Effects of an entomopathogenic fungus on the reproductive potential of Drosophila males.

While mortality is often the primary focus of pathogen virulence, non-lethal consequences, particularly for male reproductive fitness, are less understood; however, they are essential for understanding how sexual selection contributes to promoting resistance. We investigated how the fungal pathogen Metarhizium brunneum affects mating ability, fertility, and seminal fluid protein (SFP) expression of male Drosophila melanogaster paired with highly receptive virgin females in non-competitive settings. Depending on sex and dose, there was a 3-6-day incubation period after infection, followed by an abrupt onset of mortality. Meanwhile, the immune response was strongly induced already 38 h after infection and continued to increase as infection progressed. Latency to mate somewhat increased during the incubation period compared to sham-treated males, but even on Day 5 post infection >90% of infected males mated within 2 h. During the incubation period, M. brunneum infection reduced male reproductive potential (the number of offspring sired without mate limitation) by 11%, with no clear increase over time. Approaching the end of the incubation period, infected males had lower ability to convert number of mating opportunities into number of offspring. After repeated mating, infected males had lower SFP expression than sham controls, more so in males that mated with few mates 24 h earlier. Overall, despite strong activation of the immune response, males' mating ability and fertility remained surprisingly little affected by the fungal infection, even shortly before the onset of mortality. This suggests that the selection for resistance acts mainly through mortality, and the scope for fertility selection to enhance resistance in non-competing settings is rather limited.

Cis-regulatory polymorphism at fiz ecdysone oxidase contributes to polygenic evolutionary response to malnutrition in Drosophila.

We investigate the contribution of a candidate gene, fiz (fezzik), to complex polygenic adaptation to juvenile malnutrition in Drosophila melanogaster. Experimental populations maintained for >250 generations of experimental evolution to a nutritionally poor larval diet (Selected populations) evolved several-fold lower fiz expression compared to unselected Control populations. Here we show that this divergence in fiz expression is mediated by a cis-regulatory polymorphism. This polymorphism, originally sampled from a natural population in Switzerland, is distinct from a second cis-regulatory SNP previously identified in non-African D. melanogaster populations, implying that two independent cis-regulatory variants promoting high fiz expression segregate in non-African populations. Enzymatic analyses of Fiz protein expressed in E. coli demonstrate that it has ecdysone oxidase activity acting on both ecdysone and 20-hydroxyecdysone. Four of five fiz paralogs annotated to ecdysteroid metabolism also show reduced expression in Selected larvae, implying that malnutrition-driven selection favored general downregulation of ecdysone oxidases. Finally, as an independent test of the role of fiz in poor diet adaptation, we show that fiz knockdown by RNAi results in faster larval growth on the poor diet, but at the cost of greatly reduced survival. These results imply that downregulation of fiz in Selected populations was favored by selection on the nutritionally poor diet because of its role in suppressing growth in response to nutrient shortage. However, they suggest that fiz downregulation is only adaptive in combination with other changes evolved by Selected populations, which ensure that the organism can sustain the faster growth promoted by fiz downregulation.

Evolutionary adaptation to juvenile malnutrition impacts adult metabolism and impairs adult fitness in Drosophila.

Juvenile undernutrition has lasting effects on adult metabolism of the affected individuals, but it is unclear how adult physiology is shaped over evolutionary time by natural selection driven by juvenile undernutrition. We combined RNAseq, targeted metabolomics, and genomics to study the consequences of evolution under juvenile undernutrition for metabolism of reproductively active adult females of Drosophila melanogaster. Compared to Control populations maintained on standard diet, Selected populations maintained for over 230 generations on a nutrient-poor larval diet evolved major changes in adult gene expression and metabolite abundance, in particular affecting amino acid and purine metabolism. The evolved differences in adult gene expression and metabolite abundance between Selected and Control populations were positively correlated with the corresponding differences previously reported for Selected versus Control larvae. This implies that genetic variants affect both stages similarly. Even when well fed, the metabolic profile of Selected flies resembled that of flies subject to starvation. Finally, Selected flies had lower reproductive output than Controls even when both were raised under the conditions under which the Selected populations evolved. These results imply that evolutionary adaptation to juvenile undernutrition has large pleiotropic consequences for adult metabolism, and that they are costly rather than adaptive for adult fitness. Thus, juvenile and adult metabolism do not appear to evolve independently from each other even in a holometabolous species where the two life stages are separated by a complete metamorphosis.

Experimental evolution of metabolism under nutrient restriction: enhanced amino acid catabolism and a key role of branched-chain amino acids.

Periodic food shortage is a common ecological stressor for animals, likely to drive physiological and metabolic adaptations to alleviate its consequences, particularly for juveniles that have no option but to continue to grow and develop despite undernutrition. Here we study changes in metabolism associated with adaptation to nutrient shortage, evolved by replicate Drosophila melanogaster populations maintained on a nutrient-poor larval diet for over 240 generations. In a factorial metabolomics experiment we showed that both phenotypic plasticity and genetically-based adaptation to the poor diet involved wide-ranging changes in metabolite abundance; however, the plastic response did not predict the evolutionary change. Compared to nonadapted larvae exposed to the poor diet for the first time, the adapted larvae showed lower levels of multiple free amino acids in their tissues-and yet they grew faster. By quantifying accumulation of the nitrogen stable isotope 15N we show that adaptation to the poor diet led to an increased use of amino acids for energy generation. This apparent "waste" of scarce amino acids likely results from the trade-off between acquisition of dietary amino acids and carbohydrates observed in these populations. The three branched-chain amino acids (leucine, isoleucine, and valine) showed a unique pattern of depletion in adapted larvae raised on the poor diet. A diet supplementation experiment demonstrated that these amino acids are limiting for growth on the poor diet, suggesting that their low levels resulted from their expeditious use for protein synthesis. These results demonstrate that selection driven by nutrient shortage not only promotes improved acquisition of limiting nutrients, but also has wide-ranging effects on how the nutrients are used. They also show that the abundance of free amino acids in the tissues does not, in general, reflect the nutritional condition and growth potential of an animal.

Impact of Temperature on the Immune Interaction between a Parasitoid Wasp and Drosophila Host Species.

Temperature is particularly important for ectotherms, including endoparasitoid wasps that develop inside another ectotherm host. In this study, we tested the impact of three temperatures (20 °C, 25 °C and 30 °C) on the host-parasitoid immune interaction using two Drosophila host species (Drosophila melanogaster and D. yakuba) and two parasitoid lines of Leptopilina boulardi. Drosophila's immune defense against parasitoids consists of the formation of a melanized capsule surrounding the parasitoid egg. To counteract this response, Leptopilina parasitoids rely on the injection of venom during oviposition. Here, we tested the effect of temperature on parasitic success and host encapsulation capacity in response to a parasitoid egg or other foreign body. Increased temperature either promoted or did not affect the parasitic success, depending on the parasitoid-host pairs considered. The mechanisms behind the higher success seemed to vary depending on whether the temperature primarily affected the host immune response or also affected the parasitoid counter-immune response. Next, we tested the effect of parasitoid rearing temperature on its success and venom composition. Venom composition varied strongly with temperature for both parasitoid lines, partially consistent with a change in their parasitic success. Overall, temperature may have a significant impact on the host-parasitoid immune interaction.

Experimental evolution of post-ingestive nutritional compensation in response to a nutrient-poor diet.

The geometric framework of nutrition predicts that populations restricted to a single imbalanced diet should evolve post-ingestive nutritional compensation mechanisms bringing the blend of assimilated nutrients closer to physiological optimum. The evolution of such nutritional compensation is thought to be mainly driven by the ratios of major nutrients rather than overall nutritional content of the diet. We report experimental evolution of divergence in post-ingestive nutritional compensation in populations of Drosophila melanogaster adapted to diets that contained identical imbalanced nutrient ratios but differed in total nutrient concentration. Larvae from 'Selected' populations maintained for over 200 generations on a nutrient-poor diet with a 1 : 13.5 protein : carbohydrate ratio showed enhanced assimilation of nitrogen from yeasts and reduced assimilation of carbon from sucrose than 'Control' populations evolved on a diet with the same nutrient ratio but fourfold greater nutrient concentration. Compared to the Controls, the Selected larvae also accumulated less triglycerides relative to protein. This implies that the Selected populations evolved a higher assimilation rate of amino acids from the poor imbalanced diet and a lower assimilation of carbohydrates than Controls. Thus, the evolution of nutritional compensation may be driven by changes in total nutrient abundance, even if the ratios of different nutrients remain unchanged.

Rapid and Differential Evolution of the Venom Composition of a Parasitoid Wasp Depending on the Host Strain.

Parasitoid wasps rely primarily on venom to suppress the immune response and regulate the physiology of their host. Intraspecific variability of venom protein composition has been documented in some species, but its evolutionary potential is poorly understood. We performed an experimental evolution initiated with the crosses of two lines of Leptopilinaboulardi of different venom composition to generate variability and create new combinations of venom factors. The offspring were maintained for 10 generations on two strains of Drosophila melanogaster differing in resistance/susceptibility to the parental parasitoid lines. The venom composition of individuals was characterized by a semi-automatic analysis of 1D SDS-PAGE electrophoresis protein profiles whose accuracy was checked by Western blot analysis of well-characterized venom proteins. Results made evident a rapid and differential evolution of the venom composition on both hosts and showed that the proteins beneficial on one host can be costly on the other. Overall, we demonstrated the capacity of rapid evolution of the venom composition in parasitoid wasps, important regulators of arthropod populations, suggesting a potential for adaptation to new hosts. Our approach also proved relevant in identifying, among the diversity of venom proteins, those possibly involved in parasitism success and whose role deserves to be deepened.

No clear effect of admixture between two European invading outbreaks of Diabrotica virgifera virgifera in natura.

In this study, we challenged the hypothesis that admixture may have had a positive impact in the context of the European invasion of the western corn rootworm (WCR), Diabrotica virgifera virgifera, LeConte. This beetle was introduced in Europe from the USA several times since the 1980's. The multiple introductions of this major pest of cultivated corn led to the formation of two major outbreaks in North Western (NW) Italy and in Central and South Eastern (CSE) Europe that eventually merged into a secondary contact zone where insects from both outbreaks interbreed. We collected about 600 insects from this contact zone and genotyped them using 13 microsatellite markers. Three types of information were obtained from the collected individuals: (i) their survival under starvation; (ii) their admixed status, determined through a Bayesian method of genetic clustering and (iii) their mating probability, studied via the detection, isolation and genotyping of sperm in female spermathecae. Twenty six % and 12% of the individuals were assigned to the NW Italy or the CSE Europe parental types, respectively, and 23% and 39% to the F1 and backcross hybrid types, respectively. Globally, our results do not reveal any significant impact of the admixed status on the mating probability and on the choice of mating partners. However the admixed status had a sex- and sampling site-dependent effect on survival in adults under starvation. In addition sex had an effect on survival, with mortality hazard about 3 times larger in males than in females. The consequences of these findings for the evolution of the admixture zone of northern Italy are discussed.