Cryopreservation of Anopheles stephensi embryos.

The ability to cryopreserve mosquitoes would revolutionize work on these vectors of major human infectious diseases by conserving stocks, new isolates, lab-bred strains, and transgenic lines that currently require continuous life cycle maintenance. Efforts over several decades to develop a method for cryopreservation have, until now, been fruitless: we describe here a method for the cryopreservation of Anopheles stephensi embryos yielding hatch rates of ~ 25%, stable for > 5 years. Hatched larvae developed into fertile, fecund adults and blood-fed females, produced fully viable second generation eggs, that could be infected with Plasmodium falciparum at high intensities. The key components of the cryopreservation method are: embryos at 15-30 min post oviposition, two incubation steps in 100% deuterated methanol at - 7 °C and - 14.5 °C, and rapid cooling. Eggs are recovered by rapid warming with concomitant dilution of cryoprotectant. Eggs of genetically modified A. stephensi and of A. gambiae were also successfully cryopreserved. This enabling methodology will allow long-term conservation of mosquitoes as well as acceleration of genetic studies and facilitation of mass storage of anopheline mosquitoes for release programs.

Larval ecology and bionomics of Anopheles funestus in highland and lowland sites in western Kenya.

BACKGROUND: An. funestus is a major Afrotropical vector of human malaria. This study sought to investigate the larval ecology, sporozoite infection rates and blood meal sources of An. funestus in western Kenya. METHODS: Larval surveys were carried out in Bungoma (Highland) and Kombewa (lowland) of western Kenya. Aquatic habitats were identified, characterized, georeferenced and carefully examined for mosquito larvae and predators. Indoor resting mosquitoes were sampled using pyrethrum spray catches. Adults and larvae were morphologically and molecularly identified to species. Sporozoite infections and blood meal sources were detected using real-time PCR and ELISA respectively. RESULTS: Of the 151 aquatic habitats assessed, 62/80 (78%) in Bungoma and 58/71(82%) in Kombewa were positive for mosquito larvae. Of the 3,193 larvae sampled, An. funestus larvae constitute 38% (1224/3193). Bungoma recorded a higher number of An. funestus larvae (85%, 95%, CI, 8.722-17.15) than Kombewa (15%, 95%, CI, 1.33-3.91). Molecular identification of larvae showed that 89% (n = 80) were An. funestus. Approximately 59%, 35% and 5% of An. funestus larvae co-existed with An. gambiae s.l, Culex spp and An. coustani in the same habitats respectively. Of 1,221 An. funestus s.l adults sampled, molecular identifications revealed that An. funestus constituted 87% (n = 201) and 88% (n = 179) in Bungoma and Kombewa, respectively. The Plasmodium falciparum sporozoite rate of An. funestus in Bungoma and Kombewa was 2% (3/174) and 1% (2/157), respectively, and the human blood index of An. funestus was 84% (48/57) and 89% (39/44) and for Bungoma and Kombewa, respectively. CONCLUSION: Man-made ponds had the highest abundance of An. funestus larvae. Multiple regression and principal component analyses identified the distance to the nearest house as the key environmental factor associated with the abundance of An. funestus larvae in aquatic habitats. This study serves as a guide for the control of An. funestus and other mosquito species to complement existing vector control strategies.

A genetically encoded anti-CRISPR protein constrains gene drive spread and prevents population suppression.

CRISPR-based gene drives offer promising means to reduce the burden of pests and vector-borne diseases. These techniques consist of releasing genetically modified organisms carrying CRISPR-Cas nucleases designed to bias their inheritance and rapidly propagate desired modifications. Gene drives can be intended to reduce reproductive capacity of harmful insects or spread anti-pathogen effectors through wild populations, even when these confer fitness disadvantages. Technologies capable of halting the spread of gene drives may prove highly valuable in controlling, counteracting, and even reverting their effect on individual organisms as well as entire populations. Here we show engineering and testing of a genetic approach, based on the germline expression of a phage-derived anti-CRISPR protein (AcrIIA4), able to inactivate CRISPR-based gene drives and restore their inheritance to Mendelian rates in the malaria vector Anopheles gambiae. Modeling predictions and cage testing show that a single release of male mosquitoes carrying the AcrIIA4 protein can block the spread of a highly effective suppressive gene drive preventing population collapse of caged malaria mosquitoes.

Interaction of RNA viruses of the natural virome with the African malaria vector, Anopheles coluzzii.

Mosquitoes are colonized by a little-studied natural virome. Like the bacterial microbiome, the virome also probably influences the biology and immunity of mosquito vector populations, but tractable experimental models are lacking. We recently discovered two novel viruses in the virome of wild Anopheles and in colonies of the malaria vector Anopheles coluzzii: Anopheles C virus and Anopheles cypovirus. Here, we describe biological interactions between these two viruses and An. coluzzii mosquitoes. Viral abundance varies reproducibly during mosquito development. DNA forms of these viruses were not detected, and thus viral persistence is likely based on vertical transmission of RNA genomes. At least Anopheles C virus is vertically transmitted by an intraembryonic route. Relative abundance of the two viruses is inversely correlated in individual mosquitoes. One possible mechanism for this could be interactions with host immunity, and functional genomic analysis indicated differential influence of at least the Toll and JAK/STAT immune signaling pathways upon the viruses. The nonrandom distributions and interactions with host immunity suggest that these and other members of the natural virome may constitute a source of unrecognized heterogeneity in mosquito vector populations.

Pure early zygotic genes in the Asian malaria mosquito Anopheles stephensi.

BACKGROUND: The Asian malaria mosquito, Anopheles stephensi, is a major urban malaria vector in the Middle East and on the Indian subcontinent. Early zygotic transcription, which marks the maternal-to-zygotic transition, has not been systematically studied in An. stephensi or any other Anopheles mosquitoes. Improved understanding of early embryonic gene expression in An. stephensi will facilitate genetic and evolutionary studies and help with the development of novel control strategies for this important disease vector. RESULTS: We obtained RNA-seq data in biological triplicates from four early An. stephensi embryonic time points. Using these data, we identified 70 and 153 pure early zygotic genes (pEZGs) under stringent and relaxed conditions, respectively. We show that these pEZGs are enriched in functional groups related to DNA-binding transcription regulators, cell cycle modulators, proteases, transport, and cellular metabolism. On average these pEZGs are shorter and have less introns than other An. stephensi genes. Some of the pEZGs may arise de novo while others have clear non-pEZG paralogs. There is no or very limited overlap between An. stephensi pEZGs and Drosophila melanogaster or Aedes aegypti pEZGs. Interestingly, the upstream region of An. stephensi pEZGs lack significant enrichment of a previously reported TAGteam/VBRGGTA motif found in the regulatory region of pEZGs in D. melanogaster and Ae. aegypti. However, a GT-rich motif was found in An. stephensi pEZGs instead. CONCLUSIONS: We have identified a number of pEZGs whose predicted functions and structures are consistent with their collective roles in the degradation of maternally deposited components, activation of the zygotic genome, cell division, and metabolism. The pEZGs appear to rapidly turn over within the Dipteran order and even within the Culicidae family. These pEZGs, and the shared regulatory motif, could provide the promoter or regulatory sequences to drive gene expression in the syncytial or early cellular blastoderm, a period when the developing embryo is accessible to genetic manipulation. In addition, these molecular resources may be used to achieve sex separation of mosquitoes for sterile insect technique.

Proteome-wide analysis of Anopheles culicifacies mosquito midgut: new insights into the mechanism of refractoriness.

BACKGROUND: Midgut invasion, a major bottleneck for malaria parasites transmission is considered as a potential target for vector-parasite interaction studies. New intervention strategies are required to explore the midgut proteins and their potential role in refractoriness for malaria control in Anopheles mosquitoes. To better understand the midgut functional proteins of An. culicifacies susceptible and refractory species, proteomic approaches coupled with bioinformatics analysis is an effective means in order to understand the mechanism of refractoriness. In the present study, an integrated in solution- in gel trypsin digestion approach, along with Isobaric tag for relative and absolute quantitation (iTRAQ)-Liquid chromatography/Mass spectrometry (LC/MS/MS) and data mining were performed to identify the proteomic profile and differentially expressed proteins in Anopheles culicifacies susceptible species A and refractory species B. RESULTS: Shot gun proteomics approaches led to the identification of 80 proteins in An. culicifacies susceptible species A and 92 in refractory species B and catalogue was prepared. iTRAQ based proteomic analysis identified 48 differentially expressed proteins from total 130 proteins. Of these, 41 were downregulated and 7 were upregulated in refractory species B in comparison to susceptible species A. We report that the altered midgut proteins identified in naturally refractory mosquitoes are involved in oxidative phosphorylation, antioxidant and proteolysis process that may suggest their role in parasite growth inhibition. Furthermore, real time polymerase chain reaction (PCR) analysis of few proteins indicated higher expression of iTRAQ upregulated protein in refractory species than susceptible species. CONCLUSION: This study elucidates the first proteome of the midguts of An. culicifacies sibling species that attempts to analyze unique proteogenomic interactions to provide insights for better understanding of the mechanism of refractoriness. Functional implications of these upregulated proteins in refractory species may reflect the phenotypic characteristics of the mosquitoes and will improve our understandings of blood meal digestion process, parasite vector interactions and proteomes of other vectors of human diseases for development of novel vector control strategies.

Salivary gland maturation and duct formation in the African malaria mosquito Anopheles gambiae.

Mosquito-borne diseases cause one million deaths and hundreds of millions of human infections yearly. With all such diseases, the pathogen must traverse the mosquito salivary gland (SG) for transmission to a new host, making the SGs ideal targets for genetic strategies to block transmission. Prior studies have elucidated details of SG structure by light and electron microscopy and have deeply explored the salivary transcriptome and proteome. Very little is known, however, about how the unique functional architecture of mosquito SGs is achieved. Using immunohistochemistry and confocal microscopy, we address two questions regarding SGs of the malaria vector Anopheles gambiae. How does the distinct cup-shaped morphology of SG secretory cells arise? And, how does the salivary duct, the structure through which saliva and parasites exit the glands, form? We demonstrate that SG cells begin as cuboidal-shaped cells surrounding a matrix-filled lumen that mature into cup-shaped cells through the formation and fusion of a large pre-apical compartment (PAC) to the apical surface. The secretory duct begins as buds of chitin at the apical surface of individual secretory cells. Further chitin deposition connects these chitin buds to form a contiguous duct that largely separates from the apical surface during PAC fusion.

The role of microRNAs in Anopheles biology-an emerging research field.

In the last years, microRNAs (miRNAs) have been established as important post-transcriptional regulators of critical physiological processes in animals and plants. Here, we summarize what is known about miRNA biosynthesis, expression and function in the malaria vector mosquito Anopheles gambiae with a particular emphasis on the mosquito-parasite interactions. We discuss the important gaps in the current knowledge, including the potential of miRNA manipulation for future vector control strategies.

A maleness gene in the malaria mosquito Anopheles gambiae.

The molecular pathways controlling gender are highly variable and have been identified in only a few nonmammalian model species. In many insects, maleness is conferred by a Y chromosome-linked M factor of unknown nature. We have isolated and characterized a gene, Yob, for the M factor in the malaria mosquito Anopheles gambiae Yob, activated at the beginning of zygotic transcription and expressed throughout a male's life, controls male-specific splicing of the doublesex gene. Silencing embryonic Yob expression is male-lethal, whereas ectopic embryonic delivery of Yob transcripts yields male-only broods. This female-killing property may be an invaluable tool for creation of conditional male-only transgenic Anopheles strains for malaria control programs.

Physical features and chitin content of eggs from the mosquito vectors Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus: Connection with distinct levels of resistance to desiccation.

Mosquito eggs are laid in water but freshly laid eggs are susceptible to dehydration, if their surroundings dry out at the first hours of development. During embryogenesis of different mosquito vectors the serosal cuticle, an extracellular matrix, is produced; it wraps the whole embryo and becomes part of the eggshell. This cuticle is an essential component of the egg resistance to desiccation (ERD). However, ERD is variable among species, sustaining egg viability for different periods of time. While Aedes aegypti eggs can survive for months in a dry environment (high ERD), those of Anopheles aquasalis and Culex quinquefasciatus in the same condition last, respectively, for one day (medium ERD) or a few hours (low ERD). Resistance to desiccation is determined by the rate of water loss, dehydration tolerance and total amount of water of a given organism. The ERD variability observed among mosquitoes probably derives from diverse traits. We quantified several attributes of whole eggs, potentially correlated with the rate of water loss: length, width, area, volume, area/volume ratio and weight. In addition, some eggshell aspects were also evaluated, such as absolute and relative weight, weight/area relationship (herein called surface density) and chitin content. Presence of chitin specifically in the serosal cuticle as well as aspects of endochorion external surface were also investigated. Three features could be related to differences on ERD levels: chitin content, directly related to ERD, the increase in the egg volume during embryogenesis and the eggshell surface density, which were both inversely related to ERD. Although data suggest that the amount of chitin in the eggshell is relevant for egg impermeability, the participation of other yet unidentified eggshell attributes must be considered in order to account for the differences in the ERD levels observed among Ae. aegypti, An. aquasalis and Cx. quinquefasciatus.

MicroRNAs of two medically important mosquito species: Aedes aegypti and Anopheles stephensi.

MicroRNAs (miRNAs) are endogenous, single-stranded small RNAs that have important regulatory functions at the post-transcriptional level. In the present study, we characterize miRNAs in two divergent mosquito species, Aedes aegypti and Anopheles stephensi, through deep sequencing of small RNAs spanning all developmental stages. We discovered eight novel miRNAs in Ae. aegypti and 20 novel miRNAs in An. stephensi, which enabled the first systematic analysis of miRNA evolution in mosquitos. We traced the phylogenetic history of all miRNAs in both species and report a rate of 0.055-0.13 miRNA net gain per million years. Most novel miRNAs originate de novo. Duplications that produced miRNA clusters and families are more common in Ae. aegypti than in An. stephensi. We also identified arm-switch as a source of new miRNAs. Expression profile analysis identified mosquito-specific miRNAs that showed strong stage-specific expression in one or both lineages. For example, the aae-miR-2941/2946 family represents the most abundant maternally deposited and zygotically transcribed miRNAs in Ae. aegypti. miR-2943 is a highly expressed zygotic miRNA in both Ae. aegypti and An. stephensi. Such information provides the basis from which to study the function of these miRNAs in biology common to all mosquitos or unique to one particular lineage.

Embryonic development and rates of metabolic activity in early and late hatching eggs of the major malaria vector Anopheles gambiae.

Anopheles gambiae eggs generally hatch at the completion of embryo development; two-three days post oviposition. However, staggered or delayed hatching has been observed whereby a single batch of eggs shows marked variation in time-to-hatch, with some eggs hatching 18 days post oviposition or later. The mechanism enabling delayed hatch has not been clearly elucidated but is likely mediated by environmental and genetic factors that either induce diapause or slow embryo development. This study aimed to compare metabolic activity and embryonic development between eggs collected from sub-colonies of the baseline Anopheles gambiae GAH colony previously selected for early or late time-to-hatch. Egg batches from early and late hatch sub-colonies as well as from the baseline colony were monitored for hatching. For both time-to-hatch selected sub-colonies and the baseline colony the majority of eggs hatched on day two post oviposition. Nevertheless, eggs produced by the late hatch sub-colony showed a significantly longer mean time to hatch than those produced by the early hatch sub-colony. The overall proportions that hatched were similar for all egg batches. CO2 output between eggs from early and late hatch sub-colonies showed significant differences only at 3 and 7 days post oviposition where eggs from the early hatch and the late hatch sub-colony were more metabolically active, respectively. No qualitative differences were observed in embryo development between the sub-colonies. It is concluded that all viable embryos develop to maturity at the same rate and that a small proportion then enter a state of diapause enabling them to hatch later. As it has previously been shown that it is possible to at least partially select for late hatch, this characteristic is likely to involve genetic as well as environmental factors. Delayed hatching in An. gambiae is likely an adaptation to maximise reproductive output despite the increased risk of desiccation in an unstable aquatic environment.

Maternal germline-specific genes in the Asian malaria mosquito Anopheles stephensi: characterization and application for disease control.

Anopheles stephensi is a principal vector of urban malaria on the Indian subcontinent and an emerging model for molecular and genetic studies of mosquito biology. To enhance our understanding of female mosquito reproduction, and to develop new tools for basic research and for genetic strategies to control mosquito-borne infectious diseases, we identified 79 genes that displayed previtellogenic germline-specific expression based on RNA-Seq data generated from 11 life stage-specific and sex-specific samples. Analysis of this gene set provided insights into the biology and evolution of female reproduction. Promoters from two of these candidates, vitellogenin receptor and nanos, were used in independent transgenic cassettes for the expression of artificial microRNAs against suspected mosquito maternal-effect genes, discontinuous actin hexagon and myd88. We show these promoters have early germline-specific expression and demonstrate 73% and 42% knockdown of myd88 and discontinuous actin hexagon mRNA in ovaries 48 hr after blood meal, respectively. Additionally, we demonstrate maternal-specific delivery of mRNA and protein to progeny embryos. We discuss the application of this system of maternal delivery of mRNA/miRNA/protein in research on mosquito reproduction and embryonic development, and for the development of a gene drive system based on maternal-effect dominant embryonic arrest.

In depth annotation of the Anopheles gambiae mosquito midgut transcriptome.

BACKGROUND: Genome sequencing of Anopheles gambiae was completed more than ten years ago and has accelerated research on malaria transmission. However, annotation needs to be refined and verified experimentally, as most predicted transcripts have been identified by comparative analysis with genomes from other species. The mosquito midgut-the first organ to interact with Plasmodium parasites-mounts effective antiplasmodial responses that limit parasite survival and disease transmission. High-throughput Illumina sequencing of the midgut transcriptome was used to identify new genes and transcripts, contributing to the refinement of An. gambiae genome annotation. RESULTS: We sequenced ~223 million reads from An. gambiae midgut cDNA libraries generated from susceptible (G3) and refractory (L35) mosquito strains. Mosquitoes were infected with either Plasmodium berghei or Plasmodium falciparum, and midguts were collected after the first or second Plasmodium infection. In total, 22,889 unique midgut transcript models were generated from both An. gambiae strain sequences combined, and 76% are potentially novel. Of these novel transcripts, 49.5% aligned with annotated genes and appear to be isoforms or pre-mRNAs of reference transcripts, while 50.5% mapped to regions between annotated genes and represent novel intergenic transcripts (NITs). Predicted models were validated for midgut expression using qRT-PCR and microarray analysis, and novel isoforms were confirmed by sequencing predicted intron-exon boundaries. Coding potential analysis revealed that 43% of total midgut transcripts appear to be long non-coding RNA (lncRNA), and functional annotation of NITs showed that 68% had no homology to current databases from other species. Reads were also analyzed using de novo assembly and predicted transcripts compared with genome mapping-based models. Finally, variant analysis of G3 and L35 midgut transcripts detected 160,742 variants with respect to the An. gambiae PEST genome, and 74% were new variants. Intergenic transcripts had a higher frequency of variation compared with non-intergenic transcripts. CONCLUSION: This in-depth Illumina sequencing and assembly of the An. gambiae midgut transcriptome doubled the number of known transcripts and tripled the number of variants known in this mosquito species. It also revealed existence of a large number of lncRNA and opens new possibilities for investigating the biological function of many newly discovered transcripts.

Serosal cuticle formation and distinct degrees of desiccation resistance in embryos of the mosquito vectors Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus.

Given their medical importance, mosquitoes have been studied as vectors of parasites since the late 1800's. However, there are still many gaps concerning some aspects of their biology, such as embryogenesis. The embryonic desiccation resistance (EDR), already described in Aedes and Anopheles gambiae mosquitoes, is a peculiar trait. Freshly laid eggs are susceptible to water loss, a condition that can impair their viability. EDR is acquired during embryogenesis through the formation of the serosal cuticle (SC), protecting eggs from desiccation. Nevertheless, conservation of both traits (SC presence and EDR acquisition) throughout mosquito evolution is unknown. Comparative physiological studies with mosquito embryos from different genera, exhibiting distinct evolutionary histories and habits is a feasible approach. In this sense, the process of EDR acquisition of Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus at 25°C was evaluated. Completion of embryogenesis occurs in Ae. aegypti, An. aquasalis and Cx. quinquefasciatus at, respectively 77.4, 51.3 and 34.3hours after egg laying, Cx. quinquefasciatus embryonic development taking less than half the time of Ae. aegypti. In all cases, EDR is acquired in correlation with SC formation. For both Ae. aegypti and An. aquasalis, EDR and SC appear at 21% of total embryonic development, corresponding to the morphological stage of complete germ band elongation/beginning of germ band retraction. Although phylogenetically closer to Ae. aegypti than to An. aquasalis, Cx. quinquefasciatus acquires both EDR and serosal cuticle later, with 35% of total development, when the embryo already progresses to the middle of germ band retraction. EDR confers distinct egg viability in these species. While Ae. aegypti eggs demonstrated high viability when left up to 72hours in a dry environment, those of An. aquasalis and Cx. quinquefasciatus supported these conditions for only 24 and 5hours, respectively. Our data suggest that serosa development is at least partially uncoupled from embryo development and that, depending upon the mosquito species, EDR bestows distinct levels of egg viability.

A comparative analysis of transcription factor expression during metazoan embryonic development.

During embryonic development, a complex organism is formed from a single starting cell. These processes of growth and differentiation are driven by large transcriptional changes, which are following the expression and activity of transcription factors (TFs). This study sought to compare TF expression during embryonic development in a diverse group of metazoan animals: representatives of vertebrates (Danio rerio, Xenopus tropicalis), a chordate (Ciona intestinalis) and invertebrate phyla such as insects (Drosophila melanogaster, Anopheles gambiae) and nematodes (Caenorhabditis elegans) were sampled, The different species showed overall very similar TF expression patterns, with TF expression increasing during the initial stages of development. C2H2 zinc finger TFs were over-represented and Homeobox TFs were under-represented in the early stages in all species. We further clustered TFs for each species based on their quantitative temporal expression profiles. This showed very similar TF expression trends in development in vertebrate and insect species. However, analysis of the expression of orthologous pairs between more closely related species showed that expression of most individual TFs is not conserved, following the general model of duplication and diversification. The degree of similarity between TF expression between Xenopus tropicalis and Danio rerio followed the hourglass model, with the greatest similarity occuring during the early tailbud stage in Xenopus tropicalis and the late segmentation stage in Danio rerio. However, for Drosophila melanogaster and Anopheles gambiae there were two periods of high TF transcriptome similarity, one during the Arthropod phylotypic stage at 8-10 hours into Drosophila development and the other later at 16-18 hours into Drosophila development.

A unique Y gene in the Asian malaria mosquito Anopheles stephensi encodes a small lysine-rich protein and is transcribed at the onset of embryonic development.

In many organisms the Y chromosome initiates sex determination and regulates male fertility and mating behaviour. However, molecular characterization of Y genes is rare outside of a few model species because it is difficult to clone and analyse repeat-rich heterochromatic Y sequences. In insects, Y genes are only well characterized in a small number of Drosophila species. Here we report the discovery of GUY1 (gene unique to the Y), a gene unique to the Y chromosome in the Asian malaria mosquito, Anopheles stephensi, using an approach that compares Illumina sequences separately obtained from male and female genomic DNA. Experimental evidence confirmed that GUY1 is a single copy gene found only on the Y chromosome. GUY1 is transcribed at the very onset of zygotic transcription and encodes a small lysine-rich protein that forms two alpha helices and shows DNA-binding properties. Interestingly, three helix-loop-helix proteins are key factors that determine sex in the early embryo in Drosophila melanogaster. Single embryo analysis indicated that GUY1 is only transcribed in male embryos and that the GUY1 promoter is functional in the early embryos. GUY1 may be used as a paternally inherited molecular marker. Further investigation of GUY1 will contribute to the genetic approaches to control mosquito-borne diseases.

Stable and fluctuating temperature effects on the development rate and survival of two malaria vectors, Anopheles arabiensis and Anopheles funestus.

BACKGROUND: Understanding the biology of malaria vector mosquitoes is crucial to understanding many aspects of the disease, including control and future outcomes. The development rates and survival of two Afrotropical malaria vectors, Anopheles arabiensis and Anopheles funestus, are investigated here under conditions of constant and fluctuating temperatures. These data can provide a good starting point for modelling population level consequences of temperature change associated with climate change. For comparative purposes, these data were considered explicitly in the context of those available for the third African malaria vector, Anopheles gambiae. METHODS: Twenty five replicates of 20-30 eggs were placed at nine constant and two fluctuating temperatures for development rate experiments and survival estimates. Various developmental parameters were estimated from the data, using standard approaches. RESULTS: Lower development threshold (LDT) for both species was estimated at 13-14°C. Anopheles arabiensis developed consistently faster than An. funestus. Optimum temperature (Topt) and development rate at this temperature (µmax) differed significantly between species for overall development and larval development. However, Topt and µmax for pupal development did not differ significantly between species. Development rate and survival of An. funestus was negatively influenced by fluctuating temperatures. By contrast, development rate of An. arabiensis at fluctuating temperatures either did not differ from constant temperatures or was significantly faster. Survival of this species declined by c. 10% at the 15°C to 35°C fluctuating temperature regime, but was not significantly different between the constant 25°C and the fluctuating 20°C to 30°C treatment. By comparison, previous data for An. gambiae indicated fastest development at a constant temperature of 28°C and highest survival at 24°C. CONCLUSIONS: The three most important African malaria vectors all differ significantly in development rates and survival under different temperature treatments, in keeping with known distribution data, though differences among M and S molecular forms of An. gambiae likely complicate the picture. Increasing temperatures associated with climate change favour all three species, but fluctuations in temperatures are detrimental to An. funestus and may also be for An. gambiae. This may have significant implications for disease burden in areas where each species is the main malaria vector.

Investigations on the role of a lysozyme from the malaria vector Anopheles dirus during malaria parasite development.

A cDNA encoding a lysozyme was obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) from females of the malaria vector Anopheles dirus A (Diptera: Culicidae). The 623 bp lysozyme (AdLys c-1) cDNA encodes the 120 amino acid mature protein with a predicted molecular mass of 13.4 kDa and theoretical pI of 8.45. Six cysteine residues and a potential calcium binding motif that are present in AdLys c-1 are highly conserved relative to those of c-type lysozymes found in other insects. RT-PCR analysis of the AdLys c-1 transcript revealed its presence at high levels in the salivary glands both in larval and adult stages and in the larval caecum. dsRNA mediated gene knockdown experiments were conducted to examine the potential role of this lysozyme during Plasmodium berghei infection. Silencing of AdLys c-1 resulted in a significant reduction in the number of oocysts as compared to control dsGFP injected mosquitoes.

Computer simulation on disease vector population replacement driven by the maternal effect dominant embryonic arrest.

In this chapter, we present a series of computer simulations on the genetic modification of disease vectors. We compared the effectiveness of two techniques of genetic modification, transposable elements and maternal effect dominant embryonic arrest (MEDEA). A gene drive mechanism based on MEDEA is introduced in the population to confer immunity to individuals. Experimental results suggested that the genetic maternal effects could be necessary for the effectiveness of a disease control strategy based on the genetic modification of vectors.