Sexual conflict drives micro- and macroevolution of sexual dimorphism in immunity

Abstract

Background: Sexual dimorphism in immunity is believed to reflect sex differences in reproductive strategies and trade-offs between competing life history demands. Sexual selection can have major effects on mating rates and sex-specific costs of mating and may thereby influence sex differences in immunity as well as associated host-pathogen dynamics. Yet, experimental evidence linking the mating system to evolved sexual dimorphism in immunity are scarce and the direct effects of mating rate on immunity are not well established. Here, we use transcriptomic analyses, experimental evolution and phylogenetic comparative methods to study the association between the mating system and sexual dimorphism in immunity in seed beetles, where mating causes internal injuries in females.

Results: We demonstrate that female phenoloxidase (PO) activity, involved in wound healing and defence against parasitic infections, is elevated relative to males. This difference is accompanied by concomitant sex differences in the expression of genes in the prophenoloxidase activating cascade. We document substantial phenotypic plasticity in female PO activity in response to mating and show that experimental evolution under enforced monogamy (resulting in low remating rates and reduced sexual conflict relative to natural polygamy) rapidly decreases female (but not male) PO activity. Moreover, monogamous females had evolved increased tolerance to bacterial infection unrelated to mating, implying that female responses to costly mating may trade off with other aspects of immune defence, an hypothesis which broadly accords with the documented sex differences in gene expression. Finally, female (but not male) PO activity shows correlated evolution with the perceived harmfulness of male genitalia across 12 species of seed beetles, suggesting that sexual conflict has a significant influence on sexual dimorphisms in immunity in this group of insects.

Conclusions: Our study provides insights into the links between sexual conflict and sexual dimorphism in immunity and suggests that selection pressures moulded by mating interactions can lead to a sex-specific mosaic of immune responses with important implications for host-pathogen dynamics in sexually reproducing organisms.

Keywords: Callosobruchus maculatus; Experimental evolution; Immunity; Mating; Phenoloxidase; Sexual conflict; Sexual dimorphism; Sexual selection; Sexually transmitted disease; Trade-off.

Fig. 1

Sex-biased gene expression in the proPO signalling cascade. In a, schematic representation of key proteins in the proPO activating cascade, based on previous studies of insects and other invertebrates (reviewed in [54, 55, 67, 73, 78]). In b, sex-bias and effects of mating status on gene expression in the abdomen for C. maculatus orthologs from published data [79] mapped to the sequences of the functionally annotated proteins. Full results in Additional file 1: Table S1. Spätzle processing enzyme (SPE: pink) initiates cleavage of proPO (yellow) into active PO, which ultimately leads to wound healing as well as encapsulation and killing of foreign pathogens. However, SPE also regulates the production of Spätzle protein (SPZ) from proSPZ (blue), which ultimately leads to increased production of antimicrobial peptides (AMPs) via the TOLL pathway, which offers inducible immunity against pathogens, thus setting the stage for an allocation trade-off between PO activity and AMP production. Overactivation of the proPO cascade has toxic side-effects via the production of secondary metabolites, suggesting that overproduction of SPE may come at a cost to overall health. Here, production of serine protease inhibitors (serpins: grey) in the TOLL pathway exerts negative feedback and control over the cascade. b C. maculatus females show higher expression of SPE (pink) and proPO (yellow) as virgins. Males show higher expression of proSPZ (blue) and serpins (grey). Shown are individual samples and means ± 1 SD. These patterns in gene expression suggest a mechanistic basis for sex-specific immunity trade-offs between different components in the proPO activating cascade, where females are predicted to invest more in PO activity (wound healing and potentially encapsulation of pathogens transferred at mating) in their reproductive tract in response to mating, at the potential cost of reduced inducible immunity via AMP production and/or toxic side-effects of overactivation of the proPO cascade

Fig. 2

Sex-specific regulation of phenoloxidase levels. a There were significant differences in PO activity throughout development, with levels near zero detected in male (blue) and female (red) pupae and virgin adult males, but detectable levels in (unsexed = black) larvae and high levels in virgin adult females. b PO activity measured on day 3 in females mated only on day 1 (100), days 1 and 2 (110), days 1 and 3 (101), or on all days (111) (open symbols). A second experiment measured PO activity for a random set of females assigned to treatments 100 and 001 (mated only on day three) (filled symbols). Female PO activity is reduced after mating but is then quickly restored (compare also to virgin females (i.e. 000 treatment) in a. Shown are means ± 1 SE and individual observations. PO activity was corrected for body mass by including mass as a covariate in all analyses but is here displayed as raw data since the mean amount of tissue in samples was similar for all groups

Fig. 3

Microevolutionary change in PO activity during experimental evolution. PO activity measured from whole-body samples of virgin (a) and mated (b) females from polygamous (green) monogamous (orange) and male-limited (blue) evolution lines. The mating treatment significantly reduced female PO activity and male-limited and polygamous females had higher PO activity than monogamous females. Polygamous and monogamous females also differed significantly in the relationship between body mass and PO activity, suggesting that different allocation strategies evolved under the alternative mating regimes. Given are regression slopes, shaded 95% confidence limits, and individual observations. Males from the regimes did not express detectable levels of PO activity and showed no significant differences among regimes and mating treatments (Supplementary Table 1c). In the lower panels, sex differences in size-corrected PO activity is illustrated in each regime for (c) virgin and (d) reproducing beetles (means ± 1 SE and individual data points)

Fig. 4

Microevolutionary change in tolerance to bacterial infection during experimental evolution under alternative mating regimes. Response to bacterial infection was estimated by the change in mortality rate between individuals infected with two doses of bacteria and a sham control. When infected with the gram-positive bacteria B. thuringiensis, monogamous females (a, b) had significantly higher survival under infection compared with polygamous females (c, d), while virgin (a, c) and mated (b, d) females had similar responses. Shown are survival curves for each replicate evolution line (thin lines) together with mean survival (thick line) and 95% confidence limits (shaded area) based on all three replicate lines per regime and mating treatment. Virgin males (triangles) from monogamous (e) and polygamous (f) regimes did not show the strong differences seen in virgin females (circles), resulting in an apparent increase in sexual dimorphism in response to infection in the polygamy regime (compare panels e and f) (means ±1SE; lower dose = 1.0 OD, higher dose = 2.0 OD for females and 2.5 OD for males). When mated females were infected with the gram-negative bacteria, P. entomophila, which allowed assaying of in vivo bacterial counts in infected individuals, monogamous lines (g) again showed higher survival under infection compared with polygamous lines (h) (lower dose = 0.5 OD, higher dose = 1.0 OD). (i) Counts of bacterial loads in females 12 h post infection showed that difference in survival were likely not due to more efficient clearance of bacteria in monogamous lines. Means ± 1 SE per replicate line (two lines used per regime and dose) and individual data points per assay

Fig. 5

Phylogenetic covariance between harmfulness of male genital morphology and PO activity in virgin male and female seed beetles. a Female PO activity and the harmfulness of male genitalia mapped on the phylogeny of the 12 species used. Scores are given by colour from blue (high harm/PO) to red (low harm/PO). Lower panels show correlations across species between male harmfulness and male (blue open) and female (red closed) PO activity, shown as b raw tip data and c phylogenetic independent contrasts (PICs). Standard errors around each species’ mean were typically of the magnitude ~ 0.02 for male and female PO activity, and ~ 0.6 for male genital morphology. The y-axes of b and c are scaled to have the same range. Species codes represent robi = Amblycerus robinae; subf = Zabrotes subfasciatus; obte = Acanthoscelides obtectus; atro = Bruchidius atrolineatus; dich = Bruchidius dichrostachydis; tonk = Megabruchidius tonkineus; dors = Megabruchidius dorsalis; phas = Callosobruchus phaseoli; chin = Callosobruchus chinensis; subi = Callosobruchus subinnotatus; macu = Callosobruchus maculatus; anal = Callosobruchus analis