Ancestral environmental conditions can impact descendant phenotypes through a variety of epigenetic mechanisms. Previous studies on transgenerational effects in Drosophila melanogaster suggest that parental nutrition may affect the body size, developmental duration and egg size of the next generation. However, it is unknown whether these effects on phenotype remain stable across generations, or if specific generations have general responses to ancestral diet. In the current study, we examined the effect on multiple life-history phenotypes of changing diet quality across three generations. Our analysis revealed unforeseen patterns in how phenotypes respond to dietary restriction. Our generalized linear model showed that when considering only two generations, offspring phenotypes were primarily affected by their own diet, and to a lesser extent by the diet of their parents or the interaction between the two generations. Surprisingly, however, when considering three generations, offspring phenotypes were primarily impacted by their grandparents' diet and their own diet. Interactions among different generations’ diets affected development time, egg volume and pupal mass more than ovariole number or wing length. Furthermore, pairwise comparisons of diet groups from the same generation revealed commonalities in strong responses to rich versus poor diet: ovariole number, pupal mass and wing length responded more strongly to poor diet than to rich diet, while development time responded strongly to both rich and poor diets. To improve investigations into the mechanisms and consequences of transgenerational, epigenetic inheritance, future studies should closely examine how phenotypes change across a higher number of generations, and consider responses to broader variability in diet treatments.

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Eggs are an immobile, vulnerable stage of development and their success often depends on the oviposition decisions of the mother. Studies show that female animals, and sometimes males, may invest parental resources in order to increase the survival of their offspring. Here, we describe a unique form of parental investment in offspring survival. The seed beetle Mimosestes amicus may lay eggs singly, or may cover eggs with additional egg(s). This egg stacking serves to significantly reduce the mortality of the protected egg from parasitism by the parasitic wasp, Uscana semifumipennis. The smaller top eggs serve only as protective shields; they are inviable, and wasps that develop in them suffer negative fitness consequences. Further, we found egg stacking to be inducible; M. amicus increase the number of stacks they lay when parasitoids are present. However, stacking invokes a cost. When wasps are absent, beetles lay more single eggs, and produce more offspring, highlighting the adaptive value of this extraordinary example of behavioural plasticity in parental investment.

Most species pairs are isolated through the collective action of a suite of barriers. Recent work has shown that cryptic barriers such as conspecific sperm precedence can be quite strong, suggesting that they evolve quickly. However, because the strength of multiple barriers has been formally quantified in very few systems, the relative speed with which conspecific sperm precedence evolves remains unclear. Here, we measure the strength of both conspecific sperm precedence and cryptic non-competitive isolation between the hybridizing sister species, Chrysochus auratus and C. cobaltinus (Coleoptera: Chrysomelidae), and compare the strength of those barriers to the strength of other known reproductive barriers in this system. Overall, cryptic barriers in this system are weaker than other barriers, indicating that they have not evolved rapidly. Furthermore, their evolution has been asymmetric. Non-competitive barriers substantially reduce the production of hybrid offspring by C. auratus females but not by C. cobaltinus females. In multiply-mated C. cobaltinus females, heterospecific sperm outcompete conspecific sperm, as evidenced by the fact that heterospecific males sired disproportionately more offspring than predicted from the results for singly-mated females. In C. auratus females, neither sperm type has a competitive advantage. Such asymmetries explain why nearly all F1 hybrids in the field are from crosses between C. cobaltinus females and C. auratus males. We discuss these findings in terms of understanding the cost of mating 'mistakes' in the Chrysochus hybrid zone. In addition, our discovery that 95% confidence intervals for commonly-used isolation statistics can be very wide has important implications for speciation research. Specifically, to avoid biases in the interpretation of such isolation metrics, we suggest that studies should routinely include error estimates in their analyses of reproductive isolation.