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

The immune response of hemocytes of the insect Oncopeltus fasciatus against the flagellate Phytomonas serpens.

The genus Phytomonas includes parasites that are etiological agents of important plant diseases, especially in Central and South America. These parasites are transmitted to plants via the bite of an infected phytophagous hemipteran. Despite the economic impact of these parasites, many basic questions regarding the genus Phytomonas remain unanswered, such as the mechanism by which the parasites cope with the immune response of the insect vector. In this report, using a model of systemic infection, we describe the function of Oncopeltus fasciatus hemocytes in the immune response towards the tomato parasite Phytomonas serpens. Hemocytes respond to infection by trapping parasites in nodular structures and phagocytizing the parasites. In electron microscopy of hemocytes, parasites were located inside vacuoles, which appear fused with lysosomes. The parasites reached the O. fasciatus salivary glands at least six hours post-infection. After 72 hours post-infection, many parasites were attached to the salivary gland outer surface. Thus, the cellular responses did not kill all the parasites

Survival of <i>P.</i><i>serpens</i>-infected <i>O. fasciatus</i>.

<p>Groups of 25 adult insects were either infected with 5×10⁴ parasites or mock-infected with PBS and their survival was monitored twice a day. The mock-infected insects did not die due to the challenge. On the other hand, approximately 13% of the insects infected with parasites died, which was significant (P<0.05). The experiment was conducted twice.</p

The number of circulating hemocytes in <i>O.</i><i>fasciatus</i> hemolymph increases after infection.

<p>The insects (n = 24) were challenge with 5×10⁴ parasites and the cellular density of the hemocytes was determined using a Neubauer hemocytometer chamber. Each bar represents the mean ± standard error of eight samples and each sample was obtained by pooling together the hemolymph of three insects. Alternatively, the insects were mock challenged with PBS. The asterisk indicates that each one of the three bars (infected 24 h, infected 48 h and infected 72 h) has value significantly different from the value obtained for the hemocytes collected from hemolymph of non-infected insects, using One-way ANOVA with Dunnett's post test (p<0.05).</p