A mosaic of endogenous and plant-derived courtship signals in moths.

Insects rely on olfaction to guide a wide range of adaptive behaviors, including mate and food localization, mate choice, oviposition site selection, kin recognition, and predator avoidance.1 In nocturnal insects, such as moths2 and cockroaches,3 mate finding is stimulated predominantly by long-range species-specific sex pheromones, typically emitted by females. During courtship, at close range, males in most moth species emit a blend of pheromone compounds from an everted, often large, pheromone gland. While long-distance communication with sex pheromones has been remarkably well characterized in thousands of moth species,2,4 close-range chemosensory sexual communication remains poorly understood. We reveal that in the moth Chloridea virescens, the male pheromone consists of three distinct classes of compounds: de novo biosynthesized alcohols, aldehydes, acetates, and carboxylic acids that resemble the female's emissions; newly identified compounds that are unique to the male pheromone, such as aliphatic polyunsaturated hydrocarbons; and sequestered plant secondary compounds and hormone derivatives, including methyl salicylate (MeSA). Thus, males employ a mosaic pheromone blend of disparate origins that may serve multiple functions during courtship. We show that two olfactory receptors in female antennae are tuned to MeSA, which facilitates female acceptance of the male. Because MeSA is emitted by plants attacked by pathogens and herbivores,5 the chemosensory system of female moths was likely already tuned to this plant volatile, and males appear to exploit the female's preadapted sensory bias. Interestingly, while female moths (largely nocturnal) and butterflies (diurnal) diverged in their use of sensory modalities in sexual communication,6 MeSA is used by males of both lineages.

Alcohol Contributes to Attraction of Heliothis (= Chloridea) virescens Males to Females.

Female-emitted volatile sex pheromones in most moths are composed of biosynthetically related blends of fatty acid derivatives, such as aldehydes, acetate esters and alcohols. In many moths, as in the noctuid Heliothis (Chloridea) virescens, the pheromone gland contains alcohols (e.g., (Z)-11-hexadecen-1-ol, hereafter Z11-16:OH) that may serve dual functions as pheromone components as well as precursors of other pheromone components. The relative importance of Z11-16:OH to male attraction in H. virescens has been controversial. It occurs in the pheromone gland in relatively large amounts, but several studies could neither detect Z11-16:OH in gland emissions nor attribute any conspecific behavioral function to it in flight- tunnel assays. Trapping assays in the field, however, have more consistently documented that the addition of Z11-16:OH increased trap catch. Using a short section of thick film megabore column, in combination with derivatization and GC-CI-SIM-MS, we determined that Z11-16:OH is emitted from the sex pheromone gland during calling. Field trapping studies demonstrated that trap catch increased when Z11-16:OH was added to a 2-component minimal blend and to a 6-component blend. Behavioral observations in the field confirmed that more males responded to a pheromone blend that contained a low blend ratio of Z11-16:OH, but ≥5% Z11-16:OH depressed both male behavior and trap catch. We conclude that Z11-16:OH should be considered a component of the sex pheromone of H. virescens females.

Adaptive Divergence in a Defense Symbiosis Driven from the Top Down.

Most studies of adaptive radiation in animals focus on resource competition as the primary driver of trait divergence. The roles of other ecological interactions in shaping divergent phenotypes during such radiations have received less attention. We evaluate natural enemies as primary agents of diversifying selection on the phenotypes of an actively diverging lineage of gall midges on tall goldenrod. In this system, the gall of the midge consists of a biotrophic fungal symbiont that develops on host-plant leaves and forms distinctly variable protective carapaces over midge larvae. Through field studies, we show that fungal gall morphology, which is induced by midges (i.e., it is an extended phenotype), is under directional and diversifying selection by parasitoid enemies. Overall, natural enemies disruptively select for either small or large galls, mainly along the axis of gall thickness. These results imply that predators are driving the evolution of phenotypic diversity in symbiotic defense traits in this system and that divergence in defensive morphology may provide ecological opportunities that help to fuel the adaptive radiation of this genus of midges on goldenrods. This enemy-driven phenotypic divergence in a diversifying lineage illustrates the potential importance of consumer-resource and symbiotic species interactions in adaptive radiation.

Proximity of signallers can maintain sexual signal variation under stabilizing selection.

How sexual communication systems can evolve under stabilizing selection is still a paradox in evolutionary biology. In moths, females emit a species-specific sex pheromone, consisting of a blend of biochemically related components, to which males are attracted. Although males appear to exert strong stabilizing selection on female pheromone, these blends seem to have evolved rapidly, as evidenced by ~120,000 moth species. Here we propose and test a "proximity model" wherein two females that vary in their relative attractiveness to males, can both benefit from calling in close proximity to each other. In a field study, we show that (1) artificially selected unattractive females can achieve mating rates comparable to attractive females if they signal in close proximity to attractive females, and (2) attractive females benefit from higher mating rates when signalling in close proximity to unattractive females. We propose that frequency-dependent behavioural and spatial interactions can sustain signal variation within populations even when these signals are under stabilizing selection.

Exploring plant defense theory in tall goldenrod, Solidago altissima.

Understanding the evolutionary reasons for patterns of chemical defense in plants is an ongoing theoretical and empirical challenge. The goal is to develop a model that can reliably predict how defenses are distributed within the plant over space and time. This is difficult given that evolutionary, ecological, and physiological processes and tradeoffs can operate over different spatial and temporal scales. We evaluated the major predictions of two leading defense theories, the growth-differentiation balance hypothesis (GDBH) and optimal defense theory (ODT). To achieve this, enemies, fitness components, terpenoids, and protease inhibitors were measured in Solidago altissima and used to construct conventional univariate and structural equation models (SEMs). Leaf-tissue value indices extracted from an SEM revealed a strong correlation between tissue value and terpenoid defense that supports ODT. A tradeoff between serine protease inhibition and growth as well as an indirect tradeoff between growth and terpenoids manifested through galling insects supported the GDBH. Interestingly, there was a strong direct effect of terpenoids on rhizome mass, suggesting service to both storage and defense. The results support established theories but unknown genotypic traits explained much of the variation in defense, confirming the need to integrate emerging theories such as pollination constraints, defense syndromes, tolerance, mutualisms, and facilitation.

Carotenoids in unexpected places: gall midges, lateral gene transfer, and carotenoid biosynthesis in animals.

Carotenoids are conjugated isoprenoid molecules with many important physiological functions in organisms, including roles in photosynthesis, oxidative stress reduction, vision, diapause, photoperiodism, and immunity. Until recently, it was believed that only plants, microorganisms, and fungi were capable of synthesizing carotenoids and that animals acquired them from their diet, but recent studies have demonstrated that two arthropods (pea aphid and spider mite) possess a pair of genes homologous to those required for the first step of carotenoid biosynthesis. Absent in all other known animal genomes, these genes appear to have been acquired by aphids and spider mites in one or several lateral gene transfer events from a fungal donor. We report the third case of fungal carotenoid biosynthesis gene homologs in an arthropod: flies from the family Cecidomyiidae, commonly known as gall midges. Using phylogenetic analyses we show that it is unlikely that lycopene cyclase/phytoene synthase and phytoene desaturase homologs were transferred singly to an ancient arthropod ancestor; instead we propose that genes were transferred independently from related fungal donors after divergence of the major arthropod lineages. We also examine variation in intron placement and copy number of the carotenoid genes that may underlie function in the midges. This trans-kingdom transfer of carotenoid genes may represent a key innovation, underlying the evolution of phytophagy and plant-galling in gall midges and facilitating their extensive diversification across plant lineages.