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Rhodnius prolixus is the principal vector of Trypanosoma cruzi , the aetiological agent of Chagas disease in American countries. This insect is haematophagous during all life cycles and, to antagonize its haemostatic, inflammatory and immune systems, it secretes saliva while feeding on the vertebrate host's blood. Here, we investigated characteristic changes of the salivary glands (SG) that occur during insect development. Two pairs of lobules and ducts comprise the SG of R. prolixus . The organ's size increases over time, but the microanatomical structures are preserved during insect development. Both lobules have a single layer epithelium formed by binucleated cells, which surrounds the saliva reservoir. The principal lobule presents higher polysaccharide and total protein contents than the accessory lobe. A network of external muscle layers is responsible for organ contraction and saliva release. Apocrine, merocrine and holocrine secretion types occur in the secretory epithelium. Dopamine, serotonin and tyrosine-hydroxylase are neural-related molecules that regulate SG function both during and after feeding.

We describe the immature stages of Migonemyia migonei, which is the vector of Leishmania (Viannia) braziliensis, the etiological agent of cutaneous leishmaniasis in South America, and a putative vector of Leishmania infantum chagasi. Scanning Electron Microscopy (SEM) was used to refine the description of the structures of the egg, all instar larvae, and the pupa. The eggs have polygonal cells on the egg exochorion, and differences between larval and pupal chaetotaxy have been highlighted. Different sensillary subtypes-trichoidea, basiconica, coelonica and campanoformia-were observed in the larval stages. The results presented herein contribute to the taxonomy of Mg. migonei and may contribute to future studies on the phylogeny of this important vector species., Competing Interests: The authors have declared that no competing interests exist.

Background: Lutzomyia umbratilis, the vector for Leishmania guyanensis in northern South America, has been found naturally infected with L. guyanensis only in areas north of the Negro and Amazon rivers. While populations of this sand fly species are also found in areas south of these rivers, these populations have never been reported to be infected and/or transmitting L. guyanensis. However, no studies on the corresponding host-parasite interactions are available., Objectives: This study evaluated the interaction between Lu. guyanensis promastigotes and field-collected Lu. umbratilis sand flies from Rio Preto da Eva and Manacapuru, which are located to the north and south, respectively, of the Negro River., Methods: Procyclic and metacyclic attachment was quantified using an in vitro system., Findings: Low attachment of parasites to the midguts of insects collected from Manacapuru was detected. Conversely, greater binding of metacyclic parasites was observed in the midguts of insects collected from Rio Preto da Eva, and this attachment was more pronounced than that observed for procyclics (p < 0.03)., Main Conclusions: The Lu. umbratilis population from an area south of the Negro River has lower in vitro interaction with L. guyanensis. The higher attachment of L. guyanensis to midguts of insects from Rio Preto da Eva may suggest better vector competence. These findings are in accordance with previously reported epidemiological information of American cutaneous leishmaniasis (ACL) transmission in the Amazon.

Background: Parasites of the genus Leishmania cause a broad spectrum of diseases, collectively known as leishmaniasis, in humans worldwide. American cutaneous leishmaniasis is a neglected disease transmitted by sand fly vectors including Lutzomyia intermedia, a proven vector. The female sand fly can acquire or deliver Leishmania spp. parasites while feeding on a blood meal, which is required for nutrition, egg development and survival. The microbiota composition and abundance varies by food source, life stages and physiological conditions. The sand fly microbiota can affect parasite life-cycle in the vector., Methods: We performed a metagenomic analysis for microbiota composition and abundance in Lu. intermedia, from an endemic area in Brazil. The adult insects were collected using CDC light traps, morphologically identified, carefully sterilized, dissected under a microscope and the females separated into groups according to their physiological condition: (i) absence of blood meal (unfed = UN); (ii) presence of blood meal (blood-fed = BF); and (iii) presence of developed ovaries (gravid = GR). Then, they were processed for metagenomics with Illumina Hiseq Sequencing in order to be sequence analyzed and to obtain the taxonomic profiles of the microbiota., Results: Bacterial metagenomic analysis revealed differences in microbiota composition based upon the distinct physiological stages of the adult insect. Sequence identification revealed two phyla (Proteobacteria and Actinobacteria), 11 families and 15 genera; 87 % of the bacteria were Gram-negative, while only one family and two genera were identified as Gram-positive. The genera Ochrobactrum, Bradyrhizobium and Pseudomonas were found across all of the groups., Conclusions: The metagenomic analysis revealed that the microbiota of the Lu. intermedia female sand flies are distinct under specific physiological conditions and consist of 15 bacterial genera. The Ochrobactrum, Bradyrhizobium and Pseudomonas were the common genera. Our results detailing the constituents of Lu. intermedia native microbiota contribute to the knowledge regarding the bacterial community in an important sand fly vector and allow for further studies to better understand how the microbiota interacts with vectors of human parasites and to develop tools for biological control.

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly.

Background: Malaria is transmitted when an infected mosquito delivers Plasmodium sporozoites into a vertebrate host. There are many species of Plasmodium and, in general, the infection is host-specific. For example, Plasmodium gallinaceum is an avian parasite, while Plasmodium berghei infects mice. These two parasites have been extensively used as experimental models of malaria transmission. Plasmodium falciparum and Plasmodium vivax are the most important agents of human malaria, a life-threatening disease of global importance. To complete their life cycle, Plasmodium parasites must traverse the mosquito midgut and form an oocyst that will divide continuously. Mature oocysts release thousands of sporozoites into the mosquito haemolymph that must reach the salivary gland to infect a new vertebrate host. The current understanding of the biology of oocyst formation and sporozoite release is mostly based on experimental infections with P. berghei, and the conclusions are generalized to other Plasmodium species that infect humans without further morphological analyses., Results: Here, it is described the microanatomy of sporozoite escape from oocysts of four Plasmodium species: the two laboratory models, P. gallinaceum and P. berghei, and the two main species that cause malaria in humans, P. vivax and P. falciparum. It was found that sporozoites have species-specific mechanisms of escape from the oocyst. The two model species of Plasmodium had a common mechanism, in which the oocyst wall breaks down before sporozoites emerge. In contrast, P. vivax and P. falciparum sporozoites show a dynamic escape mechanism from the oocyst via polarized propulsion., Conclusions: This study demonstrated that Plasmodium species do not share a common mechanism of sporozoite escape, as previously thought, but show complex and species-specific mechanisms. In addition, the knowledge of this phenomenon in human Plasmodium can facilitate transmission-blocking studies and not those ones only based on the murine and avian models.

Salivary gland polytene chromosomes of 4th instar Anopheles darlingi Root were examined from multiple locations in the Brazilian Amazon. Minor modifications were made to existing polytene photomaps. These included changes to the breakpoint positions of several previously described paracentric inversions and descriptions of four new paracentric inversions, two on the right arm of chromosome 3 and two on the left arm of chromosome 3 that were found in multiple locations. A total of 18 inversions on the X (n = 1) chromosome, chromosome 2 (n = 7) and 3 (n = 11) were scored for 83 individuals from Manaus, Macapá and Porto Velho municipalities. The frequency of 2Ra inversion karyotypes in Manaus shows significant deficiency of heterozygotes (p < 0.0009). No significant linkage disequilibrium was found between inversions on chromosome 2 and 3. We hypothesize that at least two sympatric subpopulations exist within the An. darlingi population at Manaus based on inversion frequencies.

Background: In Brazil, dengue epidemics erupt sporadically throughout the country and it is unclear if outbreaks may initiate a sustainable transmission cycle. There are few studies evaluating the ability of Brazilian Aedes aegypti populations to transmit dengue virus (DENV). The aim of this study was to compare DENV susceptibility of field-captured Ae. aegypti populations from nine distinct geographic areas of the city of Belo Horizonte in 2009 and 2011. Infection Rate (IR), Vector Competence (VC) and Disseminated Infection Rate (DIR) were determined., Methods: Aedes aegypti eggs from each region were collected and reared separately in an insectary. Adult females were experimentally infected with DENV-2 and the virus was detected by qPCR in body and head samples. Data were analyzed with the Statistical Package for the Social Sciences version 17., Results: IR varied from 40.0% to 82.5% in 2009 and 60.0% to 100.0% in 2011. VC ranged from 25.0% to 77.5% in 2009 and 25.0% to 80.0% in 2011. DIR oscillated from 68.7% to 100.0% in 2009 and 38.4% to 86.8 in 2011. When the results were evaluated by a logistic model using IR as covariate, North, Barreiro, South-Central and Venda Nova showed the strongest association in 2009. In 2011, a similar association was observed for South-Central, Venda Nova, West and Northeast regions. Using VC as covariate, South-Central and Venda Nova showed the most relevant association in 2009. In 2011, South-Central, Venda Nova and Barreiro presented the greatest revelation associations. When DIR data were analyzed by logistic regression models, Pampulha, South-Central, Venda Nova, West, Northeast and East (2009) as well as South-Central, Venda Nova and West (2011) were the districts showing the strongest associations., Conclusions: We conclude that Ae. aegypti populations from Belo Horizonte exhibit wide variation in vector competence to transmit dengue. Therefore, vector control strategies should be adapted to the available data for each region. Further analysis should be conducted to better understand the reasons for this large variability in vector competence and how these parameters correlate with epidemiological findings in subsequent years.

Background: Anopheles darlingi is the major malaria vector in countries located in the Amazon region. Anopheles aquasalis and Anopheles albitarsis s.l. are also proven vectors in this region. Anopheles nuneztovari s.l. and Anopheles triannulatus s.l. were found infected with Plasmodium vivax; however, their status as vectors is not yet well defined. Knowledge of susceptibility of Amazon anopheline populations to Plasmodium infection is necessary to better understand their vector capacity. Laboratory colonization of An. darlingi, the main Amazon vector, has proven to be difficult and presently An. aquasalis is the only available autonomous colony., Methods: Larvae of An. darlingi, An. albitarsis s.l., An. nuneztovari s.l. and An. triannulatus s.l. were collected in the field and reared until adult stage. Adults of An. aquasalis were obtained from a well-established colony. Mosquitoes were blood-fed using a membrane-feeding device containing infected blood from malarial patients.The infection of the distinct Anopheles species was evaluated by the impact variance of the following parameters: (a) parasitaemia density; (b) blood serum inactivation of the infective bloodmeal; (c) influence of gametocyte number on infection rates and number of oocysts. The goal of this work was to compare the susceptibility to P. vivax of four field-collected Anopheles species with colonized An. aquasalis., Results: All Anopheles species tested were susceptible to P. vivax infection, nevertheless the proportion of infected mosquitoes and the infection intensity measured by oocyst number varied significantly among species. Inactivation of the blood serum prior to mosquito feeding increased infection rates in An. darlingi and An. triannulatus s.l., but was diminished in An. albitarsis s.l. and An. aquasalis. There was a positive correlation between gametocyte density and the infection rate in all tests (Z = -8.37; p < 0.001) but varied among the mosquito species. Anopheles albitarsis s.l., An. aquasalis and An. nuneztovari s.l. had higher infection rates than An. darlingi., Conclusion: All field-collected Anopheles species, as well as colonized An. aquasalis are susceptible to experimental P. vivax infections by membrane feeding assays. Anopheles darlingi, An. albitarsis s.l. and An. aquasalis are very susceptible to P. vivax infection. However, colonized An. aquasalis mosquitoes showed the higher infection intensity represented by infection rate and oocyst numbers. This study is the first to characterize experimental development of Plasmodium infections in Amazon Anopheles vectors and also to endorse that P. vivax infection of colonized An. aquasalis is a feasible laboratory model.

We evaluated the ability of dogs naturally infected with Leishmania (Leishmania) infantum chagasi to transfer the parasite to the vector and the factors associated with transmission. Thirty-eight infected dogs were confirmed to be infected by direct observation of Leishmania in lymph node smears. Dogs were grouped according to external clinical signs and laboratory data into symptomatic (n=24) and asymptomatic (n=14) animals. All dogs were sedated and submitted to xenodiagnosis with F1-laboratory-reared Lutzomyia longipalpis. After blood digestion, sand flies were dissected and examined for the presence of promastigotes. Following canine euthanasia, fragments of skin, lymph nodes, and spleen were collected and processed using immunohistochemistry to evaluate tissue parasitism. Specific antibodies were detected using an enzyme-linked immunosorbent assay. Antibody levels were found to be higher in symptomatic dogs compared to asymptomatic dogs (p=0.0396). Both groups presented amastigotes in lymph nodes, while skin parasitism was observed in only 58.3% of symptomatic and in 35.7% of asymptomatic dogs. Parasites were visualized in the spleens of 66.7% and 71.4% of symptomatic and asymptomatic dogs, respectively. Parasite load varied from mild to intense, and was not significantly different between groups. All asymptomatic dogs except for one (93%) were competent to transmit Leishmania to the vector, including eight (61.5%) without skin parasitism. Sixteen symptomatic animals (67%) infected sand flies; six (37.5%) showed no amastigotes in the skin. Skin parasitism was not crucial for the ability to infect Lutzomyia longipalpis but the presence of Leishmania in lymph nodes was significantly related to a positive xenodiagnosis. Additionally, a higher proportion of infected vectors that fed on asymptomatic dogs was observed (p=0.0494). Clinical severity was inversely correlated with the infection rate of sand flies (p=0.027) and was directly correlated with antibody levels (p=0.0379). Age and gender did not influence the transmissibility. Our data show that asymptomatic dogs are highly infective and competent for establishing sand fly infection, indicating their role in maintaining L. (L.) infantum chagasi cycle as well as their involvement in VL spreading in endemic areas., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We analyzed the development of Leishmania (Leishmania) infantum chagasi in its natural sandfly vector Lutzomyia longipalpis. In addition, we compared sandfly infections initiated with axenic amastigotes or promastigotes. Our data showed no important difference between Lu. longipalpis infection rates resulting from either type of infections. Furthermore, development of infection was equivalent in both cases. All promastigote forms were found inside the sandfly and, after blood digestion, most of the population consisted of procyclics and nectomonads. A low percentage of metacyclic forms was coincident with a high number of nectomonads during late stages of infection, but which form gives rise to metacyclic forms in L. infantum chagasi is unknown. These results also show that the promastigote infection model, at least for this situation, is suitable for obtaining of infected sandflies because it is easier and less laborious.

Background: Sandflies are vectors of Leishmania, the causative agent of leishmaniasis in mammalian hosts, including humans. The protozoan parasite is transmitted by the sandfly bite during salivation that occurs at the moment of blood feeding. The components of vector saliva include anticlotting and vasodilatory factors that facilitate blood flow and immunomodulatory factors that inhibit wound healing and quell the immune response. Not surprisingly, these factors also play important roles in the establishment of Leishmania infection. To date, the majority of knowledge that has been generated regarding the process of Leishmania infection, including L. infantum chagasi transmission has been gathered by using intradermal or subcutaneous inoculation of purified parasites., Findings: This study presents the establishment of a transmission model of Leishmania infantum chagasi by the bite of Lutzomyia longipalpis, the vector of American visceral leishmaniasis. The parasites were successfully transmitted by infected sandfly bites to mice and hamsters, indicating that both animals are good experimental models. The L. infantum chagasi dose that was transmitted in each single bite ranged from 10 to 10, 000 parasites, but 75% of the sandflies transmitted less than 300 parasites., Conclusions: The strategy for initiating infection by sandfly bite of experimental animals facilitates future investigations into the complex and dynamic mechanisms of visceral leishmaniasis. It is important to elucidate the transmission mechanism of vector bites. This model represents a useful tool to study L. infantum chagasi infection transmitted by the vector.

Background: Bacteria associated with insects can have a substantial impact on the biology and life cycle of their host. The checkerboard DNA-DNA hybridization technique is a semi-quantitative technique that has been previously employed in odontology to detect and quantify a variety of bacterial species in dental samples. Here we tested the applicability of the checkerboard DNA-DNA hybridization technique to detect the presence of Aedes aegypti-associated bacterial species in larvae, pupae and adults of A. aegypti., Findings: Using the checkerboard DNA-DNA hybridization technique we could detect and estimate the number of four bacterial species in total DNA samples extracted from A. aegypti single whole individuals and midguts. A. aegypti associated bacterial species were also detected in the midgut of four other insect species, Lutzomyia longipalpis, Drosophila melanogaster, Bradysia hygida and Apis mellifera., Conclusions: Our results demonstrate that the checkerboard DNA-DNA hybridization technique can be employed to study the microbiota composition of mosquitoes. The method has the sensitivity to detect bacteria in single individuals, as well as in a single organ, and therefore can be employed to evaluate the differences in bacterial counts amongst individuals in a given mosquito population. We suggest that the checkerboard DNA-DNA hybridization technique is a straightforward technique that can be widely used for the characterization of the microbiota in mosquito populations.

Malaria affects 300 million people worldwide every year and 450,000 in Brazil. In coastal areas of Brazil, the main malaria vector is Anopheles aquasalis, and Plasmodium vivax is responsible for the majority of malaria cases in the Americas. Insects possess a powerful immune system to combat infections. Three pathways control the insect immune response: Toll, IMD, and JAK-STAT. Here we analyze the immune role of the A. aquasalis JAK-STAT pathway after P. vivax infection. Three genes, the transcription factor Signal Transducers and Activators of Transcription (STAT), the regulatory Protein Inhibitors of Activated STAT (PIAS) and the Nitric Oxide Synthase enzyme (NOS) were characterized. Expression of STAT and PIAS was higher in males than females and in eggs and first instar larvae when compared to larvae and pupae. RNA levels for STAT and PIAS increased 24 and 36 hours (h) after P. vivax challenge. NOS transcription increased 36 h post infection (hpi) while this protein was already detected in some midgut epithelial cells 24 hpi. Imunocytochemistry experiments using specific antibodies showed that in non-infected insects STAT and PIAS were found mostly in the fat body, while in infected mosquitoes the proteins were found in other body tissues. The knockdown of STAT by RNAi increased the number of oocysts in the midgut of A. aquasalis. This is the first clear evidence for the involvement of a specific immune pathway in the interaction of the Brazilian malaria vector A. aquasalis with P. vivax, delineating a potential target for the future development of disease controlling strategies.

Interspecies variations in lipophosphoglycan (LPG) have been the focus of intense study over the years due its role in specificity during sand fly-Leishmania interaction. This cell surface glycoconjugate is highly polymorphic among species with variations in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO(4) backbone of repeat units. However, the degree of intraspecies polymorphism in LPG of Leishmania infantum (syn. Leishmania chagasi) is not known. In this study, intraspecific variation in the repeat units of LPG was evaluated in 16 strains of L. infantum from Brazil, France, Algeria and Tunisia. The structural polymorphism in the L. infantum LPG repeat units was relatively slight and consisted of three types: type I does not have side chains; type II has one β-glucose residue that branches off the disaccharide-phosphate repeat units and type III has up to three glucose residues (oligo-glucosylated). The significance of these modifications was investigated during in vivo interaction of L. infantum with Lutzomyia longipalpis, and in vitro interaction of the parasites and respective LPGs with murine macrophages. There were no consequential differences in the parasite densities in sand fly midguts infected with Leishmania strains exhibiting type I, II and III LPGs. However, higher nitric oxide production was observed in macrophages exposed to glucosylated type II LPG., (Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Midgut enzymatic activity is one of the obstacles that Leishmania must surpass to succeed in establishing infection. Trypsins are abundant digestive enzymes in most insects. We have previously described two trypsin cDNAs of L. longipalpis: one (Lltryp1) with a bloodmeal induced transcription pattern, the other (Lltryp2) with a constitutive transcription pattern. We have now characterized the expression and activity of trypsin-like proteases of Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil., Methodology and Principal Findings: In order to study trypsin expression profiles we produced antibodies against peptides specific for Lltryp1 and Lltryp2. The anti-Lltryp1-peptide antibody revealed a band of 28 kDa between 6 and 48 hours. The anti-Lltryp2 peptide antibody did not evidence any band. When proteinaceous substrates (gelatin, hemoglobin, casein or albumin) were co-polymerized in polyacrylamide gels, insect midguts obtained at 12 hours after feeding showed a unique proteolytic pattern for each substrate. All activity bands were strongly inhibited by TLCK, benzamidine and 4-amino-benzamidine, indicating that they are trypsin-like proteases. The trypsin-like activity was also measured in vitro at different time points after ingestion of blood or blood containing Leishmania infantum chagasi, using the chromogenic substrate BArhoNA. L. longipalpis females fed on blood infected with L. i. chagasi had lower levels of trypsin activity after 12 and 48 hours than non-infected insects, suggesting that the parasite may have a role in this modulation., Conclusions and Significance: Trypsins are important and abundant digestive enzymes in L. longipalpis. Protein production and enzymatic activity followed previously identified gene expression of a blood modulated trypsin gene. A decrease of enzymatic activity upon the parasite infection, previously detected mostly in Old World vectors, was detected for the first time in the natural vector-parasite pair L. longipalpis-L. i. chagasi.

Malaria affects 300 million people worldwide every year and is endemic in 22 countries in the Americas where transmission occurs mainly in the Amazon Region. Most malaria cases in the Americas are caused by Plasmodium vivax, a parasite that is almost impossible to cultivate in vitro, and Anopheles aquasalis is an important malaria vector. Understanding the interactions between this vector and its parasite will provide important information for development of disease control strategies. To this end, we performed mRNA subtraction experiments using A. aquasalis 2 and 24 hours after feeding on blood and blood from malaria patients infected with P. vivax to identify changes in the mosquito vector gene induction that could be important during the initial steps of infection. A total of 2,138 clones of differentially expressed genes were sequenced and 496 high quality unique sequences were obtained. Annotation revealed 36% of sequences unrelated to genes in any database, suggesting that they were specific to A. aquasalis. A high number of sequences (59%) with no matches in any databases were found 24 h after infection. Genes related to embryogenesis were down-regulated in insects infected by P. vivax. Only a handful of genes related to immune responses were detected in our subtraction experiment. This apparent weak immune response of A. aquasalis to P. vivax infection could be related to the susceptibility of this vector to this important human malaria parasite. Analysis of some genes by real time PCR corroborated and expanded the subtraction results. Taken together, these data provide important new information about this poorly studied American malaria vector by revealing differences between the responses of A. aquasalis to P. vivax infection, in relation to better studied mosquito-Plasmodium pairs. These differences may be important for the development of malaria transmission-blocking strategies in the Americas.

We investigated the effects of Lutzomyia longipalpis salivary glands homogenate of wild-caught and laboratory-reared vectors on the lesion evolution and immunomodulation of the infection caused by Leishmania (Leishmania) amazonensis. To compare the effect of both salivary glands homogenate (SGH), C57BL/6 mice were inoculated subcutaneously into the hind footpads or into the ear dermis with 10(6) promastigotes in the presence or not of SGH from wild-caught and laboratory-colonized sand flies. Comparing SGH groups, the lesion size was lower in mice co-inoculated with wild-caught SGH, as the parasitism and the infiltration of macrophages at the inoculation site. Wild-caught SGH also determined lower production of IL-4 and IL-10 but higher IL-12 levels compared with laboratory-reared SGH. Our findings address a probable bias by using SGH from laboratory-colonized sand flies instead of wild-caught vector SGH in studies concerning saliva effects. A possible mild influence of sand fly saliva in natural infections caused by Leishmania is also speculated, as infection is transmitted by wild and not by laboratory-reared vectors.

The effectiveness of a polyester mesh cover (evidengue), aimed at preventing the access of female Aedes aegypti mosquitoes to flowerpot saucers, was evaluated in laboratory. Two saucers of flowerpot with water were individually wrapped with the cover was placed with their respective pots in two entomological cages. One identical set of flowerpot and saucer was placed in a third cage. In each cage, 20 gravid females, fed on mouse blood, were released. Results show that the cover was effective to prevent access of females. Further tests are necessary to assess cover effectiveness as a device to prevent saucer oviposition.

In order to compare the saliva effect from wild-caught and lab-reared L. longipalpis on the development of experimental cutaneous leishmaniasis, C57BL/6 mice were inoculated subcutaneously into the hind footpads with promastigotes of L. (L.) amazonensis plus salivary gland lysate from wild-caught (SGL-W) and lab-colonized (SGL-C) vectors. Lesion sizes were significantly larger in the mice infected with both saliva compared to mice infected with parasites alone; moreover, the lesions caused by parasite+SGL-C were significantly larger than the lesions caused by parasite+SGL-W. Histopathological morphometric studies regarding the acute phase of infections showed lower numbers of polymorphonuclear cells, greater numbers of mononuclear cells and parasites in SGL-C infected mice compared to SGL-W infected mice. In the chronic phase of infection, the number of mononuclear cells was lower and the number of parasites was greater in SGL-C infected mice than SGL-W infected mice. In vitro studies showed increased infection index of macrophages infected with parasites plus saliva compared to infection with parasites alone, with no difference between the saliva infection indices. SDS-PAGE gel for SGL-C and SGL-W showed differences in the composition and quantity of protein bands, determined by densitometry. These results call attention to the experimental saliva model, which shows exacerbation of infection caused by sandfly saliva.

The specific aims of this work were to examine the antennal sensilla of Lutzomyia longipalpis (Lutz & Neiva 1912) (Diptera: Psychodidae) adults and to characterize their typology and topography, with special attention to olfactory sensilla. The surfaces of the antennal segments of Lu. longipalpis males and females were studied by scanning electron microscopy (SEM). Lu. longipalpis used in the current study were obtained from a colony originating from Lapinha Cave, Minas Gerais state, Brazil. Microtrichiae and 11 subtypes of sensilla were observed and characterized according to the following categories: five subtypes of trichoid sensilla (short, medium, long blunt-tipped, long pointed-tipped, and apical), two coeloconic sensilla (grooved and praying hands), and campaniform, chaetic, basiconic, and squamiform sensilla. SEM analyses showed few differences between males and females in the typology, topography, and quantity of antennal sensilla described. The current study is the first to identify several categories of antennal sensilla of the genus Lutzomyia and their distribution patterns. The identification of these sensillar types may be important in planning future electrophysiological studies to develop alternative measures of control and monitoring of Lu. longipalpis.

Lutzomyia (Nyssomyia) intermedia (Lutz & Neiva 1912) and Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho 1939) (Diptera: Psychodidae) are vectors of American cutaneous leishmaniasis in several endemic regions of Brazil. We analyzed the external morphological aspects of the immature stages of these two vectors by using scanning electron microscopy. In general, the larval stages of the two species are morphologically similar, although some differences were noted. Detailed examination of the eggs of both species revealed similar exchorionic ornamentations of unconnected parallel ridges. The larval head capsules are well defined, heavily sclerotized, and bear prominent chewing mouthparts. The abdominal segments are easily recognized by the presence of prolegs on their ventral surfaces. The morphology of the anal lobe on the terminal abdominal segment differs between the two species. We found the following three types of sensillae inserted on the antennae: (1) clavate basiconic; (2) small, blunt coeloconic; and (3) multipourous clavate coleoconic. In addition; five subtypes of trichoid sensillae were found on the larval body: (1) long, (2) short, (3) curved long, (4) brush-like, and (5) weakly brush-like. The caudal filaments located on the last abdominal segment were recognized as long trichoid sensillae. We observed pores on the surface of the clavate coelonic sensillae and on the caudal filaments that presumably function as chemoreceptors. The larvae of the two species show similarities in the lobular-form antennae of L1 larvae, which changes to digitiform in second instar (L2), L3, and L4. This study demonstrated that the external surface of the eggs and larvae of Lu. intermedia and Lu. whitmani are morphologically similar, but they can be distinguished by details in the microanatomy observed by scanning electron microscopy.

The correct identification of etiological agents in vector insects is crucial for epidemiological studies. Identification of flagellates in such vectors, usually by dissection of the digestive tract and microscopic observation of the contents as well as attempts at parasite isolation from insects in culture media, have proven operationally inadequate and with poor diagnostic specificity, since female sand flies are also hosts for other flagellates like Trypanosoma and Endotrypanum. Due to the efficiency and specificity of DNA target sequence amplification by polymerase chain reaction (PCR), the latter could be used to investigate the presence of Leishmania in sand flies, although the insects need to be properly stored and the Leishmania DNA extracted using appropriate methodology. This paper describes methodologies to standardize sand fly storage and Leishmania DNA extraction in such specimens as a more practical method in field studies.

Leishmaniasis is one of the most diverse and complex of all vector-borne diseases. Because it involves several overlapping species and sandfly vectors, the disease has a complex ecology and epidemiology. Adequate therapy and follow-up depend on parasitological diagnosis, but classical methods present several constraints when identifying species. We describe a polymerase chain reaction (PCR) which uses primers designed from mini-exon repetitive sequences that are specific for subgenus LeishmaniaViannia (PV), as well as sequences with specificity for genus (PG) that can distinguish between Leishmania species from other insect flagellates with minor differences in PCR products. For standardization, these PCR were tested in experimentally infected sandflies, and Leishmania infection in these insects was successfully confirmed. This methodology identified a 3.9% infection rate in field-captured phlebotomine sandflies from an endemic region in Brazil. Natural infection by Leishmania species was identified in three samples of Lutzomyia longipalpis, of which two were Leishmania (L.) chagasi and one Leishmania (L.) amazonensis. Irrespective of specific epidemiological conclusions, the method used in this study was able to identify Leishmania infections both in experimentally infected and field-captured phlebotomine sandflies, and could be a useful tool in epidemiological studies and strategic planning for the control of human leishmaniasis.

Egg hatching has been studied in Aedes aegypti (L.) through scanning electron microscopy. The first sign of egg hatching is a small protrusion on the eggshell in the anterior pole. The larval movement provokes a crack in the eggshell with the egg buster located in the dorsal head. The egg buster provokes a small transverse fissure in the eggshell that gradually increases in the chorion. Then, the rupture is completed around the eggshell. The separation of the anterior pole occurs, showing the dorsal region of the larva head with the egg buster and the cap. After sequential movements, the larva looses the cap. Finally, the first instar is ready to be free showing details of its body with the egg buster over its head. This structure is a cuticular formation, similar to a cone structure that ends in a very fine tip and emerges from a pear-like depression with high rounded borders. Our results describe the anatomy of the egg hatching process in Ae. aegypti, showing details of the participation of the egg buster.

Sandflies are vectors of several pathogens, constituting serious health problems. Lutzomyia longipalpis (Lutz & Neiva, 1912) is the main vector of Leishmania chagasi, agent of visceral leishmaniasis. They synthesize a thick bag-like structure that surrounds the bloodmeal, named peritrophic matrix (PM). One of the major roles of PM in blood-fed insects includes protection against ingested pathogens by providing a defensive barrier to their development. We used traditional and modern morphological methods as well as biochemical and immunolabeling tools to define details of the PM structure of the Lu. longipalpis sandfly, including composition, synthesis, and degradation. The kinetics of PM formation and degradation was found to be related to the ingestion and time of digestion of the bloodmeal. The midgut changes its size and morphology after the blood ingestion and during the course of digestion. A striking morphological modification takes place in the midgut epithelium after the stretching caused by the bloodmeal, revealing a population of cells that was not observed in the unfed midgut. The transmission and scanning electron microscopies were used to reveal several morphological aspects of PM formation. The PM looks thicker and well formed 24 h after the bloodmeal. Presence of chitin in the PM was demonstrated by immunolabeling with an alpha-chitin monoclonal antibody. SDS-polyacrylamide gel electrophoresis showed at least five protein bands with molecular masses of 38.7-135 kDa, induced by the protein-free diet. Mouse polyclonal antiserum was produced against PMs induced by protein-free meal and used in Western blotting, which revealed at least three associated proteins.

Measurement of the hydrolysis of specific fluorogenic substrates by spectrophotometry as well as the substrate activity-SDS-PAGE gel analysis of the chitinolytic activity in Aedes aegypti guts showed that both chitinase and beta-N-acetylglucosaminidase are present and physiologically active. Both enzymes were present even in guts from unfed insects, but the activities increased rapidly after feeding on blood or an artificial protein-free diet. Chitinase activity was predominantly of the 'endo'-type, reaching its maximum activity at 36 h and then declining to very low levels after the degradation of the peritrophic matrix (PM). Chitinase assay in gels after SDS-PAGE was a very sensitive method that allowed us to detect two chitinases with distinct molecular weights in the mosquito gut. Hydrolysis of a chitinase-specific substrate by chitinolytic activities in the mosquito guts was inhibited by allosamidin, a potent chitinase inhibitor. Allosamidin treatment led to the formation of an atypical thick PM, while the addition of exogenous chitinase completely blocked its formation. This chitinolytic system appears to operate both on the formation and degradation of the PM. Since the PM is involved in pathogen invasion, these results are important in facilitating a search for mechanisms that can block pathogen development in the mosquito vector.

DNA amplification by the polymerase chain reaction (PCR) was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae) parasites in single phlebotomine sandflies. Three phlebotomine/parasite pairs were used: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis and Lutzomyia migonei/Leishmania braziliensis, all of them incriminated in the transmission of visceral or cutaneous leishmaniasis. DNA extraction was performed with whole insects, with no need of previous digestive tract dissection or pooling specimens. The presence of either mouse blood in the digestive tract of the sandflies or the digestive tract itself did not interfere in the PCR. Infection by as few as 10 Leishmania sp. per individual were sufficient for DNA amplification with genus-specific primers. Using primers for L. braziliensis and L. mexicana complexes, respectively, it was possible to discriminate between L. braziliensis and L. amazonensis in experimentally infected vectors (L. migonei).

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.