TatD DNases of African trypanosomes confer resistance to host neutrophil extracellular traps.

African trypanosomatid parasites escape host acquired immune responses through periodic antigenic variation of their surface coat. In this study, we describe a mechanism by which the parasites counteract innate immune responses. Two TatD DNases were identified in each of Trypanosoma evansi and Trypanosoma brucei. These DNases are bivalent metal-dependent endonucleases localized in the cytoplasm and flagella of the parasites that can also be secreted by the parasites. These enzymes possess conserved functional domains and have efficient DNA hydrolysis activity. Host neutrophil extracellular traps (NETs) induced by the parasites could be hydrolyzed by native and recombinant TatD DNases. NET disruption was prevented, and the survival rate of parasites was decreased, in the presence of the DNase inhibitor aurintricarboxylic acid. These data suggest that trypanosomes can counteract host innate immune responses by active secretion of TatD DNases to degrade NETs.

Trypanosoma, Paramecium and Tetrahymena: From genomics to flagellar and ciliary structures and cytoskeleton dynamics.

Cilia and flagella play an important role in motility, sensory perception, and the life cycles of eukaryotes, from protists to humans. However, much critical information concerning cilia structure and function remains elusive. The vast majority of ciliary and flagellar proteins analyzed so far are evolutionarily conserved and play a similar role in protozoa and vertebrates. This makes protozoa attractive biological models for studying cilia biology. Research conducted on ciliated or flagellated protists may improve our general understanding of cilia protein composition, of cilia beating, and can shed light on the molecular basis of the human disorders caused by motile cilia dysfunction. The Symposium "From genomics to flagellar and ciliary structures and cytoskeleton dynamics" at ECOP2019 in Rome presented the latest discoveries about cilia biogenesis and the molecular mechanisms of ciliary and flagellum motility based on studies in Paramecium, Tetrahymena, and Trypanosoma. Here, we review the most relevant aspects presented and discussed during the symposium and add our perspectives for future research.

Pan-American Trypanosoma (Megatrypanum) trinaperronei n. sp. in the white-tailed deer Odocoileus virginianus Zimmermann and its deer ked Lipoptena mazamae Rondani, 1878: morphological, developmental and phylogeographical characterisation.

BACKGROUND: The subgenus Megatrypanum Hoare, 1964 of Trypanosoma Gruby, 1843 comprises trypanosomes of cervids and bovids from around the world. Here, the white-tailed deer Odocoileus virginianus (Zimmermann) and its ectoparasite, the deer ked Lipoptena mazamae Rondani, 1878 (hippoboscid fly), were surveyed for trypanosomes in Venezuela. RESULTS: Haemoculturing unveiled 20% infected WTD, while 47% (7/15) of blood samples and 38% (11/29) of ked guts tested positive for the Megatrypanum-specific TthCATL-PCR. CATL and SSU rRNA sequences uncovered a single species of trypanosome. Phylogeny based on SSU rRNA and gGAPDH sequences tightly cluster WTD trypanosomes from Venezuela and the USA, which were strongly supported as geographical variants of the herein described Trypanosoma (Megatrypanum) trinaperronei n. sp. In our analyses, the new species was closest to Trypanosoma sp. D30 from fallow deer (Germany), both nested into TthII alongside other trypanosomes from cervids (North American elk and European fallow, red and sika deer), and bovids (cattle, antelopes and sheep). Insights into the life-cycle of T. trinaperronei n. sp. were obtained from early haemocultures of deer blood and co-culture with mammalian and insect cells showing flagellates resembling Megatrypanum trypanosomes previously reported in deer blood, and deer ked guts. For the first time, a trypanosome from a cervid was cultured and phylogenetically and morphologically (light and electron microscopy) characterised. CONCLUSIONS: In the analyses based on SSU rRNA, gGAPDH, CATL and ITS rDNA sequences, neither cervids nor bovids trypanosomes were monophyletic but intertwined within TthI and TthII major phylogenetic lineages. One host species can harbour more than one species/genotype of trypanosome, but each trypanosome species/genotype was found in a single host species or in phylogenetically closely related hosts. Molecular evidence that L. mazamae may transmit T. trinaperronei n. sp. suggests important evolutionary constraints making tight the tripartite T. trinaperronei-WTD-deer ked association. In a plausible evolutionary scenario, T. trinaperronei n. sp. entered South America with North American white-tailed deer at the Pliocene-Pleistocene boundary following the closure of the Panama Isthmus.

Nucleolar Structure and Function in Trypanosomatid Protozoa.

The nucleolus is the conspicuous nuclear body where ribosomal RNA genes are transcribed by RNA polymerase I, pre-ribosomal RNA is processed, and ribosomal subunits are assembled. Other important functions have been attributed to the nucleolus over the years. Here we review the current knowledge about the structure and function of the nucleolus in the trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania ssp., which represent one of the earliest branching lineages among the eukaryotes. These protozoan parasites present a single nucleolus that is preserved throughout the closed nuclear division, and that seems to lack fibrillar centers. Trypanosomatids possess a relatively low number of rRNA genes, which encode rRNA molecules that contain large expansion segments, including several that are trypanosomatid-specific. Notably, the large subunit rRNA (28S-type) is fragmented into two large and four small rRNA species. Hence, compared to other organisms, the rRNA primary transcript requires additional processing steps in trypanosomatids. Accordingly, this group of parasites contains the highest number ever reported of snoRNAs that participate in rRNA processing. The number of modified rRNA nucleotides in trypanosomatids is also higher than in other organisms. Regarding the structure and biogenesis of the ribosomes, recent cryo-electron microscopy analyses have revealed several trypanosomatid-specific features that are discussed here. Additional functions of the nucleolus in trypanosomatids are also reviewed.

The marsupial trypanosome Trypanosoma copemani is not an obligate intracellular parasite, although it adversely affects cell health.

BACKGROUND: Trypanosoma cruzi invades and replicates inside mammalian cells, which can lead to chronic Chagas disease in humans. Trypanosoma copemani infects Australian marsupials and recent investigations indicate it may be able to invade mammalian cells in vitro, similar to T. cruzi. Here, T. cruzi 10R26 strain (TcIIa) and two strains of T. copemani [genotype 1 (G1) and genotype 2 (G2)] were incubated with marsupial cells in vitro. Live-cell time-lapse and fluorescent microscopy, combined with high-resolution microscopy (transmission and scanning electron microscopy) were used to investigate surface interactions between parasites and mammalian cells. RESULTS: The number of parasites invading cells was significantly higher in T. cruzi compared to either genotype of T. copemani, between which there was no significant difference. While capable of cellular invasion, T. copemani did not multiply in host cells in vitro as there was no increase in intracellular amastigotes over time and no release of new trypomastigotes from host cells, as observed in T. cruzi. Exposure of host cells to G2 trypomastigotes resulted in increased host cell membrane permeability within 24 h of infection, and host cell death/blebbing was also observed. G2 parasites also became embedded in the host cell membrane. CONCLUSIONS: Trypanosoma copemani is unlikely to have an obligate intracellular life-cycle like T. cruzi. However, T. copemani adversely affects cell health in vitro and should be investigated in vivo in infected host tissues to better understand this host-parasite relationship. Future research should focus on increasing understanding of the T. copemani life history and the genetic, physiological and ecological differences between different genotypes.

The kinetoplast DNA of the Australian trypanosome, Trypanosoma copemani, shares features with Trypanosoma cruzi and Trypanosoma lewisi.

Kinetoplast DNA (kDNA) is the mitochondrial genome of trypanosomatids. It consists of a few dozen maxicircles and several thousand minicircles, all catenated topologically to form a two-dimensional DNA network. Minicircles are heterogeneous in size and sequence among species. They present one or several conserved regions that contain three highly conserved sequence blocks. CSB-1 (10 bp sequence) and CSB-2 (8 bp sequence) present lower interspecies homology, while CSB-3 (12 bp sequence) or the Universal Minicircle Sequence is conserved within most trypanosomatids. The Universal Minicircle Sequence is located at the replication origin of the minicircles, and is the binding site for the UMS binding protein, a protein involved in trypanosomatid survival and virulence. Here, we describe the structure and organisation of the kDNA of Trypanosoma copemani, a parasite that has been shown to infect mammalian cells and has been associated with the drastic decline of the endangered Australian marsupial, the woylie (Bettongia penicillata). Deep genomic sequencing showed that T. copemani presents two classes of minicircles that share sequence identity and organisation in the conserved sequence blocks with those of Trypanosoma cruzi and Trypanosoma lewisi. A 19,257 bp partial region of the maxicircle of T. copemani that contained the entire coding region was obtained. Comparative analysis of the T. copemani entire maxicircle coding region with the coding regions of T. cruzi and T. lewisi showed they share 71.05% and 71.28% identity, respectively. The shared features in the maxicircle/minicircle organisation and sequence between T. copemani and T. cruzi/T. lewisi suggest similarities in their process of kDNA replication, and are of significance in understanding the evolution of Australian trypanosomes.

A comparative molecular and 3-dimensional structural investigation into cross-continental and novel avian Trypanosoma spp. in Australia.

BACKGROUND: Molecular and structural information on avian Trypanosoma spp. throughout Australia is limited despite their intrinsic value in understanding trypanosomatid evolution, diversity, and structural biology. In Western Australia tissue samples (n = 429) extracted from 93 birds in 25 bird species were screened using generic PCR primers to investigate the diversity of Trypanosoma spp. To investigate avian trypanosome structural biology the first 3-dimensional ultrastructural models of a Trypanosoma spp. (Trypanosoma sp. AAT) isolated from a bird (currawong, Strepera spp.) were generated using focussed ion beam milling combined with scanning electron microscopy (FIB-SEM). RESULTS: Here, we confirm four intercontinental species of avian trypanosomes in native Australian birds, and identify a new avian Trypanosoma. Trypanosome infection was identified in 18 birds from 13 different bird species (19%). A single new genotype was isolated and found to be closely related to T. culicavium (Trypanosoma sp. CC2016 B002). Other Trypanosoma spp. identified include T. avium, T. culicavium, T. thomasbancrofti, Trypanosoma sp. TL.AQ.22, Trypanosoma sp. AAT, and an uncharacterised Trypanosoma sp. (group C-III sensu Zidková et al. (Infect Genet Evol 12:102-112, 2012)), all previously identified in Australia or other continents. Serially-sectioning Trypanosoma sp. AAT epimastigotes using FIB-SEM revealed the disc-shaped kinetoplast pocket attached perpendicular to the branching mitochondrion. Additionally, the universal minicircle sequence within the kinetoplast DNA and the associated binding protein were determined in Trypanosoma sp. AAT. CONCLUSIONS: These results indicate that bird trypanosomes are relatively conserved across continents, while being locally diverse, which supports the hypothesis that bird trypanosomes exist as fewer species than described in the literature. Evidence exists that avian Trypanosoma spp. are infecting mammals and could be transmitted by haemadipsid leeches. Trypanosoma sp. AAT is most likely a separate species currently found only in Australia and the first 3-dimentional ultrastructural analysis of an avian trypanosome provides interesting information on their morphology and organelle arrangement.

Biting midges (Ceratopogonidae) as vectors of avian trypanosomes.

BACKGROUND: Although avian trypanosomes are widespread parasites, the knowledge of their vectors is still incomplete. Despite biting midges (Diptera: Ceratopogonidae) are considered as potential vectors of avian trypanosomes, their role in transmission has not been satisfactorily elucidated. Our aim was to clarify the potential of biting midges to sustain the development of avian trypanosomes by testing their susceptibility to different strains of avian trypanosomes experimentally. Moreover, we screened biting midges for natural infections in the wild. RESULTS: Laboratory-bred biting midges Culicoides nubeculosus were highly susceptible to trypanosomes from the Trypanosoma bennetti and T. avium clades. Infection rates reached 100%, heavy infections developed in 55-87% of blood-fed females. Parasite stages from the insect gut were infective for birds. Moreover, midges could be infected after feeding on a trypanosome-positive bird. Avian trypanosomes can thus complete their cycle in birds and biting midges. Furthermore, we succeeded to find infected blood meal-free biting midges in the wild. CONCLUSIONS: Biting midges are probable vectors of avian trypanosomes belonging to T. bennetti group. Midges are highly susceptible to artificial infections, can be infected after feeding on birds, and T. bennetti-infected biting midges (Culicoides spp.) have been found in nature. Moreover, midges can be used as model hosts producing metacyclic avian trypanosome stages infective for avian hosts.

Fleas and trypanosomes of peridomestic small mammals in sub-Saharan Mali.

BACKGROUND: Fleas are obligate blood-feeding ectoparasites and vectors of several bacterial zoonotic pathogens as well as trypanosomes that parasitize rodents and other small mammals. During investigations of tick- and rodent-borne diseases in Mali, West Africa, we included fleas and rodent-borne trypanosomes, both of which are poorly known in this country, but are attracting greater public health interest. METHODS: Small mammals were captured in 20 Malian villages from December 2007 to October 2011. Fleas were collected and identified to species, and thin blood smears were prepared, stained and examined microscopically for trypanosomes. RESULTS: We captured 744 small mammals, 68 (9.1 %) of which yielded fleas. Two species of fleas, Xenopsylla cheopis and Xenopsylla nubica, were collected from six species of rodents and one species of shrew. Multimammate rats, Mastomys natalensis, were hosts for 58.5 % of all fleas collected. Xenopsylla cheopis was found in the moister southern savannah while X. nubica was mostly restricted to the drier Sahel. Trypanosomes were found in 3 % of 724 blood smears, although 91 % of parasitemic animals originated from two villages where black rats (Rattus rattus) and M. natalensis were the primary hosts and X. cheopis the dominant flea. The trypanosomes were morphologically consistent with the Trypanosoma (Herpetosoma) lewisi group, flea-borne hemoflagellates that parasitize domestic rats. CONCLUSIONS: Xenopsylla cheopis and trypanosomes parasitize peridomestic rats that commingle with people in southern Mali. Given the increasing awareness of flea-borne trypanosomes as possible human pathogens, we hope our findings will stimulate future investigators to examine the potential public health significance of flea-borne trypanosomosis in West Africa.

Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes.

The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics.

Antiparasitic effect of Dinoponera quadriceps giant ant venom.

Neglected tropical diseases (NTD) are treated with toxic therapy of limited efficacy. Previously, we studied the antimicrobial effect of Dinoponera quadriceps venom (DqV) against bacteria. To continue the study, we report in this short communication the antimicrobial effect of DqV against Leishmania amazonensis and Trypanosoma cruzi. DqV inhibits the promastigote forms of L. amazonensis and all T. cruzi developmental forms, with low toxicity in host cells. DqV causes cell death in T. cruzi through necrotic and apoptotic mechanisms observed by staining the cells with annexin V-FITC (AX) and propidium iodide (PI), loss of mitochondrial membrane potential by flow cytometry analyses and confocal microscopy and morphological alterations, such as loss of membrane integrity and cell shrinkage by scanning electron microscopy (SEM). In conclusion, we suggest there is an antimicrobial effect also on parasites.

Nerolidol nanospheres increases its trypanocidal efficacy against Trypanosoma evansi: New approach against diminazene aceturate resistance and toxicity.

The aims of this study were to develop nerolidol-loaded nanospheres, and to evaluate their efficacy in vitro and in vivo against Trypanosoma evansi, as well as to determine their physicochemical properties, morphology, and any possible side effect in vitro against peripheral blood mononuclear cell (PBMC). The nanospheres showed an adequate particle size (149.5 nm), narrow particle distribution (0.117), negative zeta potential (-12.8 mV), and pH of 6.84, such as observed by transmission electron microscopy. In vitro, a trypanocidal effect of nerolidol and nanospheres containing nerolidol was observed at 0.5, 1.0, and 2.0%, i.e., both treatments showed a faster trypanocidal effect compared to chemotherapy (diminazene aceturate - D.A.). T. evansi infected mice were used to evaluate the effects of nerolidol-loaded nanospheres regarding pre-patent period, longevity, and therapeutic efficacy. Oral administration of nerolidol-loaded nanospheres at 1.0 mL/kg/day during 10 days increased mice survival (66.66%) compared to 0% and 33.33% of mice survival when treated with nerolidol in its free form and D.A., respectively. Cytotoxic study indicated that both treatments showed no side effects in vitro against PBMC, an important marker used in toxicological surveys. Therefore, nanoencapsulation increased the therapeutic efficacy of nerolidol against T. evansi, and can be used as an alternative treatment for T. evansi infection.

Developmental and Ultrastructural Characterization and Phylogenetic Analysis of Trypanosoma herthameyeri n. sp. of Brazilian Leptodactilydae Frogs.

We described the phylogenetic affiliation, development in cultures and ultrastructural features of a trypanosome of Leptodacylus chaquensis from the Pantanal biome of Brazil. In the inferred phylogeny, this trypanosome nested into the Anura clade of the basal Aquatic clade of Trypanosoma, but was separate from all known species within this clade. This finding enabled us to describe it as Trypanosoma herthameyeri n. sp., which also infects other Leptodacylus species from the Pantanal and Caatinga biomes. Trypanosoma herthameyeri multiplies as small rounded forms clumped together and evolving into multiple-fission forms and rosettes of epimastigotes released as long forms with long flagella; scarce trypomastigotes and glove-like forms are common in stationary-phase cultures. For the first time, a trypanosome from an amphibian was observed by field emission scanning electron microscopy, revealing a cytostome opening, well-developed flagellar lamella, and many grooves in pumpkin-like forms. Transmission electron microscopy showed highly developed Golgi complexes, relaxed catenation of KDNA, and a rich set of spongiome tubules in a regular parallel arrangement to the flagellar pocket as confirmed by electron tomography. Considering the basal position in the phylogenetic tree, developmental and ultrastructural data of T. herthameyeri are valuable for evolutionary studies of trypanosome architecture and cell biology.

Intercontinental distribution of a new trypanosome species from Australian endemic Regent Honeyeater (Anthochaera phrygia).

Establishing a health screening protocol is fundamental for successful captive breeding and release of wildlife. The aim of this study was to undertake a parasitological survey focusing on the presence of trypanosomes in a cohort of Regent Honeyeaters, Anthochaera phrygia, syn. Xanthomyza phrygia (Aves: Passeriformes) that are part of the breeding and reintroduction programme carried out in Australia. We describe a new blood parasite, Trypanosoma thomasbancrofti sp. n. (Kinetoplastida: Trypanosomatidae) with prevalence of 24·4% (20/81) in a captive population in 2015. The sequence of the small subunit rRNA gene (SSU rDNA) and kinetoplast ultrastructure of T. thomasbancrofti sp. n. are the key differentiating characteristics from other Trypanosoma spp. T. thomasbancrofti sp. n. is distinct from Trypanosoma cf. avium found in sympatric Noisy Miners (Manorina melanocephala). The SSU rDNA comparison suggests an intercontinental distribution of T. thomasbancrofti sp. n. and Culex mosquitoes as a suspected vector. Currently, no information exists on the effect of T. thomasbancrofti sp. n. on its hosts; however, all trypanosome-positive birds remain clinically healthy. This information is useful in establishing baseline health data and screening protocols, particularly prior to release to the wild.

A Clinical and Epidemiological Investigation of the First Reported Human Infection With the Zoonotic Parasite Trypanosoma evansi in Southeast Asia.

BACKGROUND: Trypanosomais a genus of unicellular parasitic flagellate protozoa.Trypanosoma bruceispecies and Trypanosoma cruziare the major agents of human trypanosomiasis; other Trypanosomaspecies can cause human disease, but are rare. In March 2015, a 38-year-old woman presented to a healthcare facility in southern Vietnam with fever, headache, and arthralgia. Microscopic examination of blood revealed infection with Trypanosoma METHODS: Microscopic observation, polymerase chain reaction (PCR) amplification of blood samples, and serological testing were performed to identify the infecting species. The patient's blood was screened for the trypanocidal protein apolipoprotein L1 (APOL1), and a field investigation was performed to identify the zoonotic source. RESULTS: PCR amplification and serological testing identified the infecting species as Trypanosoma evansi.Despite relapsing 6 weeks after completing amphotericin B therapy, the patient made a complete recovery after 5 weeks of suramin. The patient was found to have 2 wild-type APOL1 alleles and a normal serum APOL1 concentration. After responsive animal sampling in the presumed location of exposure, cattle and/or buffalo were determined to be the most likely source of the infection, with 14 of 30 (47%) animal blood samples testing PCR positive forT. evansi. CONCLUSIONS: We report the first laboratory-confirmed case ofT. evansiin a previously healthy individual without APOL1 deficiency, potentially contracted via a wound while butchering raw beef, and successfully treated with suramin. A linked epidemiological investigation revealed widespread and previously unidentified burden ofT. evansiin local cattle, highlighting the need for surveillance of this infection in animals and the possibility of further human cases.

Aflagellar epimastigote forms are found in axenic culture of Trypanosoma caninum.

Representatives of the genus Trypanosoma have been traditionally found in epimastigote, espheromastigote and trypomastigote flagellated forms in axenic cultures. Trypanosoma caninum is a trypanosomatid that has recently been reported infecting dogs in endemic areas of canine leishmaniasis in Brazil. It presents specific biological characteristics and it is found exclusively on healthy skin. Here, we describe the evolutive forms of this parasite showing not only the forms commonly found in culture, but also epimastigote forms with no free flagellum. The study was conducted using scanning and transmission electron microscopy and, we demonstrate that typical flagellated epimastigotes originate from forms without flagellum, although the latter may remain without differentiation in the culture. Two hypotheses are considered and discussed in this paper: (i) the aflagellated epimastigotes are a typical developmental forms of T. caninum and (ii) the emergence of these aflagellated forms could be resultant from a disturbed process during cell division caused by interfering specific proteins, which leads to inability to form and regulate the flagellum length. In any case, considering that T. caninum is a parasite that is still little studied, the information brought by our study adds data which may be useful to clarify aspects on the cell cycle of this intriguing parasite that has been found in different regions of Brazil.

Isolation and in vitro maintenance of trypanosomes from naturally infected and commercially important Brazilian fish.

Fish trypanosomes are widely distributed in commercially important fish, with high prevalence in some Brazilian species. This study provides the first record of the isolation and in vitro maintenance of trypanosomes from Brazilian fish. We produced 49 trypanosome isolates from naturally infected catfish (Hypostomus affinis and Hypostomus luetkeni), using 9 different culture media (out of 31 tested). Trypanosomes were maintained in culture for at least 15 mo and were successfully cryopreserved. Culture forms-epimastigotes and short trypomastigotes-were capable of dividing in vitro. Our study is an important step in the investigation of ultrastructure, taxonomy, and phylogeny of trypanosomes from commercially important Brazilian fish.

Trypanosoma epinepheli n. sp. (Kinetoplastida) from a farmed marine fish in China, the brown-marbled grouper (Epinephelus fuscoguttatus).

An outbreak of trypanosomosis occurred in farmed Epinephelus fuscoguttatus in Xincun Bay, province of Hainan, South China Sea. The infected fish showed loss of appetite, lethargy, emaciation, severe anemia, and splenomegaly. Light and scanning electron microscopic examination of bloodstream trypomastigotes revealed morphological features typical for small-sized marine fish trypanosomes. The trypanosome possesses a short body length (mean 22.3 µm, range 17.6-25.9 µm) and narrow body width (mean1.7 µm, range 1.3-2.0 µm), a central nucleus, a narrow but distinct undulating membrane, and a relatively long free flagellum (mean 10.1 µm, range 7.4-13.3 µm). The kinetoplast is situated at approximately one quarter of body length from posterior extremity. The division process of this trypanosome was observed in the peripheral blood of the host, and occurred by transverse constriction at a point between the kinetoplasts. Comparison of the small subunit rDNA (SSU rDNA) sequences revealed that the trypanosome from E. fuscoguttatus showed 93.4-97.1% identity with the available sequences from Trypanosoma spp. from other piscine hosts. Phylogenetic analysis supported the existence of an aquatic clade, and the present trypanosome grouped with other marine fish trypanosomes, in a subclade together with Trypanosoma senegalense. Based on the differences in morphological characteristics, host species, and molecular data, the trypanosome infecting E. fuscoguttatus is considered to be a new species, for which we propose the name Trypanosoma epinepheli n. sp.