Second Blood Meal by Female Lutzomyia longipalpis: Enhancement by Oviposition and Its Effects on Digestion, Longevity, and Leishmania Infection.

Lutzomyia longipalpis is the main vector of visceral leishmaniasis (VL) in America. Physiological and molecular mechanisms of Leishmania infection in sand flies have been studied during the first gonotrophic cycle. There are few studies about these interactions during the second gonotrophic cycle mainly because of the difficulties maintaining sand flies through sequential feeds. Here we standardized conditions to perform the second blood feed efficiently, and our results show that oviposition is an essential factor for the success of multiple feeds. We evaluated the impact of the second blood meal on longevity, protein digestion, trypsin activity, and Leishmania mexicana development within L. longipalpis gut. Mortality of blood-fed females increases after second blood meal as compared to sugar-fed females. Trypsin activity was lower during the second gonotrophic cycle. However, no difference in protein intake was observed between blood meals. There was no difference in the population size of Leishmania in the gut after both blood meals. In this work, we presented an optimized protocol for obtaining sufficient numbers of sand fly females fed on a second blood meal, and we described some physiological and parasitological aspects of the second gonotrophic cycle which might influence the vectorial competence of sand flies.

The role of promastigote secretory gel in the origin and transmission of the infective stage of Leishmania mexicana by the sandfly Lutzomyia longipalpis.

Transmission of leishmaniasis is effected by a specific developmental stage, the metacyclic promastigote. The precursors of metacyclic promastigotes were a distinct subpopulation of parasites, identified for the first time as a new stage in the life-cycle and named leptomonad promastigotes. Microdissection of infected sandflies into 4 midgut regions and foregut allowed precursor-product relationships to be established for amastigote-procyclic promastigote, procyclic-nectomonad promastigote, nectomonad-leptomonad promastigote and leptomonad-metacyclic promastigote developmental switches. Metacyclic promastigotes occurred mainly in the thoracic midgut and cardia, coincident with the accumulation of a promastigote secretory gel (PSG) plug in these anterior regions. The gel-like plug was isolated from flies with mature infections and found to contain predominantly leptomonad promastigotes. The PSG plug also contained the majority (75%) of the total metacyclic promastigote population in the sandflies, which were concentrated at the anterior pole. The PSG plug was found to be the main site of metacyclogenesis, and acted as a reservoir of leptomonad promastigotes from which metacyclic forms differentiated and migrated forward to promote the infective potential of the fly. The PSG plug occluded and distorted the midgut, forcing the stomodeal valve open and affecting the feeding success of the sandflies, such that they experienced difficulty in taking a full meal. Collectively, these data support the role of the PSG in the transmission of leishmaniasis, by conditioning the midgut environment for metacyclogenesis and altering the feeding ability of infected sandflies.

Transformation of Leishmania mexicana metacyclic promastigotes to amastigote-like forms mediated by binding of human C-reactive protein.

Infective metacyclic promastigote forms of Leishmania mexicana are introduced by the bite of sandfly vectors into their human hosts where they transform into the amastigote form. The kinetics of this process was examined in vitro in response to different combinations of temperature (26 degrees C or 32 degrees C), pH (7.2 or 5.5), and exposure to human serum. Little transformation occurred at 26 degrees C/pH 7.2, intermediate levels at 26 degrees C/pH 5.5 and 32 degrees C/pH 7.2, and the greatest response at 32 degrees C/pH 5.5. Transformation was stimulated by exposure to normal human serum, but was markedly reduced when serum previously incubated at 56 degrees C for 1 h was used (complement heat-inactivated). This stimulatory effect was reproduced by exposure to a single purified component of human serum, C-reactive protein (CRP). Binding of CRP to the whole surface of L. mexicana metacyclic promastigotes, including the flagella, was demonstrated by an indirect fluorescent antibody test. The effect of purified CRP was dose dependent and occurred using normal serum concentrations. The stimulatory effect of whole serum was oblated by CRP depletion and restored by addition of purified CRP. The effects of cAMP analogues indicated that transformation could be mediated via an adenylate cyclase cascade.

Transformation, development, and transmission of axenically cultured amastigotes of Leishmania mexicana in vitro and in Lutzomyia longipalpis.

Axenic cultures of Leishmania mexicana amastigotes were transformed to promastigotes in vitro and in vivo in Lutzomyia longipalpis. In vitro, both exponential phase and stationary phase amastigotes were capable of transforming and growing as promastigotes, but exponential phase amastigotes completed this transition more quickly. In vivo, both populations were capable of establishing infections in sand flies by membrane feeding and could be transmitted to BALB/c mice via bite. A variety of morphologic forms could be observed in vivo, including putative metacyclic promastigotes. Infection rates in sandflies with axenic amastigotes were comparable with those achieved with lesion-derived amastigotes, supporting the use of these cultured forms in studies of parasite biology.

In vitro stimulation of metacyclogenesis in Leishmania braziliensis, L. donovani, L. major and L. mexicana.

Promastigotes of Leishmania braziliensis, L. donovani, L. major and L. mexicana recently derived from tissue amastigotes were cultured in Schneider's Drosophila medium supplemented with 20% (v/v) heat-inactivated foetal calf serum and 25 micrograms gentamicin sulfate/ml at pH 5.5. These cultures produced more metacyclic promastigotes in their stationary-phase populations than others cultured at pH 7.0. Metacyclic promastigotes possessed a short (< or = 8 microns) and narrow (< or = 1.5 microns) cell body with a flagellum twice or more the length of the cell body. Promastigotes from acidic cultures were more resistant to complement-mediated lysis and more infective in vivo than those grown at neutral pH. These results demonstrate that induction of metacyclogenesis by acidic pH is a response conserved across a variety of species of Leishmania.

Phenol red method for measuring the pH of the gut contents in Lutzomyia longipalpis (Psychodidae:Diptera).

AIM: To develop a method to estimate the pH of the gut contents of female sandflies using a microcapillary feeding technique. METHODS: Female Lutzomyia longipalpis were fed with a small quantity of phenol red solution (indicator range pH 6.8-8.4) before and after a bloodmeal. The colour patterns of the gut contents were recorded by video microscopy immediately after the alimentary canal was dissected out of the sandfly body, and used to determine the pH level. RESULTS: In unfed flies the thoracic mid-gut (TMG) is normally neutral, with the pH ranging between 7.0 to 7.3; and the abdominal mid-gut(AMG) is mildly alkaline from pH 7.1 to 8.4 with the maximum pH observed at the junction with the hind-gut. The presence of sugar in the crop reduced the pH of the TMG to 6.8, and the presence of a recently ingested bloodmeal raised the pH of the TMG to 7.4. However, as bloodmeal digestion proceeded the pH of the TMG was reduced to acidic levels, pH 6.8 or below. CONCLUSION: The new method could be integrated with the investigation of metacyclogenesis of Leishmania parasites in vivo.

Expression of cysteine proteinases by metacyclic promastigotes of Leishmania mexicana.

The expression of cysteine proteinases by metacyclic promastigotes of Leishmania mexicana was investigated using gelatin polyacrylamide gel electrophoresis. Two prominent bands were detected which distinguished metacyclics from multiplicative promastigotes, lacking detectable cysteine proteinase activity, and amastigotes, with a distinct banding pattern composed of multiple enzymes. A correlation between relative activity of the metacyclic-specific bands and the prevalence of metacyclics was found both during the growth cycle in vitro as metacyclogenesis occurred, and by comparison of stationary phase populations from consecutive subpassages in vitro. Irreversible inhibition of the metacyclic activities using N-benzyloxycarbonyl-phenylalanyl-alanyl diazomethane did not inhibit metacyclic to amastigote transformation in vitro. These activities provide a useful biochemical marker for the metacyclic promastigotes of L. mexicana.

Null mutants for the lmcpa cysteine proteinase gene in Leishmania mexicana.

The parasitic protozoon Leishmania mexicana possesses an abundance of developmentally regulated cathepsin L-like cysteine proteinases expressed at highest levels in amastigotes. We recently characterised lmcpa, a single-copy gene encoding one such proteinase, LmCPa, which differs from other homologues by possessing a 3-amino-acid insertion at the amino terminal of the predicted mature proteinase. To investigate the role of LmCPa in L. mexicana, we used gene-targeting of promastigotes with hygromycin- and phleomycin-resistance markers to generate null mutants by disrupting sequentially both alleles of lmcpa. The promastigote null mutants did not differ significantly from wild-type L. mexicana in growth rate or morphology and could differentiate to metacyclics and the amastigote-like form, both of which could infect the J774G8 macrophage-like cell line. The null mutant amastigote-like form obtained from the J774G8 cells could also establish rump lesions in CBA mice. By these criteria, therefore, LmCPa appears to be non-essential although there is the possibility that LmCPa could be required during development in the sandfly, a stage not analysed here. The apparent redundancy of LmCPa in amastigotes may be due to the presence of other cysteine proteinases and has implications for the choice of candidate targets for rationally designed anti-leishmanial drugs.

Complete developmental cycle of Leishmania mexicana in axenic culture.

A complete developmental sequence of Leishmania mexicana has been produced in axenic culture for the first time. This was achieved by manipulation of media, pH and temperature conditions over a period of 16 days. All experiments were initiated with lesion amastigotes that were transformed to multiplicative promastigotes by culture in HOMEM, 10% foetal calf serum, pH 7.5, at 25 degrees C. Metacyclogenesis was induced by subpassage in Schneider's Drosophila medium, 20% foetal calf serum, pH 5.5, and the resulting forms transformed to axenically growing amastigotes by subpassage in the same medium and raising the temperature to 32 degrees C. Parasites from each day were characterized with respect to their general morphology using light microscopy of Giemsa-stained smears, and biochemically by analysis of total protein content, proteinases, nucleases and secretory acid phosphatase. The results demonstrated that the three main stages identified--amastigotes, multiplicative promastigotes and metacyclic promastigotes--each exhibited the expected suite of biochemical properties. Further, the changes in morphology observed as the developmental sequence proceeded from stage to stage were accompanied by appropriate changes in biochemical properties. These results provide both useful biochemical markers and a culture system in which to examine the regulation of differentiation and transformation during the Leishmania life-cycle.

Leishmania mexicana: induction of metacyclogenesis by cultivation of promastigotes at acidic pH.

Cultivation of recently transformed Leishmania mexicana promastigotes at pH 5.5 in Schneider's Drosophila medium supplemented with 20% fetal calf serum produced a homogeneous stationary phase population morphologically similar to metacyclic forms. The cultured forms developed functional characteristics consistent with being metacyclic: they were resistant to complement-mediated lysis, possessed a discernable surface membrane coat in transmission electron micrographs, and were highly infective to peritoneal macrophages in vitro. In contrast, growth of promastigotes at pH 7.0 produced morphologically mixed populations of stationary phase promastigotes, but including a subpopulation with metacyclic-like morphology. These results provide a method for culturing pure populations of L. mexicana metacyclics and provide evidence that metacyclics are biochemically preadapted for survival at acidic pH as amastigotes in macrophage phagolysosomes.

Characterization of developmentally-regulated nucleases in promastigotes and amastigotes of Leishmania mexicana.

The parasitic protozoon Leishmania mexicana was examined for the presence of 3'-nucleotidase/nuclease using substrate SDS-PAGE. Two activities were detected: one with an apparent molecular mass of 40 kDa, and a doublet of 29/31 kDa. The two enzymes showed differences in their levels of expression in the two life-cycle stages of parasite examined: the 29/31 kDa doublet was detected at 60-fold higher levels in the pathogenic amastigote stage, but was also expressed by promastigotes, whereas the 40 kDa activity was only detected in promastigotes. Both were capable of hydrolysing a variety of substrates including 3'-AMP and poly(A). However, the 29/31 kDa form showed a broader, unique substrate specificity in that it was also capable of digesting double-stranded RNA and DNA.

Axenic cultivation and characterization of Leishmania mexicana amastigote-like forms.

A new method is described which has made possible the long-term axenic cultivation of Leishmania mexicana amastigote-like forms in Schneider's Drosophila medium supplemented with 20% (v/v) foetal calf serum. Unlike previous methods, it utilizes direct culture of parasites obtained from the lesions of infected animals rather than adaptation of promastigotes in vitro. Ultrastructural (possession of megasomes), biochemical (cysteine proteinase activity and gelatin SDS-PAGE banding pattern) and infectivity (in vivo) data are presented which show the close similarity of the cultured forms to lesion amastigotes. The axenically cultured forms grew optimally at a temperature of 32-33 degrees C, providing further evidence for their amastigote nature. It was found that adjustment of the pH of the growth medium to 5.4 was required in order to retain the amastigote morphology of the cultured parasites. This supports the notion that leishmanial amastigotes are acidophiles.