Genomic analysis of novel Yarrowia-like yeast symbionts associated with the carrion-feeding burying beetle Nicrophorus vespilloides.

BACKGROUND: Mutualistic interactions with microbes can help insects adapt to extreme environments and unusual diets. An intriguing example is the burying beetle Nicrophorus vespilloides, which feeds and reproduces on small vertebrate carcasses. Its fungal microbiome is dominated by yeasts that potentially facilitate carcass utilization by producing digestive enzymes, eliminating cadaver-associated toxic volatiles (that would otherwise attract competitors), and releasing antimicrobials to sanitize the microenvironment. Some of these yeasts are closely related to the biotechnologically important species Yarrowia lipolytica. RESULTS: To investigate the roles of these Yarrowia-like yeast (YLY) strains in more detail, we selected five strains from two different phylogenetic clades for third-generation sequencing and genome analysis. The first clade, represented by strain B02, has a 20-Mb genome containing ~ 6400 predicted protein-coding genes. The second clade, represented by strain C11, has a 25-Mb genome containing ~ 6300 predicted protein-coding genes, and extensive intraspecific variability within the ITS-D1/D2 rDNA region commonly used for species assignments. Phenotypic microarray analysis revealed that both YLY strains were able to utilize a diverse range of carbon and nitrogen sources (including microbial metabolites associated with putrefaction), and can grow in environments with extreme pH and salt concentrations. CONCLUSIONS: The genomic characterization of five yeast strains isolated from N. vespilloides resulted in the identification of strains potentially representing new YLY species. Given their abundance in the beetle hindgut, and dominant growth on beetle-prepared carcasses, the analysis of these strains has revealed the genetic basis of a potential symbiotic relationship between yeasts and burying beetles that facilitates carcass digestion and preservation.

Effects of inbreeding on behavioural plasticity of parent-offspring interactions in a burying beetle.

Inbreeding depression is defined as a fitness decline in progeny resulting from mating between related individuals, the severity of which may vary across environmental conditions. Such inbreeding-by-environment interactions might reflect that inbred individuals have a lower capacity for adjusting their phenotype to match different environmental conditions better, as shown in prior studies on developmental plasticity. Behavioural plasticity is more flexible than developmental plasticity because it is reversible and relatively quick, but little is known about its sensitivity to inbreeding. Here, we investigate effects of inbreeding on behavioural plasticity in the context of parent-offspring interactions in the burying beetle Nicrophorus vespilloides. Larvae increase begging with the level of hunger, and parents increase their level of care when brood sizes increase. Here, we find that inbreeding increased behavioural plasticity in larvae: inbred larvae reduced their time spent associating with a parent in response to the length of food deprivation more than outbred larvae. However, inbreeding had no effect on the behavioural plasticity of offspring begging or any parental behaviour. Overall, our results show that inbreeding can increase behavioural plasticity. We suggest that inbreeding-by-environment interactions might arise when inbreeding is associated with too little or too much plasticity in response to changing environmental conditions.

Early-life effects on body size in each sex interact to determine reproductive success in the burying beetle Nicrophorus vespilloides.

Early-life conditions have been shown to have a profound effect on an animal's body size and fecundity across diverse taxa. However, less is known about how early-life effects on fecundity within each sex interact to determine reproductive success. We used experiments with burying beetles Nicrophorus vespilloides to analyse this problem. The nutritional conditions experienced by burying beetles in early life are a key determinant of adult body size in both sexes, and adult body size in turn influences male reproductive tactics. In previous work, we showed that smaller males are more effective than larger males at stimulating virgin female fecundity. In this study, we manipulated male and female body size by restricting access to food in early development. We then conducted breeding assays, in which small and large females were mated sequentially with small and large males, and then allowed to raise offspring without paternal care. We tested whether large females, which are potentially more fecund, laid even more eggs when mated with small males. We found no evidence to support this prediction. Instead, we detected only a weak non-significant trend in the predicted direction and no equivalent trend in the number of larvae produced. However, we did find that larvae attained a greater mass by the end of development when their mother was large and mated with a small male first. We suggest that large females might have evolved counter-measures that prevent exploitation by small fecundity-stimulating males, including partial filial cannibalism. By eating surplus larvae during reproduction, larger females would leave more of the carrion for their offspring to consume. This could explain why their surviving larvae are able to attain a greater mass by the time they complete their development.

Females adjust maternal hormone concentration in eggs according to male condition in a burying beetle.

In birds and other vertebrates, there is good evidence that females adjust the allocation of hormones in their eggs in response to prenatal environmental conditions, such as food availability or male phenotype, with profound consequences for life history traits of offspring. In insects, there is also evidence that females deposit juvenile hormones (JH) and ecdysteroids (ESH) in their eggs, hormones that play a key role in regulating offspring growth and metamorphosis. However, it is unclear whether females adjust their hormonal deposition in eggs in response to prenatal environmental conditions. Here we address this gap by conducting an experiment on the burying beetle Nicrophorus vespilloides, in which we manipulated the presence of the male parent and the size of the carcass used for breeding at the time of laying. We also tested for effects of the condition (i.e., body mass) of the parents. We then recorded subsequent effects on JH and ESH concentrations in the eggs. We found no evidence for an effect of these prenatal environmental conditions (male presence and carcass size) on hormonal concentration in the eggs. However, we found that females reduced their deposition of JH when mated with heavier males. This finding is consistent with negative differential allocation of maternal hormones in response to variation in the body mass of the male parent. We encourage further work to investigate the role of maternally derived hormones in insect eggs.

Effects of variation in resource acquisition during different stages of the life cycle on life-history traits and trade-offs in a burying beetle.

Individual variation in resource acquisition should have consequences for life-history traits and trade-offs between them because such variation determines how many resources can be allocated to different life-history functions, such as growth, survival and reproduction. Since resource acquisition can vary across an individual's life cycle, the consequences for life-history traits and trade-offs may depend on when during the life cycle resources are limited. We tested for differential and/or interactive effects of variation in resource acquisition in the burying beetle Nicrophorus vespilloides. We designed an experiment in which individuals acquired high or low amounts of resources across three stages of the life cycle: larval development, prior to breeding and the onset of breeding in a fully crossed design. Resource acquisition during larval development and prior to breeding affected egg size and offspring survival, respectively. Meanwhile, resource acquisition at the onset of breeding affected size and number of both eggs and offspring. In addition, there were interactive effects between resource acquisition at different stages on egg size and offspring survival. However, only when females acquired few resources at the onset of breeding was there evidence for a trade-off between offspring size and number. Our results demonstrate that individual variation in resource acquisition during different stages of the life cycle has important consequences for life-history traits but limited effects on trade-offs. This suggests that in species that acquire a fixed-sized resource at the onset of breeding, the size of this resource has larger effects on life-history trade-offs than resources acquired at earlier stages.

Interplay between age-based competitive asymmetries within the brood and direct competition between inbred and outbred offspring in a burying beetle.

Theory suggests that intraspecific competition associated with direct competition between inbred and outbred individuals should be an important determinant of the severity of inbreeding depression. The reason is that, if outbred individuals are stronger competitors than inbred ones, direct competition should have a disproportionate effect on the fitness of inbred individuals. However, an individual's competitive ability is not only determined by its inbreeding status but also by competitive asymmetries that are independent of an individual's inbreeding status. When this is the case, such competitive asymmetries may shape the outcome of direct competition between inbred and outbred individuals. Here, we investigate the interface between age-based competitive asymmetries within broods and direct competition between inbred and outbred offspring in the burying beetle Nicrophorus vespilloides. We found that inbred offspring had lower survival than outbred ones confirming that there was inbreeding depression. Furthermore, seniors (older larvae) grew to a larger size and had higher survival than juniors (younger larvae), confirming that there were age-based competitive asymmetries. Nevertheless, there was no evidence that direct competition between inbred and outbred larvae exacerbated inbreeding depression, no evidence that inbreeding depression was more severe in juniors and no evidence that inbred juniors suffered disproportionately due to competition from outbred seniors. Our results suggest that direct competition between inbred and outbred individuals does not necessarily exacerbate inbreeding depression and that inbred individuals are not always more sensitive to poor and stressful conditions than outbred ones.

Changes of gene expression but not cytosine methylation are associated with male parental care reflecting behavioural state, social context and individual flexibility.

Behaviour is often a front line response to changing environments. Recent studies show behavioural changes are associated with changes of gene expression; however, these studies have primarily focused on discrete behavioural states. We build on these studies by addressing additional contexts that produce qualitatively similar behavioural changes. We measured levels of gene expression and cytosine methylation, which is hypothesized to regulate the transcriptional architecture of behavioural transitions, within the brain during male parental care of the burying beetle Nicrophorus vespilloides in a factorial design. Male parenting is a suitably plastic behaviour because although male N. vespilloides typically do not provide direct care (i.e. feed offspring) when females are present, levels of feeding by a male equivalent to the female can be induced by removing the female. We examined three different factors: behavioural state (caring versus non-caring), social context (with or without a female mate) and individual flexibility (if a male switched to direct care after his mate was removed). The greatest number of differentially expressed genes were associated with behavioural state, followed by social context and individual flexibility. Cytosine methylation was not associated with changes of gene expression in any of the factors. Our results suggest a hierarchical association between gene expression and the different factors, but that this process is not controlled by cytosine methylation. Our results further suggest that the extent a behaviour is transient plays an underappreciated role in determining its underpinning molecular mechanisms.

Disentangling Pre- and Postnatal Maternal Age Effects on Offspring Performance in an Insect with Elaborate Maternal Care.

Maternal effect senescence has attracted much recent scientific interest. However, the age-related effects of pre- and postnatal maternal age are often conflated, as these naturally originate from the same individual. Additionally, many maternal effect senescence studies fail to account for potential biases associated with selective disappearance. Here we use a cross-fostered laboratory population of a burying beetle, Nicrophorus vespilloides, to examine both the effects of female pre- and postnatal maternal age on offspring life-history traits and the postcare outcomes of mothers while accounting for selective disappearance of postnatal caregivers. Neither pre- nor postnatal maternal age affected offspring longevity or larval weight at hatching, and postnatal age had no effect on postcare maternal outcomes except to confirm the presence of actuarial senescence. There was weak evidence for concave relationships between two larval traits (dispersal weight and survival) and the age of egg producers. Selective disappearance of caregivers had no clear effect on any of the measured offspring traits. Contrary to predictions from evolutionary theory, maternal effect senescence and reproductive effort increases do not always manifest, and current theory may be insufficient to account for the true diversity of aging patterns relating to maternal care.

Effects of Offspring and Parental Inbreeding on Parent-Offspring Communication.

There is mounting evidence that inbreeding can have complex effects on social interactions among inbred and outbred individuals. Here, we investigate effects of offspring and maternal inbreeding on parent-offspring communication in the burying beetle Nicrophorus vespilloides. We find effects of the interaction between offspring and maternal inbreeding on maternal behavior. Outbred females provided more direct care toward inbred larvae, while inbred females provided similar levels of direct care toward inbred and outbred larvae. Furthermore, we find direct and indirect effects of offspring inbreeding on offspring begging and maternal behavior, respectively. Inbred larvae spent less time begging than outbred larvae, and (outbred) females provided more direct care and less indirect care toward inbred larvae. Finally, we find effects of the interaction between offspring and maternal inbreeding on larval body mass. Inbred and outbred offspring grew to a similar size when the female was outbred, while inbred offspring were of a smaller size when the female was inbred. Our results suggest that outbred females provided more care toward inbred offspring to compensate for their poor genetic quality. Our study advances our understanding of inbreeding by showing that inbreeding can have direct effects on the behavior of inbred individuals and indirect effects on the behavior of outbred individuals and that indirect effects on outbred individuals may in turn influence the fitness of inbred individuals.

Biparental care is more than the sum of its parts: experimental evidence for synergistic effects on offspring fitness.

Despite an extensive body of theoretical and empirical literature on biparental cooperation, it is still unclear whether offspring fare equally, better or worse when receiving care by two parents versus a single parent. Some models predict that parents should withhold the amount of care they provide due to sexual conflict, thereby shifting as much of the workload as possible to their partner. This conflict should lead to offspring faring worse with two parents. Yet, other models predict that when parents care for their offspring together, their individual contributions can have synergistic (more than additive) effects on offspring fitness. Under this scenario, biparental cooperation should lead to offspring faring better with two parents. We address this fundamental question using a unique experimental design where we compared offspring fitness when the two parents worked together (biparental treatment) and when they worked separately (uniparental treatment), while keeping constant the amount of resources and number of offspring per parent across treatments. This made it possible to directly compare the biparental treatment to the sum of the male and female contributions in the uniparental treatment. Our main finding was that offspring grew larger and were more likely to survive to adulthood when reared by both parents than a single parent. This is the first empirical evidence for a synergistic effect of biparental cooperation on offspring fitness and could provide novel insights into the conditions favouring the evolution of biparental cooperation.

Differential effects of offspring and maternal inbreeding on egg laying and offspring performance in the burying beetle Nicrophorus vespilloides.

We investigate the effect of offspring and maternal inbreeding on maternal and offspring traits associated with early offspring fitness in the burying beetle Nicrophorus vespilloides. We conducted two experiments. In the first experiment, we manipulated maternal inbreeding only (keeping offspring outbred) by generating mothers that were outbred, moderately inbred or highly inbred. Meanwhile, in the second experiment, we manipulated offspring inbreeding only (keeping females outbred) by generating offspring that were outbred, moderately inbred or highly inbred. In both experiments, we monitored subsequent effects on breeding success (number of larvae), maternal traits (clutch size, delay until laying, laying skew, laying spread and egg size) and offspring traits (hatching success, larval survival, duration of larval development and average larval mass). Maternal inbreeding reduced breeding success, and this effect was mediated through lower hatching success and greater larval mortality. Furthermore, inbred mothers produced clutches where egg laying was less skewed towards the early part of laying than outbred females. This reduction in the skew in egg laying is beneficial for larval survival, suggesting that inbred females adjusted their laying patterns facultatively, thereby partially compensating for the detrimental effects of maternal inbreeding on offspring. Finally, we found evidence of a nonlinear effect of offspring inbreeding coefficient on number of larvae dispersing. Offspring inbreeding affected larval survival and larval development time but also unexpectedly affected maternal traits (clutch size and delay until laying), suggesting that females adjust clutch size and the delay until laying in response to being related to their mate.

Flexible parents: joint effects of handicapping and brood size manipulation on female parental care in Nicrophorus vespilloides.

Parental care is highly variable, reflecting that parents make flexible decisions in response to variation in the cost of care to themselves and the benefit to their offspring. Much of the evidence that parents respond to such variation derives from handicapping and brood size manipulations, the separate effects of which are well understood. However, little is known about their joint effects. Here, we fill this gap by conducting a joint handicapping and brood size manipulation in the burying beetle Nicrophorus vespilloides. We handicapped half of the females by attaching a lead weight to their pronotum, leaving the remaining females as controls. We also manipulated brood size by providing each female with 5, 20 or 40 larvae. In contrast to what we predicted, handicapped females spent more time provisioning food than controls. We also found that handicapped females spent more time consuming carrion. Furthermore, handicapped females spent a similar amount of time consuming carrion regardless of brood size, whereas controls spent more time consuming carrion as brood increased. Females spent more time provisioning food towards larger broods, and females were more likely to engage in carrion consumption when caring for larger broods. We conclude that females respond to both handicapping and brood size manipulations, but these responses are largely independent of each other. Overall, our results suggest that handicapping might lead to a higher investment into current reproduction and that it might be associated with compensatory responses that negate the detrimental impact of higher cost of care in handicapped parents.

Intraspecific Competition and Inbreeding Depression: Increased Competitive Effort by Inbred Males Is Costly to Outbred Opponents.

A recent theoretical model suggests that intraspecific competition is an important determinant of the severity of inbreeding depression. The reason for this is that intraspecific competition is density dependent, leading to a stronger negative effect on inbred individuals if they are weaker competitors than outbred ones. In support of this prediction, previous empirical work shows that inbred individuals are weaker competitors than outbred ones and that intraspecific competition often exacerbates inbreeding depression. Here, we report an experiment on the burying beetle Nicrophorus vespilloides, in which we recorded the outcome of competition over a small vertebrate carcass between an inbred or outbred male resident caring for a brood and a size-matched inbred or outbred male intruder. We found that inbred males were more successful as intruders in taking over a carcass from a male resident and were injured more frequently as either residents or intruders. Furthermore, inbred males gained less mass during the breeding attempt and had a shorter adult life span than outbred males. Finally, successful resident males reared a substantially smaller brood comprised of lighter larvae when the intruder was inbred than when it was outbred. Our results shows that inbred males increased their competitive effort, thus contradicting previous work suggesting that inbred males are weaker competitors. Furthermore, our results shows that inbred intruders impose a greater cost to resident males, suggesting that outbred individuals can suffer fitness costs as a result of competition with inbred ones.

Beyond Cuticular Hydrocarbons: Chemically Mediated Mate Recognition in the Subsocial Burying Beetle Nicrophorus vespilloides.

Burying beetles have fascinated scientists for centuries due to their elaborate form of biparental care that includes the burial and defense of a vertebrate carcass, as well as the subsequent feeding of the larvae. However, besides extensive research on burying beetles, one fundamental question has yet to be answered: what cues do males use to discriminate between the sexes? Here, we show in the burying beetle Nicrophorus vespilloides that cuticular lipids trigger male mating behavior. Previous chemical analyses have revealed sex differences in cuticular hydrocarbon (CHC) composition; however, in the current study, fractionated-guided bioassay showed that cuticular lipids, other than CHCs, elicit copulation. Chemical analyses of the behaviorally active fraction revealed 17 compounds, mainly aldehydes and fatty acid esters, with small quantitative but no qualitative differences between the sexes. Supplementation of males with hexadecanal, the compound contributing most to the statistical separation of the chemical profiles of males and females, did not trigger copulation attempts by males. Therefore, a possible explanation is that the whole profile of polar lipids mediates sex recognition in N. vespilloides.

Effects of Prior Contest Experience and Contest Outcome on Female Reproductive Decisions and Offspring Fitness.

Winning or losing a prior contest can influence the outcome of future contests, but it might also alter subsequent reproductive decisions. For example, losers may increase their investment in the current breeding attempt if losing a contest indicates limited prospects for future breeding. Using the burying beetle Nicrophorus vespilloides, we tested whether females adjust their prehatching and posthatching reproductive effort after winning or losing a contest with a same-sex conspecific. Burying beetles breed on carcasses of small vertebrates for which there is fierce intrasexual competition. We found no evidence that winning or losing a contest influenced reproductive investment decisions in this species. Instead, we show that a female's prior contest experience (regardless of its outcome) influenced the amount of posthatching care provided, with downstream consequences for the female's reproductive output; both winners and losers spent more time provisioning food to their offspring and produced larger broods than females with no contest experience. We discuss the wider implications of our findings and present a conceptual model linking contest-mediated adjustments in parental investment to population-level processes. We propose that the frequency of intraspecific contests could both influence and be influenced by population dynamics in species where contest experience influences the size and/or number of offspring produced.

Maternal effects alter the severity of inbreeding depression in the offspring.

A maternal effect is a causal influence of the maternal phenotype on the offspring phenotype over and above any direct effects of genes. There is abundant evidence that maternal effects can have a major impact on offspring fitness. Yet, no previous study has investigated the potential role of maternal effects in influencing the severity of inbreeding depression in the offspring. Inbreeding depression is a reduction in the fitness of inbred offspring relative to outbred offspring. Here, we tested whether maternal effects due to body size alter the magnitude of inbreeding depression in the burying beetle Nicrophorus vespilloides We found that inbreeding depression in larval survival was more severe for offspring of large females than offspring of small females. This might be due to differences in how small and large females invest in an inbred brood because of their different prospects for future breeding opportunities. To our knowledge, this is the first evidence for a causal effect of the maternal phenotype on the severity of inbreeding depression in the offspring. In natural populations that are subject to inbreeding, maternal effects may drive variation in inbreeding depression and therefore contribute to variation in the strength and direction of selection for inbreeding avoidance.

Asynchronous hatching provides females with a means for increasing male care but incurs a cost by reducing offspring fitness.

In species with biparental care, sexual conflict occurs because the benefit of care depends on the total amount of care provided by the two parents while the cost of care depends on each parent's own contribution. Asynchronous hatching may play a role in mediating the resolution of this conflict over parental care. The sexual conflict hypothesis for the evolution of asynchronous hatching suggests that females adjust hatching patterns in order to increase male parental effort relative to female effort. We tested this hypothesis in the burying beetle Nicrophorus vespilloides by setting up experimental broods with three different hatching patterns: synchronous, asynchronous and highly asynchronous broods. As predicted, we found that males provided care for longer in asynchronous broods whereas the opposite was true of females. However, we did not find any benefit to females of reducing their duration of care in terms of increased lifespan or reduced mass loss during breeding. We found substantial negative effects of hatching asynchrony on offspring fitness as larval mass was lower and fewer larvae survived to dispersal in highly asynchronous broods compared to synchronous or asynchronous broods. Our results suggest that, even though females can increase male parental effort by hatching their broods more asynchronously, females pay a substantial cost from doing so in terms of reducing offspring growth and survival. Thus, females should be under selection to produce a hatching pattern that provides the best possible trade-off between the benefits of increased male parental effort and the costs due to reduced offspring fitness.

Sibling competition does not exacerbate inbreeding depression in the burying beetle Nicrophorus vespilloides.

Inbreeding results from matings between relatives and can cause a reduction in offspring fitness, known as inbreeding depression. Previous work has shown that a wide range of environmental stresses, such as extreme temperatures, starvation and parasitism, can exacerbate inbreeding depression. It has recently been argued that stresses due to intraspecific competition should have a stronger effect on the severity of inbreeding depression than stresses due to harsh physical conditions. Here, we tested whether an increase in the intensity of sibling competition can exacerbate inbreeding depression in the burying beetle Nicrophorus vespilloides. We used a 2 × 3 factorial design with offspring inbreeding status (outbred or inbred) and brood size (5, 20, or 40 larvae) as the two factors. We found a main effect of inbreeding status, as inbred larvae had lower survival than outbred larvae, and a main effect of brood size, as larvae in large broods had lower survival and mass than larvae in medium-sized broods. However, there was no effect of the interaction between inbreeding status and brood size, suggesting that sibling competition did not influence the severity of inbreeding depression. Since we focused on sibling competition within homogeneous broods of either inbred or outbred larvae, we cannot rule out possible effects of sibling competition on inbreeding depression in mixed paternity broods comprising of both inbred and outbred offspring. More information on whether and when sibling competition might influence inbreeding depression can help advance our understanding of the causes underlying variation in the severity of inbreeding depression.

Beyond species recognition: somatic state affects long-distance sex pheromone communication.

Long-range sex pheromones have been subjected to substantial research with a particular focus on their biosynthesis, peripheral perception, central processing and the resulting orientation behaviour of perceivers. Fundamental to the research on sex attractants was the assumption that they primarily coordinate species recognition. However, especially when they are produced by the less limiting sex (usually males), the evolution of heightened condition dependence might be expected and long-range sex pheromones might, therefore, also inform about a signaller's quality. Here we provide, to our knowledge, the first comprehensive study of the role of a male's long-range pheromone in mate choice that combines chemical analyses, video observations and field experiments with a multifactorial manipulation of males' condition. We show that the emission of the long-distance sex pheromone of the burying beetle, Nicrophorus vespilloides is highly condition-dependent and reliably reflects nutritional state, age, body size and parasite load--key components of an individual's somatic state. Both, the quantity and ratio of the pheromone components were affected but the time invested in pheromone emission was largely unaffected by a male's condition. Moreover, the variation in pheromone emission caused by the variation in condition had a strong effect on the attractiveness of males in the field, with males in better nutritional condition, of older age, larger body size and bearing less parasites being more attractive. That a single pheromone is influenced by so many aspects of the somatic state and causes such variation in a male's attractiveness under field conditions was hitherto unknown and highlights the need to integrate indicator models of sexual selection into pheromone research.

State-dependent cooperation in burying beetles: parents adjust their contribution towards care based on both their own and their partner's size.

Handicapping experiments on species with biparental care show that a focal parent increases its contribution when its partner is handicapped. Such results are interpreted as evidence for negotiation, whereby each parent adjusts its amount of care to that of its partner. However, it is currently unclear whether the focal parent responds to a change in its handicapped partner's behaviour or state. To address this gap, we conducted an experiment on the burying beetle Nicrophorus vespilloides where we first generated different-sized males and females by varying the duration of larval development. We then used a 2 × 2 factorial design in which a small or large male was paired with a small or large female. Small females provided less direct care (food provisioning and interactions with larvae) than large females, and both males and females provided less direct care when paired with a small partner. Thus, the focal parent adjusted its contribution towards care based on both its own state and that of its partner. There was also evidence for negotiation between the two parents as the focal parent adjusted its contribution based on the amount of care by its partner. However, there was no evidence that negotiation accounted for how the focal parent responded to its partner's size. Our results have important implications for our understanding of biparental cooperation as they show that each parent adjusts its contribution not only based on the amount of care provided by its partner but also based on its own state and its partner's state.