A single mating is sufficient to induce persistent reduction of immune defense in mated female Drosophila melanogaster

Abstract

In many species, female reproductive investment comes at a cost to immunity and resistance to infection. Mated Drosophila melanogaster females are more susceptible to bacterial infection than unmated females. Transfer of the male seminal fluid protein Sex Peptide reduces female post-mating immune defense. Sex Peptide is known to cause both short- and long-term changes to female physiology and behavior. While previous studies showed that females were less resistant to bacterial infection as soon as 2.5 h and as long as 26.5 h after mating, it is unknown whether this is a binary switch from mated to unmated state or whether females can recover to unmated levels of immunity. It is additionally unknown whether repeated mating causes progressive reduction in defense capacity. We compared the immune defense of mated females when infected at 2, 4, 7, or 10 days after mating to that of unmated females and saw no recovery of immune capacity regardless of the length of time between mating and infection. Because D. melanogaster females can mate multiply, we additionally tested whether a second mating, and therefore a second transfer of seminal fluids, caused deeper reduction in immune performance. We found that females mated either once or twice before infection survived at equal proportions, both with significantly lower probability than unmated females. We conclude that a single mating event is sufficient to persistently suppress the female immune system. Interestingly, we observed that induced levels of expression of genes encoding antimicrobial peptides (AMPs) decreased with age in both experiments, partially obscuring the effects of mating. Collectively, the data indicate that being reproductively active versus reproductively inactive are alternative binary states with respect to female D. melanogaster immunity. The establishment of a suppressed immune status in reproductively active females can inform our understanding of the regulation of immune defense and the mechanisms of physiological trade-offs.

Keywords: Antimicrobial peptides; Drosophila melanogaster; Innate immunity; Reproduction-immunity trade-offs; Sex Peptide.

Figure 1:

Mating suppresses immune defense for up to 10 days after mating. (A) Design for experiments testing the persistence of immune suppression after mating. All females were age-matched and infected on the same day. The four intervals between mating and infection were 2, 4, 7, and 10 days. An additional group remained unmated throughout the experiment. (B) All mated females had significantly lower survivorship than unmated females (n=79) regardless of the interval between mating and infection (2 day interval, p = 0.001, n=90; 4 day interval, p < 0.001, n=92; 7 day interval, p = 0.026, n=87; 10 day interval, p < 0.01, n=99). There was no statistical difference in survivorship between the four mated groups. Statistical significance of all pairwise comparisons are represented by letter. The graph illustrates combined data from two biological-replicate blocks, which did not significantly differ. (C) All mated females had significantly higher bacterial loads than unmated females (n=47) regardless of interval between mating and infection (2 day interval, p = 0.025, n=51; 4 day interval, p < 0.001, n=37; 7 day interval, p < 0.001, n=49; 10 day interval, p = 0.006, n=41). Mated females had similar bacterial loads, regardless of the interval between mating and infection, except 2 day and 4 day intervals (p = 0.036). Only significant differences are represented with brackets. Each data point represents a single female. (D) Infection-induced expression of AMP genes in unmated, 2 day, 4 day, 7 day, and 10 day interval mated females. Infection of mated and unmated females induces AMP gene expression statistically equivalently (p>0.05), regardless of the interval between mating and infection. (E) No statistically significant (p>0.05) differences in AMP gene expression we observed between 4 day, 8 day, and 14 day post-eclosion females, regardless of mating status.

Figure 2:

(A) Experimental design for experiments testing the effects of a second mating event on immune suppression. All females were age matched and infected on the same day. Females mated twice were first mated to CS pBac{3xP3-EGFP, ProtB-EGFP}16B (Manier et al., 2010), then CS. Females mated once were mated only to CS males. One group of females remained unmated throughout the experiment. (B) Mated females exhibited significantly lower survivorship than unmated (n=79) females (two matings, p = 1 x 10⁻⁴, n=55 ; one mating, p = 0.005, n=76), with no statistically-significant difference in survivorship between the two matings and one mating groups (p = 0.213). Statistical significance of all pairwise comparisons are represented by letter. The graph illustrates combined data from two biological- replicate blocks. (C) Females mated once and twice both had significantly higher bacterial load than unmated (n=83) females. (two matings, p < 0.001, n=73; one mating, p = 0.003, n=75). There was no difference in bacterial load between females mated once or twice (p = 0.388). Each data point represents a single female. Plots represent combined data from two independent blocks. (D) Infection increased the expression of AMPs relative to the control gene, Actin5C. Unmated females had higher levels of Attacin A and Cecropin transcripts (standardized by Actin 5C expression) than females mated either once or twice but had lower levels of Defensin transcripts than mated females. Unmated and mated females expressed similar levels of Diptericin A transcripts, regardless of whether they were mated once or twice.