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