The Potential of Metarhizium anisopliae Blastospores to Control Aedes aegypti Larvae in the Field.

Entomopathogenic fungi are promising as an environmentally benign alternative to chemical pesticides for mosquito control. The current study investigated the virulence of Metarhizium anisopliae blastospores against Aedes aegypti under both laboratory and field conditions. Virulence bioassays of conidia and blastospores were conducted in the laboratory, while field simulation bioassays were conducted under two conditions: totally shaded (TS) or partially shaded (PS). In the first bioassay (zero h), the larvae were added to the cups shortly after the preparation of the blastospores, and in the subsequent assays, larvae were added to the cups 3, 6, 9, and 12 days later. The survival of the larvae exposed to blastospores in the laboratory was zero on day two, as was the case for the larvae exposed to conidia on the sixth day. Under TS conditions, zero survival was seen on the third day of the bioassay. Under PS conditions, low survival rates were recorded on day 7. For the persistence bioassay under PS conditions, low survival rates were also observed. Metarhizium anisopliae blastospores were more virulent to Ae. aegypti larvae than conidia in the laboratory. Blastospores remained virulent under field simulation conditions. However, virulence rapidly declined from the third day of field bioassays. Formulating blastospores in vegetable oil could protect these propagules when applied under adverse conditions. This is the first time that blastospores have been tested against mosquito larvae under simulated field conditions, and the current study could be the basis for the development of a new biological control agent.

Metarhizium anisopliae blastospores are highly virulent to adult Aedes aegypti, an important arbovirus vector.

BACKGROUND: The use of entomopathogenic fungi (EPF) for the control of adult mosquitoes is a promising alternative to synthetic insecticides. Previous studies have only evaluated conidiospores against adult mosquitoes. However, blastospores, which are highly virulent against mosquito larvae and pupae, could also be effective against adults. METHODS: Metarhizium anisopliae (ESALQ 818 and LEF 2000) blastospores and conidia were first tested against adult Aedes aegypti by spraying insects with spore suspensions. Blastospores were then tested using an indirect contact bioassay, exposing mosquitoes to fungus-impregnated cloths. Virulence when using blastospores suspended in 20% sunflower oil was also investigated. RESULTS: Female mosquitoes sprayed with blastospores or conidia at a concentration of 108 propagules ml-1 were highly susceptible to both types of spores, resulting in 100% mortality within 7 days. However, significant differences in virulence of the isolates and propagules became apparent at 107 spores ml-1, with ESALQ 818 blastospores being more virulent than LEF 2000 blastospores. ESALQ 818 blastospores were highly virulent when mosquitoes were exposed to black cotton cloths impregnated with blastospores shortly after preparing the suspensions, but virulence declined rapidly 12 h post-application. The addition of vegetable oil to blastospores helped maintain virulence for up to 48 h. CONCLUSION: The results showed that blastospores were more virulent to adult female Ae. aegypti than conidia when sprayed onto the insects or applied to black cloths. Vegetable oil helped maintain blastospore virulence. The results show that blastospores have potential for use in integrated vector management, although new formulations and drying techniques need to be investigated.

Production of conidia using different culture media modifies the virulence of the entomopathogenic fungus Metarhiziumys against Aedes aegypti larvae.

BACKGROUND & OBJECTIVES: Entomopathogenic fungi are being investigated for the biological control of a range of mosquitoes. Metarhizium conidiospores (conidia) effectively kill Aedes aegypti larvae and could be deployed as an alternative to chemical insecticides. Conidial yield and virulence of fungi when cultured on three different types of solid media, was investigated. METHODS: Three culture media were tested: a) Sabouraud dextrose agar (SDA); b) rice flour yeast agar (RYA) and c) rice grains. Conidia produced using these substrates were tested for virulence against Ae. aegypti larvae obtained from field collected eggs. Larvae (2nd - 3rd instar) were exposed to aqueous conidial suspensions and survival monitored over 7 days. Survival analysis was performed using Log-Rank and Kaplan Meier tests, while fungal growth and conidial yields were analyzed using a two-way ANOVA. RESULTS: There were only small differences between growth rates on RYA and SDA; however, ESALQ 818 showed the highest conidial yield on rice. Conidia produced on rice grains were more virulent, rapidly reducing survival rates of mosquito larvae. ESALQ 818 conidia produced on rice grains, RYA and SDA killed 100% of the larvae on the 2nd, 3rd and 4th day of exposure, respectively. IP 46 virulence of was consistently lower than ESALQ 818 for all the media tested. INTERPRETATION & CONCLUSION: The choice of culture media can influence the virulence of fungal conidia to Ae. aegypti larvae, demonstrating the importance of not only selecting the most virulent isolate but also standardizing growth conditions when screening for virulence.

Effects of a Reserve Protein on Spodoptera frugiperda Development: A Biochemical and Molecular Approach to the Entomotoxic Mechanism.

Talisin is a storage protein from Talisia esculenta seeds that presents lectin-like and peptidase inhibitor properties. These characteristics suggest that talisin plays a role in the plant defense process, making it a multifunctional protein. This work aimed to investigate the effects of chronic intake of talisin on fifth instar larvae of Spodoptera frugiperda, considered the main insect pest of maize and the cause of substantial economic losses in several other crops. The chronic intake of talisin presented antinutritional effects on the larvae, reducing their weight and prolonging the total development time of the insects. In addition, talisin-fed larvae also showed a significant reduction in the activity of trypsin-like enzymes. Midgut histology analysis of talisin-fed larvae showed alterations in the intestinal epithelium and rupture of the peritrophic membrane, possibly causing an increase of aminopeptidase activity in the midgut lumen. Talisin also proved to be resistant to degradation by the digestive enzymes of S. frugiperda. The transcription profile of trypsin, chymotrypsin and aminopeptidase genes was also analyzed through qPCR technique. Talisin intake resulted in differential expression of at least two genes from each of these classes of enzymes. Molecular docking studies indicated a higher affinity of talisin for the less expressed enzymes.

Isolation and Identification of a Potential Amylolytic Probiotic Bacterium from the Gut of Jundiá Catfish, Rhamdia quelen

ABSTRACT This study aimed to isolate a potential probiotic amylolytic strain from the gut of jundiá catfish to improve carbohydrate digestibility in fish. Two of 31 strains isolated from the foregut of Rhamdia quelen were able to grow on starch-agar medium and were considered amylolytic. The strain that presented higher amylolytic potential, based on a qualitative amylase assay, was chosen. The strain was phenotypically characterized and analysed to determine bile and pH tolerance and extracellular quantitative amylase activity. The probiotic candidate, identified as Aeromonas veronii, showed the ability to survive stresses from a range of pH and bile salt conditions and secreted an interesting enzymatic profile, which may exhibit a synergistic effect when combined with the enzymes secreted by the jundiá catfish, improving carbohydrate digestion in the host. The results demonstrated the potential of A. veronii to improve the digestion process in jundiá by providing exogenous enzymes for the breakdown and absorption of nutrients.

Entomotoxic properties of Dioclea violacea lectin and its effects on digestive enzymes of Anagasta kuehniella (Lepidoptera).

Entomotoxic plant lectins have been extensively studied in the past two decades, yet the exact mechanisms underlying their toxic effects remain unknown. This study investigated the effects of Dioclea violacea lectin (DVL) on larval development in Anagasta kuehniella. Chronic exposure of larvae (from neonates to the fourth instar) demonstrated that DVL interfered with larval growth, retarding development and decreasing larval mass without affecting survival. DVL decreased trypsin-like, chymotrypsin-like, and α-amylase activities and proved resistant to proteolysis by midgut proteases up to 24h. Shorter exposures to dietary DVL had no effect on midgut enzyme activity. Feeding fourth-instar larvae with fluorescently-labeled DVL revealed lectin binding to the peritrophic membrane.

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus.

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.

Freshwater catfish jundiá (Rhamdia quelen) larvae are prepared to digest inert feed at the exogenous feeding onset: physiological and histological assessments.

This study assessed the morphological development of jundiá larvae's digestive system and digestive proteolytic activity. Specific serine proteinases activities varied over time, with the highest peak at 12 h after hatching (AH), which corresponded to 296.38 ± 84.20 mU mg⁻¹ for trypsin and 315.45 ± 42.16 mU mg⁻¹ for chymotrypsin. Specific aspartic proteinases activities increased up to the start of weaning, oscillated during that phase, but showed a consistent increase after that, resulting in the highest specific activity at 252 h AH (7.88 ± 0.68 mU mg⁻¹). Gel assays showed different molecular forms, especially of serine proteinases. Histology showed the gastrointestinal tract development onset at 0 h AH and open mouth at 4 h AH. At 16 h AH, the following differentiation of the digestive tract was evident: oropharyngeal cavity, esophagus, liver, pancreas, stomach, and intestine. At 40 h AH, zymogen granules in the pancreas were observed, and at 48 h AH, mucus in the digestive tract and gastric glands in the stomach. Findings indicate that jundiá has a functional stomach before the end of vitelline reserves. Therefore, jundiá larvae are probably capable to digest inert feed at the exogenous feeding onset.

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.

Alpha-glucosidase promotes hemozoin formation in a blood-sucking bug: an evolutionary history.

BACKGROUND: Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: Hz formation activity of an alpha-glucosidase was investigated. Hz formation was inhibited by specific alpha-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect alpha-glucosidase was able to inhibit Hz formation. The alpha-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that alpha-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of alpha-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both alpha-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of alpha-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of alpha-glucosidase shows a high similarity to the insect alpha-glucosidases, with critical histidine and aspartic residues conserved among the enzymes. CONCLUSIONS/SIGNIFICANCE: Herein the Hz formation is shown to be associated to an alpha-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that alpha-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.

Hydrolytic enzymes of leaf-cutting ant fungi.

The production of enzymes and the colonization of leaves by Leucoagaricus gongylophorus were investigated to further understand the digestive interactions of leaf-cutting ant colonies. The enzymes detected were indicative of a saprophytic origin of this fungus, producing all the enzymes necessary for plant tissue breakdown. Enhanced activities of certain enzymes in the fungus garden extracts may be due to the particular behaviour of the adult worker ants that concentrate fungal acquired enzymes in the rectal fluid and subsequently defaecate these enzymes onto the leaves. The production of chitinases by the fungus may be an ancestral vestige of lower attines, and may have a role as agonists of invading microbes. Growth of the fungus on plant cell wall medium resulted in highest enzyme activity against pectin, reflecting the fact that polygalacturonans comprise the main matrix of the primary plant cell wall. SEM shows that L. gongylophorus does not form specialized structures for cell wall penetration, but gains access to the inner plant tissue at the cut edges of the leaf fragments. Enzymes secreted by the fungus were compared to those seen in larval and adult leaf-cutting ants, demonstrating the inter-dependence of the symbiotic relationship between the ants and their fungi.