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.     

Identification and characterization of a novel chitinase-like gene cluster (AgCht5) possibly derived from tandem duplications in the African malaria mosquito, Anopheles gambiae

Insect chitinase 5 (Cht5), a well-characterized enzyme found in the molting fluid and/or integument, is classified as a group I chitinase and is usually encoded by a single gene. In this study, a Cht5 gene cluster consisting of five different chitinase-like genes (AgCht5-1, AgCht5-2, AgCht5-3, AgCht5-4 and AgCht5-5) was identified by a bioinformatics search of the genome of Anopheles gambiae. The gene models were confirmed by cloning and sequencing of the corresponding cDNAs and gene expression profiles during insect development were determined. All of these genes are found in a single cluster on chromosome 2R. Their open reading frames (ORF) range from 1227 to 1713 bp capable of encoding putative proteins ranging in size from 409 to 571 amino acids. The identities of their cDNA sequences range from 52 to 66%, and the identities of their deduced amino acid sequences range from 38 to 53%. There are four introns for AgCht5-1, two for AgCht5-2 and AgCht5-3, only one for AgCht5-4, but none for AgCht5-5 in the genome. All five chitinase-like proteins possess a catalytic domain with all of the conserved sequence motifs, but only AgCht5-1 has a chitin-binding domain. Phylogenetic analysis of these deduced proteins along with those from other insect species suggests that AgCht5-1 is orthologous to the Cht5 proteins identified in other insect species. The differences in expression patterns of these genes at different developmental stages further support that these genes may have distinct functions. Additional searching of the genomes of two other mosquito species led to the discovery of four Cht5-like genes in Aedes aegypti and three in Culex quinquefasciatus. Thus, the presence of a Cht5 gene cluster appears to be unique to mosquito species and these genes may have resulted from gene tandem duplications.     

Isolation and characterization of Y chromosome sequences from the African malaria mosquito Anopheles gambiae

The karyotype of the African malaria mosquito Anopheles gambiae contains two pairs of autosomes and a pair of sex chromosomes. The Y chromosome, constituting approximately 10% of the genome, remains virtually unexplored, despite the recent completion of the A. gambiae genome project. Here we report the identification and characterization of Y chromosome sequences of total length approaching 150 kb. We developed 11 Y-specific PCR markers that consistently yielded male-specific products in specimens from both laboratory colony and natural populations. The markers are characterized by low sequence polymorphism in samples collected across Africa and by presence in more than one copy on the Y. Screening of the A. gambiae BAC library using these markers allowed detection of 90 Y-linked BAC clones. Analysis of the BAC sequences and other Y-derived fragments showed massive accumulation of a few transposable elements. Nevertheless, more complex sequences are apparently present on the Y; these include portions of an approximately 48-kb-long unmapped AAAB01008227 scaffold from the whole genome shotgun assembly. Anopheles Y appears not to harbor any of the genes identified in Drosophila Y. However, experiments suggest that one of the ORFs from the AAAB01008227 scaffold represents a fragment of a gene with male-specific expression.     

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.     

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.     

An active transposable element, Herves, from the African malaria mosquito Anopheles gambiae

Transposable elements have proven to be invaluable tools for genetically manipulating a wide variety of plants, animals, and microbes. Some have suggested that they could be used to spread desirable genes, such as refractoriness to Plasmodium infection, through target populations of Anopheles gambiae, thereby disabling the mosquito's ability to transmit malaria. To achieve this, a transposon must remain mobile and intact after the initial introduction into the genome. Endogenous, active class II transposable elements from An. gambiae have not been exploited as gene vectors/drivers because none have been isolated. We report the discovery of an active class II transposable element, Herves, from the mosquito An. gambiae. Herves is a member of a distinct subfamily of hAT elements that includes the hopper-we element from Bactrocera dorsalis and B. cucurbitae. Herves was transpositionally active in mobility assays performed in Drosophila melanogaster S2 cells and developing embryos and was used as a germ-line transformation vector in D. melanogaster. Herves displays an altered target-site preference from the distantly related hAT elements, Hermes and hobo. Herves is also present in An. arabiensis and An. merus with copy numbers similar to that found in An. gambiae. Preliminary data from an East African population are consistent with the element being transpositionally active in mosquitoes.     

Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito,Anopheles gambiae

Abstract Chitin synthase (CHS) is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control. However, our understanding of biochemical properties of insect CHSs has been very limited. We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito, Anopheles gambiae. Our study, which represents the first time to use a nonradioactive method to assay CHS activity in an insect species, determined the optimal conditions for measuring the enzyme activity, including pH, temperature, and concentrations of the substrate uridine diphosphate N‐acetyl‐d ‐glucosamine (UDP‐GlcNAc) and Mg⁺⁺. The optimal pH was about 6.5–7.0, and the highest activity was detected at temperatures between 37°C and 44°C. Dithithreitol is required to prevent melanization of the enzyme extract. CHS activity was enhanced at low concentration of GlcNAc, but inhibited at high concentrations. Proteolytic activation of the activity is significant both in the 500 ×g supernatant and the 40 000 ×g pellet. Our study revealed only slight in vitro inhibition of A. gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration (2.5 μmol/L) examined. There was no in vitro inhibition by polyoxin D at any concentration examined. Furthermore, we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined. Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A. gambiae.     

Development of the bi-partite Gal4-UAS system in the African malaria mosquito, Anopheles gambiae

Functional genetic analysis in Anopheles gambiae would be greatly improved by the development of a binary expression system, which would allow the more rapid and flexible characterisation of genes influencing disease transmission, including those involved in insecticide resistance, parasite interaction, host and mate seeking behaviour. The Gal4-UAS system, widely used in Drosophila melanogaster functional genetics, has been significantly modified to achieve robust application in several different species. Towards this end, previous work generated a series of modified Gal4 constructs that were up to 20 fold more active than the native gene in An. gambiae cells. To examine the Gal4-UAS system in vivo, transgenic An. gambiae driver lines carrying a modified Gal4 gene under the control of the carboxypeptidase promoter, and responder lines carrying UAS regulated luciferase and eYFP reporter genes have been created. Crossing of the Gal4 and UAS lines resulted in progeny that expressed both reporters in the expected midgut specific pattern. Although there was minor variation in reporter gene activity between the different crosses examined, the tissue specific expression pattern was consistent regardless of the genomic location of the transgene cassettes. The results show that the modified Gal4-UAS system can be used to successfully activate expression of transgenes in a robust and tissue specific manner in Anopheles gambiae. The midgut driver and dual reporter responder constructs are the first to be developed and tested successfully in transgenic An. gambiae and provide the basis for further advancement of the system in this and other insect species.     

The antennal lobe of the African malaria mosquito, Anopheles gambiae - innervation and three-dimensional reconstruction

Antibody labelling and subsequent three-dimensional reconstructions of the primary olfactory centres, the antennal lobes, of male and female African malaria mosquitoes, Anopheles gambiae, revealed 61 and 60 glomerular neuropils respectively. In addition to the small difference in number of glomeruli, sexual dimorphism was observed in both the size of the antennal lobe and of individual glomeruli. Furthermore, sexual specificity was observed within the array. Anterograde staining of afferents from peripheral olfactory organs support the reconstruction of the glomerular array. Although anterograde stainings support an organotopic organization of the antennal lobe, convergence of afferents originating from different organs into single glomeruli is observed. This finding, in both A. gambiae and A. aegypti, may shed new light upon the development and function of the olfactory system.     

New repellent effective against African malaria mosquito Anopheles gambiae: implications for vector control

Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) is a vector for Plasmodium, the causative agent of malaria. Current control strategies to reduce the impact of malaria focus on reducing the frequency of mosquito attacks on humans, thereby decreasing Plasmodium transmission. A need for new repellents effective against Anopheles mosquitoes has arisen because of changes in vector behaviour as a result of control strategies and concern over the health impacts of current repellents. The response of A. gambiae to potential repellents was investigated through an electroantennogram screen and the most promising of these candidates (1‐allyloxy‐4‐propoxybenzene, 3c {3,6}) chosen for behavioural testing. An assay to evaluate the blood‐host seeking behaviour of A. gambiae towards a simulated host protected with this repellent was then performed. The compound 3c {3,6} was shown to be an effective repellent, causing mosquitoes to reduce their contact with a simulated blood‐host and probe less at the host odour. Thus, 3c {3,6} may be an effective repellent for the control of A. gambiae.     

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.