Variation in parenting behavior is widespread across the animal kingdom, both within and between species. There are two ecotypes of the three-spined stickleback fish (Gasterosteus aculeatus) that exhibit dramatic differences in their paternal behavior. Males of the common ecotype are highly attentive fathers, tending to young from eggs to fry, while males of the white ecotype desert offspring as eggs. As the pituitary is a key regulator in the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-gonadal (HPG) axis between the brain and body, its peptides may influence parenting behaviors. Here, we utilized matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) for high-throughput peptide analysis in single cells of pituitaries from both three-spined stickleback ecotypes. Peptide mass fingerprinting was performed using an in silico generated peptide library to identify detected prohormones. Differential analysis revealed POMC-derived peptides, MCH-derived peptides, and oxytocin as significantly different between the two ecotypes, with higher oxytocin levels in the common ecotype. Interestingly, these subtle chemical differences were not captured by Leiden clustering of the cellular phenotypes. These results call for further investigation of the neurochemical basis for parenting in sticklebacks.

The mechanisms underlying the divergence of reproductive strategies between closely related species are still poorly understood. Additionally, it is unclear which selective factors drive the evolution of reproductive behavioral variation and how these traits coevolve, particularly during early divergence. To address these questions, we quantified behavioral differences in a recently diverged pair of Nova Scotian three-spined stickleback (Gasterosteus aculeatus) populations, which vary in parental care, with one population displaying paternal care and the other lacking this. We compared both populations, and a full reciprocal F1 hybrid cross, across four major reproductive stages: territoriality, nesting, courtship, and parenting. We identified significant divergence in a suite of heritable behaviors. Importantly, F1 hybrids exhibited a mix of behavioral patterns, some of which suggest sex linkage. This system offers fresh insights into the coevolutionary dynamics of reproductive behaviors during early divergence and offers support for the hypothesis that coevolutionary feedback between sexual selection and parental care can drive rapid evolution of reproductive strategies.

Oxytocin (OXT) and its homologs are known to regulate parental care in vertebrates, but it is unknown what role these neuropeptides may play in the evolutionary loss of care. Here, we compared two recently diverged ecotypes of threespine stickleback (Gasterosteus aculeatus) that differ in parental care. Males of the common ecotype provide obligate, uniparental care to their offspring, whereas males of the white ecotype abandon their offspring after fertilization. To test if OXT plays a role in the loss of care, we manipulated OXT in males of both ecotypes via intraperitoneal injection of a vehicle control, OXT single- or double-dose, or an OXT antagonist. We observed the behavioral response to injection at two time points for commons (0 and 4 days post-fertilization (dpf)) and one for whites (0 dpf). Our results suggest that, in commons, OXT promotes the onset of care but not its maintenance. Notably, commons that ultimately terminated their clutches did not respond to OXT at 0 dpf, which may have contributed to their failure to transition to a state of care. Whites responded to OXT manipulation in a different manner than commons, suggesting that the loss of care in whites is not due to a loss of sensitivity to OXT, or insufficient levels of OXT ligand, but rather an evolutionary change to the underlying parental circuit that OXT is acting on. These results provide evidence that ancient hormonal systems like OXT can contribute to losses of care over multiple timescales

The vertebrate immune system provides an impressively effective defense against parasites and pathogens. However, these benefits must be balanced against a range of costly side-effects including energy loss and risks of auto-immunity. These costs might include biomechanical impairment of movement, but little is known about the intersection between immunity and biomechanics. Here, we show that a fibrosis immune response in threespine stickleback (Gasterosteus aculeatus) has collateral effects on their locomotion. When freshwater stickleback are infected with the tapeworm parasite Schistocephalus solidus, they face an array of fitness consequences ranging from impaired body condition and fertility to an increased risk of mortality. To fight the infection, some stickleback will initiate a fibrosis immune response in which they produce excess collagenous tissue in their coelom. Although fibrosis is effective at reducing infection, some populations of stickleback actively suppress this immune response, possibly because the costs of fibrosis outweigh the benefits. Here we quantify the locomotor effects of the fibrosis immune response in the absence of parasites to investigate whether there are collateral costs of fibrosis that could help explain why some fish forego this effective defense. To do this, we induce fibrosis in stickleback and then test their C-start escape performance. Additionally, we measure the severity of fibrosis, body stiffness, and body curvature during the escape response. We were able to estimate performance costs of fibrosis by including these variables as intermediates in a structural equation model. This model reveals that among control fish without fibrosis, there is a performance cost associated with increased body stiffness. However, fish with fibrosis did not experience this cost but rather displayed increased performance with higher fibrosis severity. This result demonstrates that the adaptive landscape of immune responses can be complex with the potential for wide reaching and unexpected fitness consequences.

Closely related populations often differ in resistance to a given parasite, as measured by infection success or failure. Yet, the immunological mechanisms of these evolved differences are rarely specified. Does resistance evolve via changes to the host's ability to recognize that an infection exists, actuate an effective immune response, or attenuate that response? We tested whether each of these phases of the host response contributed to threespine sticklebacks' recently evolved resistance to their tapeworm Schistocephalus solidus. Although marine stickleback and some susceptible lake fish permit fast-growing tapeworms, other lake populations are resistant and suppress tapeworm growth via a fibrosis response. We subjected lab-raised fish from three populations (susceptible marine "ancestors," a susceptible lake population, and a resistant lake population) to a novel immune challenge using an injection of (1) a saline control, (2) alum, a generalized pro-inflammatory adjuvant that causes fibrosis, (3) a tapeworm protein extract, or (4) a combination of alum and tapeworm protein. With enough time, all three populations generated a robust fibrosis response to the alum treatments. Yet, only the resistant population exhibited a fibrosis response to the tapeworm protein alone. Thus, these populations differed in their ability to respond to the tapeworm protein but shared an intact fibrosis pathway. The resistant population also initiated fibrosis faster in response to alum, and was able to attenuate fibrosis, unlike the susceptible populations' slow but longer lasting response to alum. As fibrosis has pathological side effects that reduce fecundity, the faster recovery by the resistant population may reflect an adaptation to mitigate the costs of immunity. Broadly, our results confirm that parasite detection and immune initiation, activation speed, and immune attenuation simultaneously contribute to the evolution of parasite resistance and adaptations to infection in natural populations.

Parental care has attracted attention from both proximate and ultimate perspectives. While understanding the adaptive significance of care has been the focus of work in diverse organisms in behavioral ecology, most of what we know about the proximate mechanisms underlying parental care behavior comes from studies in mammals. Although studies on mammals have greatly improved our understanding of care, viewing parental care solely through a mammalian lens can limit our understanding. Here, we draw upon examples from non-mammalian vertebrate systems to show that in many ways mammals are the exception rather than the rule for caregiving: across vertebrates, maternal care is often not the ancestral or the most common mode of care and fathering is not derivative of mothering. Embracing the diversity of parental care can improve our understanding of both the proximate basis and adaptive significance of parental care and the affective processes involved in caregiving.

Since the New Synthesis, most migration-selection balance theory has predicted that there should be negligible differentiation over small spatial scales (relative to dispersal), because gene flow should erode any effect of divergent selection. Nevertheless, there are classic examples of microgeographic divergence, which theory suggests can arise under specific conditions: exceptionally strong selection, phenotypic plasticity in philopatric individuals, or nonrandom dispersal. Here, we present evidence of microgeographic morphological variation within lake and stream populations of threespine stickleback (Gasterosteus aculeatus). It seems reasonable to assume that a given lake or stream population of fish is well-mixed. However, we found this assumption to be untenable. We examined trap-to-trap variation in 34 morphological traits measured on stickleback from 16 lakes and 16 streams. Most traits varied appreciably among traps within populations. Both between-trap distance and microhabitat characteristics such as depth and substrate explained some of the within-population morphological variance. Microhabitat was also associated with genotype at particular loci but there was no genetic isolation by distance, implying that heritable habitat preferences may contribute to microgeographic variation. Our study adds to growing evidence that microgeographic divergence can occur across small spatial scales within individuals' daily dispersal neighborhood where gene flow is expected to be strong.

ABSTRACTClosely related populations often differ in resistance to a given parasite, as measured by infection success or failure. Yet, the immunological mechanisms of these evolved differences are rarely specified. Does resistance evolve via changes to the host’s ability to recognize that an infection exists, actuate an effective immune response, or attenuate that response? We tested whether each of these phases of the host response contributed to threespine sticklebacks’ recently evolved resistance to their tapewormSchistocephalus solidus.While marine stickleback and some susceptible lake fish permit fast-growing tapeworms, other lake populations are resistant and suppress tapeworm growth via a fibrosis response. We subjected lab-raised fish from three populations (susceptible marine ‘ancestors’, a susceptible lake, a resistant lake), to a novel immune challenge (injection of: 1) a saline control, 2) alum, a generalized pro-inflammatory adjuvant that causes fibrosis, 3) a tapeworm protein extract, and 4) a combination of alum and tapeworm protein). All three populations were capable of a robust fibrosis response to the alum treatments (but not the saline control). Yet, only the resistant population exhibited a fibrosis response to the tapeworm protein alone. Thus, these populations differed in their ability to recognize the tapeworm but shared an intact fibrosis pathway. However, the resistant population also initiated fibrosis faster, and was able to attenuate fibrosis, unlike the susceptible populations slow but longer-lasting response to alum. As fibrosis has presumed pathological side-effects, this difference may reflect adaptions to mitigate costs of immunity in the resistant population. Broadly, our results confirm that parasite detection, activation speed, and immune attenuation simultaneously contribute to adaptations to parasite infection in natural populations.IMPACT SUMMARYDramatic variation in parasite resistance is common in nature, even to the same parasite, yet we are still working to understand the mechanisms of how such differences evolve. Many evolution studies focus on the broad outcomes of infection (infected or not) when studying this question, without specifying what part of the immune response has evolved. Here, we experimentally partition different sequential stages in the host immune response (recognition, actuation, attenuation), to evaluate which stage(s) underly the evolution of host resistance to infection. This study compares three populations of threespine stickleback that naturally differ in their exposure and their ability to resist infections of a freshwater tapeworm. These include a “resistant” lake population, a “susceptible” lake population, and an ancestral marine population that is rarely exposed to the tapeworm in nature, but is susceptible when exposed in the lab. The resistant population exhibits a fibrosis immune response to infection, which has previously been linked to suppressed tapeworm growth and viability. We injected different immune challenges directly into the site of infection (peritoneal cavity) and measured the subsequent fibrosis response through time. We found that all populations were capable of producing fibrosis in response to a general immune stimulant (alum). But, only the resistant population was able to recognize and respond to tapeworm protein alone. This population also responded faster than the others, within 24 hours, and attenuated its fibrosis by 90 days post-injections whereas the other populations exhibited a slower response that did not attenuate in the study time-frame. We concluded that variation in parasite recognition, an early phase in the host response, shapes the evolution of the initiation and resolution of the physical response to infection. Broadly, our results support that parasite detection mechanisms could play a key role in the rapid evolution of parasite resistance.

BackgroundWhen threespine stickleback colonized fresh water, they repeatedly evolved reduced armour plating via changes in Eda allele frequency. This evolution is typically attributed to differential predation pressure between marine and freshwater environments. However, the chromosomal region containing Eda is associated with many other phenotypes, including schooling, antipredator behaviour, and immunity. Consequently, the evolution of armour plating may be driven by multiple selective pressures acting on Eda or linked genes.QuestionIs parasite infection associated with armour phenotype?HypothesisParasite load differs between stickleback armour plate morphs.OrganismsAn armour-polymorphic population of threespine stickleback (Gasterosteus aculeatus), and their parasites.Field siteIn June 2009 and 2012, we sampled stickleback from a single human-made salt-marsh pool in the Campbell River Estuary on Vancouver Island.MethodsWe counted macroparasites on approximately 100 fish per year and counted lateral armour plates. We used generalized linear models to test for correlations between armour morph and parasite load.ResultsMost parasite species were not associated with armour. The gill parasite Thersitina was more abundant on more fully armoured fish in both years. The nematode Eustrongylides also exhibited a marginally significant positive trend. If parasitic infections reduce stickleback fitness, this positive covariance between armour and infection would accelerate the loss of armour plating in stickleback colonizing fresh water.

Helical swimming is a common behavior in larvae of many marine invertebrate species that may facilitate either exploration or feeding. Swimming in helices may increase exposure of larvae to settlement cues localized to the seafloor by enhancing their horizontal scanning motion near potential settlement sites. Alternatively, helical swimming may increase feeding efficiency by allowing an organism to maximize time spent in vertically-constrained food patches. In this study, we investigated whether the prevalence and geometry of helical swimming in competent larvae of the eastern oyster (Crassostrea virginica) vary in response to a settlement cue or to food. We performed two experiments, one examining helical swimming behavior in larvae exposed to different concentrations of a chemical settlement cue (“Cue Experiment”) and the other examining helical swimming of fed and starved larvae in conditions with and without algal food (“Feeding Experiment”). In the Cue Experiment, the proportion of larvae swimming in helices increased with decreasing cue concentration, and helices became wider, which suggests that helices may be an exploratory behavior that is curtailed when preferred habitat is detected. In the Feeding Experiment, neither the proportion of larvae performing helices nor helix geometry varied with food availability or satiation. Our results indicate that variations in helical swimming likely enhance the ability of C. virginica larvae to detect lateral variation in waterborne cues and locate suitable habitat prior to settlement.