Gene expression remodelling and immune response during adaptive divergence in an African cichlid fish

Variation in gene expression contributes to ecological speciation by facilitating population persistence in novel environments. Likewise, immune responses can be of relevance in speciation driven by adaptation to different environments. Previous studies examining gene expression differences between recently diverged ecotypes have often relied on only one pair of populations, targeted the expression of only a subset of genes or used wild‐caught individuals. Here, we investigated the contribution of habitat‐specific parasites and symbionts and the underlying immunological abilities of ecotype hosts to adaptive divergence in lake–river population pairs of the cichlid fish Astatotilapia burtoni. To shed light on the role of phenotypic plasticity in adaptive divergence, we compared parasite and microbiota communities, immune response, and gene expression patterns of fish from natural habitats and a lake‐like pond set‐up. In all investigated population pairs, lake fish were more heavily parasitized than river fish, in terms of both parasite taxon composition and infection abundance. The innate immune response in the wild was higher in lake than in river populations and was elevated in a river population exposed to lake parasites in the pond set‐up. Environmental differences between lake and river habitat and their distinct parasite communities have shaped differential gene expression, involving genes functioning in osmoregulation and immune response. Most changes in gene expression between lake and river samples in the wild and in the pond set‐up were based on a plastic response. Finally, gene expression and bacterial communities of wild‐caught individuals and individuals acclimatized to lake‐like pond conditions showed shifts underlying adaptive phenotypic plasticity.     

Among‐lake reciprocal transplants induce convergent expression of immune genes in threespine stickleback

Geographic variation in parasite communities can drive evolutionary divergence in host immune genes. However, biotic and abiotic environmental variation can also induce plastic differences in immune function among populations. At present, there is little information concerning the relative magnitudes of heritable vs. induced immune divergence in natural populations. We examined immune gene expression profiles of threespine stickleback (Gasterosteus aculeatus) from six lakes on Vancouver Island, British Columbia. Parasite community composition differs between lake types (large or small, containing limnetic‐ or benthic‐like stickleback) and between watersheds. We observed corresponding differences in immune gene expression profiles among wild‐caught stickleback, using a set of seven immune genes representing distinct branches of the immune system. To evaluate the role of environmental effects on this differentiation, we experimentally transplanted wild‐caught fish into cages in their native lake, or into a nearby foreign lake. Transplanted individuals' immune gene expression converged on patterns typical of their destination lake, deviating from their native expression profile. Transplant individuals' source population had a much smaller effect, suggesting relatively weak genetic underpinning of population differences in immunity, as viewed through gene expression. This strong environmental regulation of immune gene expression provides a counterpoint to the large emerging literature documenting microevolution and genetic diversification of immune function. Our findings illustrate the value of studying immunity in natural environmental settings where the immune system has evolved and actively functions.     

A genetics‐based approach confirms immune associations with life history across multiple populations of an aquatic vertebrate (Gasterosteus aculeatus)

Understanding how wild immune variation covaries with other traits can reveal how costs and trade‐offs shape immune evolution in the wild. Divergent life history strategies may increase or alleviate immune costs, helping shape immune variation in a consistent, testable way. Contrasting hypotheses suggest that shorter life histories may alleviate costs by offsetting them against increased mortality, or increase the effect of costs if immune responses are traded off against development or reproduction. We investigated the evolutionary relationship between life history and immune responses within an island radiation of three‐spined stickleback, with discrete populations of varying life histories and parasitism. We sampled two short‐lived, two long‐lived and an anadromous population using qPCR to quantify current immune profile and RAD‐seq data to study the distribution of immune variants within our assay genes and across the genome. Short‐lived populations exhibited significantly increased expression of all assay genes, which was accompanied by a strong association with population‐level variation in local alleles and divergence in a gene that may be involved in complement pathways. In addition, divergence around the eda gene in anadromous fish is likely associated with increased inflammation. A wider analysis of 15 populations across the island revealed that immune genes across the genome show evidence of having diverged alongside life history strategies. Parasitism and reproductive investment were also important sources of variation for expression, highlighting the caution required when assaying immune responses in the wild. These results provide strong, gene‐based support for current hypotheses linking life history and immune variation across multiple populations of a vertebrate model.     

Distinct haplotype structure at the innate immune receptor Toll‐like receptor 2 across bank vole populations and lineages in Europe

Parasite‐mediated selection may contribute to the maintenance of genetic variation at host immune genes over long time scales. To date, the best evidence for the long‐term maintenance of immunogenetic variation in natural populations comes from studies on the major histocompatibility complex (MHC) genes, whereas evidence for such processes from other immune genes remains scarce. In the present study, we show that, despite pronounced population differentiation and the occurrence of numerous private alleles within populations, the innate immune gene Toll‐like receptor 2 (TLR2) displays a distinct haplotype structure in 21 bank vole (Myodes glareolus) populations across Europe. Haplotypes from all populations grouped in four clearly differentiated clusters, with the three main clusters co‐occurring in at least three previously described mitochondrial lineages. This pattern indicates that the distinct TLR2 haplotype structure may precede the split of the mitochondrial lineages 0.19–0.56 Mya and suggests that haplotype clusters at this innate immune receptor are maintained over prolonged time in wild bank vole populations.     

Strong and parallel salinity‐induced phenotypic plasticity in one generation of threespine stickleback

Phenotypic plasticity is a major factor contributing to variation of organisms in nature, yet its evolutionary significance is insufficiently understood. One example system where plasticity might have played an important role in an adaptive radiation is the threespine stickleback (Gasterosteus aculeatus), a fish that has diversified after invading freshwater lakes repeatedly from the marine habitat. The parallel phenotypic changes that occurred in this radiation were extremely rapid. This study evaluates phenotypic plasticity in stickleback body shape in response to salinity in fish stemming from a wild freshwater population. Using a split‐clutch design, we detected surprisingly large phenotypically plastic changes in body shape after one generation. Fish raised in salt water developed shallower bodies and longer jaws, and these changes were consistent and parallel across families. Although this work highlights the effect of phenotypic plasticity, we also find indications that constraints may play a role in biasing the direction of possible phenotypic change. The slopes of the allometric relationship of individual linear traits did not change across treatments, indicating that plastic change does not affect the covariation of traits with overall size. We conclude that stickleback have a large capacity for plastic phenotypic change in response to salinity and that plasticity and evolutionary constraints have likely contributed to the phenotypic diversification of these fish.     

Consistency of host responses to parasitic infection in the three‐spined stickleback fish infected by the diphyllobothriidean cestode Schistocephalus solidus

Among populations of the three‐spined stickleback fish in Alaska, females appear to show two forms of sterility tolerance to infection by the diphyllobothriidean cestode Schistocephalus solidus. In contrast to sticklebacks in other regions of the northern hemisphere, female fish are capable of producing clutches of eggs despite supporting large parasite burdens. Nonetheless, nutrient loss to the parasite, coupled with the energetic demands of host reproduction, eventually curtails spawning among infected females. Host females in Walby Lake experience ‘fecundity reduction’ resulting from nutrient theft as a side effect of infection. In Scout Lake, infected females show ‘fecundity compensation’, an adaptive, inducible response allowing them to increase current fecundity to compensate for reduction or loss of future reproduction. This multi‐year study of sticklebacks from each lake addresses two empirical questions for a better understanding of the dynamic interplay between host and parasite. First, is there is any annual variation within the two responses to parasitism in each host population; and, if so, is it related to parasite burden? Second, do the two host responses show consistent differences between the populations of sticklebacks despite any yearly variation in them? We found annual, intra‐population variation within the response shown by each population of stickleback which appears to have been influenced by the parasite : host mass ratio and possibly by unknown environmental conditions affecting the reproductive physiology of stickleback females. Moreover, the data support the hypothesis that ovum mass is more sensitive to parasitism (parasite burden) than clutch size in females from Walby Lake which exhibit fecundity reduction. Notwithstanding the intra‐population variation within each host response, the responses to infection occurred consistently within each respective stickleback population and appear to reflect stable, fundamental characteristics of the populations. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 958–968.     

Half the story: Thermal effects on within‐host infectious disease progression in a warming climate

Immune defense is temperature dependent in cold‐blooded vertebrates (CBVs) and thus directly impacted by global warming. We examined whether immunity and within‐host infectious disease progression are altered in CBVs under realistic climate warming in a seasonal mid‐latitude setting. Going further, we also examined how large thermal effects are in relation to the effects of other environmental variation in such a setting (critical to our ability to project infectious disease dynamics from thermal relationships alone). We employed the three‐spined stickleback and three ecologically relevant parasite infections as a “wild” model. To generate a realistic climatic warming scenario we used naturalistic outdoors mesocosms with precise temperature control. We also conducted laboratory experiments to estimate thermal effects on immunity and within‐host infectious disease progression under controlled conditions. As experimental readouts we measured disease progression for the parasites and expression in 14 immune‐associated genes (providing insight into immunophenotypic responses). Our mesocosm experiment demonstrated significant perturbation due to modest warming (+2°C), altering the magnitude and phenology of disease. Our laboratory experiments demonstrated substantial thermal effects. Prevailing thermal effects were more important than lagged thermal effects and disease progression increased or decreased in severity with increasing temperature in an infection‐specific way. Combining laboratory‐determined thermal effects with our mesocosm data, we used inverse modeling to partition seasonal variation in Saprolegnia disease progression into a thermal effect and a latent immunocompetence effect (driven by nonthermal environmental variation and correlating with immune gene expression). The immunocompetence effect was large, accounting for at least as much variation in Saprolegnia disease as the thermal effect. This suggests that managers of CBV populations in variable environments may not be able to reliably project infectious disease risk from thermal data alone. Nevertheless, such projections would be improved by primarily considering prevailing thermal effects in the case of within‐host disease and by incorporating validated measures of immunocompetence.     

Specific alleles at immune genes,rather than genome‐wide heterozygosity,are related to immunity and survival in the critically endangered Attwater's prairie‐chicken

The negative effects of inbreeding on fitness are serious concerns for populations of endangered species. Reduced fitness has been associated with lower genome‐wide heterozygosity and immune gene diversity in the wild; however, it is rare that both types of genetic measures are included in the same study. Thus, it is often unclear whether the variation in fitness is due to the general effects of inbreeding, immunity‐related genes or both. Here, we tested whether genome‐wide heterozygosity (20 990 SNPs) and diversity at nine immune genes were better predictors of two measures of fitness (immune response and survival) in the endangered Attwater's prairie‐chicken (Tympanuchus cupido attwateri). We found that postrelease survival of captive‐bred birds was related to alleles of the innate (Toll‐like receptors, TLRs) and adaptive (major histocompatibility complex, MHC) immune systems, but not to genome‐wide heterozygosity. Likewise, we found that the immune response at the time of release was related to TLR and MHC alleles, and not to genome‐wide heterozygosity. Overall, this study demonstrates that immune genes may serve as important genetic markers when monitoring fitness in inbred populations and that in some populations specific functional genes may be better predictors of fitness than genome‐wide heterozygosity.     

Vector competence and innate immune responses to dengue virus infection in selected laboratory and field‐collected Stegomyia aegypti (= Aedes aegypti)

Control of dengue virus (DenV) transmission, primarily based on strategies to reduce populations of the principle vector Stegomya aegypti (= Aedes aegypti) (Diptera: Culicidae), is difficult to sustain over time. Other potential strategies aim to manipulate characteristics such as vector competence (VC), the innate capacity of the vector to transmit the virus. Previous studies have identified genetic factors, including differential expression of apoptosis‐related genes, associated with the refractory and susceptible phenotypes in selected strains of S. aegypti from Cali, Colombia. The present study was designed to evaluate the variability of VC in selected strains against different DenV serotypes and to determine whether field‐collected mosquitoes respond similarly to selected laboratory strains in terms of enhanced or reduced expression of apoptosis‐related genes. Vector competence differed between strains, but did not differ in response to different DenV serotypes. Differences in VC were observed among mosquitoes collected from different localities in Cali. The overexpression of the pro‐apoptosis genes, caspase 16 and Aedronc, was conserved in field‐collected refractory mosquitoes and the selected laboratory refractory strain. The results suggest that the apoptosis response is conserved among all refractory mosquitoes to inhibit the development of all DenV serotypes.     

Intraguild predation leads to genetically based character shifts in the threespine stickleback

Intraguild predation is a common ecological interaction that occurs when a species preys upon another species with which it competes. The interaction is potentially a mechanism of divergence between intraguild prey (IG‐prey) populations, but it is unknown if cases of character shifts in IG‐prey are an environmental or evolutionary response. We investigated the genetic basis and inducibility of character shifts in threespine stickleback from lakes with and without prickly sculpin, a benthic intraguild predator (IG‐predator). Wild populations of stickleback sympatric with sculpin repeatedly show greater defensive armor and water column height preference. We laboratory‐raised stickleback from lakes with and without sculpin, as well as marine stickleback, and found that differences between populations in armor, body shape, and behavior persisted in a common garden. Within the common garden, we raised stickleback half‐families from multiple populations in the presence and absence of sculpin. Although the presence of sculpin induced trait changes in the marine stickleback, we did not observe an induced response in the freshwater stickleback. Behavioral and morphological trait differences between freshwater populations thus have a genetic basis and suggest an evolutionary response to intraguild predation.     

Evidence for genetic differentiation in timing of maturation among nine‐spined stickleback populations

Timing of maturation is an important life‐history trait that is likely to be subjected to strong natural selection. Although population differences in timing of maturation have been frequently reported in studies of wild animal populations, little is known about the genetic basis of this differentiation. Here, we investigated population and sex differences in timing of maturation within and between two nine‐spined stickleback (Pungitius pungitius) populations in a laboratory breeding experiment. We found that fish from the high‐predation marine population matured earlier than fish from the low‐predation pond population and males matured earlier than females. Timing of maturation in both reciprocal hybrid crosses between the two populations was similar to that in the marine population, suggesting that early timing of maturation is a dominant trait, whereas delayed timing of maturation in the pond is a recessive trait. Thus, the observed population divergence is suggestive of strong natural selection against early maturation in the piscine‐predator‐free pond population.     

Not going with the flow: Locomotor activity does not constrain immunity in a wild fish

Immunity is a central component of fitness in wild animals, but its determinants are poorly understood. In particular, the importance of locomotory activity as a constraint on immunity is unresolved. Using a piscine model (Gasterosteus aculeatus), we combined a 25‐month observational time series for a wild lotic habitat with an open flume experiment to determine the influence of locomotor activity (countercurrent swimming) on natural variation in immune function. To maximize the detectability of effects in our flume experiment, we set flow velocity and duration (10 cm/s for 48 hr) just below the point at which exhaustion would ensue. Following this treatment, we measured expression in a set of immune‐associated genes and infectious disease resistance through a standard challenge with an ecologically relevant monogenean infection (Gyrodactylus gasterostei). In the wild, there was a strong association of water flow with the expression of immune‐associated genes, but this association became modest and more complex when adjusted for thermal effects. Our flume experiment, although statistically well‐powered and based on a scenario near the limits of swimming performance in stickleback, detected no countercurrent swimming effect on immune‐associated gene expression or infection resistance. The field association between flow rate and immune expression could thus be due to an indirect effect, and we tentatively advance hypotheses to explain this. This study clarifies the drivers of immune investment in wild vertebrates; although locomotor activity, within the normal natural range, may not directly influence immunocompetence, it may still correlate with other variables that do.     

Measures of immune system status in young‐of‐the‐year winter flounder Pseudopleuronectes americanus

The immune status of young‐of‐the‐year (YOY) winter flounder Pseudopleuronectes americanus was evaluated in fish collected from six areas around Long Island, NY, U.S.A. representing more urban areas with high population density in the west, to less densely populated more rural areas in to the east. Gene expression markers for innate immunity (pleurocidin) and contaminant exposure (cytochrome P4501A; cyp1a) were measured in liver and fin of fish collected at each site. Expression of pleurocidin was significantly higher in fin than liver, but was highly variable among individuals. Some statistically significant differences in pleurocidin expression among sites were observed, although elevated levels were not associated with degree of urbanization. Expression was related in part to fish size: a positive correlation between expression and total length (L_T) of fish was observed with the largest L_T class (>125 mm) exhibiting significantly elevated pleurocidin expression as compared with fish in the smaller L_T class. This indicates that immune competency may increase with age. No site‐specific differences in cyp1a expression were observed. These data suggest that exposure to aromatic hydrocarbon contaminants is fairly widespread throughout the study area and that any differences in pleurocidin expression in YOY P. americanus are probably due to other factors. Antimicrobial activity was also measured as a functional indicator of immune response. Activity was highly variable, showing no significant site‐specific differences, and no significant correlation to pleurocidin expression. The lack of correlation between pleurocidin expression and antimicrobial activity indicates that other antimicrobial peptides may be active against the bacteria tested or that other factors are influencing antimicrobial activity. This is the first quantitative evaluation of pleurocidin expression in YOY P. americanus from an urban area. Further work is needed to characterize factors controlling pleurocidin expression, as well as other indicators of immune response in young fish.     

Natural variation in the contribution of microbial density to inducible immune dynamics

Immune responses evolve to balance the benefits of microbial killing against the costs of autoimmunity and energetic resource use. Models that explore the evolution of optimal immune responses generally include a term for constitutive immunity, or the level of immunological investment prior to microbial exposure, and for inducible immunity, or investment in immune function after microbial challenge. However, studies rarely consider the functional form of inducible immune responses with respect to microbial density, despite the theoretical dependence of immune system evolution on microbe‐ versus immune‐mediated damage to the host. In this study, we analyse antimicrobial peptide (AMP) gene expression from seven wild‐caught flour beetle populations (Tribolium spp.) during acute infection with the virulent bacteria Bacillus thuringiensis (Bt) and Photorhabdus luminescens (P.lum) to demonstrate that inducible immune responses mediated by the humoral IMD pathway exhibit natural variation in both microbe density‐dependent and independent temporal dynamics. Beetle populations that exhibited greater AMP expression sensitivity to Bt density were also more likely to die from infection, while populations that exhibited higher microbe density‐independent AMP expression were more likely to survive P. luminescens infection. Reduction in pathway signalling efficiency through RNAi‐mediated knockdown of the imd gene reduced the magnitude of both microbe‐independent and dependent responses and reduced host resistance to Bt growth, but had no net effect on host survival. This study provides a framework for understanding natural variation in the flexibility of investment in inducible immune responses and should inform theory on the contribution of nonequilibrium host‐microbe dynamics to immune system evolution.     

Male colour variation in a eurytopic African cichlid: the role of diet and hypoxia

Species that cross strong environmental gradients are expected to face divergent selective pressures that can act on sexually‐selected traits. In the present study, we examine the role of hypoxia and carotenoid availability in driving divergence in two sexually‐selected traits, male colour and reproductive behaviour, in the African cichlid Pseudocrenilabrus multicolor victoriae. Low‐dissolved oxygen (DO) (hypoxic) environments are expected to be energetically challenging; given that male nuptial colour expression and courtship displays can be costly, we expected fish in low‐DO versus high‐DO environments to differ in these traits. First, a field survey was used to describe natural variation in male nuptial colour patterns and diet across habitats divergent in DO. Next, using wild‐caught fish from a low‐DO and high‐DO habitat, we tested for differences in reproductive behaviour. Finally, a laboratory rearing experiment was used to quantify the interaction of DO and diet (low‐ versus high‐carotenoid availability) on the expression of male colour during development. In energetically challenging low‐DO environments, fish were more red and, in high‐DO environments, fish were typically brighter and more yellow. The frequency of reproductive displays in fish of low‐DO origin was 75% lower, although this had no consequence for brooding frequency (i.e. both populations produced the same number of broods on average). Our laboratory rearing study showed carotenoid availability to be important in colour production with no direct influence of DO on colour. Additionally, weak patterns of diet variation across wild populations suggest that other factors in combination with diet are contributing to colour divergence.     

Does plasticity enhance or dampen phenotypic parallelism? A test with three lake–stream stickleback pairs

Parallel (and convergent) phenotypic variation is most often studied in the wild, where it is difficult to disentangle genetic vs. environmentally induced effects. As a result, the potential contributions of phenotypic plasticity to parallelism (and nonparallelism) are rarely evaluated in a formal sense. Phenotypic parallelism could be enhanced by plasticity that causes stronger parallelism across populations in the wild than would be expected from genetic differences alone. Phenotypic parallelism could be dampened if site‐specific plasticity induced differences between otherwise genetically parallel populations. We used a common‐garden study of three independent lake–stream stickleback population pairs to evaluate the extent to which adaptive divergence has a genetic or plastic basis, and to investigate the enhancing vs. dampening effects of plasticity on phenotypic parallelism. We found that lake–stream differences in most traits had a genetic basis, but that several traits also showed contributions from plasticity. Moreover, plasticity was much more prevalent in one watershed than in the other two. In most cases, plasticity enhanced phenotypic parallelism, whereas in a few cases, plasticity had a dampening effect. Genetic and plastic contributions to divergence seem to play a complimentary, likely adaptive, role in phenotypic parallelism of lake–stream stickleback. These findings highlight the value of formally comparing wild‐caught and laboratory‐reared individuals in the study of phenotypic parallelism.     

Differences in rheotactic responses contribute to divergent habitat use between parapatric lake and stream threespine stickleback

Migration among populations is widely thought to undermine adaptive divergence, assuming gene flow arises from random movement of individuals. If individuals instead differ in dispersal behavior, phenotype‐dependent dispersal can reduce the effective rate of gene flow or even facilitate divergence. For example, parapatric populations of lake and stream stickleback tend to actively avoid dispersing into the adjoining habitat. However, the behavioral basis of this nonrandom dispersal was previously unknown. Here, we show that lake and stream stickleback exhibit divergent rheotactic responses (behavioral response to currents). During the breeding season, wild‐caught inlet stream stickleback were better than lake fish at maintaining position in currents, faced upstream more, and spent more time in low‐current areas. As a result, stream fish expended significantly less energy in currents than did lake fish. These divergent rheotactic responses likely contribute to divergent habitat use by lake and stream stickleback. Although rheotactic differences were absent in nonbreeding fish, divergent behavior of breeding‐season fish may suffice for assortative mating by breeding location. The resulting reproductive isolation between lake and stream fish may explain the fine‐scale evolutionary differentiation in parapatric stickleback populations.