The Secreted Triose Phosphate Isomerase of Brugia malayi Is Required to Sustain Microfilaria Production In Vivo

Human lymphatic filariasis is a major tropical disease transmitted through mosquito vectors which take up microfilarial larvae from the blood of infected subjects. Microfilariae are produced by long-lived adult parasites, which also release a suite of excretory-secretory products that have recently been subject to in-depth proteomic analysis. Surprisingly, the most abundant secreted protein of adult Brugia malayi is triose phosphate isomerase (TPI), a glycolytic enzyme usually associated with the cytosol. We now show that while TPI is a prominent target of the antibody response to infection, there is little antibody-mediated inhibition of catalytic activity by polyclonal sera. We generated a panel of twenty-three anti-TPI monoclonal antibodies and found only two were able to block TPI enzymatic activity. Immunisation of jirds with B. malayi TPI, or mice with the homologous protein from the rodent filaria Litomosoides sigmodontis, failed to induce neutralising antibodies or protective immunity. In contrast, passive transfer of neutralising monoclonal antibody to mice prior to implantation with adult B. malayi resulted in 60–70% reductions in microfilarial levels in vivo and both oocyte and microfilarial production by individual adult females. The loss of fecundity was accompanied by reduced IFNγ expression by CD4⁺ T cells and a higher proportion of macrophages at the site of infection. Thus, enzymatically active TPI plays an important role in the transmission cycle of B. malayi filarial parasites and is identified as a potential target for immunological and pharmacological intervention against filarial infections.     

Genetic and Environmental Factors Associated with Laboratory Rearing Affect Survival and Assortative Mating but Not Overall Mating Success in Anopheles gambiae Sensu Stricto

Anopheles gambiae sensu stricto, the main vector of malaria in Africa, is characterized by its vast geographical range and complex population structure. Assortative mating amongst the reproductively isolated cryptic forms that co-occur in many areas poses unique challenges for programs aiming to decrease malaria incidence via the release of sterile or genetically-modified mosquitoes. Importantly, whether laboratory-rearing affects the ability of An. gambiae individuals of a given cryptic taxa to successfully mate with individuals of their own form in field conditions is still unknown and yet crucial for mosquito-releases. Here, the independent effects of genetic and environmental factors associated with laboratory rearing on male and female survival, mating success and assortative mating were evaluated in the Mopti form of An. gambiae over 2010 and 2011. In semi-field enclosures experiments and despite strong variation between years, the overall survival and mating success of male and female progeny from a laboratory strain was not found to be significantly lower than those of the progeny of field females from the same population. Adult progeny from field-caught females reared at the larval stage in the laboratory and from laboratory females reared outdoors exhibited a significant decrease in survival but not in mating success. Importantly, laboratory individuals reared as larvae indoors were unable to mate assortatively as adults, whilst field progeny reared either outdoors or in the laboratory, as well as laboratory progeny reared outdoors all mated significantly assortatively. These results highlight the importance of genetic and environment interactions for the development of An. gambiae''s full mating behavioral repertoire and the challenges this creates for mosquito rearing and release-based control strategies.     

Assembly of the Genome of the Disease Vector Aedes aegypti onto a Genetic Linkage Map Allows Mapping of Genes Affecting Disease Transmission

The mosquito Aedes aegypti transmits some of the most important human arboviruses, including dengue, yellow fever and chikungunya viruses. It has a large genome containing many repetitive sequences, which has resulted in the genome being poorly assembled — there are 4,758 scaffolds, few of which have been assigned to a chromosome. To allow the mapping of genes affecting disease transmission, we have improved the genome assembly by scoring a large number of SNPs in recombinant progeny from a cross between two strains of Ae. aegypti, and used these to generate a genetic map. This revealed a high rate of misassemblies in the current genome, where, for example, sequences from different chromosomes were found on the same scaffold. Once these were corrected, we were able to assign 60% of the genome sequence to chromosomes and approximately order the scaffolds along the chromosome. We found that there are very large regions of suppressed recombination around the centromeres, which can extend to as much as 47% of the chromosome. To illustrate the utility of this new genome assembly, we mapped a gene that makes Ae. aegypti resistant to the human parasite Brugia malayi, and generated a list of candidate genes that could be affecting the trait.     

Effects of predation by the copepod Mesocyclops ogunnus on the sex ratios of mosquito Aedes albopictus

Cyclopoid copepods are important predators in many aquatic ecosystems and have been used as biological agents in successful programs to control mosquito larvae. However, the impacts of this predation on adult mosquito populations are still poorly understood. The present study compared the sex ratios and body sizes (measured as wing length) of Aedes albopictus mosquitoes emerging from recipients containing the copepod predator Mesocyclops ogunnus with control situations without this predator. We found that copepod predation significantly biased mosquito sex ratios toward females, and that both the males and females emerging from copepod-containing recipients were significantly larger than control insects. The ecological and epidemiological consequences of the changes induced by copepod predation on mosquito populations are discussed.     

The effect of selection of both sire and dam on the response of F1 generation lambs to vaccination with irradiated Trichostrongylus colubriformis larvae

Windon R. G. and Dineen J. K. (1981). The effect of selection of both sire and dam on the response of F₁ generation lambs to vaccination with irradiated Trichostrongylus colubriformis larvae. International Journal for Parasitology11: 11–18. Rams and ewes, tested for responsiveness to vaccination with irradiated T. colubriformis larvae at an early age, were mated on the basis of responder × responder and non-responder × non-responder. Progeny were vaccinated at 8 and 12 weeks of age with 20,000 irradiated larvae, treated with anthelmintic at 16 weeks and challenged with 20,000 normal larvae at 17 weeks. Faecal egg counts of progeny from responder matings were significantly lower than progeny from non-responders, and within each mating type, ewe lambs had markedly lower egg counts than ram lambs. The level of circulating complement-fixing antibodies to T. colubriformis larval extract were inversely related to egg counts. Thus, ewe progeny from responder matings had the highest serum antibody levels, non-responder ram progeny had the lowest levels and responder rams and non-responder ewes had similar intermediate levels. In vitro responses of cells stimulated with T. colubriformis L₃ antigen were greater in progeny from responder matings, whereas responses to bacterial lipopolysaccharide were higher in progeny from non-responder matings. The results confirm that the response to vaccination at an early age is genetically determined, and show that the response of progeny is most vigorously expressed when both sires and dams have been selected.     

Anopheles Midgut Epithelium Evades Human Complement Activity by Capturing Factor H from the Blood Meal

Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood.     

Assessing the effects of native and alien plant ash on mosquito abundance

Plant invasions have been linked to displacement of native vegetation and altering of fire regimes and might influence vector mosquito populations by altering habitats or nutrient inputs. Whereas wildfire effects on terrestrial ecosystems are relatively well‐studied, ash depositions into aquatic ecosystems and effects on semi‐aquatic taxa such as mosquitoes have remained overlooked. Here, we investigated mosquito colonization in water treated with ash from native plants [quinine tree (Rauvolfia caffra), Transvaal milk plum (Englerophytum magalismontanum), apple leaf (Philenoptera violacea)] and invasive alien plants [i.e., lantana (Lantana camara), guava (Psidium guajava), red river gum (Eucalyptus camaldulensis)] in containers at two ash concentrations (i.e., 1, 2 g/L). Overall, there was no statistically clear difference in colonization between ash from native and alien species. We recorded colonization by two mosquito genera (Culex spp. and Anopheles spp.), with Culex generally much more abundant than Anopheles. Few differences were identified among the plants, with statistically clear effects of ash type and concentration on larval and pupal stages. High Culex egg and larval abundances were shown in lantana and apple leaf treatments compared to controls, and milkplum versus controls for pupae of both genera. Further research is required to elucidate the influence of nutrient inputs from different ash species on vector mosquito population dynamics.     

Intermediate host for an Amblyospora sp. (microspora) infecting the mosquito, Culex annulirostris

Experiments conducted in Australia are described which show that there is an intermediate host involved in the life cycle of an Amblyospora sp. infecting the mosquito, Culex annulirostris. Microsporidian spores produced in the cyclopoid copepod, Mesocyclops albicans, are infectious to laboratory colony C. annulirostris larvae and produce developmental stages identical to those of natural Amblyospora infections in this mosquito. These larval infections result in uninucleate spores in progeny mosquitoes which, in turn, are infectious to M. albicans. Subsequent studies conducted with Amblyospora, copepods, and mosquitoes in the United States to replicate and reconfirm results obtained in Australia were successful. This is the first substantiated evidence of the involvement of intermediate hosts in the life cycles of microsporidia, and the discovery now provides us with information needed to evaluate these organisms as biological control agents for mosquitoes and other disease vectors.     

Annotated Differentially Expressed Salivary Proteins of Susceptible and Insecticide-Resistant Mosquitoes of Anopheles stephensi

Vector control is one of the major global strategies for control of malaria. However, the major obstacle for vector control is the development of multiple resistances to organochlorine, organophosphorus insecticides and pyrethroids that are currently being used in public health for spraying and in bednets. Salivary glands of vectors are the first target organ for human-vector contact during biting and parasite-vector contact prior to parasite development in the mosquito midguts. The salivary glands secrete anti-haemostatic, anti-inflammatory biologically active molecules to facilitate blood feeding from the host and also inadvertently inject malaria parasites into the vertebrate host. The Anopheles stephensi mosquito, an urban vector of malaria to both human and rodent species has been identified as a reference laboratory model to study mosquito—parasite interactions. In this study, we adopted a conventional proteomic approach of 2D-electrophoresis coupled with MALDI-TOF mass spectrometry and bioinformatics to identify putative differentially expressed annotated functional salivary proteins between An. stephensi susceptible and multiresistant strains with same genetic background. Our results show 2D gel profile and MALDI-TOF comparisons that identified 31 differentially expressed putative modulated proteins in deltamethrin/DDT resistant strains of An. stephensi. Among these 15 proteins were found to be upregulated and 16 proteins were downregulated. Our studies interpret that An. stephensi (multiresistant) caused an upregulated expression of proteins and enzymes like cytochrome 450, short chain dehyrdogenase reductase, phosphodiesterase etc that may have an impact in insecticide resistance and xenobiotic detoxification. Our study elucidates a proteomic response of salivary glands differentially regulated proteins in response to insecticide resistance development which include structural, redox and regulatory enzymes of several pathways. These identified proteins may play a role in regulating mosquito biting behavior patterns and may have implications in the development of malaria parasites in resistant mosquitoes during parasite transmission.     

Evolutionary History of Eukaryotic α-Glucosidases from the α-Amylase Family

Although some α-glucosidases from the α-amylase family (glycoside hydrolase family GH13) have been studied extensively, their exact number, organization on the chromosome, and orthology/paralogy relationship were unknown. This was true even for important disease vectors where gut α-glucosidase is known to be receptor for the Bin toxin used to control the population of some mosquito species. In some cases orthologs from related species were studied intensively, while potentially important paralogs were omitted. We have, therefore, used a bioinformatics approach to identify all family GH13 α-glucosidases from the selected species from Metazoa (including three mosquito species: Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus) as well as from Fungi in an effort to characterize their arrangement on the chromosome and evolutionary relationships among orthologs and among paralogs. We also searched for pseudogenes and genes coding for enzymatically inactive proteins with a possible new function. We have found GH13 α-glucosidases mostly in Arthropoda and Fungi where they form gene families, as a result of multiple lineage-specific gene duplications. In mosquito species we have identified 14 α-glucosidase (Aglu) genes of which only five have been biochemically characterized so far, two are putative pseudogenes and the rest remains uncharacterized. We also revealed quite a complex evolutionary history of the eukaryotic α-glucosidases probably involving multiple losses of genes or horizontal gene transfer from bacteria.     

Characterization of subcellular localization of duck enteritis virus UL51 protein

Background

Dengue virus (DENV), a mosquito borne flavivirus is an important pathogen causing more than 50 million infections every year around the world. Dengue diagnosis depends on serology, which is not useful in the early phase of the disease and virus isolation, which is laborious and time consuming. There is need for a rapid, sensitive and high throughput method for detection of DENV in the early stages of the disease. Several real-time PCR assays have been described for dengue viruses, but there is scope for improvement. The new generation TaqMan Minor Groove Binding (MGB) probe approach was used to develop an improved real time RT-PCR (qRT-PCR) for DENV in this study.

Results

The 3'UTR of thirteen Indian strains of DENV was sequenced and aligned with 41 representative sequences from GenBank. A region conserved in all four serotypes was used to target primers and probes for the qRT-PCR. A single MGB probe and a single primer pair for all the four serotypes of DENV were designed. The sensitivity of the two step qRT-PCR assay was10 copies of RNA molecules per reaction. The specificity and sensitivity of the assay was 100% when tested with a panel of 39 known positive and negative samples. Viral RNA could be detected and quantitated in infected mouse brain, cell cultures, mosquitoes and clinical samples. Viral RNA could be detected in patients even after seroconversion till 10 days post onset of infection. There was no signal with Japanese Encephalitis (JE), West Nile (WN), Chikungunya (CHK) viruses or with Leptospira, Plasmodium vivax, Plasmodium falciparum and Rickettsia positive clinical samples.

Conclusion

We have developed a highly sensitive and specific qRT-PCR for detection and quantitation of dengue viruses. The assay will be a useful tool for differential diagnosis of dengue fever in a situation where a number of other clinically indistinguishable infectious diseases like malaria, Chikungunya, rickettsia and leptospira occur. The ability of the assay to detect DENV-2 in inoculated mosquitoes makes it a potential tool for detecting DENV in field-caught mosquitoes.     

A mosquito parasite from a mosquito predator

The fungus Coelomomyces macleayae has been reported from treehole mosquito larvae of three Aedes subgenera in Australia, Fiji, and the United States. This fungus is now recorded for the first time from a mosquito of the genus Toxorhynchites, the large predatory larvae of which are of some importance in the naturalistic control of associated mosquito pests and vectors. A single parasitized larva of T. rutilus septentrionalis was collected from a magnolia treehole (previously used for A. triseriatus infection experiments) near West Lake, Louisiana, in September, 1971. Larval A. triseriatus and Orthopodomyia signifera were also present but were uninfected.     

Development of low-cost media for the culture of mosquito larvicides, Bacillus sphaericus and Bacillus thuringiensis serovar. israelensis

Mosquito larvicides like Bacillus sphaericus and Bacillus thuringiensis serovar. israelensis have been widely and effectively used in mosquito control programs, but the industrial production of these bacilli is expensive. Here we have attempted to develop three cost-effective media, based on cheap sources, potato, common sugar and bengalgram. Growth and production of the insecticidal proteins from these bacteria were satisfactory. Bioassay studies with different mosquito larvae showed considerable toxicity. Therefore the investigation suggests that potato-based culture media are more economical for the industrial production of B. sphaericus and B. thuringiensis serovar. israelensis.     

Mosquito Consumption by Insectivorous Bats: Does Size Matter?

Insectivorous bats have often been touted as biological control for mosquito populations. However, mosquitoes generally represent only a small proportion of bat diet. Given the small size of mosquitoes, restrictions imposed on prey detectability by low frequency echolocation, and variable field metabolic rates (FMR), mosquitoes may not be available to or profitable for all bats. This study investigated whether consumption of mosquitoes was influenced by bat size, which is negatively correlated with echolocation frequency but positively correlated with bat FMR. To assess this, we investigated diets of five eastern Australian bat species (Vespadelus vulturnus Thomas, V. pumilus Gray, Miniopterus australis Tomes, Nyctophilus gouldi Tomes and Chalinolobus gouldii Gray) ranging in size from 4-14 g in coastal forest, using molecular analysis of fecal DNA. Abundances of potential mosquito and non-mosquito prey were concurrently measured to provide data on relative prey abundance. Aedes vigilax was locally the most abundant mosquito species, while Lepidoptera the most abundant insect order. A diverse range of prey was detected in bat feces, although members of Lepidoptera dominated, reflecting relative abundance at trap sites. Consumption of mosquitoes was restricted to V. vulturnus and V. pumilus, two smaller sized bats (4 and 4.5 g). Although mosquitoes were not commonly detected in feces of V. pumilus, they were present in feces of 55 % of V. vulturnus individuals. To meet nightly FMR requirements, Vespadelus spp. would need to consume ~600-660 mosquitoes on a mosquito-only diet, or ~160-180 similar sized moths on a moth-only diet. Lower relative profitability of mosquitoes may provide an explanation for the low level of mosquito consumption among these bats and the absence of mosquitoes in feces of larger bats. Smaller sized bats, especially V. vulturnus, are likely to be those most sensitive to reductions in mosquito abundance and should be monitored during mosquito control activities.     

Anopheles Midgut FREP1 Mediates Plasmodium Invasion

Malaria transmission depends on sexual stage Plasmodium parasites successfully invading Anopheline mosquito midguts following a blood meal. However, the molecular mechanisms of Plasmodium invasion of mosquito midguts have not been fully elucidated. Previously, we showed that genetic polymorphisms in the fibrinogen-related protein 1 (FREP1) gene are significantly associated with Plasmodium falciparum infection in Anopheles gambiae, and FREP1 is important for Plasmodium berghei infection of mosquitoes. Here we identify that the FREP1 protein is secreted from the mosquito midgut epithelium and integrated as tetramers into the peritrophic matrix, a chitinous matrix formed inside the midgut lumen after a blood meal feeding. Moreover, we show that the FREP1 can directly bind Plasmodia sexual stage gametocytes and ookinetes. Notably, ablating FREP1 expression or targeting FREP1 with antibodies significantly decreases P. falciparum infection in mosquito midguts. Our data support that the mosquito-expressed FREP1 mediates mosquito midgut invasion by multiple species of Plasmodium parasites via anchoring ookinetes to the peritrophic matrix and enabling parasites to penetrate the peritrophic matrix and the epithelium. Thus, targeting FREP1 can limit malaria transmission.     

Molecular analysis of mitochondrial cytochrome oxidase I gene of Aedes aegypti L. mosquitoes

Aedes aegypti is the most important arboviral vector worldwide. Recent studies reported that genetic variations and gene flow among same mosquito species is responsible for different disease transmission rate. Hence, to understand the relationship between genetic diversity and disease transmission potential, study on genetic variations among mosquito populations is essential. The aim of present study was to investigate the genetic variations of Ae. aegypti targeting COI gene from nine villages of Jalna District, Maharashtra and three laboratory strains originated from Aurangabad, Delhi and transgenic OX513A strain imported from OXITEC, UK. OX513A strain consists of a self-limiting dominant lethal gene construct intended for its use in suppression of Ae. aegypti population by sustained male adult releases in the environment. Mosquito eggs from field and laboratory strains were reared to adults and identified on the basis of morphological characteristics followed by COI gene sequence. Result of MSA and haplotype analysis revealed low genetic variations among field samples and Aurangabad strain, belonged to two haplotypes (H1 and H2) except Ramkheda village represented by separate haplotype H3. Other laboratory DEL strain and transgenic OX513A have great genetic variability to all isolates and have a separate haplotypes H4 and H5. Similar results were observed in phylogenetic analysis. Our observation of phylogenies revealed close relationship among the DEL and transgenic strain OX513A with few Indian and worldwide isolates. The information on genetic variability of mosquito population could help to understand and design the strategies for risk mitigation and effective implementation of new vector control tools like genetically modified mosquitoes.     

Expression of mosquito-larvicidal toxin genes under the control of a native promoter in Enterobacter amnigenus An11

Enterobacter amnigenus An11, that can colonize the gut of mosquito larva, is an alternative toxin-producing host to be used as a mosquito control since it is able to float in the feeding zone of mosquito larvae. To produce mosquito-larvicidal toxins in this bacterium, a native promoter has been identified from its genomic DNA. The promoter exhibited consensus sequences for ?35 and ?10 regions of bacterial promoters and constitutively drove the expression of gfp. This promoter was inserted into recombinant plasmids upstream of promoter-free cyt2Aa2 from Bacillus thuringiensis and mtx2 from Bacillus sphaericus. Results demonstrated that Cyt2Aa2 and Mtx2 are constitutively produced without induction. The recombinant E. amnigenus showed toxicity against mosquito larvae, demonstrating a potential to be applied in a mosquito control program.     

Laboratory demonstration of the vertical transmission of Rift Valley fever virus by Culex tarsalis mosquitoes

Rift Valley fever virus (RVFV) is a mosquito-transmitted virus with proven ability to emerge into naïve geographic areas. Limited field evidence suggests that RVFV is transmitted vertically from parent mosquito to offspring, but until now this mechanism has not been confirmed in the laboratory. Furthermore, this transmission mechanism has allowed for the prediction of RVFV epizootics based on rainfall patterns collected from satellite information. However, in spite of the relevance to the initiation of epizootic events, laboratory confirmation of vertical transmission has remained an elusive research aim for thirty-five years. Herein we present preliminary evidence of the vertical transmission of RVFV by Culex tarsalis mosquitoes after oral exposure to RVFV. Progeny from three successive gonotrophic cycles were reared to adults, with infectious RVFV confirmed in each developmental stage. Virus was detected in ovarian tissues of parental mosquitoes 7 days after imbibing an infectious bloodmeal. Infection was confirmed in progeny as early as the first gonotrophic cycle, with infection rates ranging from 2.0–10.0%. Virus titers among progeny were low, which may indicate a host mechanism suppressing replication.