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.

Predator-Induced Plasticity on Warning Signal and Larval Life-History Traits of the Aposematic Wood Tiger Moth, Arctia plantaginis

Predator-induced plasticity in life-history and antipredator traits during the larval period has been extensively studied in organisms with complex life-histories. However, it is unclear whether different levels of predation could induce warning signals in aposematic organisms. Here, we investigated whether predator-simulated handling affects warning coloration and life-history traits in the aposematic wood tiger moth larva, Arctia plantaginis. As juveniles, a larger orange patch on an otherwise black body signifies a more efficient warning signal against predators but this comes at the costs of conspicuousness and thermoregulation. Given this, one would expect that an increase in predation risk would induce flexible expression of the orange patch. Prior research in this system points to plastic effects being important as a response to environmental changes for life history traits, but we had yet to assess whether this was the case for predation risk, a key driver of this species evolution. Using a full-sib rearing design, in which individuals were reared in the presence and absence of a non-lethal simulated bird attack, we evaluated flexible responses of warning signal size (number of orange segments), growth, molting events, and development time in wood tiger moths. All measured traits except development time showed a significant response to predation. Larvae from the predation treatment developed a more melanized warning signal (smaller orange patch), reached a smaller body size, and molted more often. Our results suggest plasticity is indeed important in aposematic organisms, but in this case may be complicated by the trade-off between costly pigmentation and other life-history traits.

Molecular phylogeny of the Lichen Tiger Moths (Lepidoptera: Erebidae: Arctiinae: Lithosiini) of the Western Hemisphere

Few molecular-based studies have tested the monophyly of the Lithosiini subtribes proposed by Bendib and Minet (2000), and those have been limited by meager taxon sampling. Several studies have suggested some subtribes are not monophyletic as they are currently defined. We conducted a molecular phylogenetic study of representatives of the North American lithosiine fauna which are currently classified within the subtribes Acsalina, Lithosiina, Cisthenina and Eudesmiina, the latter having never been included in a molecular-based analysis before. Based on analyses of cytochrome oxidase subunit I (COI), ribosomal protein S5 (RPS5) and the large subunit 28S ribosomal DNA (28S), we assign some of the North American genera to subtribe for the first time and re-assign others contrary to previous placements. Then, we discuss the morphological characters that Bendib and Minet (2000) proposed to define subtribes and re-consider them in the context of our inferred phylogeny. We report high support for a monophyletic Lithosiina+ Agylla + Inopsis + Gnamptonychia, three genera being unplaced or of uncertain placement (Agylla) by Bendib and Minet (2000). We remove Gardinia from the subtribe Lithosiina and place it in the Cisthenina, along with Eudesmia, formerly placed in its own subtribe, the Eudesmiina. Two other genera, Bruceia and Ptychoglene, not previously assigned to a subtribe are found to be members of the Cisthenina. We remove Clemensia from the Cisthenina and report it, along with the neotropical Pronola, as part of undefined clade. After these changes, our phylogeny shows strong support for the monophyly of Cisthenina + Gardinia + Eudesmia + Bruceia + Ptychoglene. We find Acsala anomala occurs on a long branch by itself, confirming the uniqueness of this species and its placement in a monotypic subtribe. Finally, we confirm that Afrida exegens, sometimes considered a member of the Lithosiini, is not even an erebid, but rather it is a member of the Nolidae, as proposed by Holloway (1998) and Kitching and Rawlins (1998).

Survival Sounds in Insects: Diversity, Function, and Evolution

Insect defense sounds have been reported for centuries. Yet, aside from the well-studied anti-bat sounds of tiger moths, little is understood about the occurrence, function, and evolution of these sounds. We define a defense sound as an acoustic signal (air- or solid-borne vibration) produced in response to attack or threat of attack by a predator or parasitoid and that promotes survival. Defense sounds have been described in 12 insect orders, across different developmental stages, and between sexes. The mechanisms of defensive sound production include stridulation, percussion, tymbalation, tremulation, and forced air. Signal characteristics vary between species, and we discuss how morphology, the intended receiver, and specific functions of the sounds could explain this variation. Sounds can be directed at predators or non-predators, and proposed functions include startle, aposematism, jamming, and alarm, although experimental evidence for these hypotheses remains scant for many insects. The evolutionary origins of defense sounds in insects have not been rigorously investigated using phylogenetic methodology, but in most cases it is hypothesized that they evolved from incidental sounds associated with non-signaling behaviors such as flight or ventilatory movements. Compared to our understanding of visual defenses in insects, sonic defenses are poorly understood. We recommend that future investigations focus on testing hypotheses explaining the functions and evolution of these survival sounds using predator-prey experiments and comparative phylogenetics.

Extreme duty cycles in the acoustic signals of tiger moths: Sexual and natural selection operating in parallel

Sound production in tiger moths (Erebidae: Arctiinae) plays a role in natural selection. Some species use tymbal sounds as jamming signals avoiding bat predation. High duty cycle signals have the greatest efficacy in this regard. Tiger moth sounds can also be used for intraspecific communication. Little is known about the role of sound in the mating behavior of jamming species or the signal preferences underlying mate choice. We recorded sound production during the courtship of two high duty cycle arctiines, Bertholdia trigona and Carales arizonensis. We characterized variation in their acoustic signals, measured female preference for male signals that vary in duty cycle, and performed female choice experiments to determine the effect of male duty cycle on the acceptance of male mates. Although both species produced sound during courtship, the role of acoustic communication appears different between the species. Bertholdia trigona was acoustically active in all intraspecific interactions. Females preferred and ultimately mated with males that produced higher duty cycles. Muted males were never chosen. In C. arizonensis however, sound emissions were limited during courtship and in some successful matings no sound was detected. Muted and clicking males were equally successful in female mate-choice experiments, indicating that acoustic communication is not essential for mating in C. arizonensis. Our results suggest that in B. trigona natural and sexual selection may work in parallel, to favor higher duty cycle clicking.

Emperors, admirals and giants, zebras, tigers and woolly bears: casting a broader net in exploring heparin effects on Lepidoptera wing patterns

Background: Studies of heparin effects on Lepidoptera wing patterns have been restricted to a small number of species. I report observations from experiments on a broader range of taxa, including first results from swallowtails, tiger moths and microlepidoptera. Methods: Heparin injections were made in prepupae and pupae of Junonia coenia (common buckeyes), Agraulis vanillae (gulf fritillaries), Heliconius charithonia (zebra longwings), Asterocampa clyton (tawny emperors), Danaus plexippus (monarchs), Vanessa atalanta (red admirals); Heraclides cresphontes (giant swallowtails), Pterourus troilus (spicebush swallowtails), Protographium marcellus (zebra swallowtails), Battus polydamas (polydamas swallowtails); Hypercompe scribonia (giant leopard moths), Estigmene acrea (acrea moths), Hyphantria cunea (fall webworm moths), Utetheisa ornatrix (ornate bella moths); Glyphodes sibillalis (mulberry leaftier). Results: Heparin sometimes altered the entire pattern in a dramatic way, sometimes caused changes locally. In buckeyes, the previous heparin study conducted on pupae was compared to injections made at a prepupal stage. In gulf fritillaries, zebra longwings and tawny emperors, the dramatic changes occurred throughout their wings, while in monarchs, changes were restricted to wing margins. Changes achieved in red admirals, show that heparin action is unrelated to the original color. In swallowtails, transformations were restricted to border system, indicating higher levels of stability and compartmentalization of wing patterns. In mulberry leaftier, changes were restricted to the marginal bands. In tiger moths, elongation of black markings led to merging of spots; in the ornate bella moth, it was accompanied by an expansion of the surrounding white bands, and results were compared to the effects of colder temperatures. Conclusions: Using pharmaceutical intervention demonstrates that there are many similarities and some very significant differences in the ways wing patterns are formed in different Lepidoptera lineages. By creating a range of variation one can demonstrate how one pattern can easily evolve into another, aiding in understanding of speciation and adaptation processes.

Emperors, admirals and giants, zebras, tigers and woolly bears: casting a broader net in exploring heparin effects on Lepidoptera wing patterns

Background: Studies of heparin effects on Lepidoptera wing patterns have been restricted to a small number of species. I report observations from experiments on a broader range of taxa, including first results from swallowtails, tiger moths and microlepidoptera. Methods: Heparin injections were made in prepupae and pupae of Junonia coenia (common buckeyes), Agraulis vanillae (gulf fritillaries), Heliconius charithonia (zebra longwings), Asterocampa clyton (tawny emperors), Danaus plexippus (monarchs), Vanessa atalanta (red admirals); Heraclides cresphontes (giant swallowtails), Pterourus troilus (spicebush swallowtails), Protographium marcellus (zebra swallowtails), Battus polydamas (polydamas swallowtails); Hypercompe scribonia (giant leopard moths), Estigmene acrea (acrea moths), Hyphantria cunea (fall webworm moths), Utetheisa ornatrix (ornate bella moths); Glyphodes sibillalis (mulberry leaftier). Results: Heparin sometimes altered the entire pattern in a dramatic way, sometimes caused changes locally. In buckeyes, the previous heparin study conducted on pupae was compared to injections made at a prepupal stage. In gulf fritillaries, zebra longwings and tawny emperors, the dramatic changes occurred throughout their wings, while in monarchs, changes were restricted to wing margins. Changes achieved in red admirals, show that heparin action is unrelated to the original color. In swallowtails, transformations were restricted to border system, indicating higher levels of stability and compartmentalization of wing patterns. In mulberry leaftier, changes were restricted to the marginal bands. In tiger moths, elongation of black markings led to merging of spots; in the ornate bella moth, it was accompanied by an expansion of the surrounding white bands, and results were compared to the effects of colder temperatures. Conclusions: Using pharmaceutical intervention demonstrates that there are many similarities and some very significant differences in the ways wing patterns are formed in different Lepidoptera lineages. By creating a range of variation one can demonstrate how one pattern can easily evolve into another, aiding in understanding of speciation and adaptation processes.

Vanewrightia gen. nov.—A Highly Variable Taxon of Neotropical Ctenuchina (Lepidoptera: Erebidae: Arctiinae: Arctiini) Revealed by Behavioral Traits

A series of different-looking tiger moths was collected at pyrrolizidine alkaloid baits in the daytime in Peru. They proved to be variants of a new genus, Vanewrightia gen. nov., and a new species, Vanewrightia kiesela sp. nov., both described here. This species presents a striking example of extensive intraspecific variation within a population. Its discovery demonstrates the importance of sampling moths by means other than collecting with light, of studying patterns of wing undersides, and of the value of barcoding. Specimens found in collections considered here potentially to represent further species of the new genus are documented and discussed; Vanewrightia subflavescens (Kaye, 1911) comb. nov., and Vanewrightia patawaensis (Cerda, 2017) comb. nov. are established; we propose Epidesma parva (Rothschild, 1912) as a junior synonym of E. aurimacula (Schaus, 1905). The newly recognized intraspecific variation greatly challenges delimitation of morphospecies and uncovers uncertainties in the taxonomy of Epidesma Hübner, [1819]. The occurrence of an oblique forewing band in many Lepidoptera and the stunning similarity in overall appearance of variants of Vanewrightia with unrelated taxa, in particular notodontid moths (Josiini) and Chamaelimnas C. & R. Felder, [1885] butterflies (Riodinidae), are discussed in the context of mimicry and crypsis, and some perspectives for further research are suggested.