Preliminary description of a new entomoparasitic nematode infecting Lutzomyia longipalpis sand fly,the vector of visceral leishmaniasis in the New World

Phlebotomine sandflies are vectors of important pathogens world-wide, including Leishmania spp. in the Neotropics. Entomoparasites have been described from phlebotomines, including virus, bacteria, protozoa, fungi, nematodes, and mites, some of which are capable of killing the host. In the present study, interference, fluorescence, and scanning electron microscopies were used for the first time to detect and morphologically characterize a new entomoparasite infecting Lutzomyia longipalpis. Several filiform larvae and eggs in different stages were encountered in the abdomen of female and male insects. Pairs of large egg-bearing nematodes found within cyst-like structures or free in the hemocel accompanied by larvae could be the adult sexual stages. This entomoparasite infects sand flies naturally in the field. We believe that stress caused by the colonization procedure produced an increase in the infection rate among sand flies affecting their development. These findings could be applied to future biological control studies of sand fly vectors.     

Midgut pH profile and protein digestion in the larvae of Lutzomyia longipalpis (Diptera: Psychodidae)

The sand fly Lutzomyia longipalpis is the vector of Leishmania infantum, the etiological agent of American visceral leishmaniasis. Despite its importance, until now the internal anatomy of the immature forms has never been described and little is known about their digestive processes. In nature, sand fly larvae feed on organic detritus in the soil, constantly ingesting large amounts of material. The objective of this study was to describe the anatomy of the gut and the pH of the gut lumen, as well as to investigate the proteases responsible for protein digestion. The larvae have a short gut with a prominent, well-developed midgut. Ingestion of food containing indicator dyes permitted the gut pH to be measured. A pH gradient was observed, varying from >9 in the anterior midgut to 6.5-7.0, in the posterior midgut. The endoproteolytic enzymes are secreted in the anterior midgut and are able to digest azocasein over a large pH range, specially at pH 11. Studies with various inhibitors indicated that the digestive endoproteases are trypsin- and chymotrypsin-like enzymes. These results were confirmed by using the substrates BApNA and N-CBZ-L-PpNA, specific for trypsin and chymotrypsin, respectively. Aminopeptidases were also investigated with p-nitroaniline-derived substrates. These enzymes are located in the posterior midgut, bound to the membranes and functioning at an optimal pH of 6.5-8.0. The results presented here are consistent with the current proposal that proteins are digested to peptides in the anterior midgut inside the endoperitrophic space and subsequently undergo digestion in the ectoperitrophic space of the posterior midgut.     

Trypanosoma thecadactyli sp. n. from Forest Geckoes in Panama,and its Development in the Sandfly Lutzomyia trinidadensis (Newstead) (Diptera,Psychodidae)*

SYNOPSIS. Trypanosoma thecadactyli sp. n. is described from the forest gecko Thecadactylus rapicaudus in Panama. Blood smears from 18 of 25 (72%) geckoes sampled were positive for the new trypanosome. The flagellate, ～22 μm long, is ovoid or roughly triangular in shape and has a posterior cytoplasmic projection. Tree buttresses, rock crevices, and caves are microhabitats shared by T. rapicaudus and the phlebotomine sandfly Lutzomyia trinidadensis. Approximately 20% of the wild-caught and dissected L. trinidadensis were infected with flagellates. Twenty-four of 45 (53%) laboratory-reared L. trinidadensis allowed to feed on parasitized geckoes developed heavy infections of the mid- and hindgut which persisted up to 2 weeks, the longest postprandial period before dissection. Transmission probably occurs as a result of geckoes feeding on infected sandflies in nature. The distribution of L. trinidadensis, from Mexico to Brazil, approximates that of the gecko. Laboratory results and ecologic observations implicate L. trinidadensis as a potential vector of T. thecadactyli.     

Ultrastructural description of the life cycle of Nosema monorchis n. sp. (Microspora, Nosematidae), hyperparasite of Monorchis parvus (Digenea, Monorchiidae), intestinal parasite of Diplodus annularis (Pisces, Teleostei)

Life cycle stages of a new species of the genus Nosema Naegeli, 1857 (Microspora, Nosematidae), were examined by light and electron microscopy. It parasitizes the gut and the uterus of the digenean Monorchis parvus (Monorchiidae), in Diplodus annularis (Pisces, Teleostei). All stages were in close contact with the cytoplasm of the host cell and were probably all diplokaryotic. The divisions of meronts and sporonts were recognizable by the formation of spindle plaques at the surface of the nucleus. Spores were oval, measured 3.2±0.3×2.5±0.2 μm on ultrathin sections, and had a polar filament with 16–17 coils. The polaroplast presented two parts: an anterior region with closely packed lamellae and a posterior part with wider lamellae. This Nosema species is compared with the other microsporidian parasites of digeneans. This new species is named Nosema monorchis n. sp., after the generic name of its host.     

Ultrastructure of Triangulamyxa amazonica n. gen. and n. sp. (Myxozoa, Myxosporea), a parasite of the Amazonian freshwater fish, Sphoeroides testudineus (Teleostei, Tetrodontidae)

Triangulamyxa amazonica n. gen. and n. sp. (Myxozoa, Ortholineidae), found in the lumen of the intestine of the freshwater fish Sphoeroides testudineus, is described. The fish were collected from the Amazon River near the city of Algodoal, State of the Pará, Brazil. Numerous irregular plasmodia containing different stages of sporogony, including spores, were observed. The plasmodia were lying free in the lumen or had slender pseudopodia-like cytoplasmic processes in contact with intestinal epithelial cells with microvilli projections. Spores, which are equilaterally triangular in valvar view with rounded pointed ends and ellipsoidal in transverse section, are 8.5 μm long, 7.6 μm wide, and 3.8 μm thick. The anterior end of the spores contains two equal drop-shaped polar capsules measuring 2.6 μm in length, each having an isofilar polar filament with 5–6 turns. The characteristics of the spore shape, the spore wall structure and its ridge organization, the plasmodial characteristics and the identity of the host suggest that the parasite is a new genus and species, which is herein designated T. amazonica.     

Phyllodiaptomus praedictus n. sp. (Copepoda,Calanoida) from Thailand

Phyllodiaptomus praedictus n. sp. is described from the Bangkok area. It might be endemic to Thailand. It belongs to the blanci-group of species, and is most closely related to P. annae and P. wellekensae.     

Evaluation of the fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes), a potential biological control agent of Lutzomyia longipalpis (Diptera, Psychodidae)

Visceral leishmaniasis is a zoonosis whose primary vector in Brazil is the sandfly Lutzomyia longipalpis Lutz & Neiva. Presently, efforts to control the vector have not been effective in reducing the prevalence of disease. A possible alternative to current strategies is the biological control of the vector using entomopathogenic fungi. This study evaluates the effects of the fungus, Beauveria bassiana (Bals.) Vuilleman, in different developmental stages of L. longipalpis. Five concentrations of the fungus were utilized ranging from 10⁴ to 10⁸ conidia/ml, with appropriate controls. The unhatched eggs, larvae and dead adults exposed to B. bassiana were sown to reisolate the fungus. The fungus was subsequently identified by polymerase chain reaction (PCR) and DNA sequencing. Exposure to B. bassiana reduced the number of eggs that hatched by 59% (P < 0.01). The longevity of infected adults was 5 days, significantly lower than that of the negative control which was 7 days (P < 0.001). The longevity of the adult sandfly exposed to the positive chemical (pyrethroid, cypermetherin) control was less than 1 day. The effects of fungal infection on the hatching of eggs laid by infected females were also significant and dose-dependent (P < 0.05). With respect to fungal post-infection growth parameters, only germination and sporulation were significantly higher than the fungi before infection (P < 0.001). The identity of the reisolated fungus was confirmed by automated DNA sequencing post-passage in all insect stages. These data show that B. bassiana has good pathogenic potential, primarily on L. longipalpis larvae and adults. Consequently, the use of this fungus in sandfly control programs has potential in reducing the use of chemical insecticides, resulting in benefits to humans and the environment.     

Eodiaptomus shihi n. sp. (Copepoda,Calanoida) from central India

Eodiaptomus shihi n. sp. is described from the material collected from Gandhisagar reservoir, River Narmada and a hillpool in the Madhya Pradesh State of central India. The salient features of this new species include: in female, the left metasomal wing is strongly developed, and the endopodite in leg 5 is 2-segmented and longer than first exopodite-segment; in male, the right caudal ramus is armed with a tooth-like chitinous structure near ventro-distal region, and in right leg 5, the basis is devoid of any hyaline lobe on inner margin and the first exopodite-segment is produced into a large spinous process at disto-outer corner.     

Cryptosporidium molnari n. sp. (Apicomplexa: Cryptosporidiidae) infecting two marine fish species,Sparus aurata L. and Dicentrarchus labrax L

Cryptosporidium molnari n. sp. is described from two teleost fish, the gilthead sea bream (Sparus aurata L.) and the European sea bass (Dicentrarchus labrax L.). The parasite was found mainly in the stomach epithelium and seldom in the intestine. Oocysts were almost spherical, with four naked sporozoites and a prominent residuum, and measured 3.23-5.45 x 3.02-5.04 (mean 4.72 x 4.47) microm in the type host, gilthead sea bream (shape index 1-1.17, mean 1.05). Sporulation was endogenous, as fully sporulated oocysts were found within the fish, both in the stomach epithelium and lumen, and in faeces. Oocysts and other stages of C. molnari fit most of the diagnostic features of the genus Cryptosporidium, but differ from hitherto described species, including piscine ones. All stages were located within a host contributed parasitophorous vacuole lined by a double host microvillar membrane. Merogonial and gamogonial stages appeared in the typical extracytoplasmic position, whereas oogonial and sporogonial stages were located deeply within the epithelium. Ultrastructural features, including the characteristic contact zone of the parasite with the host epithelial surface, were mostly coincident with those of other Cryptosporidium spp. Mitochondria were found in dividing meronts, merozoites, microgamonts and sporozoites. Pathological effects were more evident in gilthead sea bream, which also exhibited a clearly higher prevalence (24.4 versus 4.64% in sea bass). External clinical signs, consisting of whitish faeces, abdominal swelling and ascites, were rarely observed, in contrast with important histopathological damage. The wide zones of epithelium invaded by oogonial and sporogonial stages appeared necrotic, with abundant cell debris, and sloughing of epithelial cells, which detached to the lumen. No inflammation reaction was observed and the cellular reaction was limited to the cells involved in the engulfing of intraepithelial stages and debris, probably macrophages.     

Genetic diversity and phylogenetic position of the subclass Astomatia (Ciliophora) based on a sampling of six genera from West African oligochaetes (Glossoscolecidae, Megascolecidae), including description of the new genus Paraclausilocola n. gen

To more confidently assess phylogenetic relationships among astome ciliates, we obtained small subunit (SSU) rRNA sequences from nine species distributed in six genera and three families: Almophrya bivacuolata, Eudrilophrya complanata, Metaracoelophrya sp. 1, Metaracoelophrya sp. 2, Metaracoelophrya intermedia, Metaradiophrya sp., Njinella prolifera, Paraclausilocola constricta n. gen., n. sp., and Paraclausilocola elongata n. sp. The two new species in the proposed new clausilocolid genus Paraclausilocola n. gen. are astomes with no attachment apparatus, two files of contractile vacuoles, and an arc-like anterior suture that has differentiations of thigmotactic ciliature on the anterior ends of the left kineties of the upper surface. Phylogenetic analyses were undertaken using neighbor-joining, Bayesian inference, maximum likelihood, and maximum parsimony. The nine species of astomes formed a strongly supported clade, showing the subclass Astomatia to be monophyletic and a weakly supported sister clade to the scuticociliates. There were two strongly supported clades within the astomes. However, genera assigned to the same family were found in different clades, and genera assigned to the same order were found in both clades. Thus, astome taxa appear to be paraphyletic when morphology is used to assign species to genera.     

Arctodiaptomus (Rhabdodiaptomus) michaeli n.sp. (Copepoda,Calanoida) from Kashmir,India

Arctodiaptomus (Rhabdodiaptomus) michaeli n.sp. is described from the material collected in Alapathar Lake and Marsar Lake of Kashmir, India.     

The ultrastructure and reproduction of Amphiamblys capitellides (Microspora, Metchnikovellidae), a parasite of the gregarine Ancora sagittata (Apicomplexa, Lecudinidae), with redescription of the species and comments on the taxonomy

The ultrastructural cytology and reproduction of the hyperparasitic microsporidium Amphiamblys capitellides (Caullery and Mesnil, 1897) is described. Merogonial reproduction was not observed. The sporogony comprises two sequences: a sac-bound sporogony in close contact with the cytoplasm of the host and a free sporogony in parasitophorous vacuoles. The free sporogony, which probably precedes the sac-bound, yields a small number of rounded spores. The sac-bound sporogony is polysporoblastic, generating two rows of elongated spores. All stages have isolated nuclei. Both spore types have an extrusion apparatus of the metchnikovellidean type, with a polar sac devoid of anchoring disc, a polar filament with one manubroid and one bulbous part, and a posterior semicircular membrane fold enclosing rounded or tubular structures. Hosts are gregarines of the species Ancora sagittata living in the intestine of polychaetes of the genus Capitella, probably the species Capitella giardi. The cytology, life cycle and classification are discussed. The species is redescribed and the diagnosis of the genus Amphiamblys Caullery and Mesnil, 1914 is emended.     

Tasmanomysis oculata n.g. n.sp. from Tasmanian waters (Crustacea: Mysidacea: Mysini)

Tasmanomysis oculata n.g. n.sp. belonging to the Tribe Mysini is described from the coastal waters of southern Tasmania. This new genus superficially resembles the monospecific genus Arthromysis Colosi,1924 known only from the Straits of Magellan. The form of the pleopods in the male together with the segmentation of the thoracic legs and shape of the telson clearly distinguish the two genera.     

The sandfly fauna,anthropophily and the seasonal activities of Pintomyia spinicrassa (Diptera: Psychodidae: Phlebotominae) in a focus of cutaneous leishmaniasis in northeastern Colombia

This study was conducted to identify the sandfly fauna and the anthropophilic species in a coffee-growing area of Villanueva, Norte de Santander, Colombia, a focus of American cutaneous leishmaniasis, and to analyse the relationship between the most frequent species and rainfall, relative humidity and temperature, with the aim of contributing to epidemiological surveillance in the area. Sandfly collections were performed fortnightly between February 2006-September 2007 using automatic light traps, Shannon traps, protected human bait and aspiration in resting places. A total of 7,051 sandflies belonging to 12 species were captured. Pintomyia spinicrassa (95.7%) predominated. Pintomyia oresbia and Lutzomyia sp. of Pichinde were found in the state of Norte de Santander for the first time. Pi. spinicrassa, Pintomyia nuneztovari, Micropygomyia venezuelensis, Lutzomyia (Helcocyrtomyia) scorzai and Lu. (Helcocyrtomyia) sp. were captured on the protected human bait. A significant association between Pi. spinicrassa abundance and the total rainfall and the average temperature and humidity 10 days before the collection was observed. The dominance of Pi. spinicrassa, a recognised vector of Leishmania braziliensis, especially during the dry periods, indicates that the risk of parasite transmission may increase.     

Lecane segersi n.sp. (Rotifera, Lecanidae) from Thailand

Lecane segersi n.sp. collected from a swamp in Udornthani province, northeastern part of Thailand, is described and figured. It is closely related to the common, warm-stenotherm Lecane papuana (Murray), but distinguished by the presence of inwardly directed antero-lateral spines.