Selection on the joint actions of pairs leads to divergent adaptation and coadaptation of care-giving parents during pre-hatching care.

The joint actions of animals in partnerships or social groups evolve under both natural selection from the wider environment and social selection imposed by other members of the pair or group. We used experimental evolution to investigate how jointly expressed actions evolve upon exposure to a new environmental challenge. Our work focused on the evolution of carrion nest preparation by pairs of burying beetles Nicrophorus vespilloides, a joint activity undertaken by the pair but typically led by the male. In previous work, we found that carrion nest preparation evolved to be faster in experimental populations without post-hatching care (No Care: NC lines) than with post-hatching care (Full Care: FC lines). Here, we investigate how this joint activity evolved. After 15 generations of experimental evolution, we created heterotypic pairs (NC females with FC males and NC males with FC females) and compared their carrion nest making with homotypic NC and FC pairs. We found that pairs with NC males prepared the nest more rapidly than pairs with FC males, regardless of the female's line of origin. We discuss how social coadaptations within pairs or groups could act as a post-mating barrier to gene flow.

Parental effects on inbreeding depression in a beetle with obligate parental care.

Inbreeding depression occurs when individuals who are closely related mate and produce offspring with reduced fitness. Although inbreeding depression is a genetic phenomenon, the magnitude of inbreeding depression can be influenced by environmental conditions and parental effects. In this study, we tested whether size-based parental effects influence the magnitude of inbreeding depression in an insect with elaborate and obligate parental care (the burying beetle, Nicrophorus orbicollis). We found that larger parents produced larger offspring. However, larval mass was also influenced by the interaction between parental body size and larval inbreeding status: when parents were small, inbred larvae were smaller than outbred larvae, but when parents were large this pattern was reversed. In contrast, survival from larval dispersal to adult emergence showed inbreeding depression that was unaffected by parental body size. Our results suggest that size-based parental effects can generate variation in the magnitude of inbreeding depression. Further work is needed to dissect the mechanisms through which this might occur and to better understand why parental size influences inbreeding depression in some traits but not others.

Can age-related changes in parental care modulate inbreeding depression? A test using the burying beetle, Nicrophorus orbicollis.

Parental care has been shown to reduce the magnitude of inbreeding depression in some species with facultative care. However, parents often vary in the quality or amount of care they provide to their offspring, and it is less clear whether this variation also impacts the magnitude of inbreeding depression. Here, we tested whether age-related changes in parental care modulate the expression of inbreeding depression in the burying beetle, Nicrophorus orbicollis. Consistent with previous studies, we found that older parents produced larger broods of offspring than younger parents without sacrificing mean larval mass. Inbreeding depression was evident in several fitness-related traits: brood size at dispersal, the proportion of the brood that survived to eclosion, and mean age at death were all reduced in inbred broods compared with outbred broods. Surprisingly, inbred offspring were heavier at dispersal than outbred offspring. This was likely due to reduced sibling competition in inbred broods. Despite evidence for age-related changes in parental investment and the existence of inbreeding depression, there was no evidence that an interaction between the two influenced any of the traits we measured. Our results suggest that age-related changes in parental care may be too slight to influence the expression of inbreeding depression.

Polyphenisms and polymorphisms: Genetic variation in plasticity and color variation within and among bluefin killifish populations.

The presence of stable color polymorphisms within populations begs the question of how genetic variation is maintained. Consistent variation among populations in coloration, especially when correlated with environmental variation, raises questions about whether environmental conditions affect either the fulcrum of those balanced polymorphisms, the plastic expression of coloration, or both. Color patterns in male bluefin killifish provoke both types of questions. Red and yellow morphs are common in all populations. Blue males are more common in tannin-stained swamps relative to clear springs. Here, we combined crosses with a manipulation of light to explore how genetic variation and phenotypic plasticity shape these patterns. We found that the variation in coloration is attributable mainly to two axes of variation: (1) a red-yellow axis with yellow being dominant to red, and (2) a blue axis that can override red-yellow and is controlled by genetics, phenotypic plasticity, and genetic variation for phenotypic plasticity. The variation among populations in plasticity suggests it is adaptive in some populations but not others. The variation among sires in plasticity within the swamp population suggests balancing selection may be acting not only on the red-yellow polymorphism but also on plasticity for blue coloration.

Patients With Idiopathic Pulmonary Fibrosis Admitted to the ICU With Acute Respiratory Failure-A Reevaluation of the Risk Factors and Outcomes.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) patients admitted to the ICU with acute respiratory failure (ARF) are known to have a poor prognosis. However, the majority of the studies published to date are older and had small sample sizes. Given the advances in ICU care since the publication of these studies, we sought to reevaluate the outcomes and risk factors associated with mortality in these patients. METHODS: Retrospective study using a large multi-center ICU database. We identified 411 unique patients with IPF admitted with ARF between 2014-2015. RESULTS: Of all IPF patients admitted to the ICU with ARF, 81.3% required mechanical ventilation (MV): 48.9% invasive and 32.4% non-invasive alone. The hospital mortality rate was 34.5% for all patients; 48.8% in patients requiring invasive MV, 21.8% in those requiring non-invasive MV and 19.5% with no MV. In multiple regression analyses, age, APACHE score, invasive MV, and hyponatremia at admission were associated with increased mortality whereas post-op status was associated with lower mortality. In patients requiring invasive MV, baseline PaO2/FiO2 ratio was also predictive of mortality. Non-pulmonary organ failures were present in less than 20% of the patients. CONCLUSIONS: Although the overall mortality rate for IPF patients admitted to the ICU with ARF has improved, the mortality rates for patients requiring invasive MV remains high at approximately 50%. Older age, high APACHE score, and low baseline PaO2/FiO2 ratio are factors predictive of increased mortality in this population.

Larval environmental conditions influence plasticity in resource use by adults in the burying beetle, Nicrophorus vespilloides.

Recent studies have shown that intraspecific patterns of phenotypic plasticity can mirror patterns of evolutionary diversification among species. This appears to be the case in Nicrophorus beetles. Within species, body size is positively correlated with the size of carrion used to provision larvae and parental performance. Likewise, among species, variation in body size influences whether species exploit smaller or larger carrion and the extent to which larvae depend on parental care. However, it is unclear whether developmental plasticity in response to carcass size, parental care, or both underlie transitions to new carcass niches. We examined this by testing whether variation in the conditions experienced by Nicrophorus vespilloides larvae influenced their ability to breed efficiently upon differently sized carcasses as adults. We found that the conditions experienced by larvae during development played a critical role in determining their ability to use large carcasses effectively as adults. Specifically, individuals that developed with parental care and on large carcasses were best able to convert the resources on a large carcass into offspring when breeding themselves. Our results suggest that parentally induced plasticity can be important in the initial stages of niche expansion.

Adaptation to monogamy influences parental care but not mating behavior in the burying beetle, Nicrophorus vespilloides.

The mating system is expected to have an important influence on the evolution of mating and parenting behaviors. Although many studies have used experimental evolution to examine how mating behaviors evolve under different mating systems, this approach has seldom been used to study the evolution of parental care. We used experimental evolution to test whether adaptation to different mating systems involves changes in mating and parenting behaviors in populations of the burying beetle, Nicrophorus vespilloides. We maintained populations under monogamy or promiscuity for six generations. This manipulation had an immediate impact on reproductive performance and adult survival. Compared to monogamy, promiscuity reduced brood size and adult (particularly male) survival during breeding. After six generations of experimental evolution, there was no divergence between monogamous and promiscuous populations in mating behaviors. Parents from the promiscuous populations (especially males) displayed less care than parents from the monogamous populations. Our results are consistent with the hypothesis that male care will increase with the certainty of paternity. However, it appears that this change is not associated with a concurrent change in mating behaviors.

Intraspecific variation in resource use is not explained by population persistence or seasonality.

Populations of generalist grazers often contain genotypes with "powerful" and "efficient" strategies. Powerful genotypes grow rapidly on rich-quality resources, but slowly on poorer-quality ones, while efficient genotypes grow relatively better on poorer resources but cannot exploit richer resources as well. Via a "power-efficiency" trade-off, variation in resource quality could maintain genetic diversity. To evaluate this mechanism, we sampled six populations of the freshwater cladoceran Daphnia pulicaria. In persisting (year-round) populations, Daphnia consume resources that vary in quality, whereas in non-persisting (spring-only) populations, Daphnia primarily encounter rich-quality resources. We hypothesized that non-persisting populations harbor no efficient clones (hence should show lower growth on poor-quality resources). Although individuals from non-persisting populations remained smaller than individuals from persisting populations, no evidence arose for a trade-off between powerful and efficient strategies. In fact, growth rates on the two diets were positively correlated (instead of negatively, as predicted). Furthermore, in the persisting populations, we predicted that clonal selection from spring to summer should shift the distribution of genotypes from powerful (specialists on richer spring resources) to efficient (poorer, summer resources). Genetic composition of populations shifted from spring to summer, but not toward more efficient genotypes. Therefore, in these lakes, maintenance of variation among genotypes must stem from more complicated factors than population persistence patterns or seasonal shifts in resource quality alone.

Balancing Container Closure Integrity and Aesthetics for a Robust Aseptic or Sterile Vial Packaging System.

Container closure integrity (CCI) is one of the requirements for a sterile packaging system. For vial-based systems, the capping process is a critical step in creating and ensuring an adequate seal with acceptable CCI. Container closure integrity tests (CCITs) such as the dye ingress and the helium leak rate are two methods among many that, in the appropriate scenario, help to challenge this required attribute. The use of locked-in stopper compression (compression under the crimp seal post capping) enables correlation of these methods to CCI and seal quality. In fact, the overall acceptability of a seal can be evaluated using quantitative and qualitative methods. Usually lost in these assessments is the existence of seal cosmetics as an essential additional seal quality attribute. Unacceptable cosmetic quality can have a major impact on manufacturing (reduced batch output, high yield cost, etc.) and user (perceived low quality, brand image, potential injury, etc.) experiences. Interestingly, the aesthetics of a seal is also impacted by the capping process which is quite complicated because the acceptance criteria for aesthetics of a seal is subjective. Ultimately, this affects commercial manufacturing efficiency and CCI. Here, we present a simple methodology for package selection and evaluated multiple package configurations using locked-in stopper compression (through residual seal force, RSF) measurements and seal aesthetics analyses (using a semi-quantitative aesthetics scale). The integrity of the seals was analyzed using multiple CCIT methods. We determined that component dimensions such as the seal length play a major role in obtaining proper seal aesthetics and integrity. This can ultimately enable the selection of robust packaging components that provide an adequate range of manufacturing conditions without cosmetic defects. A failure to do this could result in high rejects during drug product visual inspection culminating in low batch yield, high costs or could pose harm to patients if suitable CCI is not achieved.LAY ABSTRACT: One common container closure system for parenteral drug products includes a glass vial, rubber stopper, and aluminum crimp seal. The capping process, in which the elastomeric closure is compressed against the vial by means of an aluminum crimp seal, is key to ensuring an optimal seal from both an aesthetic and CCI perspective. Ensuring a robust capping process must include a deep and necessary understanding of the interconnection between the selected components, desired aesthetics of the seal, stopper compression, residual seal force, and CCI; the way in which the capper is configured (sealing parameters) will play a part in addition to the "style" used in manufacturing. Previous published studies have focused on capping process controls to only ensure CCI. Here, we present a useful methodology for selecting appropriate components and capping process parameters using a scaled-down approach to achieve elegant seal quality and CCI simultaneously. Dimensional analysis and capping design of experiments (DOEs) were conducted on lab-scale equipment that was representative of commercial configurations. The seals made from these studies were analyzed using residual seal force, helium leak, and dye ingress methods. The results and their implications were discussed with regard to the operating principle of the rail-type capping machine.

Parental care and sibling competition independently increase phenotypic variation among burying beetle siblings.

Several recent hypotheses suggest that parental care can influence the extent of phenotypic variation within populations; however, there have been few tests of these ideas. We exploited the facultative nature of posthatching parental care in the burying beetle, Nicrophorus vespilloides, to test whether parental care influences the expression of phenotypic variation in an important fitness trait (body size). We found that parental care and brood size (which influences sibling competition) had positive and independent effects on variation in body size. First, the mean coefficient of variation (CV) of body size was significantly greater in broods that received care than in those that did not. Second, CV body size increased with brood size in both parental care treatments. These results are not consistent with predictions from recent hypotheses that predict parental care will reduce phenotypic variation among siblings. The positive effects of parental care and brood size on phenotypic variation that we observed are likely due to sibling competition for access to provisioning parents and competition for limiting resources contained in the breeding carcass. Our results suggest that future theory linking parental care to the generation and maintenance of phenotypic variation must integrate the nature of interactions among family members.

A sustained change in the supply of parental care causes adaptive evolution of offspring morphology.

Although cooperative social interactions within species are considered an important driver of evolutionary change, few studies have experimentally demonstrated that they cause adaptive evolution. Here we address this problem by studying the burying beetle Nicrophorus vespilloides. In this species, parents and larvae work together to obtain nourishment for larvae from the carrion breeding resource: parents feed larvae and larvae also self-feed. We established experimentally evolving populations in which we varied the assistance that parents provided for their offspring and investigated how offspring evolved in response. We show that in populations where parents predictably supplied more care, larval mandibles evolved to be smaller in relation to larval mass, and larvae were correspondingly less self-sufficient. Previous work has shown that antagonistic social interactions can generate escalating evolutionary arms races. Our study shows that cooperative interactions can yield the opposite evolutionary outcome: when one party invests more, the other evolves to invest less.

Adaptation to a novel family environment involves both apparent and cryptic phenotypic changes.

Cryptic evolution occurs when evolutionary change is masked by concurrent environmental change. In most cases, evolutionary changes in the phenotype are masked by changing abiotic factors. However, evolutionary change in one trait might also be masked by evolutionary change in another trait, a phenomenon referred to as evolutionary environmental deterioration. Nevertheless, detecting this second type of cryptic evolution is challenging and there are few compelling examples. Here, we describe a likely case of evolutionary environmental deterioration occurring in experimental burying beetle (Nicrophorus vespilloides) populations that are adapting to a novel social environment that lacks post-hatching parental care. We found that populations rapidly adapted to the removal of post-hatching parental care. This adaptation involved clear increases in breeding success and larval density (number of dispersing larvae produced per gram of breeding carcass), which in turn masked a concurrent increase in the mean larval mass across generations. This cryptic increase in larval mass was accomplished through a change in the reaction norm that relates mean larval mass to larval density. Our results suggest that cryptic evolution might be commonplace in animal families, because evolving trophic and social interactions can potentially mask evolutionary change in other traits, like body size.

Cooperative interactions within the family enhance the capacity for evolutionary change in body size.

Classical models of evolution seldom predict the rate at which populations evolve in the wild. One explanation is that the social environment affects how traits change in response to natural selection. Here, we determine how social interactions between parents and offspring, and among larvae, influence the response to experimental selection on adult size. Our experiments focus on burying beetles (Nicrophorus vespilloides), whose larvae develop within a carrion nest. Some broods exclusively self-feed on the carrion while others are also fed by their parents. We found populations responded to selection for larger adults but only when parents cared for their offspring. We also found populations responded to selection for smaller adults too, but only by removing parents and causing larval interactions to exert more influence on eventual adult size. Comparative analyses revealed a similar pattern: evolutionary increases in species size within the genus Nicrophorus are associated with the obligate provision of care. Synthesising our results with previous studies, we suggest that cooperative social environments enhance the response to selection whereas excessive conflict can prevent further directional selection.

A gene associated with social immunity in the burying beetle Nicrophorus vespilloides.

Some group-living species exhibit social immunity, where the immune response of one individual can protect others in the group from infection. In burying beetles, this is part of parental care. Larvae feed on vertebrate carcasses which their parents smear with exudates that inhibit microbial growth. We have sequenced the transcriptome of the burying beetle Nicrophorus vespilloides and identified six genes that encode lysozymes-a type of antimicrobial enzyme that has previously been implicated in social immunity in burying beetles. When females start breeding and producing antimicrobial anal exudates, we found that the expression of one of these genes was increased by approximately 1000 times to become one of the most abundant transcripts in the transcriptome. Females varied considerably in the antimicrobial properties of their anal exudates, and this was strongly correlated with the expression of this lysozyme. We conclude that we have likely identified a gene encoding a key effector molecule in social immunity and that it was recruited during evolution from a function in personal immunity.

A limit on the extent to which increased egg size can compensate for a poor postnatal environment revealed experimentally in the burying beetle, Nicrophorus vespilloides.

It is often assumed that there is a positive relationship between egg size and offspring fitness. However, recent studies have suggested that egg size has a greater effect on offspring fitness in low-quality environments than in high-quality environments. Such observations suggest that mothers may compensate for poor posthatching environments by increasing egg size. In this paper we test whether there is a limit on the extent to which increased egg size can compensate for the removal of posthatching parental care in the burying beetle, Nicrophorus vespilloides. Previous experiments with N. vespilloides suggest that an increased egg size can compensate for a relatively poor environment after hatching. Here, we phenotypically engineered female N. vespilloides to produce large or small eggs by varying the amount of time they were allowed to feed on the carcass as larvae. We then tested whether differences between these groups in egg size translated into differences in larval performance in a harsh postnatal environment that excluded parental care. We found that females engineered to produce large eggs did not have higher breeding success, and nor did they produce larger larvae than females engineered to produce small eggs. These results suggest that there is a limit on the extent to which increased maternal investment in egg size can compensate for a poor posthatching environment. We discuss the implication of our results for a recent study showing that experimental N. vespilloides populations can adapt rapidly to the absence of posthatching parental care.

Using Experimental Evolution to Study Adaptations for Life within the Family.

Parents of many species provision their young, and the extent of parental provisioning constitutes a major component of the offspring's social environment. Thus, a change in parental provisioning can alter selection on offspring, resulting in the coevolution of parental and offspring traits. Although this reasoning is central to our evolutionary understanding of family life, there is little direct evidence that selection by parents causes evolutionary change in their offspring. Here we use experimental evolution to examine how populations of burying beetles adapt to a change in posthatching parental provisioning. We measured the performance of larvae descended from lab populations that had been maintained with and without posthatching parental care (Full Care and No Care populations). We found that adaptation to the absence of posthatching care led to rapid and consistent changes in larval survival in the absence of care. Specifically, larvae from No Care populations had higher survival in the absence of care than larvae from Full Care populations. Other measures of larval performance, such as the ability of larvae to consume a breeding carcass and larval mass at dispersal, did not differ between the Full Care and No Care populations. Nevertheless, our results show that populations can adapt rapidly to a change in the extent of parental care and that experimental evolution can be used to study such adaptation.

Parental care masks a density-dependent shift from cooperation to competition among burying beetle larvae.

Studies of siblings have focused mainly on their competitive interactions and to a lesser extent on their cooperation. However, competition and cooperation are at opposite ends on a continuum of possible interactions and the nature of these interactions may be flexible with ecological factors tipping the balance toward competition in some environments and cooperation in others. Here we show that the presence of parental care and the density of larvae on the breeding carcass change the outcome of sibling interactions in burying beetle broods. With full parental care there was a strong negative relationship between larval density and larval mass, consistent with sibling competition for resources. In the absence of care, initial increases in larval density had beneficial effects on larval mass but further increases in larval density reduced larval mass. This likely reflects a density-dependent shift between cooperation and competition. In a second experiment, we manipulated larval density and removed parental care. We found that the ability of larvae to penetrate the breeding carcass increased with larval density and that feeding within the carcass resulted in heavier larvae than feeding outside the carcass. However, larval density did not influence carcass decay.

Differences in offspring size predict the direction of isolation asymmetry between populations of a placental fish.

Crosses between populations or species often display an asymmetry in the fitness of reciprocal F1 hybrids. This pattern, referred to as isolation asymmetry or Darwin's Corollary to Haldane's Rule, has been observed in taxa from plants to vertebrates, yet we still know little about which factors determine its magnitude and direction. Here, we show that differences in offspring size predict the direction of isolation asymmetry observed in crosses between populations of a placental fish, Heterandria formosa. In crosses between populations with differences in offspring size, high rates of hybrid inviability occur only when the mother is from a population characterized by small offspring. Crosses between populations that display similarly sized offspring, whether large or small, do not result in high levels of hybrid inviability in either direction. We suggest this asymmetric pattern of reproductive isolation is due to a disruption of parent-offspring coadaptation that emerges from selection for differently sized offspring in different populations.

Large-scale natural disturbance alters genetic population structure of the sailfin molly, Poecilia latipinna.

Many inferences about contemporary rates of gene flow are based on the assumption that the observed genetic structure among populations is stable. Recent studies have uncovered several cases in which this assumption is tenuous. Most of those studies have focused on the effects that regular environmental fluctuations can have on genetic structure and gene flow patterns. Occasional catastrophic disturbances could also alter either the distribution of habitat or the spatial distribution of organisms in a way that affects population structure. However, evidence of such effects is sparse in the literature because it is difficult to obtain. Hurricanes, in particular, have the potential to exert dramatic effects on population structure of organisms found on islands or coral reefs or in near shore and coastal habitats. Here we draw on a historic genetic data set and new data to suggest that the genetic structure of sailfin molly (Poecilia latipinna) populations in north Florida was altered dramatically by an unusually large and uncommon type of storm surge associated with Hurricane Dennis in 2005. We compare the spatial pattern of genetic variation in these populations after Hurricane Dennis to the patterns described in an earlier study in this same area. We use comparable genetic data from another region of Florida, collected in the same two periods, to estimate the amount of change expected from typical temporal variation in population structure. The comparative natural history of sailfin mollies in these two regions indicates that the change in population structure produced by the storm surge is not the result of many local extinctions with recolonization from a few refugia but emerged from a pattern of mixing and redistribution.

Embryonic IGF2 expression is not associated with offspring size among populations of a placental fish.

In organisms that provision young between fertilization and birth, mothers and their developing embryos are expected to be in conflict over embryonic growth. In mammalian embryos, the expression of Insulin-like growth factor II (IGF2) plays a key role in maternal-fetal interactions and is thought to be a focus of maternal-fetal conflict. Recent studies have suggested that IGF2 is also a focus of maternal-fetal conflict in placental fish in the family Poeciliidae. However, whether the expression of IGF2 influences offspring size, the trait over which mothers and embryos are likely to be in conflict, has not been assessed in a poeciliid. We tested whether embryonic IGF2 expression varied among four populations of a placental poeciliid that display large and consistent differences in offspring size at birth. We found that IGF2 expression varied significantly among embryonic stages with expression being 50% higher in early stage embryos than late stage embryos. There were no significant differences among populations in IGF2 expression; small differences in expression between population pairs with different offspring sizes were comparable in magnitude to those between population pairs with the same offspring sizes. Our results indicate that variation in IGF2 transcript abundance does not contribute to differences in offspring size among H. formosa populations.