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Progress in Biological Control
Jean-Michel Mérillon
Kishan Gopal Ramawat Editors
Plant
Defence:
Biological
Control
Second Edition
Progress in Biological Control
Volume 22
Series Editors
Heikki M. T. Hokkanen
Department of Environmental and Biological Sciences, University of Eastern
Finland, Kuopio, Finland
Yulin Gao
Institute of Plant Protection, Chinese Academy of Agricultural Sciences,
Beijing, China
Biological control of pests, weeds, and plant and animal diseases utilising their
natural antagonists is a well-established but rapidly evolving field of applied
ecology. Despite its documented applications and systematic development efforts
for longer than a century, biological control still remains a grossly underexploited
method of pest management. Its untapped potential represents the best hope to
providing lasting, environmentally sound, and socially acceptable control of most
problem pests in agriculture, and of invasive alien organisms threatening global
biodiversity. Based on the overwhelmingly positive features of biological control,
it is the prime candidate in the search for reducing dependency on chemical
pesticides. Public demand for finding solutions based on biological control is the
main driving force in the rapid developments in the various strategies of utilising
natural enemies for controlling noxious organisms. This book series is intended to
accelerate these developments through exploring the progress made within the
various aspects of biological control, and via documenting these advances to the
benefit of fellow scientists, students, public officials, and the public at large. Each of
the books in this series is expected to provide a comprehensive, authoritative
synthesis of the topic, likely to stand the test of time.
This Springer imprint is published by the registered company Springer Nature Switzerland AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface to Second Edition
We are pleased to present the second revised edition of Plant Defence: Biological
Control. Production of a second edition in itself is evidence that the first one was a
great success. The work on biological control agents is preferred due to their envi-
ronmental friendly role. Firstly, the use of biodegradable and eco-friendly control
agents is a necessity as continuous long-term use of chemical pesticides has detri-
mental effect on soil health and consequently residue present in plants has long-
term effect on human health. Secondly, search for safe alternative agents like
biological control of diseases still continues. This endeavour this is a timely compi-
lation of state-of-the-art information dealing with various aspects of biological con-
trol agents. The present book is organised into four parts: I. Biology of Plant
Defence, II. Use of Natural Compounds, III. Use of Biological Agents, and
IV. Market and Commercialization. Most of the chapters are new and a few are
updated.
The book will be useful to upper students studying crop protection, agricultural
sciences, applied entomology, plant pathology and plant sciences. Biological and
agricultural research scientists in biotechnology, forestry, plant pathology and post-
harvest technology, crop management and environmental sciences, agrochemical
and crop protection industries, and in academia will find this book very useful. The
editors wish to thank all contributors as well as the staff at Springer for their coop-
eration in completion of this book.
v
Preface to First Edition
Approximately 6.6 billion humans inhabit the Earth. Notably, the human population
has grown nearly tenfold over the past three centuries and has increased by a factor
of four in the last. Therefore, demand for food, feed and fodder is ever increasing.
Plant diseases worldwide are responsible for billions of dollars’ worth of crop
losses every year. Productivity of crops is at risk due to the incidence of pests,
pathogens and animal pests. Crop losses to pests can be substantial and may be
reduced by various control activities. Estimates of crop loss are available for major
food and cash crops at the world level. Among crops, the total loss potential of pests
worldwide varies from 25 to 40%. Globally, enormous crop losses are caused by
plant diseases, which can occur from the time of seed sowing to harvesting and stor-
age. Important historical evidences of plant disease epidemics are Irish Famine due
to late blight of potato (Ireland, 1845), Bengal famine due to brown spot of rice
(India, 1942) and Coffee rust (Sri Lanka, 1967). Such epidemics have left their
effect on the economy of the affected countries and deep scars in the memory of
human civilization.
Plant diseases, caused primarily by fungal and bacterial pathogens, cause losses
to agricultural and horticultural crops every year. These losses can result in reduced
food supplies, poorer quality of agricultural products, economic hardship for grow-
ers and processors, and ultimately higher prices for consumers. For many diseases,
traditional chemical control methods are not always economical nor are they effec-
tive, and fumigation as well as other chemical control methods may have unwanted
health, safety and environmental risks.
Biological control involves use of beneficial microorganisms, such as specialised
fungi and bacteria, to attack and control plant pathogens and diseases they cause.
Biological control offers an environmental friendly approach to the management of
plant diseases and can be incorporated into cultural and physical controls and lim-
ited chemical uses for an effective integrated pest management system. Due to the
high cost of synthetic pesticides and concerns over environmental pollution associ-
ated with the continuous use of these chemicals, there is a renewed interest in the
use of botanicals and biological control agents for crop protection. Benefits and
risks are always associated with new technologies and their utilization. These types
vii
viii Preface to First Edition
8 By-Products from Pine: A Prospective Tool for Pest Biocontrol �������� 193
Julien Gabaston, Josep Valls, Caroline Rouger, Tristan Richard,
Pierre Waffo-Teguo, Stéphanie Cluzet, and Jean-Michel Mérillon
9 Stilbenoid-Enriched Grape Cane Extracts for the Biocontrol
of Grapevine Diseases������������������������������������������������������������������������������ 215
Kevin Billet, Magdalena Anna Malinowska, Thibaut Munsch,
Marianne Unlubayir, Thomas Dugé de Bernonville,
Sébastien Besseau, Vincent Courdavault, Audrey Oudin,
Olivier Pichon, Marc Clastre, Nathalie Giglioli-Guivarc’h,
and Arnaud Lanoue
Index������������������������������������������������������������������������������������������������������������������ 473
Part I
Biology of Plant Defence
Chapter 1
Biological and Molecular Control Tools
in Plant Defense
1.1 Introduction
To cope with pathogens and herbivorous pests, plants have evolved sophisticated
defense mechanisms broadly classified as passive or constitutive and active or
inducible (Fig. 1.1). Passive or constitutive defense mechanisms are constitutively
expressed and provide protection from initial invasion or attack [4–6]. Against
pathogens, these may include physical barriers, such as wax layers [7], cuticle [8]
and cell wall [9], as well as preformed chemical compounds with antimicrobial
(generically called phytoanticipins) and lytic effects [4, 10]. If these preformed bar-
riers are overcome, pathogens can still be confronted by inducible host plant defense
mechanisms, which prevent further colonization or pathogen spread [4]. Similarly,
arthropods are confronted with an array of constitutive and/or inducible plant
1 Biological and Molecular Control Tools in Plant Defense 5
Fig. 1.1 Global overview of plant defense responses against herbivores and pathogens. Herbivore-,
pathogen- or damage-associated molecular patterns (HAMPs, PAMPs and DAMPs, respectively)
are recognized by pattern recognition receptors (PRRs) and lead to pattern-triggered immunity
(PTI). Effector-like molecules from herbivores and pathogens can suppress PTI and result to
effector-triggered susceptibility (ETS). The recognition of these molecules by plant resistance pro-
teins (R proteins) lead to effector-triggered immunity (ETI) that, in the case of pathogens, often
culminates in hypersensitive response (HR). Uncharacterized elements are indicated by dashed
lines. Defense mechanisms (passive and active defense) operating during herbivore attack and
pathogen infestation are indicated on the right
defenses such as physical traits (trichomes, wax layers, etc.) and chemicals (toxins,
anti-digestive compounds, secondary metabolites) that aim at killing, deterring or
retarding the population growth of pests [5]. Plants can also defend themselves indi-
rectly by emitting volatile compounds that attract the natural enemies of herbivores
[11, 12]. As with pathogens, inducible plant defenses against herbivores are initi-
ated upon recognition of the attacker and downstream activation of defense signal-
ling [13, 14]. Compared to constitutive defenses, induced plant responses are
considered to be cost-saving, preventing auto-intoxication and more advantageous
as they can be tailored to the attacker after specific cues recognition by the plant
[15–17].
The first defensive line of plant immunity relies on the perception of pathogen- or
damage-associated molecular patterns (PAMPs and DAMPs, respectively) by recep-
tors called pattern recognition receptors (PRRs) localized on the plant plasma mem-
brane [18] (Fig. 1.1). All plant PRRs identified to date belong to receptor-like
kinases (RLKs) or receptor-like proteins (RLPs) [19]. RLKs are proteins with an
extracellular domain involved in the perception of signal molecules (i.e., PAMPs/
DAMPs), and additionally of a transmembrane domain and an intracellular kinase
6 M. L. Pappas et al.
domain, which amplify or transduce these signals into the cell, respectively [20].
RLPs have a similar structural organization but lack the intracellular kinase domain
[20]. Recent studies suggest that sensing of PAMPs/DAMPs could be also happen-
ing through membrane lipids [21]. PAMPs comprise a diverse array of structural
components of the pathogen, such as bacterial flagellin, fungal cell wall-derived
chitins and glucans, as well as pathogen-specific lipopolysaccharides, proteins, pep-
tidoglycan, elongation factors (e.g., EF-Tu) or microbial nucleic acids [19, 20, 22].
DAMPs are molecules of plant origin released upon pathogen-induced cell damage,
and include mainly cell wall or cytosolic proteins, peptides, nucleotides, and amino
acids [23].
The recognition of PAMPs/DAMPs by PPRs can activate the immune plant
response, a process collectively termed ‘pattern-triggered immunity’ (PTI) [24]. In
this process, a complex network of signalling events is activated, leading to a series
of cellular and physiological responses. Such signalling events include, for instance,
the rapid generation of cytosolic Ca2+ and reactive oxygen species (ROS) or reac-
tive nitrogen species, ion efflux, protein phosphorylation, activation of Ca2+-
dependent protein kinases (CDPKs) and mitogen-activated protein kinases
(MAPKs), increased biosynthesis of phytohormones, and transcriptional repro-
gramming [20, 25]. This complex signaling network leads to the establishment of a
number of plant defense responses, such as plasmodesmata closure to inhibit
molecular exchanges among cells, stomatal closure to limit pathogen entry, produc-
tion of antimicrobial compounds (e.g., phytoalexins) and generation of ROS either
to signal downstream defenses or inhibit growth of pathogens, callose deposition to
provide a physical barrier for pathogen attacks, and accumulation of pathogenesis-
related proteins such as lytic enzymes (chitinases, glucanases, and proteases) [20].
In general, PTI is sufficient to fight off most pathogens, in particular host non-
adapted pathogens [18]. However, some pathogens have developed strategies to
evade PTI and for these, plant initiates a second layer of inducible defense, termed
as Effector-Triggered Immunity (ETI), resulting in an incompatible reaction [26]
(Fig. 1.1). In general, ETI activation results from the intracellular recognition of
pathogen effector molecules by plant resistance proteins (R proteins) [26]. These
effectors, synthetized by the pathogen and injected into the host cell cytosol, have
an important function in pathogenesis [27]; some enhance pathogen virulence and
suppress PTI, while others aid pathogens to propagate on their host by reprogram-
ming host cell metabolism and physiology, causing effector-triggered susceptibility
(ETS) [27]. Plants, in turn, recognize these effectors by receptor R proteins in a
specific manner [28]. Recognition by R proteins can be mediated either through
direct physical interaction with the effector (ligand-receptor model) or indirectly by
detecting modifications on other host proteins caused by effector activity (guard
model) [29]. Most of the R proteins identified so far belong to the nucleotide bind-
ing leucine-rich repeat (NB-LRR) type [26]. In comparison with PTI, ETI is a stron-
ger and more efficient response, and often culminates in hypersensitive response
(HR), a type of programmed cell death that limits the spread of the pathogen from
infection sites [24]. Several studies suggest that ETI utilizes the same defense sig-
nalling network as PTI, but in distinct ways, emitting stronger and longer-lasting
1 Biological and Molecular Control Tools in Plant Defense 7
responses [29]. In general, ETI restores and amplifies PTI basal transcriptional pro-
grams and antimicrobial defences [24]. Both PTI and ETI can induce immune
responses against pathogens on uninfected distal tissues [30]. Among the diverse
chemical signals identified so far, the plant hormone salicylic acid (SA), has been
found to play an important role in systemic resistance that provides broad spectrum
and long-lasting protection to future infections [30]. Establishment of systemic
resistance involves the generation of signals in the damaged tissue, and their further
transport via vascular system to sites further from the injury location.
As with pathogens efficiently evading PTI, many arthropods have evolved a vari-
ety of strategies to cope with plant defenses including behavioural adaptations and
mechanisms to decrease exposure (e.g. via detoxification or sequestration) or sensi-
tivity (e.g. via target-site sensitivity) to defense compounds [34]. Furthermore, cer-
tain herbivores are known to be able to manipulate sink source flows or to suppress
plant defenses [14, 34–36, 44]. Similar to pathogen effectors, effector-like mole-
cules from herbivores, specifically those secreted via their saliva into the host plant
are presumed to also interfere with PTI and lead to ETS [36, 37]. However, as with
HAMPs and PRRs, our knowledge on herbivore effectors is still limited.
Plants are surrounded by multiple threats that they must face by responding effec-
tively to survive. After specific attacker’s recognition, plants need to re-organize all
immune machinery to counteract the attack. The speed and intensity of the response
will determine the final output. As described above, at first, plants may use constitu-
tive defense barriers, and if those are not efficient enough, inducible defenses are
activated to defeat pathogens and pests. To mount an efficient response, plants need
to sense “the non-self”. Different stimuli can prepare plants to gain these inducible
defenses and set plants’ immunity in a manner that they can respond in a shorter time
and more efficiently to pathogen/pest attack [45, 46] (Fig. 1.2). Upon perception of
appropriate stimuli (‘sense of danger’) different physiological and molecular
changes, timely and quantitatively, prepare defenses for future attacks, resulting in
incompatible interactions. Those changes taking place between the sensing of the
stimuli and the presence of the challenge are known as the ‘priming state’ [46]
(Fig. 1.2). During this phase, the plant adapts its immune responses by learning from
experience.
Distinct stimuli may trigger the priming state, like beneficial organisms, arthro-
pods, pathogens, and avirulent bacteria, as well as chemical compounds or even
abiotic cues that may stimulate the production of active compounds. A silent time-
frame comes until the challenge shows up (the ‘priming phase’). Hence, when the
plant is exposed to a subsequent stress, it is sensitized to respond faster and with
higher intensity, and this is the so-called ‘post-challenge primed state’. In this phase,
there is an enhancement in the response following perception of danger and signal
transduction. For example, sour orange citrus displays constitutive priming against
the two-spotted spider mite Tetranychus urticae due to a high level of flavonoids and
a faster activation of the oxylipin pathway [47].
Among the different stimuli, there are genes that confer constitutive priming. For
instance, a mutation in the gene NRT2.1 that functions as a transceptor in Arabidopsis
confers constitutive priming against the pathogen Pseudomonas syringae pv tomato
DC3000 [48]. The knockdown of NRT2.1 allows a lower sensitivity to the toxin
coronatine, preventing the plant from the effector manipulation. Another example of
constitutive priming in Arabidopsis is generated by the mutant edr1 (ENHANCE
1 Biological and Molecular Control Tools in Plant Defense 9
Fig. 1.2 Intervals of action in priming defenses. Different stimuli in plants can produce a transient
and small response that tend to equilibrate afterwards. Priming inducers may range from biological
(MIR, beneficial microorganisms and arthropods, avirulent bacteria) to chemical (BABA, I3CA)
or genetic inputs (for example, downregulation of NRT2.1, OPC3 or EDR1). When plant defenses
go to basal levels, a memory window lasts until the threat appears. This period is the so-called
‘priming phase’. Along this phase, different players have been described, such as changes in pri-
mary and secondary metabolism, although this is dependent on the interaction between the priming
inducer and the plant species. Then, after the attack of a pathogen/pest, the post-challenge primed
phase starts. At this stage, primed plants (dark continuous blue line) respond faster and stronger to
the challenge than non-primed plants (grey continuous line). Different mechanisms may orches-
trate and coordinate a horizontal response to overcome the infection/attack. The intensity of the
response in the long term depends on the interaction between plant -pathogen/pest- priming
inducer, and may be associated with changes in the chromatin and histone modifications. Stressful
memories can be transmitted to the offspring (transgenerational phase) through epigenetic modifi-
cations if the presence of the stress persists along time (blue dashed line corresponds to response
intensity of plants that are still primed and grey dashed line, to the ones that have not been primed
before). The dark blue squares show the names of the priming periods of priming and light blue
squares show the type of defense responses (“silent”, active responses or transgenerational)
ascorbic acid, also shows constitutive priming of PR1 and SA [53]. Thus, these
genes may function as nodes that balance plant decisions relative to growth, abiotic
stress tolerance or resistance to biotic insults. Loss of function mutants of these
genes may be constitutively prepared for hyperactivation of defense responses with-
out costs in plant fitness.
Despite the pre-challenge phase has been described in the past as uneventful and
without fitness cost, now it is known to be associated with several molecular
changes. Subtle changes during that phase may be translated into fitness cost, that it
can be compensated by the final result when a threat appears [54]. A plant strategy
during this “silent” phase (Fig. 1.2) is the accumulation of hormone and metabolite
conjugates that will be hydrolysed to their active form upon a challenge. Following
certain priming stimuli such as β-aminobutyric acid (BABA) and avirulent bacteria,
the two main glycosylated forms of SA (SAG and SGE) are accumulated [55].
Other glucose conjugates of phytoanticipins also accumulate at this stage, such as
the aliphatic and indolic glucosinolates [56] or benzoxazinoids [45], which are
sequestered in the vacuole allowing their faster release upon pathogen/herbi-
vore attack.
An open debate is whether changes and induced resistance by beneficial organ-
isms may be mediated by defense priming [54] (TIPS). Among them, Arbuscular
Mycorrhizal Fungi (AMF) were shown to protect a wide range spectrum of plant
species against pathogen insults [57]. Reasonably, since AMF symbiosis and inter-
actions with beneficial microorganisms take place before the challenge, there are
obvious metabolic changes in the symbiont. Mycorrhiza-Induced Resistance (MIR)
is a particular defense priming since in the priming phase, there is a whole molecu-
lar and metabolic dialogue between the plant and AMF leading to the symbiosis. In
fact, priming during MIR is under consideration since it may be tissue dependent.
MIR is effective against several root and foliar pathogens and current studies aim to
elucidate the changes in the priming phase related to MIR.
Since carbon–rich compounds, amino acids and lipids are the main metabolites
exchanged between AMF and the host plant, AM symbiosis is expected to impact
primary metabolism. Several metabolites related to carbon metabolism were accu-
mulated in AM-Lotus japonicusplants before challenge [58]. Tomato plants colo-
nized by Rhizoglomus irregularis (formerly Glomus intraradices) showed enhanced
OPDA content and up-regulation of LOX-D gene expression level in the priming
phase [59]. Changes in the pre-challenge priming state usually targets the primary
metabolism, such as sugar and amino acid pathways, not only in AM priming but
also with other priming stimuli. Using qPCR and mutant approaches, an ABA-
dependent regulation of starch degradation after BABA and I3CA priming was
shown [60], and the sugar-derivative glycerol-3-phosphate has been reported as a
key signal in the azelaic acid-induced systemic immunity and priming [61].
1 Biological and Molecular Control Tools in Plant Defense 11
Amino acids are the precursors of many secondary metabolites that can partici-
pate in the subsequent defense responses. Pastor et al. [62] reported changes in
Arabidopsis primary metabolism, mainly in tricarboxylic acid (TCA) metabolites
such as citrate, fumarate, malate and 2-oxoglutarate as well as an enhanced bio-
synthesis of phenylpropanoid pathway following BABA priming before chal-
lenge. In the same study, authors compared changes occurring after BABA and
P. syringae pv tomato (PstAVRpt2) priming treatment and found that pathways
that were up-regulated after BABA priming were repressed after PstAVRpt2 treat-
ment. BABA is a water-soluble chemical compound that is rapidly distributed
throughout the plant while the bacteria use the plant sensing mechanisms to coor-
dinate the interaction between themselves and the plant. The different responses
to these two priming stimuli recorded by the authors, highlighted that not only
plant species but also the nature of the stimulus is important for the priming
response. Hence, priming is a horizontal phenomenon that triggers multiple meta-
bolic pathways shortly after infection/attack, resulting in enhanced defensive
responses.
The spatiotemporal input of priming has been recently revisited as the ‘internal’ and
‘external’ strategies of plant defense [63]. As part of the internal plant defense
responses, priming is a mechanism regulating the boosted defense reaction upon
challenge along with systemic acquired resistance [46]. This internal response in
primed plants, the so called ‘post-challenge primed state’, ranges from hours after
challenge to longer period, which may also be extended to the progeny [46, 64, 65]
(Fig.1.2). This transgenerational, epigenetically regulated defense priming may be
fixed along evolution terms by genetic adaptations, leading to ETI. Conversely,
defense priming regulates boosted responses during the external strategies that are
based, on the one hand, on interactions with microbes at the root or shoot level that
trigger the well-known induced systemic resistance [46, 66] (ISR) and, on the other
hand, on recruitment of natural enemies, the so-called ‘induced indirect defense’.
During herbivory, VOCs are released within the first few hours after attack and
attraction of natural enemies takes place at shorter term [67]. In a longer term, prim-
ing by beneficial microbes leads to the formation of disease-suppressive microbi-
omes [68, 69] that may protect plants through antibiosis, competition and induced
resistance [70–72].
As regards internal strategies, several mechanisms were shown to be involved
during the post-challenge priming state (Fig. 1.2). One of the first responses of
primed plants after PAMPs perception is stronger production of H2O2, preceding an
earlier and stronger callose accumulation [45]. Surprisingly, primed plants that are
effectively protected by this battery of early responses do not trigger, or even down
regulate, subsequent immune responses [73]. When the activation of subsequent
12 M. L. Pappas et al.
The so-called ‘external strategies’ of plants are long been known. The study of
beneficial insects that are attracted by plants following herbivory can be useful in
Integrated Pest Management (IPM) programs. Plants in response to HAMPs release
HIPVs that improve the recruitment of beneficial arthropods [67, 83]. Importantly,
external strategies of plants can be enhanced via priming, for example, when plants
are exposed to appropriate stimuli. In fact, several interesting studies in phyloge-
netically distant plant species such as maize and citrus show similar outputs when
susceptible plants are exposed to VOCs [84, 85]. Maize plants exposed to VOCs
released by plants treated by caterpillar regurgitant were more efficient to mount
effective defenses against Spodoptera littoralis [84]. In addition, maize plants
primed with VOCs were more attractive to the parasitic wasp Cotesia marginiven-
tris while control plants and plants only treated with VOCs did not result in a sig-
nificant attraction. Similarly, mite-susceptible citrus genotypes can express
resistance after priming by VOCs released by resistant citrus attacked by the spider
mite T. urticae. VOCs-mediated priming results in enhanced resistance against spi-
der mites and priming of JA-dependent responses [85]. Thus, priming against her-
bivores, either by stimulating direct (internal) or indirect (external) defenses, is
another example of adaptive immune responses of plants [86, 87]. Notably, plants
are not only able to be attractive to aboveground beneficial arthropods but also to
beneficial microbes present in the rhizosphere [88]. It is well-known that plants
exposed to phosphorous deficiency are more attractive to mycorrhizal fungi by the
release of strigolactones at the very early stages of the mycorrhizal symbiosis,
which at a later stage ends up in MIR that is also mediated via priming [57, 59].
has evolved as a major antiviral defense mechanism [93, 99]. Recently, it has been
suggested that RNAi is also involved in antifungal defense, since plants send sRNAs
into fungal pathogens in order to target essential fungal genes, as cotton does against
Verticillium dahliae, Arabidopsis against Botrytis cinerea and wheat against
Fusarium graminearum [100–102].
The tremendous gene silencing potential of RNAi has not skipped the attention
of plant biotechnologists. During the last two decades, plant scientists have trans-
formed a plethora of plants expressing dsRNAs against various viruses, fungi,
oomycetes, insects, mites and nematodes, all resulting in very high levels of plant
defense against each corresponding target [98, 103–108]. Common denominator in
all these approaches was the use of a transgene consisting of an invertedly repeated
cDNA that, upon transcription, would generate dsRNA molecules that would trig-
ger RNAi against the selected target. However, since the use of transgenes, trans-
genic plants and genetically modified organisms (GMOs) in general have been met
with considerable public and scientific concern, plant biologists have lately resorted
to GMO-free RNAi approaches by simply exogenously applying dsRNAs and
sRNAs inplants against various pests and pathogens using methods such as high-
pressure spraying and trunk injection [109–111] (Fig. 1.3). RNAi-based biopesti-
cides, consisting solely of dsRNA and/or sRNA molecules, could exhibit an
extremely specific mode of action since they require only 21 nt homology with their
target, thus aiming specific regions of specific genes in specific species, practically
eliminating undesired off-target effects. Importantly, according to the 40th annual
meeting of the Toxicology Forum, the exogenous application of RNA molecules
pose no threat to human health even when present in diet [107]. Not surprisingly,
the non-GMO, non-toxic and highly specific character of RNA-based tools has ren-
dered them a vital importance in modern crop protection platforms [112, 113].
Viruses cause epidemics on almost all agronomical important crops, posing a seri-
ous threat to global food security and being responsible for yield losses roughly
estimated to cost worldwide more than 30 billion USD annually [114]. Most plant
viruses exhibit a single stranded RNA genome and replicate in plant cell cytoplasm
through dsRNA intermediates, thus serving as targets for host RNAi machinery.
Hence, a well-established strategy involves pre-treating of plants with dsRNAs/
sRNAs designed to target specific viral regions (e.g. coat or movement protein) in
order to resist imminent viral infection (Fig. 1.3). Indeed, leaf spraying and/or
mechanical inoculation of RNAi molecules targeting viral sequences resulted in
1 Biological and Molecular Control Tools in Plant Defense 15
Fig. 1.3 Transgene-free RNA-based molecular control tools in plant defense involve the exogenous
application of in vitro and/or in vivo transcribed dsRNA molecules in plants with the objective to
trigger RNAi against (1) plant/weed genes, (2) viruses/viroids, (3) fungi/oomycetes and (4) insects/
mites. In cases (1) and (2), the exogenously applied dsRNA needs to be efficiently taken up by the
plant cell in order to be processed by plant DCLs into siRNAs that will target for degradation the
corresponding transcripts in the cytoplasm. To achieve efficient delivery inside the plant cell, the
dsRNA needs to be applied by high-pressure spraying which allows the mechanical disruption of
the plant cell wall. In cases (3) and (4), the exogenously applied dsRNA is supposed to trigger RNAi
not inside the plant cell but inside the fungal and/or insect cell. To increase RNAi efficiency inside
the fungal and insect cells, the applied dsRNA needs to avoid processing by plant DCLs and,
instead, be processed solely by the fungal or insect Dicers into siRNAs which will target the corre-
sponding fungal or insect mRNAs for degradation. To achieve this, the exogenous dsRNA needs to
be applied by trunk injection and/or petiole absorption, since by these two methods the dsRNA is
transported exclusively through the plant xylem and apoplast (where no plant DCLs are present) to
distant tissues and are thus accessible to be taken up by the plant tissue-penetrating fungi and by the
chewing and/or xylem sap-feeding insects. However, trunk injection and petiole uptake are not suit-
able in the case of phloem-sap feeding insects (e.g. aphids) since in that case the xylem-residing
dsRNA would be inaccessible to them. In the latter case, high pressure spraying of dsRNA would
be more advisable, since it allows the symplastic delivery of RNA molecules to systemic tissues.
Image adopted by permission from Dalakouras et al. [110]. Copyright American Society of Plant
Biologists
significant viral resistance (1) in N. benthamiana (against Pepper Mild Mottle Virus,
Tobacco Etch Virus, Alfalfa Mosaic Virus, Tobacco Mosaic Virus), (2) in N. taba-
cum (against Tobacco Mosaic Virus, Potato Virus Y, Cucumber Mosaic Virus), (3) in
Cucumis sativus (against Zucchini Yellow Mosaic Virus), (4) in Vigna unguiculate
(against Bean Common Mosaic Virus), (5) in Zea mays (against Sugarcane Mosaic
Virus), (6) in Carica papaya (against Papaya Ringspot Virus) and (7) in Pisum sati-
vum (against Pea Seed-borne Mosaic Virus) [115–124]. Closely related to viruses
are viroids which are non-encapsidated, non-coding, circular, single stranded RNA
pathogens [125]. Similar to antiviral applications, mechanical inoculation in
16 M. L. Pappas et al.
Knowledge on priming during the last 5–6 years has grown exponentially and many
published studies have paid attention to the mechanisms underlying this adaptive
immune response [46, 63, 141]. Most studies focus on model plant species covering
fundamental aspects of priming and, research in applied aspects of priming in com-
mon crops has received much less attention. Reasonably, since the availability of
molecular tools in common crops is less abundant, most research data of priming in
crops such as potato, wheat, barley, cowpea or citrus refer to yield improvement,
disease phenotypes or pest resistance and sometimes, hormonal or metabolic imbal-
ances during post-challenge primed state [46, 87, 142, 143].
Accordingly, our knowledge on the mechanisms underlying biocontrol priming
in crops is scarce. In many cases, the application of the triggering priming agent,
either a chemical or a beneficial organism, is reported not to display a benefit on
crop growth, until a disease infestation or insect attack. In barley, it was shown that
saccharin treatments did not increase plant growth, although primed plants increased
grain yield in the presence of the fungus Rhynchosporium secalis [144]. Seemingly,
plant colonization by AMF has rather variable outputs in terms of growth [145].
Despite these limitations, the low or non-existent benefits of priming sensing during
the priming phase counterweights the benefits following disease or insect attack.
In semi-field experiments, priming triggered by mycorrhizal symbiosis was
shown to be functional in potato against the herbivore Trichoplusia ni [142].
Although mycorrhization had no effects on potato growth, it effectively reduced
larval weight that may be explained by enhanced JA-dependent responses. In stud-
ies on priming in citrus trees, sour orange rootstock was found to display constitu-
tive priming against spider mites [47, 85]. Interestingly, rootstock resistance is
transmitted to the scion, therefore these findings can be applied to commercial vari-
eties to stimulate plant immunity in the field. Another unexplored field aspect is the
improvement of IPM strategies by using citrus plants that are more attractive to
natural enemies. Recently, it was shown that sour orange recruits more efficiently
the generalist predatory mite Euseius stipulatus that may improve the efficiency of
pest control in agriculture [146]. Priming has also been shown in a context of treat-
ments with natural extracts such as mint volatiles that were proven to confer
enhanced defenses in field trials on soybean against both the herbivore Spodoptera
litura and the fungus Phakopsora pachyrhizi [147]. Therefore, defense priming
known as ‘green vaccination’ has been proposed as the perfect match to IPM strate-
gies which, following appropriate field experimentation, could be transferred to
applied science [148].
18 M. L. Pappas et al.
by the induction of PR1 gene among others [168, 169]. Interestingly, the strain
F. oxysporum 47 (Fo47) could not protect these plants from foliar infection by
B. cinerea. Perhaps the induction of SA in plants colonized by Fo47 blocks other
defenses that influence other diseases. This fungus may act at several levels like the
production of VOCs, plant growth promotion, antibiosis and mycoparasitism in
vitro, induced resistance, also by competition at the root site [170].
Other beneficial microorganisms that are emerging as potential biocontrol agents,
are strains belonging to the Rhizobia genus. Traditionally, this genus has been con-
sidered an essential player in nitrogen fixation and uptake by the plant. Nevertheless,
evidence suggests additional roles in plant defense regarding root diseases.
Rhizobium bacteria can produce and release proteolytic enzymes and parasite fungi
in the rhizosphere such as pathogenic strains of F. oxysporum [171]. Also, Rhizobium
leguminosarum strain Rl was able to protect chickpea against the pathogen F. oxys-
porum f. sp. ciceris (Foc) [172]. This protection is also present against other micro-
organisms (bacteria, viruses) and nematodes, via ISR [173]. Additional responses
like emissions of antimicrobial VOCs, siderophore production, competition and
changes in volatile plant compounds are also contributing to plant defense by
Rhizobium [173].
Besides beneficial soil microbes, zoophytophagous predators such as the mirids
M. pygmaeus, N. tenuis and Orius laevigatus have been shown to induce plant
defenses against herbivorous pests via their phytophagy [83, 174–178]. Exposing
plants to M. pygmaeus negatively affected the performance of the two spotted spider
mite T. urticae in tomato and the western flower thrips Frankliniella occidentalis in
pepper [174, 175, 178]. These negative effects against pests were attributed to the
increased accumulation of transcripts and the activity of proteinase inhibitors (PI) in
the mirid-exposed tomato plants [175], and to the activation of the JA-related
responses in pepper plants [178]. Furthermore, tomato and pepper plants exposed to
N. tenuiswerefound to be more attractive to predator conspecifics [179] and to the
parasitoid Encarsia formosa, a biological control agent of whiteflies [176]. This
indirect plant defense response was related to changes in the volatile blend released
by the mirid-exposed plants, via the activation of ABA and JA signaling pathways
[176]. Notably, mirid-induced plants were shown to be less attractive to key pests
such as the tomato leaf miner T. absoluta, the whitefly Bemisia tabaci, the western
flower thrips F. occidentalisand the two-spotted spider mite T. urticae [83, 176, 177,
180, 181]. Overall, the above studies suggest that zoophytophagous predators may
serve as ‘plant vaccination agents’ at the early stages of the establishment of a crop
directly affecting herbivores via predation and indirectly, via the induction of direct
and indirect plant defense responses, eventually enhancing their overall biocontrol
efficiency [174, 182].
Interestingly, zoophytophagous predators have been recently shown to positively
interact with beneficial soil microbes to the benefit of their host plant. The coloniza-
tion of tomato plants with a root restricted endophyte, the non-pathogenic stain FsK
was shown to result in reduced feeding symptoms (necrotic rings on leaves and
stems) by the zoophytophagous predator N. tenuis possibly via the upregulation of
the ethylene and JA pathways [165], and to alter volatile blend emission by tomato
20 M. L. Pappas et al.
Most of chemical priming inducers are natural compounds isolated from challenged
plants, or compounds mimicking the structures of natural immune inducers. They
do not have in vitro antimicrobial activity, and target the main defense-related phy-
tohormone pathways. The first chemical inducers of priming studied were SA and
synthetic SA analogues such as 2,6-dichloroisonicotinic acid (INA) and
thiadiazole-7-carbothioic acid (BTH). Both were shown to prime parsley cells to
resist Phytophtora sojae [186]. Accumulation of SA is a common trait in SAR and
mediates the activation of a set of pathogenesis-related (PR) genes. Mono- and di-
chloro substituted SA and fluoro-SA derivatives were found to induce PR proteins
in tobacco against TMV infection [187, 188]. While SA regulates defense against
biotrophic pathogens, JA and MeJA control mainly the immune responses against
necrotrophic pathogens and herbivores. JA and several synthetic JA mimics have
been shown to induce priming by activating JA signaling and defense responses in
different plant species (reviewed by Zhou and Wang [189]). In most cases, when
phytohormone analogues are used as priming agents, it is concentration that deter-
mines whether priming or direct defenses are displayed by the plant [49].
Besides the main phytohormones and their analogues, several chemical com-
pounds such as BABA and Indol-3-carboxylic acid (I3CA) are known to prime the
plants to cope with environmental and biotic stresses [190, 191]. Among these
chemical inducers, BABA-IR has the widest protection spectrum; it has been shown
to protect about forty plant species including mono- and dicotyledonous against
several pathogens and pests, including viruses, Protista, bacteria, oomycetes, fungi
and arthropods being effective in a wide range of applications (foliar spray, soil
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Fig. 313.—Diodon maculatus.
THIRD SUB-CLASS—CYCLOSTOMATA.
Skeleton cartilaginous and notochordal, without ribs and without
real jaws. Skull not separate from the vertebral column. No limbs.
Gills in the form of fixed sacs, without branchial arches, six or seven
in number on each side. One nasal aperture only. Heart without
bulbus arteriosus. Mouth anterior, surrounded by a circular or
subcircular lip, suctorial. Alimentary canal straight, simple, without
coecal appendages, pancreas or spleen. Generative outlet
peritoneal. Vertical fins rayed.
The Cyclostomes are most probably a very ancient type.
Unfortunately the organs of these creatures are too soft to be
preserved, with the exception of the horny denticles with which the
mouth of some of them is armed. And, indeed, dental plates, which
are very similar to those of Myxine, are not uncommon in certain
strata of Devonian and Silurian age (see p. 193). The fishes
belonging to this sub-class may be divided into two families—
First Family—Petromyzontidæ.
Body eel-shaped, naked. Subject to a metamorphosis; in the
perfect stage with a suctorial mouth armed with teeth, simple or
multicuspid, horny, sitting on a soft papilla. Maxillary, mandibulary,
lingual, and suctorial teeth may be distinguished. Eyes present (in
mature animals). External nasal aperture in the middle of the upper
side of the head. The nasal duct terminates without perforating the
palate. Seven branchial sacs and apertures on each side behind the
head; the inner branchial ducts terminate in a separate common
tube. Intestine with a spiral valve. Eggs small. The larvæ without
teeth, and with a single continuous vertical fin.
“Lampreys” are found in the rivers and on the coasts of the
temperate regions of the northern and southern hemispheres. Their
habits are but incompletely known, but so much is certain that at
least some of them ascend rivers periodically, for the purpose of
spawning, and that the young pass several years in rivers, whilst
they undergo a metamorphosis (see p. 170). They feed on other
fishes, to which they suck themselves fast, scraping off the flesh with
their teeth. Whilst thus engaged they are carried about by their
victim; Salmon have been captured in the middle course of the Rhine
with the Marine Lamprey attached to them.
FOURTH SUB-CLASS—LEPTOCARDII.
Skeleton membrano-cartilaginous and notochordal, ribless. No
brain. Pulsating sinuses in place of a heart. Blood colourless.
Respiratory cavity confluent with the abdominal cavity; branchial
clefts in great number, the water being expelled by an opening in
front of the vent. Jaws none.
This sub-class is represented by a single family (Cirrostomi) and
by a single genus (Branchiostoma);[48] it is the lowest in the scale of
fishes, and lacks so many characteristics, not only of this class, but
of the vertebrata generally, that Hæckel, with good reason,
separates it into a separate class, that of Acrania. The various parts
of its organisation have been duly noticed in the first part of this
work.
The “Lancelet” (Branchiostoma lanceolatum, see Fig. 28, p. 63),
seems to be almost cosmopolitan within the temperate and tropical
zones. Its small size, its transparency, and the rapidity with which it
is able to bury itself in the sand, are the causes why it escapes so
readily observation, even at localities where it is known to be
common. Shallow, sandy parts of the coasts seem to be the places
on which it may be looked for. It has been found on many localities of
the British, and generally European coasts, in North America, the
West Indies, Brazil, Peru, Tasmania, Australia, and Borneo. It rarely
exceeds a length of three inches. A smaller species, in which the
dorsal fringe is distinctly higher and rayed, and in which the caudal
fringe is absent, has been described under the name of
Epigionichthys pulchellus; it was found in Moreton Bay.
APPENDIX.
DIRECTIONS FOR COLLECTING AND PRESERVING FISHES.
Whenever practicable fishes ought to be preserved in spirits.
To insure success in preserving specimens the best and
strongest spirits should be procured, which, if necessary, can be
reduced to the strength required during the journey with water or
weaker spirit. Travellers frequently have great difficulties in procuring
spirits during their journey, and therefore it is advisable, especially
during sea voyages, that the traveller should take a sufficient
quantity with him. Pure spirits of wine is best. Methylated spirits may
be recommended on account of their cheapness; however,
specimens do not keep equally well in this fluid, and very valuable
objects, or such as are destined for minute anatomical examination,
should always be kept in pure spirits of wine. If the collector has
exhausted his supply of spirits he may use arrack, cognac, or rum,
provided that the fluids contain a sufficient quantity of alcohol.
Generally speaking, spirits which, without being previously heated,
can be ignited by a match or taper, may be used for the purposes of
conservation. The best method to test the strength of the spirits is
the use of a hydrometer. It is immersed in the fluid to be measured,
and the deeper it sinks the stronger is the spirit. On its scale the
number 0 signifies what is called proof spirit, the lowest degree of
strength which can be used for the conservation of fish for any length
of time. Spirits, in which specimens are packed permanently, should
be from 40 to 60 above proof. If the hydrometers are made of glass
they are easily broken, and therefore the traveller had better provide
himself with three or four of them, their cost being very trifling.
Further, the collector will find a small distilling apparatus very useful.
By its means he is able not only to distil weak and deteriorated spirits
or any other fluid containing alcohol, but also, in case of necessity, to
prepare a small quantity of drinkable spirits.
Of collecting vessels we mention first those which the collector
requires for daily use. Most convenient are four-sided boxes made of
zinc, 18 in. high, 12 in. broad, and 5 in. wide. They have a round
opening at the top of 4 in. diameter, which can be closed by a strong
cover of zinc of 5 in. diameter, the cover being screwed into a raised
rim round the opening. In order to render the cover air-tight, an
indiarubber ring is fixed below its margin. Each of these zinc boxes
fits into a wooden case, the lid of which is provided with hinges and
fastenings, and which on each side has a handle of leather or rope,
so that the box can be easily shifted from one place to another.
These boxes are in fact made from the pattern of the ammunition
cases used in the British army, and extremely convenient, because a
pair can be easily carried strapped over the shoulders of a man or
across the back of a mule. The collector requires at least two, still
better four or six, of these boxes. All those specimens which are
received during the day are deposited in them, in order to allow them
to be thoroughly penetrated by the spirit, which must be renewed
from time to time. They remain there for some time under the
supervision of the collector, and are left in these boxes until they are
hardened and fit for final packing. Of course, other more simple
vessels can be used and substituted for the collecting boxes. For
instance, common earthenware vessels, closed by a cork or an
indiarubber covering, provided they have a wide mouth at the top,
which can be closed so that the spirit does not evaporate, and which
permits of the specimens being inspected at any moment without
trouble. Vessels in which the objects are permanently packed for the
home journey are zinc boxes of various sizes, closely fitting into
wooden cases. Too large a size should be avoided, because the
objects themselves may suffer from the superimposed weight, and
the risk of injury to the case increases with its size. It should hold no
more than 18 cubic feet at most, and what, in accordance with the
size of the specimens, has to be added in length should be deducted
in depth or breadth. The most convenient cases, but not sufficient for
all specimens, are boxes 2 feet in length, 1½ foot broad, and 1 foot
deep. The traveller may provide himself with such cases ready
made, packing in them other articles which he wants during his
journey; or he may find it more convenient to take with him only the
zinc plates cut to the several sizes, and join them into boxes when
they are actually required. The requisite wooden cases can be
procured without much difficulty almost everywhere. No collector
should be without the apparatus and materials for soldering, and he
should be well acquainted with their use. Also a pair of scissors to
cut the zinc plates are useful.
Wooden casks are not suitable for the packing of specimens
preserved in spirits, at least not in tropical climates. They should be
used in cases of necessity only, or for packing of the largest
examples, or for objects preserved in salt or brine.
Very small and delicate specimens should never be packed
together with larger ones, but separately, in small bottles.
Mode of preserving.—All fishes, with the exception of very large
ones (broad kinds exceeding 3–4 feet in length; eel-like kinds more
than 6 feet long), should be preserved in spirits. A deep cut should
be made in the abdomen between the pectoral fins, another in front
of the vent, and one or two more, according to the length of the fish,
along the middle line of the abdomen. These cuts are made partly to
remove the fluid and easily decomposing contents of the intestinal
tract, partly to allow the spirit quickly to penetrate into the interior. In
large fleshy fishes several deep incisions should be made with the
scalpel into the thickest parts of the dorsal and caudal muscles, to
give ready entrance to the spirits. The specimens are then placed in
one of the provisional boxes, in order to extract, by means of the
spirit, the water of which fishes contain a large quantity. After a few
days (in hot climates after 24 or 48 hours) the specimens are
transferred into a second box with stronger spirits, and left therein for
several days. A similar third and, in hot climates sometimes a fourth,
transfer is necessary. This depends entirely on the condition of the
specimens. If, after ten or fourteen days of such treatment the
specimens are firm and in good condition, they may be left in the
spirits last used until they are finally packed. But if they should be
soft, very flexible, and discharge a discoloured bloody mucus, they
must be put back in spirits at least 20° over proof. Specimens
showing distinct signs of decomposition should be thrown away, as
they imperil all other specimens in the same vessel. Neither should
any specimen in which decomposition has commenced when found,
be received for the collecting boxes, unless it be of a very rare
species, when the attempt may be made to preserve it separately in
the strongest spirits available. The fresher the specimens to be
preserved are, the better is the chance of keeping them in a perfect
condition. Specimens which have lost their scales, or are otherwise
much injured, should not be kept. Herring-like fishes, and others with
deciduous scales, are better wrapped in thin paper or linen before
being placed in spirits.
The spirits used during this all-important process of preservation
loses, of course, gradually in strength. As long as it keeps 10° under
proof it may still be used for the first stage of preservation, but
weaker spirits should be re-distilled; or, if the collector cannot do this,
it should be at least filtered through powdered charcoal before it is
mixed with stronger spirits. Many collectors are satisfied with
removing the thick sediment collected at the bottom of the vessel,
and use their spirits over and over again without removing from it by
filtration the decomposing matter with which it has been
impregnated, and which entirely neutralises the preserving property
of the spirits. The result is generally the loss of the collection on its
journey home. The collector can easily detect the vitiated character
of his spirits by its bad smell. He must frequently examine his
specimens; and attention to the rules given, with a little practice and
perseverance, after the possible failure of the first trial, will soon
insure to him the safety of his collected treasures. The trouble of
collecting specimens in spirits is infinitely less than that of preserving
skins or dry specimens of any kind.
When a sufficient number of well-preserved examples have been
brought together, they should be sent home by the earliest
opportunity. Each specimen should be wrapped separately in a piece
of linen, or at least soft paper; the specimens are then packed as
close as herrings in the zinc case, so that no free space is left either
at the top or on the sides. When the case is full, the lid is soldered
on, with a round hole about half an inch in diameter near one of the
corners. This hole is left in order to pour the spirit through it into the
case. Care is taken to drive out the air which may remain between
the specimens, and to surround them completely with spirits, until
the case is quite full. Finally, the hole is closed by a small square lid
of tin being soldered over it. In order to see whether the case keeps
in the spirit perfectly, it is turned upside down and left over night.
When all is found to be securely fastened, the zinc case is placed
into the wooden box and ready for transport.
Now and then it happens in tropical climates that collectors are
unable to keep fishes from decomposition even in the strongest
spirits without being able to detect the cause. In such cases a
remedy will be found in mixing a small quantity of arsenic or
sublimate with the spirits; but the collector ought to inform his
correspondent, or the recipient of the collection, of this admixture
having been made.
In former times fishes of every kind, even those of small size,
were preserved dry as flat skins or stuffed. Specimens thus prepared
admit of a very superficial examination only, and therefore this
method of conservation has been abandoned in all larger museums,
and should be employed exceptionally only, for instance on long
voyages overland, during which, owing to the difficulty of transport,
neither spirits nor vessels can be carried. To make up as much as
possible for the imperfection of such specimens, the collector ought
to sketch the fish before it is skinned, and to colour the sketch if the
species is ornamented with colours likely to disappear in the dry
example. Collectors who have the requisite time and skill, ought to
accompany their collections with drawings coloured from the living
fishes; but at the same time it must be remembered that, valuable as
such drawings are if accompanied by the originals from which they
were made, they can never replace the latter, and possess a
subordinate scientific value only.
Very large fishes can be preserved as skins only; and collectors
are strongly recommended to prepare in this manner the largest
examples obtainable, although it will entail some trouble and
expense. So very few large examples are exhibited in museums, the
majority of the species being known from the young stage only, that
the collector will find himself amply recompensed by attending to
these desiderata.
Scaly fishes are skinned thus: with a strong pair of scissors an
incision is made along the median line of the abdomen from the
foremost part of the throat, passing on one side of the base of the
ventral and anal fins, to the root of the caudal fin, the cut being
continued upwards to the back of the tail close to the base of the
caudal. The skin of one side of the fish is then severed with the
scalpel from the underlying muscles to the median line of the back;
the bones which support the dorsal and caudal are cut through, so
that these fins remain attached to the skin. The removal of the skin
of the opposite side is easy. More difficult is the preparation of the
head and scapulary region; the two halves of the scapular arch
which have been severed from each other by the first incision are
pressed towards the right and left, and the spine is severed behind
the head, so that now only the head and shoulder bones remain
attached to the skin. These parts have to be cleaned from the inside,
all soft parts, the branchial and hyoid apparatus, and all smaller
bones, being cut away with the scissors or scraped off with the
scalpel. In many fishes, which are provided with a characteristic
dental apparatus in the pharynx (Labroids, Cyprinoids), the
pharyngeal bones ought to be preserved, and tied with a thread to
the specimen. The skin being now prepared so far, its entire inner
surface as well as the inner side of the head are rubbed with
arsenical soap; cotton-wool, or some other soft material is inserted
into any cavities or hollows, and finally a thin layer of the same
material is placed between the two flaps of the skin. The specimen is
then dried under a slight weight to keep it from shrinking.
The scales of some fishes, as for instance of many kinds of
herrings, are so delicate and deciduous that the mere handling
causes them to rub off easily. Such fishes may be covered with thin
paper (tissue-paper is the best), which is allowed to dry on them
before skinning. There is no need for removing the paper before the
specimen has reached its destination.
Scaleless Fishes, as Siluroids and Sturgeons, are skinned in the
same manner, but the skin can be rolled up over the head; such
skins can also be preserved in spirits, in which case the traveller
may save to himself the trouble of cleaning the head.
Some Sharks are known to attain to a length of 30 feet, and
some Rays to a width of 20 feet. The preservation of such gigantic
specimens is much to be recommended, and although the difficulties
of preserving fishes increase with their size, the operation is
facilitated, because the skins of all Sharks and Rays can easily be
preserved in salt and strong brine. Sharks are skinned much in the
same way as ordinary fishes. In Rays an incision is made not only
from the snout to the end of the fleshy part of the tail, but also a
second across the widest part of the body. When the skin is removed
from the fish, it is placed into a cask with strong brine mixed with
alum, the head occupying the upper part of the cask; this is
necessary, because this part is most likely to show signs of
decomposition, and therefore most requires supervision. When the
preserving fluid has become decidedly weaker from the extracted
blood and water, it is thrown away and replaced by fresh brine. After
a week’s or fortnight’s soaking the skin is taken out of the cask to
allow the fluid to drain off; its inner side is covered with a thin layer of
salt, and after being rolled up (the head being inside) it is packed in a
cask, the bottom of which is covered with salt; all the interstices and
the top are likewise filled with salt. The cask must be perfectly water-
tight.
Of all larger examples of which the skin is prepared, the
measurements should be taken before skinning so as to guide the
taxidermist in stuffing and mounting the specimens.
Skeletons of large osseous fishes are as valuable as their skins.
To preserve them it is only necessary to remove the soft parts of the
abdominal cavity and the larger masses of muscle, the bones being
left in their natural continuity. The remaining flesh is allowed to dry
on the bones, and can be removed by proper maceration at home.
The fins ought to be as carefully attended to as in a skin, and of
scaly fishes so much of the external skin ought to be preserved as is
necessary for the determination of the species, as otherwise it is
generally impossible to determine more than the genus.
A few remarks may be added as regards those Faunæ, which
promise most results to the explorer, with some hints as to desirable
information on the life and economic value of fishes.
It is surprising to find how small the number is of the freshwater
faunæ which may be regarded as well explored; the rivers of Central
Europe, the Lower Nile, the lower and middle course of the Ganges,
and the lower part of the Amazons are almost the only fresh waters
in which collections made without discrimination would not reward
the naturalist. The oceanic areas are much better known; yet almost
everywhere novel forms can be discovered and new observations
made. Most promising and partly quite unknown are the following
districts:—the Arctic Ocean, all coasts south of 38° lat. S., the Cape
of Good Hope, the Persian Gulf, the coasts of Australia (with the
exception of Tasmania, New South Wales, and New Zealand), many
of the little-visited groups of Pacific islands, the coasts of north-
eastern Asia north of 35° lat. N., and the western coasts of North and
South America.
No opportunity should be lost to obtain pelagic forms, especially
the young larva-like stages of development abounding on the
surface of the open ocean. They can be obtained without difficulty by
means of a small narrow meshed net dragged behind the ship. The
sac of the net is about 3 feet deep, and fastened to a strong brass-
ring 2 or 2½ feet in diameter. The net is suspended by three lines
passing into the strong main line. It can only be used when the
vessel moves very slowly, its speed not exceeding three knots an
hour, or when a current passes the ship whilst at anchor. To keep the
net in a vertical position the ring can be weighted at one point of its
circumference; and by using heavier weights two or three drag-nets
can be used simultaneously at different depths. This kind of fishing
should be tried at night as well as day, as many fishes come to the
surface only after sunset. The net must not be left long in the water,
from 5 to 20 minutes only, as delicate objects would be sure to be
destroyed by the force of the water passing through the meshes.
Objects found floating on the surface, as wood, baskets,
seaweed, etc., deserve the attention of the travellers, as they are
generally surrounded by small fishes or other marine animals.
It is of the greatest importance to note the longitude and latitude
at which the objects were collected in the open ocean.
Fishing in great depths by means of the dredge, can be practised
only from vessels specially fitted out for the purpose; and the
success which attended the “Challenger,” and North American Deep-
sea explorations, has developed Deep-sea fishing into such a
speciality that the requisite information can be gathered better by
consulting the reports of those expeditions than from a general
account, such as could be given in the present work.
Fishes offer an extraordinary variety with regard to their habits,
growth, etc., so that it is impossible to enumerate in detail the points
of interest to which the travellers should pay particular attention.
However, the following hints may be useful.
Above all, detailed accounts are desirable of all fishes forming
important articles of trade, or capable of becoming more generally
useful than they are at present. Therefore, deserving of special
attention are the Sturgeons, Gadoids, Thyrsites and Chilodactylus,
Salmonoids, Clupeoids. Wherever these fishes are found in sufficient
abundance, new sources may be opened to trade.
Exact observations should be made on the fishes the flesh of
which is poisonous either constantly or at certain times and certain
localities; the cause of the poisonous qualities as well as the nature
of the poison should be ascertained. Likewise the poison of fishes
provided with special poison-organs requires to be experimentally
examined, especially with regard to its effects on other fishes and
animals generally.
All observations directed to sex, mode of propagation, and
development, will have special interest: thus those relating to
secondary sexual characters, hermaphroditism, numeric proportion
of the sexes, time of spawning and migration, mode of spawning,
construction of nests, care of progeny, change of form during growth,
etc.
If the collector is unable to preserve the largest individuals of a
species that may come under his observation he should note at least
their measurements. There are but few species of fishes of which the
limit of growth is known.
The history of Parasitic Fishes is almost unknown, and any
observations with regard to their relation to their host as well as to
their early life will prove to be valuable; nothing is known of the
propagation of fishes even so common as Echeneis and Fierasfer,
much less of the parasitic Freshwater Siluroids.
The temperature of the blood of the larger freshwater and marine
species should be exactly measured.
Many pelagic and deep-sea fishes are provided with peculiar
small round organs of a mother-of-pearl colour, distributed in series
along the side of the body, especially along the abdomen. Some
zoologists consider these organs as accessory eyes, others (and it
appears to us with better reason) as luminous organs. They deserve
an accurate microscopic examination made on fresh specimens; and
their function should be ascertained from observation of the living
fishes, especially also with regard to the question, whether or not the
luminosity (if such be their function) is subject to the will of the fish.