Project description:Globally invasive Aedes aegypti mosquitoes disseminate numerous arboviruses that impact human health. One promising method to control Ae. aegypti populations is transinfection with the intracellular bacterium Wolbachia pipientis, a symbiont that naturally infects ~40-52% of insects but is normally absent from Ae. aegypti. Transinfection of Ae. aegypti with the wMel Wolbachia strain induces cytoplasmic incompatibility (CI), allowing infected individuals to rapidly invade native populations. Further, wMel Wolbachia-infected females display refractoriness to medically relevant arboviruses. Thus, wMel Wolbachia-infected Ae. aegypti are being released in several areas to replace native populations, thereby suppressing disease transmission by this species. Wolbachia is reported to have minimal effects on Ae. aegypti fertility, but its influence on other reproductive processes is unknown. Female insects undergo several post-mating physiological and behavioral changes required for optimal fertility. Post-mating responses (PMRs) in female insects are typically elicited by receipt of male seminal fluid proteins (SFPs) transferred with sperm during mating, but can be modified by other factors, such as adult age, nutritional status, and microbiome composition. To assess how Wolbachia infection influences Ae. aegypti female PMRs, we collected wMel Wolbachia-infected Ae. aegypti from the field in Medellín, Colombia and introduced the bacterium into our laboratory strain. We found that Wolbachia influences female fecundity, fertility, and re-mating incidence. Further, we observed that Wolbachia significantly extends longevity of virgin females. Changes in female PMRs are not due to defects in sperm transfer by infected males, or sperm storage by infected females. Using proteomic methods to examine the seminal proteome of infected males, we found that Wolbachia infection has a moderate effect on SFP composition. However, we identified 125 Wolbachia proteins that are paternally transferred to females by infected males. Surprisingly, the CI factor proteins (Cifs), were not detected in the ejaculates of Wolbachia-infected males. Our findings indicate that Wolbachia infection of Ae. aegypti alters female post-mating responses, potentially influencing control programs that utilize Wolbachia-infected individuals.

Project description:Female Aedes aegypti mosquitoes impose a severe global public health burden as primary vectors of multiple viral and parasitic pathogens. Under optimal environmental conditions, Aedes aegypti females have access to human hosts that provide blood proteins for egg development, conspecific males that provide sperm for fertilization, and freshwater that serves as an egg-laying substrate suitable for offspring survival. As global temperatures rise, Aedes aegypti females are faced with climate challenges, like intense droughts and intermittent precipitation, which create unpredictable and suboptimal conditions for the egg-laying step of their reproductive cycle. Aedes aegypti mosquitoes nonetheless show remarkable reproductive resilience, but how they achieve this is unknown. Here we show that under drought-like conditions simulated in the laboratory, mated, blood-fed Aedes aegypti females carrying mature eggs retain them in their ovaries for extended periods, while maintaining the viability of these eggs until they can be deposited in freshwater. Using transcriptomic and proteomic profiling of Aedes aegypti ovaries, we identify two previously uncharacterized genes – here named tweedledee and tweedledum – that show ovary-enriched, temporally-restricted expression during egg retention. These genes are mosquito-specific, linked within a syntenic locus, and rapidly evolving under positive selection, raising the possibility that they serve an adaptive function. Using loss-of-function mutagenesis to disrupt both genes, we show that, tweedledee and tweedledum, which encode secreted proteins, are specifically required for extended retention of viable eggs, such as during intermittent precipitation or drought. These results highlight an elegant example of taxon-restricted genes at the heart of an important adaptation that equips Aedes aegypti females with “insurance” to, when contextually appropriate, flexibly extend their reproductive sequence without losing reproductive capacity, thus allowing this species to exploit diverse and unpredictable habitats.

Project description:Seminal fluid contains some of the fastest evolving proteins currently known. These seminal fluid proteins (Sfps) play crucial roles in reproduction, such as supporting sperm function, and – particularly in insects – modifying female physiology and behaviour. Identification of Sfps in small animals is challenging, and often relies on samples taken from the female reproductive tract after mating. A key pitfall of this method is that it might miss Sfps that are of low abundance due to dilution in the female-derived sample or rapid processing in females. Here we present a new and complimentary method, which provides added sensitivity to Sfp identification. We applied label-free quantitative proteomics to Drosophila melanogaster male reproductive tissue – where Sfps are unprocessed, and highly abundant – and quantified Sfps before and immediately after mating, to infer those transferred during copulation. We also analysed female reproductive tracts immediately before and after copulation to confirm the presence and abundance of known and candidate Sfps, where possible. Results were cross-referenced with transcriptomic and sequence databases to improve confidence in Sfp detection. Our data was consistent with 124 previously reported Sfps. We found 8 high-confidence novel candidate Sfps, which were both depleted in mated versus unmated males and identified within the reproductive tract of mated but not virgin females. We also identified 31 more candidates that are likely Sfps based on their abundance, known expression and predicted characteristics, and revealed that four proteins previously identified as Sfps are at best minor contributors to the ejaculate. The estimated copy numbers for our candidate Sfps were lower than for previously identified Sfps, supporting the idea that our technique provides a deeper analysis of the Sfp proteome than previous studies. Our results demonstrate a novel, high-sensitivity approach to the analysis of seminal fluid proteomes, whose application will further our understanding of reproductive biology.

Project description:Female Aedes aegypti mosquitoes impose a severe global public health burden as primary vectors of multiple viral and parasitic pathogens. Under optimal environmental conditions, Aedes aegypti females have access to human hosts that provide blood proteins for egg development, conspecific males that provide sperm for fertilization, and freshwater that serves as an egg-laying substrate suitable for offspring survival. As global temperatures rise, Aedes aegypti females are faced with climate challenges, like intense droughts and intermittent precipitation, which create unpredictable and suboptimal conditions for the egg-laying step of their reproductive cycle. Aedes aegypti mosquitoes nonetheless show remarkable reproductive resilience, but how they achieve this is unknown. Here we show that under drought-like conditions simulated in the laboratory, mated, blood-fed Aedes aegypti females carrying mature eggs retain them in their ovaries for extended periods, while maintaining the viability of these eggs until they can be deposited in freshwater. Using transcriptomic and proteomic profiling of Aedes aegypti ovaries, we identify two previously uncharacterized genes – here named tweedledee and tweedledum – that show ovary-enriched, temporally-restricted expression during egg retention. These genes are mosquito-specific, linked within a syntenic locus, and rapidly evolving under positive selection, raising the possibility that they serve an adaptive function. Using loss-of-function mutagenesis to disrupt both genes, we show that, tweedledee and tweedledum, which encode secreted proteins, are specifically required for extended retention of viable eggs, such as during intermittent precipitation or drought. These results highlight an elegant example of taxon-restricted genes at the heart of an important adaptation that equips Aedes aegypti females with “insurance” to, when contextually appropriate, flexibly extend their reproductive sequence without losing reproductive capacity, thus allowing this species to exploit diverse and unpredictable/chaotic/changing habitats.

			Action	DRS
	P1014_Hemolymph_Band_A.mgf	Mgf
	P1014_Hemolymph_Band_A.raw	Raw
	P1014_Hemolymph_Band_B.mgf	Mgf
	P1014_Hemolymph_Band_B.raw	Raw
	P1014_Hemolymph_Band_C.mgf	Mgf

Dataset Information

Expanded characterization and localization of male seminal fluid proteins within the female reproductive tract of the dengue vector mosquito Aedes aegypti

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