Female mosquitoes undergo multiple rounds of reproduction known as gonotrophic cycles. A gonotrophic cycle spans the period from blood meal intake to egg laying. Nutrients from vertebrate host blood are necessary for completing egg development. During oogenesis, a female pre-packages mRNA into her oocytes, and these maternal transcripts drive the first two hours of embryonic development before zygotic genome activation. In this study, we profiled transcriptional changes in 1-2 hour-old Aedes aegypti embryos across two gonotrophic cycles. We found that homeotic genes which are regulators of embryogenesis are downregulated in embryos from the second gonotrophic cycle. Interestingly, embryos produced by Ae. aegypti females progressively reduced their ability to hatch as the number of gonotrophic cycles increased. We show that this fertility decline is due to increased reproductive output and not the mosquitoes’ age. Moreover, we found a similar decline in fertility and fecundity across three gonotrophic cycles in Ae. albopictus. Our results are useful for predicting mosquito population dynamics to inform vector control efforts.

Transcriptomic data processing and analysis

RNA sequencing (RNA-seq) data for 1-2hrs old wild-type Ae. aegypti embryos were retrieved from the transcriptome reported in our previous study [1]. Each library read was trimmed to remove the adapter sequence and then mapped to gene models from the AaegL5.0 genome. Upon mapping, transcript level abundance was determined from mapped reads using tximport. Principal component and differential gene expression analyses were performed on read counts using R’s shiny DEBrowser. Differential expression was calculated using Deseq2 at a corrected FDR of α < 0.01. Gene ontology (GO) enrichment was performed on differentially expressed genes at α < 0.01 using the bioinformatics resources on vectorbase.org, and redundant or obsolete GO terms were removed.  

Fecundity and fertility assays

To determine fecundity, the number of eggs deposited per female, 5–7-day old, mated females were starved on water overnight and then provided with a blood meal to repletion as described above. Fifty engorged females were transferred to a fresh cage and maintained on a 10% sucrose solution. 72-96hrs post blood meal, females were cold-anaesthetized and individually prepared for oviposition in a 24-well plate as previously described. Females were allowed to lay eggs over a period of 24 hours and individuals who failed to lay eggs were removed from the experiment. Upon egg collection, females were returned to the rearing cage with unlimited access to 10% sucrose solution and allowed to recover for 24hrs after which they were prepared for the second round of egg collection. Egg papers were kept moist for an additional 3-5 days post-collection and the number of eggs laid by individual females was subsequently counted. To determine fertility, the percentage of eggs that hatched into larvae, 5–7-day old eggs were hatched in individual 50mL cups containing DI water with dissolved TetraMin powder over a period of 3 days after which hatched larvae were counted. The same protocol was used in all experiments except in the delayed gonotrophic assay where some females were provided with their first blood meal at 20 days old. All experiments were conducted in triplicate with different mosquito-rearing cohorts.

1. David OG, Sanchez KM, Arce A V., Costa-da-Silva AL, Bellantuono AJ, DeGennaro M. 2023 Fertility decline in female mosquitoes is regulated by the orco olfactory co-receptor. iScience 26, 106883. (doi:10.1016/j.isci.2023.106883)