Age, sex, and mating status discrimination in the sand fly Lutzomyia longipalpis using near infra-red spectroscopy (NIRS).

BACKGROUND: Understanding aspects related to the physiology and capacity of vectors is essential for effectively controlling vector-borne diseases. The sand fly Lutzomyia longipalpis has great importance in medical entomology for disseminating Leishmania parasites, the causative agent of Leishmaniasis, one of the main neglected diseases listed by the World Health Organization (WHO). In this respect, it is necessary to evaluate the transmission potential of this species and the success of vector control interventions. Near-infrared spectroscopy (NIRS) has been used to estimate the age of mosquitoes in different conditions (laboratory, semi-field, and conservation), taxonomic analysis, and infection detection. However, no studies are using NIRS for sand flies. METHODS: In this study, we developed analytic models to estimate the age of L. longipalpis adults under laboratory conditions, identify their copulation state, and evaluate their gonotrophic cycle and diet. RESULTS: Sand flies were classified with an accuracy of 58-82% in 3 age groups and 82-92% when separating them into young (<8 days) or old (>8 days) insects. The classification between mated and non-mated sandflies was 98-100% accurate, while the percentage of hits of females that had already passed the first gonotrophic cycle was only 59%. CONCLUSIONS: We consider the age and copula estimation results very promising, as they provide essential aspects of vector capacity assessment, which can be obtained quickly and at a lower cost with NIRS.

Digestive α-L-fucosidase activity in Rhodnius prolixus after blood feeding: effect of secretagogue and nutritional stimuli.

Introduction: Rhodnius prolixus (Hemiptera: Reduviidae) is an important vector of Trypanosoma cruzi, the causative agent of Chagas Disease. This insect is a model for the study of insect physiology, especially concerning the digestion of blood. Among the enzymes produced in the midgut of R. prolixus after blood feeding there is a α-L-fucosidase activity. There are very few studies on α-L-fucosidase of insects, and the role of R. prolixus α-L-fucosidase is still not clear. In this work, we tested if the mechanism for production of this enzyme is similar to the observed for proteases, a secretatogue mechanism that respond to the protein contents of the meal. Methods: We tested if specific proteins or sugars elicit this response, which may help to understand the nature of the physiological substrate for this enzyme. Results: In general, our results showed that the Anterior Midgut was the only midgut fraction that responds to the blood meal in terms of α-L-fucosidase production. Besides that, this response was not triggered by midgut distension or by ingestion of the blood cell fraction. Conversely, the enzyme was produced after feeding with the plasma fraction. However, the production of α-L-fucosidase was also triggered by different biochemical stimuli, as protein or fucoidan ingestion. Discussion: This suggested that the production of the enzyme in the anterior midgut was a general physiological response under control of different convergent signals. Besides that, the comparison between different treatments for artificial blood feeding showed that heparinated blood was the choice with minor side effects for the study of the midgut α-L-fucosidase, when compared to defibrinated or citrated blood.

Structure and expression of Rhodnius prolixus GH18 chitinases and chitinase-like proteins: Characterization of the physiological role of RpCht7, a gene from subgroup VIII, in vector fitness and reproduction.

Chitinases are enzymes responsible for the hydrolysis of glycosidic linkages within chitin chains. In insects, chitinases are typically members of the multigenic glycoside hydrolase family 18 (GH18). They participate in the relocation of chitin during development and molt, and in digestion in detritivores and predatory insects, and they control the peritrophic membrane thickness. Chitin metabolism is a promising target for developing vector control strategies, and knowledge of the roles of chitinases may reveal new targets and illuminate unique aspects of their physiology and interaction with microorganisms. Rhodnius prolixus is an important vector of Chagas disease, which is caused by the parasite Trypanosoma cruzi. In this study, we performed annotation and structural characterization of nine chitinase and chitinase-like protein genes in the R. prolixus genome. The roles of their corresponding transcripts were studied in more depth; their physiological roles were studied through RNAi silencing. Phylogenetic analysis of coding sequences showed that these genes belong to different subfamilies of GH18 chitinases already described in other insects. The expression patterns of these genes in different tissues and developmental stages were initially characterized using RT-PCR. RNAi screening showed silencing of the gene family members with very different efficiencies. Based on the knockdown results and the general lack of information about subgroup VIII of GH18, the RpCht7 gene was chosen for phenotype analysis. RpCht7 knockdown doubled the mortality in starving fifth-instar nymphs compared to dsGFP-injected controls. However, it did not alter blood intake, diuresis, digestion, molting rate, molting defects, sexual ratio, percentage of hatching, or average hatching time. Nevertheless, female oviposition was reduced by 53% in RpCht7-silenced insects, and differences in oviposition occurred within 14-20 days after a saturating blood meal. These results suggest that RpCht7 may be involved in the reproductive physiology and vector fitness of R. prolixus.

Effects of anti-Leishmania compounds in the behavior of the sand fly vector Lutzomyia longipalpis.

BACKGROUND: Leishmaniasis is an infectious parasitic disease caused by pathogens of the genus Leishmania transmitted through the bite of adult female sand flies. To reduce case numbers, it is necessary to combine different control approaches, especially those aimed at the sand fly vectors. Innovative forms of control with the use of attractive sugar baits explored the fact that adult sand flies need to feed on sugars of plant origin. Leishmania parasites develop in the gut of sand flies, interacting with the sugars in the diet of adults. Recent studies have shown that sugar baits containing plant-derived compounds can reduce sand fly survival, the number of parasites per gut, and the percentage of infected sand flies. Several synthetic compounds produced from naphthoquinones and pterocarpans have anti-parasitic activity on Leishmania amazonensis and/or Leishmania infantum in cell culture. This work aimed to assess the inclusion of these compounds in sugar baits for blocking transmission, targeting the development of the Leishmania parasite inside the sand fly vector. RESULTS: We evaluated the attractant or repellent properties of these compounds, as well as of the reference compound N,N'-diethyl-m-toluamide (DEET), in sugar baits. We also observed changes in feeding preference caused by these compounds, looking for anti-feeding or stimulation of ingestion. Pterocarpanquinone L4 and pentamidine showed attractant and repellent properties, respectively. CONCLUSION: Based on the effects in feeding preference and intake volume, pterocarpanquinone L6, and the pyrazole-derived compound P8 were chosen as the most promising compounds for the future development of anti-Leishmania sugar baits. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

P2X7 Receptor Triggers Lysosomal Leakage Through Calcium Mobilization in a Mechanism Dependent on Pannexin-1 Hemichannels.

The P2X7 receptor is a critical purinergic receptor in immune cells. Its activation was associated with cathepsin release into macrophage cytosol, suggesting its involvement in lysosomal membrane permeabilization (LMP) and leakage. Nevertheless, the mechanisms by which P2X7 receptor activation induces LMP and leakage are unclear. This study investigated cellular mechanisms associated with endosomal and lysosomal leakage triggered by P2X7 receptor activation. We found that ATP at 500 µM and 5 mM (but not 50 µM) induced LMP in non-stimulated peritoneal macrophages. This effect was not observed in P2X7-deficient or A740003-pretreated macrophages. We found that the P2X7 receptor and pannexin-1 channels mediate calcium influx that might be important for activating specific ion channels (TRPM2 and two-pore channels) on the membranes of late endosomes and lysosomes leading to LMP leakage and consequent cathepsin release. These findings suggest the critical role of the P2X7 receptor in inflammatory and infectious diseases via lysosomal dysfunction.

Remote conferences in parasitology and entomology during the pandemic: eliminate or maintain in a post-pandemic world? That is the dilemma!

The COVID-19 pandemic brought enormous challenges for health, scientists and academic world two years ago. Social isolation and the inabilities of face-to-face activities generated the emergence of many educational and scientific initiatives. Remote activities gave information and brought company and affection to people which allowed students and professionals from different parts of the world to integrate. In this report we are showing the experience from three initiatives in South America of scientific dissemination in infectious diseases. We discuss the scope of having a permanent practice for access and integration in science using remote communication, which can give great benefits in unequal societies.

High larvicidal efficacy of yeast-encapsulated orange oil against Aedes aegypti strains from Brazil.

BACKGROUND: Botanical substances such as essential oils (EOs) have demonstrated insecticidal properties and are a valid option for vector control. However, free EOs are unreliable as mosquito larvicides due their easy degradation by environmental exposure to ultraviolet light and higher temperatures. Here, we assessed the efficacy of a mosquito larvicide based on orange oil in a yeast-based delivery system against Aedes aegypti strains with different resistance status towards chemical neurotoxic insecticides. This larvicide preparation was physicochemically characterized in a previous report. METHODS: Larvae of four Ae. aegypti strains from different regions of Brazil and different resistance profiles for deltamethrin (pyrethroid) and temephos (organophosphate) were tested against yeast-encapsulated orange oil (YEOO) in laboratory conditions for measurement of LC50 and LC90 values. The same assays were performed with the Belo Horizonte strain under environmental conditions (natural light and temperature). The resistance profiles of these strains were compared to the Rockefeller reference strain in all conditions. RESULTS: YEOO was found to be a highly active larvicide (LC50 < 50 mg/L) against all Ae. aegypti strains tested in both laboratory conditions (LC50 = 8.1-24.7 mg/L) and environmental conditions with natural light and temperature fluctuation (LC50 = 20.0-49.9 mg/L). Moreover, all strains were considered susceptible (RR < 5) to YEOO, considering resistance ratios calculated based on the Rockefeller strain. The resistance ratios were only higher than 2.5 for LC90-95 of Belo Horizonte in the laboratory, probably due the higher heterogeneity associated with older egg papers (> 5 months). CONCLUSION: YEOO demonstrates high larvicidal activity against Ae. aegypti strains with resistant phenotypes for deltamethrin (PY) and temephos (OP). This larvicidal activity suggests the potential for the development of YEOO as an alternative intervention to synthetic insecticides in integrated vector management programs, for populations with resistance to commonly used insecticides.

Characterization of Glycoside Hydrolase Families 13 and 31 Reveals Expansion and Diversification of α-Amylase Genes in the Phlebotomine Lutzomyia longipalpis and Modulation of Sandfly Glycosidase Activities by Leishmania Infection.

Sugar-rich food sources are essential for sandflies to meet their energy demands, achieving more prolonged survival. The digestion of carbohydrates from food is mainly realized by glycoside hydrolases (GH). To identify genes coding for α-glycosidases and α-amylases belonging to Glycoside Hydrolase Family 13 (GH13) and Glycoside Hydrolase Family 31 (GH31) in Lutzomyia longipalpis, we performed an HMMER search against its genome using known sequences from other dipteran species. The sequences retrieved were classified based on BLASTP best hit, analysis of conserved regions by alignment with sequences of proteins with known structure, and phylogenetic analysis comparing with orthologous proteins from other dipteran species. Using RT-PCR analysis, we evaluated the expression of GH13 and GH31 genes, in the gut and rest of the body of females, in four different conditions: non-fed, sugar-fed, blood-fed, and Leishmania mexicana infected females. L. longipalpis has GH13/31 genes that code for enzymes involved in various aspects of sugar metabolism, as carbohydrate digestion, storage, and mobilization of glycogen reserves, proteins involved in transport, control of N-glycosylation quality, as well as others with a putative function in the regulation of myogenesis. These proteins are representatives of GH13 and GH31 families, and their roles seem to be conserved. Most of the enzymes seem to be active with conserved consense sequences, including the expected catalytic residues. α-amylases also demonstrated the presence of calcium and chloride binding sites. L. longipalpis genome shows an expansion in the α-amylase gene family, with two clusters. In contrast, a retraction in the number of α-glucosidases occurred. The expansion of α-amylases is probably related to the specialization of these proteins for different substrates or inhibitors, which might correlate with the higher diversity of plant foods available in the natural habitat of L. longipalpis. The expression of α-glucosidase genes is higher in blood-fed females, suggesting their role in blood digestion. Besides that, in blood-fed females infected with the parasite Leishmania mexicana, these genes were also modulated. Glycoside Hydrolases from families 13 and 31 are essential for the metabolism of L. longipalpis, and GH13 enzymes seem to be involved in the interaction between sandflies and Leishmania.

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.

Determination of Chitin Content in Insects: An Alternate Method Based on Calcofluor Staining.

Chitin is an aminopolysaccharide present in yeast cells and arthropod cuticle and is one of the most abundant biopolymers. The conventional methods for the quantitation of chitin content in biological samples are based on its hydrolysis (acid or enzymatic), and the assessment of the byproduct, glucosamine. However, previously described methodologies are time-consuming, laborious, low throughput, and not applicable to insect samples in many cases. Here we describe a new approach to chitin content quantitation based on calcofluor fluorescent brightener staining of samples, followed by microplate fluorescence readings. Calcofluor is a specific chitin stain commonly used for topological localization of the polymer. The protocol was tested in three important disease vector species, namely Lutzomyia longipalpis, Aedes aegypti, and Rhodnius prolixus, and then compared to a classic colorimetric chitin assessment method. Results show that chitin content in the tested insects can vary largely in a range of 8-4600 micrograms of chitin per insect, depending on species, sex, and instar. Comparisons between measurements from the previous protocol and calcofluor method showed statistically significant differences in some samples. However, the difference might be due to interference in the classic method from non-chitin sources of glucosamine and reducing agents. Furthermore, chitinase hydrolysis reduces the total chitin mass estimated between 36 and 74%, consolidating the fluorescent measurements as actual stained chitin in the same extent that was observed with the standard protocol. Therefore, the calcofluor staining method revealed to be a fast and reliable technique for chitin quantitation in homogenized insect samples.

Characterization of the Temporal Pattern of Blood Protein Digestion in Rhodnius prolixus: First Description of Early and Late Gut Cathepsins.

Rhodnius prolixus is one important vector for the parasite Trypanosoma cruzi in Latin America, where Chagas disease is a significant health issue. Although R. prolixus is a model for investigations of vector-parasite interaction and transmission, not much has been done recently to further comprehend its protein digestion. In this work, gut proteolysis was characterized using new fluorogenic substrates, including optimum pH, inhibition profiles, and tissue and temporal expression patterns. Each protease possessed a particular tissue prevalence and activity cycle after feeding. Cathepsin L had a higher activity in the posterior midgut lumen, being characterized by a plateau of high activities during several days in the intermediate phase of digestion. Cathepsin D showed high activity levels in the tissue homogenates and in the luminal content of the posterior midgut, with a single peak 5 days after blood feeding. Aminopeptidases are highly associated with the midgut wall, where the highest activity is located. Assays with proteinaceous substrates as casein, hemoglobin, and serum albumin revealed different activity profiles, with some evidence of biphasic temporal proteolytic patterns. Cathepsin D genes are preferentially expressed in the anterior midgut, while cathepsin L genes are mainly located in the posterior portion of the midgut, with specific sets of genes being differently expressed in the initial, intermediate, or late phases of blood digestion. Significance Statement This is the first description in a non-dipteran hematophagous species of a sequential protease secretion system based on midgut cathepsins instead of the most common insect digestive serine proteases (trypsins and chymotrypsins). The midgut of R. prolixus (Hemiptera) shows a different temporal expression of proteases in the initial, intermediate, and late stages of blood digestion. In this respect, a different timing in protease secretion may be an example of adaptative convergence in blood-sucking vectors from different orders. Expanding the knowledge about gut physiology in triatomine vectors may contribute to the development of new control strategies, aiming the blocking of parasite transmission.

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.

Characterization of α-Glucosidases From Lutzomyia longipalpis Reveals Independent Hydrolysis Systems for Plant or Blood Sugars.

Lutzomyia longipalpis is the main vector of Leishmania infantum and exploits different food sources during development. Adults have a diet rich in sugars, and females also feed on blood. The sugar diet is essential for maintaining longevity, infection, and Leishmaniasis transmission. Carbohydrases, including α-glucosidases, are the main enzymes involved in the digestion of sugars. In this context, we studied the modulation of α-glucosidase activities in different feeding conditions and compartments of Lutzomyia longipalpis females, in order to characterize in detail their roles in the physiology of this insect. All tissues showed activity against MUαGlu and sucrose, with highest activities in the midgut and crop. Activity was 1,000 times higher on sucrose than on MUαGlu. Basal activities were observed in non-fed insects; blood feeding induced activity in the midgut contents, and sugar feeding modulated activity in midgut tissues. α-glucosidase activity changed after female exposure to different sugar concentrations or moieties. α-glucosidases from different tissues showed different biochemical properties, with an optimum pH around 7.0-8.0 and K M between 0.37 and 4.7 mM, when MUαGlu was used as substrate. Using sucrose as substrate, the optimum pH was around 6.0, and K M ranges between 11 and 800 mM. Enzymes from the crop and midgut tissues showed inhibition in high substrate concentrations (sucrose), with K I ranging from 39 to 400 mM, which explains the high K M values found. Chromatographic profiles confirmed that different α-glucosidases are been produced in L. longipalpis in different physiological contexts, with the distinction of at least four α-glucosidases. The results suggest that some of these enzymes are involved in different metabolic processes, like digestion of plant sugars, digestion of blood glycoproteins or glycolipids, and mobilization of energetic storages during starvation.

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.

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

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

Penicillium citrinum UFV1 ß-glucosidases: purification, characterization, and application for biomass saccharification.

BACKGROUND: ß-Glucosidases are components of the cellulase system, a family of enzymes that hydrolyze the ß-1,4 linkages of cellulose. These proteins have been extensively studied due to the possibility of their use in various biotechnological processes. They have different affinities for substrates (depending on their source) and their activities can be used for saccharification of different types of biomass. In this context, the properties and the synergistic capacity of ß-glucosidases from different organisms, to supplement the available commercial cellulase cocktails, need a comprehensive evaluation. RESULTS: Two ß-glucosidases belonging to GH3 family were secreted by Penicillium citrinum UFV. PcßGlu1 (241 kDa) and PcßGlu2 (95 kDa) presented acidic and thermo-tolerant characteristics. PcßGlu1 showed Michaelis-Menten kinetics for all substrates tested with Km values ranging from 0.09 ± 0.01 (laminarin) to 1.7 ± 0.1 mM (cellobiose, C2) and kcat values ranging from 0.143 ± 0.005 (laminarin) to 8.0 ± 0.2 s-1 (laminaribiose, Lb). PcßGlu2 showed substrate inhibition for 4-methylumbelliferyl-ß-d-glucopyranoside (MUßGlu), p-nitrophenyl-ß-d-glucopyranoside (pNPßGlu), cellodextrins (C3, C4, and C5), N-octil-ß-d-glucopyranoside, and laminaribiose, with Km values ranging from 0.014 ± 0.001 (MUßGlu) to 0.64 ± 0.06 mM (C2) and kcat values ranging from 0.49 ± 0.01 (gentiobiose) to 1.5 ± 0.2 s-1 (C4). Inhibition constants (Ki) for PcßGlu2 substrate inhibition ranged from 0.69 ± 0.07 (MUßGlu) to 10 ± 1 mM (Lb). Glucose and cellobiose are competitive inhibitors of PcßGlu1 and PcßGlu2 when pNPßGlu is used as a substrate. For PcßGlu1 inhibition, Ki = 1.89 ± 0.08 mM (glucose) and Ki = 3.8 ± 0.1 mM (cellobiose); for PcßGlu2, Ki = 0.83 ± 0.05 mM (glucose) and Ki = 0.95 ± 0.07 mM (cellobiose). The enzymes were tested for saccharification of different biomasses, individually or supplementing a Trichoderma reesei commercial cellulose preparation. PcßGlu2 was able to hydrolyze banana pseudostem and coconut fiber with the same efficiency as the T. reesei cocktail, showing significant synergistic properties with T. reesei enzymes in the hydrolysis of these alternative biomasses. CONCLUSIONS: The ß-glucosidases from P. citrinum UFV1 present different enzymatic properties from each other and might have potential application in several biotechnological processes, such as hydrolysis of different types of biomass.