Phylogenetic and morphological characterization of trypanosomes from Brazilian armoured

17 Pages·2015·3.6 MB·English

by  Moara Lemos

Lemosetal.Parasites&Vectors (2015) 8:573
DOI10.1186/s13071-015-1193-7
RESEARCH Open Access
Phylogenetic and morphological
characterization of trypanosomes from
Brazilian armoured catfishes and leeches
reveal high species diversity, mixed
infections and a new fish trypanosome
species
Moara Lemos1,6, Bruno R. Fermino2, Cíntia Simas-Rodrigues3, Luísa Hoffmann4,5, Rosane Silva4,5, Erney P. Camargo2,
Marta M. G. Teixeira2 and Thaïs Souto-Padrón1,6*
Abstract
Background: SeveralTrypanosomaspeciestransmittedbyleechesinfectmarineandfreshwaterfishworldwide.Todate,
allSouthAmericanfishtrypanosomespeciesidentifiedhavebeenbasedonunreliablemorphologicalparameters.We
recentlyisolatedandculturedtrypanosomesfromtheBrazilianarmouredcatfishesHypostomusluetkeniandH.affinis.
Here,wereportthefirstphylogeneticanalysesofSouthAmerican(Brazilian)trypanosomesisolatedfromfish,andfrom
leechesremovedfromthesefish.Wealsoanalysedmorphologicallyandmorphometricallythedifferentformsoffish,
leechandculturedtrypanosomes.
Methods:V7V8SSUrRNAandgGAPDHsequenceswereusedforphylogeneticanalysisofBrazilianfishandleech
trypanosomes.Trypanosomesfromcultures,fishbloodandleechsampleswerealsocharacterizedmorphologicallyand
morphometricallybylightandelectronmicroscopy.
Results:Inbloodsmearsfromfishhightrypanosomeprevalence(90–100%)andparasitemia(0.9-1.0x102)wereobserved.
PhylogeneticrelationshipsusingSSUrRNAandgGAPDHshowedthat,despiterelevantsequencedivergence,allBrazilian
fish(andderivedcultures)andleechtrypanosomesclusteredtogetherintoasingleclade.TheBraziliancladeclustered
withEuropean,NorthAmericanandAfricanfishtrypanosomes.Basedonsequenceanalysis,weuncoveredanewspecies
ofBrazilianfishtrypanosome,Trypanosomaabelin.sp.Trypanosomaabeliculturescontainedpleomorphicepimastigotes,
smalltrypomastigotesandraresphaeromastigotes.UltrastructuralfeaturesofT.abeliincludedacytostome-cytopharynx
complexinepi-andtrypomastigotes,acompactrod-likekinetoplast,lysosome-relatedorganelles(LROs)and
multivesicularbodies.Trypanosomesfoundinfishbloodsmearsandleechsampleswerehighlypleomorphic,in
agreementwithsequencedatasuggestingthatcatfishesandleechesoftenhavemixedtrypanosomeinfections.
(Continuedonnextpage)
*Correspondence:[email protected]
1DepartamentodeMicrobiologiaGeral,InstitutodeMicrobiologiaPaulode
Góes,CentrodeCiênciasdaSaúde,UniversidadeFederaldoRiodeJaneiro,
BlocoI,sala019.Av.CarlosChagasFilho,373,IlhadoFundão,Riodejaneiro
21941-902,Brazil
6InstitutoNacionaldeCiênciaeTecnologiaemBiologiaEstruturale
Bioimagens,CentrodeCiênciasdaSaúde,blocoI,RiodeJaneiro,Brazil
Fulllistofauthorinformationisavailableattheendofthearticle
©2015Lemosetal.OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0
InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinkto
theCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver
(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Lemosetal.Parasites&Vectors (2015) 8:573 Page2of17
(Continuedfrompreviouspage)
Conclusions:Trypanosomaabelin.sp.is the first trypanosome from South American fishes isolated in culture,
positioned in phylogenetic trees and characterized at the ultrastructural level. Trypanosoma abeli n. sp. is
highly prevalent in H. luetkeni and H. affinis armoured catfish from the Atlantic Forest biome, and in other
catfish species from the Amazon and the Pantanal. Sequencing data suggested that Brazilian catfish often
have mixed trypanosome infections, highlighting the importance of molecular characterization to identify
trypanosome species in fishes and leeches.
Keywords: Trypanosoma, Fish, Catfish, Leech, Culture, Phylogeny, Taxonomy, Morphology, Ultrastructure,
Mixed infections
Background Giemsa-stained bloodstream trypomastigote forms of fish
Trypanosomes are widespread and highly prevalent in trypanosomes [24–29],comparatively lessisknown about
freshwater and marine fishes, including teleost and elas- theirultrastructure,sinceonlyafewspecieswereanalysed
mobranch species. Since the initial report of trypano- atthislevel,eitherinculture[19,30],orinvivo,infish[5,
somes in the blood of trouts from the species Salmo 31,32]andleechsamples[33,34].
fario [1], more than 200 species of fish trypanosomes Nevertheless, all descriptions of Brazilian fish trypano-
have been identified under the taxonomic criteria of some species available to date were based exclusively on
morphology, and geographical and host origin [2, 3]. Al- morphologyandhosttaxonomycriteria[3,12,14,35–37].
though most infected fish are asymptomatic, fish tryp- Thesecriteriaareinsufficientforspeciesidentification,be-
anosomiasis can be severe at high parasitemias, and cause some species are morphologically indistinguishable
symptoms include anaemia, leukocytosis, hypoglycemia (aside from intra-specific pleomorphism), and not host-
and splenomegaly [4, 5]. Aquatic leeches are both hosts specific, with a high frequency of mixed infections
and vectors of fish trypanosomes [6, 7]. Despite the re- detected in molecular studies [11, 38–41]. Barcoding by
ports of trypanosomes infecting leech-parasitizing fishes variable SSU rRNA sequences is capable of distinguishing
[7–10], host-vector relationships were only recently new trypanosome species/genotypes from previously
demonstrated by molecular comparison of trypanosomes knownones[11,41–47].
from fishandleechesremovedfrom thesame fish[11]. The number of phylogenetic studies of fish trypano-
In Brazil, more than 60 species of trypanosomes somes has increased in recent years, and these studies
were recorded in marine and freshwater fishes [3] in- have focused on trypanosomes removed directly from
cluding loricariid (catfish) species such as Trypano- the blood of European [38, 41, 48, 49], African [11, 39]
soma hypostomi [12], Trypanosoma chagasi and and Asian [5, 40, 50] marine and freshwater fishes.
Trypanosoma guaibensis [13] and Trypanosoma lopesi These trypanosomes segregate, in general, in two groups
[14]. Catfishes belong to the order Siluriformes, which within a main clade [5, 11, 38, 40, 41, 49, 50]. However,
comprises more than ~3,093 species of freshwater molecular phylogenetic analysis currently adopted for
and salt water fishes inhabiting every continent except the description of trypanosome species is still lacking for
Antarctica, although more than 50 % of all catfish Brazilian fish trypanosomes. These limitations render
species, including all members of Loricariidae (>680 necessary a molecular revision of the profusion of fish
species), live in the tropical Americas. Loricariidae trypanosome species, including all of those described in
species vary largely in size, inhabit almost all fresh- Brazil beforethisstudy.
water habitats and are mostly bottom feeders. The In this study, specimens of the ornamental armoured
genus Hypostomus is native to South America and catfishes H. luetkeni and H. affinis were captured in The
comprises of large armoured catfish species used ex- Atlantic Forest biome of Southeast Brazil and examined
tensively as ornamental fish and food [15–17]. to assess trypanosome prevalence and parasitemia. We
After the initialdescription offishtrypanosome cultiva- used phylogenetic analysis and both light and electron
tion by Thomson [18], several authors reported culturing microscopy to characterize trypanosomes from blood
ofavariety oftrypanosomesfromthe bloodof freshwater samples, cultures and leeches removed from catfish.
and marine fish species from Europe, North America and Also, the phylogenetic analysis included novel data on
Africa [7, 18–22]. However, only recently trypanosomes additional trypanosome isolates from other loricariid
fromSouthAmerican(Brazilian)fish–thecatfishH.luet- fishes captured in northeast (Amazonia) and central
keni and H. affinis - were established in culture [23], des- (Pantanal) Brazil, aiming to assess the genetic diversity.
pite the numerous reports of fish trypanosomes in Brazil. By integrating the phylogeny of fish trypanosomes with
While there are many species descriptions based on the morphological and ultrastructural features of
Lemosetal.Parasites&Vectors (2015) 8:573 Page3of17
cultured parasites, our findings enabled the description V7V8regionofSSUrRNAgeneswasperformedusingthe
of Trypanosoma abeli n. sp. of Brazilian armoured primers 609 F (5’CACCCGCGGTAATTCCAGC3’) and
catfishes. 706R (5’TCTGAGACTGTAACCTCAA3’), and the
primers GAP3F (5’GTGAAGGCGCAGCGCAAC3’) and
Methods GAP5R (5’CCGAGGATGYCCTTCATG3’) were used for
Fishandleechcollection,studiedarea,parasitemiaand the amplification of gGAPDH sequences. The PCR reac-
trypanosomeprevalence tion conditions employed for the two sequences were de-
Armoured catfish, identified as Hypostomus affinis and tailed previously [45]. Amplified DNA fragments were
Hypostomusluetkeni,werecapturedinthecityofGuarani, cloned in pGEM-T-easy, and 3–5 clones were sequenced
state of Minas Gerais, Brazil (21°21’S, 43°02’W), in the for each sample, except for culture L4100, for which an
banks of Pomba River, a 300-km long affluent of the additional 15 clones were sequenced, aiming at detecting
Paraiba do Sul River basin that extendsthroughthe States mixed trypanosome infections. Sequences were deposited
of São Paulo, Minas Gerais and Rio de Janeiro, in the in GenBank under the accession numbers shown in
AtlanticForestbiome.Fishcapturewasperformedaccord- Table 1. For comparative purposes, DNA from blood try-
ing to procedures D-075 of the National Forests Institute panosomes of loricariid fishes captured in the Amazonia
(IEF)andN°24402–1oftheBrazilianInstituteofEnviron- and the Pantanal biomes (Fermino et al., in preparation)
ment and Renewable Natural Resources (IBAMA). If wereincludedinthisstudy(Table1).
present, leeches were removed manually from the body Trypanosome SSU rRNA and gGAPDH gene se-
surfaceandtheoralandbranchialcavitiesofcapturedfish. quences determined in this study were aligned with
Leechesweremaceratedandsectionedinlongitudinaland those from several fish trypanosomes available in Gen-
transversaldirectionsfortrypanosomedetection.Forblood Bank. Two alignments were created: One comprising
sample collection, fish were anaesthetized and blood was SSU rRNA sequences (800~bp of variableV7V8 region
collected by cardiac puncture, and blood from the heart, of SSU rRNA) from Brazilian fish trypanosomes in cul-
liver and kidney were used for smears. Only blood from tures and trypanosomes from leeches, aligned with se-
cardiac puncture was used for trypanosome hemoculture, quences from freshwater and marine fish trypanosomes
performedasdescribedpreviously[23]. fromEurope,AfricaandAsia;trypanosomesfromcroco-
Trypanosome prevalence was determined by light mi- dilians, lizards and snakes were used as outgroup. The
croscopy examination of stained blood smears, and para- second alignment was composed of gGAPDH sequences
sitaemia was determined by direct counting of (~600 bp) of fish trypanosomes obtained from cultures,
trypanosome cells in fish blood using a hemocytometer. blood samples and leeches taken from captured fishes.
Samples of leeches macerated in sterile PBS were exam- This alignment includes GenBank sequences from a var-
ined by light microscopy for the presence of trypano- iety of ‘Aquatic clade’ trypanosomes (found in fishes and
somes. Fish blood and leech samples were processed in other aquatic and semi-aquatic vertebrates), as well as
immediatelyforelectronmicroscopy(seeSEMandTEM). those from trypanosomes outside the Aquatic clade, and
other non-trypanosome trypanosomatids, which were
Hemoculturesoffishtrypanosomes used as outgroups. The complete list of samples, host
Here, we used 16 cultured fish trypanosome isolates and geographic origin, and Genbank accession numbers
whose in vitro cultivation was first described by Lemos of all sequences determined in this work are shown in
& Souto-Padrón [23]. Isolates were cultured in three dif- Table1.Phylogenetic analysisofthenewsequences(bar-
ferent biphasic media: a) Ponsele medium overlaid with codes) were done by maximum parsimony using V7V8
Eagle’s Basal Medium (BME) (CULTILAB, Brazil) di- SSU rRNA sequences (dendrogram of Aquatic clade try-
luted to different concentrations; b) blood agar base panosomes), and maximum likelihood (ML), maximum
(BAB) (HIMEDIA, Brazil) overlaid with BME diluted to parsimony (MP) and Bayesian inference (BI) for infer-
50 %, and c) NNN blood agar base overlaid with Fish ences of phylogenetic trees based on gGAPDH se-
ringer’s medium. All media were supplemented with quences,asdescribedpreviously [43,44,46,47].
10 % heat-inactivated fetal calf serum (FCS) and cultures
were maintained at22°C. Lightmicroscopyandmorphometry
Smears of fish blood samples, leech gut contents and
DNAextraction,PCRamplification,sequencingand cultures were fixed with methanol, stained with Giemsa,
phylogeneticanalysis and examined using aZeiss Axioplan IIlight microscope
DNA was extracted from trypanosome cultures in the equipped with a Color View XS digital camera. Morph-
exponential phase of growth using phenol-chloroform, ometry analysis of the different developmental forms
and from fish blood andleech samples usingthe method was performed using the AnalySIS® soft, using images of
describedbyFerminoetal.[44].PCRamplificationofthe 80 bloodstream trypomastigotes (20 images per
Lemosetal.Parasites&Vectors (2015) 8:573 Page4of17
Table1Host,geographicorigin,andSSUrRNAandgGAPDHsequencesoffishandleechtrypanosomeisolates
Isolate Host City/Coordenates SSUrRNA gGAPDH Organism
Cultures
H27100a H.affinis RioPomba-MG/21°27’S43°18’W KR052821 T.abeli
L450 H.luetkeni RioPomba-MG/21°27’S43°18’W KR048309 T.abeli
L460 H.luetkeni RioPomba-MG/21°27’S43°18’W KR048306 T.sp.
L4100 H.luetkeni RioPomba-MG/21°27’S43°18’W KR048310 KR048292 T.abeli
L6NBA H.luetkeni RioPomba-MG/21°27’S43°18’W KR048307 T.abeli
L7fisha H.luetkeni RioPomba-MG/21°27’S43°18’W KR048308 T.abeli
P130 H.affinis RioPomba-MG/21°27’S43°18’W KR048293 T.abeli
P1100 H.affinis RioPomba-MG/21°27’S43°18’W KR048294 T.abeli
P250 H.affinis RioPomba-MG/21°27’S43°18’W KR048295 T.abeli
P350 H.affinis RioPomba-MG/21°27’S43°18’W KR048296 T.abeli
P3100 H.affinis RioPomba-MG/21°27’S43°18’W KR048297 T.abeli
P450 H.affinis RioPomba-MG/21°27’S43°18’W KR048298 T.abeli
P4100 H.affinis RioPomba-MG/21°27’S43°18’W KR048299 T.abeli
P560 H.affinis RioPomba-MG/21°27’S43°18’W KR048300 T.abeli
P5100 H.affinis RioPomba-MG/21°27’S43°18’W KR048301 T.abeli
PO8R H.luetkeni RioPomba-MG/21°27’S43°18’W KR048302 T.abeli
Blood
BSC100 L.anisitsi Miranda-MS/19°57’S57°011W KR048304 KR048287 T.sp.
CRCPE03 H.affinis C.doRioClaro-MG/20°98’S46°11’W KR048305 T.sp.
H27ab H.affinis RioPomba-MG/21°27’S43°18’W KR048291 T.sp.
L7ab H.luetkeni RioPomba-MG/21°27’S43°18’W KR048290 T.sp.
BSC451 Ancistrussp. AmazonBasin KR048303 T.abeli
Leech
SSH2b Haementeriasp RioPomba-MG/21°27’S43°18’W KR052820 T.sp.
TSC11 Haementeriasp Miranda-MS/19°57’S57°011W KR048288 T.sp.
TSC13 Haementeriasp Miranda-MS/19°57’S57°011W KR048289 T.sp.
aSequencesobtainedfromculturesandbloodsamples
bmixedinfections
developmental form). Data were analysed by descriptive ScanningElectronMicroscopy(SEM)andTransmission
statistics and Kruskal-Wallis variance analysis (p<0.05 ElectronMicroscopy(TEM)
wasconsideredstatisticallysignificant,usingtheBioEstat Blood and axenic trypanosome cultures were fixed in
software,version 5.0). 2.5 % glutaraldehyde in 0.1-M cacodylate buffer (pH 7.2)
containing 5 mM calcium chloride and 3.7 % sucrose.
For SEM, cells were washed and adhered to glass cover-
Fluorescencemicroscopy slips previously coated with 0.1 % of poly-L-lysine and
Trypanosomes in the logarithmic phase of growth were processed (dehydrated, critical point dried and coated),
washed in BME at 4 °C and adhered to glass coverslips according Lemos et al. [51]. Leeches were sectioned in
coated with 0.1 % of poly-L-lysine in PBS (pH 7.2), for the longitudinal and transverse directions, fixed and crit-
20 min. Then, cells were washed, fixed in methanol at ical point dried for SEM (as described above), adhered
20 °C, and incubated at 22 °C with 1 μg/mL Hoechst toglasscoverslips usingadhesivetape, andcoated witha
H33342 (Molecular Probes) in BME, for 15 min. Cover- 20-nm-thick gold layer, using a Bal-Tec CPD030 sput-
slips were mounted using 0.2 M N-propyl-gallate tering device. SEM samples were examined in a
(Sigma, USA) in glycerol and 0.01 M PBS (pH 7.2), and QUANTA 250 (Fei Company) Scanning Electron
analyzed by epifluorescence microscopy using a Zeiss Microscope operated at 15 kV.
Axioplan II light microscope equipped with a Color For TEM, flagellates from blood samples, cultures and
ViewXS digitalcamera. leeches were fixed as described above and post-fixed for
Lemosetal.Parasites&Vectors (2015) 8:573 Page5of17
30 min (in the dark) in 1 % osmium tetroxide (OsO ), mouth and, occasionally, to the pectoral fins of
4
0.8 % potassium ferrocyanide and 5 mM calcium chlor- armoured catfish, at all seasons. The prevalence of try-
ide, in 0.1 M cacodylate buffer (pH 7.3). Then, samples panosomes in leeches was 81 % and 75 %, for leeches
were washed, dehydrated in a series of acetone solutions taken from H. affinis and H. luetkeni, respectively. The
of ascending concentrations, and embedded in Polybed numberoftrypanosomesinfectingleecheswashigh,ran-
812 resin. Ultrathin sections were stained with uranyl ging from1.6to1.1×104parasites/mlofleechmacerate.
acetate and lead citrate, and observed in a FEI Morgagni Our data showing high trypanosome prevalence in
F268 Transmission Electron Microscope (Eindhoven, leeches infecting fish is in line with that reported in pre-
TheNetherlands),operatingat80kV. vious studies: 61 % of Actinobdella inequiannulata
leeches were infected by T. catostomi [57] and 60–100 %
Results and discussion of Johanssonia arctica leeches were infected with T.
Prevalenceoftrypanosomesinarmouredcatfishesandin murmanensis [58]. Importantly, these leech species were
leechesremovedfromthesefishes capable of transmitting trypanosomes to fish, by feeding
In this study, Giemsa-stained blood smears from 40 [10]. High prevalence was also reported in the leech Z.
specimens of H. affinis were all positive for trypano- arugamensis, considered the vector of Trypanosoma
somes by microscopy, yielding 100 % prevalence, with nudigobii [11]. In Brazil, a single report related the pres-
an average of 0.9×102 parasites/ml. The prevalence of ence of trypanosomes in freshwater leeches (from the
trypanosomes in 10 specimens of H. luetkeni was 90 %, species Batracobdella gemmata) removed from H. punc-
and the average parasitaemia was 1×102 parasites/ml. tatus catfish [59]; however, no prevalence data was re-
Similar levels of parasitaemia were detected in blood portedinthat study.
samples collected from the heart, liver and kidneys, for
both fish species (Additional file 1). The fish trypano- Cultureoffishtrypanosomes
some T. cobitis concentrates in the visceral circulation, The isolation and in vitro maintenance of fish trypano-
mainly of the kidney of the hosts Cottus gobio, Phoxinus someshasbeenachallengesincetheinitialdescriptionof
phoxinus, Nemacheilus barbatulus and Gobio gobio [9], T. danilewskyi isolation [18]. Recently, we isolated try-
whileT.mioxocephaliconcentratesintheheartofMyox- panosomes from Brazilian fish for the first time, success-
ocephalus octodecimspinosus [52]. These studies sug- fully culturing these parasites in haemocultures [23]. In
gested that blood samples obtained from the heart and the present study we analysed 16 of these culture isolates
kidneys may allow improved sensitivity for the detection obtainedfrom6H.affinisand4H.luetkenispecimens.
of fish trypanosomes, since these may be a preferred in-
fectionsitefor sometrypanosomes. BarcodingofBrazilianfishandleechtrypanosomesand
The first reports of fish trypanosomes in Brazil date phylogeneticrelationships
from the early 20th century, and surveys performed in Most molecular and phylogenetic studies of fish trypano-
severalregionsdescribed diversetrypanosomes in catfish somes relied on SSU rRNA sequences, and show a major
[35, 53, 54]. Splendore [12] was the first to provide data clade harbouring all fish trypanosomes, divided in two
on the prevalence (9.5 %) of Trypanosoma hypostomi in cladesofspeciesfrommarineorfreshwaterfishes.Thepo-
blood smears from Hypostomus aurogutatus caught in sitioning of trypanosomes from turtles and platypuses was
theTietê River (SP). Bara and Serra-Freire [13] reported unresolved,buttheyalwaysclusteredcloselyrelatedtofish
high prevalences of Trypanosoma chagasi (95 %) and trypanosomesinthephylogenetictrees[5,11,38–41,50].
low of Trypanosoma guaibensis (7 %) in Hypostomus In the present study, PCR-amplified trypanosome SSU
punctatus. Several armoured fish species captured in the rRNA and gGAPDH sequences obtained directly from
hydrological basin of the Guamá River (in the state of fish blood samples had to be cloned before sequencing
Pará, in Northern Brazil) showed variable prevalence of duetothepresenceofmorethanonesequencepersam-
trypanosome infection; the prevalence in Hypostomus sp. ple, indicating that the Brazilian armoured catfishes ex-
was of 20 %, and these fish showed the highest parasit- amined in the present study had mixed trypanosome
aemia and haematological changes, but no evidence of infections (Table 1). Although other trypanosomespecies
disease [55]. Here, despite relevant parasitaemia, no were identified by sequence analyses, the lack at this mo-
macro pathological signs were observed in H. affinis and ment of appropriate type-materials prevented their de-
H.luetkeni infectedwith trypanosomes. scription as additional species. According to the V7V8
The leeches removed from H. affinis and H. luetkeni SSU rRNA analysis, sequences from Brazilian fish and
were identified morphologically as Haementeria sp. and leech trypanosomes examined in this study clustered to-
molecularly as Haementeria brasiliensis [56]. On aver- gether in a clade with small heterogeneity (average of
age, two and nine leeches per fish (for H. affinis and H. ~0.8 %); however, relevant sequence divergences (up to
luetkeni, respectively) were found attached to the skin, 3.8 %) separated this clade from available sequences from
Lemosetal.Parasites&Vectors (2015) 8:573 Page6of17
European, Asian and African fish trypanosomes Aside from yielding highly consistent alignments,
(Fig. 1). The closest relatives of the Brazilian fish gGAPDH sequences are much more polymorphic than
trypanosome was T. sp. K&A [60] from the European SSU rRNA sequences for most trypanosome clades,
freshwater leech Piscicola geometra, and a trypano- resulting in phylogenies with considerably improved
some from European percae (Perca fluviatilis) [41] resolution [43, 44, 46, 47, 61]. Indeed, our analysis
(Fig. 1). Sequences of trypanosomes from diverse fish showed an average of 3.8 % gGAPDH sequence diver-
orders and families, including catfishes from other gence within the clade formed by the Brazilian trypano-
parts of the world, did not cluster with those from somes of fishes and leeches, compared with ~0.8 % of
Brazilian loricariid catfish, or from leeches that were divergence for V7V8 SSU rRNA sequences. The phylo-
attached to these fish (Fig. 1). Other catfish trypano- genetic inferences based on gGAPDH strongly sup-
somes included in the phylogenetic analysis were iso- ported the positioning of the trypanosomes found in
lated from widespread species of Clariidae (Africa), Brazilian Hypostomus catfishes, as well as of the whole
Siluridae (Europa) and Bagridae (Asia) [38, 39, 41]. assemblageoffishtrypanosomes (Fig. 2).
Fig.1Barcodingandphylogeneticrelationshipsoffishtrypanosomes.PhylogeneticanalysisinferredbymaximumparsimonybasedonV7V8SSU
rRNAsequences(barcodes)offishtrypanosomesfromBrazilianarmouredcatfish(Loricariidae),orfromleechestakenfromthesefishes.Notethe
inclusionofanewspecies,Trypanosomaabelin.sp.establishedinculture.Freshwaterandmarinefishtrypanosomesfromdifferentcontinents
wereincludedintheanalysis,aswellasothertrypanosomespeciesoftheAquaticclade.Numbersatnodesarebootstrapvaluesderivedfrom
100replicates.GenBankaccessionnumbersofSSUrRNAsequencesdeterminedinthisstudyarelistedinTable1.Codesinparenthesisare
GenBankaccessionnumbersavailableforpreviouslydescribedspecies
Lemosetal.Parasites&Vectors (2015) 8:573 Page7of17
Fig.2PhylogenetictreeoffishtrypanosomeswiththepositioningofTrypanosomaabelin.sp.:Maximumlikelihoodphylogenetictreeinferredfrom
gGAPDHsequencesofTrypanosomaabeli(cultures)andothertrypanosomesfromBrazilianloricariidsandleeches,includingsequencesfromEuropean,
AfricanandAsianfishtrypanosomes,aswellasturtleandplatypustrypanosomesplacedwithintheAquaticclade.Sequencesfromothertrypanosomes
andnon-trypanosometrypanosomatidswereusedasoutgroupsinthephylogenetictree(−Ln=−7078.905197).Numbersonbranchesrepresent
bootstrapsupport(>50)estimatedwith500pseudoreplicatesinRAxML,usingGTRGAMMA.GenBankaccessionnumbersofgGAPDHsequences
determinedinthisstudyarelistedinTable1.CodesinparenthesisareGenBankaccessionnumbersavailableforpreviouslydescribedspecies
To better assess the genetic diversity, we compared identified bysequencingalarger number ofclonedPCR-
gGAPDH sequences obtained from blood samples and amplified DNA sequences obtained from blood samples
cultures of trypanosomes from Brazilian catfishes and ofeachfishspecimen.
leeches removed from these fishes. We detected se- All trypanosome gGAPDH sequences from Brazilian
quence polymorphisms among different blood samples armoured catfish obtained in this study clustered to-
from the same fish specimen (not shown), but also be- gether in phylogenetic inferences (Fig. 2), supporting the
tween samples from fishes and their respective leeches, existence of a separate clade exclusive for trypanosomes
and between samples from different specimens of the from Brazilian catfishes and from leeches taken from
same fish species. Importantly, blood samples frequently these fishes. The clade of Brazilian catfish trypanosomes
harboured different sequences, indicating mixed tryp- was nested into the “Aquatic clade” composed by try-
anosome infections, in contrast to cultured isolates, panosomes from anurans, fishes, turtles, platypuses and
from which, in general, only one sequence was recov- aquatic leeches [38, 61–63]. In agreement with previous
ered, likely due to selection by culturing (Table 1). It ap- phylogenetic studies [38, 39, 41], all fish trypanosomes
pears likely that more mixed samples would be nested into a single clade that also harboured
Lemosetal.Parasites&Vectors (2015) 8:573 Page8of17
trypanosomes from turtles and platypuses; this complex In the absence of cultures, new fish trypanosome spe-
branching pattern needs the inclusion of more trypano- cies have been described based on the combination of
somesfor improvedresolution. morphological and molecular phylogenetic data from
In our study, no sequence from trypanosomes found blood flagellates [5, 11, 40, 50]. Our finding that fish try-
in leeches completely matched those found in the corre- panosomes often harbour mixed infections, combined
sponding fish blood samples. In contrast, a recent study with similar data from previous studies [40, 41], suggests
reported identical SSU rRNA sequences for trypano- that this approach requires a careful assessment to ex-
somes in marine fishes and leeches taken from the same clude the possibility of mixed infections, which preclude
fishes, confirming host-vector relationships [11]. Our the unambiguous association between trypanosome se-
findings support the existence of a large repertoire of quences and parasite morphotypes found in fish blood
trypanosome species and genotypes infecting Brazilian andleechsamples.
catfishes and leeches, including relevant divergences dis-
tinguishing trypanosomes found in sympatric Hyposto- Morphologicalanddevelopmentalanalysesof
mus fishes and within the same fish specimens. Trypanosomaabelin.sp.incultures
Therefore, results from the present study reinforce the In primary hemocultures, bloodstream trypomastigotes
need for molecular characterization using sensitive ap- (Fig. 3, a and b) transformed into short trypomastigotes
proaches in order to evaluate species repertoire before (Fig. 3c) and, within two days, into epimastigotes
ascertaining any parasite-vector association between (Fig. 3d).Establishedcultures inthelogarithmic phaseof
trypanosomespeciesinfectingthesehosts. growth consisted mostly of epimastigotes that ranged in
Taken together, our phylogenetic analyses using V7V8 shapeandsizefromsmallandoften“pear-shaped”(Fig.3
SSU rRNA and gGAPDH sequences support the iden- d and e) to elongated and slender (Fig. 3f), with a long
tification of a clade of new trypanosome species from flagellum, a rod-like kinetoplast and an oval nucleus.
Brazilian loricariid fishes differing from previously Small (Fig. 3c) and elongated (Fig. 3g) trypomastigotes,
molecularly characterized fish trypanosomes, including as well as spheromastigotes with a round kinetoplast
those from other catfish species (Figs. 1 and 2). This and a short free flagellum (Fig. 3, h and i), appeared in
clade may be associated with the evolutionary history stationary-phase cultures. We also observed dividing
of the Loricariidae restricted to Central and South epimastigotes (Fig. 3, j-m) and trypomastigotes (Fig. 3,
America [15, 16]. In contrast, phylogenetic studies of n-t). In dividing trypomastigotes that had not reached
fish trypanosomes from Europe, Africa and Asia did the stage of cytokinesis, the two kinetoplasts located
not support geographical structure or host-restrictions considerably posterior to the nuclei (Fig. 3, p and q). In
at species, genus or family levels [11, 38, 41]. Further cells that seemed to be at later stages in cell division, the
studies are required to verify whether trypanosomes kinetoplasts and nuclei appeared closer to each other
from other Neotropical fish families also cluster (Fig. 3 r and s), but probably separated once again later
within this clade. incytokinesis (Fig. 3t).
Measurements of culture forms (type material) are still
DescriptionofTrypanosomaabelin.sp.inBrazilian important for species description and must be included
armouredcatfishes in the taxonomic section. Thus, we performed a detailed
Fishtrypanosomescharacterizedinthisstudysharedsimi- morphometric analysis of the different cell types found
lar sequences regardless of host species (H. affinis and H. in cultures of isolate L4100 of T. abeli n. sp. (Additional
luetkeni). Within the group of sequences obtained, we file 2). Slenderand pyriform epimastigotes had 19.8±6.3
identified unique SSU rRNA and gGAPDH sequences (as and 13.3±5.4 μm in body length along cell midline, re-
determined by BLASTsearches and positioning in phylo- spectively, while trypomastigotes were longer, with body
genetic trees) that supported the description of a new lengths along cell midline of 34.4±1.6 and 22.8±5 μm,
trypanosome species – hereby named Trypanosoma abeli forlong andshort forms,respectively.
n.sp.(Figs.1and2).Here,we characterized this species
using as type material the cultured isolate L4100, which Scanningelectronmicroscopy(SEM)offishand
showed a single trypanosome sequence in the analysis of leechtrypanosomes
sequences from 15 clones of the PCR-amplified DNA. In The SEM analysis of T. abeli n. sp. highlighted the mor-
addition to H. affinis and H. luetkeni captured in South- phological diversity of cultured epimastigotes (Fig. 4, a
east Brazil, in the Atlantic Forest biome, isolates of T. and b) and trypomastigotes (Fig. 4, c and d), corroborat-
abeli n. sp. sharing identical or very similar sequences ing the light microscopy findings. The pleomorphism of
were found in loricariids from other genera captured in bloodstream trypanosomes from H. affinis and H. luet-
Amazonia (Ancistrus sp.) and The Pantanal (Liposarcus keniwasclearinSEMimages(Fig. 4e-g).Trypanosomes
anisistis)biomes(Figs.1and2). from fish blood and leeches were, in fact, too
Lemosetal.Parasites&Vectors (2015) 8:573 Page9of17
Fig.3(Seelegendonnextpage.)
pleomorphic (possibly due, in part, to mixed infections) to allow identification of the forms that corresponded to
Lemosetal.Parasites&Vectors (2015) 8:573 Page10of17
(Seefigureonpreviouspage.)
Fig.3LightmicroscopyanalysisofTrypanosomaabelin.sp.culturedinvitro.ParasiteswerevisualizedbyGiemsastaining(a-g,j,k,n,o,r-t),
differentialinterferencecontrast(h,l,andp),andfluorescencemicroscopyusingHoechstH33342(i,m,andq).Elongated(a)andshortand
broad(b)bloodstreamtrypomastigotes.Shorttrypomastigotes(c)and‘pyriform’(i.e.“pearshaped”)epimastigotes(dande)andwerethefirst
developmentalformsobservedinprimarycultures.Althoughtrypomastigotes(g)wereagainobservedinstationaryphase,aswellas
spheromastigotes(h and i), with a round kinetoplast (arrowhead in i) and short flagellum (arrow in h). Cultures contained dividing
epimastigotes (j-m) and trypomastigotes (n-t) at different stages of cell division, with duplicated single-copy structures, including the fla-
gellum (F), the nucleus (N) and the kinetoplast (k). G, granules; V, vacuoles. Scale bar, 10 μm
the bloodstream trypomastigotes of T. abeli. Flagellates remains unknown, and hypotheses suggest that they are
found in the stomach caeca of leeches (Fig. 4h-j) were formed by viral particles, or may be associated with the
similar to those observed in cultures of T. abeli, and endocyticorexocyticpathways[34,68].
comprisedofepimastigote(Fig.4h),elongatedtrypomas- Both epimastigotes and bloodstream trypomastigotes of
tigote (Fig.4i) andspheromastigote (Fig. 4j)forms. T.abelin.sp.displayedstructuresmorphologicallysimilar
to acidocalcisomes, an acidic organelle rich calcium and
UltrastructuralanalysisofTrypanosomaabelin.sp.by polyphosphates and involved in osmoregulation, in some
transmissionelectronmicroscopy(TEM) protozoa [69]. These structures have been described in
To examine in detail the ultrastructure of T. abeli n. sp., other fish trypanosomes (Trypanosoma pseudobagri and
we analysed log-phase cultures of this parasite by trans- T. danilewskyi) [31, 32], and in Trypanosoma fallisi from
mission electron microscopy (TEM). When observed by anurans [70], T. serpentis and T. cascavelli from snakes
TEM, T. abeli n. sp. trypomastigotes and epimastigotes [43],T.terenaandT.ralphifromcaymans[44],aswellas
had a rod-like kinetoplast containing a compact array of in several trypanosomes of mammals, including T. cruzi,
kDNA fibrils (Fig. 5, a and c) similar to that observed in TrypanosomaerneyiandTrypanosomabrucei[46,71].
the kinetoplast of bloodstream trypomastigotes found in The cytostome-cytopharynx complex - an invagination
H. affinis and H. luetkeni (Fig. 5 b). The kinetoplast pat- of plasma membrane penetrating deep into the cyto-
tern observed in T. abeli was similar to that of fresh- plasm and sometimes reaching the perinuclear region -
water and marine fish trypanosomes, such as T. wasobservedintrypomastigotes(Fig.5d, h)andepimas-
pseudobagre and T. triglae [31, 32], but differed from tigotes (not shown) of T. abeli. In some images, the
that of T. cruzi, which exhibits a round kinetoplast with cytostome appears to ‘open’ into the flagellar pocket
arelatively‘loose’kDNAnetwork [64]. (not shown), differently from that observed in other try-
Numerouslipidbodies withvariablesizes(from 50nm panosomes [30, 31, 34, 43, 44, 47, 64, 70, 72]. The cyto-
to 1.5 μm) were observed in the cytoplasm of T. abeli n. pharynx was first observed in epimastigotes and
sp.(Fig.5c,d,f),andwealsoobservedsmallerlipidbod- trypomastigotes of the fish trypanosomes T. raiae, T.
ies that appeared to fuse with (or bud from) larger ones danilewskyi and T. cobitis [30, 31, 34], and a cytostome-
(Fig. 5f). At the posterior region,T. abeli n. sp. had large cytopharynxcomplex was also observed in epimastigotes
membrane-bound structures reminiscent of lysosomes- of trypanosomes from anurans, snakes and crocodilians
related organelles (LROs), containing linear membrane [43, 44, 70]. The cytostome-cytopharynx complex has
profiles and small vesicles (Fig. 5e) immersed in an also been observed in epimastigotes of T. cruzi and in
electron-dense matrix (Fig. 5c). These putative LROs other species of the subgenus Schyzotrypanum; however,
were morphologically similar to those of T. cruzi (re- trypomastigotes of these species lacked this complex [46,
ferred to as ‘reservosomes’) [65, 66], which contain rod- 72, 73]. Thus far, the presence of the cytostome-
shaped electron-lucent bodies [67] also observed in T. cytopharynx complex in blood and culture trypomasti-
abeli n.sp. (Fig.5e). gotesappearstobeacommonandexclusivecharacteristic
Epimastigotes of T. abeli n. sp. had multivesicular bod- offishtrypanosomes[31],as confirmedherebyourTEM
ies near the flagellar pocket containing numerous vesicles analysisofT.abelin.sp.
of regular size (~80 nm) and with an electron-dense
matrix (Fig. 5g). Multivesicular bodies were also observed Morphologyfeaturesofbloodtrypomastigotesfoundin
in Trypanosoma terena from cayman [44],Trypanosoma H.affinisandH.luetkeni
serpentis and Trypanosoma cascaveli from snakes [43] Bloodstream trypomastigotes from both H. affinis and
and Trypanosoma cobitis from fish [34]; however, they H. luetkeni were highly polymorphic (Fig. 6), and the
had different localization and internal vesicles of different number and proportion of morphotypes varied between
size compared with theT. abeli n. sp. multivesicular bod- blood samples. The morphological variation observed
ies. The nature of these structures in trypanosomatids here, if used as the sole taxonomic criterion, would