Oogenesis in the malaria mosquito Anopheles gambiae

Summary Oogenesis has been followed with the electron microscope in 2 strains of the malaria mosquito Anopheles gambiae, from the emergence of the adult (oocytes at leptonema) till shortly before the oocytes are ready for oviposition. After pachynema the chromosomes form a karyosphere and a fibrous capsule develops around it. Work on other mosquitoes suggests that the capsule may be related to the synaptonemal complexes. Both Anopheles strains contain at some time an extrachromosomal (not DNA-containing) body comparable to the karyosphere in size. Clusters of granules are present at the surface of the nucleolus and free in the nucleoplasm. Tentative results indicate that they may contain DNA. During oogenesis the nucleolus becomes very large, mainly because of proliferation of the nucleolonema. Towards the end of oocyte development the nucleus assumes the large canoe-shape also seen in Aedes and Culex. Nucleolonema traverse the entire nucleus, and modified granular clusters are found throughout.     

冈比亚按蚊嗅觉结合蛋白候选基因cDNA的克隆、鉴定及其表达型分析

疟蚊主要依靠嗅觉发现寄主。非洲疟蚊冈比亚按蚊Anopheles gambiae是一种嗜吸人血的疟疾传播媒介昆虫。该文作者基于其全基因组序列,采用RT-PCR和标准分子克隆技术获得2个嗅觉结合蛋白候选基因agLZ3788和agLZ9988。测序分析结果表明,它们具有嗅觉结合蛋白的标志性结构域。进一步采用半定量RT-PCR技术研究了它们的空间表达型,结果发现它们不但在雌蚊触角中表达,也在其他部位(尤其是蚊虫足部)有强的表达。这一发现说明疟蚊嗅觉结合蛋白可能具有更广的功能,也为进一步重组表达和功能研究提供了重要依据。     

Thioredoxin reductase from the malaria mosquito Anopheles gambiae.

The mosquito, Anopheles gambiae, is an important vector of Plasmodium falciparum malaria. Full genome analysis revealed that, as in Drosophila melanogaster, the enzyme glutathione reductase is absent in A. gambiae and functionally substituted by the thioredoxin system. The key enzyme of this system is thioredoxin reductase-1, a homodimeric FAD-containing protein of 55.3 kDa per subunit, which catalyses the reaction NADPH + H+ + thioredoxin disulfide-->NADP+ + thioredoxin dithiol. The A. gambiae trxr gene is located on chromosome X as a single copy; it represents three splice variants coding for two cytosolic and one mitochondrial variant. The predominant isoform, A. gambiae thioredoxin reductase-1, was recombinantly expressed in Escherichia coli and functionally compared with the wild-type enzyme isolated in a final yield of 1.4 U.ml(-1) of packed insect cells. In redox titrations, the substrate A. gambiae thioredoxin-1 (Km=8.5 microm, kcat=15.4 s(-1) at pH 7.4 and 25 degrees C) was unable to oxidize NADPH-reduced A. gambiae thioredoxin reductase-1 to the fully oxidized state. This indicates that, in contrast to other disulfide reductases, A. gambiae thioredoxin reductase-1 oscillates during catalysis between the four-electron reduced state and a two-electron reduced state. The thioredoxin reductases of the malaria system were compared. A. gambiae thioredoxin reductase-1 shares 52% and 45% sequence identity with its orthologues from humans and P. falciparum, respectively. A major difference among the three enzymes is the structure of the C-terminal redox centre, reflected in the varying resistance of catalytic intermediates to autoxidation. The relevant sequences of this centre are Thr-Cys-Cys-SerOH in A. gambiae thioredoxin reductase, Gly-Cys-selenocysteine-GlyOH in human thioredoxin reductase, and Cys-X-X-X-X-Cys-GlyOH in the P. falciparum enzyme. These differences offer an interesting approach to the design of species-specific inhibitors. Notably, A. gambiae thioredoxin reductase-1 is not a selenoenzyme but instead contains a highly unusual redox-active Cys-Cys sequence.     

Identification of one capa and two pyrokinin receptors from the malaria mosquito Anopheles gambiae

We cloned the cDNA of three evolutionarily related G protein-coupled receptors from the malaria mosquito Anopheles gambiae and functionally expressed them in Chinese hamster ovary cells. One receptor, Ang-Capa-R, was only activated by the two Anopheles capa neuropeptides Ang-capa-1 (GPTVGLFAFPRVamide) and Ang-capa-2 (pQGLVPFPRVamide) with EC(50) values of 8.6x10(-9)M and 3.3x10(-9)M, respectively, but not by any other known mosquito neuropeptide. The second receptor, Ang-PK-1-R, was selectively activated by the Anopheles pyrokinin-1 peptides Ang-PK-1-1 (AGGTGANSAMWFGPRLamide) and Ang-PK-1-2 (AAAMWFGPRLamide) with EC(50) values of 3.3x10(-8)M and 2.5x10(-8)M, respectively, but not by mosquito capa or pyrokinin-2 peptides. For the third receptor, Ang-PK-2-R, the most potent ligands were the pyrokinin-2 peptides Ang-PK-2-1 (DSVGENHQRPPFAPRLamide) and Ang-PK-2-2 (NLPFSPRLamide) with EC(50) values of 5.2x10(-9)M and 6.4x10(-9)M, respectively. However, this receptor could also be activated by the two pyrokinins-1, albeit with lower potency (EC(50): 2-5x10(-8)M). Because Ang-capa-1 and -2 and Ang-PK-1-1 are located on one preprohormone and the other peptides on another prohormone, these results imply a considerable crosstalk between the capa, pyrokinin-1 and pyrokinin-2 systems. Gene structure and phylogenetic tree analyses showed that Ang-Capa-R was the orthologue of the Drosophila capa receptor CG14575, Ang-PK-1-R the orthologue of the Drosophila pyrokinin-1 receptor CG9918, and Ang-PK-2-R the orthologue of the Drosophila pyrokinin-2 receptors CG8784 and CG8795. This is the first report on the functional characterization and crosstalk properties of capa and pyrokinin receptors in mosquitoes.     

Comparative genome analysis of the yellow fever mosquito Aedes aegypti with Drosophila melanogaster and the malaria vector mosquito Anopheles gambiae

An in silico comparative genomics approach was used to identify putative orthologs to genetically mapped genes from the mosquito, Aedes aegypti, in the Drosophila melanogaster and Anopheles gambiae genome databases. Comparative chromosome positions of 73 D. melanogaster orthologs indicated significant deviations from a random distribution across each of the five A. aegypti chromosomal regions, suggesting that some ancestral chromosome elements have been conserved. However, the two genomes also reflect extensive reshuffling within and between chromosomal regions. Comparative chromosome positions of A. gambiae orthologs indicate unequivocally that A. aegypti chromosome regions share extensive homology to the five A. gambiae chromosome arms. Whole-arm or near-whole-arm homology was contradicted with only two genes among the 75 A. aegypti genes for which orthologs to A. gambiae were identified. The two genomes contain large conserved chromosome segments that generally correspond to break/fusion events and a reciprocal translocation with extensive paracentric inversions evident within. Only very tightly linked genes are likely to retain conserved linear orders within chromosome segments. The D. melanogaster and A. gambiae genome databases therefore offer limited potential for comparative positional gene determinations among even closely related dipterans, indicating the necessity for additional genome sequencing projects with other dipteran species.     

Endogenous siRNAs and piRNAs derived from transposable elements and genes in the malaria vector mosquito Anopheles gambiae

Background

The siRNA and piRNA pathways have been shown in insects to be essential for regulation of gene expression and defence against exogenous and endogenous genetic elements (viruses and transposable elements). The vast majority of endogenous small RNAs produced by the siRNA and piRNA pathways originate from repetitive or transposable elements (TE). In D. melanogaster, TE-derived endogenous siRNAs and piRNAs are involved in genome surveillance and maintenance of genome integrity. In the medically relevant malaria mosquito Anopheles gambiae TEs constitute 12-16% of the genome size. Genetic variations induced by TE activities are known to shape the genome landscape and to alter the fitness in An. gambiae.

Results

Here, using bioinformatics approaches we analyzed the small RNA data sets from 6 libraries formally reported in a previous study and examined the expression of the mixed germline/somatic siRNAs and piRNAs produced in adult An. gambiae females. We characterized a large population of TE-derived endogenous siRNAs and piRNAs, which constitutes 56-60% of the total siRNA and piRNA reads in the analysed libraries. Moreover, we identified a number of protein coding genes producing gene-specific siRNAs and piRNAs that were generally expressed at much lower levels than the TE-associated small RNAs. Detailed sequence analysis revealed that An. gambiae piRNAs were produced by both “ping-pong” dependent (TE-associated piRNAs) and independent mechanisms (genic piRNAs). Similarly to D. melanogaster, more than 90% of the detected piRNAs were produced from TE-associated clusters in An. gambiae. We also found that biotic stress as blood feeding and infection with Plasmodium parasite, the etiological agent of malaria, modulated the expression levels of the endogenous siRNAs and piRNAs in An. gambiae.

Conclusions

We identified a large and diverse set of the endogenously derived siRNAs and piRNAs that share common and distinct aspects of small RNA expression across insect species, and inferred their impact on TE and gene activity in An. gambiae. The TE-specific small RNAs produced by both the siRNA and piRNA pathways represent an important aspect of genome stability and genetic variation, which might have a strong impact on the evolution of the genome and vector competence in the malaria mosquitoes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1436-1) contains supplementary material, which is available to authorized users.     

A behavioral mechanism underlying ecological divergence in the malaria mosquito Anopheles gambiae

Disruptive selection mediated by predation on aquatic immature stages has been proposed as a major force driving ecological divergence and fostering speciation between the M and S molecular forms of the African malaria mosquito, Anopheles gambiae. In the dry savannahs of West Africa where both molecular forms co-occur, the S form thrives in temporary pools filled with rainwater, whereas the M form preferentially breeds in permanent freshwater habitats where predator pressure is higher. Here, we explored the proximal mechanisms by which predation may contribute to habitat segregation between molecular forms using progeny of female mosquitoes captured in Burkina Faso. We show that the S form suffers higher predation rates than the M form when simultaneously exposed to the widespread predator, Anisops jaczewskii in an experimental arena. Furthermore, behavioral plasticity induced by exposure to the predator was observed in the M form, but not in the S form, and may partially explain its habitat use and ecological divergence from the S form. We discuss the role of adaptive phenotypic plasticity in allowing successful colonization of a new ecological niche by the M form and highlight further research areas that need to be addressed for a better understanding of the ultimate mechanisms underlying ecological speciation in this pest of major medical importance.     

Odor coding in the maxillary palp of the malaria vector mosquito Anopheles gambiae

BACKGROUND: Many species of mosquitoes, including the major malaria vector Anopheles gambiae, utilize carbon dioxide (CO(2)) and 1-octen-3-ol as olfactory cues in host-seeking behaviors that underlie their vectorial capacity. However, the molecular and cellular basis of such olfactory responses remains largely unknown. RESULTS: Here, we use molecular and physiological approaches coupled with systematic functional analyses to define the complete olfactory sensory map of the An. gambiae maxillary palp, an olfactory appendage that mediates the detection of these compounds. In doing so, we identify three olfactory receptor neurons (ORNs) that are organized in stereotyped triads within the maxillary-palp capitate-peg-sensillum population. One ORN is CO(2)-responsive and characterized by the coexpression of three receptors that confer CO(2) responses, whereas the other ORNs express characteristic odorant receptors (AgORs) that are responsible for their in vivo olfactory responses. CONCLUSIONS: Our results describe a complete and highly concordant map of both the molecular and cellular olfactory components on the maxillary palp of the adult female An. gambiae mosquito. These results also facilitate the understanding of how An. gambiae mosquitoes sense olfactory cues that might be exploited to compromise their ability to transmit malaria.     

Molecular identification of a myosuppressin receptor from the malaria mosquito Anopheles gambiae

The insect myosuppressins (X1DVX2HX3FLRFamide) are neuropeptides that generally block insect muscle activities. We have used the genomic sequence information from the malaria mosquito Anopheles gambiae Genome Project to clone a G protein-coupled receptor that was closely related to the two previously cloned and characterized myosuppressin receptors from Drosophila [Proc. Natl. Acad. Sci. USA 100 (2003) 9808]. The mosquito receptor cDNA was expressed in Chinese hamster ovary cells and was found to be activated by low concentrations of Anopheles myosuppressin (TDVDHVFLRFamide; EC50, 1.6 x 10(-8)M). The receptor was not activated by a library of 35 other insect neuropeptides and monoamines, including neuropeptides that resembled myosuppressin in their C-terminal moiety, such as PDRNFLRFamide (Anopheles FMRFamide-3), other Anopheles FMRFamide peptides, or neuropeptide F-like peptides, showing that the receptor was quite selective for myosuppressin. These results also showed that the myosuppressin receptor needs a much larger portion than the C-terminal FLRFamide sequence for its activation. The insect myosuppressins are often grouped together with the insect FMRFamides under the name FaRPs (FMRFamide-related peptides). However, this is not justified anymore, because the insect myosuppressin receptor/ligand couple is both functionally and evolutionarily fully unrelated to the insect FMRFamide receptor/ligand couple. To our knowledge, this is the first report on the molecular identification of a mosquito neuropeptide receptor.     

Characterization of the Hox gene cluster in the malaria vector mosquito, Anopheles gambiae

The Hox genes play a central role in regulating development and are involved in the specification of cell fates along the anteroposterior axis. In insects and vertebrates, these genes are clustered and organized in an arrangement that is largely conserved across evolutionary lineages. By exploiting the sequence conservation of the homeobox, orthologues of the Hox genes Sex combs reduced (Scr), fushi tarazu (ftz), Antennapedia (Antp), Ultrabithorax (Ubx), and abdominal-A (abd-A) have been isolated from the malaria vector mosquito, Anopheles gambiae. These genes were first identified in Drosophila, where they achieve a high level of functional complexity, in part, by the use of alternative promoters, polyadenylation sites, and splicing to generate different protein isoforms. Preliminary analyses of the Anopheles Hox genes suggest that they do not achieve their functional complexity in the same manner. Using a combination of in situ hybridization to polytene chromosomes and chromosome walking, the Anopheles Hox genes have been localized to a single cluster in the region 19D-E on chromosome 2R, a situation distinct from that of Drosophila where the Hox complex is split into two clusters. This study, therefore, provides a framework for future comparative analyses of the structure, organization, and expression of developmental regulatory genes between the lower and higher Diptera. Moreover, the genes that have been isolated enhance the genetic and physical maps of chromosome 2R in this medically important mosquito species.     

Time-of-day specific changes in metabolic detoxification and insecticide resistance in the malaria mosquito Anopheles gambiae

Mosquitoes exhibit ∼24 h rhythms in physiology and behavior, regulated by the cooperative action of an endogenous circadian clock and the environmental light:dark cycle. Here, we characterize diel (observed under light:dark conditions) time-of-day changes in metabolic detoxification and resistance to insecticide challenge in Anopheles gambiae mosquitoes. A better understanding of mosquito chronobiology will yield insights into developing novel control strategies for this important disease vector. We have previously identified >2000 rhythmically expressed An. gambiae genes. These include metabolic detoxification enzymes peaking at various times throughout the day. Especially interesting was the identification of rhythmic genes encoding enzymes capable of pyrethroid and/or DDT metabolism (CYP6M2, CYP6P3, CYP6Z1, and GSTE2). We hypothesized that these temporal changes in gene expression would confer time-of-day specific changes in metabolic detoxification and responses to insecticide challenge. An. gambiae mosquitoes (adult female Pimperena and Mali-NIH strains) were tested by gene expression analysis for diel rhythms in key genes associated with insecticidal resistance. Biochemical assays for total GST, esterase, and oxidase enzymatic activities were undertaken on time-specific mosquito head and body protein lysates. To determine for rhythmic susceptibility to insecticides by survivorship, mosquitoes were exposed to DDT or deltamethrin across the diel cycle. We report the occurrence of temporal changes in GST activity in samples extracted from the body and head with a single peak at late-night to dawn, but no rhythms were detected in oxidase or esterase activity. The Pimperena strain was found to be resistant to insecticidal challenge, and subsequent genomic analysis revealed the presence of the resistance-conferring kdr mutation. We observed diel rhythmicity in key insecticide detoxification genes in the Mali-NIH strain, with peak phases as previously reported in the Pimperena strain. The insecticide sensitive Mali-NIH strain mosquitoes exhibited a diel rhythm in survivorship to DDT exposure and a bimodal variation to deltamethrin challenge. Our results demonstrate rhythms in detoxification and pesticide susceptibility in An. gambiae mosquitoes; this knowledge could be incorporated into mosquito control and experimental design strategies, and contributes to our basic understanding of mosquito biology.