Developmental heat stress disrupts male-induced female post-mating responses in the dengue vector mosquito, Aedes aegypti

Technical Summary: Developmental Heat Stress and Male-Induced Female Post-Mating Responses in Aedes aegypti

Problem Statement
The dengue vector Aedes aegypti is expanding its global range due to urbanization and rising global temperatures. While elevated temperatures during larval development are known to reduce adult body size and accelerate development, their specific impact on male reproductive fitness and the induction of female post-mating responses (PMRs) remains poorly understood. Male-derived seminal fluid proteins (SFPs) are critical for triggering female PMRs, which include increased egg production, inhibition of remating (refractoriness), and sperm storage. In vector control programs utilizing Wolbachia-infected males (to induce cytoplasmic incompatibility or suppress populations), the success of these strategies relies heavily on the ability of released males to mate successfully and induce strong, long-term female refractoriness. This study investigates whether developmental heat stress compromises these male-mediated functions, potentially undermining control efficacy.

Methodology
The study utilized three strains of A. aegypti: the laboratory Thai strain, the ThaiWolb strain (Thai background infected with Wolbachia wMel), and a DsRed strain (expressing fluorescent markers for sperm tracking).

• Rearing Conditions: Larvae were reared at control (27°C) or elevated temperatures (32°C and 35°C). 37°C was identified as lethal. Adult males were tested at 2 and 4 days post-emergence to assess recovery time.
• Assays:
  • Developmental Metrics: Larval development time and adult wing length (as a proxy for body size) were measured.
  • Mating Success: The proportion of females inseminated within a 2-hour window was recorded.
  • Female PMRs: Fecundity (egg count) and fertility (hatch rate) were assessed. Remating incidence was tested by allowing females to mate with a second male (DsRed strain) and checking for the presence of DsRed sperm.
  • Sperm Dynamics: Sperm transfer (bursa) and storage (spermathecae) were quantified via dissection and staining. Sperm competitive ability was determined by analyzing the paternity (P1 vs. P2) of offspring from multiply mated females using GFP-labeled larvae.
  • Statistical Analysis: Generalized linear mixed-effects models (GLMMs) were employed, accounting for hatch identity as a random effect and testing interactions between developmental temperature, male age, and strain.

Key Results

1. Developmental and Morphological Effects: Elevated rearing temperatures (32°C and 35°C) significantly accelerated larval development and reduced adult wing length. Males reared at 35°C were significantly smaller than those at 32°C.
2. Mating Success: Heat stress significantly reduced mating success in 2-day-old males reared at 35°C (51.8% insemination vs. ~92% in controls). However, by 4 days post-emergence, mating success in heat-stressed males recovered to control levels.
3. Fecundity and Fertility: Contrary to expectations, developmental heat stress did not significantly reduce the fecundity or fertility (hatch rate) of females mated to heat-stressed males in the uninfected Thai strain. Mean hatch rates remained above 83% across all groups.
4. Disruption of Female Refractoriness: A major finding was the significant increase in female remating incidence when females were initially mated to heat-stressed males.
   • In 2-day-old males, remating rates jumped from ~20% (27°C) to ~59% (35°C).
   • In 4-day-old males, remating rates remained elevated for heat-stressed males (32°C and 35°C) compared to controls, indicating a persistent defect in inducing refractoriness.
5. Sperm Transfer and Storage: Males reared at 35°C transferred significantly fewer sperm to females, and females stored significantly less sperm. This reduction persisted regardless of male age.
6. Sperm Competitive Ability: Females initially mated to heat-stressed males (35°C) subsequently used significantly more sperm from a second, competitor male to fertilize their eggs (lower P1 scores). This indicates a reduction in the competitive ability of sperm from heat-stressed males.
7. Impact of Wolbachia:
   • Wolbachia infection altered baseline reproductive outcomes (e.g., generally higher remating rates in mates of infected males).
   • However, the response to heat stress was largely similar between infected and uninfected strains. Heat stress still impaired mating success, increased remating, and reduced sperm transfer in Wolbachia-infected males.
   • Notably, fertility in mates of Wolbachia-infected males reared at 35°C was higher than at 27°C or 32°C, consistent with heat-induced reduction in Wolbachia density and a subsequent weakening of cytoplasmic incompatibility.

Key Contributions

• Mechanistic Insight: The study demonstrates that developmental heat stress impairs the male's ability to induce specific female PMRs (refractoriness) and transfer sufficient sperm, even when basic fertility (egg hatching) remains intact.
• Persistence of Defects: It distinguishes between transient defects (mating success, which recovers with age) and persistent defects (sperm quantity, storage, and competitive ability, which do not recover).
• Vector Control Implications: The findings suggest that heat stress during larval development in laboratory rearing facilities could produce males that are less effective at suppressing wild populations because they fail to prevent remating and lose sperm competition.

Significance and Claims
The authors claim that these findings improve the understanding of mosquito reproduction under warming environmental conditions. They argue that while heat stress may not eliminate fertility in single-mating scenarios, it significantly disrupts the complex male-induced behaviors required for successful vector control. Specifically, the reduced ability of heat-stressed males to induce female refractoriness and their diminished sperm competitive ability could reduce the efficacy of population suppression and replacement programs that rely on the release of laboratory-reared males. The study concludes that as global temperatures rise, thermal stress in mosquito habitats may alter reproductive interactions, necessitating a re-evaluation of control strategies to account for these physiological deficits.