The evolution of life span and aging in response to dietary macronutrients in male and female decorated crickets.

Dietary macronutrients regulate life span and aging, yet little is known about their evolutionary effects. Here, we examine the evolutionary response of these traits in decorated crickets (Gryllodes sigillatus) maintained on diets varying in caloric content and protein-to-carbohydrate ratio. After 37 generations, each population was split: half remained on the evolution diet, and half switched to a standardized diet. Crickets lived longer and aged slower when evolving on high-calorie (both sexes) and carbohydrate-biased (females only) diets and had lower baseline mortality on high-calorie (females only) diets. However, on the standardized diet, crickets lived longer when evolving on high-calorie diets (both sexes), aged slower on high-calorie (females only) and carbohydrate-biased (both sexes) diets, and had lower baseline mortality on high-calorie (males only) and protein-biased (both sexes) diets. Life span was longer, and baseline mortality was lower when provided with the evolution vs. the standardized diet, but the aging rate was comparable. Moreover, life span was longer, aging slower (females only), and baseline mortality was lower (males only) compared to our evolved baseline, suggesting varying degrees of dietary adaptation. Collectively, we show dietary components influence the evolution of life span and aging in different ways and highlight the value of combining experimental evolution with nutritional geometry.

Beauty or function? The opposing effects of natural and sexual selection on cuticular hydrocarbons in male black field crickets.

Although many theoretical models of male sexual trait evolution assume that sexual selection is countered by natural selection, direct empirical tests of this assumption are relatively uncommon. Cuticular hydrocarbons (CHCs) are known to play an important role not only in restricting evaporative water loss but also in sexual signalling in most terrestrial arthropods. Insects adjusting their CHC layer for optimal desiccation resistance is often thought to come at the expense of successful sexual attraction, suggesting that natural and sexual selection are in opposition for this trait. In this study, we sampled the CHCs of male black field crickets (Teleogryllus commodus) using solid-phase microextraction and then either measured their evaporative water loss or mating success. We then used multivariate selection analysis to quantify the strength and form of natural and sexual selection targeting male CHCs. Both natural and sexual selection imposed significant linear and stabilizing selection on male CHCs, although for very different combinations. Natural selection largely favoured an increase in the total abundance of CHCs, especially those with a longer chain length. In contrast, mating success peaked at a lower total abundance of CHCs and declined as CHC abundance increased. However, mating success did improve with an increase in a number of specific CHC components that also increased evaporative water loss. Importantly, this resulted in the combination of male CHCs favoured by natural selection and sexual selection being strongly opposing. Our findings suggest that the balance between natural and sexual selection is likely to play an important role in the evolution of male CHCs in T. commodus and may help explain why CHCs are so divergent across populations and species.

Transcriptomic responses of females to consumption of nuptial food gifts as a potential mediator of sexual conflict in decorated crickets.

Nuptial food gift provisioning by males to females at mating is a strategy in many insects that is thought to be shaped by sexual conflict or sexual selection, as it affords males access to a female's physiology. While males often attempt to use these gifts to influence female behaviour to their own advantage, females can evolve counter mechanisms. In decorated crickets, the male's nuptial gift comprises part of the spermatophore, the spermatophylax, the feeding on which deters the female from prematurely terminating sperm transfer. However, ingested compounds in the spermatophylax and attachment of the sperm-containing ampulla could further influence female physiology and behaviour. We investigated how mating per se and these two distinct routes of potential male-mediated manipulation influence the female transcriptomic response. We conducted an RNA sequencing experiment on gut and head tissues from females for whom nuptial food gift consumption and receipt of an ejaculation were independently manipulated. In the gut tissue, we found that females not permitted to feed during mating exhibited decreased overall gene expression, possibly caused by a reduced gut function, but this was countered by feeding on the spermatophylax or a sham gift. In the head tissue, we found only low numbers of differentially expressed genes, but a gene co-expression network analysis revealed that ampulla attachment and spermatophylax consumption independently induce distinct gene expression patterns. This study provides evidence that spermatophylax feeding alters the female post-mating transcriptomic response in decorated crickets, highlighting its potential to mediate sexual conflict in this system.

Ovarian apoptosis is regulated by carbohydrate intake but not by protein intake in speckled cockroaches.

When the likelihood of reproducing successfully is low, any prior investment in developing oocytes may be wasted. One means of recouping this investment is oosorption - where ova are absorbed and resources salvaged so they can be re-allocated to other traits. Food-limited female speckled cockroaches (Nauphoeta cinerea) appear to use this strategy. However, it is unclear if total food intake or the availability of specific nutrients induces this process. Here, we used the geometric framework of nutrition to determine how protein, carbohydrate and energy intake affect levels of ovarian apoptosis and necrosis (controlled versus uncontrolled cell death) in the terminal oocytes of female N. cinerea. We then compare the effects of nutrient intake on apoptosis (a key step towards oosorption) and offspring production to better understand the relationship between diet, apoptosis and female fitness. We found that even when food was abundant, females experienced high levels of apoptosis if their diet lacked carbohydrate. Necrosis was reduced when energy intake was high, but largely irrespective of nutrient ratio. Offspring production peaked on a low protein, high carbohydrate nutrient ratio (1P:7.96C), similar to that which minimized apoptosis (1P:7.34C) but not in the region of nutrient space that minimized necrosis. Thus, females consuming an ideal nutrient blend for reproduction can invest heavily in their current brood without needing to salvage nutrients from developing ova. However, offspring production was more dependent on carbohydrate consumption than apoptosis was, suggesting that the importance of carbohydrate in reproduction goes beyond regulating oosorption. This reliance on carbohydrate for female reproduction may reflect the unusual reproductive and nutritional physiology of speckled cockroaches; attributes that make this species an exciting model for understanding how diet regulates reproduction.

Evolution of immune function in response to dietary macronutrients in male and female decorated crickets.

Although dietary macronutrients are known to regulate insect immunity, few studies have examined their evolutionary effects. Here, we evaluate this relationship in the cricket Gryllodes sigillatus by maintaining replicate populations on four diets differing in protein (P) to carbohydrate (C) ratio (P- or C-biased) and nutritional content (low- or high-nutrition) for >37 generations. We split each population into two; one maintained on their evolution diet and the other switched to their ancestral diet. We also maintained populations exclusively on the ancestral diet (baseline). After three generations, we measured three immune parameters in males and females from each population. Immunity was higher on P-biased than C-biased diets and on low- versus high-nutrition diets, although the latter was most likely driven by compensatory feeding. These patterns persisted in populations switched to their ancestral diet, indicating genetic divergence. Crickets evolving on C-biased diets had lower immunity than the baseline, whereas their P-biased counterparts had similar or higher immunity than the baseline, indicating that populations evolved with dietary manipulation. Although females exhibited superior immunity for all assays, the sexes showed similar immune changes across diets. Our work highlights the important role that macronutrient intake plays in the evolution of immunity in the sexes.

Mapping sex differences in the effects of protein and carbohydrates on lifespan and reproduction in Drosophila melanogaster: is measuring nutrient intake essential?

Understanding how diet affects reproduction and survival is a central aim in evolutionary biology. Although this relationship is likely to differ between the sexes, we lack data relating diet to male reproductive traits. One exception to this general pattern is Drosophila melanogaster, where male dietary intake was quantified using the CApillary FEeder (CAFE) method. However, CAFE feeding reduces D. melanogaster survival and reproduction, so may distort diet-fitness outcomes. Here, we use the Geometric Framework of Nutrition to create nutrient landscapes that map sex-specific relationships between protein, carbohydrate, lifespan and reproduction in D. melanogaster. Rather than creating landscapes with consumption data, we map traits onto the nutrient composition of forty agar-based diets, generating broad coverage of nutrient space. We find that male and female lifespan was maximised on low protein, high carbohydrate blends (~ 1P:15.9C). This nutrient ratio also maximised male reproductive rates, but females required more protein to maximise daily fecundity (1P:1.22C). These results are consistent with CAFE assay outcomes. However, the approach employed here improved female fitness relative to CAFE assays, while effects of agar versus CAFE feeding on male fitness traits depended on the nutrient composition of experimental diets. We suggest that informative nutrient landscapes can be made without measuring individual nutrient intake and that in many cases, this may be preferable to using the CAFE approach. The most appropriate method will depend on the question and species being studied, but the approach adopted here has the advantage of creating nutritional landscapes when dietary intake is hard to quantify.

Macronutrient intake and simulated infection threat independently affect life history traits of male decorated crickets.

Nutritional geometry has advanced our understanding of how macronutrients (e.g., proteins and carbohydrates) influence the expression of life history traits and their corresponding trade-offs. For example, recent work has revealed that reproduction and immune function in male decorated crickets are optimized at very different protein:carbohydrate (P:C) dietary ratios. However, it is unclear how an individual's macronutrient intake interacts with its perceived infection status to determine investment in reproduction or other key life history traits. Here, we employed a fully factorial design in which calling effort and immune function were quantified for male crickets fed either diets previously demonstrated to maximize calling effort (P:C = 1:8) or immune function (P:C = 5:1), and then administered a treatment from a spectrum of increasing infection cue intensity using heat-killed bacteria. Both diet and a simulated infection threat independently influenced the survival, immunity, and reproductive effort of males. If they called, males increased calling effort at the low infection cue dose, consistent with the terminal investment hypothesis, but interpretation of responses at the higher threat levels was hampered by the differential mortality of males across infection cue and diet treatments. A high protein, low carbohydrate diet severely reduced the health, survival, and overall fitness of male crickets. There was, however, no evidence of an interaction between diet and infection cue dose on calling effort, suggesting that the threshold for terminal investment was not contingent on diet as investigated here.

Multivariate stabilizing sexual selection and the evolution of male and female genital morphology in the red flour beetle.

Male genitals are highly divergent in animals with internal fertilization. Most studies attempting to explain this diversity have focused on testing the major hypotheses of genital evolution (the lock-and-key, pleiotropy, and sexual selection hypotheses), and quantifying the form of selection targeting male genitals has played an important role in this endeavor. However, we currently know far less about selection targeting female genitals or how male and female genitals interact during mating. Here, we use formal selection analysis to show that genital size and shape is subject to strong multivariate stabilizing sexual selection in both sexes of the red flour beetle, Tribolium castaneum. Moreover, we show significant sexual selection on the covariance between the sexes for specific aspects of genital shape suggesting that male and female genitalia also interact to determine the successful transfer of a spermatophore during mating. Our work therefore highlights the important role that both male and female genital morphologies play in determining mating success and that these effects can occur independently, as well as through their interaction. Moreover, it cautions against the overly simplistic view that the sexual selection targeting genital morphology will always be directional in form and restricted primarily to males.

Interactions Between Mitochondrial Haplotype and Dietary Macronutrient Ratios Confer Sex-Specific Effects on Longevity in Drosophila melanogaster.

Recent studies have demonstrated that modifications to the ratio of dietary macronutrients affect longevity in a diverse range of species. However, the degree to which levels of natural genotypic variation shape these dietary effects on longevity remains unclear. The mitochondria have long been linked to the aging process. The mitochondria possess their own genome, and previous studies have shown that mitochondrial genetic variation affects longevity in insects. Furthermore, the mitochondria are the sites in which dietary nutrients are oxidized to produce adenosine triphosphate, suggesting a capacity for dietary quality to mediate the link between mitochondrial genotype and longevity. Here, we measured longevity of male and female fruit flies, across a panel of genetic strains of Drosophila melanogaster, which vary only in their mitochondrial haplotype, when fed one of the two isocaloric diets that differed in their protein-to-carbohydrate ratio. The mitochondrial haplotype affected the longevity of flies, but the pattern of these effects differed across the two diets in males, but not in females. We discuss the implications of these results in relation to an evolutionary theory linking maternal inheritance of mitochondria to the accumulation of male-harming mitochondrial mutations, and to the theory exploring the evolution of phenotypic plasticity to novel environments.

Genotype-by-sex-by-diet interactions for nutritional preference, dietary consumption, and lipid deposition in a field cricket.

Changes in feeding behaviour, especially the overconsumption of calories, has led to a rise in the rates of obesity, diabetes, and other associated disorders in humans and a range of animals inhabiting human-influenced environments. However, understanding the relative contribution of genes, the nutritional environment, and their interaction to dietary intake and lipid deposition in the sexes still remains a major challenge. By combining nutritional geometry with quantitative genetics, we determined the effect of genes, the nutritional environment, and their interaction on the total nutritional preference (TP), total diet eaten (TE), and lipid mass (LM) of male and female black field crickets (Teleogryllus commodus) fed one of four diet pairs (DPs) differing in the ratio of protein to carbohydrate and total nutritional content. We found abundant additive genetic variance for TP, TE, and LM in both sexes and across all four DPs, with significant genetic correlations between TE and TP and between TP and LM in males. We also found significant genotype-by-DP and genotype-by-sex-by-DP interactions for each trait and significant genotype-by-sex interactions for TE and LM. Complex interactions between genes, sex, and the nutritional environment, therefore, play an important role in nutrient regulation and lipid deposition in T. commodus. This finding may also help explain the increasing rate of obesity and the maintenance of sex differences in obesity observed across many animal species, including humans.

The Geometry of Nutrient Space-Based Life-History Trade-Offs: Sex-Specific Effects of Macronutrient Intake on the Trade-Off between Encapsulation Ability and Reproductive Effort in Decorated Crickets.

Life-history theory assumes that traits compete for limited resources, resulting in trade-offs. The most commonly manipulated resource in empirical studies is the quantity or quality of diet. Recent studies using the geometric framework for nutrition, however, suggest that trade-offs are often regulated by the intake of specific nutrients, but a formal approach to identify and quantify the strength of such trade-offs is lacking. We posit that trade-offs occur whenever life-history traits are maximized in different regions of nutrient space, as evidenced by nonoverlapping 95% confidence regions of the global maximum for each trait and large angles (θ) between linear nutritional vectors and Euclidean distances (d) between global maxima. We then examined the effects of protein and carbohydrate intake on the trade-off between reproduction and aspects of immune function in male and female Gryllodes sigillatus. Female encapsulation ability and egg production increased with the intake of both nutrients, whereas male encapsulation ability increased with protein intake but calling effort increased with carbohydrate intake. The trade-offs between traits was therefore larger in males than in females, as demonstrated by significant negative correlations between the traits in males, nonoverlapping 95% confidence regions, and larger estimates of θ and d. Under dietary choice, the sexes had similar regulated intakes, but neither optimally regulated nutrient intake for maximal trait expression. We highlight the fact that greater consideration of specific nutrient intake is needed when examining nutrient space-based trade-offs.

Age-dependent variation in the terminal investment threshold in male crickets.

The terminal investment hypothesis proposes that decreased expectation of future reproduction (e.g., arising from a threat to survival) should precipitate increased investment in current reproduction. The level at which a cue of decreased survival is sufficient to trigger terminal investment (i.e., the terminal investment threshold) may vary according to other factors that influence expectation for future reproduction. We test whether the terminal investment threshold varies with age in male crickets, using heat-killed bacteria to simulate an immune-inducing infection. We measured calling effort (a behavior essential for mating) and hemolymph antimicrobial activity in young and old males across a gradient of increasing infection cue intensity. There was a significant interaction between the infection cue and age in their effect on calling effort, confirming the existence of a dynamic terminal investment threshold: young males reduced effort at all infection levels, whereas old males increased effort at the highest levels relative to naïve individuals. A lack of a corresponding decrease in antibacterial activity suggests that altered reproductive effort is not traded against investment in this component of immunity. Collectively, these results support the existence of a dynamic terminal investment threshold, perhaps accounting for some of the conflicting evidence in support of terminal investment.

Little evidence for intralocus sexual conflict over the optimal intake of nutrients for life span and reproduction in the black field cricket Teleogryllus commodus.

There is often large divergence in the effects of key nutrients on life span (LS) and reproduction in the sexes, yet nutrient intake is regulated in the same way in males and females given dietary choice. This suggests that the sexes are constrained from feeding to their sex-specific nutritional optima for these traits. Here, we examine the potential for intralocus sexual conflict (IASC) over optimal protein and carbohydrate intake for LS and reproduction to constrain the evolution of sex-specific nutrient regulation in the field cricket, Teleogryllus commodus. We show clear sex differences in the effects of protein and carbohydrate intake on LS and reproduction and strong positive genetic correlations between the sexes for the regulated intake of these nutrients. However, the between-sex additive genetic covariance matrix had very little effect on the predicted evolutionary response of nutrient regulation in the sexes. Thus, IASC appears unlikely to act as an evolutionary constraint on sex-specific nutrient regulation in T. commodus. This finding is supported by clear sexual dimorphism in the regulated intake of these nutrients under dietary choice. However, nutrient regulation did not coincide with the nutritional optima for LS or reproduction in either sex, suggesting that IASC is not completely resolved in T. commodus.

Dietary choice for a balanced nutrient intake increases the mean and reduces the variance in the reproductive performance of male and female cockroaches.

Sexual selection may cause dietary requirements for reproduction to diverge across the sexes and promote the evolution of different foraging strategies in males and females. However, our understanding of how the sexes regulate their nutrition and the effects that this has on sex-specific fitness is limited. We quantified how protein (P) and carbohydrate (C) intakes affect reproductive traits in male (pheromone expression) and female (clutch size and gestation time) cockroaches (Nauphoeta cinerea). We then determined how the sexes regulate their intake of nutrients when restricted to a single diet and when given dietary choice and how this affected expression of these important reproductive traits. Pheromone levels that improve male attractiveness, female clutch size and gestation time all peaked at a high daily intake of P:C in a 1:8 ratio. This is surprising because female insects typically require more P than males to maximize reproduction. The relatively low P requirement of females may reflect the action of cockroach endosymbionts that help recycle stored nitrogen for protein synthesis. When constrained to a single diet, both sexes prioritized regulating their daily intake of P over C, although this prioritization was stronger in females than males. When given the choice between diets, both sexes actively regulated their intake of nutrients at a 1:4.8 P:C ratio. The P:C ratio did not overlap exactly with the intake of nutrients that optimized reproductive trait expression. Despite this, cockroaches of both sexes that were given dietary choice generally improved the mean and reduced the variance in all reproductive traits we measured relative to animals fed a single diet from the diet choice pair. This pattern was not as strong when compared to the single best diet in our geometric array, suggesting that the relationship between nutrient balancing and reproduction is complex in this species.

Protein and carbohydrate intake influence sperm number and fertility in male cockroaches, but not sperm viability.

It is commonly assumed that because males produce many, tiny sperm, they are cheap to produce. Recent work, however, suggests that sperm production is not cost-free. If sperm are costly to produce, sperm number and/or viability should be influenced by diet, and this has been documented in numerous species. Yet few studies have examined the exact nutrients responsible for mediating these effects. Here, we quantify the effects of protein (P) and carbohydrate (C) intake on sperm number and viability in the cockroach Nauphoeta cinerea, as well as the consequences for male fertility. We found the intake of P and C influenced sperm number, being maximized at a high intake of diets with a P : C ratio of 1 : 2, but not sperm viability. The nutritional landscapes for male fertility and sperm number were closely aligned, suggesting that sperm number is the major determinant of male fertility in N. cinerea. Under dietary choice, males regulate nutrient intake at a P : C ratio of 1 : 4.95, which is midway between the ratios needed to maximize sperm production and pre-copulatory attractiveness in this species. This raises the possibility that males regulate nutrient intake to balance the trade-off between pre- and post-copulatory traits in this species.

Testing the Effects of DL-Alpha-Tocopherol Supplementation on Oxidative Damage, Total Antioxidant Protection and the Sex-Specific Responses of Reproductive Effort and Lifespan to Dietary Manipulation in Australian Field Crickets (Teleogryllus commodus).

The oxidative stress theory predicts that the accumulation of oxidative damage causes aging. More generally, oxidative damage could be a cost of reproduction that reduces survival. Both of these hypotheses have mixed empirical support. To better understand the life-history consequences of oxidative damage, we fed male and female Australian field crickets (Teleogryllus commodus) four diets differing in their protein and carbohydrate content, which have sex-specific effects on reproductive effort and lifespan. We supplemented half of these crickets with the vitamin E isoform DL-alpha-tocopherol and measured the effects of nutrient intake on lifespan, reproduction, oxidative damage and antioxidant protection. We found a clear trade-off between reproductive effort and lifespan in females but not in males. In direct contrast to the oxidative stress theory, crickets fed diets that improved their lifespan had high levels of oxidative damage to proteins. Supplementation with DL-alpha-tocopherol did not significantly improve lifespan or reproductive effort. However, males fed diets that increased their reproductive investment experienced high oxidative damage to proteins. While this suggests that male reproductive effort could elevate oxidative damage, this was not associated with reduced male survival. Overall, these results provide little evidence that oxidative damage plays a central role in mediating life-history trade-offs in T. commodus.