Phylogenetic and morphological characterization of trypanosomes from Brazilian armoured catfishes and leeches reveal high species diversity, mixed infections and a new fish trypanosome species

Abstract\ud \ud Background\ud Several Trypanosoma species transmitted by leeches infect marine and freshwater fish worldwide. To date, all South American fish trypanosome species identified have been based on unreliable morphological parameters. We recently isolated and cultured trypanosomes from the Brazilian armoured catfishes Hypostomus luetkeni and H. affinis. Here, we report the first phylogenetic analyses of South American (Brazilian) trypanosomes isolated from fish, and from leeches removed from these fish. We also analysed morphologically and morphometrically the different forms of fish, leech and cultured trypanosomes.\ud \ud \ud Methods\ud V7V8 SSU rRNA and gGAPDH sequences were used for phylogenetic analysis of Brazilian fish and leech trypanosomes. Trypanosomes from cultures, fish blood and leech samples were also characterized morphologically and morphometrically by light and electron microscopy.\ud \ud \ud Results\ud In blood smears from fish high trypanosome prevalence (90–100 %) and parasitemia (0.9-1.0x102) were observed. Phylogenetic relationships using SSU rRNA and gGAPDH showed that, despite relevant sequence divergence, all Brazilian fish (and derived cultures) and leech trypanosomes clustered together into a single clade. The Brazilian clade clustered with European, North American and African fish trypanosomes. Based on sequence analysis, we uncovered a new species of Brazilian fish trypanosome, Trypanosoma abeli n. sp. Trypanosoma abeli cultures contained pleomorphic epimastigotes, small trypomastigotes and rare sphaeromastigotes. Ultrastructural features of T. abeli included a cytostome-cytopharynx complex in epi- and trypomastigotes, a compact rod-like kinetoplast, lysosome-related organelles (LROs) and multivesicular bodies. Trypanosomes found in fish blood smears and leech samples were highly pleomorphic, in agreement with sequence data suggesting that catfishes and leeches often have mixed trypanosome infections.\ud \ud \ud Conclusions\ud \ud Trypanosoma abeli n. 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.We are grateful to Mr. Alcir Pereira de Souza, Ms. Alessandra Simões de\ud Toledo Pereira and Mrs. Tereza da Silva Lemos for their valuable support\ud with the fieldwork, and to Dr. José Carlos Oliveira and Valter M. AzevedoSantos\ud for fish identification. DNA sequencing and phylogenetic analyses\ud done in USP were supported by grants from CNPq and CAPES to MMGT. ML\ud is a postdoctoral fellow funded by CNPq, BRF is a PhD student funded by\ud CAPES, CSR is PhD student funded by CNPq, and LH is postdoctoral fellow\ud funded by FAPERJ

The surface charge of trypanosomatids

The surface charge of trypanosomatids was evaluated by means of the binding of cationic particles, as visualized by electron microscopy and by direct measurements of the electrophoretic mobility of cells. The results obtained indicate that most of the trypanosomatids exhibit a negatively charged surface whose value is species specific and varies according to the developmental stages. Sialic acids associated with glycoproteins, glycolipids and phosphate groups are the major components responsible for the net negative surface charge of the trypanosomatids

Immunocytochemical localisation of calreticulin in Trypanosoma cruzi

Calreticulin, a Ca(2+) chaperone, is found in many different locations in various eukaryotic cells, including lumen of the endoplasmic reticulum, the cell surface, perinuclear areas and cytosolic granules. In the present study, a polyclonal antibody against calreticulin was used for the immunocytochemical localisation of the protein in Trypanosoma cruzi. Labelling was observed in the endoplasmic reticulum, Golgi complex, reservosomes, flagellar pocket, cell surface, cytosol, nucleus and kinetoplast. Significant differences in labelling were observed among the three evolutive forms of the protozoan. The functional role of calreticulin in T. cruzi is discussed.Fil: Souto Padrón, Thaïs. Universidade Federal do Rio de Janeiro; BrasilFil: Labriola, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: de Souza, Wanderley. Universidade Federal do Rio de Janeiro; Brasi

<i>Apis mellifera</i>venom induces different cell death pathways in<i>Trypanosoma cruzi</i>

SUMMARYChagas disease chemotherapy is based on drugs that exhibit toxic effects and have limited efficacy, such as Benznidazole. Therefore, research into new chemotherapeutic agents from natural sources needs to be exploited.Apis melliferavenom consists of many biologically active molecules and has been reported to exhibit remarkable anti-cancer effects, often promoting an apoptosis-like death phenotype. This study demonstrates thatA. melliferavenom can affect the growth, viability and ultrastructure of allTrypanosoma cruzidevelopmental forms, including intracellular amastigotes, at concentrations 15- to 100-fold lower than those required to cause toxic effects in mammalian cells. The ultrastructural changes induced by the venom in the different developmental forms led us to hypothesize the occurrence of different programmed cell death pathways. Autophagic cell death, characterized by the presence of autophagosomes-like organelles and a strong monodansyl cadaverine labelling, appears to be the main death mechanism in epimastigotes. In contrast, increased TUNEL staining, abnormal nuclear chromatin condensation and kDNA disorganization was observed in venom-treated trypomastigotes, suggesting cell death by an apoptotic mechanism. On the other hand, intracellular amastigotes presented a heterogeneous cell death phenotype profile, where apoptosis-like death seemed to be predominant. Our findings confirm the great potential ofA. melliferavenom as a source for the development of new drugs for the treatment of neglected diseases such as Chagas disease.</jats:p