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Butterflies, like many insects, use gustatory and olfactory cues innately to assess the suitability of an oviposition site and are able to associate colours and leaf shapes with an oviposition reward. Studies on other insects have demonstrated that the quality of the reward is a crucial factor in forming associative memory. We set out to investigate whether the large cabbage white Pieris brassicae (Linnaeus) has the ability to associate an oviposition experience with a neutral olfactory cue. In addition, we tested whether the strength of this association is dependent on the gustatory response to the glucosinolate sinigrin, which is a known oviposition stimulus for P. brassicae. Female butterflies were able to associate a neutral odour with an oviposition experience after a single oviposition experience, both in a greenhouse and in a semi-natural outdoor setting. Moreover, butterflies performed best when trained with concentrations of sinigrin that showed the strongest response by specific gustatory neurons on the forelegs. Our study provides novel insight into the role of both gustatory and olfactory cues during oviposition learning in lepidopterans and contributes to a better understanding of how these insects might be able to adapt to a rapidly changing environment.

Butterflies, like many insects, use gustatory and olfactory cues innately to assess the suitability of an oviposition site and are able to associate colours and leaf shapes with an oviposition reward. Studies on other insects have demonstrated that the quality of the reward is a crucial factor in forming associative memory. We set out to investigate whether the large cabbage white Pieris brassicae (Linnaeus) has the ability to associate an oviposition experience with a neutral olfactory cue. In addition, we tested whether the strength of this association is dependent on the gustatory response to the glucosinolate sinigrin, which is a known oviposition stimulus for P. brassicae. Female butterflies were able to associate a neutral odour with an oviposition experience after a single oviposition experience, both in a greenhouse and in a semi-natural outdoor setting. Moreover, butterflies performed best when trained with concentrations of sinigrin that showed the strongest response by specific gustatory neurons on the forelegs. Our study provides novel insight into the role of both gustatory and olfactory cues during oviposition learning in lepidopterans and contributes to a better understanding of how these insects might be able to adapt to a rapidly changing environment.

The olfactory system of adult lepidopterans is among the best described neuronal circuits. However, comparatively little is known about the organization of the olfactory system in the larval stage of these insects. Here, we explore the expression of olfactory receptors and the organization of olfactory sensory neurons in caterpillars of Pieris brassicae, a significant pest species in Europe and a well-studied species for its chemical ecology. To describe the larval olfactory system in this species, we first analyzed the head transcriptome of third-instar larvae (L3) and identified 16 odorant receptors (ORs) including the OR coreceptor (Orco), 13 ionotropic receptors (IRs), and 8 gustatory receptors (GRs). We then quantified the expression of these 16 ORs in different life stages, using qPCR, and found that the majority of ORs had significantly higher expression in the L4 stage than in the L3 and L5 stages, indicating that the larval olfactory system is not static throughout caterpillar development. Using an Orco-specific antibody, we identified all olfactory receptor neurons (ORNs) expressing the Orco protein in L3, L4, and L5 caterpillars and found a total of 34 Orco-positive ORNs, distributed among three sensilla on the antenna. The number of Orco-positive ORNs did not differ among the three larval instars. Finally, we used retrograde axon tracing of the antennal nerve and identified a mean of 15 glomeruli in the larval antennal center (LAC), suggesting that the caterpillar olfactory system follows a similar design as the adult olfactory system, although with a lower numerical redundancy. Taken together, our results provide a detailed analysis of the larval olfactory neurons in P. brassicae, highlighting both the differences as well as the commonalities with the adult olfactory system. These findings contribute to a better understanding of the development of the olfactory system in insects and its life-stage-specific adaptations.

Neuroparasitism concerns the hostile take-over of a host’s nervous system by a foreign invader, in order to alter the behaviour of the host in favour of the parasite. One of the most remarkable cases of parasite-induced host behavioural manipulation comprises the changes baculoviruses induce in their caterpillar hosts. Baculoviruses may manipulate caterpillar behaviour in two ways: hyperactivity (increased movement in the horizontal plane) and/or tree-top disease (movement to elevated levels in the vertical plane). Those behavioural changes are followed by liquefaction and death of the caterpillar. In Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Spodoptera exigua caterpillars, an enzymatic active form of the virally encoded protein tyrosine phosphatase (PTP) is needed for the expression of hyperactivity from 3 days post infection (dpi). Using eGFP-expressing recombinant AcMNPV strains, we show that infection of the caterpillar’s central nervous system (CNS) can be observed primarily from 3 dpi onwards. In addition, we demonstrate that the structural and enzymatic function of PTP does not play a role in infection of the CNS. Instead we show that the virus entered the CNS via the trachea, progressing caudally to frontally through the CNS and that the infection progressed from the outermost cell layers towards the inner cell layers of the CNS, in a PTP independent manner. These findings help to further understand parasitic manipulation and the mechanisms by which neuroparasites infect the host nervous system to manipulate host behaviour.

The two videos are the supplementary videos of the Chapter 3 of the doctoral thesis "Odorant receptors in larvae and adults of a butterfly species: from identification to ecological significance". Supplementary video V1 describes the morphology of Pieris brassicae L3 caterpillar head and Orco-positive neurons housed by three olfacotry sensillum. Orco-positive neurons are stained by Orco anti-body. Supplementary video V2 shows all glomeruli in the larval antennal center of Pieris brassicae larvae, glomeruli are stained by axon tracing. Data was collected usingLeica Stellaris 8 confocal microscope.

Future food farming technology faces challenges that must integrate the core goal of keeping the global temperature increase within 1.5 °C without reducing food security and nutrition. Here, we show that boosting the production of insects and earthworms based on food waste and livestock manure to provide food and feed in China will greatly contribute to meeting the country’s food security and carbon neutrality pledges. By substituting domestic products with mini-livestock (defined as earthworms and insects produced for food or feed) protein and utilizing the recovered land for bioenergy production plus carbon capture and storage, China’s agricultural sector could become carbon-neutral and reduce feed protein imports to near zero. This structural change may lead to reducing greenhouse gas emissions by 2,350 Tg CO2eq per year globally when both domestic and imported products are substituted. Overall, the success of mini-livestock protein production in achieving carbon neutrality and food security for China and its major trading partners depends on how the substitution strategies will be implemented and how the recovered agricultural land will be managed, e.g., free use for afforestation and bioenergy or by restricting this land to food crop use. Using China as an example, this study also demonstrates the potential of mini-livestock for decreasing the environmental burden of food production in general.

Sensory processes have often been argued to play a central role in the selection of ecological niches and in the formation of new species. Butterflies are among the best studied animal groups with regards to their evolutionary and behavioral ecology and thereby offer an attractive system to investigate the role of chemosensory genes in sympatric speciation. We focus on two Pieris butterflies with overlapping host-plant ranges: P. brassicae and P. rapae. Host-plant choice in lepidopterans is largely based on their olfactory and gustatory senses. Although the chemosensory responses of the two species have been well characterized at the behavioral and physiological levels, little is known about their chemoreceptor genes. Here, we compared the chemosensory genes of P. brassicae and P. rapae to investigate whether differences in these genes might have contributed to their evolutionary separation. We identified a total of 130 and 122 chemoreceptor genes in the P. brassicae genome and antennal transcriptome, respectively. Similarly, 133 and 124 chemoreceptors were identified in the P. rapae genome and antennal transcriptome. We found some chemoreceptors being differentially expressed in the antennal transcriptomes of the two species. The motifs and gene structures of chemoreceptors were compared between the two species. We show that paralogs share conserved motifs and orthologs have similar gene structures. Our study therefore found surprisingly few differences in the numbers, sequence identities and gene structures between the two species, indicating that the ecological differences between these two butterflies might be more related to a quantitative shift in the expression of orthologous genes than to the evolution of novel receptors as has been found in other insects. Our molecular data supplement the wealth of behavioral and ecological studies on these two species and will thereby help to better understand the role of chemoreceptor genes in the evolution of lepidoptera

The COVID-19 pandemic has illustrated the need for the development of fast and reliable testing methods for novel, zoonotic, viral diseases in both humans and animals. Pathologies lead to detectable changes in the volatile organic compound (VOC) profile of animals, which can be monitored, thus allowing the development of a rapid VOC-based test. In the current study, we successfully trained honeybees (Apis mellifera) to identify SARS-CoV-2 infected minks (Neovison vison) thanks to Pavlovian conditioning protocols. The bees can be quickly conditioned to respond specifically to infected mink's odours and could therefore be part of a wider SARS-CoV-2 diagnostic system.We tested two different training protocols to evaluate their performance in terms of learning rate, accuracy and memory retention.We designed a non-invasive rapid test in which multiple bees are tested in parallel on the same samples. This provided reliable results regarding a subject's health status. Using the data from the training experiments, we simulated a diagnostic evaluation trial to predict the potential efficacy of our diagnostic test, which yielded a diagnostic sensitivity of 92% and specificity of 86%. We suggest that a honeybee-based diagnostics can offer a reliable and rapid test that provides a readily available, low-input addition to the currently available testing methods. A honeybee-based diagnostic test might be particularly relevant for remote and developing communities that lack the resources and infrastructure required for mainstream testing methods.

Pheromones are pivotal to sexual communication in insects. These chemical signals are processed by sexually dimorphic circuitries in the antennal lobe (AL) of the insect brain. However, there is limited understanding of how these circuitries form during AL development. Our review addresses this issue by comparing how circuitries develop throughout the growth processes of peripheral and deutocerebral neurons in various insect orders. Olfactory sensory neurons (OSNs) expressing novel pheromone receptors are eligible candidates to initiate new sexually-dimorphic circuitries when these OSNs survive programmed cell death and match the physiological properties of pheromone-sensing sensilla. The probability of these OSNs forming new glomeruli is largely determined by the degree of glia-OSN interactions and projection neuron (PN) prepatterning. The relative contribution of either of these processes determines the degree of evolutionary neuroplasticity, which is particularly prevalent in those species with complex ALs lacking specific macroglomerular structures. The extent of sexual dimorphism is determined by sex-determination genes, such as Doublesex and Fruitless, that regulate factors inducing OSN programmed cell death. Currently, these mechanisms are largely unexplored. This review, therefore, aims to provide a solid foundation for ongoing research into the evolution of AL sexual dimorphism and formation of pheromone circuitries in the light of insect sex determination.

Most flowering plants depend on animal pollination for successful sexual reproduction. Floral signals such as color, shape, and odor are crucial in establishing this (often mutualistic) interaction. Plant and pollinator phenotypes can vary temporally but also spatially, thus creating mosaic-like patterns of local adaptations. Here, we investigated natural variation in floral morphology, flower volatile emission, and phenology in four accessions of a self-compatible wild tobacco, Nicotiana attenuata, to assess how these traits match the sensory perception of a known pollinator, the hawkmoth Manduca sexta. These accessions differ in floral traits and also in their habitat altitudes. Based on habitat temperatures, the accession occurring at the highest altitude (California) is less likely to be visited by M. sexta, while the others (Arizona, Utah 1, and Utah 2) are known to receive M. sexta pollinations. The accessions varied significantly in flower morphologies, volatile emissions, flower opening, and phenology, traits likely important for M. sexta perception and floral handling. In wind tunnel assays, we assessed the seed set of emasculated flowers after M. sexta visitation and of natural selfed and hand-pollinated selfed flowers. After moth visitations, plants of two accessions (Arizona and Utah 2) produced more capsules than the other two, consistent with predictions that accessions co-occurring with M. sexta would benefit more from the pollination services of this moth. We quantified flower and capsule production in four accessions in a glasshouse assay without pollinators to assess the potential for self-pollination. The two Utah accessions set significantly more seeds after pollen supplementation compared with those of autonomous selfing flowers, suggesting a greater opportunistic benefit from efficient pollinators than the other two. Moreover, emasculated flowers of the accession with the most exposed stigma (Utah 2) produced the greatest seed set after M. sext

Parasitic wasps and their larval hosts are intimately connected by an array of behavioral adaptations and counter-adaptations. This co-evolution has led to highly specific, natural variation in learning rates and memory consolidation in parasitoid wasps. Similarly, the hosts of the parasitoids show specific sensory adaptations as well as non-associative learning strategies for parasitoid avoidance. However, these neuronal and behavioral adaptations of both hosts and wasps have so far been studied largely apart from each other. Here we argue that a parallel investigation of the nervous system in wasps and their hosts might lead to novel insights into the evolution of insect behavior and the neurobiology of learning and memory.

Flower signaling and orientation are key characteristics, which determine a flower’s pollinator guild. However, many flowers actively move during their daily cycle, changing both their detectability and accessibility to pollinators. The flowers of the wild tobacco Nicotiana attenuata orientate their corolla upwards at sunset and downwards after sunrise. Here, we investigated the effect of different flower orientations on a major pollinator of N. attenuata, the hawkmoth Manduca sexta. We found that although flower orientation influenced the flight altitude of the moth in respect to the flower, it did not alter its overall attractiveness. These behavioral observations were consistent with the finding that orientation did not systematically change the spatial distribution of floral volatiles, which are major attractants for moths. Moreover, moths invested the same amount of time into probing flowers at different orientations, even though they were only able to feed and take up pollen from horizontally and upward oriented flowers, but not from downward facing flowers. The orientation of the flower was hence crucial for a successful interaction between N. attenuata and its hawkmoth pollinator. Additionally, we determined potential adverse effects of exposing flowers at different orientations to natural daylight levels, finding that anther temperature of upward oriented flowers was more than 7°C higher than for downward oriented flowers. This increase in temperature likely caused the significantly reduced germination success that was observed for pollen grains from upward oriented flowers in comparison to those of downward and horizontally oriented flowers. These results highlight the importance of flower reorientation to ensure pollen protection, while maintaining the association between flower signals and flower accessibility necessary for a successful interaction of the plant with its insect pollinators.