Mikael A. Carlsson

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.

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.

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.

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 inputper seaffects 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.

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.

Among insects, sexual pheromones are typically mixtures of two to several components, all of which are generally required to elicit a behavioural response. Here we show for the first time that a complete blend of sexual pheromone components is needed to elicit a response also in a butterfly. Males of the Green-veined White, Pieris napi, emit an aphrodisiac pheromone, citral, from wing glands. This pheromone is requisite for females to accept mating with a courting male. Citral is a mixture of the two geometric isomers geranial (E-isomer) and neral (Z-isomer) in an approximate 1:1 ratio. We found that both these compounds are required to elicit acceptance behaviour, which indicates synergistic interaction between processing of the isomers. Using functional Ca2+ imaging we found that geranial and neral evoke significantly different but overlapping glomerular activity patterns in the antennal lobe, which suggests receptors with different affinity for the two isomers. However, these glomeruli were intermingled with glomeruli responding to ,for example, plant-related compounds, i.e. no distinct subpopulation of pheromone-responding glomeruli as in moths and other insects. In addition, these glomeruli showed lower specificity than pheromone-activated glomeruli in moths. We could, however, not detect any mixture interactions among four identified glomeruli, indicating that the synergistic effect may be generated at a higher processing level. Furthermore, correlations between glomerular activity patterns evoked by the single isomers and the blend did not change over time.

Summary: When females mate with multiple partners, the risk of sperm competition depends on female mating history. To maximize fitness, males should adjust their mating investment according to this risk. In polyandrous butterflies, males transfer a large, nutritious ejaculate at mating. Larger ejaculates delay female remating and confer an advantage in sperm competition. We test whether male ejaculate size in the butterfly Pieris napi (Lepidoptera) varies with female mating history and thus sperm competition, and whether males assess sperm competition using the male‐transferred anti‐aphrodisiac methyl salicylate (MeS) as a cue. Both sexes responded physiologically to MeS in a dose‐dependent manner. Males, however, were more sensitive to MeS than females. Ejaculates transferred by males mating with previously mated females were on average 26% larger than ejaculates transferred by males mating with virgin females, which conforms to sperm competition theory and indicates that males tailored their reproductive investment in response to sperm competition. Furthermore, ejaculates transferred by males mating with virgin females with artificially added MeS were also 26% larger than ejaculates transferred to control virgin females. Male‐transferred anti‐aphrodisiac pheromone not only functions as a male deterrent, but also carries information on female mating history and thus allows males to assess sperm competition.

Four forms of short neuropeptide F (sNPF1–4), derived from the gene snpf, have been identified in Drosophila and are known to act on a single G-protein-coupled receptor (sNPFR). Several functions have been suggested for sNPFs in Drosophila, including the regulation of feeding and growth in larvae, the control of insulin signalling and the modulation of neuronal circuits in adult flies. Furthermore, sNPF has been shown to act as a nutritional state-dependent neuromodulator in the olfactory system. The role of sNPF in the larval nervous system is less well known. To analyse sites of action of sNPF in the larva, we mapped the distribution of sNPF- and sNPFR-expressing neurons. In particular, we studied circuits associated with chemosensory inputs and systems involved in the regulation of feeding, including neurosecretory cell systems and the hypocerebral ganglion. We employed a combination of immunocytochemistry and enhancer trap and promoter Gal4 lines to drive green fluorescent protein. We found a good match between the distribution of the receptor and its ligand. However, several differences between the larval and adult systems were observed. Thus, neither sNPF nor its receptor was found in the olfactory (or other sensory) systems in the larva and cells producing insulin-like peptides did not co-express sNPFR, as opposed to results from adults. Moreover, sNPF was expressed in a subpopulation of Hugin cells (second-order gustatory neurons) only in adult flies. We propose that the differences in sNPF signalling between the developmental stages is explained by differences in their feeding behaviour.