A versatile inhibitor of digestive enzymes in Aedes aegypti larvae selected from a pacifastin (TiPI) phage display library.

In Brazil, the major vector of arboviruses is Aedes aegypti, which can transmit several alpha and flaviviruses. In this work, a pacifastin protease inhibitor library was constructed and used to select mutants for Ae. aegypti larvae digestive enzymes. The library contained a total of 3.25 × 105 cfu with random mutations in the reactive site (P2-P2'). The most successfully selected mutant, TiPI6, a versatile inhibitor, was able to inhibit all three Ae. aegypti larvae proteolytic activities, trypsin-like, chymotrypsin-like and elastase-like activities, with IC50 values of 0.212 nM, 0.107 nM and 0.109 nM, respectively. In conclusion, the TiPI mutated phage display library was shown to be a useful tool for the selection of an inhibitor of proteolytic activities combined in a mix. TiPI6 is capable of controlling all three digestive enzyme activities present in the larval midgut extract. To our knowledge, this is the first time that one inhibitor containing a Gln at the P1 position showed inhibitory activity against trypsin, chymotrypsin, and elastase-like activities. TiPI6 can be a candidate for further larvicidal studies.

Rotenoids from Clitoria fairchildiana R. Howard (Fabaceae) seeds affect the cellular metabolism of larvae of Aedes aegypti L. (Culicidae).

Non-domesticated species may represent a treasure chest of defensive molecules which must be investigated and rescued. Clitoria fairchildiana R. Howard is a non-domesticated Fabacea, native from the Amazonian Forest whose seeds are exquisitely refractory to insect predation. Secondary metabolites from these seeds were fractionated by different organic solvents and the CH2Cl2 fraction (CFD - Clitoria fairchildiana dichloromethane fraction), as the most toxic to 3rd instar Aedes aegypti larvae (LC50 180 PPM), was subjected to silica gel chromatography, eluted with a gradient of CH2Cl2: MeOH and sub fractioned in nine fractions (CFD1 - CFD9). All obtained fractions were tested in their toxicity to the insect larvae. Two rotenoids, a 11α-O-ß-D-glucopyranosylrotenoid and a 6-deoxyclitoriacetal 11-O-n-glucopyranoside, were identified in the mixture of CFD 7.4 and CFD 7.5, and they were toxic (LC50 120 PPM) to 3rd instar Ae. aegypti larvae, leading to exoskeleton changes, cuticular detachment and perforations in larval thorax and abdomen. These C. fairchildiana rotenoids interfered with the acidification process of cell vesicles in larvae midgut and caused inhibition of 55% of V-ATPases activity of larvae treated with 80 PPM of the compounds, when compared to control larvae. The rotenoids also led to a significant increase in the production of reactive oxygen species (ROS) in treated larvae, especially in the hindgut region of larvae intestines, indicating a triggering of an oxidative stress process to these insects.

Neem oil increases the persistence of the entomopathogenic fungus Metarhizium anisopliae for the control of Aedes aegypti (Diptera: Culicidae) larvae.

BACKGROUND: The entomopathogenic fungus Metarhizium anisopliae is a candidate for the integrated management of the disease vector mosquito Aedes aegypti. Metarhizium anisopliae is pathogenic and virulent against Ae. aegypti larvae; however, its half-life is short without employing adjuvants. Here, we investigated the use of neem oil to increase virulence and persistence of the fungus under laboratory and simulated field conditions. METHODS: Neem was mixed with M. anisopliae and added to recipients. Larvae were then placed in recipients at 5-day intervals for up to 50 days. Survival rates were evaluated 7 days after exposing larvae to each treatment. The effect of neem on conidial germination following exposure to ultraviolet radiation was evaluated under laboratory conditions. Statistical tests were carried out using ANOVA and regression analysis. RESULTS: Laboratory bioassays showed that the fungus alone reduced survival to 30% when larvae were exposed to the treatment as soon as the suspension had been prepared (time zero). A mixture of fungus + neem resulted in 11% survival at time zero. The combination of fungus + neem significantly reduced larval survival rates even when suspensions had been maintained for up to 45 days before adding larvae. For simulated-field experiments 1% neem was used, even though this concentration is insecticidal, resulting in 20% survival at time zero. However, this toxic effect was reduced over time. When used alone under simulated-field conditions the fungus rapidly lost virulence. The formulation fungus + neem effectively maintained fungal virulence, with larval survival rates significantly reduced for up to 45 days after preparation of the suspensions. The effective half-life of the fungus or neem when used separately was 6 and 13 days, respectively. The half-life of fungus formulated in 1% neem was 34 days. Conidia suspended in neem maintained high levels of germination even following a 2-h exposure to ultraviolet radiation. CONCLUSIONS: A combination of the entomopathogenic fungus M. anisopliae with neem oil effectively increases the half-life and virulence of the fungus when tested against Ae. aegypti larvae, even under simulated field conditions. Neem oil also protected the fungus from the damaging effects of ultraviolet radiation.

Hypometabolic strategy and glucose metabolism maintenance of Aedes aegypti egg desiccation.

The mosquito Aedes aegypti is vector of several viruses including yellow fever virus, dengue virus chikungunya virus and Zika virus. One of the major problems involving these diseases transmission is that A. aegypti embryos are resistant to desiccation at the end of embryogenesis, surviving and remaining viable for several months inside the egg. Therefore, a fine metabolism control is essential to support these organisms throughout this period of resistance. The carbohydrate metabolism has been shown to be of great importance during arthropod embryogenesis, changing dramatically in order to promote growth and differentiation and in periods of resistance. This study investigated fundamental aspects of glucose metabolism in three stages of A. aegypti egg development: pre-desiccated, desiccated, and rehydrated. The activities of regulatory enzymes in carbohydrate metabolism such as pyruvate kinase, hexokinase and glucose 6-phosphate dehydrogenase were evaluated. We show that these activities were reduced in A. aegypti desiccated eggs, suggesting a decreased activity of glycolytic and pentose phosphate pathway. In contrast, gluconeogenesis increased in desiccated eggs, which uses protein as substrate to synthesize glucose. Accordingly, protein amount decreased during this stage, while glucose levels increased. Glycogen content, a major carbohydrate reserve in mosquitoes, was evaluated and shown to be lower in desiccated and rehydrated eggs, indicating it was used to supply energy metabolism. We observed a reactivation of carbohydrate catabolism and an increased gluconeogenesis after rehydration, suggesting that controlling glucose metabolism was essential not only to survive the period of desiccation, but also for subsequent larvae hatch. Taken together, these results contribute to a better understanding of metabolism regulation in A. aegypti eggs during desiccation periods. Such regulatory mechanisms enable higher survival rate and consequently promote virus transmission by these important disease vectors, making them interesting subjects in the search for novel control methods.

Functional characterization of a serine protease inhibitor modulated in the infection of the Aedes aegypti with dengue virus.

During feeding with blood meal, female Aedes aegypti can transmit infectious agents, such as dengue, yellow fever, chikungunya and Zika viruses. Dengue virus causes human mortality in tropical regions of the world, and there is no specific treatment or vaccine with maximum efficiency being used for these infections. In the vector-virus interaction, the production of several molecules is modulated by both mosquitoes and invading agents. However, little information is available about these molecules in the Ae. aegypti mosquito during dengue infection. Inhibitors of the pacifastin family have been described to participate in the immune response of insects and Pac2 is the only gene of this family present in Ae. aegypti being then chosen for investigation. Pac2 was expressed in E. coli, purified and analyzed by mass spectrometry and SDS-PAGE. The Pac2 transcript was detected by qPCR, and its protein levels were assessed by Western blotting. The inhibitory activity of Pac2 was measured using its Ki, IC50 and zymography. Mosquito infections with DENV were introduced with the Brazilian ACS-46 DENV-2 strain propagated in C6/36 cells. In the present work, we showed that it is possibly involved in the interaction of the mosquitoes with the dengue virus. The Pac2 transcript was detected in larvae and in both the salivary gland and midgut of Ae. aegypti females, while the native protein was identified in females 3 h post-blood meal. Pac2 is a strong inhibitor of trypsin-like and thrombin-like proteases, which are present in 4th instar larvae midgut and females 24 h after blood meal. During DENV infection, up regulation of Pac2 expression occurs in the salivary gland and midgut. Pac2 is the first Pacifastin inhibitor member described in mosquitoes. Our results suggest that Pac2 acts on mosquito serine proteases, mainly the trypsin-like type, and is under transcriptional control by virus infection signals to allow its survival in the vector or by the mosquito as a defense mechanism against virus infection.

Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvae.

BACKGROUND: Dengue, transmitted primarily by the bites of infected Aedes aegypti L., is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies are primarily based on controlling the vector, using insecticides, but the appearance of resistance poses new challenges. Recently, highly selective protease inhibitors by phage display were obtained for digestive enzymes of the 4th instar larvae (L4) midgut. These mutants were not confirmed as a larvicide due to the low yield of the expression of these inhibitors. In the present study, chimera molecules were constructed based on the mutations at positions P1-P4' selected previously. The T6, T23 and T149 mutants were mixed with another Kunitz inhibitor, domain 1 of the inhibitor boophilin (D1). METHODS: The chimeras T6/D1, T149/D1 and T23/D1 were expressed at high levels in P. pastoris yeast, purified by ionic exchange chromatography and their homogeneity was analyzed by SDS-PAGE. The chimera inhibitors were assayed against larval trypsin, chymotrypsin and elastase using specific chromogenic substrates. The inhibitors were assayed for their larvicide potential against L4. RESULTS: The chimeras exhibited strong inhibitory activities against the larval digestive enzymes in a dose-dependent manner. T6/D1, T149/D1 and T23/D1 exhibited strong larvicidal activity against L4 of Ae. aegypti with inhibitor concentrations in the µM range. A synergistic increase in mortality was observed when a mixture of the three chimeric inhibitors was tested. CONCLUSIONS: The strategy for constructing the chimeric inhibitors was successful. The chimeras showed strong larvicidal activity against Ae. aegypti. In the future, our findings can be used to design synthetic inhibitors for larvae digestive enzymes as an alternative method to control the dengue vector.

Toxicity of hydrolyzed vicilins toward Callosobruchus maculatus and phytopathogenic fungi.

Studies have shown that vicilins (7S storage proteins) from seeds were able to bind to the surface of the Callosobruchus maculatus larval midgut and to the peritrophic matrices of the midguts of Diatraea saccharalis and Tenebrio molitor , inhibiting larval development. Vicilins were also shown to inhibit yeast growth and bind to yeast cells through the association with chitin-containing structures. The present work studies the association of peptides from vicilins of genotypes of Vigna unguiculata (susceptible and resistant to bruchid) with acetylated chitin and the toxicity of vicilin fragments and chitin-binding vicilin fragments to C. maculatus and phytopathogenic fungi. Hydrolysis of vicilins with alpha-chymotrypsin results in a complex mixture of fragments that were separated by chitin-affinity chromatography. Chitin-binding peptides from both genotypes were toxic to C. maculatus larvae, and alpha-chymotrypsin-hydrolyzed vicilins were deleterious to the above insect and to Fusarium oxysporum , Colletotrichum musae , and Saccharomyces cerevisiae fungi.

First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): new perspectives for an insect-bacteria association.

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.

First isolation of microorganisms from the gut diverticulum of Aedes aegypti (Diptera: Culicidae): new perspectives for an insect-bacteria association

We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.