Molecular characteristics and putative functions of reproduction-associated proteins of Anopheles gambiae mosquito

Abstract

In Anopheles gambiae mosquito vector of malaria, mating is mediated by transfer of a plug (mating plug protein) from males to females. In the females, the constituents of the plug include male accessory glands (MAGs) proteins and female specific proteins that influence ovulation, oviposition, lifetime refractoriness to mating and other post-mating behaviors in the females. Manipulation of the plug can significantly affect the reproduction potential and concomitant population density of the vector. Investigations were conducted to establish in the mosquito 1) putative structural properties of MAGs proteins (transglutaminase and plugin) mediating successful interaction for mating-plug formation in the male An. gambiae 2) putative interacting proteins potentially mediating the mating-plug formation and post-mating phenotype, 3) mating responsive molecular factors in the proteins, 4) influence of the interactions among the proteins on mating responses and 5) patterns and roles of swarms in mating responses in females. Structural properties of An. gambiae MAGs (transglutaminase and plugin) proteins were identified using Protein-Predict software and type of selection pressure among related orthologs in different taxa was determined through phylogenetic analysis. Orthologs of the An. gambiae plug proteins in Drosophila melanogaster were obtained from STRING database and their interactions (protein-protein) predicted using CYTOScape software. Phylogeny among key D. melanogaster interacting protein homolog equivalents in An. gambiae (interologs) was determined by maximum likelihood phylogenetic method. Pathway analyses of the interologs in Reactome database was used to identify proteins involed in plug formation and post-mating phenotype. Expression of mRNA for genes of the proteins in males (MAGs and testes) and females (atria, ovary and spermatheca) reproductive tissues was determined by RT-qPCR, and identification of the genes verified through sequencing of the amplicons and western blot analyses. Role of the genes in mating responses in the mosquito was verified through RNAi technique. Patterns and roles of swarms in mating responses in females were determined through cage releases and observations in a semi–field setting. Plugin was predicted to be non-structural and under positive selection among the MAGs proteins, and also identified as the main protein in mating plug formation on the protein-protein interaction network. Plugin was also shown to putatively interact with TRYPSIN-LIKE SERINE PROTEASE (AGAP005195) in the female atria after mating through a putative two response regulatory pathway. Plugin (AGAP009368), TRYPSIN-LIKE SERINE PROTEASE (AGAP005195) and CATHEPSIN B (AGAP004533) appeared to interact and influence postmating effects in the females. CATHEPSIN B (AGAP004533) was co-expressed and regulated by TRYPSIN-LIKE vii SERINE PROTEASE (AGAP005195) in the female. TRYPSIN-LIKE SERINE PROTEASE (AGAP005195) specifically interacted with plugin on the mating plug and was transported to the spermatheca after mating, implicating it in postmating events within the females. Knockdown of CATHEPSIN B suggested that plugin was alternatively spliced in the male. The knockdown implicates CATHEPSIN B and TRYPSIN-LIKE SERINE PROTEASE in a common reproductive pathway in females, supported by mating propensity phenotype observed in the females. A swarming window of about 30 minutes was identified for An. gambiae mosquito where 41.3% of females were successfully inseminated. Interaction between CATHEPSIN B and plugin in the male then CATHEPSIN B and TRYPSIN-LIKE SERINE PROTEASE in the female produces successful interactions which are involved in reproductive pathways within the male and the female mosquitoes hence potentially and significantly affecting male and female reproductive processes. Detailed molecular analysis through high throughput interventions can potentially elucidate actual binding sites between CATHEPSIN B and its partner in the male (plugin) and female TRYPSIN-LIKE SERINE PROTEASE mosquitoes which can be exploited for vector control interventions.