Kenneth M. Fedorka

Post-mating disparity between potential and realized immune response in Drosophila melanogaster

Reproductive costs are an essential component of evolutionary theory. For instance, an increase in reproduction is generally coupled with a decrease in immunocompetence shortly after mating. However, recent work in Drosophila melanogaster suggests that the potential to mount an immune response, as measured by the levels of immune gene expression, increases after mating. These data are in contrast to previous studies, which suggest that mating can reduce a flys ability to survive an actual bacterial challenge (realized immunity). This pattern may be driven by some aspect of mating, independent of resource limitation, which reduces immune function by inhibiting the effective deployment of immune gene products. Though several studies have examined both the potential and the realized immunity after mating, none have examined these immune measures simultaneously. Here, we examined the link between the potential and the realized immunity in a sterile mutant of D. melanogaster. Shortly after mating, we found that female immune gene expression was high, but survival against infection was low. Surprisingly, this pattern was reversed within 24 h. Thus, estimates of immunity based on gene expression do not appear to reflect an actual ability to defend against pathogens in the hours following copulation. We discuss the possible mechanisms that may account for this pattern.

Perceived sperm competition intensity influences seminal fluid protein production prior to courtship and mating.

Sperm competition is a potent postcopulatory selective force where sperm from rival males compete to fertilize a limited set of ova. Considering that sperm production is costly, we expect males to strategically allocate sperm in accordance with the level of competition. Accordingly, previous work has examined a males strategic allocation in terms of sperm number. However, the seminal fluid proteins (Sfps) transferred along with sperm may also play a crucial role in competition. Surprisingly, the strategic allocation of Sfps has remained largely unexplored. Using Drosophila melanogaster, we examined the expression of three seminal fluid and four spermatogenesis genes in response to perceived sperm competition intensity by manipulating male density in a pre‐mating and courtship environment. In the pre‐mating environment, we found that males modified Sfp ratios by reducing the production of two spfs when potential rivals were present, while one Sfp and all spermatogenesis genes remained unaltered. In the courtship environment, males did not modify spermatogenesis or Sfp production in response to either rival males or female presence. Our data suggest that perceived competition in the pre‐mating environment places a significant influence on Sfp allocation, which may be a general trend in promiscuous animal systems with internal fertilization.

Thermal environment shapes cuticle melanism and melanin-based immunity in the ground cricket Allonemobius socius

The thermal melanin hypothesis posits that ectothermic individuals of larger size or from colder environments exhibit darker cuticles due to melanin’s efficacy in absorbing solar radiation. However, melanin is also a crucial component of arthropod immunity. Thus, thermal selection for increased cuticle darkness may profoundly influence melanin-based immune function. In this study, we address the relationships between the thermal environment (season length), cuticular melanism and two aspects of melanin-based immunity across nine thermally distinct populations of the cricket Allonemobiussocius. We found that season length (i.e. degree days) and body size had a positive association with cuticle melanism in both sexes across populations, supporting the thermal melanism hypothesis. Despite their smaller size, males were found to have darker cuticles and superior melanin-based immunity. This pattern may be the result of additional selection on males due to sex-specific temperature-dependent activities, such as male calling song. Perhaps most interestingly, we found that short season length populations (i.e. colder) exhibited a greater phenoloxidase activity (aspect of the melanin-based immune system) in addition to darker cuticles in both sexes. This pattern is consistent with direct thermal selection on cuticular color, coupled with indirect selection on melanin-based immunity due to pleiotropy. Thus, thermal selection on cuticle darkness appears to indirectly shape the evolution of pathogen resistance in this system, and potentially for other terrestrial arthropod systems whose ranges encompass a significant thermal gradient.

Seasonality influences cuticle melanization and immune defense in a cricket: support for a temperature-dependent immune investment hypothesis in insects

SUMMARY To improve thermoregulation in colder environments, insects are expected to darken their cuticles with melanin via the phenoloxidase cascade, a phenomenon predicted by the thermal melanin hypothesis. However, the phenoloxidase cascade also plays a significant role in insect immunity, leading to the additional hypothesis that the thermal environment indirectly shapes immune function via direct selection on cuticle color. Support for the latter hypothesis comes from the cricket Allonemobius socius, where cuticle darkness and immune-related phenoloxidase activity increase with latitude. However, thermal environments vary seasonally as well as geographically, suggesting that seasonal plasticity in immunity may also exist. Although seasonal fluctuations in vertebrate immune function are common (because of flux in breeding or resource abundance), seasonality in invertebrate immunity has not been widely explored. We addressed this possibility by rearing crickets in simulated summer and fall environments and assayed their cuticle color and immune function. Prior to estimating immunity, crickets were placed in a common environment to minimize metabolic rate differences. Individuals reared under fall-like conditions exhibited darker cuticles, greater phenoloxidase activity and greater resistance to the bacteria Serratia marcescens. These data support the hypothesis that changes in the thermal environment modify cuticle color, which indirectly shapes immune investment through pleiotropy. This hypothesis may represent a widespread mechanism governing immunity in numerous systems, considering that most insects operate in seasonally and geographically variable thermal environments.

Immune activation decreases sperm viability in both sexes and influences female sperm storage

All animals are under the constant threat of pathogenic infection. However, little is known regarding the influence of acute infection on sperm viability, particularly in female insects. This information is crucial for our understanding of mating and immune system coevolution, considering that females store sperm and serve as the site of sperm competition. Using the fruitfly, Drosophila melanogaster, we examined the influence of infection on sperm viability and storage. Twenty-four hours after haemocoel inoculation with a pathogen mimic (peptidoglycan, PGN) both sexes exhibited reduced sperm viability, indicating that systemic immune activation played a significant role in gamete survival. Surprisingly, sperm death did not appear to result from a reproductive-immune system trade-off, considering that sperm survived 24 h in vitro once removed from their somatic resources. Instead, our results are most consistent with death owing to immune effector collateral damage. We also examined the potential for sexually transmitted pathogens to influence sperm storage. Females mated with ‘infected’ males (created by dipping genitalia into a PGN solution) exhibited a higher proportion of empty sperm stores 48 h after mating compared to their controls. Remarkably, these data indicate that females may increase their fitness by removing ‘infected’ ejaculates from storage over time.

The influence of male age and simulated pathogenic infection on producing a dishonest sexual signal.

In recent years, studies have shown that reproductive effort decelerates in response to pathogenic infection. If infection substantially reduces a hosts residual reproductive value (RRV), however, then an acceleration of effort may instead occur (e.g. terminal investment). Reproductive acceleration would theoretically allow hosts to maintain or exaggerate their sexual signal upon infection. This would create a deceptive message from the perspective of the chooser, who may unwittingly copulate with an infected mate to their detriment. Using the cricket Allonemobius socius, we assessed the potential for reduced RRV to accelerate male reproductive effort and create a dishonest signal. RRV was manipulated through male age and simulated pathogenic insult. Reproductive effort was measured as calling song energetics, mating success, latency to mate and nuptial gift size. We show that males adopted either an accelerated or decelerated reproductive strategy upon infection, and that this decision was probably mediated by RRV. Moreover, males who accelerated their effort produced a dishonest signal by increasing their song energetics while providing fewer paternal resources (i.e. smaller gifts). Our study is one of the few to document the existence of dishonest signals and relate dishonesty to a potential reduction in female fitness, underscoring the conflict inherent in sexual reproduction.

Sex-specific variation in the emphasis, inducibility and timing of the post-mating immune response in Drosophila melanogaster

Ecological immunology attempts to explain variation in immune function. Much of this work makes predictions about how potential hosts should invest in overall immunity. However, this ‘overall’ perspective under-emphasizes other critical aspects, such as the specificity, inducibility and timing of an immune response. Here, we investigate these aspects by examining gene regulation across several immune system components in both male and female Drosophila melanogaster prior to and after mating. To elucidate potentially important temporal dynamics, we also assayed several genes over time. We found that males and females emphasized different components of their immune system, however overall investment was similar. Specifically, the sexes emphasized different gene paralogues within major gene families, and males tended to invest more in gram-negative defence. By contrast, the inducibility of the immune response was both transient (lasting approx. 24 hours) and equal between the sexes. Furthermore, mating tended to induce humoral gene upregulation, while cell-mediated genes were unaffected. Within the humoral system, gram-negative bacterial defence genes exhibited a greater inducibility than those associated with fungal or gram-positive bacterial defence. Our results suggest that variation in the effectiveness of the immune response between the sexes may be driven by differences in emphasis rather than overall investment.

Thermoregulatory strategy may shape immune investment in Drosophila melanogaster

As temperatures change, insects alter the amount of melanin in their cuticle to improve thermoregulation. However, melanin is also central to insect immunity, suggesting that thermoregulatory strategy may indirectly impact immune defense by altering the abundance of melanin pathway components (a hypothesis we refer to as thermoregulatory-dependent immune investment). This may be the case in the cricket Allonemobius socius, where warm environments (both seasonal and geographical) produced crickets with lighter cuticles and increased pathogen susceptibility. Unfortunately, the potential for thermoregulatory strategy to influence insect immunity has not been widely explored. Here we address the relationships between temperature, thermoregulatory strategy and immunity in the fruit fly Drosophila melanogaster. To this end, flies from two separate Canadian populations were reared in either a summer- or autumn-like environment. Shortly after adult eclosion, flies were moved to a common environment where their cuticle color and susceptibility to a bacterial pathogen (Pseudomonas aeruginosa) were measured. As with A. socius, individuals from summer-like environments exhibited lighter cuticles and increased pathogen susceptibility, suggesting that the thermoregulatory–immunity relationship is evolutionarily conserved across the hemimetabolous and holometabolous clades. If global temperatures continue to rise as expected, then thermoregulation might play an important role in host infection and mortality rates in systems that provide critical ecosystem services (e.g. pollination), or influence the prevalence of insect-vectored disease (e.g. malaria).

Male Mate Choice for Unparasitized Females in Drosophila melanogaster

Parasite-mediated sexual selection theory suggests that females may choose male partners who possess a relatively low pathogen load in order to maximize their reproductive investment (e.g. by avoiding pathogens or producing pathogen-resistant offspring). However, males might also maximize their reproductive investment by preferring unparasitized females; even in non-sex-role-reversed systems where female choice predominates. We address this hypothesis in the fruit fly Drosophila melanogaster where female choice plays a major selective role. Females in this system exhibit reduced fecundity when parasitized, creating the opportunity for simultaneous parasite-mediated male choice. To address our hypothesis, virgin females were either prick-injected with a sub-lethal dose of the gram-negative bacteria Pseudomonas aeuginosa (pricked-parasitized group, PP), prick-injected with sterile media (pricked-unparasitized group, PU) or unpricked and unparasitized (UU group). Twenty-four hours post-infection, females were decapitated to minimize the confounding effects of female behavior on male preference (decapitated females will stand upright, but lack reproductive behavior). Males were then assigned to one of two different two-choice trials (experiment A: PU versus UU females or experiment B: PP versus UU females) and their courtship behavior monitored. In experiment A, males exhibited no courtship preference for either female. However, experiment B males preferentially courted UU females more often than PP females. These results suggest that males prefer unparasitized mates, which could play a significant role in shaping disease resistance in this system. These data also provide some of the first evidence that males prefer unparasitized females in systems that do not exhibit sex-role reversal.

Y-linked variation for autosomal immune gene regulation has the potential to shape sexually dimorphic immunity.

Sexually dimorphic phenotypes arise from the differential expression of male and female shared genes throughout the genome. Unfortunately, the underlying molecular mechanisms by which dimorphic regulation manifests and evolves are unclear. Recent work suggests that Y-chromosomes may play an important role, given that Drosophila melanogaster Ys were shown to influence the regulation of hundreds of X and autosomal genes. For Y-linked regulatory variation (YRV) to facilitate sexually dimorphic evolution, however, it must exist within populations (where selection operates) and influence male fitness. These criteria have seldom been investigated, leaving the potential for dimorphic evolution via YRV unclear. Interestingly, male and female D. melanogaster differ in immune gene regulation. Furthermore, immune gene regulation appears to be influenced by the Y-chromosome, suggesting it may contribute to dimorphic immune evolution. We address this possibility by introgressing Y-chromosomes from a single wild population into an isogenic background (to create Y-lines) and assessing immune gene regulation and bacterial defence. We found that Y-line males differed in their immune gene regulation and their ability to defend against Serratia marcescens. Moreover, gene expression and bacterial defence were positively genetically correlated. These data indicate that the Y-chromosome has the potential to shape the evolution of sexually dimorphic immunity in this system.