Juvenile Hormone Suppresses Resistance to Infection in Mated Female Drosophila melanogaster

Abstract

Hormonal signaling provides metazoans with the ability to regulate development, growth, metabolism, immune defense, and reproduction in response to internal and external stimuli. The use of hormones as central regulators of physiology makes them prime candidates for mediating allocation of resources to competing biological functions (i.e., hormonal pleiotropy) [1]. In animals, reproductive effort often results in weaker immune responses (e.g., [2-4]), and this reduction is sometimes linked to hormone signaling (see [5-7]). In the fruit fly, Drosophila melanogaster, mating and the receipt of male seminal fluid proteins results in reduced resistance to a systemic bacterial infection [8, 9]. Here, we evaluate whether the immunosuppressive effect of reproduction in female D. melanogaster is attributable to the endocrine signal juvenile hormone (JH), which promotes the development of oocytes and the synthesis and deposition of yolk protein [10, 11]. Previous work has implicated JH as immunosuppressive [12, 13], and the male seminal fluid protein Sex Peptide (SP) activates JH biosynthesis in female D. melanogaster after mating [14]. We find that transfer of SP activates synthesis of JH in the mated female, which in turn suppresses resistance to infection through the receptor germ cell expressed (gce). We find that mated females are more likely to die from infection, suffer higher pathogen burdens, and are less able to induce their immune responses. All of these deficiencies are rescued when JH signaling is blocked. We argue that hormonal signaling is important for regulating immune system activity and, more generally, for governing trade-offs between physiological processes.

Keywords: endocrine; evolution; hormone; immune defense; insect immunity; mating; providencia; reproduction; trade-off; tradeoff.

Figure 1. Juvenile hormone is immunosuppressive

(A) mRNA expression of antimicrobial peptide genes 8 hours after an injection with heat-killed P. rettgeri relative to CO₂ controls. Unmated females were exposed to methoprene, acetone, or CO₂ and mated females were exposed to CO₂ (Repeated-measures ANOVA, p < 0.0001, Treatment group: F_3,52 = 27.24, p < 0.0001). Tukey’s HSD was performed within each gene. Means with the same letter are not significantly different (p > 0.05) and error bars represent standard errors of the mean (SEM). (B) Bacterial load of individual unmated females that received acetone or methoprene (ANOVA, Treatment: F_{3, 72} = 13.92, p < 0.0001). Means with the same letter are not significantly different (p > 0.05) and error bars represent one SEM. None of the sterile wound treatments (dashed lines) experienced mortality events. n = 19 ± 1; two replicates. (C) Survivorship of unmated females subsequent to methoprene exposure and injection with sterile medium (PBS, dotted lines) or P. rettgeri (solid lines) (Cox, Chemical (infected only): $X_3^2$ = 35.98, p < 0.0001). n = 50 ± 6; two replicates.

Figure 2. Transgenic males lacking the N-terminus of Sex Peptide do not elicit immunosuppression in recipient females

(A) jhamt mRNA expression in females 10 hours after mating to SP genotypes relative to unmated females (ANOVA, Status: F_{2, 6} = 24.87, p = 0.00124). Bars represent the mean ± SEM. Means with the same letter are not significantly different (p > 0.05); three replicates. (B) Amino acid sequences of Sex Peptide; SP^QQ: the R₇K₈ trypsin cleavage site has been changed to Q₇Q₈; SP^Δ2-7: N-terminal amino acids (E₂-R₇) deleted; SP^WT: wildtype. (C) Bacterial load of individual females that had been infected with P. rettgeri subsequent to mating with males of different SP genotypes (ANOVA, mating status: F_{4, 189} = 13.91, p < 0.0001). Means with the same letter are not significantly different (p > 0.05) and error bars represent one SEM. n = 40 ± 6; three replicates. (D) Infection survivorship subsequent to mating with males of different SP genotypes (Cox, mating status: $X_4^2$ = 23.37, p = 0.00011). Letters indicate levels of significance. n =110 ± 15; three replicates.

Figure 3. Genetic ablation of JH biosynthesis rescues virgin-levels of resistance

(A)(B) Bacterial load (CFU) of individual CA+ and CA-ablated females subsequent to mating and infection. Mean bacterial load of unmated and mated females within a genotype were compared with a Wilcoxon test. (A) DTI, n = 60 ± 10; four replicates. (B) NIPP1, n = 30 ± 5; three replicates. Error bars represent one SEM. (C)(D) Survivorship of CA+ and CA− females subsequent to mating and infection. Survivorship was compared within a genotype using a log-rank test. (C) DTI, n > 120; five replicates. (D) NIPP1, n = 48 ± 15; three replicates. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001

Figure 4. RNAi-mediated knockdown of gce, a JH receptor mediates the effect of JH on immunity

(A)(B) Bacterial load (CFU) within Met-RNAi or gce-RNAi females and their controls, respectively. Mean bacterial load of unmated and mated females within a genotype were compared with a Wilcoxon test. Error bars represent one SEM. n = 29 ± 2; three replicates. (C)(D) Infection survivorship of Met-RNAi or gce-RNAi females and their controls, respectively. Survivorship was compared within a genotype using a log-rank test. n = 105 ± 15; four replicates. (E)(F) Infection survivorship of unmated Met-RNAi or gce-RNAi females exposed to methoprene (MP+) or acetone (MP−). Survivorship was compared within a genotype using a log-rank test. In the absence of infection, methoprene did not impact survivorship. (E) Met, n = 80 ± 15; three replicates. (F) gce, n =120 ± 20; three replicates. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001