Azadirachtin interferes with basal immunity and microbial homeostasis in the Rhodnius prolixus midgut.

Rhodnius prolixus is an insect vector of two flagellate parasites, Trypanosoma rangeli and Trypanosoma cruzi, the latter being the causative agent of Chagas disease in Latin America. The R. prolixus neuroendocrine system regulates the synthesis of the steroid hormone ecdysone, which is essential for not only development and molting but also insect immunity. Knowledge for how this modulates R. prolixus midgut immune responses is essential for understanding interactions between the vector, its parasites and symbiotic microbes. In the present work, we evaluated the effects of ecdysone inhibition on R. prolixus humoral immunity and homeostasis with its microbiota, using the triterpenoid natural product, azadirachtin. Our results demonstrated that azadirachtin promoted a fast and lasting inhibitory effect on expression of both RpRelish, a nuclear factor kappa B transcription factor (NF-kB) component of the IMD pathway, and several antimicrobial peptide (AMP) genes. On the other hand, RpDorsal, encoding the equivalent NF-kB transcription factor in the Toll pathway, and the defC AMP gene were upregulated later in azadirachtin treated insects. The treatment also impacted on proliferation of Serratia marcescens, an abundant commensal bacterium. The simultaneous administration of ecdysone and azadirachtin in R. prolixus blood meals counteracted the azadirachtin effects on insect molting and also on expression of RpRelish and AMPs genes. These results support the direct involvement of ecdysone in regulation of the IMD pathway in the Rhodnius prolixus gut.

Nitric oxide effects on Rhodnius prolixus's immune responses, gut microbiota and Trypanosoma cruzi development.

The immune system of Rhodnius prolixus comprehends the synthesis of different effectors that modulate the intestinal microbiota population and the life cycle of the parasite Trypanosoma cruzi inside the vector midgut. One of these immune responses is the production of reactive nitrogen species (RNS) derived by the action of nitric oxide synthase (NOS). Therefore, we investigated the effects of L-arginine, the substrate for nitric oxide (NO) production and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of NOS, added in the insect blood meal. We analyzed the impact of these treatments on the immune responses and development of intestinal bacteria and parasites on R. prolixus nymphs. The L-arginine treatment in R. prolixus nymphs induced a higher NOS gene expression in the fat body and increased NO production, but reduced catalase and antimicrobial activities in the midgut. As expected, L-NAME treatment reduced NOS gene expression in the fat body. In addition, L-NAME treatment diminished catalase activity in the hemolymph and posterior midgut reduced phenoloxidase activity in the anterior midgut and increased the antimicrobial activity in the hemolymph. Both treatments caused a reduction in the cultivatable intestinal microbiota, especially in insects treated with L-NAME. However, T. cruzi development in the insect's digestive tract was suppressed after L-arginine treatment and the opposite was observed with L-NAME, which resulted in higher parasite counts. Therefore, we conclude that induction and inhibition of NOS and NO production are associated with other R. prolixus humoral immune responses, such as catalase, phenoloxidase, and antibacterial activities in different insect organs. These alterations reflect on intestinal microbiota and T. cruzi development.

Development of Leishmania mexicana in Lutzomyia longipalpis in the absence of sugar feeding.

The leishmaniases are caused by Leishmania parasites and transmitted through the bites of phlebotomine sand flies. During parasite development inside the vector's midgut, promastigotes move towards the stomodeal valve, a mechanism that is crucial for transmission. It has been reported that the sugar meal acquired by sand flies during feeding between bloodmeals is essential for the development and migration of parasites. We demonstrated that the distribution of Leishmania mexicana parasites was affected by the sugar meals obtained by the sand flies. Promastigote migration towards the cardia region seems to be only partially based on the stimuli provided by sugar molecules. In the absence of sugars, significant amounts of parasites developed in the hindgut. In addition, sugar meals were important for the survival of sand flies, especially during blood digestion, presumably supporting their energy requirements.

Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels.

Glycoside Hydrolases (GHs) are enzymes able to recognize and cleave glycosidic bonds. Insect GHs play decisive roles in digestion, in plant-herbivore, and host-pathogen interactions. GH activity is normally measured by the detection of a release from the substrate of products as sugars units, colored, or fluorescent groups. In most cases, the conditions for product release and detection differ, resulting in discontinuous assays. The current protocols result in using large amounts of reaction mixtures for the obtainment of time points in each experimental replica. These procedures restrain the analysis of biological materials with limited amounts of protein and, in the case of studies regarding small insects, implies in the pooling of samples from several individuals. In this respect, most studies do not assess the variability of GH activities across the population of individuals from the same species. The aim of this work is to approach this technical problem and have a deeper understanding of the variation of GH activities in insect populations, using as models the disease vectors Rhodnius prolixus (Hemiptera: Triatominae) and Lutzomyia longipalpis (Diptera: Phlebotominae). Here we standardized continuous assays using 4-methylumbelliferyl derived substrates for the detection of α-Glucosidase, ß-Glucosidase, α-Mannosidase, N-acetyl-hexosaminidase, ß-Galactosidase, and α-Fucosidase in the midgut of R. prolixus and L. longipalpis with results similar to the traditional discontinuous protocol. The continuous assays allowed us to measure GH activities using minimal sample amounts with a higher number of measurements, resulting in data that are more reliable and less time and reagent consumption. The continuous assay also allows the high-throughput screening of GH activities in small insect samples, which would be not applicable to the previous discontinuous protocol. We applied continuous GH measurements to 90 individual samples of R. prolixus anterior midgut homogenates using a high-throughput protocol. α-Glucosidase and α-Mannosidase activities showed the normal distribution in the population. ß-Glucosidase, ß-Galactosidase, N-acetyl-hexosaminidase, and α-Fucosidase activities showed non-normal distributions. These results indicate that GHs fluorescent-based high-throughput assays apply to insect samples and that the frequency distribution of digestive activities should be considered in data analysis, especially if a small number of samples is used.

Genome Wide Mapping of Peptidases in Rhodnius prolixus: Identification of Protease Gene Duplications, Horizontally Transferred Proteases and Analysis of Peptidase A1 Structures, with Considerations on Their Role in the Evolution of Hematophagy in Triatominae.

Triatominae is a subfamily of the order Hemiptera whose species are able to feed in the vertebrate blood (i.e., hematophagy). This feeding behavior presents a great physiological challenge to insects, especially in Hemipteran species with a digestion performed by lysosomal-like cathepsins instead of the more common trypsin-like enzymes. With the aim of having a deeper understanding of protease involvement in the evolutionary adaptation for hematophagy in Hemipterans, we screened peptidases in the Rhodnius prolixus genome and characterized them using common blast (NCBI) and conserved domain analyses (HMMER/blast manager software, FAT, plus PFAM database). We compared the results with available sequences from other hemipteran species and with 18 arthropod genomes present in the MEROPS database. Rhodnius prolixus contains at least 433 protease coding genes, belonging to 71 protease families. Seven peptidase families in R. prolixus presented higher gene numbers when compared to other arthropod genomes. Further analysis indicated that a gene expansion of the protease family A1 (Eukaryotic aspartyl protease, PF00026) might have played a major role in the adaptation to hematophagy since most of these peptidase genes seem to be recently acquired, are expressed in the gut and present putative secretory pathway signal peptides. Besides that, most R. prolixus A1 peptidases showed high frequencies of basic residues at the protein surface, a typical structural signature of Cathepsin D-like proteins. Other peptidase families expanded in R. prolixus (i.e., C2 and M17) also presented significant differences between hematophagous (higher number of peptidases) and non-hematophagous species. This study also provides evidence for gene acquisition from microorganisms in some peptidase families in R. prolixus: (1) family M74 (murein endopeptidase), (2) family S29 (Hepatitis C virus NS3 protease), and (3) family S24 (repressor LexA). This study revealed new targets for studying the adaptation of these insects for digestion of blood meals and their competence as vectors of Chagas disease.

Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion.

The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females) or blood feeders (females only), and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves ß-1,3-glucanases, chitinases, and digestive lysozymes. In this work, the transcripts of digestive ß-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18, and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes.

Caracterização molecular e bioquímica de carboidrases digestivas em larvas de Lutzomyia longipalpis / Molecular and biochemical characterization of digestive carbohydrases in larvae of Lutzomyia longipalpis

Lutzomyia longipalpis é o principal vetor da leishmaniose visceral no Novo Mundo. Em flebotomíneos as fases adultas e imaturas exploram diferentes fontes alimentares. Os adultos se alimentam de seiva vegetal (no caso de machos e fêmeas) ou sangue (no caso das fêmeas) e as larvas crescem em material orgânico em decomposição, principalmente de origem vegetal ou fezes de animais. Com relação à digestão das larvas, poucos trabalhos vêm sendo desenvolvidos, entretanto, atualmente é sugerido na literatura que as larvas se alimentam de microorganismos contidos em sua dieta. No presente trabalho foram caracterizadas sob o ponto de vista bioquímico as carboidrases digestivas presentes em larvas de L. longipalpis. Ensaios bioquímicos mostraram atividade enzimática no homogenato do tubo digestivo de Beta-1,3-glucanase, quitinase, lisozima, amilase, Alfa e Beta-glicosidase, Alfa e Beta-manosidase, N-acetilglicosaminidase e sialidase. Beta-1,3-glucanase e amilase foram as hidrolases com as maiores atividades, estando localizadas majoritariamente no espaço endoperitrófico. As enzimas lisozima/quitinase, Alfa- e Beta-glicosidases, N-acetil-glicosaminidase mostraram maiores atividades enzimáticas no epitélio intestinal do tubo digestivo de larvas de L. longipalpis, enquanto sialidase e Beta-manosidade apresentaram atividades equiparadas entre conteúdo luminal e epitélio intestinal no tubo digestivo das fases larvais. Cromatografia de filtração em gel e propriedades cinéticas mostraram que as isoformas solúveis de todas as enzimas encontradas nos tubos digestivos das formas larvais de L. longipalpis são diferentes das isoformas encontradas na dieta. O pirosequenciamento utilizando 454 Genome Sequencer FLX (Roche Applied Science) originou cerca de 223.093 reads e a montagem desses gerou 2883 transcritos, os quais 97% foram anotados. A anotação dos transcritos encontrados no transcriptoma mostou a preseça de enzimas cruciais para a digestão de proteínas, carboidratos e lipídios, bem como enzimas importantes para a detoxificação de xenobióticos, estresse oxidativo, resposta imune além da presença de peritrofinas, mucinas e hemocianinas. A análise pelo GO classificou 2195 isotigs em três categorias: função molecular, processos biológicos e componentes celulares, onde a maioria dos transcritos foi caracterizado como atividade catalítica (função molecular), processos metabólicos (processo biológico) e célula (componentes celulares). Tendo em vista a ingestão de microorganismos na dieta alimentar de larvas de flebotomíneo, o presente trabalho pesquisou na biblioteca de EST de L. longipalpis membros das enzimas glicosídeo hidrolases pertencentes às famílias 16 (Beta-1,3-glucanases), 18 (quitinases) e 22 (lisozimas), onde foi encontrado um total de 3, 5 e 1 sequências, respectivamente. Os transcritos NSFM-24g06 e NSFM-140g04 foram expressos no tubo digestivo das larvas quando comparados à expressão na carcaça. Outros transcritos foram mais expressos na carcaça e/ou cabeça ou não foram expressos em nenhum tecido das formas larvais. Diante dos dados obtidos nesse trabalho é sugerido que o perfil de carboidrases encontradas no tubo digestivo de larvas de L. longipalpis é coerente com o hábitat de vida detritívoro e provavelmente participa da digestão de bactérias e fungos. Além disso, propomos que os transcritos NSFM-24g06 and NSFM-140g04 correspondem a atividade de Beta-1,3-glucanase e quitinase digestivas os quais podem representar importantes alvos para supressão ou inibição da digestão desse vetor.