Microorganism-Based Larval Diets Affect Mosquito Development, Size and Nutritional Reserves in the Yellow Fever Mosquito Aedes aegypti (Diptera: Culicidae).

BACKGROUND: Mosquito larvae feed on organic detritus from the environment, particularly microorganisms comprising bacteria, protozoa, and algae as well as crustaceans, plant debris, and insect exuviae. Little attention has been paid to nutritional studies in Aedes aegypti larvae. OBJECTIVES: We investigated the effects of yeast, bacteria and microalgae diets on larval development, pupation time, adult size, emergence, survivorship, lifespan, and wing morphology. MATERIALS AND METHODS: Microorganisms (or Tetramin® as control) were offered as the only source of food to recently hatched first instar larvae and their development was followed until the adult stage. Protein, carbohydrate, glycogen, and lipid were analyzed in single larvae to correlate energetic reserve accumulation by larva with the developmental rates and nutritional content observed. FITC-labeled microorganisms were offered to fourth instar larvae, and its ingestion was recorded by fluorescence microscopy and quantitation. RESULTS AND DISCUSSION: Immature stages developed in all diets, however, larvae fed with bacteria and microalgae showed a severe delay in development rates, pupation time, adult emergence and low survivorship. Adult males emerged earlier as expected and had longer survival than females. Diets with better nutritional quality resulted in adults with bigger wings. Asaia sp. and Escherichia coli resulted in better nutrition and developmental parameters and seemed to be the best bacterial candidates to future studies using symbiont-based control. The diet quality was measured and presented different protein and carbohydrate amounts. Bacteria had the lowest protein and carbohydrate rates, yeasts had the highest carbohydrate amount and microalgae showed the highest protein content. Larvae fed with microalgae seem not to be able to process and store these diets properly. Larvae were shown to be able to process yeast cells and store their energetic components efficiently. CONCLUSION: Together, our results point that Ae. aegypti larvae show high plasticity to feed, being able to develop under different microorganism-based diets. The important role of Ae. aegypti in the spread of infectious diseases requires further biological studies in order to understand the vector physiology and thus to manage the larval natural breeding sites aiming a better mosquito control.

Biochemical and Functional Characterization of Glycoside Hydrolase Family 16 Genes in Aedes aegypti Larvae: Identification of the Major Digestive ß-1,3-Glucanase.

Insect ß-1,3-glucanases belong to Glycoside Hydrolase Family 16 (GHF16) and are involved in digestion of detritus and plant hemicellulose. In this work, we investigated the role of GHF16 genes in Aedes aegypti larvae, due to their detritivore diet. Aedes aegypti genome has six genes belonging to GHF16 (Aae GH16.1 - Aae GH16.6), containing two to six exons. Sequence analysis suggests that five of these GHF16 sequences (Aae GH16.1, 2, 3, 5, and 6) contain the conserved catalytic residues of this family and correspond to glucanases. All genomes of Nematocera analyzed showed putative gene duplications corresponding to these sequences. Aae GH16.4 has no conserved catalytic residues and is probably a ß-1,3-glucan binding protein involved in the activation of innate immune responses. Additionally, Ae. aegypti larvae contain significant ß-1,3-glucanase activities in the head, gut and rest of body. These activities have optimum pH about 5-6 and molecular masses between 41 and 150 kDa. All GHF16 genes above showed different levels of expression in the larval head, gut or rest of the body. Knock-down of AeGH16.5 resulted in survival and pupation rates lower than controls (dsGFP and water treated). However, under stress conditions, severe mortalities were observed in AeGH16.1 and AeGH16.6 knocked-down larvae. Enzymatic assays of ß-1,3-glucanase in AeGH16.5 silenced larvae exhibited lower activity in the gut and no change in the rest of the body. Chromatographic activity profiles from gut samples after GH16.5 silencing showed suppression of enzymatic activity, suggesting that this gene codes for the digestive larval ß-1,3-glucanase of Ae. aegypti. This gene and enzyme are attractive targets for new control strategies, based on the impairment of normal gut physiology.

Microorganism-based larval diets affect mosquito development, size and nutritional reserves in the yellow fever mosquito aedes aegypti (Diptera: Culicidae)

Background: Mosquito larvae feed on organic detritus from the environment, particularly microorganisms comprising bacteria, protozoa, and algae as well as crustaceans, plant debris, and insect exuviae. Little attention has been paid to nutritional studies in Aedes aegypti larvae. Objectives: We investigated the effects of yeast, bacteria and microalgae diets on larval development, pupation time, adult size, emergence, survivorship, lifespan, and wing morphology. Materials and Methods: Microorganisms (or Tetramin® as control) were offered as the only source of food to recently hatched first instar larvae and their development was followed until the adult stage. Protein, carbohydrate, glycogen, and lipid were analyzed in single larvae to correlate energetic reserve accumulation by larva with the developmental rates and nutritional content observed. FITC-labeled microorganisms were offered to fourth instar larvae, and its ingestion was recorded by fluorescence microscopy and quantitation. Results and Discussion: Immature stages developed in all diets, however, larvae fed with bacteria and microalgae showed a severe delay in development rates, pupation time, adult emergence and low survivorship. Adult males emerged earlier as expected and had longer survival than females. Diets with better nutritional quality resulted in adults with bigger wings. Asaia sp. and Escherichia coli resulted in better nutrition and developmental parameters and seemed to be the best bacterial candidates to future studies using symbiont-based control. The diet quality was measured and presented different protein and carbohydrate amounts. Bacteria had the lowest protein and carbohydrate rates, yeasts had the highest carbohydrate amount and microalgae showed the highest protein content. Larvae fed with microalgae seem not to be able to process and store these diets properly. Larvae were shown to be able to process yeast cells and store their energetic components efficiently. Conclusion: Together, our results point that Ae. aegypti larvae show high plasticity to feed, being able to develop under different microorganism-based diets. The important role of Ae. aegypti in the spread of infectious diseases requires further biological studies in order to understand the vector physiology and thus to manage the larval natural breeding sites aiming a better mosquito control.

Microorganism-based larval diets affect mosquito development, size and nutritional reserves in the yellow fever mosquito aedes aegypti (Diptera: Culicidae)

Background: Mosquito larvae feed on organic detritus from the environment, particularly microorganisms comprising bacteria, protozoa, and algae as well as crustaceans, plant debris, and insect exuviae. Little attention has been paid to nutritional studies in Aedes aegypti larvae. Objectives: We investigated the effects of yeast, bacteria and microalgae diets on larval development, pupation time, adult size, emergence, survivorship, lifespan, and wing morphology. Materials and Methods: Microorganisms (or Tetramin® as control) were offered as the only source of food to recently hatched first instar larvae and their development was followed until the adult stage. Protein, carbohydrate, glycogen, and lipid were analyzed in single larvae to correlate energetic reserve accumulation by larva with the developmental rates and nutritional content observed. FITC-labeled microorganisms were offered to fourth instar larvae, and its ingestion was recorded by fluorescence microscopy and quantitation. Results and Discussion: Immature stages developed in all diets, however, larvae fed with bacteria and microalgae showed a severe delay in development rates, pupation time, adult emergence and low survivorship. Adult males emerged earlier as expected and had longer survival than females. Diets with better nutritional quality resulted in adults with bigger wings. Asaia sp. and Escherichia coli resulted in better nutrition and developmental parameters and seemed to be the best bacterial candidates to future studies using symbiont-based control. The diet quality was measured and presented different protein and carbohydrate amounts. Bacteria had the lowest protein and carbohydrate rates, yeasts had the highest carbohydrate amount and microalgae showed the highest protein content. Larvae fed with microalgae seem not to be able to process and store these diets properly. Larvae were shown to be able to process yeast cells and store their energetic components efficiently. Conclusion: Together, our results point that Ae. aegypti larvae show high plasticity to feed, being able to develop under different microorganism-based diets. The important role of Ae. aegypti in the spread of infectious diseases requires further biological studies in order to understand the vector physiology and thus to manage the larval natural breeding sites aiming a better mosquito control.

Repertório de serina proteases como componentes do degradoma de Leishmania spp / Serine protease repertoire as components of Leishmania spp degradation

Esta tese faz uma abordagem in silico e experimental sobre as serina proteases de Leishmania spp. Na primeira etapa do estudo são descritas as serina proteases como parte do degradoma destes parasitos. Os genes de serina protease (n = 26 a 28) em Leishmania spp que codificam proteínas com uma ampla faixa de massa molecular, são descritos juntamente com seus graus de sintenia cromossômica e alélica. Em meio a 17 serina proteases putativas de Leishmania spp apenas 18 % possuem duas funções descritas experimentalmente como propriedades para remover o peptídeo sinal de pro-proteínas secretoras (clã SF), como maturases de outras proteínas (clã SB) e com atividades similares as metacaspase (clã SC). Análises in silico indicaram que o conjunto de serina proteases de Leishmania spp pode estabelecer interrelações funcionais com outras proteínas de Leishmania spp significando atuações essenciais na fisiologia do parasito. Padrões de redes distintos podem ocorrer e a complexidade destas redes difere entre as espécies relacionadas ao subgênero Viannia e Leishmania: Leishmania (V.) braziliensis (n = 215); Leishmania (L.) mexicana (n = 130); L. (L.) donovani (n=56) Leishmania (L.) major (n = 45); Leishmania (L.) infantum (n = 23). O fato das serina proteases de L. (V.) braziliensis indicarem mais possibilidades de interações com outras proteínas do parasito foi a motivação para segunda etapa deste estudo

Esta etapa consistiu em uma abordagem experimental e in silico investigando as serina proteases deste parasito, incluindo atividade enzimática de serina proteases de frações subcelulares obtidas por cromatografia de afinidade com benzamidina, reações em cadeia da polimerase com transcrição reversa e ensaios in silico de localização subcelular de subtilisinas. Promastigotas apresentaram serina proteases com atividade gelatinolítica na fração citosólica (43 kDa a 170 kDa) e na fração membrana (67 kDa a 170 kDa). Ambas as frações também demostraram propriedades de catálise sobre os substratos peptídicos N-benziloxicarbonil-L-fenilalanil-L-arginina 7-amino-4-metilcumarina (fração citosólica = 392 ± 30 mol.min-1 mg of protein-1; fração de membrana = 53 ± 5 mol.min-1 mg of protein-1) e N-succinil-L-alanina-L-fenilalanina-L-lisina 7-amino-4-metilcumarina (fração citosólica = 252 ± 20 mol.min-1 mg of protein-1; fração de membrana = 63.6 ± 6.5 mol.min-1 mg of protein-1) com diferentes perfis de especificidade demonstrada pela inibição com aprotinina (19 % a 80 %) e fluoreto de fenilmetilsulfonil (3 % a 69 %), dependendo da fração subcelular e do substrato. A expressão dos genes das subtilisinas (LbrM.13.0860 e LbrM28.270) e da triparedoxina peroxidase (LbrM.15.1080) também foram observadas, com a detecção dos transcritos de 200 pb, 162 pb e 166 pb, respectivamente. Ensaios subsequentes indicaram que a enzima codificada pelo gene LbrM.13.0860 tem localização no citosol e a codificada pelo gene LbrM.28.2570 na membrana do parasito. Coletivamente, os dados aqui obtidos indicam relevantes contribuições das serina proteases na fisiologia da Leishmania spp, incluindo as subtilisinas das promastigotas de L. (V.) braziliensis. (AU)

Understanding serine proteases implications on Leishmania spp lifecycle.

Serine proteases have significant functions over a broad range of relevant biological processes to the Leishmania spp lifecycle. Data gathered here present an update on the Leishmania spp serine proteases and the status of these enzymes as part of the parasite degradome. The serine protease genes (n = 26 to 28) in Leishmania spp, which encode proteins with a wide range of molecular masses (35 kDa-115 kDa), are described along with their degrees of chromosomal and allelic synteny. Amid 17 putative Leishmania spp serine proteases, only ∼18% were experimentally demonstrated, as: signal peptidases that remove the signal peptide from secretory pre-proteins, maturases of other proteins and with metacaspase-like activity. These enzymes include those of clans SB, SC and SF. Classical inhibitors of serine proteases are used as tools for the characterization and investigation of Leishmania spp. Endogenous serine protease inhibitors, which are ecotin-like, can act modulating host actions. However, crude or synthetic based-natural serine protease inhibitors, such as potato tuber extract, Stichodactyla helianthus protease inhibitor I, fukugetin and epoxy-α-lapachone act on parasitic serine proteases and are promising leishmanicidal agents. The functional interrelationship between serine proteases and other Leishmania spp proteins demonstrate essential functions of these enzymes in parasite physiology and therefore their value as targets for leishmaniasis treatment.

Digestion of Yeasts and Beta-1,3-Glucanases in Mosquito Larvae: Physiological and Biochemical Considerations.

Aedes aegypti larvae ingest several kinds of microorganisms. In spite of studies regarding mosquito digestion, little is known about the nutritional utilization of ingested cells by larvae. We investigated the effects of using yeasts as the sole nutrient source for A. aegypti larvae. We also assessed the role of beta-1,3-glucanases in digestion of live yeast cells. Beta-1,3-glucanases are enzymes which hydrolyze the cell wall beta-1,3-glucan polyssacharide. Larvae were fed with cat food (controls), live or autoclaved Saccharomyces cerevisiae cells and larval weight, time for pupation and adult emergence, larval and pupal mortality were measured. The presence of S. cerevisiae cells inside the larval gut was demonstrated by light microscopy. Beta-1,3-glucanase was measured in dissected larval samples. Viability assays were performed with live yeast cells and larval gut homogenates, with or without addition of competing beta-1,3-glucan. A. aegypti larvae fed with yeast cells were heavier at the 4th instar and showed complete development with normal mortality rates. Yeast cells were efficiently ingested by larvae and quickly killed (10% death in 2 h, 100% in 48 h). Larvae showed beta-1,3-glucanase in head, gut and rest of body. Gut beta-1,3-glucanase was not derived from ingested yeast cells. Gut and rest of body activity was not affected by the yeast diet, but head homogenates showed a lower activity in animals fed with autoclaved S. cerevisiae cells. The enzymatic lysis of live S. cerevisiae cells was demonstrated using gut homogenates, and this activity was abolished when excess beta-1,3-glucan was added to assays. These results show that live yeast cells are efficiently ingested and hydrolyzed by A. aegypti larvae, which are able to fully-develop on a diet based exclusively on these organisms. Beta-1,3-glucanase seems to be essential for yeast lytic activity of A. aegypti larvae, which possess significant amounts of these enzyme in all parts investigated.

Diferenças na expressão gênica e na forma de direcionamento da região COOH-terminal de císteina-proteinase B de Leishmania (Leishmania) amazonensis / Differences in gene expression and direction of the COOH-Terminal region of cysteine proteinase B of Leishmania (Leishmania) amazonensis

A Leishmania (Leishmania) amazonensis apresenta mecanismos de adaptação guiados porsuas proteinases. Neste contexto, destacam-se as cisteína proteinases (CPs) onde acisteína proteinase B (CPB) é a CP mais estudada dentre as CPs deste parasito. O focodeste trabalho é a região COOH-terminal da CPB (cyspep), gerada a partir doprocessamento final desta proteinase. O estudo foi conduzido em cinco fases: obtenção dacyspep recombinante (rcyspep); avaliação da antigenicidade da rcyspep em camundongosexperimentalmente infectados com L.(L.) amazonensis; diferenciação in vitro dospromastigotas em amastigotas; avaliação da expressão do gene cpb ao longo dadiferenciação do parasito; e detecção da cyspep nas formas promastigotas e amastigotas doparasito. O polipeptídeo rcyspep, de 14 kDa, foi obtida em sistema pET28-a e purificadopara posterior produção de anticorpos policlonais específicos em camundongos. Observouseque, durante a infecção em camundongos, há uma resposta imune humoral contra orcyspep, caracterizada por um aumento da detecção de anti-cyspep quando comparado comanimais de controle (p <0,05). Nos ensaios de diferenciação constatamos três padrõesmorfológicos ao logo de 96 horas de análise, em uma cinética temporal: formas alongadascom flagelo livre, arredondados com flagelo e arredondadas sem flagelo por microscopia.Adicionalmente a cultura foi analisada por citometria de fluxo demonstrando uma migraçãogradual da população da região R1 para região R2 do dotplot indicando uma diminuição notamanho e aumento da granulosidade celular. Observamos que ao longo destadiferenciação ocorre um aumento da quantidade de transcritos do gene cpb, nos tempos de48 horas (1,3 ×), 72 horas (3,2 ×) e 96 horas (3,4 ×) nas preparações de cDNA dosparasitos, quando comparada com os resultados de quantificação realizados compreparações de cDNA dos promastigotas...

Leishmania (Leishmania) amazonensis presents adaptive mechanisms guided by theirproteinases. In this context, we highlight the cysteine proteinases (CPs) where the cysteineproteinase B (CPB) is the most studied CP among these parasites. The focus of this work isthe COOH-terminal region of CPB (cyspep), generated during the final processing stage ofthis proteinase. Our study comprises five main phases: obtaining of a recombinant cyspeppolypeptide (rcyspep); evaluation of the antigenicity of rcyspep in mice experimentallyinfected with L. (L.) amazonensis; in vitro differentiation of promastigotes into amastigotes;evaluation of cpb gene expression throughout morphological differentiation of the parasite;and detection of cyspep in promastigotes and amastigotes of L. (L.) amazonensis. Thercyspep polypeptide, with 14 kDa, was obtained using a pET28-a system and, subsequently,specific polyclonal antibodies were produced by inoculation in mice. We observed that,during mice infection, there is a humoral immune response against rcyspep, characterized byan increase in detection of anti-cyspep when compared to control animals (p<0.05). Duringdifferentiation assays three morphological patterns could be observed over 96 hours ofobservation: elongated shapes with free flagellum, rounded shapes with free flagellum androunded shapes without visible flagellum. The data point to a gradual migration of thepopulation of parasites from region R1 to region R2 into the dotplot indicating a decrease insize and increase in cell granularity. We observed that alongside the morphologicaldifferentiation there is an increase in the amount of cpb gene transcripts, in time of 48 hours(1.3 ×), 72 hours (3.2 ×) and 96 hours (× 3.4), compared with quantitation results obtainedwith cDNA of promastigotes...

Beta-1, 3-glucanases e digestão de leveduras em larvas de Aedes aegypti Linnaeus (Diptera: Culicidae) / Beta-1,3-glucanases and digestion of yeasts in Aedes aegypti Linnaeus, 1762 (Diptera: Culicidae) larvae: physiological and molecular aspects

Beta-1,3-glucanases estão envolvidas na digestão de insetos que utilizam como fonte nutricional detritos ou plantas. Nesse trabalho, nós investigamos o papel dos genes da família 16 das glicosídeo hidrolases (GHF 16) em larvas de Ae. aegypti e sua possível participação na digestão de leveduras. O genoma de Ae. aegypti contém seis genes (Aae GH16.1 – Aae GH16.6) que codificam para proteínas da família GH16, contendo de 2 a 6 éxons. Análises filogenéticas sugerem que em Nematoceros ocorreram duplicações em alguns genes que contêm a região catalítica conservada nessa família de proteínas (Aae GH16. 1, 2, 3, 5, 6)> Essas proteínas são similares a outras proteínas de insetos com atividades glucanásicas. A proteína Aae GH16. 4 parece estar relacionada a proteínas ligantes de beta-1,3-glucana, não possuindo os resíduos catalíticos conservados e peptídeo sinal. Beta-1,3-glucanases e proteínas ligantes de beta-glucana são proteínas homólogas e parecem ter sido originadas a partir do mesmo gene ancestral, antes da diversificação dos holometábolos. Larvas de Ae. aegypt possuem atividades de beta-1,3-glucanases na cabeça, tubo digestivo e resto do corpo. As atividades encontradas no tubo digestivo não foram atribuídas à dieta das larvas. Essas atividades possuem uma faixa de pH ótimo entre 5-9 e massas moleculares de 41 kDa a 150kDa. Larvas de Ae. aegypt se desenvolvem a partir da eclosão dos ovos até a fase adulta em uma dieta exclusiva contendo leveduras Ustilago sp (vivas ou autoclavadas), porém esta condição não provocou uma indução das atividades de beta-1,3-glucanasesO homogenato do tubo digestivo das larvas parece lisar a parede das leveduras após 4 h de incubação. Todos os genes da família GH16 foram expressos em todos os tecidos e apresentaram diferentes padrões de expressão. Larvas silenciadas para os genes AeGH 16.5 da GHF 16 apresentaram fenótipos de curvas de sobrevivência e taxas de pupação inferiores aos controles...

Sob condições de estresse, o fenótipo observado nas larvas se agrava consideravelmente, principalmente nos genes AeGH16.1 e AeGH16.6. Nos experimentos de ensaios enzimáticos de beta-1,3-glucanases, insetos silenciados para o gene AeGH16. 5 apresentaram uma atividade muito inferior comparados aos silenciados para AeGH16.6 ou aos grupos controles (GFP e água) em amostras de tubo digestivo. Ensaios nesses grupos utilizando o resto do corpo não sofreram alterações. O perfil cromatográfico do tubo digestivo também foi analisado, e novamente insetos silenciados para AeGH16.5 apresentaram um pico de atividade menor que os demais, sugerindo que esse gene codifica a beta-1,3-glucanase intestinal em larvas de Ae. aegypt...