Limited effects of a glyphosate-based herbicide on the behaviour and immunity of males from six populations of the European earwig.

The use of herbicides on crops often results in unintentional, low-dose exposure of non-target organisms, such as insects. While these exposures are increasingly known to alter the survival and physiology of insects, it remains unclear whether these effects can vary between populations and modify other fitness-related traits, such as behaviour and immunity. Here, we addressed these questions by testing the effects of sublethal exposure to a glyphosate-based herbicide (GBH) on the behaviour and immunity of European earwig males from six natural populations. We exposed each male to a dose of a common GBH (Roundup©) that was either recommended for crops, five times lower than that recommended for crops, or to a control solution. Twenty-four hours later, we measured the activity, boldness, and aggregation of each male. We then exposed them to an entomopathogenic fungus, monitored their survival for 6 weeks, and measured the immune response of the survivors. We found a condition-dependent effect of GBH exposure on male activity. Exposure to low doses induced a positive association between activity and weight, which was not observed in the high-dose and control groups. However, GBH had no effect on any of the other measured traits. All these results were consistent across the six populations tested, although we did find population-specific differences in almost all measurements on males. Further research is now needed to better understand the dose-response to GBH on male activity and its biological impact, as well as to evaluate the effectiveness of detoxification processes in this species. Overall, these results emphasise the importance of investigating the effects of herbicides on insects to expand our general understanding of the use and potential risks of plant protection products in integrated pest management programs.

The Biology and Social Life of Earwigs (Dermaptera).

Earwigs are often known for the forceps-like appendage at the end of their abdomen, urban legends about them crawling into human ears, and their roles as pest and biological control agents. However, they are much less known for their social life. This is surprising, as many of the 1,900 species of earwigs show social behaviors toward eggs, juveniles, and adults. These behaviors typically occur during family and group living, which may be obligatory or facultative, last up to several months, and involve only a few to several hundred related or unrelated individuals. Moreover, many individuals can alternate between solitary and group living during their life cycle, an ability that probably prevailed during the emergence of social life. In this review, I detail the diversity of group living and social behavior in earwigs and show how further developing this knowledge in Dermaptera can improve our general understanding of the early evolution of social life in insects.

Exposure to pyriproxyfen (juvenile hormone agonist) does not alter maternal care and reproduction in the European earwig.

Sublethal exposure to pesticides can alter the survival and reproduction of a wide range of non-target organisms. However, it remains unclear whether this exposure can alter behaviours that are often essential for long-term population dynamics and maintenance, such as parental care. In this study, we tested the effect of pyriproxyfen exposure (an insect growth regulator) on maternal care in the European earwig, an insect that is both used in pest control in pip-fruit orchards and considered a pest in stone fruit orchards. We exposed 424 females at doses either 10 times lower, equivalent or 10 times higher than normal application rates in French orchards. As maternal care can change over the weeks of family life, we exposed the earwig mothers at five different days before and after egg hatching. We then measured the expression of ten forms of maternal care towards eggs and juveniles, six non-caring behaviours, eggs and juvenile development, metabolic reserves in mothers at egg hatching and females' production of a terminal clutch. First, our results revealed that the three tested doses of pyriproxyfen were non-lethal and confirmed that maternal care decreased throughout both pre- and post-hatching family life. However, we did not detect any effect of pyriproxyfen on maternal care and non-care behaviours, eggs and juvenile development, quantities of lipids, proteins and glycogen in mothers at egg hatching, and on the production of a future clutch. Overall, these findings suggest that the maximal doses of pyriproxyfen authorized in French orchards is likely to have limited effects on the short- and long-term maintenance of populations of the European earwig and raises fundamental questions about the nature of the link between juvenile hormone and parental care in insects.

Earwig mothers consume the feces of their juveniles during family life.

Many animals consume the feces of their conspecifics. This allo-coprophagy can have benefits, such as access to nutrients and symbionts, but also risks for consumers, mainly due to direct contact with pathogens that develop on feces. In the European earwig Forficula auricularia, mothers and juveniles live in nests lined with their feces. This surprising habit allows juveniles to consume the feces of their siblings during family life and provides them with nutritional benefits when mothers provide low care. However, it was unclear whether earwig mothers also practice allo-coprophagy, and whether this behavior is motivated by their nutritional needs. Here, we set up four types of experimental families in which we manipulated the nutritional needs of mothers and/or juveniles and measured the effects on the production of feces by the juveniles, and the consumption of these feces by the mothers. Our results first show that fed juveniles produced more feces pellet in presence of fed compared to food-deprived mothers. We also found that, overall, about 50% of the mothers consumed juveniles feces. This consumption was both more likely and larger when the feces were produced by fed compared to food-deprived juveniles, while the proportion of feces pellets eaten was larger in food-deprived compared to fed mothers. Overall, our results reveal that allo-coprophagy involves every family member and suggest that it can have both nutritional and non-nutritional benefits for earwig mothers. Allo-coprophagy could thus favor the maintenance of mothers in the nest and, more generally, promote the early evolution of family life.

Effects of temperature, fungal infection and weight on intermoult duration and survival of starving earwig larvae.

Moulting is a cornerstone of arthropods development. It can be determined by numerous factors such as body mass, temperature, and immunity. However, the effects of these factors can be dependent on each other, so that it is often difficult to predict whether and how they shape moulting, and whether their effects are additive or interactive. In this study, we addressed these questions by testing the effects of body mass, ambient temperature, fungal infection and their interaction on intermoult duration and survival in starved juveniles of the European earwig Forficula auricularia. We recorded the date of moult and death of a total of 207 earwig juveniles that were weighed, exposed to different doses of the entomopathogenic fungus Metarizium brunneum and then maintained at either 20 °C or 24 °C. Our results first reveal that juveniles moulted earlier when they were heavy compared to light on the day of exposure, as well as earlier when maintained at 24 °C compared to 20 °C. By contrast, pathogen exposure did not affect the moulting date. We also found that nymphs died faster when they were light compared to heavy on the day of exposure, when they were exposed to high (106 and 107 spores/ml) compared to low (104, 105 and 0 spores/ml) pathogen concentrations, and when they were maintained at 24 °C compared to 20 °C. We detected no sign of interaction between temperature, fungal infection and body mass on both moulting and survival. Overall, these findings shed light on the limited importance of infection on moulting in starved juveniles, and reveal that weight, temperature, and infection have additive effects on their survival. More generally, this study emphasizes that the three tested factors do not necessarily interact to shape key physiological processes in an insect.

Alteration of gut microbiota with a broad-spectrum antibiotic does not impair maternal care in the European earwig.

The microbes residing within the gut of an animal host often increase their own fitness by modifying their host's physiological, reproductive and behavioural functions. Whereas recent studies suggest that they may also shape host sociality and therefore have critical effects on animal social evolution, the impact of the gut microbiota on maternal care remains unexplored. This is surprising, as this behaviour is widespread among animals, often determines the fitness of both juveniles and parents, and is essential in the evolution of complex animal societies. Here, we tested whether life-long alterations of the gut microbiota with rifampicin-a broad-spectrum antibiotic-impair pre- and post-hatching maternal care in the European earwig. Our results first confirm that rifampicin altered the mothers' gut microbial communities and indicate that the composition of the gut microbiota differs before and after egg care. Contrary to our predictions, however, the rifampicin-induced alterations of the gut microbiota did not modify pre- or post-hatching care. Independent of maternal care, rifampicin increased the females' faeces production and resulted in lighter eggs and juveniles. By contrast, rifampicin altered none of the other 21 physiological, reproductive and longevity traits measured over the 300 days of a female's lifetime. Overall, these findings reveal that altering the gut microbiota with a large spectrum antibiotic such as rifampicin does not necessarily affect host sociality. They also emphasize that not all animals have evolved a co-dependence with their microbiota and call for caution when generalizing the central role of gut microbes in host biology.

Sublethal exposure to deltamethrin stimulates reproduction and has limited effects on post-hatching maternal care in the European earwig.

Although pesticides are typically used to limit pest population, the diversity and nature of their unintentional effects on non-target organisms remain unclear. Better understanding these effects requires to carry out risk assessments on key physiological and behavioral processes specific to beneficial insects. In this study, we addressed this question by exposing mothers of the European earwig (a beneficial insect) to two sublethal doses of deltamethrin (a common pesticide in agriculture) during family life and measured the short- and long-term effects on a series of behavioral, physiological, and reproductive traits. Somewhat surprisingly, our results first revealed that high and low doses of deltamethrin enhanced mothers' future reproduction by augmenting their likelihood to produce a second clutch, shortening the number of days until its production, and increasing the resulting number of eggs and their hatching rate. Conversely, the high dose of deltamethrin was detrimental, as it limited maternal brood defence, and reduced food consumption and expression of self-grooming. Finally, other traits were independent of deltamethrin exposure, such as three proxies of family interactions (i.e., distance to the brood, occurrence, and duration of mother-offspring contacts), mothers' walking distance, and mother weight gain during family life. Our study overall demonstrates that sublethal exposure to a pesticide such as deltamethrin can have both positive and negative effects on non-target beneficial insects. It thus emphasizes that focusing on narrow parameters can lead to misleading conclusions about the unintended impacts of pesticides in treated agro-ecosystems and call for better considering this parameters diversity in integrated pest management programs.

Reproductives and eggs trigger worker vibration in a subterranean termite.

In insect societies, the presence of reproductives or eggs has been shown to shape several biological traits in the colony members. Social interactions are one of these traits that involve modification of the communication system of the entire colony. Many studies described the role of chemical compounds and dominance behaviors in the presence of reproductive but vibratory behaviors received very few investigations. Yet, vibratory behaviors are ideal candidates, particularly for subterranean species like termites, as they could be quickly transmitted through the substrate and could be very diversified (origin, modulation). Here, we investigated whether the presence of reproductives/eggs affects the vibratory behavior (body-shaking) of workers in the subterranean termite Reticulitermes flavipes. Our results reveal that the presence of reproductives or eggs triggers an increase of workers' body-shaking, independent of their colony of origin after 24 hr. We hypothesize that vibratory communication could be used to transfer information about the presence of reproductives and eggs to the entire colony, suggesting that vibratory behaviors could serve as an important yet neglected mediator of social regulation.

Sublethal exposure to deltamethrin impairs maternal egg care in the European earwig Forficula auricularia.

The application of pesticides typically leads to lethal and sublethal exposure of non-target insects. Whereas our current understanding of these sublethal effects typically focuses on reproductive and physiological parameters, recent works emphasize that sublethal effects on behaviors such as maternal care could be of major importance in non-target species. However, it remained unknown whether these sublethal effects occur in insects. Here, we tested if exposure to sublethal doses of deltamethrin - a pyrethroid insecticide commonly used in crops - alters the expression of maternal egg care in females of the European earwig Forficula auricularia, a predator insect and pest control. Our results first reveal that deltamethrin exposure impaired the expression of three forms of maternal egg care: It decreased the likelihood of mothers to gather their otherwise scattered clutch of eggs, increased the time during which the female abandoned the clutch after a predator attack and reduced egg grooming duration. These sublethal effects did not reflect a lower activity of deltamethrin-exposed females, as these females increased their expression of self-grooming, and deltamethrin exposure did not affect females' exploration and mobility. Finally, we found that the negative effects of deltamethrin on egg care did not modify egg development, hatching rate and juvenile weight, possibly due to the transient effects of deltamethrin on maternal behaviors. Overall, our results reveal that sublethal exposure to a pesticide may diminish maternal egg care in a natural pest control and call for the integration of this measurement in assays on pesticides application.

Offspring reverse transcriptome responses to maternal deprivation when reared with pathogens in an insect with facultative family life.

Offspring of species with facultative family life are able to live with and without parents (i.e. to adjust to extreme changes in their social environment). While these adjustments are well understood on a phenotypic level, their genetic underpinnings remain surprisingly understudied. Investigating gene expression changes in response to parental absence may elucidate the genetic constraints driving evolutionary transitions between solitary and family life. Here, we manipulated maternal presence to observe gene expression changes in the fat body of juvenile European earwigs, an insect with facultative family life. Because parents typically protect offspring against pathogens, expression changes were recorded in pathogen-free and pathogen-exposed environments. We found that manipulating maternal presence changed the expression of 154 genes, including several metabolism and growth-related genes, and that this change depended on pathogen presence. Specifically, localization and cell transporter genes were downregulated in maternal absence without pathogens but upregulated with pathogens. At least one immunity gene (pathogenesis-related protein 5) was affected by pathogen exposure regardless of maternal presence. Overall, our findings explicate how offspring adjust to parental deprivation on a molecular level and reveal that such adjustments heavily depend on pathogens in the environment. This emphasizes the central role of pathogens in family life evolution.

Friend or foe? The apparent benefits of gregarine (Apicomplexa: Sporozoa) infection in the European earwig.

Studying the costs and benefits of host-parasite interactions is of central importance to shed light on the evolutionary drivers of host life history traits. Although gregarines (Apicomplexa: Sporozoa) are one of the most frequent parasites in the gut of invertebrates, the diversity of its potential impacts on a host remains poorly explored. In this study, we addressed this gap in knowledge by investigating the prevalence of natural infections by the gregarine Gregarina ovata and testing how these infections shape a large set of morphological, behavioural and physiological traits in the European earwig Forficula auricularia. Our results first show that G. ovata was present in 76.8% of 573 field-sampled earwigs, and that its prevalence was both higher in males compared with females and increased between July and September. The load of G. ovata in the infected individuals was higher in males than females, but this sex difference vanished during the season. Our experiments then surprisingly revealed apparent benefits of G. ovata infections. Food-deprived hosts survived longer when they exhibited high compared with low gregarine loads. Moreover, the presence of gregarines was associated with a reduced phenoloxidase activity, indicating a lower immune resistance or a higher immune tolerance of the infected hosts. By contrast, we found no effect of G. ovata presence and number on earwigs' development (eye distance, forceps length), activity, food consumption or resistance against a fungal pathogen. Overall, our findings suggest that G. ovata could be involved in a mutualistic relationship with the European earwig. Given the ubiquitous presence of gregarines among invertebrates, our data also suggest that this common member of insect gut flora could have a broad and positive role in the life history of many host species.

Variations in seasonal (not mean) temperatures drive rapid adaptations to novel environments at a continent scale.

The recent development of human societies has led to major, rapid, and often inexorable changes in the environment of most animal species. Over the last decades, a growing number of studies formulated predictions on the modalities of animal adaptation to novel or changing environments, questioning how and at what speed animals should adapt to such changes, discussing the levels of risks imposed by changes in the mean and/or variance of temperatures on animal performance, and exploring the underlying roles of phenotypic plasticity and genetic inheritance. These fundamental predictions, however, remain poorly tested using field data. Here, we tested these predictions using a unique continental-scale data set in the European earwig Forficula auricularia L., a univoltine insect introduced in North America one century ago. We conducted a common garden experiment, in which we measured 13 life-history traits in 4,158 field-sampled earwigs originating from 19 populations across North America. Our results first demonstrate that 10 of the 13 measured life-history traits are associated with two sets of variations in seasonal temperatures, that is, winter-summer and autumn-spring. We found, however, no association with the overall mean monthly temperatures of the invaded locations. Furthermore, our use of a common garden setup reveals that the observed patterns of variation in earwigs' life-history traits are not mere plastic responses to their current environment, but are either due to their genetic background and/or to the environmental conditions they experienced during early life development. Overall, these findings provide continent-scale support to the claims that adaptation to thermal changes can occur quickly (in less than 100 generations), even in insects with long life cycles, and emphasize the importance of variation in seasonal temperature over mean population temperatures in climate adaptation.

Efficient but occasionally imperfect vertical transmission of gut mutualistic protists in a wood-feeding termite.

Although mutualistic associations between animals and microbial symbionts are widespread in nature, the mechanisms that have promoted their evolutionary persistence remain poorly understood. A vertical mode of symbiont transmission (from parents to offspring) is thought to ensure partner fidelity and stabilization, although the efficiency of vertical transmission has rarely been investigated, especially in cases where hosts harbour a diverse microbial community. Here we evaluated vertical transmission rates of cellulolytic gut oxymonad and parabasalid protists in the wood-feeding termite Reticulitermes grassei. We sequenced amplicons of the 18S rRNA gene of protists from 24 colonies of R. grassei collected in two populations. For each colony, the protist community was characterized from the gut of 14 swarming reproductives and from a pool of 10 worker guts. A total of 98 operational taxonomic units belonging to 13 species-level taxa were found. The vertical transmission rate was estimated for each protist present in a colony based on its frequency among the reproductives. The results revealed that transmission rates were high, with an average of 0.897 (±0.164) per protist species. Overall, the protist community did not differ between reproductive sexes, suggesting that both the queen and the king could contribute to the gut microbiota of the offspring. A positive relationship between the transmission rate of protists and their prevalence within populations was also detected. However, transmission rates alone do not explain the prevalence of protists. In conclusion, these findings reveal key forces behind a conserved, multispecies mutualism, raising further questions on the roles of horizontal transfer and negative selection in shaping symbiont prevalence.

Joint effects of group sex-ratio and Wolbachia infection on female reproductive success in the terrestrial isopod Armadillidium vulgare.

BACKGROUND: In species that reproduce with sexual reproduction, males and females often have opposite strategies to maximize their own fitness. For instance, males are typically expected to maximize their number of mating events, whereas an excessive number of mating events can be costly for females. Although the risk of sexual harassment by males and resulting costs for females are expected to increase with the proportion of males, it remains unknown whether and how parasitic distorters of a host population's sex-ratio can shape this effect on the fitness of females. Here, we addressed this question using Armadillidium vulgare and its parasite Wolbachia that alters the sex-ratio of a population. We set up Wolbachia-free and Wolbachia-infected females in experimental groups exhibiting 100, 80, 50% or 20% females for 1 year, during which we measured changes in survival, fertility and fecundity. RESULTS: Wolbachia infection shaped the effects of both population sex-ratio and reproductive season on female fecundity. Compared to Wolbachia-free females, Wolbachia-infected females were less likely to be gravid in populations exhibiting an excess of females and did not exhibit the otherwise negative effect of seasons on this likelihood. Group sex-ratio and Wolbachia infection have independent effects on other measured traits. Male-biased populations had females both exhibiting the lowest survival rate after 6 months and producing the smallest number of offspring, independent of Wolbachia infection. Conversely, Wolbachia-infected females had the lowest likelihood of producing at least one offspring, independent of group sex-ratio. Wolbachia infection had no effect on female survival rate. CONCLUSIONS: We demonstrated that male-biased sex-ratio and the presence of Wolbachia are costly for females due to sexual harassment by males and bacterial infection, respectively. Interestingly, Wolbachia infection triggers another negative effect. This effect does not come from an excess of males and its associated sexual harassment of females but instead from a lack of males and the associated risk for females of remaining unmated. Overall, these findings highlight the importance of social pressures and infection on female fitness and provide insights into our general understanding of the joint and opposite effects of these two parameters in the evolution of reproductive strategies.

Unbalanced biparental care during colony foundation in two subterranean termites.

Parental care is a major component of reproduction in social organisms, particularly during the foundation steps. Because investment into parental care is often costly, each parent is predicted to maximize its fitness by providing less care than its partner. However, this sexual conflict is expected to be low in species with lifelong monogamy, because the fitness of each parent is typically tied to the other's input. Somewhat surprisingly, the outcomes of this tug-of-war between maternal and paternal investments have received important attention in vertebrate species, but remain less known in invertebrates. In this study, we investigated how queens and kings share their investment into parental care and other social interactions during colony foundation in two termites with lifelong monogamy: the invasive species Reticulitermes flavipes and the native species R. grassei. Behaviors of royal pairs were recorded during six months using a non-invasive approach. Our results showed that queens and kings exhibit unbalanced investment in terms of grooming, antennation, trophallaxis, and vibration behavior. Moreover, both parents show behavioral differences toward their partner or their descendants. Our results also revealed differences among species, with R. flavipes exhibiting shorter periods of grooming and antennation toward eggs or partners. They also did more stomodeal trophallaxis and less vibration behavior. Overall, this study emphasizes that despite lifelong monogamy, the two parents are not equally involved in the measured forms of parental care and suggests that kings might be specialized in other tasks. It also indicates that males could play a central, yet poorly studied role in the evolution and maintenance of the eusocial organization.

The other facets of family life and their role in the evolution of animal sociality.

Family life forms an integral part of the life history of species across the animal kingdom and plays a crucial role in the evolution of animal sociality. Our current understanding of family life, however, is almost exclusively based on studies that (i) focus on parental care and associated family interactions (such as those arising from sibling rivalry and parent-offspring conflict), and (ii) investigate these phenomena in the advanced family systems of mammals, birds, and eusocial insects. Here, we argue that these historical biases have fostered the neglect of key processes shaping social life in ancestral family systems, and thus profoundly hamper our understanding of the (early) evolution of family life. Based on a comprehensive survey of the literature, we first illustrate that the strong focus on parental care in advanced social systems has deflected scrutiny of other important social processes such as sibling cooperation, parent-offspring competition and offspring assistance. We then show that accounting for these neglected processes - and their changing role over time - could profoundly alter our understanding of the origin and subsequent evolution of family life. Finally, we outline how this 'diachronic' perspective on the evolution of family living provides novel insights into general processes driving the evolution of animal sociality. Overall, we infer that the explicit consideration of thus-far neglected facets of family life, together with their study across the whole diversity of family systems, are crucial to advance our understanding of the processes that shape the evolution of social life.

Social immunity: why we should study its nature, evolution and functions across all social systems.

Mounting defences against pathogens is a necessity for all animals. Although these defences have long been known to rely on individual processes such as the immune system, recent studies have emphasized the importance of social defences for group-living hosts. These defences, called social immunity, have been mostly studied in eusocial insects such as bees, termites and ants, and include, for instance, mutual cleaning and waste management. Over the last few years, however, a growing number of works called for a broader exploration of social immunity in non-eusocial species. In this review, we summarize the rationales of this call and examine why it may provide major insights into our current understanding of the role of pathogens in social evolution. We start by presenting the original conceptual framework of social immunity developed in eusocial insects and shed light on its importance in highly derived social systems. We then clarify three major misconceptions possibly fostered by this original framework and demonstrate why they made necessary the shift towards a broader definition of social immunity. Because a broader definition still needs boundaries, we finally present three criteria to discriminate what is a form of social immunity, from what is not. Overall, we argue that studying social immunity across social systems does not only provide novel insights into how pathogens affect the evolution of eusociality, but also of the emergence and maintenance of social life from a solitary state. Moreover, this broader approach offers new scopes to disentangle the common and specific anti-pathogen defences developed by eusocial and non-eusocial hosts, and to better understand the dependent and independent evolutionary drivers of social and individual immunity.

Extended winters entail long-term costs for insect offspring reared in an overwinter burrow.

Winter imposes an ecological challenge to animals living in colder climates, especially if these adverse conditions coincide with reproduction and offspring rearing. To overcome this challenge, some insects burrow in the soil to protect adults, larvae, or eggs from negative effects of winter. However, whether this protection is effective against any long-term consequences of changes in winter duration is unclear. Here, we investigated the long-term effects of winter length variation on eggs of the European earwig Forficula auricularia. In this insect, females construct and maintain a burrow between late autumn and spring, in which they provide extensive forms of care to their eggs and then juveniles. We experimentally maintained earwig females under two winter durations of either four or six weeks and examined the resulting effects in terms of 1) hatching date, 2) developmental time of juveniles until adulthood, 3) adult mass at emergence, and 4) investment of adult offspring females in three key immune parameters: hemocyte concentration, phenoloxidase, and prophenoloxidase activities. Because earwigs' resistance against pathogens relies on their social environment, effects of winter length on immunity were tested on females exposed to different social environments: with familiar conspecifics, unfamiliar conspecifics, or in isolation. Our results reveal that after the winter treatments, eggs reared in short winters hatched earlier and the emerging juveniles reached adulthood faster than juveniles from eggs exposed to long winters. We also showed that prophenoloxidase was 30% higher in females from the long compared to short winter treatment, regardless of social environment. Finally, we found that hemocyte counts where twice as high in short compared to long winter females, but only with unfamiliar conspecifics. Overall, our study reveals that maintaining and caring for eggs in a burrow does not prevent the costs associated with increased winter duration.

Age, sex, mating status, but not social isolation interact to shape basal immunity in a group-living insect.

Immunity is a crucial but costly trait. Individuals should therefore adjust their investment into immunity to their condition and infection risks, which are often determined by their age, sex, mating status and social environment. However, whether and how these four key factors can interact to shape basal immunity remains poorly understood. Here, we tested the simultaneous effects of these factors on hemocyte concentration and phenoloxidase activity in adults of the European earwig. We found that hemocyte concentration increased with age, and that this increase was stronger in males. We also found an age-dependent increase in phenoloxidase activity in males and virgin females, but not in mated females. However, the two immune parameters were independent of social isolation. Overall, our results reveal that a complex interplay between age, sex and mating status determines basal immunity and stress the importance of interactions in our understanding of immune investment.