Female fruit flies (Drosophila melanogaster) oviposit at communal sites where the larvae may cooperate or compete for resources depending on group size. This offers a model system to determine how females assess quantitative social information. We show that the concentration of pheromones found on a substrate increases linearly with the number of adult flies that have visited that site. Females prefer oviposition sites with pheromone concentrations corresponding to an intermediate number of previous visitors, whereas sites with low or high concentrations are unattractive. This dose-dependent decision is based on a blend of 11-cis-Vaccenyl Acetate (cVA) indicating the number of previous visitors and heptanal (a novel pheromone deriving from the oxidation of 7-Tricosene), which acts as a dose-independent co-factor. This response is mediated by detection of cVA by odorant receptor neurons Or67d and Or65a, and at least five different odorant receptor neurons for heptanal. Our results identify a mechanism allowing individuals to transform a linear increase of pheromones into a non-linear behavioral response.

Diapause, a general shutdown of developmental pathways, is a vital adaptation allowing insects to adjust their life cycle to adverse environmental conditions such as winter. Diapause in the pupal stage is regulated by the major developmental hormones prothoracicotropic hormone (PTTH) and ecdysone. Termination of pupal diapause in the butterfly Pieris napi depends on low temperatures; therefore, we study the temperature-dependence of PTTH secretion and ecdysone sensitivity dynamics throughout diapause, with a focus on diapause termination. While PTTH is present throughout diapause in the cell bodies of two pairs of neurosecretory cells in the brain, it is absent in the axons, and the PTTH concentration in the haemolymph is significantly lower during diapause than during post diapause development, indicating that the PTTH signaling is reduced during diapause. The sensitivity of pupae to ecdysone injections is dependent on diapause stage. While pupae are sensitive to ecdysone during early diapause initiation, they gradually lose this sensitivity and become insensitive to non-lethal concentrations of ecdysone about 30 days into diapause. At low temperatures, reflecting natural overwintering conditions, diapause termination propensity after ecdysone injection is precocious compared to controls. In stark contrast, at high temperatures reflecting late summer and early autumn conditions, sensitivity to ecdysone does not return. Thus, here we show that PTTH secretion is reduced during diapause, and additionally, that the low ecdysone sensitivity of early diapause maintenance is lost during termination in a temperature dependent manner. The link between ecdysone sensitivity and low-temperature dependence reveals a putative mechanism of how diapause termination operates in insects that is in line with adaptive expectations for diapause.

Many insects that live in temperate zones spend the cold season in a state of dormancy, referred to as diapause. As the insect must rely on resources that were gathered before entering diapause, keeping a low metabolic rate is of utmost importance. Organs that are metabolically expensive to maintain, such as the brain, can therefore become a liability to survival if they are too large. Insects that go through diapause as adults generally do so before entering the season of reproduction. This order of events introduces a conflict between maintaining low metabolism during dormancy and emerging afterward with highly developed sensory systems that improve fitness during the mating season. We investigated the timing of when investments into the olfactory system are made by measuring the volumes of primary and secondary olfactory neuropils in the brain as they fluctuate in size throughout the extended diapause life-period of adult Polygonia c-album butterflies. Relative volumes of both olfactory neuropils increase significantly during early adult development, indicating the importance of olfaction to this species, but still remain considerably smaller than those of nondiapausing conspecifics. However, despite butterflies being kept under the same conditions as before the dormancy, their olfactory neuropil volumes decreased significantly during the postdormancy period. The opposing directions of change in relative neuropil volumes before and after diapause dormancy indicate that the investment strategies governing structural plasticity during the two life stages could be functionally distinct. As butterflies were kept in stimulus-poor conditions, we find it likely that investments into these brain regions rely on experience-expectant processes before diapause and experience-dependent processes after diapause conditions are broken. As the shift in investment strategies coincides with a hard shift from premating season to mating season, we argue that these developmental characteristics could be adaptations that mitigate the trade-off between dormancy survival and reproductive fitness.

Insect brains are known to express a high degree of experience-dependent structural plasticity. One brain structure in particular, the mushroom body (MB), has been attended to in numerous studies as it is implicated in complex cognitive processes such as olfactory learning and memory. It is, however, poorly understood to what extent sensory input per se affects the plasticity of the mushroom bodies. By performing unilateral blocking of olfactory input on immobilized butterflies, we were able to measure the effect of passive sensory input on the volumes of antennal lobes (ALs) and MB calyces. We showed that the primary and secondary olfactory neuropils respond in different ways to olfactory input. ALs show absolute experience-dependency and increase in volume only if receiving direct olfactory input from ipsilateral antennae, while MB calyx volumes were unaffected by the treatment and instead show absolute age-dependency in this regard. We therefore propose that cognitive processes related to behavioural expressions are needed in order for the calyx to show experience-dependent volumetric expansions. Our results indicate that such experience-dependent volumetric expansions of calyces observed in other studies may have been caused by cognitive processes rather than by sensory input, bringing some causative clarity to a complex neural phenomenon.

Insects searching for resources are exposed to a complexity of mixed odours, often involving both attractant and repellent substances. Understanding how insects respond to this complexity of cues is crucial for understanding consumer-resource interactions, but also to develop novel tools to control harmful pests. To advance our understanding of insect responses to combinations of attractive and repellent odours, we formulated three qualitative hypotheses; the response-ratio hypothesis, the repellent-threshold hypothesis and the odour-modulation hypothesis. The hypotheses were tested by exposing Drosophila melanogaster in a wind tunnel to combinations of vinegar as attractant and four known repellents; benzaldehyde, 1-octen-3-ol, geosmin and phenol. The responses to benzaldehyde, 1-octen-3-ol and geosmin provided support for the response-ratio hypothesis, which assumes that the behavioural response depends on the ratio between attractants and repellents. The response to phenol, rather supported the repellent-threshold hypothesis, where aversion only occurs above a threshold concentration of the repellent due to overshadowing of the attractant. We hypothesize that the different responses may be connected to the localization of receptors, as receptors detecting phenol are located on the maxillary palps whereas receptors detecting the other odorants are located on the antennae.

The Queensland fruit fly, Bactrocera tryoni, is considered one of the worst horticultural pests in Australia attacking a large variety of fruit crops. To defeat pest insects, olfactory attractants have been developed and widely used in lure and kill strategies. Male B. tryoni are strongly attracted to the compound raspberry ketone and its synthetic analog, cuelure. Despite the strong behavioral response, a recent study failed to show any activation of antennal receptors to cuelure. Therefore, we hypothesized that cuelure may be detected by an accessory olfactory organ, the maxillary palp. Combining behavioral and physiological experiments we clearly demonstrate that male flies, but not female flies, primarily use the maxillary palps and not the antennae to detect and respond to cuelure. Furthermore, regardless of satiety status, male flies always preferred cuelure over a sugar rich source, unless the maxillary palps were excised.

An ovipositing insect experiences many sensory challenges during her search for a suitable host plant. These sensory challenges become exceedingly pronounced when host range increases, as larger varieties of sensory inputs have to be perceived and processed in the brain. Neural capacities can be exceeded upon information overload, inflicting costs on oviposition accuracy. One presumed generalist strategy to diminish information overload is the acquisition of a focused search during its lifetime based on experiences within the current environment, a strategy opposed to a more genetically determined focus expected to be seen in relative specialists. We hypothesized that a broader host range is positively correlated with mushroom body (MB) plasticity, a brain structure related to learning and memory. To test this hypothesis, butterflies with diverging host ranges (Polygonia c-album, Aglais io and Aglais urticae) were subjected to differential environmental complexities for oviposition, after which ontogenetic MB calyx volume differences were compared among species. We found that the relative generalist species exhibited remarkable plasticity in ontogenetic MB volumes; MB growth was differentially stimulated based on the complexity of the experienced environment. For relative specialists, MB volume was more canalized. All in all, this study strongly suggests an impact of host range on brain plasticity in Nymphalid butterflies.

Diapause is an important escape mechanism from seasonal stress in many insects. A certain minimum amount of time in diapause is generally needed in order for it to terminate. The mechanisms of time-keeping in diapause are poorly understood, but it can be hypothesized that a well-developed neural system is required. However, because neural tissue is metabolically costly to maintain, there might exist conflicting selective pressures on overall brain development during diapause, on the one hand to save energy and on the other hand to provide reliable information processing during diapause. We performed the first ever investigation of neural development during diapause and non-diapause (direct) development in pupae of the butterfly Pieris napi from a population whose diapause duration is known. The brain grew in size similarly in pupae of both pathways up to 3 days after pupation, when development in the diapause brain was arrested. While development in the brain of direct pupae continued steadily after this point, no further development occurred during diapause until temperatures increased far after diapause termination. Interestingly, sensory structures related to vision were remarkably well developed in pupae from both pathways, in contrast with neuropils related to olfaction, which only developed in direct pupae. The results suggest that a well-developed visual system might be important for normal diapause development.

Many animals adjust their reproductive behaviour according to nutritional state and food availability. Drosophila females for instance decrease their sexual receptivity following starvation. Insulin signalling, which regulates many aspects of insect physiology and behaviour, also affects reproduction in females. We show that insulin signalling is involved in the starvation-induced reduction in female receptivity. More specifically, females mutant for the insulin-like peptide 5 (dilp5) were less affected by starvation compared to the other dilp mutants and wild-type flies. Knocking-down the insulin receptor, either in all fruitless-positive neurons or a subset of these neurons dedicated to the perception of a male aphrodisiac pheromone, decreased the effect of starvation on female receptivity. Disrupting insulin signalling in some parts of the brain, including the mushroom bodies even abolished the effect of starvation. In addition, we identified fruitless-positive neurons in the dorso-lateral protocerebrum and in the mushroom bodies co-expressing the insulin receptor. Together, our results suggest that the interaction of insulin peptides determines the tuning of female sexual behaviour, either by acting on pheromone perception or directly in the central nervous system.

Neighboring resources can affect insect oviposition behavior when the complexity of sensory information obscures information about host resource availability in heterogeneous resource patches. These effects are referred to as associational effects and are hypothesized to occur through constraints in the sensory processing of the insect during host search, resulting into suboptimal resource use. Because the possibilities to study these constraints on naturally occurring animals are limited, we instead used sensory mutants of Drosophila melanogaster to determine the importance of sensory information in the occurrence of associational effects. We found that oviposition was mainly governed by non-volatile chemical cues and less by volatile cues. Moreover, the loss of gustatory sensilla resulted in random resource selection and eliminated associational effects. In conclusion, our study shows that associational effects do not necessarily depend on constraints in the sensory evaluation of resource quality, but may instead be a direct consequence of distinctive selection behavior between different resources at small scales.