Growth of an Insect Trypanosomatid at 37 C in a Defined Medium*

SYNOPSIS. A defined medium for an insect trypanosomatid, “Leptomonas pessoai” (probably a member of the genus Herpetomonas) isolated from the reduviid Zelus leucogrammus, allows growth up to 37 C. No marked differences were evident for growth at 37 C. The requirements for amino acids, vitamins, purine, and hemin, and pH range were like those established for Crithidia fasciculata. Again like C. fasciculata, it remains alive at 4 C for at least 3 months in a çlycerolated defined medium.     

Mechanism of Acquired Immunity Induced by “Leptomonas pessoai” against Trypanosoma cruzi in Mice*

Living culture forms of “Leptomonas pessoai” cross protected mice against T. cruzi challenge infection. Circulating antibodies have been detected in the immunized mice by immunodiffusion analysis, passive hemagglutination, complement fixation test and antibody binding assay; these antibodies cross reacted with T. cruzi extracts. A cellular immune response was indicated by leucocyte migration inhibition using L. pessoai and T. cruzi antigens, strongly suggesting a role for cell-mediated immunity in the mechanism of protection induced by L. pessoai.     

Demonstration of Concanavalin A Receptors on Leptomonas pessoai Cell Membrane*§

SYNOPSIS. Leptomonas pessoai promastigotes cultivated in a synthetic medium were agglutinated with concanavalin A (Con A). Agglutination was predominantly of the flagellar-flagellar type, and was inhibited with sucrose or α-methyl-D-mannoside. Cell surface polysaccharides and/or glycoproteins were demonstrated by several cytochemical methods at the fine-structural level. A Con A-horseradish peroxidase-diaminobenzidine (DAB) technic was used to detect Con A receptors in the pellicular membrane of the flagellar pocket region and in the flagellar membrane proper. Somewhat less of the Con A-DAB reaction product was observed in the pellicular membrane enclosing the rest of the cell.     

Leptomonas lactosovorans n. sp., a Lactose-Utilizing Trypanosomatid: Description and Nutritional Requirements1

Leptomonas lactosovorans n. sp. is described from the gut of the reduviid hemipteran Zelurus martinsi. It can be grown in a defined medium. The requirements for amino acids, vitamins, purine, and heme were similar to those established for Crithida fasciculata. Growth is stimulated by aeration. Generation time is longer than for any other insect trypanosomatid described; its utilization of lactose as a carbon source is a unique feature among trypanosomatids.     

First Defined Media for Leptomonas spp. from Insects*

SYNOPSIS. Defined media for Leptomonas collosoma, L. mirabilis, and L. sp. from Dysdercus are described. In addition to factors required by Crithidia spp., including a source of Crithidia factor, all 3 organisms require glycine. Leptomonas collosoma and L. sp. from Dysdercus require choline. All trypanosomatids thus far studied require calcium pantothenate but L. collosoma has an especially large requirement. Leptomonas mirabilis but not L. sp. from Dysdercus can synthesize methionine from homocysteine thiolactone during growth. Homocysteine thiolactone is toxic to L. sp. from Dysdercus. Methionine plus vitamin B₁₂ partly annul homocysteine thiolactone toxicity but only if glycine is also present.     

Leptomonas seymouri sp. n. from the cotton stainer Dysdercus suturellus*

SYNOPSIS. Leptomonas seymouri sp. n., isolated from Dysdercus suturellus (Hemiptera: Pyrrhocoridae) from Florida, is described and distinguished from other species of Leptomonas from closely related hosts.     

Trypanosomatidae Isolated from Zelus leucogrammus (Perty, 1834) (Hemiptera,Reduviidae), with a Discussion on Flagellates of Insectivorous Bugs*

The insectivorous bug Zelus leucogrammus (Perty, 1834) (Hemiptera, Reduviidae) was found to harbor several species of trypanosomatids of the genera Leptomonas, Crithidia, Herpetomonas and Blastocrithidia. However, laboratory bred nymphs given sterile food were infected only by Blastocrithidia, the other flagellates appearing in their intestinal tract when they were fed on other insects. The Blastocrithidia was thought to belong to a single species and to be a parasite proper of Z. leucogrammus (and possibly other Hemiptera), producing luxuriant infections in the intestinal tract of the bugs captured in nature and spreading easily among isolated populations through cannibalism and ingestion of cysts; it was also abundant in the genital tract of gravid females but transovarian transmission was not proved. This Blastocrithidia experimentally infected Triatoma infestans and is very close to B. triatomae Cerisola, del Prado, Rohwedder and Bozzini, 1971. The other trypanosomatids, never numerous in naturally infected Z. leucogrammus, are acquired by the bug from many different insects, chiefly Diptera, on which it was seen to feed. Herpetomonas muscarum and C. luciliae, the common trypanosomatids among several muscoid Diptera, were isolated both from flies and from the bugs fed on them.     

High Prevalence and Endemism of Trypanosomatids on a Small Caribbean Island

We describe the monoxenous trypanosomatids parasitizing true bugs and flies on the island of Curaçao. Out of 248 examined true bugs belonging to 17 species, 93 individuals were found to be infected (overall 38% prevalence) by at least one trypanosomatid species (referred to as typing units; TUs). Out of 80 flies, six were infected. All detected trypanosomatids were compared based on their 18S rRNA sequences with TUs parasitizing bugs and flies described from mainland South America, allowing us to assess their diversity and distribution. Besides Leptomonas pyrrhocoris and Leptomonas seymouri, two known species of the subfamily Leishmaniinae, our analysis revealed six new TUs falling into the groups ‘jaculum’, Blastocrithidia and Herpetomonas. Moreover, two new members of the genus Phytomonas and three new TUs belonging to the monophyletic group designated as ‘new clade II’ sensu Mol. Phylogenet. Evol, 69, 255 (2013) were isolated. The detected trypanosomatids were characterized by moderate diversity (13 TUs) species richness. Out of nine and four TUs from the heteropteran and dipteran hosts, respectively, 11 TUs have not been encountered before. Although a sampling bias may partially affect the comparison between trypanosomatid communities on Curaçao and the mainland, the high proportion of unique TUs from the former location suggests that the prominent role of islands in increasing the global diversity of macroscopic organisms may also extend to their protistan parasites.     

Two New Species of Trypanosomatid Parasites Isolated from Heteroptera in Costa Rica

ABSTRACT. Two new trypanosomatid species (Euglenozoa, Kinetoplastea) isolated from the intestinal tract of heteropteran insect hosts were described based on molecular phylogenetic analyses of Spliced Leader (SL) RNA gene repeats, glycosomal glyceraldehyde phosphate dehydrogenase, and small subunit ribosomal RNA genes, as well as by morphology. Leptomonas barvae n. sp., from a mirid host Collaria oleosa, was found to represent one of the closest monoxenous (one host) relatives of the dixenous (two hosts) parasitic genus Leishmania. This finding further supports the origin of these dixenous parasites from monoxenous progenitors in the Neotropics. Blastocrithidia largi n. sp., from a largid host Largus cinctus, is among a few members of this genus available in culture. The species is a close relative of Blastocrithidia triatomae and is a member of a new monophyletic phylogenetic group characterized by formation of straphanger cysts.     

Ultrastructural study of the relations between Leptomonas oncopelti (Noguchi and Tilden), protozoa trypanosomatidae,and the rectal wall of adults of Oncopeltus fasciatus dallas,hemiptera lygaeidae

A laboratory colony of Oncopeltus fasciatus was found to be infected by Leptomonas oncopelti. The flagellates form a carpet attached to the cuticular intima of the rectal glands of adult bugs. The epithelial cells of these glands are characterized by infolded apical plasma membranes associated with mitochondria; the overlying cuticular intima shows endocuticular canals. The Leptomonas are attached by hemidesmosomes, located most often at the tip of the flagella. The Protozoa multiply by budding and the resultant straphangers cling to the parental flagellum. Adhesion of the flagellates to the cuticular lining is so strong that detaching flagellates carry with them the outer part of the epicuticle. Epicuticle repair presumably occurs through the endocuticular canals.     

Electrophoretic Analysis of Endonuclease-Generated Fragments of k-DNA,of Esterase Isoenzymes,and of Surface Proteins as Aids for Species Identification of Insect Trypanosomatids1

In order to verify the applicability of biochemical methods for species identification of Trypanosomatidae, 13 species of monoxenic trypanosomatids plus the heteroxenous Trypanosoma cruzi were comparatively analyzed by three different biochemical methods. Insect trypanosomatids examined were: Crithidia acanthocephali, C. fasciculata (three varieties), C. luciliae luciliae, C. luciliae thermophila, C. deanei, C. oncopelti, Herpetomonas muscarum muscarum, H. megaseliae, H. samuelpessoai, H. mariadeanei, Leptomonas seymouri, L. collosoma, L. samueli, and Blastocrithidia culicis. Also included in the survey were aposymbiotic strains of C. deanei and C. oncopelti. Methods used were: electrophoretic profiling of endonuclease-generated fragments of k-DNA, esterase isoenzymes profiling, and polyacrylamide-gel electrophoresis (SDS-PAGE) of radioiodinated cell surface proteins. Interspecific but not intraspecific differences were detected by all three methods among the 13 monoxenic species examined. Thus, it is concluded that these methods can be successfully used, in addition to classical criteria, for species identification of insect trypanosomatids.     

Intranuclear Parasitism of the Ciliate Euplotes by a Trypanosomatid Flagellate

A trypanosomatid flagellate, Leptomonas sp., develops and multiplies in the macronucleus (only) of natural and laboratory-reared populations of the ciliate Euplotes. Up to 90% of the natural populations of Euplotes in our test pond had such nuclear infections. Laboratory infections were transmitted to this ciliate by feeding it liberated parasites. Paramecium resisted infections. All laboratory-induced infections were lethal to Euplotes, while control clones of the uninfected ciliates remained viable. This leptomonad, unlike Leptomonas karyophilus (found in Paramecium), shows no leishmanial forms in its several ciliate hosts and shows a varied pattern of locomotion.     

Larval habitats of sand flies in rural areas of southern Brazil

We report the results of an investigation of natural larval sand fly habitats in the Recanto Marista, Doutor Camargo municipality, Paraná state, Brazil, from May, 2010 to August, 2012. We used Alencar emergence traps (AT), experimental traps (ET), and soil samples incubated in a biochemical oxygen demand incubator. Eight sand flies were collected with ATs. One specimen was collected with an ET and 21 were collected in soil samples. The collected species were Brumptomyia brumpti, Micropygomyia ferreirana, Migonemyia bursiformis, Migonemyia migonei, Nyssomyia neivai, Nyssomyia whitmani, and Pintomyia pessoai. The laval habitats of sand flies were located in the Recanto Marista, especially between tree roots, but the number of adults that emerged in the traps and soil samples was small despite the high density of sand flies that has been recorded in the Recanto Marista.     

Conserved repeats in the kinetoplast maxicircle divergent region of Leishmania sp. and Leptomonas seymouri

The maxicircle control region [also termed divergent region (DR)] composed of various repeat elements remains the most poorly studied part of the kinetoplast genome. Only three extensive DR sequences demonstrating no significant similarity were available for trypanosomatids (Leishmania tarentolae, Crithidia oncopelti, Trypanosoma brucei). Recently, extensive DR sequences have been obtained for Leishmania major and Trypanosoma cruzi. In this work we have sequenced DR fragments of Leishmania turanica, Leishmania mexicana, Leishmania chagasi and two monogenetic trypanosomatids Leptomonas seymouri and Leptomonas collosoma. With the emergence of the additional extensive sequences some conserved features of DR structure become evident. A conserved palindromic sequence has been revealed in the DRs of the studied Leishmania species, L. seymouri, and T. cruzi. The overall DR structure appears to be similar in all the Leishmania species, their relative L. seymouri, and T. brucei: long relatively GC-rich repeats are interspersed with clusters of short AT-rich repeats. C. oncopelti, L. collosoma, and T. cruzi have a completely different DR structure. Identification of conserved sequences and invariable structural features of the DR may further our understanding of the functioning of this important genome fragment.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at Nucleotide sequence data reported in this paper are available in the GenBank™, EMBL and DDBJ databases under the accession numbers DQ107351, DQ107352, DQ107354-DQ107358, DQ239759-DQ239765, DQ492251-DQ492256.     

Five Trypanosomatid Species of Insects Distinguished by Isoenzymes*

SYNOPSIS Blastocrithidia culicis, Crithidia deanei, Crithidia fasciculata, Herpetomonas samuelpessoai, Leptomonas seymouri and Leishmania tarentolae grown in cultures were compared by electrophoretic mobility for isoenzymes in 6 enzymes. All species were found distinct in these characteristics. Endosymbiotic C. deanei, which was identical to the aposymbiotic C. deanei in 5 enzymes, had an extra band in aspartate aminotransferase. No differences in isoenzymes were found between members of one species maintained in 2 different culture media.     

Fine structure and cytochemistry of peroxisomes (microbodies) in Leptomonas samueli

Summary Leptomonas samueli possesses in its cytoplasm a membrane-bounded organelle which can reach a length of 2.8 m and a diameter of 0.2 m. Catalase activity, which is inhibited by 3-amino-1, 2, 4-triazole, was detected at the ultrastructural level in the matrix of the organelle by using an alkaline diaminobenzidine medium. Freeze-fracture studies showed the presence of a large number of intramembranous particles on both the P and the E faces of the membrane of the organelle. Based on these data as well as on previous observations, it is suggested that the trypanosomatids possess an organelle that can be considered to be a peroxisome.     

Guidelines for the Description of New Species of Lower Trypanosomatids1

Morphological, cultural, and biochemical criteria that have been used in describing lower trypanosomatids, genera Blastocrithidia, Crithidia, Leptomonas, Herpetomonas, Rhynchoidomonas, and Phytomonas are reviewed. Kinetoplast structure, carbohydrate utilization, electrophoretic mobilities of isoenzymes, and kDNA fingerprinting are among the recommended criteria for species differentiation. Temperature, pH, and osmolarity tolerance are useful growth parameters. Generic placement may be assisted by the determination of nitrogenous excretion products and ornithine-arginine cycle enzymes.