Field and experimental evidence of a new caiman trypanosome species closely phylogenetically related to fish trypanosomes and transmitted by leeches.

Bruno R Fermino,Marta Campaner,Luiz Eduardo R Tavares,Cátia D De-Paula,Priscilla Soares,Laerte B Viola,Fernando Paiva,Carmen S.A Takata,Robson C Ferreira,Erney P Camargo,Marta M.G Teixeira,Robinson Botero-Arias

doi:10.1016/j.ijppaw.2015.10.005

Abstract

Trypanosoma terena and Trypanosoma ralphi are known species of the South American crocodilians Caiman crocodilus, Caiman yacare and Melanosuchus niger and are phylogenetically related to the tsetse-transmitted Trypanosoma grayi of the African Crocodylus niloticus. These trypanosomes form the Crocodilian clade of the terrestrial clade of the genus Trypanosoma. A PCR-survey for trypanosomes in caiman blood samples and in leeches taken from caimans revealed unknown trypanosome diversity and frequent mixed infections. Phylogenies based on SSU (small subunit) of rRNA and gGAPDH (glycosomal Glyceraldehyde Phosphate Dehydrogenase) gene sequences revealed a new trypanosome species clustering with T. terena and T. ralphi in the crocodilian clade and an additional new species nesting in the distant Aquatic clade of trypanosomes, which is herein named Trypanosoma clandestinus n. sp. This new species was found in Caiman yacare, Caiman crocodilus and M. niger from the Pantanal and Amazonian biomes in Brazil. Large numbers of dividing epimastigotes and unique thin and long trypomastigotes were found in the guts of leeches (Haementeria sp.) removed from the mouths of caimans. The trypanosomes recovered from the leeches had sequences identical to those of T. clandestinus of caiman blood samples. Experimental infestation of young caimans (Caiman yacare) with infected leeches resulted in long-lasting T. clandestinus infections that permitted us to delineate its life cycle. In contrast to T. terena, T. ralphi and T. grayi, which are detectable by hemoculturing, microscopy and standard PCR of caiman blood, T. clandestinus passes undetected by these methods due to very low parasitemia and could be detected solely by the more sensitive nested PCR method. T. clandestinus n. sp. is the first crocodilian trypanosome known to be transmitted by leeches and positioned in the aquatic clade closest to fish trypanosomes. Our data show that caimans can host trypanosomes of the aquatic or terrestrial clade, sometimes simultaneously.

Highlights

Flagellates of the genus Trypanosoma (Euglenozoa, Kinetoplastea, Trypanosomatidae) are obligate parasites of all vertebrate classes and are distributed into two major phylogenetic lineages: the Terrestrial clade composed of trypanosomes of mammals, snakes, lizards, crocodilians and birds and the Aquatic clade containing trypanosomes of aquatic leeches and aquatic or semi-aquatic hosts and, oddly, a trypanosome of chameleon (Stevens et al, 2001; Hamilton et al, 2005, 2007).B.R
The analyses of V7V8 SSU rRNA and gGAPDH trypanosome sequences obtained from blood or tissue samples from 34 caimans revealed that 23 (68%) of the caimans presented sequences from a single species, whereas 11 (32%) presented mixed infections with two to four trypanosome species; T. terena and T. ralphi were detected in ~50% of the mixed infections
Samples sharing a new sequence formed a clade (Cay03) that nested together with T. terena and T. ralphi in the previously described Crocodilian Clade (Fermino et al, 2013); the other new sequences formed the new Clandestinus clade, which were positioned within the Aquatic clade of Trypanosoma (Table 1, Fig. 1)

Summary

Introduction

Flagellates of the genus Trypanosoma (Euglenozoa, Kinetoplastea, Trypanosomatidae) are obligate parasites of all vertebrate classes and are distributed into two major phylogenetic lineages: the Terrestrial clade composed of trypanosomes of mammals, snakes, lizards, crocodilians and birds and the Aquatic clade containing trypanosomes of aquatic leeches and aquatic (fishes) or semi-aquatic (chelonians, anurans and platypus) hosts and, oddly, a trypanosome of chameleon (Stevens et al, 2001; Hamilton et al, 2005, 2007).B.R. Available phylogenetic data show very close phylogenetic relationships between the African T. grayi from Crocodylus niloticus and the Brazilian T. ralphi from caimans, as well as between the Brazilian T. terena from caimans and an unnamed African trypanosome from O. tetraspis. These South American and African crocodilian trypanosomes form the major Crocodilian clade comprising the clades Terena, Ralphi and Grayi; each clade harbors a number of closely related genotypes (Fermino et al, 2013). The phylogenetic relationships between the crocodilian trypanosomes concur with the worldwide transoceanic dispersal of Crocodylus during the Miocene that ended in either South America or Africa (Oaks, 2011)

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Conclusion