Mate recognition is crucial for reproductive isolation and for maintaining species integrity. Chemosensory-mediated sexual communication with pheromones is an essential component of mate recognition in moths. Confronted with sex pheromone stimuli released from conspecific and closely related heterospecific females, which partially overlap in chemical composition, male moths are under strong selection to recognize compatible mates. Here, we investigated the role of pheromone signals in premating communication in the sibling species Spodoptera littoralis and S. litura (Lepidoptera, Noctuidae). Further, we measured the reproductive consequence of conspecific vs. heterospecific matings. Both species use Z 9,E11-14:Ac as the major pheromone compound, and the 11-component blend found in pheromone glands of S. littoralis comprises the compounds found in S. litura. Accordingly, S. littoralis and S. litura males readily responded to conspecific and heterospecific calling females in no-choice behavioral tests. In contrast, in a dual-choice test, S. littoralis males choose conspecific calling females, whereas S. litura males did not discriminate between conspecific and heterospecific females. In S. littoralis females, heterospecific matings had a negative fitness effect as compared to conspecific matings. Female longevity, egg-laying and hatching of larvae were significantly reduced by matings with heterospecific males. Reciprocal crossings, between S. litura females and S. littoralis males, were prevented by genital morphology, which is consistent with reduced heterospecific attraction of S. littoralis males in a dual-choice assay. On the other hand, matings between S. littoralis females and S. litura males, under a no-choice situation, show that interspecific matings occur in zones of geographical overlap and corroborate the idea that mate quality, in these closely related species, is a continuous and not a categorical trait.

The insect olfactory system discriminates odor signals of different biological relevance, which drive innate behavior. Identification of stimuli that trigger upwind flight attraction toward host plants is a current challenge, and is essential in developing new, sustainable plant protection methods, and for furthering our understanding of plant-insect interactions. Using behavioral, analytical and electrophysiological studies, we here show that both females and males of the Egyptian cotton leafworm, Spodoptera littoralis (Lepidoptera, Noctuidae), use blends of volatile compounds to locate their host plant, cotton, Gossypium hirsutum (Malvales, Malvaceae). Female S. littoralis were engaged in upwind orientation flight in a wind tunnel when headspace collected from cotton plants was delivered through a piezoelectric sprayer. Although males took off toward cotton headspace significantly fewer males than females flew upwind toward the sprayed headspace. Subsequent assays with antennally active synthetic compounds revealed that a blend of nonanal, (Z)-3 hexenyl acetate, (E)-β-ocimene, and (R)-(+)-limonene was as attractive as cotton headspace to females and more attractive to males. Two compounds, 4,8-dimethyl-1,3(E),7-nonatriene (DMNT) and (R)-(−)-linalool, both known plant defense compounds may have reduced the flight attraction of both females and males; more moths were attracted to blends without these two compounds, however, other compounds such as benzaldehyde may also be behavioral antagonists. Our findings provide a platform for further investigations on host plant signals mediating innate behavior, and for the development of novel insect plant protection strategies against S. littoralis.

20Mate recognition in animals evolves during niche adaptation and involves habitat and 21 social olfactory signals. Drosophila melanogaster is attracted to fermenting fruit for 22 feeding and egg-laying. We show that, in addition, female flies release a pheromone (Z)-23 , that elicits flight attraction in both sexes. The biosynthetic 24 precursor of Z4-11Al is the cuticular hydrocarbon (Z,Z)-7,11-heptacosadiene (7, 25 which is known to afford reproductive isolation between D. melanogaster and D. 26 simulans. A pair of alternatively spliced receptors, Or69aB and Or69aA, is tuned to Z4-27 11Al and to food olfactory cues, respectively. These receptors are co-expressed in the 28 same olfactory sensory neurons, and feed into a neural circuit mediating species-specific, 29 long-range communication: the close relative D. simulans does not respond. That Or69aA 30 and Or69aB have adopted dual olfactory traits highlights the interplay of habitat and 31 social signals in mate finding. These olfactory receptor genes afford a collaboration 32 between natural and sexual selection, which has the potential to drive phylogenetic 33 divergence. 34 Keywords 35 pheromone, kairomone, olfaction, reproductive isolation 36 Introduction 37 Volatile insect pheromones transmit species-specific messages over a distance. Premating 38 communication with pheromones facilitates and accelerates mate-finding, and reduces 39 predation risk and energy expenditure, which is particularly adapative in short-lived 40 insects (1,2). 41Sexual communication subserves mate-finding and reproduction. The sensory drive 42 hypothesis predicts that mate recognition in animals evolves during niche adaptation and 43 that premating sexual communication involves olfactory specialization to both social 44 signals and habitat cues (3). Pheromones are released into an atmosphere that is filled 45 with environmental, habitat-related olfactory cues, some of which signal mating sites and 46 food sources. The response to pheromones and habitat odourants (kairomones) is under 47Drosophila species, they cannot account for species-specific communication (18)(19)(20). 62Interspecific matings of D. melanogaster with sibling species are inhibited by the female-63 produced cuticular hydrocarbon (Z,Z)-7,11-heptacosadiene (7,11-HD), which is not 64 volatile and perceived through gustatory receptors only at close range (21-23). This 65 raises the question whether Drosophila uses, in addition, volatile pheromone signals that 66 mediate specific mate recognition at a distance. 67We have identified the first long-range, species-specific pheromone in D. melanogaster. A 68 pair of spliced olfactory receptors, feeding into the same neural circuit, has developed a 69 dual affinity to this pheromone and to environmental semiochemicals, encoding adult and 70 larval food. A blend of this pheromone and a food odourant specifically attracts D. 71 melanogaster, but not the close relative D. simulans. This becomes an excellent paradigm 72 to study the interaction of social signals and hab...