Multimodal Aposematic Defenses Through the Predation Sequence

Aposematic organisms warn predators of their unprofitability using a combination of defenses, including visual warning signals, startling sounds, noxious odors, or aversive tastes. Using multiple lines of defense can help prey avoid predators by stimulating multiple senses and/or by acting at different stages of predation. We tested the efficacy of three lines of defense (color, smell, taste) during the predation sequence of aposematic wood tiger moths (Arctia plantaginis) using blue tit (Cyanistes caeruleus) predators. Moths with two hindwing phenotypes (genotypes: WW/Wy = white, yy = yellow) were manipulated to have defense fluid with aversive smell (methoxypyrazines), body tissues with aversive taste (pyrrolizidine alkaloids) or both. In early predation stages, moth color and smell had additive effects on bird approach latency and dropping the prey, with the strongest effect for moths of the white morph with defense fluids. Pyrrolizidine alkaloid sequestration was detrimental in early attack stages, suggesting a trade-off between pyrrolizidine alkaloid sequestration and investment in other defenses. In addition, pyrrolizidine alkaloid taste alone did not deter bird predators. Birds could only effectively discriminate toxic moths from non-toxic moths when neck fluids containing methoxypyrazines were present, at which point they abandoned attack at the consumption stage. As a result, moths of the white morph with an aversive methoxypyrazine smell and moths in the treatment with both chemical defenses had the greatest chance of survival. We suggest that methoxypyrazines act as context setting signals for warning colors and as attention alerting or "go-slow" signals for distasteful toxins, thereby mediating the relationship between warning signal and toxicity. Furthermore, we found that moths that were heterozygous for hindwing coloration had more effective defense fluids compared to other genotypes in terms of delaying approach and reducing the latency to drop the moth, suggesting a genetic link between coloration and defense that could help to explain the color polymorphism. Conclusively, these results indicate that color, smell, and taste constitute a multimodal warning signal that impedes predator attack and improves prey survival. This work highlights the importance of understanding the separate roles of color, smell and taste through the predation sequence and also within-species variation in chemical defenses.Peer reviewe

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.Peer reviewe

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

Polymorphic 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.Peer reviewe

Lisämateriaali julkaisuun: 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 20000 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 categorisation 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. This dataset contains supplementary data to the article that handles the issue described above. For detailed description of the material, methods, and results of the study, see the article

Heterozygote advantage and pleiotropy contribute to intraspecific color trait variability

The persistence of intrapopulation phenotypic variation typically requires some form of balancing selection since drift and directional selection eventually erode genetic variation. Heterozygote advantage remains a classic explanation for the maintenance of genetic variation in the face of selection. However, examples of heterozygote advantage, other than those associated with disease resistance are rather uncommon. Across most of its distribution, males of the aposematic moth Arctia plantaginis have two hindwing phenotypes determined by a heritable one locus-two allele polymorphism (genotypes: WW/Wy = white morph, yy = yellow morph). Using genotyped moths we show that the presence of one or two copies of the yellow allele affects several life-history traits. Reproductive output of both males and females, and female mating success are negatively affected by two copies of the yellow allele. Females carrying one yellow allele (i.e. Wy) have higher fertility, hatching success, and offspring survival than either homozygote, thus leading to strong heterozygote advantage. Our results indicate strong female contribution especially at the postcopulatory stage in maintaining the color polymorphism. The interplay between heterozygote advantage, yellow allele pleiotropic effect and morph-specific predation pressure may exert balancing selection on the color locus, suggesting that color polymorphism may be maintained through complex interactions between natural and sexual selection.peerReviewe

Colour polymorphism associated with a gene duplication in male wood tiger moths

Colour is often used as an aposematic warning signal, with predator learning expected to lead to a single colour pattern within a population. However, there are many puzzling cases where aposematic signals are also polymorphic. The wood tiger moth, Arctia plantaginis, displays bright hindwing colours associated with unpalatability, and males have discrete colour morphs which vary in frequency between localities. In Finland, both white and yellow morphs can be found, and these colour morphs also differ in behavioural and life-history traits. Here, we show that male colour is linked to an extra copy of a yellow family gene that is only present in the white morphs. This white-specific duplication, which we name valkea, is highly upregulated during wing development. CRISPR targeting valkea resulted in editing of both valkea and its paralog, yellow-e, and led to the production of yellow wings. We also characterise the pigments responsible for yellow, white and black colouration, showing that yellow is partly produced by pheomelanins, while black is dopamine-derived eumelanin. Our results add to a growing number of studies on the genetic architecture of complex and seemingly paradoxical polymorphisms, and the role of gene duplications and structural variation in adaptive evolution.peerReviewe

Colour polymorphism associated with a gene duplication in male wood tiger moths

Colour is often used as an aposematic warning signal, with predator learning expected to lead to a single colour pattern within a population. However, there are many puzzling cases where aposematic signals are also polymorphic. The wood tiger moth, Arctia plantaginis, displays bright hindwing colours associated with unpalatability, and males have discrete colour morphs which vary in frequency between localities. In Finland, both white and yellow morphs can be found, and these colour morphs also differ in behavioural and life-history traits. Here, we show that male colour is linked to an extra copy of a yellow family gene that is only present in the white morphs. This white-specific duplication, which we name valkea, is highly upregulated during wing development. CRISPR targeting valkea resulted in editing of both valkea and its paralog, yellow-e, and led to the production of yellow wings. We also characterise the pigments responsible for yellow, white, and black colouration, showing that yellow is partly produced by pheomelanins, while black is dopamine-derived eumelanin. Our results add to a growing number of studies on the genetic architecture of complex and seemingly paradoxical polymorphisms, and the role of gene duplications and structural variation in adaptive evolution.Peer reviewe

Colour polymorphism associated with a gene duplication in male wood tiger moths.

Peer reviewed: TrueAcknowledgements: We thank Alma Oksanen, Kaisa Suisto, and the greenhouse staff for insect rearing, and Elisa Salmivirta and Sari Viinikainen for lab assistance. Thanks to Emeritus Prof. Shosuke Ito for kindly providing pheomelanin standards, Bodo Wilts for advice on the pigment analyses, and James Barnett for help with the spectrophotometry. We thank Muktai Kuwalekar, Claudius Kratochwil, Rachel Blow, Ian Warren, Tom Generalovic, and Joe Hanly for providing equipment and advice regarding the CRISPR injections. Funding This work was supported by the Academy of Finland grants to MB (#343356) and JM (projects 345091 and 328474), and a Biotechnology and Biological Sciences Research Council (BBSRC) grant to CJ (046_BB_V0145X_1).Colour is often used as an aposematic warning signal, with predator learning expected to lead to a single colour pattern within a population. However, there are many puzzling cases where aposematic signals are also polymorphic. The wood tiger moth, Arctia plantaginis, displays bright hindwing colours associated with unpalatability, and males have discrete colour morphs which vary in frequency between localities. In Finland, both white and yellow morphs can be found, and these colour morphs also differ in behavioural and life-history traits. Here, we show that male colour is linked to an extra copy of a yellow family gene that is only present in the white morphs. This white-specific duplication, which we name valkea, is highly upregulated during wing development. CRISPR targeting valkea resulted in editing of both valkea and its paralog, yellow-e, and led to the production of yellow wings. We also characterise the pigments responsible for yellow, white, and black colouration, showing that yellow is partly produced by pheomelanins, while black is dopamine-derived eumelanin. Our results add to a growing number of studies on the genetic architecture of complex and seemingly paradoxical polymorphisms, and the role of gene duplications and structural variation in adaptive evolution