Herbivory is a key innovation in insects, yet has only evolved in onethird of living orders. The evolution of herbivory likely involves major behavioral changes mediated by remodeling of canonical chemosensory modules. Herbivorous flies in the genus Scaptomyza (Drosophilidae) are compelling species in which to study the genomic architecture linked to the transition to herbivory because they recently evolved from microbe-feeding ancestors and are closely related to Drosophila melanogaster. We found that Scaptomyza flava, a leaf-mining specialist on plants in the family (Brassicaceae), was not attracted to yeast volatiles in a four-field olfactometer assay, whereas D. melanogaster was strongly attracted to these volatiles. Yeast-associated volatiles, especially short-chain aliphatic esters, elicited strong antennal responses in D. melanogaster, but weak antennal responses in electroantennographic recordings from S. flava. We sequenced the genome of S. flava and characterized this species' odorant receptor repertoire. Orthologs of odorant receptors, which detect yeast volatiles in D. melanogaster and mediate critical host-choice behavior, were deleted or pseudogenized in the genome of S. flava. These genes were lost step-wise during the evolution of Scaptomyza. Additionally, Scaptomyza has experienced gene duplication and likely positive selection in paralogs of Or67b in D. melanogaster. Olfactory sensory neurons expressing Or67b are sensitive to green-leaf volatiles. Major trophic shifts in insects are associated with chemoreceptor gene loss as recently evolved ecologies shape sensory repertoires.plant-herbivore interactions | gene loss | olfaction | Drosophila melanogaster | Scaptomyza flava U nderstanding the origins and consequences of trophic shifts, especially the transition to herbivory, has been a central problem in evolutionary biology. The paleontological record suggests that evolutionary transitions to herbivory have been rare in insects (1), and the first transitions to herbivory in vertebrates occurred long after the colonization of land (2). However, species radiations result from herbivorous transitions in insects and vertebrates, suggesting that herbivory is a key innovation (3, 4). Identifying functional genomic changes associated with the evolutionary transition to herbivory could yield insight into the mechanisms that have driven their success. However, the origins of the most diverse clades of herbivorous insects are ancient and date to the Jurassic or earlier (5), limiting meaningful genomic comparisons. In contrast, herbivory has evolved more times in Diptera than in any other order (3). The Drosophilidae is an excellent system to study the evolution of herbivory from a functional genomic perspective because it includes several transitions to herbivory, and the genomic model Drosophila melanogaster (6, 7).The transition to herbivory involves adaptations in physiology (8-10), morphology (11), and behavior (12). The evolution of sensory repertoires could reinforce or even precipitate these adapt...

SUMMARY Drosophila melanogaster (Meigen) detects and uses many volatiles for its survival. Carbon dioxide (CO2) is detected in adults by a special class of olfactory receptor neurons, expressing the gustatory receptor Gr21a. The behavioral responses to CO2 were investigated in a four-field olfactometer bioassay that is new for Drosophila. We determined (1) whether the sensitivity of this response changes with odor context, and (2) if it depends on sex and life stage. When CO2 was added to ambient air in one field and tested against ambient air in the three other fields, individually observed adults avoided CO2 (0.1-1%above ambient), but did not respond to a low rise of 0.02%. We relate this behavior to measurements of CO2 production in bananas and flies. When 0.02% CO2 was combined with the odor of apple cider vinegar in one field of the olfactometer and tested against ambient air in the three other fields, the addition of CO2 did not affect the attractiveness of apple cider vinegar alone. However, this combination of CO2 and vinegar became repellent when it was tested against vinegar at ambient CO2 concentrations in the three other fields. This `odor background effect' was female-specific, revealing a sexually dimorphic behavior. The new assay allowed us to test larvae under similar conditions and compare their behavior to that of adults. Like adults, they avoided CO2, but with lower sensitivity. Larvae lacking neurons expressing Gr21a lost their avoidance behavior to CO2, but kept their positive response to vinegar odor. Hence, Gr21a-expressing neurons mediate similar behaviors in larvae and adults.

Behavioural responses of animals to volatiles in their environment are generally dependent on context. Most natural odours are mixtures of components that can each induce different behaviours when presented on their own. We have investigated how a complex of two olfactory stimuli is evaluated by Drosophila flies in a free-flying two-trap choice assay and how these stimuli are encoded in olfactory receptor neurons. We first observed that volatiles from apple cider vinegar attracted flies while carbon dioxide (CO2) was avoided, confirming their inherent positive and negative values. In contradiction with previous results obtained from walking flies in a four-field olfactometer, in the present assay the addition of CO2 to vinegar increased rather than decreased the attractiveness of vinegar. This effect was female-specific even though males and females responded similarly to CO2 and vinegar on their own. To test whether the female-specific behavioural response to the mixture correlated with a sexual dimorphism at the peripheral level we recorded from olfactory receptor neurons stimulated with vinegar, CO2 and their combination. Responses to vinegar were obtained from three neuron classes, two of them housed with the CO2-responsive neuron in ab1 sensilla. Sensitivity of these neurons to both CO2 and vinegar per se did not differ between males and females and responses from female neurons did not change when CO2 and vinegar were presented simultaneously. We also found that CO2-sensitive neurons are particularly well adapted to respond rapidly to small concentration changes irrespective of background CO2 levels. The ability to encode temporal properties of stimulations differs considerably between CO2- and vinegar-sensitive neurons. These properties may have important implications for in-flight navigation when rapid responses to fragmented odour plumes are crucial to locate odour sources. However, the flies’ sex-specific response to the CO2-vinegar combination and the context-dependent hedonics most likely originate from central rather than peripheral processing.

We compared food choice and the initial response to deterrent treated diet between fifth instars of Helicoverpa armigera, a polyphagous generalist pest, and Bombyx mori, an oligophagous specialist beneficial. Bombyx mori was more behaviorally sensitive to salicin than to caffeine. The relative sensitivities were reversed for H. armigera, which was tolerant to the highest levels of salicin found in natural sources but sensitive to caffeine. A single gustatory receptor neuron (GRN) in the medial styloconic sensillum of B. mori was highly sensitive to salicin and caffeine. The styloconic sensilla of H. armigera did not respond consistently to either of the bitter compounds. Phagostimulants also were tested. Myo-inositol and sucrose were detected specifically by two GRNs located in B. mori lateral styloconic sensillum, whereas, in H. armigera, sucrose was sensed by a GRN in the lateral sensillum, and myo-inositol by a GRN in the medial sensillum. Myo-inositol responsiveness in both species occurred at or below 10−3 mM, which is far below the naturally occurring concentration of 1 mM in plants. Larval responses to specific plant secondary compounds appear to have complex determinants that may include host range, metabolic capacity, and gustatory repertoire.Electronic supplementary materialThe online version of this article (doi:10.1007/s10886-013-0303-2) contains supplementary material, which is available to authorized users.