Diet influences resource allocation in chemical defence but not melanin synthesis in an aposematic moth.

For animals that synthesise their chemical compounds de novo, resources, particularly proteins, can influence investment in chemical defences and nitrogen-based wing colouration such as melanin. Competing for the same resources often leads to trade-offs in resource allocation. We manipulated protein availability in the larval diet of the wood tiger moth, Arctia plantaginis, to test how early life resource availability influences relevant life history traits, melanin production and chemical defences. We expected higher dietary protein to result in more effective chemical defences in adult moths and a higher amount of melanin in the wings. According to the resource allocation hypothesis, we also expected individuals with less melanin to have more resources to allocate to chemical defences. We found that protein-deprived moths had a slower larval development, and their chemical defences were less unpalatable for bird predators, but the expression of melanin in their wings did not differ from that of moths raised on a high-protein diet. The amount of melanin in the wings, however, unexpectedly correlated positively with chemical defences. Our findings demonstrate that the resources available in early life have an important role in the efficacy of chemical defences, but melanin-based warning colours are less sensitive to resource variability than other fitness-related traits.

Not just the sum of its parts: Geographic variation and nonadditive effects of pyrazines in the chemical defence of an aposematic moth.

Chemical defences often vary within and between populations both in quantity and quality, which is puzzling if prey survival is dependent on the strength of the defence. We investigated the within- and between-population variability in chemical defence of the wood tiger moth (Arctia plantaginis). The major components of its defences, SBMP (2-sec-butyl-3-methoxypyrazine) and IBMP (2-isobutyl-3-methoxypyrazine), are volatiles that deter bird attacks. We hypothesized that (1) variation in the chemical defences of male wood tiger moths reflects the local predation pressure; (2) observed differences in quantity and quality of defence among populations have a genetic basis; and (3) increasing concentrations of SBMP and IBMP will elicit greater aversive reactions in predators, with the two pyrazines having an additive effect on predators' avoidance. We found that (1) the chemical defence of wild moths partly reflects local predator selection: high predation pressure populations (Scotland and Georgia) had stronger chemical defences, but not lower variance, than the low-predation populations (Estonia and Finland). (2) Based on the common garden results, both genetic and environmental components seem to influence the strength of chemical defence in moth populations; and (3) IBMP alone did not provide protection against bird predators but worked against bird attacks only when combined with SBMP, and while SBMP was more effective at higher concentrations, IBMP was not. Altogether this suggests that, when it comes to pyrazine concentration, more is not always better, highlighting the importance of testing the efficacy of chemical defence and its components with relevant predators, as extrapolating from chemical data may be less than straightforward.

Linking Predator Responses to Alkaloid Variability in Poison Frogs.

Many chemically-defended/aposematic species rely on diet for sequestering the toxins with which they defend themselves. This dietary acquisition can lead to variable chemical defenses across space, as the community composition of chemical sources is likely to vary across the range of (an aposematic) species. We characterized the alkaloid content of two populations of the Dyeing Poison Frog (Dendrobates tinctorius) in northeastern French Guiana. Additionally, we conducted unpalatability experiments with naive predators, Blue Tits (Cyanistes caeruleus), using whole-skin secretion cocktails to assess how a model predator would respond to the defense of individuals from each population. While there was some overlap between the two D. tinctorius populations in terms of alkaloid content, our analysis revealed that these two populations are markedly distinct in terms of overall alkaloid profiles. Predator responses to skin secretions differed between the populations. We identified 15 candidate alkaloids (including three previously undescribed) in seven classes that are correlated with predator response in one frog population. We describe alkaloid profile differences between populations for D. tinctorius and provide a novel method for assessing unpalatability of skin secretions and identifying which toxins may contribute to the predator response. In one population, our results suggest 15 alkaloids that are implicated in predator aversive response. This method is the first step in identifying the causal link between alkaloids and behavioral responses of predators, and thus makes sense of how varying alkaloid combinations are capable of eliciting consistent behavioral responses, and eventually driving evolutionary change in aposematic characters (or characteristics).

The Effect of Predator Population Dynamics on Batesian Mimicry Complexes.

AbstractUnderstanding Batesian mimicry is a classic problem in evolutionary biology. In Batesian mimicry, a defended species (the model) is mimicked by an undefended species (the mimic). Prior theories have emphasized the role of predator behavior and learning as well as evolution in model-mimic complexes but have not examined the role of population dynamics in potentially governing the relative abundances and even persistence of model-mimic systems. Here, we examined the effect of the population dynamics of predators and alternative prey on the prevalence of warning-signaling prey composed of models and mimics. Using optimal foraging theory and signal detection theory, we found that the inclusion of predator and alternative prey population dynamics could reverse traditional theoretical predictions: as alternative prey increase in numbers, mimics suffer because larger populations of predators are maintained, resulting in apparent competition. Under some circumstances, apparent competition affects model populations as well, although not as severely as it affects mimics. Our results bear on the intriguing puzzle that in nature warning signals are relatively scarce, yet experiments suggest that such signals can be highly advantageous. The availability of alternative prey and numerical responses by predators can overwhelm advantages observed in experiments to keep warning signals in model-mimic systems relatively scarce.

Genetic colour variation visible for predators and conspecifics is concealed from humans in a polymorphic moth.

The definition of colour polymorphism is intuitive: genetic variants express discretely coloured phenotypes. This classification is, however, elusive as humans form subjective categories or ignore differences that cannot be seen by human eyes. We demonstrate an example of a 'cryptic morph' in a polymorphic wood tiger moth (Arctia plantaginis), a phenomenon that may be common among well-studied species. We used pedigree data from nearly 20,000 individuals to infer the inheritance of hindwing colouration. The evidence supports a single Mendelian locus with two alleles in males: WW and Wy produce the white and yy the yellow hindwing colour. The inheritance could not be resolved in females as their hindwing colour varies continuously with no clear link with male genotypes. Next, we investigated if the male genotype can be predicted from their phenotype by machine learning algorithms and by human observers. Linear discriminant analysis grouped male genotypes with 97% accuracy, whereas humans could only group the yy genotype. Using vision modelling, we also tested whether the genotypes have differential discriminability to humans, moth conspecifics and their bird predators. The human perception was poor separating the genotypes, but avian and moth vision models with ultraviolet sensitivity could separate white WW and Wy males. We emphasize the importance of objective methodology when studying colour polymorphism. Our findings indicate that by-eye categorization methods may be problematic, because humans fail to see differences that can be visible for relevant receivers. Ultimately, receivers equipped with different perception than ours may impose selection to morphs hidden from human sight.

Weak warning signals can persist in the absence of gene flow.

Aposematic organisms couple conspicuous warning signals with a secondary defense to deter predators from attacking. Novel signals of aposematic prey are expected to be selected against due to positive frequency-dependent selection. How, then, can novel phenotypes persist after they arise, and why do so many aposematic species exhibit intrapopulation signal variability? Using a polytypic poison frog (Dendrobates tinctorius), we explored the forces of selection on variable aposematic signals using 2 phenotypically distinct (white, yellow) populations. Contrary to expectations, local phenotype was not always better protected compared to novel phenotypes in either population; in the white population, the novel phenotype evoked greater avoidance in natural predators. Despite having a lower quantity of alkaloids, the skin extracts from yellow frogs provoked higher aversive reactions by birds than white frogs in the laboratory, although both populations differed from controls. Similarly, predators learned to avoid the yellow signal faster than the white signal, and generalized their learned avoidance of yellow but not white. We propose that signals that are easily learned and broadly generalized can protect rare, novel signals, and weak warning signals (i.e., signals with poor efficacy and/or poor defense) can persist when gene flow among populations, as in this case, is limited. This provides a mechanism for the persistence of intrapopulation aposematic variation, a likely precursor to polytypism and driver of speciation.

Safety in Numbers: How Color Morph Frequency Affects Predation Risk in an Aposematic Moth.

AbstractPolymorphic warning signals in aposematic systems are enigmatic because predator learning should favor the most common form, creating positive frequency-dependent survival. However, many populations exhibit variation in warning signals. There are various selective mechanisms that can counter positive frequency-dependent selection and lead to temporal or spatial warning signal diversification. Examining these mechanisms and their effects requires first confirming whether the most common morphs are favored at both local and regional scales. Empirical examples of this are uncommon and often include potentially confounding factors, such as a lack of knowledge of predator identity and behavior. We tested how bird behavior influences the survival of three coexisting morphs of the aposematic wood tiger moth Arctia plantaginis offered to a sympatric predator (great tit Parus major) at different frequencies. We found that although positive frequency-dependent selection is present, its strength is affected by predator characteristics and varying prey profitability. These results highlight the need to understand predator foraging in natural communities with variable prey defenses in order to better examine how behavioral interactions shape evolutionary outcomes.

Antibiotics accelerate growth at the expense of immunity.

Antibiotics have long been used in the raising of animals for agricultural, industrial or laboratory use. The use of subtherapeutic doses in diets of terrestrial and aquatic animals to promote growth is common and highly debated. Despite their vast application in animal husbandry, knowledge about the mechanisms behind growth promotion is minimal, particularly at the molecular level. Evidence from evolutionary research shows that immunocompetence is resource-limited, and hence expected to trade off with other resource-demanding processes, such as growth. Here, we ask if accelerated growth caused by antibiotics can be explained by genome-wide trade-offs between growth and costly immunocompetence. We explored this idea by injecting broad-spectrum antibiotics into wood tiger moth (Arctia plantaginis) larvae during development. We follow several life-history traits and analyse gene expression (RNA-seq) and bacterial (r16S) profiles. Moths treated with antibiotics show a substantial depletion of bacterial taxa, faster growth rate, a significant downregulation of genes involved in immunity and significant upregulation of growth-related genes. These results suggest that the presence of antibiotics may aid in up-keeping the immune system. Hence, by reducing the resource load of this costly process, bodily resources may be reallocated to other key processes such as growth.

Hard to catch: experimental evidence supports evasive mimicry.

Most research on aposematism has focused on chemically defended prey, but the signalling difficulty of capture remains poorly explored. Similar to classical Batesian and Müllerian mimicry related to distastefulness, such 'evasive aposematism' may also lead to convergence in warning colours, known as evasive mimicry. A prime candidate group for evasive mimicry are Adelpha butterflies, which are agile insects and show remarkable colour pattern convergence. We tested the ability of naive blue tits to learn to avoid and generalize Adelpha wing patterns associated with the difficulty of capture and compared their response to that of birds that learned to associate the same wing patterns with distastefulness. Birds learned to avoid all wing patterns tested and generalized their aversion to other prey to some extent, but learning was faster with evasive prey compared to distasteful prey. Our results on generalization agree with longstanding observations of striking convergence in wing colour patterns among Adelpha species, since, in our experiments, perfect mimics of evasive and distasteful models were always protected during generalization and suffered the lowest attack rate. Moreover, generalization on evasive prey was broader compared to that on distasteful prey. Our results suggest that being hard to catch may deter predators at least as effectively as distastefulness. This study provides empirical evidence for evasive mimicry, a potentially widespread but poorly understood form of morphological convergence driven by predator selection.

Geographic mosaic of selection by avian predators on hindwing warning colour in a polymorphic aposematic moth.

Warning signals are predicted to develop signal monomorphism via positive frequency-dependent selection (+FDS) albeit many aposematic systems exhibit signal polymorphism. To understand this mismatch, we conducted a large-scale predation experiment in four countries, among which the frequencies of hindwing warning coloration of the aposematic moth, Arctia plantaginis, differ. Here we show that selection by avian predators on warning colour is predicted by local morph frequency and predator community composition. We found +FDS to be the strongest in monomorphic Scotland and lowest in polymorphic Finland, where the attack risk of moth morphs depended on the local avian community. +FDS was also found where the predator community was the least diverse (Georgia), whereas in the most diverse avian community (Estonia), hardly any models were attacked. Our results support the idea that spatial variation in predator communities alters the strength or direction of selection on warning signals, thus facilitating a geographic mosaic of selection.

Biased predation could promote convergence yet maintain diversity within Müllerian mimicry rings of Oreina leaf beetles.

Müllerian mimicry is a classic example of adaptation, yet Müller's original theory does not account for the diversity often observed in mimicry rings. Here, we aimed to assess how well classical Müllerian mimicry can account for the colour polymorphism found in chemically defended Oreina leaf beetles by using field data and laboratory assays of predator behaviour. We also evaluated the hypothesis that thermoregulation can explain diversity between Oreina mimicry rings. We found that frequencies of each colour morph were positively correlated among species, a critical prediction of Müllerian mimicry. Predators learned to associate colour with chemical defences. Learned avoidance of the green morph of one species protected green morphs of another species. Avoidance of blue morphs was completely generalized to green morphs, but surprisingly, avoidance of green morphs was less generalized to blue morphs. This asymmetrical generalization should favour green morphs: indeed, green morphs persist in blue communities, whereas blue morphs are entirely excluded from green communities. We did not find a correlation between elevation and coloration, rejecting thermoregulation as an explanation for diversity between mimicry rings. Biased predation could explain within-community diversity in warning coloration, providing a solution to a long-standing puzzle. We propose testable hypotheses for why asymmetric generalization occurs, and how predators maintain the predominance of blue morphs in a community, despite asymmetric generalization.

Appearance before performance? Nutritional constraints on life-history traits, but not warning signal expression in aposematic moths.

Trade-offs have been shown to play an important role in the divergence of mating strategies and sexual ornamentation, but their importance in explaining warning signal diversity has received less attention. In aposematic organisms, allocation costs of producing the conspicuous warning signal pigmentation under nutritional stress could potentially trade-off with life-history traits and maintain variation in warning coloration. We studied this with an aposematic herbivore Arctia plantaginis (Arctiidae), whose larvae and adults show extensive variation in aposematic coloration. In larvae, less melanic coloration (i.e. larger orange patterns) produces a more efficient warning signal against predators, whereas high amounts of melanism (smaller orange pattern) enhance thermoregulation, correlate with better immunity and make individuals harder to detect for naïve predators. We conducted a factorial rearing experiment with larvae originating from lines selected for either small or large orange signal size, which were reared on an artificial diet that had either low or high protein content. Protein content of the diet is critical for melanin production. We measured the effects of diet on individual coloration, life-history traits, immune defence and reproductive output. We also compared the responses to dietary conditions between the small and large larval signal genotypes. Protein content of the diet did not affect warning coloration in the larval stage, but larval signal sizes differed significantly among selection lines, confirming that its variation is mainly genetically determined. In adults, signal line or diet did not affect coloration in hindwings, but males' forewings had more melanin on the high than on low protein diet. Contrary to coloration, diet quality had a stronger impact on life-history traits: individuals developed for longer had smaller hindwing sizes in females and lower immune defence on the low protein content diet compared with the high. These costs were higher for more melanic larval signal genotypes in terms of development time and female hindwing size. We conclude that low plasticity in warning signal characteristics makes signal expression robust under varying dietary conditions. Therefore, variation in diet quality is not likely to constrain signal expression, but can have a bigger impact on performance.

Social learning within and across predator species reduces attacks on novel aposematic prey.

To make adaptive foraging decisions, predators need to gather information about the profitability of prey. As well as learning from prey encounters, recent studies show that predators can learn about prey defences by observing the negative foraging experiences of conspecifics. However, predator communities are complex. While observing heterospecifics may increase learning opportunities, we know little about how social information use varies across predator species. Social transmission of avoidance among predators also has potential consequences for defended prey. Conspicuous aposematic prey are assumed to be an easy target for naïve predators, but this cost may be reduced if multiple predators learn by observing single predation events. Heterospecific information use by predators might further benefit aposematic prey, but this remains untested. Here we test conspecific and heterospecific information use across a predator community with wild-caught blue tits (Cyanistes caeruleus) and great tits (Parus major). We used video playback to manipulate social information about novel aposematic prey and then compared birds' foraging choices in 'a small-scale novel world' that contained novel palatable and aposematic prey items. We expected that blue tits would be less likely to use social information compared to great tits. However, we found that both blue tits and great tits consumed fewer aposematic prey after observing a negative foraging experience of a demonstrator. In fact, this effect was stronger in blue tits compared to great tits. Interestingly, blue tits also learned more efficiently from watching conspecifics, whereas great tits learned similarly regardless of the demonstrator species. Together, our results indicate that social transmission about novel aposematic prey occurs in multiple predator species and across species boundaries. This supports the idea that social interactions among predators can reduce attacks on aposematic prey and therefore influence selection for prey defences.

De novo Synthesis of Chemical Defenses in an Aposematic Moth.

Many animals protect themselves from predation with chemicals, both self-made or sequestered from their diet. The potential drivers of the diversity of these chemicals have been long studied, but our knowledge of these chemicals and their acquisition mode is heavily based on specialist herbivores that sequester their defenses. The wood tiger moth (Arctia plantaginis, Linnaeus, 1758) is a well-studied aposematic species, but the nature of its chemical defenses has not been fully described . Here, we report the presence of two methoxypyrazines, 2-sec-butyl-3-methoxypyrazine and 2-isobutyl-3-methoxypyrazine, in the moths' defensive secretions. By raising larvae on an artificial diet, we confirm, for the first time, that their defensive compounds are produced de novo rather than sequestered from their diet. Pyrazines are known for their defensive function in invertebrates due to their distinctive odor, inducing aversion and facilitating predator learning. While their synthesis has been suspected, it has never previously been experimentally confirmed. Our results highlight the importance of considering de novo synthesis, in addition to sequestration, when studying the defensive capabilities of insects and other invertebrates.

Coinfection outcome in an opportunistic pathogen depends on the inter-strain interactions.

BACKGROUND: In nature, organisms are commonly coinfected by two or more parasite strains, which has been shown to influence disease virulence. Yet, the effects of coinfections of environmental opportunistic pathogens on disease outcome are still poorly known, although as host-generalists they are highly likely to participate in coinfections. We asked whether coinfection with conspecific opportunistic strains leads to changes in virulence, and if these changes are associated with bacterial growth or interference competition. We infected zebra fish (Danio rerio) with three geographically and/or temporally distant environmental opportunist Flavobacterium columnare strains in single and in coinfection. Growth of the strains was studied in single and in co-cultures in liquid medium, and interference competition (growth-inhibiting ability) on agar. RESULTS: The individual strains differed in their virulence, growth and ability for interference competition. Number of coinfecting strains significantly influenced the virulence of infection, with three-strain coinfection differing from the two-strain and single infections. Differences in virulence seemed to associate with the identity of the coinfecting bacterial strains, and their pairwise interactions. This indicates that benefits of competitive ability (production of growth-inhibiting compounds) for virulence are highest when multiple strains co-occur, whereas the high virulence in coinfection may be independent from in vitro bacterial growth. CONCLUSIONS: Intraspecific competition can lead to plastic increase in virulence, likely caused by faster utilization of host resources stimulated by the competitive interactions between the strains. However, disease outcome depends both on the characteristics of individual strains and their interactions. Our results highlight the importance of strain interactions in disease dynamics in environments where various pathogen genotypes co-occur.

How to fight multiple enemies: target-specific chemical defences in an aposematic moth.

Animals have evolved different defensive strategies to survive predation, among which chemical defences are particularly widespread and diverse. Here we investigate the function of chemical defence diversity, hypothesizing that such diversity has evolved as a response to multiple enemies. The aposematic wood tiger moth (Arctia plantaginis) displays conspicuous hindwing coloration and secretes distinct defensive fluids from its thoracic glands and abdomen. We presented the two defensive fluids from laboratory-reared moths to two biologically relevant predators, birds and ants, and measured their reaction in controlled bioassays (no information on colour was provided). We found that defensive fluids are target-specific: thoracic fluids, and particularly 2-sec-butyl-3-methoxypyrazine, which they contain, deterred birds, but caused no aversive response in ants. By contrast, abdominal fluids were particularly deterrent to ants, while birds did not find them repellent. Our study, to our knowledge, is the first to show evidence of a single species producing separate chemical defences targeted to different predator types, highlighting the importance of taking into account complex predator communities in studies on the evolution of prey defence diversity.

Colour and luminance contrasts predict the human detection of natural stimuli in complex visual environments.

Much of what we know about human colour perception has come from psychophysical studies conducted in tightly-controlled laboratory settings. An enduring challenge, however, lies in extrapolating this knowledge to the noisy conditions that characterize our actual visual experience. Here we combine statistical models of visual perception with empirical data to explore how chromatic (hue/saturation) and achromatic (luminant) information underpins the detection and classification of stimuli in a complex forest environment. The data best support a simple linear model of stimulus detection as an additive function of both luminance and saturation contrast. The strength of each predictor is modest yet consistent across gross variation in viewing conditions, which accords with expectation based upon general primate psychophysics. Our findings implicate simple visual cues in the guidance of perception amidst natural noise, and highlight the potential for informing human vision via a fusion between psychophysical modelling and real-world behaviour.

Deimatism: a neglected component of antipredator defence.

Deimatic or 'startle' displays cause a receiver to recoil reflexively in response to a sudden change in sensory input. Deimatism is sometimes implicitly treated as a form of aposematism (unprofitability associated with a signal). However, the fundamental difference is, in order to provide protection, deimatism does not require a predator to have any learned or innate aversion. Instead, deimatism can confer a survival advantage by exploiting existing neural mechanisms in a way that releases a reflexive response in the predator. We discuss the differences among deimatism, aposematism, and forms of mimicry, and their ecological and evolutionary implications. We highlight outstanding questions critical to progress in understanding deimatism.

Lepidopteran species have a variety of defence strategies against bacterial infections.

The insect immune system has versatile ways of coping with microbial insults. Currently, innate immune priming has been described in several invertebrates, and the first insights into its mechanistic basis have been described. Here we studied infections with two different strains of Serratia marcescens bacteria in two different Lepidopteran hosts. The results reveal fundamental differences between the two hosts, a well-known model organism Galleria mellonella and a non-model species Arctia plantaginis. They differ in their strategies for resisting oral infections; priming their defences against a recurring sepsis; and upregulating immunity related genes as a response to the specific pathogen strains. The two bacterial strains (an environmental isolate and an entomopathogenic isolate) differ in their virulence, use of extracellular proteases, survival in the larval gut, and in the immune response they evoke in the hosts. This study explores the potential mechanistic explanations for both host and pathogen specific characters that significantly affect the outcome of Gram-negative bacterial infection in Lepidopteran larvae. The results highlight the need to pay greater attention to the differences between model and non-model hosts, and closely related pathogen strains, in immunological studies.

Intensive aquaculture selects for increased virulence and interference competition in bacteria.

Although increased disease severity driven by intensive farming practices is problematic in food production, the role of evolutionary change in disease is not well understood in these environments. Experiments on parasite evolution are traditionally conducted using laboratory models, often unrelated to economically important systems. We compared how the virulence, growth and competitive ability of a globally important fish pathogen, Flavobacterium columnare, change under intensive aquaculture. We characterized bacterial isolates from disease outbreaks at fish farms during 2003-2010, and compared F. columnare populations in inlet water and outlet water of a fish farm during the 2010 outbreak. Our data suggest that the farming environment may select for bacterial strains that have high virulence at both long and short time scales, and it seems that these strains have also evolved increased ability for interference competition. Our results are consistent with the suggestion that selection pressures at fish farms can cause rapid changes in pathogen populations, which are likely to have long-lasting evolutionary effects on pathogen virulence. A better understanding of these evolutionary effects will be vital in prevention and control of disease outbreaks to secure food production.