Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol.

106(3): 301-307, May 2011 301

Morphological and molecular description of Blastocrithidia cyrtomeni

sp. nov. (Kinetoplastea: Trypanosomatidae) associated with
Cyrtomenus bergi Froeschner (Hemiptera: Cydnidae) from Colombia
Ana Milena Caicedo1/+, Gerardo Gallego2, Jaime Eduardo Muñoz3, Harold Suárez2,
Andrés Gerardo Torres4, Humberto Carvajal5, Fanny Caro De Carvajal5,
Andrés Mauricio Posso3, Dmitriv Maslov6, James Montoya-Lerma1
1
Biology Department 5Parasitology Laboratory, Universidad del Valle, Cali, Valle, Colombia 2Biodiversity and Biotechnology Program,
International Center of Tropical Agriculture, Cali, Valle, Colombia 3Molecular Biology Laboratory, Universidad Nacional de Colombia,
Palmira, Valle, Colombia 4Microscopy Laboratory, Universidad del Cauca, Popayán, Cauca, Colombia
6
Department of Biology, University of California, Riverside, California, USA

A new trypanosomatid species, Blastocrithidia cyrtomeni, is herein described using morphological and molecu-
lar data. It was found parasitising the alimentary tract of the insect host Cyrtomenus bergi, a polyphagous pest. The
morphology of B. cyrtomeni was investigated using light and transmission microscopy and molecular phylogeny
was inferred from the sequences of spliced leader RNA (SL rRNA) - 5S rRNA gene repeats and the 18S small subunit
(SSU) rRNA gene. Epimastigotes of variable size with straphanger cysts adhering to the middle of the flagellum were
observed in the intestinal tract, hemolymph and Malpighian tubules. Kinetoplasts were always observed anterior
to the nucleus. The ultrastructure of longitudinal sections of epimastigotes showed the flagellum arising laterally
from a relatively shallow flagellar pocket near the kinetoplast. SL RNA and 5S rRNA gene repeats were positive in
all cases, producing a 0.8-kb band. The amplicons were 797-803 bp long with > 98.5% identity, indicating that they
originated from the same organism. According to the sequence analysis of the SL-5S rRNA gene repeats and the
18S SSU rRNA gene, B. cyrtomeni is different from all other known species or isolates of Trypanosomatidae. Both
analyses indicate that among known species, it is most closely related to Blastocrithidia triatomae.

Key words: SL rRNA - 5S rRNA - SSU rRNA - trypanosomatids - Cyrtomenus - Blastocrithidia cyrtomeni

Kinetoplastid flagellates (Euglenozoa and Kineto- tive positions of the nucleus and kinetoplast have been
plastea) are a mixed group of free-living organisms and used to develop the current taxonomic system (Hoare
mono and dixenous parasites that colonise a variety of eu- 1964, Hoare & Wallace 1966, Wallace 1966).
karyotes, preferentially insects (Wallace 1966, Fernandes Although morphological characteristics are often re-
et al. 1993, Vickerman 1994, Podlipaev et al. 2004). The liable for identifying a genus, they are inadequate for
most studied kinetoplastids belong to the family Trypano- identifying the species, mainly due to the high variabil-
somatidae, which is characterised by a single flagellum ity of morphology and their sensitivity to environmen-
and a relatively small kinetoplast, resembling a part of a tal factors or culture conditions (Merzlyak et al. 2001).
single-branched mitochondrion of the cell containing a As stated by Yurchenko et al. (2006), the validity of
large mass of mitochondrial DNA (McGhee & Cosgrove most of the described trypanosomatid species is ques-
1980, Maslov et al. 1996, 2001, Merzlyak et al. 2001). tionable, with the notable exception of those of medical
Trypanosomatids of the genera Trypanosoma, En- and veterinary importance (Trypanosoma and Leishma-
dotrypanum and Leishmania are dixenous parasites of nia), which have been studied extensively. Moreover, as
vertebrates (Fernandes et al. 1993, Merzlyak et al. 2001, shown by recent molecular phylogenetic studies (Maslov
Podlipaev 2001). The remaining members of the family et al. 1996, Merzlyak et al. 2001, Podlipaev et al. 2004,
include monoxenous parasites of invertebrates (genera Simpson et al. 2006, Yurchenko et al. 2006), most mor-
Blastocrithidia, Crithidia, Leptomonas, Herpetomonas phological genera are polyphyletic.
and Wallaceina) and dixenous parasites of plants and in- One of the main concerns in the field of trypanoso-
sects (genus Phytomonas) (Dollet et al. 2000, Maslov et matid research is the scarcity of information about the
al. 2001, Podlipaev et al. 2004). Since the early 1960s, diversity of this group. It has been estimated that of the
life-cycle information combined with morphological approximately one million insect species described, only
features such as cell shape and dimension and the rela- ~2,500 have been examined for the potential presence
of trypanosomatids (Momen 2001, Podlipaev 2001). For
instance, of approximately 23,000 identified species of
Hemiptera, one of the most studied orders, only 500-600
have been examined for flagellate parasites (Teixeira et al.
Financial support: COLCIENCIAS (021-2005)
2000). Moreover, the current system does not take into ac-
+ Corresponding author: anamcaicedo@yahoo.com count genetic and evolutionary parameters; consequently,
Received 24 October 2010 a great number of synonyms or descriptions of organisms
Accepted 26 January 2011 of dubious origin have been generated (Podlipaev 2001).
online | memorias.ioc.fiocruz.br
302 Blastocrithidia in C. bergi • Ana Milena Caicedo et al.

To overcome the aforementioned taxonomic limi- ent series, the cells were embedded in resin. Thin ultra-
tations, biochemical, ultrastructural, serological and microtomed sections (0.7-0.8 µm) were stained with lead
nutritional approaches to discriminate and define taxa citrate and uranyl acetate and examined under a JEOL
were proposed 20 years ago (Wallace et al. 1983). The JEM 1010 microscope.
most recent characterisations and analyses of biodiver-
DNA extraction, polymerase chain reaction (PCR)
sity of new trypanosomatid species are based on the
amplification and sequencing - Pooled gut and hemo-
use of molecular methods and phylogenetic analysis of
lymph samples from five infected insects were homoge-
gene sequences such as spliced leader RNA (SL rRNA),
nised and the debris was removed by centrifugation.
5S rRNA and glycosomal glyceraldehyde phosphate
Total DNA was extracted according to Rotureau et al.
dehydrogenase, which have proven to be powerful for
(2005) and Westenberger et al. (2004).
discriminating among genera and species (Podlipaev
The SL rRNA gene repeats were amplified with
et al. 2004, Yurchenko et al. 2006, Maslov et al. 2007,
the primers ME-1 5’-TTCTGTACTTTATTGGTA and
Svobodová et al. 2007). Recently, small subunit (SSU)
ME-2 5’-CAATAAAGTACAGAAACTG (Podlipaev
rRNA gene-based phylogenies have been used to estab-
et al. 2004). The 5S rRNA gene repeats were amplified
lish major natural groups within a family (Merzlyak et
with the primers 5S-L 5’-CCGTCCGATTTGTGAAGT-
al. 2001). Although useful in individual cases, these new
TAAGC and 5S-R 5’-TAACTTCACAAATCGGACG-
criteria have not been applied uniformly (Podlipaev et
GGAT. Amplifications were done with Taq polymerase.
al. 2004, Yurchenko et al. 2006). Therefore, it is highly
The thermal cycling profile for both 5S rRNA and SL
advisable to use and compare at least two of these meth-
RNA genes consisted of the following steps: initial de-
ods when a new species is described.
naturation at 94ºC for 2 min, 40 cycles of denaturing at
In recent studies, we observed that Cyrtomenus bergi
94ºC for 30 s, annealing at 50ºC for 30 s and extension at
Froeschner, a polyphagous Cydnidae pest, is naturally
72ºC for 30 s, followed by a final extension at 72ºC for
infected with trypanosomatid flagellates in their salivary
12 min. SSU rRNA genes were amplified with the primers
glands, intestinal tract and hemolymph (AM Caicedo,
S-762 5’-GACTTTTGCTTCCTCTA(A/T)TG and S-763
unpublished observations). In this study, we report the
5’-CATATGCTTGTTTCAAGGAC (Maslov et al. 1996).
isolation of the trypanosomatid parasites from C. bergi
Taq DNA polymerase was used for amplification and ther-
as well as their morphological description and molecular
mal cycling conditions were as follows: initial denaturation
characterisation. Based on these analyses, this organism
at 95ºC for 5 min, 5 cycles at 95ºC for 1 min, annealing at
has been identified as a new species of Blastocrithidia.
45ºC for 30 s and extension at 65ºC for 1 min, 35 cycles at
Materials and Methods 95ºC for 1 min, 50ºC for 30 s and 72ºC for 1 min, and a
Maintenance of insect hosts - Approximately 5,000 final extension at 65ºC for 30 min (Maslov et al. 1996).
specimens of the burrowing bug C. bergi were collected in The amplified SL and 5S products were extracted
onion fields in Pereira (Risaralda, Colombia) from 2004- from agarose gel using a QIAquick gel extraction kit
2008. The insects were maintained in an insectarium (23 (QUIGEN, Valencia, CA) and cloned into the pGEM-T
± 2ºC, relative humidity 65 ± 5%, L12:D12) in sterilised Easy vector (Promega, Madison, WI). The clones were
loamy clay soil and sand, mixed in a 3:1 proportion, at a sequenced at a commercial facility (Macrogen, Korea).
moisture level approximating field conditions (33.5% gra- The SSU rRNA gene amplification product was gel-
vimetric soil water) and fed on sprouting maize kernels. purified by the same procedure and directly sequenced
using a set of conserved internal primers (Maslov et al.
Light microscopy - Hemolymph taken from fifth 1996) at the University of California-Riverside IIGB
instar nymphs and adults was re-suspended in phos- Core Instrumentation facility.
phate-buffered saline (PBS). Drops of the suspension The sequences were deposited in GenBankTM under
were smeared on slides, methanol dried and stained the following accessions: SL RNA gene and 5S rRNA
with Wright-Giemsa at room temperature (RT) for 24 h. gene repeat unit: FJ916991 and FJ916990 and SSU rRNA
Slides were examined at 100x with a Nikon Microphot. gene: FJ916992.
Measures and photographs of different morph types
Phylogenetic analyses - Prior to the analyses of SL
were taken using an NIS elements Nikon camera (v. 2.3).
RNA gene repeats, the regions corresponding to the am-
Wright-Giemsa-stained microscope slides, representing
plification primers and most of the hypervariable inter-
gut smears of the original host infected with the Try-
genic regions were excluded (Westenberger et al. 2004).
panosomatidae isolate and described herein as Blas-
The remaining ~150 nt, including the exon, the intron
tocrithidia cyrtomeni, were deposited with the Depart-
and a relatively conserved sequence at positions -100
ment of Microbiology of the Universidad del Valle, Cali
to -1 upstream of the exon, were aligned using CLUST-
(Valle de Cauca Province, Colombia).
ALX v. 1.81 (Thompson et al. 1997). Cluster analysis
Electron microscopy - Flagellates collected from the was performed using the neighbour-joining method and
mid and hindgut of C. bergi adults were centrifuged at Kimura 2-parameter distances and PAUP* 4.0, beta ver-
10,000 g, washed in 0.1 M PBS and fixed in 2.5% glutar- sion (Swofford 1998). The phylogenetic analyses of the
aldehyde in 5 mM HCl and 0.1 M cacodylate buffer, first SSU rRNA gene sequences included the most conserved
for 1 h at RT and then for at least 24 h at 4ºC. Samples regions, which were selected arbitrarily from the initial
were post-fixed in 2% osmium tetroxide in the same buf- multiple alignment generated by CLUSTAL. The best-
fer for 2 h at RT. After dehydration in an ethanol gradi- fitting model of the sequence evolution was selected us-
 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(3), May 2011 303

ing Modeltest, v. 3.06 (Posada & Crandall 1998), which Furthermore, the presence of flagellar cysts (strap-
corresponded to the general time-reversible model with hangers) was observed in Forms I and III, with groups of
a proportion of invariable sites I = 0.5029 and the gamma four-five cysts adhering to the middle of the flagellum
distribution shape parameter Г = 0.5416. The analyses (Figs 1A, 5). In addition, relatively small cells, appar-
involved maximum likelihood, distance and parsimony ently generated by unequal division, were also observed
implemented with PAUP* 4.0 (Swofford 1998). as flagellar attachments (Figs 1A, 5), suggesting that
they represent immature cysts. Cyst forms varied from
Results small and round, to bacillus-like cells, and to those with
Trypanosomatid infection (100% prevalence) was anterior pointed ends. In all forms, the kinetoplast was
detected in C. bergi adult populations collected in onion anterior to the nucleus. The width of the mature strap-
fields in La Florida, Pereira. However, no obvious mor- hangers ranged from 2.42-4.43 µm, the length ranged
phological or behavioural symptoms of infection were from 5.86-11.93 µm.
observed in individual hosts. Light microscopy revealed Transmission (TEM) and scanning (SEM) electron
trypanosomatids within the intestinal tract, hemolymph microscopy - The ultrastructure of longitudinal sec-
and Malpighian tubules. Attempts were made to estab- tions of epimastigotes isolated from midgut showed that
lish a stable culture starting from hemolymph samples in the flagellum arises laterally from a relatively shallow
several media, including NNN, LIT and Schneider, but flagellar pocket near the kinetoplast. The typical disk-
these were not successful. shaped kinetoplast was located next to the bottom of the
Light microscopy - Hemolymph and midgut smears flagellar pocket (Fig. 6). We observed the longitudinal
revealed epimastigotes of variable size with straphanger division of epimastigotes with kinetoplasts and bi-fla-
cysts adhering to the middle of the flagellum. The cells gellum (Fig. 7). Few promastigotes were seen with the
varied in size and shape, from slender to rounded forms flagellum arising in front of the kinetoplast in the midg-
ut (Fig. 8). Different forms of amastigotes with some
with short and long flagella. Some promastigotes could
acidocalcisomes and basophilic granules were also ob-
also be seen (Figs 1A, B, 8). The kinetoplast was always
served throughout the cell body (Fig. 9). The flagellum
observed anterior to the nucleus.
was supported by a nonprominent paraflagellar rod (Fig.
Three forms of epimastigotes were seen: Form I, with 10). The parasites were attached to the intestine by the
a short flagellum and rounded body (Fig. 2A, B), Form II, flagellum and different forms of parasites were observed
with a slender body, pointed ends, a short flagellum and (Fig. 11). In general, the nucleus was found mainly in
two to three spiral twists in the body (Fig. 3), and Form III, the middle or close to the posterior end of the cell body,
representing apparently dividing epimastigotes, with a sec- covering most of the cell width. The apparent absence of
ond long flagellum separated from the cell body (Fig. 4). an undulated membrane, one of the features of epimas-
The morphometry of the three main morph types tigotes, is noteworthy.
observed is summarised in Table I. The most noticeable
difference between Forms I and III was flagellum length,
with the latter being longer than the former. These forms
also showed at least two-three twists, and the posterior end
of Form I was more pointed than Form III (Figs 1A, 3, 4).

Figs 1-5: morphology of Blastocrithidia cyrtomeni sp. nov. from in-

fected hosts as revealed by light microscopy. 1A: different forms (small
body with straphangers with typical division forms); 1B: slender with
needle-like posterior end with short flagella; 2A: epi and promastigote Figs 6-11: transmission electronic microscopy of Blastocrithidia cyr-
forms (up); 2B: slender body, short flagellum, body with three spiral tomeni sp. nov. in midgut. 6: flagellar pocket (FP), kinetoplast (K) and
twists; 3: rounded forms with short flagellum; 4: groups of epimastig- flagellum (F) of epimastigote; 7: K and bi-F of epimastigote in division;
otes with straphangers at the middle of the flagellum; 5: a cell with a 8: FP, K and F of promastigote; 9: nucleus (N) and K of amastigotes; 10:
second flagellum, apparently during an early stage of cell division. longitudinal section of F; 11: different forms of amastigotes in midgut.
304 Blastocrithidia in C. bergi • Ana Milena Caicedo et al.

Using SEM, it was possible to observe numerous Yurchenko et al. 2006, 2008) (data not shown). In all
parasites adhered to the intestinal wall by their flagella analyses, the C. bergi parasites were found to be closely
(Fig. 12). Typical elongated epimastigotes with strap- associated with B. triatomae (the only available represen-
hanger cysts were also observed on the midgut of C. tative of the aforementioned clade), with 100% bootstrap
bergi adults (Fig. 13). support in all the methods used (Fig. 16).
Based on the presence of epimastigotes and flagel-
PCR amplification and phylogenetic analysis - DNA
lar cysts and on the molecular phylogenetic analyses,
was extracted from the midgut of C. bergi adults, fourth
we concluded that the parasite from C. bergi is a new
and fifth instar nymphs and eggs. PCR amplification of
trypanosomatid species that should be assigned to the
SL RNA gene repeats was positive in all cases, produc-
genus Blastocrithidia.
ing a 0.8 kb band corresponding to a monomeric repeat
unit, often accompanied by higher molecular weight Taxonomic summary - Class: Kinetoplastea Honig-
bands, possibly representing repeat dimers and trimers. berg 1963 emends. Vickerman 1976. Subclass: Metaki-
Similarly, PCR amplification with 5S rRNA primers re- netoplastina Vickerman 2004. Order: Trypanosomatida
sulted in a monomeric amplicon of the same size and Kent 1880 stat. nov. Hollande 1952. Family: Trypanoso-
higher molecular weight products that were apparently matidae Doflein 1951. B. cyrtomeni sp. nov. Caicedo, Gal-
oligomeric (Fig. 14). lego, Muñoz, Montoya, Carvajal & Maslov et al. 2009.
Cloning and sequencing of the SL RNA and 5S rRNA
Type host - Salivary glands, intestinal tract and hemo-
monomeric amplicons showed that they have the same re-
lymph of C. bergi Froeschner, 1960 (Hemiptera: Cydnidae).
peat unit, wherein individual SL RNA genes were inter-
spersed with 5S rRNA genes, forming a linkage arrange- Type locality - La Florida, Pereira, Risaralda Provin-
ment often observed in trypanosomatids (Westenberger et ce, Colombia (4º45’38.65’’N 75º36’57.49’’W).
al. 2004). Individual repeats, derived from more than 30 Type data - Microscope slides (MUV 001-006), des-
sequenced amplicons, were 797-803 bp long and > 98.5% ignated as hapantotype, were deposited in the type col-
identical to each other, indicating that they all originated lection of the School of Microbiology, Universidad del
from the same organism (Thomas et al. 2005). Valle, Cali, Colombia.
Seven individual SL repeat units were compared with
the database of the repeats available from other Trypano- Genus assignment - The presence of epimastigotes
somatidae (Maslov et al. 2007) using neighbour-joining and promastigotes defines the placement of the new spe-
cluster analysis. The trypanosomatid from C. bergi clus- cies in the genus Blastocrithidia Laird, 1959.
tered with members of the B. triatomae-Blastocrithidia
leptocoridis clade (not shown). A part of this analysis,
covering the Blastocrithidia group, is shown in Fig. 15.
The repeats from the C. bergi parasite are unique and
clearly distinct from B. triatomae (59.2% identity level)
and B. leptocoridis (55.8% identity). The C. bergi parasite
is most closely related to the organisms from Typing Unit
25, an unnamed trypanosomatid species found in two spe-
cies of Leptoscelis (Hemiptera: Coreidae) from Costa Rica
and Ecuador. However, the relatively low repeat identity
level (~63%) can clearly separate these two organisms.
The identification of the new parasite as Blastocrithidia
was verified by the analyses of SSU rRNA gene sequenc-
es and comparison with selected members of the major Figs 12-13: scanning electronic microscopy of Blastocrithidia cyrtome-
known phylogenetic clades of the Trypanosomatidae. The ni sp. nov. in midgut of Cyrtomenus bergi adults. Most elongated forms
general topology of the resulting trees was analogous to adhered by flagellum to the intestine wall (12). Epimastigotes form
those derived in previous studies (Merzlyak et al. 2001, with straphanger cysts and round body cell with long flagellum (13).

Table I
Morphometric analysis of Blastocrithidia cyrtomeni - midgut

Parasite

Length (µ) Width (µ) Posterior-nucleus (µ) Kinetoplast-nucleus (µ) Flagellum (µ)
Epimastigotes Form mean SE (n) mean SE (n) mean SE (n) mean SE (n) mean SE (n)

I 24.10 ± 8.24 (33) 4.24 ± 1.43 (33) 11.24 ± 6.28 (33) 4.37 ± 2.50 (33) 28.09 ± 13.18 (33)
II 53.55 ±12.92 (30) 3.62 ±0.64 (30) 20.70 ± 0.96 (30) 10.23 ± 3.60 (30) 36.33 ± 9.59 (30)
III 50.2 ±4.92 (31) 3.24 ±0.44 (31) 27.8 ± 4.63 (31) 10.07 ± 2.56 (31) 46.61 ± 9.23 (31)
Cyst and straphangers 9.36 ±1.5 (70) 3.50 ± 0.40 (70) - - -
 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(3), May 2011 305

Species characters - The species is mainly defined Remarks - Epimastigotes often display straphanger
by molecular phylogenetic criteria. The new species is cysts adhered to the middle of the flagellum in clusters
clearly distinguished from the other known trypanoso- of two-four. The species has proven to be refractory to
matids, including the closely related B. triatomae, by the cultivation in the media tested.
SL RNA - 5S rRNA gene repeat sequences (GenBankTM
Etymology - The species name is derived from the
accessions FJ916991, FJ916990).
insect host.
DISCUSSION
The new species of Blastocrithidia Laird, 1959,
named B. cyrtomeni sp. nov., was isolated from hemo-
lymph and the intestinal tract of C. bergi. Identification
of this trypanosomatid was based on morphological
descriptions using light and electron microscopic tech-
niques and supported by analyses of molecular markers
and phylogeny (Maslov et al. 1996, 2007, Merzlyak et al.
2001, Yurchenko et al. 2006).
The predominant form infecting fifth instar nymph
and adults of C. bergi was elongated epimastigotes with
straphanger cysts adhered to the middle of the flagellum.
This characteristic is mentioned by Wallace (1966) in
only five of thirty species of Blastocrithidia described:

Fig. 14: amplification of the 5S rRNA (A) and spliced leader RNA (B)
genes of Blastocrithidia cyrtomeni sp. nov. M: molecular marker
[1 kb plus DNA Ladder (Invitrogen)]; 1: midgut of female; 2: midgut
of female; 3: egg; 4: midgut of female; 5: midgut of the fifth instar
nymph; 6: midgut of fourth instar nymph; 7: in vitro.

Fig. 16: bootstrap majority consensus maximum likelihood small sub-

Fig. 15: neighbor-joining analyses of the conserved regions of the unit rRNA gene tree of the selected Trypanosomatidae with emphasis
spliced leader RNA gene repeats from Blastocrithidia cyrtomeni along on the position of Blastocrithidia cyrtomeni sp. nov. The tree (-Ln
with its closest relatives, which include Blastocrithidia triatomae, = 9403.73836) was inferred by assuming the general time-reversible
Blastocrithidia leptocoriodis and several unnamed Blastocorithidia model of sequence evolution with I = 0.5029 and Г= 0.5416. The boot-
spp represented by the respective Typing Units. The unit designations strap percent values shown were derived from 100 replicates with
(in black boxes) correspond to the trypanosomatid samples described maximum likelihood (1st value), 1,000 replicates with minimum evo-
by Maslov et al. (2007) and Westenberger et al. (2004). The tree is lution (2nd value) and 1,000 replicates with parsimony (3rd value).
unrooted. The bar represents 0.01 substitutions per site. Asterisks mean bootstrap values below 50%.
306 Blastocrithidia in C. bergi • Ana Milena Caicedo et al.

Blastocrithidia familiaris Gibbs (1950), B. leptocoridis Table II

McCulloch (1915), Blastocrithidia ortheae Uribe (1926), Dimensions of Blastocrithidia species
Blastocrithidia sandoni Gibbs (1951) and Blastocrithidia
euchisti Hanson and McGhee, 1961 (Cerisola et al. 1971). Body length Flagellum length
In B. triatomae, Cerisola et al. (1971) mentioned the Species (µ) (µ)
cysts as a remarkable feature, describing them as small
bodies of variable size hanging from the flagellum in Blastocrithidia gerridis 17.5 70 (48.6) 1.75-2.7 (2.3)
most specimens. Blastocrithidia gallardoi 30-38 39-90
As mentioned by Merzlyak et al. (2001), the origi- Blastocrithidia triatomae 14.8-32 (25) 19.5-28.5 (22.5)
nal identification of Blastocrithidia gerricola from the
gut of Gerris lacustris was based on the epimastigotes Blastocrithidia lituri, 20 -
observed with some rare promastigotes. This is also the Blastocrithidia nalipi,
Blastocrithidia spinigeri,
case with B. cyrtomeni, with epimastigotes being the
Blastocrithidia vacuolata
predominant forms and few promastigotes observed.
We did not observe the “cytoplasmatic bridge” men- Blastocrithidia apiomerusi 34 20
tioned by Schaub and Böker (1986) in their microscopic Blastocrithidia cyrtomeni 53.5 ± 12.9 36.3 ± 9.5
studies of B. triatomae, nor were we able to find cysts
connected to the posterior end of epimastigotes as re- source: Cerisola et al. (1971).
ported by the authors in three cases.
The posterior end of members of the Blastocrithidia
species is a variable feature. In B. cyrtomeni, it was ob- level. We found a special case of direct transmission
served as needle-like in light SEM and TEM images (Figs (transovarial), in which the infection is transferred by
1B, 13). In contrast, Schaub and Böker (1986) observed a parent to its unborn offspring. Therefore, despite the
that the broad posterior end was rolled up in a spiral in fact that B. cyrtomeni has an extremely high rate of di-
the first SEM images of B. triatomae and B. gerridis. rect reproduction within the host, it is unlikely that this
B. cyrtomeni has nearly the same body length as B. microparasite is a pathogen.
gerridis (Wallace 1966) and is almost double the average Using PCR amplification of SL rRNA genes from 16
size of B. triatomae (Cerisola et al. 1971). The flagel- samples of parasites isolated from the intestinal tract and
lum length is the most noticeable difference among these eggs of C. bergi, it was possible to identify B. cyrtomeni
three species; it is shortest in B. gerridis and longest in sp. nov. using the approach proposed by Maslov et al.
B. cyrtomeni (Table II). (2007), who established that a single repeat of SL is suf-
Common features among these three Blastocrithidia ficient to serve as a marker for a natural clone or species
species are the position of the kinetoplast, which is ante- of trypanosomatid and to distinguish among closely re-
rior to the nucleus, and the width of the nucleus, which lated trypanosomatids.
is almost the same as that of the body (Wallace 1966, It is critical to establish the presence and prevalence
Cerisola et al. 1971). of these monoxenous trypanosomatids in other insect
An undulated membrane in the epimastigotes was species and to evaluate in greater detail whether the
not observed. Thus far, only small forms of B. leptotry- association between insect pests and trypanosomatids
panoides (Hollande 1922) have been reported without an could be regulating the action of microorganisms used
undulating membrane (Wallace 1966). for biological control.
In Leptomonas wallacei, a trypanosomatid from the ACKNOWLEDGEMENTS
lygaeid bug Oncopeltus fasciatus, straphanger cysts ad- To Manuel Castrillón, for measuring the parasites, to Meleny
here to the beginning of the base of the flagellar pocket Ramírez, for helping to obtain permanent slides, to Efren, for
(Romeiro et al. 2001). The dimensions of the straphang- transmission microscopy pictures, to José Arroyabe, for scan-
ers of B. cyrtomeni are almost three times larger in ning microscopy pictures, to Nydia Betancourth, for helping with
length and width (9.36 and 3.50 µm, respectively), than the figures, and to Dra Tina Trenczek and Dr Gustavo Vallejo,
those of B. triatomae (3.4 and 1.9 µm, respectively) (Ce- for the first instructions at the beginning of the research.
risola et al. 1971). REFERENCES
Although we observed oval cells of B. cyrtomeni ad-
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