potentially extend to wild organisms (Naylor et al., 2005).
The use of net pen or cages systems involves a higher risk for disease transmission because
there is no impermeable barrier between the farm and the aquatic environment (Huntington
et al., 2006). There is therefore a strong potential for contamination between reared
organisms and the wildlife of the surrounding environment.
Many species of wild fish are attracted by the presence of the cages, which provide them
food and shelter (Diamant et al., 2000), often succeeding to get into cages, passing through
the mesh. Farmed species may be able to escape in the environment too. This phenomenon
may have important biological implications (e.g. in terms of genetic contamination) on wild
populations. In addition, escaped individuals can carry infections they acquired at the
culture systems (Conseil International pour L'Exploration Scientifique de la Mer
Mdetirrane [CIESM], 2007).
Farmed fish stocks may prove receptive to pathogens from the surrounding environment
too. The exchange of pathogens between farmed and wild fish has been repeatedly observed
in different geographical regions (Diamant et al., 2000; Kent, 2000; Mc Vicar, 1997; Nowak et
al., 2004; Paperna, 1998; Sepulveda et al., 2004). Bacterial infections by Mycobacterium
marinum and Streptococcus iniae, which are common among farmed fish stocks, have been
described to cause mortality even in wild fish (Colorni et al., 2002).
Spread of Pathogens from Marine Cage Aquaculture
A Potential Threat for Wild Fish Assemblages Under Protection Regimes?
405
Pathological events in populations of wild abalone (Alstatt et al., 1996) suggest a potential
susceptibility that makes them able to influence or be influenced by, reared individuals.
Such pathogen exchange relationship seems to occur frequently between farmed and wild
fish populations, as in the case of a disease known as Infectious Hematopoietic Necrosis, which
affects mainly salmons, and which appears to be transmitted between wild and farmed
specimens in both directions (Naylor et al., 2003).
Transmission of pathogens from aquaculture to wild fish can occur at different stages of the
life cycle: infection can start from the earliest stages of reproduction and larval rearing
(hatchery) and spread in the later stages (growing) contaminating wild stocks and causing
dangerous epidemics (Naylor et al., 2005).
Krkoek et al. (2011) studied the effects of sea lice (Lepeophtheirus salmonis) from salmon
farming facilities on populations of wild salmon (Onchorhynchus gorbusha and O. keta),
noting that juveniles of these species, when parasitized, show a reduced ability to escape
predators that consume selectively parasitized salmon. Therefore, the mortality of juvenile
wild salmon may be higher than previously believed and this has important implications for
the development of appropriate conservation policies.
The importation of feed for aquaculture can also be considered vehicle of sources of
pollutants and of pathogens between fish stocks very distant from each other (Dalton, 2004).
Parasites and pathogens can therefore play an important role for fish stocks, reducing both
the number and yield, increasing mortality, altering the reproductive potential, affecting the
structure of the population size and/or reducing the commercial value of harvested stocks
(Dobson & May, 1987; Kuris & Lafferty, 1992).
Being able to influence the survival of the host and his reproductive success, pathogens
should be taken into great consideration in the design and management of MPAs
(McCallum et al., 2005). Also, even if the survival or reproduction are not affected, as in the
case of infections by Anisakis simplex and Pseudoterranova decipiens, pathogens may pose a
problem for fisheries management-oriented reserves, because the infected hosts have a
lower market value and can represent a health risk when consumed as food by humans.
3. The Viral Nervous Necrosis (VNN)
The genus Betanodavirus, family Nodaviridae is a widespread and well-known viral
pathogen in the field of fish farming. It is the etiologic agent responsible for a severe disease
known as Viral Encephalopathy and Retinopathy (VER), or Viral Nervous Necrosis (VNN). It
affects more than 40 species of marine fish worldwide (Panzarin et al., 2010). VNN disease is
regarded as one of the most devastating viral infections among marine fish.
Affected fish, particularly juvenile forms, show spiralling swimming, darkened colour and
hyper inflated swim bladders. Internal disease signs include pale livers, empty digestive
tracts and intestines filled with brownish fluid. In affected fish the virus propagates in the
eye, brain and distal spinal cord causing marked vacuolations leading to encephalopathy
and retinopathy (Fig. 1). Such lesions in the Central Nervous System and in the retina
(nervous tissue of the eye) have always been typical of larval and juvenile stages in the sea
bass (Bovo et al., 1996; Breuil et al., 1991) and in other species sensitive to nodavirosis
(Glazebrook et al., 1990; Grotmol et al., 1997; Mori et al., 1991; Nguyen et al., 1996). For this
Health and Environment in Aquaculture
406
Fig. 1. Histological preparation of a sea bass larva (age 45 days) with the typical vacuolar
necrosis of the brain tissue and in the retina (indicated by white and black arrows,
respectively). After the collection, the sample has been readily fixed in 10% neutral buffered
formalin for not more than 7 days. Thereafter it has been included in paraffin to be cut in to
sections of 5 m and then colored for histological analysis. The dye used in this histological
section is hematoxylin-eosin.
reason histological analyses have often been performed in monitoring programs to evaluate
the spread of the disease in the hatcheries (Sweetman et al 1996).
The virus also multiplies in the gonad, livers, kidney, stomach and intestine (Grotmol et al.,
2000; Munday et al., 2002).
Experiments with Pseudocaranx dentex (Arimoto et al., 1992; Mushiake et al., 1994; Nishizawa
et al., 1996) have shown that the virus transmission can occur either vertically, from
broodstock to larvae through the eggs or sperm, either horizontally, through the contact
between healthy fish and infected larvae (Arimoto et al., 1993). Experimental contamination
with infected tissue homogenates (Arimoto et al., 1993; Glazebrook et al., 1990; Grotmol et
al., 1999; Tanaka et al., 1998) or addition of purified virus from infected individuals (Nguyen
et al., 1996) or produced in SSN-1 cells (Pducasse et al., 1999) is also able to transmit the
virus. The ability of the betanodavirus to resist to a wide pH range, varying from 2 to 9
(Frerichs et al., 1996), and its resistance in sea water at 15 C for more than one year (Frerichs
et al., 2000) increases the probability of horizontal transmission (Munday et al., 2002). The
transmission of the betanodavirus from infected, asymptomatic specimens is also possible.
Some Authors (e.g. Castric et al., 2001) have shown how specimens of Sparus aurata
Spread of Pathogens from Marine Cage Aquaculture
A Potential Threat for Wild Fish Assemblages Under Protection Regimes?
407
experimentally infected but apparently healthy, are able to transmit the virus to individuals
of Dicentrarchus labrax placed in the same cage.
The finding, in Norway (Aspehaug et al., 1999), of adults of Atlantic halibut (Hippoglossus
hippoglossus) positive for betanodavirus suggests a possible danger of using wild animals as
breeding stock.
There is obviously also the possible risk of transmission occurring in the opposite direction,
namely that the virus originating from aquaculture facilities can infect wild healthy
individuals. Supports to this hypothesis, in addition to the above mentioned evidences,
which show that betanodavirus can spread both horizontally and vertically within a system,
are provided by the detection of the virus in several wild marine species, although the
infection in the examined subjects was asymptomatic (Barker et al., 2002; Gagn et al., 2004;
Gomez et al., 2004; Gomez et al., 2008; Thiry et al., 2004).
Recent studies carried out along the Italian coast have identified the etiologic agent
responsible for the disease in wild organisms belonging to a wide panel of species. From a
virological examination conducted by Maltese et al. (2005) on samples from the northern
Adriatic and the Strait of Sicily, the presence of viruses has been detected in the sea bass
(Dicentrarchus labrax), gilthead seabream (Sparus aurata), white grouper (Epinephelus aeneus),
dusky grouper (Epinephelus marginatus), red mullet (Mullus barbatus), axillary seabream
(Pagellus acarne), poor cod (Trisopterus minutus) and in the black goby (Gobius niger). The
study highlighted a possible widespread of the virus in nature, the role of S. aurata as an
asymptomatic carrier and the high sensitivity of the genus Epinephelus, for which episodes of
mortality in other geographical areas have been reported. The study also confirmed the
considerable sensitivity of D. labrax to the betanodavirus, which had already been proven in
the context of intensive farming.
4. VNN and protected populations: A limited effectiveness of MPAs?
The two areas considered for sampling are the MPA of Porto Cesareo, located along the
southwestern coast of Apulia (Ionian Sea, 4015' N - 1752' E) and the MPA of Torre
Guaceto, located along the east coast of Apulia (Adriatic Sea, 4043' N - 1748' E). Both areas
are characterized by the past presence of extensive aquaculture activities within or close to
the reserve boundaries. The survey focused mainly on specimens of D. labrax but, whenever
possible, considered, as a preliminary screening, some other species of commercial interest
for which positive serology for the betanodavirus has been already reported in literature.
For all specimens, samples of brain and gonadal tissue, the main target organs of the virus
were taken in double aliquot for DNA extraction and biomolecular analysis. Virological
analyses were performed by the laboratories of the Istituto Zooprofilattico Sperimentale
delle Venezie (Padua, Italy). Samples were analysed through real-time TaqMan PCR. This
technique proved to be highly sensitive, is suitable for a wide range of organic matrices and
is able to detect the four betanodavirus genotypes currently recognized. The experimental
protocol followed the procedure described in Panzarin et al. (2010). More particularly,
samples were processed for RNA extraction using the NucleoSpin RNA II (Macherey-
Nagel GmbH & C., Dren, Germany). Real-time PCR was performed using the LightCycler
2.0 system and carried out in 20 l with LightCycler TaqMan Master (Roche
Diagnostics GmbH, Mannheim, Germany), 0.9 M of each primer, 0.75 M of probe and 5 l
Health and Environment in Aquaculture
408
of cDNA template. The thermal prole consisted of a 10-min incubation at 95C followed by
45 cycles of 10 s denaturation at 95C, 35 s annealing at 58C and 1 s elongation at 72C,
followed by an additional 30-s cooling step at 40C.
The analysis involved the collection of 28 specimens, 9 of which coming from the MPA of
Torre Guaceto and 19 from the MPA of Porto Cesareo. Results are summarised in Table 1.
Several specimens resulted positive to the VNN test. The individuals showing positivity to
the molecular analyses were mostly represented by the sea bass (Dicentrarchus labrax) but
the presence of the virus was also detected in the scorpion fish (Scorpaena scrofa), the gray
mullet (Mugil sp.), the goby (Gobius sp.) and the comber (Serranus cabrilla). All positive
individuals appeared asymptomatic.
Fish species No. of examined fish No. of positive samples
Torre Guaceto MPA
Dicentrarchus labrax 4 3
Diplodus sargus 5 0
Porto Cesareo MPA
Dicentrarchus labrax 3 3
Scorpaena scrofa 3 1
Mugil sp. 3 1
Gobius sp. 2 1
Serranus cabrilla 2 1
Synodus saurus 2 0
Serranus scriba 1 0
Sarpa salpa 2 0
Oblada melanura 1 0
Table 1. Summary of VNN tests performed in the two Marine Protected Areas (MPAs)
5. Discussion
The lack of clinical signs in infected fish specimens observed in this study is consistent with
the results obtained in other recent studies of wild marine fauna (Baeck et al., 2007; Gomez
et al., 2004), thus confirming how rare are the observations of clinical forms in wild fish
populations (but see Gomez et al., 2009).
Some authors argue that most infections in wildlife are presumably latent and they do not
evolve into a pathological condition until the fish becomes stressed (Barker et al., 2002).
Barker et al. (2002) firstly reported piscine nodavirus in wild winter flouder Pleuronectes
americanus in Passamaquoddy Bay (New Brunswick, Canada) considering the possibility that
the virus could be transferred between wild winter flounder and sea-caged halibut or cod.
Baeck et al. (2007) detected nodavirus in 21 different apparently healthy wild marine fish
collected in coastal areas of Korea, close to aquaculture facilities and Gomez et al. (2004) reported
Spread of Pathogens from Marine Cage Aquaculture
A Potential Threat for Wild Fish Assemblages Under Protection Regimes?
409
high prevalence for betanodavirus in apparently healthy cultured and wild marine fish near
mariculture areas in Japan. In both studies, sampling areas were located near aquaculture
facilities and the authors support the hypothesis of an horizontal transmission of the virus.
The results here reported, although preliminary, make us suppose that the distribution of
the betanodavirus is currently extending and not confined to marine cage aquaculture. The
genetic characterization of isolated viral strains is currently under investigation and will
likely help determining more precisely the range of dispersion and residence time of these
possible sources of contamination, thus improving our understanding about the
epidemiological mechanisms of the disease and consequently our ability to control it.
Our findings also confirm that MPAs are not able to cope with this particular form of
impact, which clearly acts at a scale larger than that covered by the measures of protection
(Allison et al., 1998).
The specimens considered, although asymptomatic, might still be reservoirs of infection, by
acting as virus carriers, and further favoring the spread of the pathogen. The ecological role of
these asymptomatic carriers in nature remains however unclear and the possible consequences
on natural population still remain to be clarified. This is not an easy task, however, as disease
disorders are difficult to observe in the wild because affected individuals are rapidly removed
from the population by predators (Gozlan et al., 2006). These consequences are nevertheless
conceivable since in some reared species such as the sea bass, the virus causes, in addition to
the above-described damages to the nervous system, failure in reproduction (often
sterilization), reduced hatching rate and high larval mortality.
In the view of the impact caused by the over-exploitation of marine resources and given the
current trend of decline of commercially important fish stocks, increased mortalities and
failure in reproduction due to diseases should be carefully considered and adequately
addressed, especially in sound quantification of the effectiveness of MPAs.
Attempts to protect biodiversity through MPAs and/or restore ecosystem functioning (e.g.,
through the protection of over-exploited species that generate positive community-wide
effect) might be frustrated and/or biased in their quantification by external pressures.
Given the wide spread of this virus and its possible consequences in asymptomatic forms it
should be important to include the issue of pathogens in disease surveillance program and
monitoring of biological health of each MPA as well as any area that has or has had a
significant number of intensive aquaculture plants.
6. Acknowledgments
The research leading to these results has received funding from the European Communitys
seventh Framework programme (FP7 / 2007 2013) under Grant Agreement No. 266445 for
the project Vectors of Change in the Oceans and Seas Marine Life, Impact on Economic
Sectors (VECTORS).
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