Pheromones are central to the mating systems of a wide range of organisms, and reproductive isolation between closely related species is often achieved by subtle differences in pheromone composition. In insects and moths in particular, the use of structurally similar components in different blend ratios is usually sufficient to impede gene flow between taxa. To date, the genetic changes associated with variation and divergence in pheromone signals remain largely unknown. Using the emerging model system Ostrinia, we show the functional consequences of mutations in the protein-coding region of the pheromone biosynthetic fatty-acyl reductase gene pgFAR. Heterologous expression confirmed that pgFAR orthologs encode enzymes exhibiting different substrate specificities that are the direct consequences of extensive nonsynonymous substitutions. When taking natural ratios of pheromone precursors into account, our data reveal that pgFAR substrate preference provides a good explanation of how species-specific ratios of pheromone components are obtained among Ostrinia species. Moreover, our data indicate that positive selection may have promoted the observed accumulation of nonsynonymous amino acid substitutions. Site-directed mutagenesis experiments substantiate the idea that amino acid polymorphisms underlie subtle or drastic changes in pgFAR substrate preference. Altogether, this study identifies the reduction step as a potential source of variation in pheromone signals in the moth genus Ostrinia and suggests that selection acting on particular mutations provides a mechanism allowing pheromone reductases to evolve new functional properties that may contribute to variation in the composition of pheromone signals.

Females of the solitary endoparasitoid Cotesia vestalis respond to a blend of volatile organic compounds (VOCs) released from plants infested with larvae of their host, the diamondback moth (Plutella xylostella), which is an important pest insect of cruciferous plants. We investigated the flight response of female parasitoids to the cruciferous plant Brassica rapa, using two-choice tests under laboratory conditions. The parasitoids were more attracted to plants that had been infested for at least 6 hr by the host larvae compared to intact plants, but they did not distinguish between plants infested for only 3 hr and intact plants. Although parasitoids preferred plants 1 and 2 days after herbivory (formerly infested plants) over intact plants they also preferred plants that had been infested for 24 hr over formerly infested plants. This suggests that parasitoids can distinguish between the VOC profiles of currently and formerly infested plants. We screened for differences in VOC emissions among the treatments and found that levels of benzyl cyanide and dimethyl trisulfide significantly decreased after removal of the host larvae, whereas terpenoids and their related compounds continued to be released at high levels. Benzyl cyanide and dimethyl trisulfide attracted parasitoids in a dose-dependent manner, whereas the other compounds were not attractive. These results suggest that nitrile and sulfide compounds temporarily released from plants under attack by host larvae are potentially more effective attractants for this parasitoid than other VOCs that are continuously released by host-damaged plants.

Bacteria of the genus Spiroplasma are widely found in plants and arthropods. Some of the maternally transmitted Spiroplasma endosymbionts in arthropods are known to kill young male hosts (male killing). Here, we describe a new case of Spiroplasma-induced male killing in a moth, Ostrinia zaguliaevi. The all-female trait caused by Spiroplasma was maternally inherited for more than 11 generations but was spontaneously lost in several lineages. Antibiotic treatment eliminated the Spiroplasma infection and restored the 1:1 sex ratio. The survival rates and presence/absence of the W chromosome in the embryonic and larval stages of O. zaguliaevi showed that males were selectively killed, exclusively during late embryogenesis in all-female broods. Based on phylogenetic analyses of 16S rRNA, dnaA and rpoB gene sequences, the causative bacteria were identified as Spiroplasma belonging to the tick symbiont Spiroplasma ixodetis clade. Electron microscopy confirmed bacterial structures in the follicle cells and follicular sheath of adult females. Although many congeneric Ostrinia moths harbor another sex ratio-distorting bacterium (Wolbachia), only O. zaguliaevi harbors Spiroplasma.

Powdery mildews (Erysiphales) are economically important plant pathogens that attack many agricultural crops. Conventional management strategies involving fungicide application face challenges, including the evolution of resistance and concerns over impacts on non-target organisms, that call for investigation of more sustainable alternatives. Mycophagous ladybird beetles (Coleoptera: Coccinellidae) feed on powdery mildew and have considerable potential as biological control agents; however, the foraging ecology and behavior of these beetles is not well understood. Here we document the olfactory cues presented by squash plants (Cucurbita moschata) infected by powdery mildew (Podosphaera sp.) and the behavioral responses of twenty-spotted ladybird beetles (Psyllobora vigintimaculata) to these cues. Volatile analyses through gas chromatography revealed a number of volatile compounds characteristic of infected plants, including 3-octanol and its analogues 1-octen-3-ol and 3-octanone. These compounds are typical “moldy” odorants previously reported in volatiles collected from other fungi. In addition, infected plants exhibited elevated emissions of several compounds also observed in collections from healthy leaves, including linalool and benzyl alcohol, which are reported to have anti-fungal properties. In Y-tube choice assays, P. vigintimaculata beetles displayed a significant preference for the odors of infected plants compared to those of healthy plants. Moreover, beetles exhibited strong attraction to one individual compound, 1-octen-3-ol, which was the most abundant of the characteristic fungal compounds identified. These results enhance our understanding of the olfactory cues that guide foraging by mycophagous insects and may facilitate the development of integrated disease-management strategies informed by an understanding of underlying ecological mechanisms.