Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases.

BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.

Lundep, a sand fly salivary endonuclease increases Leishmania parasite survival in neutrophils and inhibits XIIa contact activation in human plasma.

Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET) were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule (Lundep) from the salivary glands of Lutzomyia longipalpis. Previous analysis of the sialotranscriptome of Lu. longipalpis showed the potential presence of an endonuclease. Indeed, not only was the cloned cDNA (Lundep) shown to encode a highly active ss- and dsDNAse, but also the same activity was demonstrated to be secreted by salivary glands of female Lu. longipalpis. Lundep hydrolyzes both ss- and dsDNA with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate)- or parasite-induced NETs by treatment with recombinant Lundep or salivary gland homogenates increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive (mutagenized) Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation.

Reactive oxygen species-mediated immunity against Leishmania mexicana and Serratia marcescens in the sand phlebotomine fly Lutzomyia longipalpis.

Phlebotomine sand flies are the vectors of medically important Leishmania. The Leishmania protozoa reside in the sand fly gut, but the nature of the immune response to the presence of Leishmania is unknown. Reactive oxygen species (ROS) are a major component of insect innate immune pathways regulating gut-microbe homeostasis. Here we show that the concentration of ROS increased in sand fly midguts after they fed on the insect pathogen Serratia marcescens but not after feeding on the Leishmania that uses the sand fly as a vector. Moreover, the Leishmania is sensitive to ROS either by oral administration of ROS to the infected fly or by silencing a gene that expresses a sand fly ROS-scavenging enzyme. Finally, the treatment of sand flies with an exogenous ROS scavenger (uric acid) altered the gut microbial homeostasis, led to an increased commensal gut microbiota, and reduced insect survival after oral infection with S. marcescens. Our study demonstrates a differential response of the sand fly ROS system to gut microbiota, an insect pathogen, and the Leishmania that utilize the sand fly as a vehicle for transmission between mammalian hosts.

Disruption of the peritrophic matrix by exogenous chitinase feeding reduces fecundity in Lutzomyia longipalpis females.

Lutzomyia longipalpis is the most important vector of visceral leishmaniasis in Brazil. When female sandflies feed on blood, a peritrophic matrix (PM) is formed around the blood bolus. The PM is secreted by midgut cells and composed of proteins, glycoproteins and chitin microfibrils. The PM functions as both a physical barrier against pathogens present in the food bolus and blood meal digestion regulator. Previous studies of mosquitoes and sandflies have shown that the absence of a PM, resulting from adding an exogenous chitinase to the blood meal, accelerates digestion. In the present study, we analysed biological factors associated with the presence of a PM in L. longipalpis females. Insects fed blood containing chitinase (BCC) accelerated egg-laying relative to a control group fed blood without chitinase. However, in the BCC-fed insects, the number of females that died without laying eggs was higher and the number of eggs laid per female was lower. The eggs in both groups were viable and generated adults. Based on these data, we suggest that the absence of a PM accelerates nutrient acquisition, which results in premature egg production and oviposition; however, the absence of a PM reduces the total number of eggs laid per female. Reduced fecundity in the absence of a PM may be due to inefficient nutrient conversion or the loss of the protective role of the PM.

Proteophosphoglycan confers resistance of Leishmania major to midgut digestive enzymes induced by blood feeding in vector sand flies.

Leishmania synthesize abundant phosphoglycan-containing molecules made up of [Gal-Man-PO(4)] repeating units, including the surface lipophosphoglycan (LPG), and the surface and secreted proteophosphoglycan (PPG). The vector competence of Phlebotomus duboscqi and Lutzomyia longipalpis sand flies was tested using L. major knockout mutants deficient in either total phosphoglycans (lpg2(-) or lpg5A(-)/5B(-)) or LPG alone (lpg1(-)) along with their respective gene add-back controls. Our results confirm that LPG, the major cell surface molecule of Leishmania promastigotes known to mediate attachment to the vector midgut, is necessary to prevent the loss of infection during excretion of the blood meal remnants from a natural vector, P. duboscqi, but not an unnatural vector, L. longipalpis. Midgut digestive enzymes induced by blood feeding pose another potential barrier to parasite survival. Our results show that 36-72 h after the infective feed, all parasites developed well except the lpg2(-) and lpg5A(-)/5B(-) mutants, which showed significantly reduced survival and growth. Protease inhibitors promoted the early survival and growth of lpg2(-) in the blood meal. PPG was shown to be the key molecule conferring resistance to midgut digestive enzymes, as it prevented killing of lpg2(-) promastigotes exposed to midgut lysates prepared from blood-fed flies. The protection was not associated with inhibition of enzyme activities, but with cell surface acquisition of the PPG, which appears to function similar to mammalian mucins to protect the surface of developing promastigotes against proteolytic damage.

Mechanisms of pH control in the midgut of Lutzomyia longipalpis: roles for ingested molecules and hormones.

Control of the midgut pH in Lutzomyia longipalpis enables the insect's digestive system to deal with different types of diet. Phlebotomines must be able to suddenly change from a condition adequate to process a sugar diet to one required to digest blood. Prior to blood ingestion, the pH in the midgut is maintained at ∼6 via an efficient mechanism. In the abdominal midgut, alkalization to a pH of ∼8 occurs as a consequence of the loss of CO(2) from blood (CO(2) volatilization) and by a second mechanism that is not yet characterized. The present study aimed to characterize the primary stimuli, present in the blood, that are responsible for shutting down the mechanism that maintains a pH of 6 and switching on that responsible for alkalization. Our results show that any ingested protein could induce alkalization. Free amino acids, at the concentrations found in blood, were ineffective at inducing alkalization, although higher concentrations of amino acids were able to induce alkalization. Aqueous extracts of midgut tissue containing putative hormones from intestinal endocrine cells slightly alkalized the midgut lumen when applied to dissected intestines, as did hemolymph collected from blood-fed females. Serotonin, a hormone that is possibly released in the hemolymph after hematophagy commences, was ineffective at promoting alkalization. The carbonic anhydrase (CA) enzyme seems to be involved in alkalizing the midgut, as co-ingestion of acetazolamide (a CA inhibitor) with proteins impaired alkalization efficiency. A general model of alkalization control is presented.

First report of an Onchocercidae worm infecting Psychodopygus carrerai carrerai sandfly, a putative vector of Leishmania braziliensis in the Amazon.

Sandflies are insects of public health interest due to their role as vectors of parasites of the genus Leishmania, as well as other pathogens. Psychodopygus carrerai carrerai is considered an important sylvatic vector of Leishmania (Viannia) braziliensis in Amazonia. In this study, sandflies were collected in a forested area in the Xapuri municipality, in the State of Acre (Northern Brazil). Two Ps. carrerai carrerai females were found parasitized with a larval form of a filarial worm, one in the labium of the proboscis, the other after the head was squashed, suggesting they were infective larvae. Sandflies were identified through morphological characters as well as amplification and sequencing of the cytochrome oxidase gene (COI). This was the first sequence obtained for Ps. carrerai carrerai for this marker. The obtained nematodes were also characterized through direct sequencing of a fragment of COI and 12S genes, both mitochondrial, and ITS1, a nuclear marker. Phylogenetic analyses revealed that the filarial nematodes belong to a species without sequences for these markers in the database, part of family Onchocercidade and closely related to genus Onchocerca (12S tree). Although sandfly infection with nematodes including members of the Onchocercidae has been reported in the Old World, this is the first report of sandfly infection by a member of the Onchocercidae family in the New World, to the best of our knowledge. Considering that the phylogenetic relationships and location in the insect, it can be expected that this is a parasite of mammals and the transmission cycle should be clarified.

Galactosamine reduces sandfly gut protease activity through TOR downregulation and increases Lutzomyia susceptibility to Leishmania.

In sandflies, males and females feed on carbohydrates but females must get a blood meal for egg maturation. Using artificial blood meals, this study aimed to understand how galactosamine interferes with sandfly digestive physiology. We also used galactosamine to manipulate the digestive physiology of Lutzomyia longipalpis to investigate its influence on sandfly digestion and Leishmania development within their insect vectors. Galactosamine was capable to reduce Lu. longipalpis trypsinolytic activity in a dose-dependent manner. This effect was specific to galactosamine as other similar sugars were not able to affect sandfly trypsin production. An excess of amino acids supplemented with the blood meal and 15 mM galactosamine was able to abrogate the reduction of the trypsinolytic activity caused by galactosamine, suggesting this phenomenon may be related to an impairment of amino acid detection by sandfly enterocytes. The TOR inhibitor rapamycin reduces trypsin activity in the L. longipalpis midgut. Galactosamine reduces the phosphorylation of the TOR pathway repressor 4EBP, downregulating TOR activity in the gut of L. longipalpis. Galactosamine reduces sandfly oviposition, causes an impact on sandfly longevity and specifically reduces sandfly gut proteases whereas increasing α-glycosidase activity. The administration of 15 and 30 mM galactosamine increased the number of promastigote forms of Le. mexicana and Le. infantum in galactosamine-treated L. longipalpis. Our results showed that galactosamine influences amino acid sensing, reduces sandfly gut protease activity through TOR downregulation, and benefits Leishmania growth within the Lu. longipalpis gut.

Inhibitor of cysteine peptidase does not influence the development of Leishmania mexicana in Lutzomyia longipalpis.

It has been proposed that the natural cysteine peptidase inhibitor ICP of Leishmania mexicana protects the protozoan parasite from insect host proteolytic enzymes, thereby promoting survival. To test this hypothesis, L. mexicana mutants deficient in ICP were evaluated for their ability to develop in the sand fly Lutzomyia longipalpis. No significant differences were found between the wild-type parasites, two independently derived ICP-deficient mutants, or mutants overexpressing ICP; all lines developed similarly in the sand fly midgut and produced heavy late-stage infections. In addition, recombinant L. mexicana ICP did not inhibit peptidase activity of the midgut extracts in vitro. We conclude that ICP has no major role in promoting survival of L. mexicana in the vectorial part of its life cycle in L. longipalpis.

Phlebotomine sandflies (Diptera: Psychodidae) of the genus Sergentomyia in Marrakech region, Morocco.

We reported the results of an entomological investigation in Marrakech area, in the aim to study the present Sergentomyia species composition. One hundred thirty seven sandflies were collected by sticky papers and they comprised three sub-genera: Parrotomyia (43.1%), Sergentomyia (36.5%), and Grassomyia (20.4%). Four species were identified; Sergentomyia (Parrotomyia) africana Newstead (43.1%) followed by S. (Grassomyia) dreyfussi Parrot, S. (Sergentomyia) fallax Parrot, and S. (S.) minuta Rondani accounted for 20.4%, 19.7%, and 16.8%, respectively. Ecological study subdivides these species into rural species (S. africana and S. dreyfussi) and ubiquitous species (S. minuta and S. fallax) which were collected in both urban and rural areas. Enzymatic analysis identified three monomorphic loci (alphaGPDH, ICD, and ME) and six polymorphic loci (PGI, HK, FUM, MDH2, 6PGD, and ACO) in the four species. At FUM and ACO loci, some alleles appeared to be fixed in each species. Morphological (counts of cibarial teeth) and isoenzymatic analysis of wild populations of S. minuta parroti from Morocco and of S. minuta minuta from continental Europe (France, Spain, and Portugal) was carried out. Morphological results showed significant differences between France and Portugal populations and south Spain populations. In contrast, there was no significant difference between northern and southern Moroccan populations. Genetic variability showed a separation between northern and southern European populations and S. minuta from Andalusia clustered with Moroccan populations.

The midgut transcriptome of Lutzomyia longipalpis: comparative analysis of cDNA libraries from sugar-fed, blood-fed, post-digested and Leishmania infantum chagasi-infected sand flies.

BACKGROUND: In the life cycle of Leishmania within the alimentary canal of sand flies the parasites have to survive the hostile environment of blood meal digestion, escape the blood bolus and attach to the midgut epithelium before differentiating into the infective metacyclic stages. The molecular interactions between the Leishmania parasites and the gut of the sand fly are poorly understood. In the present work we sequenced five cDNA libraries constructed from midgut tissue from the sand fly Lutzomyia longipalpis and analyzed the transcripts present following sugar feeding, blood feeding and after the blood meal has been processed and excreted, both in the presence and absence of Leishmania infantum chagasi. RESULTS: Comparative analysis of the transcripts from sugar-fed and blood-fed cDNA libraries resulted in the identification of transcripts differentially expressed during blood feeding. This included upregulated transcripts such as four distinct microvillar-like proteins (LuloMVP1, 2, 4 and 5), two peritrophin like proteins, a trypsin like protein (Lltryp1), two chymotrypsin like proteins (LuloChym1A and 2) and an unknown protein. Downregulated transcripts by blood feeding were a microvillar-like protein (LuloMVP3), a trypsin like protein (Lltryp2) and an astacin-like metalloprotease (LuloAstacin). Furthermore, a comparative analysis between blood-fed and Leishmania infected midgut cDNA libraries resulted in the identification of the transcripts that were differentially expressed due to the presence of Leishmania in the gut of the sand fly. This included down regulated transcripts such as four microvillar-like proteins (LuloMVP1,2, 4 and 5), a Chymotrypsin (LuloChym1A) and a carboxypeptidase (LuloCpepA1), among others. Upregulated midgut transcripts in the presence of Leishmania were a peritrophin like protein (LuloPer1), a trypsin-like protein (Lltryp2) and an unknown protein. CONCLUSION: This transcriptome analysis represents the largest set of sequence data reported from a specific sand fly tissue and provides further information of the transcripts present in the sand fly Lutzomyia longipalpis. This analysis provides the detailed information of molecules present in the midgut of this sand fly and the transcripts potentially modulated by blood feeding and by the presence of the Leishmania parasite. More importantly, this analysis suggests that Leishmania infantum chagasi alters the expression profile of certain midgut transcripts in the sand fly during blood meal digestion and that this modulation may be relevant for the survival and establishment of the parasite in the gut of the fly. Moreover, this analysis suggests that these changes may be occurring during the digestion of the blood meal and not afterwards.

Gene silencing in phlebotomine sand flies: Xanthine dehydrogenase knock down by dsRNA microinjections.

Lutzomyia longipalpis are vectors of medically important visceral leishmaniasis in South America. Blood-fed adult females digest large amounts of protein, and xanthine dehydrogenase is thought to be a key enzyme involved in protein catabolism through the production of urate. Large amounts of heme are also released during digestion with potentially damaging consequences, as heme can generate oxygen radicals that damage lipids, proteins and nucleic acids. However, urate is an antioxidant that may prevent such oxidative damage produced by heme. We investigated xanthine dehydrogenase by developing the RNAi technique for sand flies and used this technique to knock down the Lu. longipalpis xanthine dehydrogenase gene to evaluate its role in survival of adult females after blood feeding. The gene sequence of Lu. longipalpis xanthine dehydrogenase is described together with expression in different life cycle stages and RNAi knock down. Semi-quantitative RT-PCR of xanthine dehydrogenase expression showed a significant increase in expression after bloodmeal ingestion. Microinjection of dsRNA via the thorax of 1-day-old adult female sand flies resulted in approximately 40% reduction of xanthine dehydrogenase gene expression in comparison to flies injected with a control dsRNA. A significant reduction of urate in the whole body and excretions of Lu. longipalpis was observed after dsRNA xanthine dehydrogenase microinjection and feeding 96h later on rabbit blood. Sand flies injected with XDH dsRNA also exhibit significantly reduced life span in comparison with the mock-injected group when fed on sucrose or when rabbit blood fed, showing that urate could be indeed an important free radical scavenger in Lu. longipalpis. The demonstration of xanthine dehydrogenase knock down by dsRNA microinjection, low mortality of microinjected insects and the successful bloodfeeding of injected insects demonstrated the utility of RNAi as a tool for functional analysis of genes in phlebotomine sand flies.

Discrete arrays of liquid-crystal-supported proteolipid monolayers as phantom cell surfaces.

The phospholipid bilayers of living cell membranes exist almost universally in a liquid state. This enables motion and spatial reorganization of membrane components on multiple length scales, which is an essential feature of many biological processes. There is great interest in the development of molecularly defined interfaces between synthetic materials and living cells. To this end, there is a need for solid substrate materials that can be derivatized with fluid, membrane-like interfaces. Herein, we describe array fabrication of discrete liquid-crystal areas supporting phospholipid monolayer membranes, and characterize the interactions with several different membrane surface proteins [avidin series, cholera toxin, green fluorescent protein (GFP), intercellular adhesion molecule (ICAM) and major histocompatibility complex (MHC)]. Three different linkage strategies (biotin, nickel chelating lipids complexing with histidine, and the choleratoxin binding unit (CTB) associating with G(M1) are evaluated. Additionally, experiments with live immunological T cells forming active synapses at the interface exhibit the specific nature of the surface.

[Genetic structure in five Phlebotominae (Lutzomyia spp.), townsendi series, verrucarum group, in Colombia (Diptera: Prychodidae)]. / Estructura genética en cinco especies de flebótomos (Lutzomyia spp.) de la serie townsendi, grupo verrucarum, en Colombia (Diptera: Prychodidae).

Sixteen isoenzyme patterns were analyzed for five Colombian Lutzomyia species. The average unbiased expected heterozygosity levels ranged from 0.098 (Lu. youngi) to 0.215 (Lu. torrvida). The five species samples, taken all the isoenzymes employed, were significantly deviated from the Hardy-Weinberg equilibrium by homozygous excess with classical as well as Markov chain exact tests. Possible causes: (1) Wahlund effect within populations due to subdivision and/or sampling. Endogamy could be discarded because these loci were affected by highly different levels of homozygous excess. (2) Null alleles could be not discarded, at least for some isoenzymes. The hierarchical Wright's F analysis showed high and significant values for each parameter. The average F(IT) value was 0.655 with a conspicous homozygous excess at a global level (all species taken together); the average F(IS) value was significantly positive (0.515) as well, with homozygous excess within each species. The genetic heterogeneity between the fives species was noteworthy (F(ST) 0.288), indicating clear genetic differentiation. The more related species pairs were Lu. longiflocosa-Lu. torvida (0.959) and Lu torvida-Lu. spinicrassa (0.960); while Lu. torvida-Lu. youngi (0.805) and Lu. quasitownsendi-Lu. youngi (0.796) were the most divergent (Nei's genetic identity matrix). UPGMA and Wagner algorithms showed that the most divergent species was Lu. youngi, whereas the most related were Lu. longiflocosa-Lu. torvida and Lu torvida-Lu. spinicrassa. A spatial autocorrelation analysis (Moran's I index) revealed a very weak, or inexistent spatial structure, which means that the speciation events between these species were independent from the geographic distances from where they currently live.

The salivary hyaluronidase and apyrase of the sand fly Sergentomyia schwetzi (Diptera, Psychodidae).

Current knowledge of sand fly salivary components has been based solely on Lutzomyia and Phlebotomus species which feed mainly on mammals; their hyaluronidases and apyrases were demonstrated to significantly affect blood meal intake and transmission of vector-borne pathogens. Members of the third sand fly genus Sergentomyia preferentially feed on reptiles but some of them are considered as Leishmania and arboviruses vectors; however, nothing is known about their salivary components that might be relevant for pathogens transmission. Here, marked hyaluronidase and apyrase activities were demonstrated in the saliva of a Sergentomyia schwetzi colony maintained on geckos. Hyaluronidase of S. schwetzi cleaved hyaluronan as the prominent substrate, and was active over a broad pH range from 4.0 to 8.0, with a sharp peak at pH 5.0. SDS PAGE zymography demonstrated the monomeric character of the enzyme, which remained active in reducing conditions. The apparent molecular weight of 43 kDa was substantially lower than in any sand fly species tested so far and may indicate relatively low grade of the glycosylation of the enzyme. The apyrase of S. schwetzi was typical strictly Ca2+ dependent Cimex-family apyrase. It was active over a pH range from 6.5 to 9.0, with a peak of activity at pH 8.5, and had an ATPase/ADPase ratio of 0.9. The apyrase activity increased during the first 3 days post-emergence, then reached a plateau and remained relatively constant until day 8. In comparison with a majority of Phlebotomus and Lutzomyia species tested to date, both the hyaluronidase and apyrase activities of S. schwetzi were relatively low, which may reflect an adaptation of this sand fly to blood feeding on non-mammalian hosts.

Transmission blocking sugar baits for the control of Leishmania development inside sand flies using environmentally friendly beta-glycosides and their aglycones.

BACKGROUND: The sand fly Lutzomyia longipalpis is the main vector of American visceral leishmaniasis, a disease caused by parasites of the genus Leishmania. Adults of this insect feed on blood (females only) or sugar from plant sources, but their digestion of carbohydrates is poorly studied. Beta-glycosides as esculin and amygdalin are plant compounds and release toxic compounds as esculetin and mandelonitrile when hydrolyzed. Beta-glucosidase and trehalase are essential enzymes in sand fly metabolism and participate in sugar digestion. It is therefore possible that the toxic portions of these glycosides, released during digestion, affect sand fly physiology and the development of Leishmania. RESULTS: We tested the oral administration to sand flies of amygdalin, esculin, mandelonitrile, and esculetin in the sugar meal. These compounds significantly decreased the longevity of Lutzomyia longipalpis females and males. Lutzomyia longipalpis adults have significant hydrolytic activities against esculin and feeding on this compound cause changes in trehalase and ß-glucosidase activities. Female trehalase activity is inhibited in vitro by esculin. Esculin is naturally fluorescent, so its ingestion may be detected and quantified in whole insects or tissue samples stored in methanol. Mandelonitrile neither affected the amount of sugar ingested by sand flies nor showed repellent activity. Our results show that mandelonitrile significantly reduces the viability of L. amazonensis, L. braziliensis, L. infantum and L. mexicana, in a concentration-dependent manner. Esculetin caused a similar effect, reducing the number of L. infantum and L. mexicana. Female L. longipalpis fed on mandelonitrile had a reduction in the number of parasites and prevalence of infection after seven days of infection with L. mexicana, either by counting in a Neubauer chamber or by qPCR assays. CONCLUSIONS: Glycosides have significant effects on L. longipalpis longevity and metabolism and also affect the development of parasites in culture and inside the insect. These observations might help to conceptualize new vector control strategies using transmission blocking sugar baits.

Structural characterization of acetylcholinesterase 1 from the sand fly Lutzomyia longipalpis (Diptera: Psychodidae).

Acetylcholinesterase (AChE) plays a key role in cholinergic impulse transmission, and it is the target enzyme for organophosphorus and carbamate insecticides. Two genes, AceI and AceII, have been characterized from different insect species, and point mutations in either gene can lead to significant resistance to these classes of insecticides. In this report, we describe the partial characterization of the AceI gene from Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae), and we show that the possibility exists for the development of a resistant phenotype to organophosphates and carbamates in sand flies. Our results point to the presence of a single AceI gene in L. longipalpis (LlAce1) and that AChE activity is inhibited by organophosphorus at a concentration of 5 x 10(-5) M. Regarding insecticide resistance, analysis of the truncated LlAce1 cDNA suggests that a single missense mutation leading to a glycine-to-serine substitution at amino acid position 119 (G119S) may arise in L. longipalpis, similar to what has been detected in Anopheles gambiae s.s. Another missense mutation involved in resistant phenotypes, F331W, detected in Culex tritaeniorhynchus Giles, is less likely to occur in L. longipalpis, because it faces codon constraint in this sand fly species. Comparison of the three-dimensional structures of the deduced amino acid sequence of the truncated LLAChE1 with that of An. gambiae and Cx. tritaeniorhynchus also suggests that similar structural modifications due to the missense amino acid changes in the active site gorge are detected in all three insects.

Unexpected diversity of sandflies (Diptera: Psychodidae) in tourist caves in Northern Thailand.

Certain species of Phlebotomine sandflies (Diptera: Psychodidae) are vectors of the protozoa which causes leishmaniasis. Sandflies are found breeding in enclosed places like caves. Thailand is a popular tourist destination, including for ecotourism activities like caving, which increases the risk of contact between tourists and sandflies. Surveillance of sandflies is important for monitoring this risk but identification of species based on morphology is challenged by phenotypic plasticity and cryptic diversity. DNA barcodes have been used for the identification of sandflies in Thailand. We collected sandflies using CDC light trap from four tourist caves in Northern Thailand. Female sandflies were provisionally sorted into 13 morphospecies and 19 unidentified specimens. DNA was extracted from the thorax and legs of sandflies and the DNA barcode region of cytochrome c oxidase I mtDNA amplified and sequenced. The specimens were sorted into 22 molecular operational taxonomic units (MOTU) based on the 145 DNA barcodes, which is significantly more than the morphospecies. Several of the taxa thought to be present in multiple caves, based on morphospecies sorting, split into cave-specific MOTU which likely represent cryptic species. Several MOTU reported in an earlier study from Wihan Cave, Thailand, were also found in these caves. This supports the use of DNA barcodes to investigate species diversity of sandflies and their useful role in surveillance of sandflies in Thailand.

Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut.

BACKGROUND: In vector-borne diseases such as leishmaniasis, the sand fly midgut is considered to be an important site for vector-parasite interaction. Digestive enzymes including serine peptidases such as trypsin and chymotrypsin, which are secreted in the midgut are one of the obstacles for Leishmania in establishing a successful infection. The presence of some natural inhibitors of serine peptidases (ISPs) has recently been reported in Leishmania. In the present study, we deciphered the role of these ISPs in the survival of Leishmania donovani in the hostile sand fly midgut environment. METHODS: In silico and co-immunoprecipitation studies were performed to observe the interaction of L. donovani ISPs with trypsin and chymotrypsin. Zymography and in vitro enzyme assays were carried out to observe the inhibitory effect of purified recombinant ISPs of L. donovani (rLdISPs) on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of ISPs in the amastigote to promastigote transition stages were studied by semi-quantitative RT-PCR and Western blot. The role of LdISP on the survival of ISP overexpressed (OE) and ISP knocked down (KD) Leishmania parasites inside the sand fly gut was investigated by in vitro and in vivo cell viability assays. RESULTS: We identified two ecotin-like genes in L. donovani, LdISP1 and LdISP2. In silico and co-immunoprecipitation results clearly suggest a strong interaction of LdISP molecules with trypsin and chymotrypsin. Zymography and in vitro enzyme assay confirmed the inhibitory effect of rLdISP on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of LdISP2 was found to be strongly associated with the amastigote to promastigote phase transition. The activities of the digestive enzymes were found to be significantly reduced in the infected sand flies when compared to uninfected. To our knowledge, our study is the first report showing the possible reduction of chymotrypsin activity in L. donovani infected sand flies compared to uninfected. Interestingly, during the early transition stage, substantial killing was observed in ISP2 knocked down (ISP2KD) parasites compared to wild type (WT), whereas ISP1 knocked down (ISP1KD) parasites remained viable. Therefore, our study clearly indicates that LdISP2 is a more effective inhibitor of serine peptidases than LdISP1. CONCLUSION: Our results suggest that the lack of ISP2 is detrimental to the parasites during the early transition from amastigotes to promastigotes. Moreover, the results of the present study demonstrated for the first time that LdISP2 has an important role in the inhibition of peptidases and promoting L. donovani survival inside the Phlebotomus argentipes midgut.

The invertebrate growth factor/CECR1 subfamily of adenosine deaminase proteins.

Adenosine deaminase (ADA) catalyzes the hydrolysis of adenosine to inosine. Its lack determines severe combined immunodeficiency in mammals, possibly due to accumulation of extracellular adenosine, which induces apoptosis in lymphocytes (Franco et al., 1998). Thus, presence of normal levels of ADA leads to normal growth and proliferation of lymphocytes. Several vertebrate and microbial ADA amino-acid sequences are known, with substantial similarity to each other. On the other hand, there are invertebrate growth factors as well as a candidate gene for the human cat eye syndrome (CECR1) (Riazi et al., 2000. Genomics 64, 277-285), which share substantial similarity to each other, and also to ADA. In this study, we report the expression and ADA enzymatic activity of a cDNA from the salivary glands of Lutzomyia longipalpis, a blood-sucking insect, with substantial similarity to insect growth factors and to human CECR1. We also demonstrate the existence of a subfamily of the adenosine deaminase family characterized by their unique amino-terminal region. Both Drosophila melanogaster and humans have both types of adenosine deaminases. Results indicate that these invertebrate proteins previously annotated as growth factors, as well as the human CECR1 gene product, may exert their actions through adenosine depletion. The different roles played by each type of adenosine deaminase in humans and Drosophila remains to be fully investigated.