Biosynthesis of mosquito vitellogenin

Vitellogenin (Vg), the hemolymph precursor to the major yolk protein in mosquitoes, is synthesized in the fat body of blood-fed females. Mosquito Vg consists of two subunits with Mr = 200,000 and 66,000. Here, we demonstrate that both the Vg subunits are first synthesized as a single precursor. The identity of this Vg precursor was confirmed by immunoprecipitation with subunit-specific monoclonal antibodies. In cell-free translation of fat body poly (A)+ RNA, the Vg precursor had Mr = 224,000 which increased to 240,000 in the presence of canine pancreatic microsomal membranes. A precursor with Mr = 250,000 was immunoprecipitated in microsomal fractions isolated from rat bodies. With in vitro pulse labeling, the 250-kDa precursor could be detected in homogenates of fat bodies from blood-fed mosquitoes only during the first few hours accumulation of the Vg precursor was achieved by an in vitro stimulation of Vg synthesis in previtellogenic fat bodies cultured with an insect hormone, 20-hydroxyecdysone. The 250-kDa precursor was glycosylated and to a much lesser degree phosphorylated. Treatment of fat bodies with tunicamycin yielded the precursor with Mr = 226,000 which was neither glycosylated nor phosphorylated. The reduction in molecular mass of the 250-kDa Vg precursor and of both mature Vg subunits combined was similar after digestion with endoglycosidase H, indicating that glycosylation is completed prior to cleavage of the Vg precursor. In vitro pulse-chase experiments revealed rapid proteolytic cleavage of the 250-kDa precursor to two polypeptides with Mr = 190,000 and 62,000 which transformed into mature Vg subunits of 200- and 66-kDa as the last step prior to Vg secretion. This last step in Vg processing was inhibited by an ionophore, monensin, and therefore occurred in the Golgi complex. Sulfation as an additional, previously unknown, modification of mosquito Vg was revealed by the incorporation of sodium [35S]sulfate into both Vg subunits. Since sulfation of Vg was predominantly blocked by monensin, the final maturation of Vg subunits in the Golgi complex is, at least in part, due to this modification.     

Gene expression patterns of Helicoverpa armigera gut proteases

Relative quantification of reported gut proteinase cDNAs from Helicoverpa armigera larvae fed on various host plants (cotton, chickpea, pigeonpea, tomato and okra), non-host plant PIs (winged bean, bitter gourd, ground nut, and capsicum) and during larval development has been carried out using semi-quantitative RT-PCR. Five trypsin-like and three chymotrypsin-like proteinases were categorized as insensitive or sensitive to most of the proteinase inhibitors (PIs) and insensitive/sensitive to specific PIs based on their expression analysis. These results were supported by amino acid sequence analysis, where diverged amino acids were observed in the regions, which are reported to be involved in typical trypsin-trypsin inhibitor interactions and critical for proteinase inhibitor resistance. Among exopeptidases (five aminopeptidase and three carboxypeptidase), HaAmi4 and HaAmi5 of aminopeptidase and HaCar1 of carboxypeptidase exhibited considerable differential expression. Elastase and cathepsin B-like proteinases were expressed at negligible levels. The proteases identified in the study would be ideal candidates for further interactions studies with protease inhibitors to understand the structural reasons of protease inhibitor insensitivity.     

The control of gut motility

Gut motility in non-mammalian vertebrates as in mammals is controlled by the presence of food, by autonomic nerves and by hormones. Feeding and the presence of food initiates contractions of the stomach wall and subsequently gastric emptying, peristalsis, migrating motor complexes and other patterns of motility follow. This overview will give examples of similarities and differences in control systems between species. Gastric receptive relaxation occurs in fish and is an enteric reflex. Cholecystokinin reduces the rate of gastric emptying in fish as in mammals. Inhibitory control of peristalsis is exerted, e.g. by VIP, PACAP, NO in fish and amphibians, while excitatory stimuli arise from nerves releasing tachykinins, acetylcholine or serotonin (5-HT). In crocodiles, we have found the presence of the same nerve types, although the effects on peristalsis have not been studied. Recent studies on signal transduction in the gut smooth muscle of fish and amphibians suggest that external Ca2+ is of great importance, but not the only source of Ca2+ recruitment in tachykinin-, acetylcholine- or serotonin-induced contractions of rainbow trout and Xenopus gastrointestinal smooth muscle. The effect of acetylcholine involves reduction of cAMP-levels in the smooth muscle cells. It is concluded that, in general, the control systems in non-mammalian vertebrates are amazingly similar between species and animal groups and in comparison with mammals.     

Larval mosquito control through deployment of xiphidiocercariae

Larval trematodes (xiphidiocercariae) have been found to encyst in mosquito larvae. When the number of cysts is high, this results in the death of the mosquito larva. It is suggested that populating waters containing mosquito larvae with xiphidiocercariae can be developed as a mosquito control measure.     

Use of cyclopoid copepods for mosquito control

The New Orleans Mosquito Control Board mass produces Mesocyclops longisetus and Macrocyclops albidus for introduction to mosquito breeding sites as a routine part of control operations. Mesocyclops longisetus is used in tires that collect rainwater; M. albidus is used in temporary pools. Field trials in a Spartina marsh, rice fields, and residential roadside ditches in Louisiana suggest that M. longisetus and M. albidus could be of use to control larvae of Anopheles spp. and Culex quinquefasciatus. Mesocyclops longisetus has proved to be effective for Aedes aegypti control in cisterns, 55-gallon drums, and other domestic containers in Honduras.     

Wetlands and mosquito control in the twenty-first century

Wetlands Ecology and Management - This synthesis is a short introduction to the Wetlands and Mosquito Control special issue of Wetlands Ecology and Management. The geographic extent of the articles...     

Isolation and in vitro identification of proteinase inhibitors from soybean seeds inhibiting helicoverpa gut proteases

Helicoverpa armigera is a polyphagous pest damaging vast numbers of different crops leading to decrease in total production. Use of Bt transgenic to control H. armigera has worked well but has increased resistance against Bt in H. armigera and controversies about the Bt transgenic making it imperative to find another strategy to control attack. Soybean is a nonhost plant for H. armigera; reason could be laid in the defense system of the soybean. Proteinase Inhibitor (PIs) have been extensively studied for development of resistance against insect pest. Two cultivars developed by our university were investigated for the presence of proteinase inhibitors namely, MAUS-158 and MAUS-61. Partially purified inhibitors were showed inhibition of total protease activity of gut extract by 91.34±1.49 and 89.95±0.96% by MAUS-158 and MAUS-61, respectively. While inhibition of trypsin like proteases were found between 65 and 71% and inhibition of chymotrypsin like proteases ranges between 40 and 42%. The partial purification study shows stability of PIs up to 60°C. Soybean PIs are also showing more prominent inhibition pattern against trypsin than chymotrypsin.     

Biosynthesis of 130-kilodalton mosquito larvicide in the cyanobacterium Agmenellum quadruplicatum PR-6

The 130-kilodalton mosquito larvicidal gene, cloned from Bacillus thuringiensis var. israelensis, was introduced into the cyanobacterium Agmenellum quadruplicatum PR-6 by plasmid transformation. Transformed cells synthesized 130-kilodalton delta-endotoxin protein and showed mosquito larvicidal activity. Results demonstrate a potential use of a cyanobacterium for biological control of mosquitoes.     

A serpin from the gut bacterium Bifidobacterium longum inhibits eukaryotic elastase-like serine proteases

Serpins form a large class of protease inhibitors involved in regulation of a wide spectrum of physiological processes. Recently identified prokaryotic members of this protein family may provide a key to the evolutionary origins of the unique serpin fold and the associated inhibitory mechanism. We performed a biochemical characterization of a serpin from Bifidobacterium longum, an anaerobic Gram-positive bacterium that naturally colonizes human gastrointestinal tract. The B. longum serpin was shown to efficiently inhibit eukaryotic elastase-like proteases with a stoichiometry of inhibition close to 1. Porcine pancreatic elastase and human neutrophil elastase were inhibited with the second order association constants of 4.7 x 10(4) m(-1) s(-1) and 2.1 x 10(4) m(-1) s(-1), respectively. The B. longum serpin is expected to be active in the gastrointestinal tract, because incubation of the purified recombinant serpin with mouse feces produces a stable covalent serpin-protease adduct readily detectable by SDS-PAGE. Bifidobacteria may encounter both pancreatic elastase and neutrophil elastase in their natural habitat and protection against exogenous proteolysis may play an important role in the interaction between these commensal bacteria and their host.     

Dynamic gut microbiome across life history of the malaria mosquito Anopheles gambiae in Kenya

The mosquito gut represents an ecosystem that accommodates a complex, intimately associated microbiome. It is increasingly clear that the gut microbiome influences a wide variety of host traits, such as fitness and immunity. Understanding the microbial community structure and its dynamics across mosquito life is a prerequisite for comprehending the symbiotic relationship between the mosquito and its gut microbial residents. Here we characterized gut bacterial communities across larvae, pupae and adults of Anopheles gambiae reared in semi-natural habitats in Kenya by pyrosequencing bacterial 16S rRNA fragments. Immatures and adults showed distinctive gut community structures. Photosynthetic Cyanobacteria were predominant in the larval and pupal guts while Proteobacteria and Bacteroidetes dominated the adult guts, with core taxa of Enterobacteriaceae and Flavobacteriaceae. At the adult stage, diet regime (sugar meal and blood meal) significantly affects the microbial structure. Intriguingly, blood meals drastically reduced the community diversity and favored enteric bacteria. Comparative genomic analysis revealed that the enriched enteric bacteria possess large genetic redox capacity of coping with oxidative and nitrosative stresses that are associated with the catabolism of blood meal, suggesting a beneficial role in maintaining gut redox homeostasis. Interestingly, gut community structure was similar in the adult stage between the field and laboratory mosquitoes, indicating that mosquito gut is a selective eco-environment for its microbiome. This comprehensive gut metatgenomic profile suggests a concerted symbiotic genetic association between gut inhabitants and host.     

Development of an efficient recombinant mosquito densovirus-mediated RNA interference system and its preliminary application in mosquito control

The Aedes aegypti densovirus (AeDNV) has potential as a delivery vector for foreign nucleic acids into mosquito cells. In this study, we investigated the ability of plasmids containing recombinant viral transducing genome to induce RNA interference (RNAi) effects in C6/C36 cells. We then evaluated the efficiency of a recombinant AeDNV vector to induce RNAi in Aedes albopictus larvae. We found that the expression of V-ATPase was inhibited by up to 90% at 96 h post-transfection in transfected C6/C36 cells. In addition, the bioinsecticidal activities of various RNAi-expressing AeDNV vectors used to infect Ae. albopictus larvae were also tested. We found that when Ae. albopictus larvae were infected with recombinant AeDNV, expression of V-ATPase was downregulated by nearly 70% compared to controls. Furthermore, the median survival time bioassays demonstrated that recombinant AeDNV caused more serious pathogenic effects than the wild type virus. This is the first report showing that recombinant virus plasmid and corresponding recombinant AeDNV can be used as an effective in vitro and in vivo RNAi delivery system, respectively.     

Microbial synthesis of spherical nanosilver and nanogold for mosquito control

Biosynthesis of metal nanoparticles using microorganisms is an important area of research in nanobiotechnology, which is an emerging eco-friendly science of well-defined sizes, shapes and controlled monodispersity. The present study proposed a green process for the extracellular production of silver (Ag) and gold (Au) nanoparticles (NPs) using the soil fungi Chrysosporium keratinophilum and Verticillium lecanii. The synthesized NPs were formed fairly uniform with spherical shape determined by Transmission Electron Microscope (TEM) and confirmed by Scanning Electron Microscope (SEM). Elemental analysis on single particle was carried by EDX analysis. The results were further supported by UV-vis spectrophotometry. In addition, we have also investigated the effect of synthesized AgNPs and AuNPs against the larvae and pupae of Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti. The efficacy test was performed at different concentrations for periods of different lengths by the probit analysis. The larvae and pupae of Cx. quinquefasciatus, An. stephensi and Ae. aegypti were found highly susceptible to the synthesized AgNPs than the AuNPs. The larvae of Cx. quinquefasciatus and Ae. aegypti were found to be more susceptible to the AgNPs and AuNPs synthesized using the C. keratinophilum and V. lecanii compared with the larvae of An. stephensi. The pupae of Ae. aegypti have shown higher mortality against the synthesized AgNPs than the pupa of Cx. quinquefasciatus, while no adverse effects could be observed in the pupa of An. stephensi. By this approach, it is suggested that this rapid synthesis of nanoparticles would be useful for developing a biological process for mosquito control.