Demasculinization of the Anopheles gambiae X chromosome

Background: In a number of organisms sex-biased genes are non-randomly distributed between autosomes and the shared sex chromosome X (or Z). Studies on Anopheles gambiae have produced conflicting results regarding the underrepresentation of male-biased genes on the X chromosome and it is unclear to what extent sexual antagonism, dosage compensation or X-inactivation in the male germline, the evolutionary forces that have been suggested to affect the chromosomal distribution of sex-biased genes, are operational in Anopheles. Results: We performed a meta-analysis of sex-biased gene expression in Anopheles gambiae which provides evidence for a general underrepresentation of male-biased genes on the X-chromosome that increased in significance with the observed degree of sex-bias. A phylogenomic comparison between Drosophila melanogaster, Aedes aegypti and Culex quinquefasciatus also indicates that the Anopheles X chromosome strongly disfavours the evolutionary conservation of male-biased expression and that novel male-biased genes are more likely to arise on autosomes. Finally, we demonstrate experimentally that transgenes situated on the Anopheles gambiae X chromosome are transcriptionally silenced in the male germline. Conclusion: The data presented here support the hypothesis that the observed demasculinization of the Anopheles X chromosome is driven by X-chromosome inactivation in the male germline and by sexual antagonism. The demasculinization appears to be the consequence of a loss of male-biased expression, rather than a failure in the establishment or the extinction of male-biased genes. Keywords: Anopheles gambiae, demasculinization, germline x-chromosome inactivation, sexual antagonism, dosage compensation

Evaluation of the vector competence of a native UK mosquito Ochlerotatus detritus (Aedes detritus) for dengue, chikungunya and West Nile viruses.

BACKGROUND To date there has been no evidence of mosquito-borne virus transmission of public health concern in the UK, despite the occurrence of more than 30 species of mosquito, including putative vectors of arboviruses. The saltmarsh mosquito Ochlerotatus detritus [syn. Aedes (Ochlerotatus) detritus] is locally common in parts of the UK where it can be a voracious feeder on people. METHODS Here, we assess the competence of O. detritus for three major arboviruses: dengue virus (DENV), chikungunya virus (CHIKV) and West Nile virus (WNV) using adult mosquitoes reared from wild, field-obtained immatures. RESULTS We demonstrate laboratory competence for WNV at 21 °C, with viral RNA detected in the mosquito's saliva 17 days after oral inoculation. By contrast, there was no evidence of laboratory competence of O. detritus for either DENV or CHIKV. CONCLUSIONS To our knowledge, this is the first study to demonstrate competence of a UK mosquito for WNV and confirms that O. detritus may present a potential risk for arbovirus transmission in the UK and that further investigation of its vector role in the wild is required

Efficient ΦC31 integrase–mediated site-specific germline transformation of Anopheles gambiae

Current transgenic methodology developed for mosquitoes has not been applied widely to the major malaria vector Anopheles gambiae, which has proved more difficult to genetically manipulate than other mosquito species and dipteran insects. In this protocol, we describe ΦC31-mediated site-specific integration of transgenes into the genome of A. gambiae. The ΦC31 system has many advantages over 'classical' transposon-mediated germline transformation systems, because it allows integration of large transgenes at specific, characterized genomic locations. Starting from a general protocol, we have optimized steps from embryo collection to co-injection of transgene-containing plasmid and in vitro-produced ΦC31 integrase mRNA. We also provide tips for screening transgenic larvae. The outlined procedure provides robust transformation in A. gambiae, resulting in homozygous transgenic lines in ∼2-3 months

High susceptibility of wild Anopheles funestus to infection with natural Plasmodium falciparum gametocytes using membrane feeding assays.

BACKGROUND Anopheles funestus is a major vector of malaria in sub-Saharan Africa. However, because it is difficult to colonize, research on this mosquito species has lagged behind other vectors, particularly the understanding of its susceptibility and interactions with the Plasmodium parasite. The present study reports one of the first experimental infections of progeny from wild-caught An. funestus with the P. falciparum parasite providing a realistic avenue for the characterisation of immune responses associated with this infection. METHODS Wild-fed resting An. funestus females were collected using electric aspirators and kept in cages for four days until they were fully gravid and ready to oviposit. The resulting eggs were reared to adults F1 mosquitoes under insectary conditions. Three to five day-old An. funestus F1 females were fed with infected blood taken from gametocyte carriers using an artificial glass-parafilm feeding system. Feeding rate was recorded and fed mosquitoes were dissected at day 7 to count oocysts in midguts. Parallel experiments were performed with the known Plasmodium-susceptible An. coluzzii Ngousso laboratory strain, to monitor our blood handling procedures and infectivity of gametocytes. RESULTS The results revealed that An. funestus displays high and similar level of susceptibility to Plasmodium infection compared to An. coluzzii, and suggest that our methodology produces robust feeding and infection rates in wild An. funestus progeny. The prevalence of infection in An. funestus mosquitoes was 38.52 % (range 6.25-100 %) and the median oocyst number was 12.5 (range 1-139). In parallel, the prevalence in An. coluzzii was 39.92 % (range 6.85-97.5 %), while the median oocyst number was 32.1 (range 1-351). CONCLUSIONS Overall, our observations are in line with the fact that both species are readily infected with P. falciparum, the most common and dangerous malaria parasite in sub-Saharan Africa, and since An. funestus is widespread throughout Africa, malaria vector control research and implementation needs to seriously address this vector species too. Additionally, the present work indicates that it is feasible to generate large number of wild F1 infected An. funestus mosquitoes using membrane feeding assays, which can be used for comprehensive study of interactions with the Plasmodium parasite

Cytochrome P450 associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae.

The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of (14)C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An gambiae

Analysis of apyrase 5' upstream region validates improved Anopheles gambiae transformation technique

<p>Abstract</p> <p>Background</p> <p>Genetic transformation of the malaria mosquito <it>Anopheles gambiae </it>has been successfully achieved in recent years, and represents a potentially powerful tool for researchers. Tissue-, stage- and sex-specific promoters are essential requirements to support the development of new applications for the transformation technique and potential malaria control strategies. During the <it>Plasmodium </it>lifecycle in the invertebrate host, four major mosquito cell types are involved in interactions with the parasite: hemocytes and fat body cells, which provide humoral and cellular components of the innate immune response, midgut and salivary glands representing the epithelial barriers traversed by the parasite during its lifecycle in the mosquito.</p> <p>Findings</p> <p>We have analyzed the upstream regulatory sequence of the <it>An. gambiae </it>salivary gland-specific <it>apyrase </it>(<it>AgApy</it>) gene in transgenic <it>An. gambiae </it>using a <it>piggyBac </it>transposable element vector marked by a <it>3xP3 </it>promoter:<it>DsRed </it>gene fusion. Efficient germ-line transformation in <it>An. gambiae </it>mosquitoes was obtained and several integration events in at least three different G₀families were detected. <it>LacZ </it>reporter gene expression was analyzed in three transgenic lines/groups, and in only one group was tissue-specific expression restricted to salivary glands.</p> <p>Conclusion</p> <p>Our data describe an efficient genetic transformation of <it>An. gambiae </it>embryos. However, expression from the selected region of the <it>AgApy </it>promoter is weak and position effects may mask tissue- and stage- specific activity in transgenic mosquitoes.</p

Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the φC31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces φC31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the φC31 integrase. We report two representative results of φC31mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. φC31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the φC31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors

New insecticide screening platforms indicate that Mitochondrial Complex I inhibitors are susceptible to cross-resistance by mosquito P450s that metabolise pyrethroids

Fenazaquin, pyridaben, tolfenpyrad and fenpyroximate are Complex I inhibitors offering a new mode of action for insecticidal malaria vector control. However, extended exposure to pyrethroid based products such as long-lasting insecticidal nets (LLINs) has created mosquito populations that are largely pyrethroid-resistant, often with elevated levels of P450s that can metabolise and neutralise diverse substrates. To assess cross-resistance liabilities of the Complex I inhibitors, we profiled their susceptibility to metabolism by P450s associated with pyrethroid resistance in Anopheles gambiae (CYPs 6M2, 6P3, 6P4, 6P5, 9J5, 9K1, 6Z2) and An. funestus (CYP6P9a). All compounds were highly susceptible. Transgenic An. gambiae overexpressing CYP6M2 or CYP6P3 showed reduced mortality when exposed to fenpyroximate and tolfenpyrad. Mortality from fenpyroximate was also reduced in pyrethroid-resistant strains of An. gambiae (VK7 2014 and Tiassalé 13) and An. funestus (FUMOZ-R). P450 inhibitor piperonyl butoxide (PBO) significantly enhanced the efficacy of fenpyroximate and tolfenpyrad, fully restoring mortality in fenpyroximate-exposed FUMOZ-R. Overall, results suggest that in vivo and in vitro assays are a useful guide in the development of new vector control products, and that the Complex I inhibitors tested are susceptible to metabolic cross-resistance and may lack efficacy in controlling pyrethroid resistant mosquitoes

Functional genetic validation of key genes conferring insecticide resistance in the major African malaria vector

Resistance in to members of all 4 major classes (pyrethroids, carbamates, organochlorines, and organophosphates) of public health insecticides limits effective control of malaria transmission in Africa. Increase in expression of detoxifying enzymes has been associated with insecticide resistance, but their direct functional validation in is still lacking. Here, we perform transgenic analysis using the GAL4/UAS system to examine insecticide resistance phenotypes conferred by increased expression of the 3 genes-, , and -most often found up-regulated in resistant We report evidence in that organophosphate and organochlorine resistance is conferred by overexpression of GSTE2 in a broad tissue profile. Pyrethroid and carbamate resistance is bestowed by similar overexpression, and confers only pyrethroid resistance when overexpressed in the same tissues. Conversely, such overexpression increases susceptibility to the organophosphate malathion, presumably due to conversion to the more toxic metabolite, malaoxon. No resistant phenotypes are conferred when either gene overexpression is restricted to the midgut or oenocytes, indicating that neither tissue is involved in insecticide resistance mediated by the candidate P450s examined. Validation of genes conferring resistance provides markers to guide control strategies, and the observed negative cross-resistance due to gives credence to proposed dual-insecticide strategies to overcome pyrethroid resistance. These transgenic -resistant lines are being used to test the "resistance-breaking" efficacy of active compounds early in their development

Cytochrome P450associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae.

The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of 14C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An. gambiae.Fil: Balabanidou, Vasileia. Foundation for Research and Technology-Hellas; Grecia. Universidad de Creta; GreciaFil: Kampouraki, Anastasia. Universidad de Creta; GreciaFil: Mac Lean, Marina. University of Nevada; Estados UnidosFil: Blomquist, Gary J.. University of Nevada; Estados UnidosFil: Tittiger, Claus. University of Nevada; Estados UnidosFil: Juarez, Marta Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Mijailovsky, Sergio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Chalepakis, George. Universidad de Creta; GreciaFil: Anthousi, Amalia. Universidad de Creta; GreciaFil: Lynd, Amy. Liverpool School of Tropical Medicine; Reino UnidoFil: Antoine, Sanou. Liverpool School of Tropical Medicine; Reino UnidoFil: Hemingway, Janet. Liverpool School of Tropical Medicine; Reino UnidoFil: Ranson, Hilary. Liverpool School of Tropical Medicine; Reino UnidoFil: Lycett, Gareth J.. Liverpool School of Tropical Medicine; Reino UnidoFil: Vontas, John. Foundation for Research and Technology-Hellas; Grecia. Agricultural University of Athens; Greci