Coinfections are common in nature and include hosts simultaneously or sequentially exposed to different genotypes. We investigated the potential for simultaneous and sequential exposures to impact host fitness by exposing Caenorhabditis elegans hosts to different bacterial strains of Serratia marcescens . We found that in simultaneous exposures a highly virulent strain induced high mortality regardless of coexposure. In sequential exposures, the same highly virulent strain induced high mortality if it was the first strain introduced in a coexposure. Overall, we find host outcomes after coinfections may depend on particular parasite genotype combinations and infection timing.

Widespread antibiotic usage has resulted in the rapid evolution of drug-resistant bacterial pathogens. Resolving how pathogens respond to antibiotics under different contexts is critical for understanding disease emergence. It remains unclear how interactions between hosts and antibiotics impact pathogen evolution. Here, we evolved Staphylococcus aureus, a major bacterial pathogen, varying exposure to host and antibiotics to tease apart the contributions of these selective pressures on pathogen adaptation. After 12 passages, S. aureus evolving in Caenorhabditis elegans nematodes exposed to a sub-minimum inhibitory antibiotic concentration became highly virulent, regardless of whether the ancestral pathogen was methicillin-resistant (MRSA) or methicillin-sensitive (MSSA). Host and antibiotic selected for reduced drug susceptibility in MSSA while increasing MRSA total growth outside hosts. We identified mutations in genes involved in regulatory networks linking virulence and metabolism, suggesting that rapid adaptation to infect hosts may have pleiotropic effects. Mutations that arose in these genes were also enriched in clinical isolates associated with systemic infections in humans. Despite evolving in similar environments, MRSA and MSSA populations—differing only in the presence of an intact accessory gene—proceeded on divergent evolutionary paths, with MSSA populations exhibiting more similarities across replicates. Our results underscore the importance of the host context as a driver of virulence and antibiotic resistance.

Mixed-mating strategies can maximize the benefits and limit the costs of both self-fertilization and outcrossing. In addition to ecological conditions and population dynamics, the economics of mixed mating are determined by individual self-compatibility, i.e. the proportion of self-fertilization events that result in viable offspring. In gynodioecious (hermaphrodites and females) and androdioecious (hermaphrodites and males) species, self-compatibility of hermaphrodites dictates the reproductive potential of the other sex and can exert strong selection on maintenance of the non-hermaphroditic sex. Mangrove rivulus fish populations are androdioecious, and males result from hermaphrodites changing sex. Hermaphrodites overwhelmingly reproduce through internal self-fertilization, but occasionally oviposit unfertilized eggs, which males can fertilize externally. We tested the hypotheses that self-compatibility and fecundity would vary with age and as a function of genotypic variation in propensities for sex change. We reveal that fecundity and self-compatibility vary within individuals across ontogeny and among genotypes with different propensities to change sex. Hermaphrodites from genotypes that frequently change sex were significantly less fecund and self-compatible than hermaphrodites from genotypes that rarely change sex. These differences in self-compatibility and fecundity have the potential to drive mating strategy evolution in mangrove rivulus, specifically the fitness of males and associated spatiotemporal variation in sex ratios within and among populations.

Despite substantial costs, biparental sex is the dominant mode of reproduction across plant and animal taxa. The Red Queen hypothesis (RQH) posits that coevolutionary interactions with parasites can favor biparental sex in hosts, despite the costs. In support of the RQH, previous studies found that coevolutionary interactions with virulent bacterial parasites maintained high outcrossing rates in populations of the androdioecious nematode host Caenorhabditis elegans . Here we test three non-mutually exclusive mechanisms that could explain how coevolving parasites maintain outcrossing rates in C. elegans hosts: 1) short-term parasite exposure induces plastic increases in the hosts' propensity to outcross, 2) hosts evolve increased outcrossing propensity in response to selection imposed by coevolving parasites, and 3) outcrossed offspring incur less parasite-mediated fitness loss than selfed offspring, increasing host male frequencies and opportunities for outcrossing. We find no evidence that parasites cause plastic or evolved changes in host outcrossing propensity. However, parental outcrossing significantly increases survival of host offspring in the F2 generation when exposed to a coevolving parasite. Hence, coevolving parasites maintain outcrossing in host populations by selecting against selfed offspring, rather than by inducing changes in the propensity to outcross.

Theory predicts that organisms should diversify their offspring when faced with a stressful environment. This prediction has received empirical support across diverse groups of organisms and stressors. For example, when encountered by Caenorhabditis elegans during early development, food limitation (a common environmental stressor) induces the nematodes to arrest in a developmental stage called dauer and to increase their propensity to outcross when they are subsequently provided with food and enabled to develop to maturity. Here we tested whether food limitation first encountered during late development/early adulthood can also induce increased outcrossing propensity in C. elegans. Previously well-fed C. elegans increased their propensity to outcross when challenged with food limitation during the final larval stage of development and into early adulthood, relative to continuously well-fed (control) nematodes. Our results thus support previous research demonstrating that the stress of food limitation can induce increased outcrossing propensity in C. elegans. Furthermore, our results expand on previous work by showing that food limitation can still increase outcrossing propensity even when it is not encountered until late development, and this can occur independently of the developmental and gene expression changes associated with dauer.

Mixed mating, a reproduction strategy utilized by many plants and invertebrates, optimizes the cost to benefit ratio of a labile mating system. One type of mixed mating includes outcrossing with conspecifics and self-fertilizing one’s own eggs. The mangrove rivulus fish ( Kryptolebias marmoratus) is one of two vertebrates known to employ both self-fertilization (selfing) and outcrossing. Variation in rates of outcrossing and selfing within and among populations produces individuals with diverse levels of heterozygosity. I designed an experiment to explore the consequences of variable heterozygosity across four ecologically relevant conditions of salinity and water availability (10‰, 25‰, and 40‰ salinity, and twice daily tide changes). I report a significant increase in mortality in the high salinity (40‰) treatment. I also report significant effects on fecundity measures with increasing heterozygosity. The odds of laying eggs decreased with increasing heterozygosity across all treatments, and the number of eggs laid decreased with increasing heterozygosity in the 10‰ and 25‰ treatments. Increasing heterozygosity also was associated with a reduction liver mass and body condition in all treatments. My results highlight the fitness challenges that accompany living in mangrove forests ecosystem and provide the first evidence for outbreeding depression on reproductive and condition-related traits.

Mate choice has the potential to drive phenotypic evolution because it can determine traits that increase an individual’s likelihood to reproduce (courtship behaviors, elaborate ornamentation). These traits, however, can also be detrimental for health or survival, often antagonizing the evolution of extreme phenotypes. Mangrove rivulus fish ( Kryptolebias marmoratus ) develop as self-fertilizing simultaneous hermaphrodites. Hermaphrodites overwhelmingly self-fertilize their eggs internally, but occasionally oviposit unfertilized eggs. Some individuals change sex to male after sexual maturity, essentially forgoing the reproductive assurance of selfing. In a continuing effort to understand how sex change to male is maintained this species, I designed an experiment to determine whether males act as choosers to increase their likelihood of finding unfertilized eggs for reproduction. I hypothesized that males would prefer to associate with younger hermaphrodites when given a dichotomous choice, as they lay a greater proportion of unfertilized eggs compared to older hermaphrodites. The males in this study did not show a preference for either the younger or older hermaphrodite but exhibited greater within individual variance across subtrials than among individual variation. I discuss alternative hypotheses concerning male mate choice in mangrove rivulus, which may illuminate hypotheses to be tested in this and other hermaphroditic species. ### Competing Interest Statement The authors have declared no competing interest.

Mixed mating strategies offer the benefits of both self-fertilizing one’s own eggs (selfing) and outcrossing, while limiting the costs of both methods. The economics of mixed mating is further determined by individual self-compatibility. In gynodioecious (hermaphrodites, females) and androdioecious (hermaphrodites, males) species, the level of self-compatibility of the hermaphrodites also acts as a selection pressure on the fitness of the other sex. Mangrove rivulus fish populations are comprised of selfing hermaphrodites and males that result from hermaphrodites changing sex. Although hermaphrodites overwhelmingly reproduce through internal selfing, they occasionally oviposit unfertilized eggs. Males can externally fertilize these eggs. Here, we reveal that fecundity and self-compatibility varies within individuals across ontogeny until about 365 days post hatch, and among individuals derived from lineages that vary in their propensity to change sex. Hermaphrodites from high sex changing lineages were significantly less fecund and self-compatible than hermaphrodites from low sex changing lineages. These differences in self-compatibility and fecundity have the potential to drive evolutionary changes on mating strategy and the fitness of males in populations of the mangrove rivulus. This study also illustrates the importance of including lineage variation when estimating the costs and benefits of mixed mating strategies.

Sequential hermaphroditism (sex change) is understood to be a strategy that maximizes lifetime reproduction in systems where one sex confers highest fitness early in life, and the other later in life. This strategy is evolutionarily stable despite costs to growth, survival, or current reproduction. Few studies have examined advantages of sex change outside of reproduction. The mangrove rivulus fish, Kryptolebias marmoratus, presents a unique system in which to study non-reproductive consequences of sex change because reproductive opportunity decreases significantly with sex change. In natural conditions, individuals develop as self-fertilizing simultaneous hermaphrodites. Some individuals change sex to male at various points after sexual maturity, even in isolation, essentially foregoing future reproductive assurance. In a large-scale experiment that examined fitness differences among individuals exposed to ecologically relevant environmental challenges, we found that individuals that change sex from hermaphrodite to male had overwhelmingly greater chances of survival compared to those that remained hermaphrodite. Furthermore, hermaphrodites derived from lineages with higher propensities to change sex experienced greater survival advantages by changing sex. Our results indicate that sex change may be a survival strategy, one with genotype-dependent consequences.

The mangrove rivulus fish (Kryptolebias marmoratus) is an emerging model for research in the biological sciences. The species is androdioecious, with populations consisting predominantly of self-fertilizing hermaphrodites and a low abundance of males, but no females. Males arise either through environmental sex determination at the embryonic stage or environmentally driven sex change from hermaphrodite at the adult stage. Marked sexual dimorphisms in external morphological characters accompany gonadal transitions, including the loss of a caudal ocellus (eyespot') and the development of orange color in males. How well external morphological features predict internal gonadal morphology, however, has not been systematically examined across the geographical range of this species. Our goal was to identify reliable external morphological predictors of animals possessing an ovotestis (hermaphrodite) or a testis (male) and to see if the strength of prediction is consistent throughout rivulus' geographical range. We demonstrate that the state of the ocellus and, to a greater extent, orange coloration are key predictors of internal gonadal state. However, the degree to which these characteristics are predictive of internal gonadal state was region-specific, with the ocellus and orange status of animals from Bahamas and Belize, respectively, having a lower probability of forecasting internal gonadal state than those from other regions. These results will help to streamline laboratory studies by allowing unambiguous assignment of sex, and will assist field studies by providing greater opportunity to determine sex ratios in wild populations.

Although the importance of postexercise nutrient ingestion timing has been investigated for glycogen metabolism, little is known about similar effects for protein dynamics. Each subject ( n = 10) was studied twice, with the same oral supplement (10 g protein, 8 g carbohydrate, 3 g fat) being administered either immediately (EARLY) or 3 h (LATE) after 60 min of moderate-intensity exercise. Leg blood flow and circulating concentrations of glucose, amino acids, and insulin were similar for EARLY and LATE. Leg glucose uptake and whole body glucose utilization (d-[6,6-2H2]glucose) were stimulated threefold and 44%, respectively, for EARLY vs. LATE. Although essential and nonessential amino acids were taken up by the leg in EARLY, they were released in LATE. Although proteolysis was unaffected, leg (l-[ ring-2H5]phenylalanine) and whole body (l-[1-13C]leucine) protein synthesis were elevated threefold and 12%, respectively, for EARLY vs. LATE, resulting in a net gain of leg and whole body protein. Therefore, similar to carbohydrate homeostasis, EARLY postexercise ingestion of a nutrient supplement enhances accretion of whole body and leg protein, suggesting a common mechanism of exercise-induced insulin action.

Whole body oxidative rates of labeled substrates are often measured by collecting expired air and determining the amount of labeled CO(2) that is produced. However, the CO(2) produced may not be completely recovered under all circumstances, and there is a wide variation in values reported under different experimental conditions ( approximately 50-100%). The potential contribution of specific organs to this variation has not been defined. In vivo studies using healthy, postabsorptive, multicatheterized conscious canines were conducted to determine gastrointestinal tract, hepatic, hindlimb, and renal recoveries of NaH(14)CO(3) during a 180-min constant infusion [0.022+/-0.002 (SE) microCi x kg(-1) x min(-1)]. Before the constant infusion period, a bolus infusion of NaH(14)CO(3) (1.76+/-0.16 microCi/kg) was given, and the rate of decay in blood was measured over a 15-min period to determine pool size. The pool size for the distribution of (14)CO(2) was approximately 80% of the total body pool (16.0+/-1.7 liters). Whole body recovery was 97.2+/-6.7%. The recoveries across the liver, gut, leg, and kidney were 99.9+/-1.3, 98.0 +/-1.4, 96.7+/-2.6, and 99.9+/-2.1%, respectively. In conclusion, hepatic, gastrointestinal tract, hindlimb, and renal recoveries of CO(2) in vivo were near 100%, suggesting that CO(2) loss is not greater in gluconeogenic organs and that corrections for incomplete recovery of CO(2), when measuring oxidation of substrates across these organs under normal postabsorptive conditions, would be very minor.

704 While increased amino acid availability after exercise has been suggested to enhance muscle protein synthesis, the effects of post-exercise protein intake on leg glucose uptake and plasma urea are not well defined. Prior to study, catheters were inserted into a femoral vein and a heated (arterialized) hand vein. Each study consisted of a 30-min basal period, a 60-min exercise period (bicycle; 60% VO2max), and a 180-min recovery period, during which one of four treatments was randomly administered: NO = 0 g carbohydrate, 0 g protein; CHO = 8,0; PRO = 8,10; or 3PRO = 24,30 (*p<0.05 for NO vs CHO, PRO, or 3PRO). (Table)TableCompared to NO, CHO increased plasma glucose, but did not significantly increase leg glucose uptake. However, PRO increased plasma glucose and amino acids, while enhancing leg glucose uptake. Conversely, plasma urea concentrations were not increased with the intake of either 10 or 30 g of protein. Therefore, while post-exercise supplementation of protein benefits the recovery of leg glucose stores, it does not cause uremia in humans.