Conserved and distinct morphological aspects of the salivary glands of sand fly vectors of leishmaniasis: an anatomical and ultrastructural study.

BACKGROUND: Sand flies are vectors of Leishmania spp., the causative agents of leishmaniasis in vertebrates, including man. The sand fly saliva contains powerful pharmacologically active substances that prevent hemostasis and enhance Leishmania spp. infections. On the other hand, salivary proteins can protect vaccinated mice challenged with parasites. Therefore, sand fly salivary proteins are relevant for the epidemiology of leishmaniasis and can be a potential target for a vaccine against leishmaniasis. Despite this, studies on sand fly salivary glands (SGs) are limited. METHODS: The present study analyzes, in detail, the morphology, anatomy and ultrastructure of the SGs of sand fly vectors of the genera Lutzomyia and Phlebotomus. We used histology, transmission and scanning electron microscopy and lectin labeling associated with confocal laser microscopy. RESULTS: The SGs have conserved and distinct morphological aspects according to the distinct sand fly species. Each SG has a single rounded lobe constituting of c.100-120 secretory cells. The SG secretory cells, according to their ultrastructure and lectin binding, were classified into five different subpopulations, which may differ in secretory pathways. CONCLUSIONS: To the best of our knowledge, these morphological details of sand fly salivary glands are described for the first time. Further studies are necessary to better understand the role of these different cell types and better relate them with the production and secretion of the saliva substances, which has a fundamental role in the interaction of the sand fly vectors with Leishmania.

Ultrastructure of the Antennae and Sensilla of Nyssomyia intermedia (Diptera: Psychodidae), Vector of American Cutaneous Leishmaniasis.

The antennal sensilla and the antenna of females Nyssomyia intermedia, one of the main vectors of American cutaneous leishmaniasis, were studied by scanning electron microscopy. The main goal was to characterize the quantity, typology, and topography of the sensilla with particular attention to the olfactory types. The insects were captured in the city of Corte de Pedra, State of Bahia, Brazil, by CDC-type light traps and raised in a laboratory as a new colony. Fourteen well-differentiated sensilla were identified, among six cuticular types: trichoidea, campaniformia, squamiformia, basiconica, chaetica, and coeloconica. Of these, six sensilla were classified as olfactory sensilla due to their specific morphological features. Smaller noninnervated pilosities of microtrichiae type were also evidenced by covering all antennal segments. The antennal segments differ in shapes and sizes, and the amount and distribution of types and subtypes of sensilla. This study may foment future taxonomic and phylogenetic analysis for a better evolutionary understanding of the sand flies. Besides, it may assist the targeting of future electrophysiological studies by Single Sensillum Recording, and aim to develop alternative measures of monitoring and control of this vector.

Exploring Lutzomyia longipalpis Sand Fly Vector Competence for Leishmania major Parasites.

Lutzomyia longipalpis sand flies are the major natural vector of Leishmania infantum parasites, responsible for transmission of visceral leishmaniasis in the New World. Several experimental studies have demonstrated the ability of Lu. longipalpis to sustain development of different Leishmania species. However, no study had explored in depth the potential vector competence of Lu. longipalpis for Leishmania species other than L. infantum. Here, we show that Lu. longipalpis is a competent vector of L. major parasites, being able to acquire parasites from active cutaneous leishmaniasis lesions, sustain mature infections, and transmit them to naive hosts, causing disease.

Characterization of the complete mitogenome of Anopheles aquasalis, and phylogenetic divergences among Anopheles from diverse geographic zones.

Whole mitogenome sequences (mtDNA) have been exploited for insect ecology studies, using them as molecular markers to reconstruct phylogenies, or to infer phylogeographic relationships and gene flow. Recent Anopheles phylogenomic studies have provided information regarding the time of deep lineage divergences within the genus. Here we report the complete 15,393 bp mtDNA sequences of Anopheles aquasalis, a Neotropical human malaria vector. When comparing its structure and base composition with other relevant and available anopheline mitogenomes, high similarity and conserved genomic features were observed. Furthermore, 22 mtDNA sequences comprising anopheline and Dipteran sibling species were analyzed to reconstruct phylogenies and estimate dates of divergence between taxa. Phylogenetic analysis using complete mtDNA sequences suggests that A. aquasalis diverged from the Anopheles albitarsis complex ~28 million years ago (MYA), and ~38 MYA from Anopheles darlingi. Bayesian analysis suggests that the most recent ancestor of Nyssorhynchus and Anopheles + Cellia was extant ~83 MYA, corroborating current estimates of ~79-100 MYA. Additional sampling and publication of African, Asian, and North American anopheline mitogenomes would improve the resolution of the Anopheles phylogeny and clarify early continental dispersal routes.

Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification.

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.

The Midgut Muscle Network of Anopheles aquasalis (Culicidae, Anophelinae): Microanatomy and Structural Modification After Blood Meal and Plasmodium vivax (Haemosporida, Plasmodiidae) Infection.

The mosquito midgut is divided into two regions named anterior midgut (AMG) and posterior midgut (PMG). The midgut expands intensely after the blood ingestion to accommodate a large amount of ingested food. To efficiently support the bloodmeal-induced changes, the organization of the visceral muscle fibers has significant adjustments. This study describes the spatial organization of the Anopheles aquasalis (Culicidae, Anophelinae) midgut muscle network and morphological changes after bloodmeal ingestion and infection with Plasmodium vivax (Haemosporida, Plasmodiidae). The midgut muscle network is composed of two types of fibers: longitudinal and circular. The two types of muscle fibers are composed of thick and thin filaments, similar to myosin and actin, respectively. Invagination of sarcoplasm membrane forms the T-system tubules. Sarcoplasmic reticulum cisternae have been observed in association with these invaginations. At different times after the bloodmeal, the fibers in the AMG are not modified. A remarkable dilation characterizes the transitional area between the AMG and the PMG. In the PMG surface, after the completion of bloodmeal ingestion, the stretched muscle fibers became discontinued. At 72 h after bloodmeal digestion, it is possible to observe the presence of disorganized muscle fibers in the midgut regions. The Plasmodium oocyst development along the basal layer of the midgut does not have a significant role in the visceral musculature distribution. This study provides features of the visceral musculature at different blood feeding times of An. aquasalis and shows important changes in midgut topography including when the mosquitoes are infected with P. vivax.

Promising approach to reducing Malaria transmission by ivermectin: Sporontocidal effect against Plasmodium vivax in the South American vectors Anopheles aquasalis and Anopheles darlingi.

BACKGROUND: The mosquito resistance to the insecticides threatens malaria control efforts, potentially becoming a major public health issue. Alternative methods like ivermectin (IVM) administration to humans has been suggested as a possible vector control to reduce Plasmodium transmission. Anopheles aquasalis and Anopheles darlingi are competent vectors for Plasmodium vivax, and they have been responsible for various malaria outbreaks in the coast of Brazil and the Amazon Region of South America. METHODS: To determine the IVM susceptibility against P. vivax in An. aquasalis and An. darlingi, ivermectin were mixed in P. vivax infected blood: (1) Powdered IVM at four concentrations (0, 5, 10, 20 or 40 ng/mL). (2) Plasma (0 hours, 4 hours, 1 day, 5, 10 and 14 days) was collected from healthy volunteers after to administer a single oral dose of IVM (200 µg/kg) (3) Mosquitoes infected with P. vivax and after 4 days was provided with IVM plasma collected 4 hours post-treatment (4) P. vivax-infected patients were treated with various combinations of IVM, chloroquine, and primaquine and plasma or whole blood was collected at 4 hours. Seven days after the infective blood meal, mosquitoes were dissected to evaluate oocyst presence. Additionally, the ex vivo effects of IVM against asexual blood-stage P. vivax was evaluated. RESULTS: IVM significantly reduced the prevalence of An. aquasalis that developed oocysts in 10 to 40 ng/mL pIVM concentrations and plasma 4 hours, 1 day and 5 days. In An. darlingi to 4 hours and 1 day. The An. aquasalis mortality was expressively increased in pIVM (40ng/mL) and plasma 4 hours, 1, 5 10 and 14 days post-intake drug and in An. darlingi only to 4 hours and 1 day. The double fed meal with mIVM by the mosquitoes has a considerable impact on the proportion of infected mosquitoes for 7 days post-feeding. The oocyst infection prevalence and intensity were notably reduced when mosquitoes ingested blood from P. vivax patients that ingested IVM+CQ, PQ+CQ and IVM+PQ+CQ. P. vivax asexual development was considerably inhibited by mIVM at four-fold dilutions. CONCLUSION: In conclusion, whole blood spiked with IVM reduced the infection rate of P. vivax in An. aquasalis and An. darlingi, and increased the mortality of mosquitoes. Plasma from healthy volunteers after IVM administration affect asexual P. vivax development. These findings support that ivermectin may be used to decrease P. vivax transmission.

Zika virus infection modulates the bacterial diversity associated with Aedes aegypti as revealed by metagenomic analysis.

Zika is a re-emerging infection that has been considered a major threat to global public health. Currently at least 100 countries are at risk of Zika virus (ZIKV) transmission. Aedes aegypti is the main mosquito vector in the Americas. This vector is exposed to, and interacts symbiotically with a variety of microorganisms in its environment, which may result in the formation of a lifetime association. Here, the unknown effect that ZIKV exerts on the dynamic bacterial community harbored by this mosquito vector was investigated using a metagenomic analysis of its microbiota. Groups of Ae. aegypti were experimentally fed on sugar, blood and blood mixed with ZIKV, and held for 3 to 7 days after blood meal and eggs development respectively. The infected groups were processed by qPCR to confirm the presence of ZIKV. All groups were analyzed by metagenomics (Illumina Hiseq Sequencing) and 16S rRNA amplicon sequences were obtained to create bacterial taxonomic profiles. A core microbiota and exclusive bacterial taxa were identified that incorporate 50.5% of the predicted reads from the dataset, with 40 Gram-negative and 9 Gram-positive families. To address how ZIKV invasion may disturb the ecological balance of the Ae. aegypti microbiota, a CCA analysis coupled with an explanatory matrix was performed to support the biological interpretation of shifts in bacterial signatures. Two f-OTUs appeared as potential biomarkers of ZIKV infection: Rhodobacteraceae and Desulfuromonadaceae. Coincidentally, both f-OTUs were exclusively present in the ZIKV- infected blood-fed and ZIKV- infected gravid groups. In conclusion, this study shows that bacterial symbionts act as biomarkers of the insect physiological states and how they respond as a community when ZIKV invades Ae. aegypti. Basic knowledge of local haematophagous vectors and their associated microbiota is relevant when addressing transmission of vector-borne infectious diseases in their regional surroundings.

Aedes (Stegomyia) albopictus' dynamics influenced by spatiotemporal characteristics in a Brazilian dengue-endemic risk city.

Brazil reported the majority of the dengue cases in Americas during the last two decades, where the occurrence of human dengue cases is exclusively attributed to the Aedes (Stegomyia) aegypti (Linnaeus). Nowadays, other recognized Dengue virus (DENV) vector in Asian countries, Aedes (Stegomyia) albopictus (Skuse), has been detected in more than half of the 5565 Brazilian municipalities. Therefore, the aim of the present study was to investigate the presence of, and determine the Ae. albopictus' dynamics influenced by spatiotemporal characteristics in a dengue-endemic risk city of Belo Horizonte, Minas Gerais State's capital. Aedes albopictus were collected across four consecutive DENV transmission seasons from 2010 to 2014. These mosquitoes were caught in three selected districts, which had been reported in the previous ten years as having high mosquito densities and an elevated concentration of human dengue cases during epidemic seasons. All field-caught Ae. albopictus was individually processed by real-time RT-PCR, to research the DENV presence. The third season (p<0.05) and the Pampulha district (p<0.05) had the highest proportions of field-caught Ae. albopictus, respectively. The second season had the highest proportion of DENV-infected field-caught females (p<0.05), but there was no difference among the proportions of DENV-infected Ae. albopictus when comparing the collection in the three districts (p=0.98). Minimum (p=0.004) and maximum (p<0.0001) temperature were correlated with the field-caught Ae. albopictus in four different periods and districts. In the generalized linear model of Poisson, the field-caught DENV-infected Ae. albopictus (p=0.005), East district (p=0.003), minimum temperature (p<0.0001) and relative humidity (p=0.001) remained associated with the total number of human dengue cases. Our study demonstrated that the number of field-caught DENV-infected Ae. albopictus was inversed correlated with the number of human dengue cases. Our study raises the possibility that the DENV circulating in mosquitoes Ae. albopictus is happening in non-epidemic periods, showing that this species may be keeping only the presence of the virus in nature. Further long-term studies are necessary to better understand the role of Ae. albopictus in DENV transmission and or its vectorial competence in Belo Horizonte and in other endemic cities in Brazil and in the New World countries.

Species-specific escape of Plasmodium sporozoites from oocysts of avian, rodent, and human malarial parasites.

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.

Distinct variation in vector competence among nine field populations of Aedes aegypti from a Brazilian dengue-endemic risk city.

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.

Experimental Plasmodium vivax infection of key Anopheles species from the Brazilian Amazon.

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.

Development of Leishmania (Leishmania) infantum chagasi in its natural sandfly vector Lutzomyia longipalpis.

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.

The transmission of Leishmania infantum chagasi by the bite of the Lutzomyia longipalpis to two different vertebrates.

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.

Use of the checkerboard DNA-DNA hybridization technique for bacteria detection in Aedes aegypti (Diptera:Culicidae) (L.).

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.

The JAK-STAT pathway controls Plasmodium vivax load in early stages of Anopheles aquasalis infection.

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.

Anopheles aquasalis Infected by Plasmodium vivax displays unique gene expression profiles when compared to other malaria vectors and plasmodia.

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.

Ultrastructural comparison of external morphology of immature stages of Lutzomyia (Nyssomyia) intermedia and Lutzomyia (Nyssomyia) whitmani (Diptera: Psychodidae), vectors of cutaneous leishmaniasis, by scanning electron microscopy.

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.

Presence of chitinase and beta-N-acetylglucosaminidase in the Aedes aegypti. a chitinolytic system involving peritrophic matrix formation and degradation.

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.