Crustacean cardioactive peptide (CCAP) is a highly conserved arthropod neurohormone that is involved in ecdysis, hormone release and the modulation of muscle contractions. Here, we determined the CCAP gene structure in the malaria mosquito Anopheles gambiae, assessed the developmental expression of CCAP and its receptor and determined the role that CCAP plays in regulating mosquito cardiac function. RACE sequencing revealed that the A. gambiae CCAP gene encodes a neuropeptide that shares 100% amino acid identity with all sequenced CCAP peptides, with the exception of Daphnia pulex. Quantitative RT-PCR showed that expression of CCAP and the CCAP receptor displays a bimodal distribution, with peak mRNA levels in second instar larvae and pupae. Injection of CCAP revealed that augmenting hemocoelic CCAP levels in adult mosquitoes increases the anterograde and retrograde heart contraction rates by up to 28%, and increases intracardiac hemolymph flow velocities by up to 33%. Partial CCAP knockdown by RNAi had the opposite effect, decreasing the mosquito heart rate by 6%. Quantitative RT-PCR experiments showed that CCAP mRNA is enriched in the head region, and immunohistochemical experiments in newly eclosed mosquitoes detected CCAP in abdominal neurons and projections, some of which innervated the heart, but failed to detect CCAP in the abdomens of older mosquitoes. Instead, in older mosquitoes CCAP was detected in the pars lateralis, the subesophageal ganglion and the corpora cardiaca. In conclusion, CCAP has a potent effect on mosquito circulatory physiology, and thus heart physiology in this dipteran insect is under partial neuronal control.
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that it is not one of the active components of these oils, although synergistic effects cannot be excluded. Sesamin and dihydronitidine were both inactive against adult An. gambiae females. No reports have been found on previous investigations on the toxicity of these compounds against An. gambiae adults. However, sesamin is known for syner- gistic effects together with pyrethroid insecticides which was reported in a study against houseflies in the early 1940s , and has since then also been found to exert synergistic activities in other biological systems, e.g. as an- tihypertensive agent together with vitamin E in rats . The mechanisms behind the synergistic effects are not known in detail. One possibility is that sesamin (a well- known inhibitor of CYP enzymes due partly to its methylene dioxy structure [30, 31]) inhibits cytochrome P450-dependent monooxygenases. The CYP enzymes are present both in insects and mammals and are in- volved in the metabolism of exogenous compounds, as well as insecticides. Resistance to insecticides may de- velop due to increased monooxygenase activity which will result in lower insecticide uptake. Therefore, inhib- ition of CYP450s may be favourable in counteracting insecticide resistance and CYP inhibitors may also increase the uptake and work synergistically with insecticides that
Individual embryos were karyotyped by polymerase chain reaction (PCR) using primers 124678F2 (5’-TTTGAGCATGTGTTTAAAGG-3’) and 124678R2 (5’- AGGTTTTGCCGACTACAAT-3’) that target satellite DNAs AgX367 and AgY477 located on the X and the Y chromosomes, respectively (40). Their monomers have highly similar sequences, but differ in length due to a 110 bp indel. As a result, a 477 bp long male-specific PCR product can be easily differentiated from a 367 bp long non-sex- specific product on an agarose gel, allowing unequivocal discrimination between male (XY) and female (XX) samples (fig. S1). Owing to the highly repetitive nature of the satellite DNA targets, trace amounts of DNA template suffice to obtain robust PCR products. In addition to the two diagnostic fragments, faint PCR products corresponding to longer regions (dimers, trimers, or tetramers) of the same satellite species, as well as a shorter male-specific fragment corresponding to another minor Y-linked satellite species belonging to the AgY477 family, are sometimes amplified. We found that in some A. gambiae lines sexing with the above primers may not be 100% effective. In a proportion of female individuals from such lines we could detect a faint male band, apparently because rare recombination events between the X and the Y chromosome satellite DNA regions could have led to a transfer of AgY477 copies into the AgX367 arrays.
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For all four chromosome arms, linkage disequilibrium is substantially higher in the Kilifi population of An. gam- biae than in any of the other populations. In these other populations linkage disequilibrium is at background lev- els (i.e., equal to that between polymorphisms on differ- ent chromosomes – Fig. 2d) at distances of 1–10 kb or greater, whereas for An. gambiae from Kilifi this only occurs at distances greater than 1 Mb. Linkage disequi- librium between polymorphisms in the same RADseq tag location (length 110 bp) were also analysed, though there are many fewer such pairs of polymorphisms and so data
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Fig. 2. Yob is a protein-coding sex determination gene controlling dsx splicing. (A) Reverse transcription (RT)-PCR analysis of dsx splicing pattern in the A. gambiae Sua5.1 cells transfected with in vitro synthesized Yob mRNA, as compared with control non-transfected cells. M and F, A. gambiae male and female pupae; (-), negative control. Similar results were obtained in three independent experiments. (B) Digital representation of the relative amounts of dsx splice forms shown in panel A. (C) RT-PCR analysis of ribosomal protein S7 transcript levels used as a sample loading control. (D) Splicing pattern of dsx in Sua5.1 cells transfected with native Yob transcripts, non-productive forms of Yob, either lacking the methionine start codon, or containing a premature stop codon, and in non-transfected control cells. The data shown are mean + s. d. *P < 0.001, one-sided Mann–Whitney test.
The dorsal-ventral patterning of the Drosophila embryo is controlled by a well-defined gene regulation network. We wish to understand how changes in this network produce evolutionary diversity in insect gastrulation. The present study focuses on the dorsal ectoderm in two highly divergent dipterans, the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae. In D. melanogaster, the dorsal midline of the dorsal ectoderm forms a single extra-embryonic membrane, the amnioserosa. In A. gambiae, an expanded domain forms two distinct extra-embryonic tissues, the amnion and serosa. The analysis of approximately 20 different dorsal-ventral patterning genes suggests that the initial specification of the mesoderm and ventral neurogenic ectoderm is highly conserved in flies and mosquitoes. By contrast, there are numerous differences in the expression profiles of genes active in the dorsal ectoderm. Most notably, the subdivision of the extra-embryonic domain into separate amnion and serosa lineages in A. gambiae correlates with novel patterns of gene expression for several segmentation repressors. Moreover, the expanded amnion and serosa anlage correlates with a broader domain of Dpp signaling as compared with the D. melanogaster embryo. Evidence is presented that this expanded signaling is due to altered expression of the sog gene.
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Nevertheless, it would be useful to explore whether greater persistence could be achieved with alternative products. Organophosphates like temephos, appear to be less useful since they rarely show much persistence compared with microbials. Moreover, organophosphates can have a negative impact on non-target organisms and need careful resistance management. Microbial larvicides have several advantages over other mosquito control agents. This includes environmental safety and safety for human consumption; for instance when applied in drinking water , in addition to its high efficacy thus makes them powerful vector control agents in Africa and other parts of the tropics.
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Tests were carried out in an open sunlit area within icipe-TOC campus that had been cleared of vegetation. Artificial ponds were created by sinking 40 plastic tubs, (diameter 0.42 m, depth 10 cm) into the ground. Ponds were arranged 1.5 m apart in eight rows with each row having five ponds. Each plastic tub was filled with 8 L of unchlorinated water and 2 L of soil to provide suitable biotic and abiotic parameters for mosquito larvae. Artifi- cial ponds were used because tests were implemented during the dry season when natural breeding habitats of An. gambiae are often limited in number [36-38]. These tests were also conducted with insectary-reared An. gambiae s.s. and An. arabiensis larvae due to the low density of vectors in the study area during the dry sea- son . Both species were tested in parallel. Batches of 50 third-instar larvae were introduced into each pond before AMF was applied into treatment ponds; 20 ponds contained An. gambiae s.s. and 20 ponds contained An. arabiensis. The ponds were assigned into treatments and controls by lottery. Twenty ponds (10 per species) were treated with AMF at the manufacturer’s recommended dose of 1 ml/m 2 . Since the surface area of water in each pond was 0.14 m 2 , a volume of 0.14 ml (140 μl) was applied at the edge of the pond using a micropip- ette. The remaining 20 ponds (10 per species) were left untreated and served as controls. After AMF application an emergence trap modified from Fillinger et al.  was placed on top of each pond to prevent adult mos- quitoes escaping and to avoid natural colonization of ponds by wild mosquitoes. A cone-shaped frame made
maintained at 26-27°C and 85-95% relative humidity (RH) with access to 9% glucose solution ad libitum. Sur- vival was monitored daily for a maximum of 28 d. Dead mosquitoes were collected individually and put onto moist filter paper in Petri dishes, sealed with parafilm, and incubated at 26-27°C and 85-95% RH for 3-4 d, after which they were examined for evidence of fungal sporula- tion basing on colour of their conidia. Metarhizium anisop- liae yields green conidia, whereas, B. bassiana yields white conidia. Similar bioassay procedure was carried out for 30-50 mosquitoes in control groups, except that they were exposed to untreated surfaces. During all of the bioassays, four replicates were used for each experimental factor. Four different bioassays were conducted to study the effect of 1) concentration, 2) co-formulation 3) exposure time and 4) persistence on infection and survival of A. gambiae s.s.
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Background: Indoor residual spraying (IRS) of households with insecticide is a principal malaria vector control intervention in Zanzibar. In 2006, IRS using the pyrethroid lambda-cyhalothrine was introduced in Zanzibar. Following detection of pyrethroid resistance in 2010, an insecticide resistance management plan was proposed, and IRS using bendiocarb was started in 2011. In 2014, bendiocarb was replaced by pirimiphos methyl. This study investigated the residual efficacy of pirimiphos methyl (Actellic® 300CS) sprayed on common surfaces of human dwellings in Zanzibar. Methods: The residual activity of Actellic 300CS was determined over 9 months through bioassay tests that measured the mortality of female Anopheles mosquitoes, exposed to sprayed surfaces under a WHO cone. The wall surfaces included; mud wall, oil or water painted walls, lime washed wall, un-plastered cement block wall and stone blocks. Insecticide susceptibility testing was done to investigate the resistance status of local malaria vectors against Actellic 300CS using WHO protocols; Anopheline species were identified using PCR methods.
nucleotide polymorphisms (SNPs) were identified, a subset of which were sampled for variation revealing de- viations from Hardy-Weinberg equilibrium among six field-collected populations. Restriction-site associated DNA marker (RAD) sequencing is another genome- reduction strategy that can provide efficient population genomic data for non-model species. RAD sequencing of Ae. aegypti specimens from around the world suggests that a single subspeciation event occurred within the do- mesticated form in Africa; the mosquitoes then dis- persed globally along commercial trade routes . Both studies validate the likelihood that these methodologies will be useful for assessing population genetic structures in non-model vector species, with or without genome assemblies. Finally, it is important to note that the Well- come Trust (via its Sanger Institute) has established the Anopheles gambiae 1000 genomes (Ag1000G) consortium (https://www.malariagen.net/projects/vector/ag1000g) to provide a global repository for WGS data collected from wild-caught mosquitoes across Africa, thereby providing a catalog of genetic variation across natural vector popula- tions. Given its scope and the otherwise nearly impossible access to such datasets, Ag1000G is almost certain to become an increasingly important resource for the ana- lysis of vector competence and vectorial capacity in An. gambiae.
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Previous attempts have been made to develop expo- sure-free sampling tools for collecting indoor or outdoor biting mosquitoes. These techniques include but are not limited to the bed net trap , tent traps [39–41], the CDC light traps , and the mosquito magnet (MM) trap [43–45]. While these methods have shown promise in some settings, most have limitations that restrict their large-scale application, and/or bias collection towards mosquito species with particular phenotypes that may misrepresent the community of mosquitoes attracted to people . Recently, there has been renewed interest in exploring the use of electrocuting surfaces as a means of sampling malaria vectors [47–49]. This approach was originally developed for trapping tsetse flies outdoors , but later adapted to sample mosquitoes drawn towards a host odour source [51, 52]. This trap works by placing a live host in a sealed tent and piping their odour out to an electrocuting net (E-Net) approximately 10 m away that kills mosquitoes on contact. Such E-Nets have already shown promise when used to investigate host species’ preferences and odour responses of the Afri- can vector species [51, 52]. As a potential improvement, the use of commercially available ‘bug-zapping’ devices, which can sample insects in the immediate proximity of a host has been explored with some promise, indicating they can achieve a relative sampling efficiency of up to 50 % of the HLC in one study . However, given these devices were developed for large flies, their suitability for trapping African malaria vectors is unclear. Further work is required to develop an electrocuting trap that is opti- mized for malaria vectors, can meet the performance of the HLC, is suitable for use inside and outdoors, can be used safely in close proximity of humans, and is durable under field conditions. Here, a mosquito electrocuting trap (MET) was designed, developed and field-tested. This trap was custom designed to sample host-seeking African malaria vectors, with the aim of meeting all per- formance targets defined above.
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In the village of Dielmo in Senegal, An. arabiensis, An. coluzzii and An. gambiae s.s. occur in sympatry through- out the year, although with seasonal fluctuation in their relative frequencies [27,38]. The prevalence of F1 hybrids between An. gambiae and An. coluzzii (ie, formerly referred to as M/S hybrids) was also shown to vary both geographically and temporally throughout their common distribution area in West and Central Africa [10,11,25,26]. The ecological determinants of such a dynamic hybridization process are currently unknown and may be under the control of a few genes map- ping in areas of high genomic divergence between An. coluzzii and An. gambiae that have been called ‘speciation islands’ [2,3,22]. This is the case of Or39 lying in a genomic region of high differentiation on chromosome 2R. Indeed, our results detected high levels of genetic differentiation throughout this gene among the sympatric mosquito populations we have sampled, with fixed differences in coding regions resulting in amino-acid changes in the encoded protein. There was however no sign of diversifying selection acting on this gene, at least in our limited dataset which resulted in low statistical power of neutrality tests. Meanwhile, the three species explored in this study exhibited different ma- ture peptides. Most of the aminoacid changes observed between species were located on the intracellular domains of the protein, and might indeed reflect random changes accumulated since lineage splitting within the An. gam- biae s.l. complex. However, the nucleotide sequence of the first, and longest (i.e., 72 aminoacids) extracellular domain of the protein was different between species, and polymorphic within the An. gambiae and An. arabien- sis samples. At this stage of our analysis, we can only speculate on the role these amino acid changes may play for the perception of different olfactory stimuli by the mosquitoes through specific ligand-receptor interactions,
The most obvious determinant of male mating com- petitiveness is their capacity to mate with wild individ- uals from the target population. Mating competitiveness can be tracked by examining the progeny of wild females captured after the releases in order to check if released and wild individuals mated randomly [48–50]. In the past, and despite the best efforts to maintain adequate phenotypic quality, non-random mating patterns have often been detected and held responsible for low effect- ive mating ratios and the poor results of several mos- quito release projects [50–53]. Given how little was and is currently known of the complex mosquito mating phenotype it is not surprising that hit or miss results are the best that can be hoped for . Rearing techniques aiming at protecting the mosquito mating phenotype in the laboratory do not go further than the occasional genetic strain refreshing scheme and crude measure- ments of fitness. This is simply because we currently do not know precisely what phenotypic characteristics make for a sexually more attractive mosquito under natural swarm-like conditions. Nor do we know what makes a mosquito capable of differentiating conspecifics from other individuals in a mixed swarm. Given the complex- ity and size of anopheline malaria vector populations in Africa, these knowledge gaps may prove crucial for the cost effectiveness and success of release programmes.
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Several tolerance mechanisms avoid chronic activation of the immune system by gut microorganisms and participate in maintaining gut homeostasis, but they may also impair the mosquito immune defence against the parasite. Notably, PGRPLB is a negative regulator of the Imd pathway preventing systemic antimicrobial re- sponses to the microbiota, which participates in a higher tolerance to Plasmodium infection . Its expression is induced by the microbiota [24, 31]. Immunomodulatory Peroxidase (IMPer) and Dual oxidase (Duox) enzymes, induced by the blood meal in a microbiota-independent manner [24, 31], are involved in the formation of a dityr- osine network between the gut epithelium and the peri- trophic matrix . This reduction in permeability protects both the microbiota and Plasmodium by preventing the activation of gut immunity. Whether the microbiota-induced peritrophic matrix  further reduces the diffusion of immune elicitors and is also
Sequence analysis of ONNV RNA extracted from mosquito bodies and heads indicated that codon usage at this position corresponded to that of the virus administered in the blood meal. These results suggest that while selection on ONNV variants is occurring, it is not significant enough to bring about a change in codon usage at the position between nsP3 and nsP4. Thus, the primary mutation pressures contributing to the evolution of this mechanism may be present in the vertebrate host. Nevertheless, evolutionary forces appear to have main- tained in nature alphavirus genomes possessing both sense and opal codons at this particular locus. This suggests that both codons are associated with a fitness advantage but at different times in the transmission cycle. Our results demonstrate a clear fitness advantage in the mosquito host when an opal stop codon is present at this locus in the ONNV genome. Our results also suggest that this fitness advantage is primarily re- lated to an effect on mosquito infectivity. Further understand- ing the molecular basis of the fitness advantage may provide valuable insights into understanding how alphaviruses establish persistent infections in the mosquito host.
been described in the salivary glands of ticks and fleas, respectively (Bowman et al., 1997; Cheeseman et al., 2001). While abundant in the saliva of the human-feeding Culex quinquefasciatus mosquito, such salivary activity is lacking in two other anthropophilic mosquitoes (Aedes aegypti and Anopheles gambiae). PAF is an important physiologic agonist in aggregation of blood of some mammals such as the rabbit (Cazenave et al., 1979), and also for bird thrombocytes (Cox, 1985). Indeed, PAF is considered a ‘strong’ agonist in rabbit platelets, where it induces platelet activation at picomolar concentrations, independently of other mediators (Braquet et al., 1984). PAF also participates as a pro-aggregatory molecule after platelet stimulation with collagen, thrombin and other inducers (Braquet et al., 1987). In contrast, the role of PAF as an aggregating agent in human platelets is less remarkable. In fact, platelet aggregation by PAF in these cell types requires
Since the QTL detected in this study were based on the phenotype of infection intensities, as established by number of oocysts on day 8 postinfection, the loci could be associated with several biological mechanisms that reduce the number of oocysts. Such mechanisms in- clude destruction of oocysts in ways similar to intra- cellular lytic responses in the midgut (V ernick et al. 1995), humoral responses against oocysts (P askewitz et al. 1988), and mosquito defensin, an antibacterial peptide that destroys malaria parasites if a concurrent bacteria infection primes the mosquito’s immune sys- tem (L owenberger et al. 1999; L ambrechts et al. 2004). Several functional candidate gene analyses have impli- cated a number of genes and mechanisms that affect the ookinete and oocyst stages of development. Examples include thio-ester-containing proteins, leucin-rich pro- tein, and c-type lectins (CTL4 and CTLMA2) that have been shown to influence refractoriness (B landin et al. 2004; O sta et al. 2004). The knowledge and tools de- veloped in such studies are useful in analyzing natural vector–parasite systems.
throughout Cameroon . On the whole results pre- sented in this study shows a rise in frequency of resis- tant kdr alleles (e.g., 1014F and 1014S) in both molecular forms compared to previous studies related to the frequency of these alleles in the country [22,23,27]. However, these frequencies, particularly that of the 1014F recorded in the M molecular form are lower compared to that obtained in a recent survey carried out in Cameroon where the frequency of this allele was 68% in Douala and 44% in Yaounde . These alleles were not detected in An. arabiensis nor An. melas speci- mens. None of them has not yet been identified in An. melas whereas several studies reported their presence in An. arabiensis in Kenya , Sudan [62,63] and Camer- oon . The absence of these alleles in the present samples suggests their recent introduction in An. ara- biensis in Cameroon where they seem to occur at a very low frequency. Within An. gambiae s.s, both 1014F and 1014S alleles were previously reported in Cameroon [27,64]. In neighbouring countries, at least one of these alleles has been found, e.g. in Equatorial Guinea , Gabon , Central Africa Republic , Chad  and Nigeria  emphasizing the spread of the kdr muta- tions in Central Africa . However, again, referring to results based on cross sectional studies calls for caution and care must be taken in predicting absence of these alleles in a given zone/area of the country.
B. bassiana . Increasing the exposure time beyond 6 h and/or concentration did increase mortality of An. stephensi  and other arthropods [35,36]. When the concentration tested against An. gambiae was increased, no effect of exposure time was observed, though only rel- atively short exposure times were tested (15 min - 6 h). However, longer exposure times (24 h, 48 h and continu- ous) of An. gambiae to M. anisopliae were tested by Scholte et al  and at high concentrations and no difference between the exposure times tested was observed. Formu- lations of either M. anisopliae or B. bassiana could, there- fore, be used with dissemination tools/surfaces that target host seeking (short contact) as well as resting (long con- tact) mosquitoes.
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