Hymenopteran egg parasitoids of the genus Trichogramma are among the tiniest insects on Earth (< 0.5 mm in size, 8 µg) and are important biological control agents of moth pests. The existence of sex pheromones has been demonstrated in Trichogramma species, but none has yet been fully characterized. Here, we report absolute configuration, improved synthesis and behavioral activity of the sex pheromone of T. turkestanica, making it one of the smallest insects with a fully characterized sex pheromone. Enantioselective synthesis and chiral gas chromatography were used to determine the absolute configuration of the previously identified, stereochemically complex dienol (2E,4E,6S,8S,10S)-4,6,8,10-tetramethyltrideca-2,4-dien-1-ol and to newly establish the same stereochemistry for the corresponding diene (2E,4E,6S,8S,10S)-4,6,8,10-tetramethyltrideca-2,4-diene. In a 3:1 mixture of dienol and diene applied to two solvent-washed wasps (dummies), these compounds triggered attraction, arrestment, and courtship (“casting”) in males at doses, as little as 670 attograms per dummy. This pheromone could be studied to monitor T. turkestanica field populations, with the aim to improve the efficiency of biocontrol strategies.

Insect herbivores such as caterpillars are under strong selection pressure from natural enemies, especially parasitoid wasps. Although the role of olfaction in host-plant seeking has been investigated in great detail in parasitoids and adult lepidopterans, the caterpillar olfactory system and its significance in tri-trophic interactions remain poorly understood. In this study, we investigated the olfactory system of Pieris brassicae caterpillars and the importance of olfactory information in the interactions among this herbivore, its host-plant Brassica oleracea, and its primary natural enemy Cotesia glomerata. To examine the role of olfaction, we utilized CRISPR/Cas9 to knockout (KO) the odorant receptor co-receptor (Orco). This KO impaired olfactory detection and primary processing in the brain. Orco KO caterpillars exhibited reduced weight and lost preference for their optimal food plants. Interestingly, the KO caterpillars also experienced reduced weight when challenged by the parasitoid C. glomerata whose ovipositor had been removed, and the mortality of the KO caterpillars under the attack of unmanipulated parasitoids increased. We then investigated the behavior of P. brassicae caterpillars in response to volatiles from plants attacked by conspecific caterpillars and volatiles from plants on which the caterpillars were themselves attacked by C. glomerata. After analyzing the volatile compounds involved in these interactions, we concluded that olfactory information enables caterpillars to locate suitable food sources more efficiently as well as to select enemy-free spaces. Our results reveal the crucial role of olfaction in caterpillar feeding and natural-enemy avoidance, highlighting the significance of chemoreceptor genes in shaping ecological interactions.

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.

Parasitoids lay their eggs in or on a host, usually another insect. During foraging, parasitoids can encounter insects that differ in terms of host suitability and quality. At one extreme end of this spectrum are non‐hosts that are unsuitable for offspring development. Non‐hosts are generally ignored but parasitization does occur and occasionally also results in egg deposition. Here, the authors investigate how oviposition in a non‐host influences subsequent foraging behaviour of a parasitoid and whether this is mediated by learning. The study system consists of the endoparasitoid Cotesia glomerata and the presumed non‐host caterpillar Mamestra brassicae. In the presence of Pieris brassicae hosts and/or their traces (frass), C. glomerata inserted its ovipositor into M. brassicae caterpillars. Eggs were deposited, but all eggs disappeared within 96 h, confirming the non‐host status of M. brassicae. In contrast to the expectation, there was no memory retention after oviposition in a non‐host and parasitoids did not alter their behaviour with respect to non‐host contacts and ovipositions. Instead, C. glomerata became more motivated to forage on a non‐host infested leaf. The authors propose that egg deposition in non‐hosts by C. glomerata might be due to their high egg load, which is thought to make parasitoids less selective on host quality, especially when they have few reproductive opportunities. In such cases, fitness costs to individual females are low. Egg deposition in non‐hosts might ultimately lead to host range expansion if parasitoids overcome the defence response of non‐hosts over evolutionary time.

Animals can alter their foraging behavior through associative learning, where an encounter with an essential resource (e.g., food or a reproductive opportunity) is associated with nearby environmental cues (e.g., volatiles). This can subsequently improve the animal's foraging efficiency. However, when these associated cues are encountered again, the anticipated resource is not always present. Such an unrewarding experience, also called a memory-extinction experience, can change an animal's response to the associated cues. Although some studies are available on the mechanisms of this process, they rarely focus on cues and rewards that are relevant in an animal's natural habitat. In this study, we tested the effect of different types of ecologically relevant memory-extinction experiences on the conditioned plant volatile preferences of the parasitic wasp Cotesia glomerata that uses these cues to locate its caterpillar hosts. These extinction experiences consisted of contact with only host traces (frass and silk), contact with nonhost traces, or oviposition in a nonhost near host traces, on the conditioned plant species. Our results show that the lack of oviposition, after contacting host traces, led to the temporary alteration of the conditioned plant volatile preference in C. glomerata, but this effect was plant species-specific. These results provide novel insights into how ecologically relevant memory-extinction experiences can fine-tune an animal's foraging behavior. This fine-tuning of learned behavior can be beneficial when the lack of finding a resource accurately predicts current, but not future foraging opportunities. Such continuous reevaluation of obtained information helps animals to prevent maladaptive foraging behavior.

Dynamic conditions in nature have led to the evolution of behavioural traits that allow animals to use information on local circumstances and adjust their behaviour accordingly, for example through learning. Although learning can improve foraging efficiency, the learned information can become unreliable as the environment continues to change. This could lead to potential fitness costs when memories holding such unreliable information persist. Indeed, persistent unreliable memory was found to reduce the foraging efficiency of the parasitoid Cotesia glomerata under laboratory conditions. Here, we evaluated the effect of such persistent unreliable memory on the foraging behaviour of C. glomerata in the field. This is a critical step in studies of foraging theory, since animal behaviour evolved under the complex conditions present in nature. Existing methods provide little detail on how parasitoids interact with their environment in the field, therefore we developed a novel multi-camera system that allowed us to trace parasitoid foraging behaviour in detail. With this multi-camera system, we studied how persistent unreliable memory affected the foraging behaviour of C. glomerata when these memories led parasitoids to plants infested with non-host caterpillars in a semi-field set-up. Our results demonstrate that persistent unreliable memory can lead to maladaptive foraging behaviour in C. glomerata under field conditions and increased the likelihood of oviposition in the non-host caterpillar Mamestra brassica. Furthermore, these time- and egg-related costs can be context dependent, since they rely on the plant species used. These results provide us with new insight on how animals use previously obtained information in naturally complex and dynamic foraging situations and confirm that costs and benefits of learning depend on the environment animals forage in. Although behavioural studies of small animals in natural habitats remain challenging, novel methods such as our multi-camera system contribute to understanding the nuances of animal foraging behaviour.

Most endogenous viruses, an important proportion of eukaryote genomes, are doomed to slowly decay. Little is known, however, on how they evolve when they confer a benefit to their host. Bracoviruses are essential for the parasitism success of parasitoid wasps, whose genomes they integrated ~103 million years ago. Here we show, from the assembly of a parasitoid wasp genome, for the first time at a chromosomal scale, that symbiotic bracovirus genes spread to and colonized all the chromosomes. Moreover, large viral clusters are stably maintained suggesting strong evolutionary constraints. Genomic comparison with another wasps revealed that this organization was already established ~53 mya. Transcriptomic analyses highlight temporal synchronization of viral gene expression, leading to particle production. Immune genes are not induced, however, indicating the virus is not perceived as foreign by the wasp. This recognition suggests that no conflicts remain between symbiotic partners when benefits to them converge.

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.

The Drosophila olfactory system provides an excellent model to elucidate the neural circuits that control behaviors elicited by environmental stimuli. Despite significant progress in defining olfactory circuit components and their connectivity, little is known about the mechanisms that transfer the information from the primary antennal olfactory receptor neurons to the higher order brain centers. Here, we show that the Dystrophin Dp186 isoform is required in the olfactory system circuit for olfactory functions. Using two-photon calcium imaging, we found the reduction of calcium influx in olfactory receptor neurons (ORNs) and also the defect of GABA(A) mediated inhibitory input in the projection neurons (PNs) in Dp186 mutation. Moreover, the Dp186 mutant flies which display a decreased odor avoidance behavior were rescued by Dp186 restoration in the Drosophila olfactory neurons in either the presynaptic ORNs or the postsynaptic PNs. Therefore, these results revealed a role for Dystrophin, Dp 186 isoform in gain control of the olfactory synapse via the modulation of excitatory and inhibitory synaptic inputs to olfactory projection neurons.

Nasonia, a genus of parasitoid wasps, is a promising model system in the study of developmental and evolutionary genetics, as well as complex traits such as learning. Of these "jewel wasps", the species Nasonia vitripennis is widely spread and widely studied. To accelerate neuroscientific research in this model species, fundamental knowledge of its nervous system is needed. To this end, we present an average standard brain of recently eclosed naive female N. vitripennis wasps obtained by the iterative shape averaging method. This "Jewel Wasp Standard Brain" includes the optic lobe (excluding the lamina), the anterior optic tubercle, the antennal lobe, the lateral horn, the mushroom body, the central complex, and the remaining unclassified neuropils in the central brain. Furthermore, we briefly describe these well-defined neuropils and their subregions in the N. vitripennis brain. A volumetric analysis of these neuropils is discussed in the context of brains of other insect species. The Jewel Wasp Standard Brain will provide a framework to integrate and consolidate the results of future neurobiological studies in N. vitripennis. In addition, the volumetric analysis provides a baseline for future work on age- and experience-dependent brain plasticity. (C) 2019 The Authors. Published by Elsevier Ltd.

Heritable genetic variation in relative brain size can underlie the relationship between brain performance and the relative size of the brain. We used bidirectional artificial selection to study the consequences of genetic variation in relative brain size on brain morphology, cognition and longevity in Nasonia vitripennis parasitoid wasps. Our results show a robust change in relative brain size after 26 generations of selection and six generations of relaxation. Total average neuropil volume of the brain was 16% larger in wasps selected for relatively large brains than in wasps selected for relatively small brains, whereas the body length of the large-brained wasps was smaller. Furthermore, the relative volume of the antennal lobes was larger in wasps with relatively large brains. Relative brain size did not influence olfactory memory retention, whereas wasps that were selected for larger relative brain size had a shorter longevity, which was even further reduced after a learning experience. These effects of genetic variation on neuropil composition and memory retention are different from previously described effects of phenotypic plasticity in absolute brain size. In conclusion, having relatively large brains may be costly for N. vitripennis, whereas no cognitive benefits were recorded.

An extreme reduction in body size has been shown to negatively impact the memory retention level of the parasitic wasp Nasonia vitripennis. In addition, N. vitripennis and Nasonia giraulti, closely related parasitic wasps, differ markedly in the number of conditioning trials required to form long-term memory. These differences in memory dynamics may be associated with differences in the dopaminergic neurons in the Nasonia brains. Here, we used dopamine immunoreactivity to identify and count the number of cell bodies in dopaminergic clusters of normal- and small-sized N. vitripennis and normal-sized N. giraulti. We counted in total a maximum of approximately 160 dopaminergic neurons per brain. These neurons were present in 9 identifiable clusters (D1a, D1b, D2, D3, D4a, D4b, D5, D6 and D7). Our analysis revealed that N. giraulti had fewer cells in the D2 and D4a clusters but more in D4b, compared with normal-sized N. vitripennis. In addition, we found fewer cells in the D5 and D7 cluster of small-sized N. vitripennis compared to normal-sized N. vitripennis. A comparison of our findings with the literature on dopaminergic clusters in the fruit fly Drosophila melanogaster and the honey bee Apis mellifera indicates that clusters D2, D3 and D5 may play a role in memory formation in Nasonia wasps. The results from both the species comparison and the size comparison are therefore of high interest and importance for our understanding of the complex intricacies that underlie the memory dynamics of insects.

Despite the vast body of theoretical and empirical literature dealing with parasitoid learning, this knowledge has thus far rarely been exploited for manipulating the efficacy of augmentative biological pest control. This may be due to the fact that most studies on learning behavior were performed under laboratory conditions, whereas field trials remain scarce. However, the few studies that did investigate parasitoid foraging success under (semi-)field conditions show strong learning effects. Using so-called "parasitoid olfactory conditioning" (POC), parasitoids can be trained to become more efficient in the different phases involved in the process of host searching and host acceptance. POC can thus result in a "foraging efficacy gain", defined as the difference between the number of naive and conditioned parasitoids that need to be released to reach a certain parasitization level of the target-pest in the crop environment. This "gain" increases with an improved parasitoid learning ability and depends on the interplay between the parasitoid, crop, target-pest species and parasitoid rearing method. Moreover, the "foraging efficacy gain" depends on the technical implementation of POC, as this will determine the strength, duration and stability of the learning-induced behavioral change. In this perspective paper we will discuss (a) the conditions that can enhance the "foraging efficacy gain," (b) the possible approaches to implementation of POC and their costs and benefits, and (c) a stepwise approach to develop appropriate POC methods for the optimization of biological pest control.

1. Parasitoids are known to utilise learning of herbivore‐induced plant volatiles (HIPVs) when foraging for their herbivorous host. In natural situations these hosts share food plants with other, non‐suitable herbivores (non‐hosts). Simultaneous infestation of plants by hosts and non‐hosts has been found to result in induction of HIPVs that differ from host‐infested plants. Each non‐host herbivore may have different effects on HIPVs when sharing the food plant with hosts, and thus parasitoids may learn that plants with a specific non‐host herbivore also contain the host.2. This study investigated the adaptive nature of learning by a foraging parasitoid that had acquired oviposition experience on a plant infested with both hosts and different non‐hosts in the laboratory and in semi‐field experiments.3. In two‐choice preference tests, the parasitoid Cotesia glomerata shifted its preference towards HIPVs of a plant–host–non‐host complex previously associated with an oviposition experience. It could, indeed, learn that the presence of its host is associated with HIPVs induced by simultaneous feeding of its host Pieris brassicae and either the non‐host caterpillar Mamestra brassicae or the non‐host aphid Myzus persicae. However, the learned preference found in the laboratory did not translate into parasitisation preferences for hosts accompanying non‐host caterpillars or aphids in a semi‐field situation.4. This paper discusses the importance of learning in parasitoid foraging, and debates why observed learned preferences for HIPVs in the laboratory may cancel out under some field experimental conditions.

Background: Insects are important models to study learning and memory formation in both an ecological and neuroscience context due to their small size, behavioral flexibility and ecological diversity. Measuring memory retention is often done through simple time-consuming set-ups, producing only a single parameter for conditioned behavior. We wished to obtain higher sample sizes with fewer individuals to measure olfactory memory retention more efficiently. New method: The high-throughput individual T-maze uses commercially available tracking software, Ethovision XT (R), in combination with a Perspex stack of plates as small as 18 x 18 cm, which accommodates 36 olfactory T-mazes, where each individual wasp could choose between two artificial odors. Various behavioral parameters, relevant to memory retention, were acquired in this set-up; first choice, residence time, giving up time and zone entries. From these parameters a performance index was calculated as a measure of memory retention. Groups of 36 wasps were simultaneously tested within minutes, resulting in efficient acquisition of sufficiently high sample sizes. Results: This system was tested with two very different parasitic wasp species, the larval parasitoid Cotesia glomerata and the pupal parasitoid Nasonia vitripennis, and has proven to be highly suitable for testing memory retention in both these species. Comparison with existing methods: Unlike other bioassays, this system allows for both high-throughput and recording of detailed individual behavior. Conclusions: The high-throughput individual T-maze provides us with a standardized high-throughput, labor-efficient and cost-effective method to test various kinds of behavior, offering excellent opportunities for comparative studies of various aspects of insect behavior.

We are only starting to understand how variation in cognitive ability can result from local adaptations to environmental conditions. A major question in this regard is to what extent selection on cognitive ability in a specific context affects that ability in general through correlated evolution. To address this question, we performed artificial selection on visual associative learning in female Nasonia vitripennis wasps. Using appetitive conditioning in which a visual stimulus was offered in association with a host reward, the ability to learn visual associations was enhanced within 10 generations of selection. To test for correlated evolution affecting this form of learning, the ability to readily form learned associations in females was also tested using an olfactory instead of a visual stimulus in the appetitive conditioning. Additionally, we assessed whether the improved associative learning ability was expressed across sexes by color-conditioning males with a mating reward. Both females and males from the selected lines consistently demonstrated an increased associative learning ability compared to the control lines, independent of learning context or conditioned stimulus. No difference in relative volume of brain neuropils was detected between the selected and control lines.

Small animals usually have relatively larger brains than large animals. This allometric brain-body size scaling is described by Haller's rule. However, one of the smallest known insects, Trichogramma evanescens, a parasitic wasp, shows brain isometry, leading to similar relative brain sizes in small and large conspecifics. The somewhat larger Nasonia vitripennis parasitic wasp displays diphasic brain-body size scaling with isometry in small individuals and allometry in large individuals. These two species may have undersized brains for small wasps, with reduced cognitive abilities. Here, we induced intraspecific body size variation in genetically identical T. evanescens and N. vitripennis and examined cognitive trade-offs of brain scaling. We compared visual and olfactory memory retention between small and large conspecifics. Results showed that diphasic brain scaling affected memory retention levels in N. vitripennis, whereas isometric brain scaling did not affect memory retention in T. evanescens. The two species may experience different evolutionary pressures that have shaped the cognitive consequences of isometric brain-body size scaling. A possible trade-off of brain isometry in T. evanescens could be present in brain properties other than memory performance. In contrast, it may be more adaptive for N. vitripennis to invest in other aspects of brain performance, at the cost of memory retention. (C) 2018 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Parasitic wasps are known to improve their foraging efficiency after learning of herbivore-induced plant volatiles (HIPVs) upon encountering their hosts on these plants. However, due to spatial and temporal variation of herbivore communities, learned HIPV cues can become unreliable, no longer correctly predicting host presence. Little is known about the potential fitness costs when memories holding such unreliable information persist. Here we studied how persistent memory, containing unreliable information, affects the foraging efficiency for hosts in Cotesia glomerata. Wasps were conditioned to associate one of two types of HIPVs with either P. brassicae frass, 1 single oviposition in P. brassicae, 3 ovipositions in P. brassicae spaced in time or they were kept unconditioned. The following day, wasps were allowed to forage in a wind tunnel, in an environment that either conflicted or was congruent with their learned plant experience. The foraging environment consisted of host (P. brassicae) and non-host (Mamestra brassicae) infested plants. The conflicting environment had non-hosts on the conditioned plant species and hosts on the non-conditioned plant species, whereas the congruent environment had hosts on the conditioned plant species and non-hosts on the unconditioned plant species. Wasps had to navigate through five non-host infested plants to reach the host-infested plant. Since C. glomerata wasps do not distinguish between HIPVs induced by host and non-host caterpillars, the conflicting foraging situation caused a prediction error, by guiding wasps to non-host infested plants. Especially wasps given 3 spaced oviposition experiences, tested in a conflicting situation, spent significantly more time on non-host infested plants and showed a high tendency to oviposit in the non-hosts. As a result, they took significantly more time to find their hosts. Conditioned wasps, which were tested in a congruent situation, were more responsive than unconditioned wasps, but there was no difference in foraging efficiency between these two groups in the wasps that showed a response. We conclude that persistent memories, such as formed after 3 experiences spaced in time, can lead to maladaptive foraging behavior if the contained information becomes unreliable.

Haller's rule states that brains scale allometrically with body size in all animals, meaning that relative brain size increases with decreasing body size. This rule applies both on inter- and intraspecific comparisons. Only 1 species, the extremely small parasitic wasp Trichogramma evanescens, is known as an exception and shows an isometric brain-body size relation in an intraspecific comparison between differently sized individuals. Here, we investigated if such an isometric brain-body size relationship also occurs in an intraspecific comparison with a slightly larger parasitic wasp, Nasonia vitripennis, a species that may vary 10-fold in body weight upon differences in levels of scramble competition during larval development. We show that Nasonia exhibits diphasic brain-body size scaling: larger wasps scale allometrically, following Haller's rule, whereas the smallest wasps show isometric scaling. Brains of smaller wasps are, therefore, smaller than expected and we hypothesized that this may lead to adaptations in brain architecture. Volumetric analysis of neuropil composition revealed that wasps of different sizes differed in relative volume of multiple neuropils. The optic lobes and mushroom bodies in particular were smaller in the smallest wasps. Furthermore, smaller brains had a relatively smaller total neuropil volume and larger cellular rind than large brains. These changes in relative brain size and brain architecture suggest that the energetic constraints on brain tissue outweigh specific cognitive requirements in small Nasonia wasps.