The olfactory system of Pieris brassicae caterpillars: from receptors to glomeruli.

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.

Memory extinction and spontaneous recovery shaping parasitoid foraging behavior.

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.

Multi-camera field monitoring reveals costs of learning for parasitoid foraging behaviour.

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.

Chromosomal scale assembly of parasitic wasp genome reveals symbiotic virus colonization.

Endogenous viruses form an important proportion of eukaryote genomes and a source of novel functions. How large DNA viruses integrated into a genome evolve when they confer a benefit to their host, however, remains unknown. Bracoviruses are essential for the parasitism success of parasitoid wasps, into whose genomes they integrated ~103 million years ago. Here we show, from the assembly of a parasitoid wasp genome at a chromosomal scale, that bracovirus genes colonized all ten chromosomes of Cotesia congregata. Most form clusters of genes involved in particle production or parasitism success. Genomic comparison with another wasp, Microplitis demolitor, revealed that these clusters were already established ~53 mya and thus belong to remarkably stable genomic structures, the architectures of which are evolutionary constrained. Transcriptomic analyses highlight temporal synchronization of viral gene expression without resulting in immune gene induction, suggesting that no conflicts remain between ancient symbiotic partners when benefits to them converge.

A neuronal arms race: the role of learning in parasitoid-host interactions.

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.

Species- and size-related differences in dopamine-like immunoreactive clusters in the brain of Nasonia vitripennis and N. giraulti.

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.

The Jewel Wasp Standard Brain: Average shape atlas and morphology of the female Nasonia vitripennis brain.

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 naïve 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.

No gains for bigger brains: Functional and neuroanatomical consequences of relative brain size in a parasitic wasp.

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.

Dystrophin is required for normal synaptic gain in the Drosophila olfactory circuit.

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 GABAA 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.

Automated high-throughput individual tracking system for insect behavior: Applications on memory retention in parasitic wasps.

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®, in combination with a Perspex stack of plates as small as 18 × 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.

Selection for associative learning of color stimuli reveals correlated evolution of this learning ability across multiple stimuli and rewards.

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.

Nasonia Parasitic Wasps Escape from Haller's Rule by Diphasic, Partially Isometric Brain-Body Size Scaling and Selective Neuropil Adaptations.

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.

Maximized complexity in miniaturized brains: morphology and distribution of octopaminergic, dopaminergic and serotonergic neurons in the parasitic wasp, Trichogramma evanescens.

The parasitic wasp, Trichogramma evanescens, is an extremely small insect, with a body length as small as 0.3 mm. To facilitate this miniaturization, their brains may have evolved to contain smaller neural components and/or reduced neural complexity than larger insects. Here, we study whether the size and number of neurons are reduced in the miniaturized brain of T. evanescens, focusing on neurons that express serotonin (5HT), octopamine (OA) and dopamine (DA). We provide the first description of the distribution, projection patterns and number of 5HT-, OA- and DA-like immunoreactive cell bodies in T. evanescens and compare our observations with descriptions of much larger insects. The brains of T. evanescens contain comparable numbers of monoaminergic neurons to those of larger insects. Serotonergic neurons appear to be especially conserved; most of the clusters contain a similar number of neurons to those described in Apis mellifera and Drosophila melanogaster. This maintained complexity may have been facilitated by miniaturization of neuron size. However, many dopaminergic and some octopaminergic neuron clusters in T. evanescens contain fewer neurons than in larger insects. Modification of the complexity of these monoaminergic systems may have been necessary to maintain neuron functionality during brain miniaturization in T. evanescens. Our results reveal some of the evolutionary adaptations that may enable behavioural and cognitive complexity with respect to miniaturized brains.

Effects of Isometric Brain-Body Size Scaling on the Complexity of Monoaminergic Neurons in a Minute Parasitic Wasp.

Trichogramma evanescens parasitic wasps show large phenotypic plasticity in brain and body size, resulting in a 5-fold difference in brain volume among genetically identical sister wasps. Brain volume scales linearly with body volume in these wasps. This isometric brain scaling forms an exception to Haller's rule, which states that small animals have relatively larger brains than large animals. The large plasticity in brain size may be facilitated by plasticity in neuron size, in the number of neurons, or both. Here, we investigated whether brain isometry requires plasticity in the number and size of monoaminergic neurons that express serotonin (5HT), octopamine (OA), and dopamine (DA). Genetically identical small and large T. evanescens appear to have the same number of 5HT-, OA-, and DA-like immunoreactive cell bodies in their brains, but these cell bodies differ in diameter. This indicates that brain isometry can be facilitated by plasticity in the size of monoaminergic neurons, rather than plasticity in numbers of monoaminergic neurons. Selection pressures on body miniaturization may have resulted in the evolution of miniaturized neural pathways that allow even the smallest wasps to find suitable hosts. Plasticity in the size of neural components may be among the mechanisms that underlie isometric brain scaling while maintaining cognitive abilities in the smallest individuals.

The complexity of learning, memory and neural processes in an evolutionary ecological context.

The ability to learn and form memories is widespread among insects, but there exists considerable natural variation between species and populations in these traits. Variation manifests itself in the way information is stored in different memory forms. This review focuses on ecological factors such as environmental information, spatial aspects of foraging behavior and resource distribution that drive the evolution of this natural variation and discusses the role of different genes and neural networks. We conclude that at the level of individual, population or species, insect learning and memory cannot be described as good or bad. Rather, we argue that insects evolve tailor-made learning and memory types; they gate learned information into memories with high or low persistence. This way, they are prepared to learn and form memory to optimally deal with the specific ecologies of their foraging environments.

How to escape from Haller's rule: Olfactory system complexity in small and large Trichogramma evanescens parasitic wasps.

While Haller's rule states that small animals have relatively larger brains, minute Trichogramma evanescens Westwood (Hymenoptera: Trichogrammatidae) parasitic wasps scale brain size linearly with body size. This linear brain scaling allows them to decrease brain size beyond the predictions of Haller's rule, and is facilitated by phenotypic plasticity in brain size. In the present study we addressed whether this plasticity resulted in adaptations to the complexity of the morphology of the olfactory system of small and large T. evanescens. We used confocal laser scanning microscopy to compare size and number of glomeruli in the antennal lobe in the brain, and scanning electron microscopy to compare length and number of olfactory sensilla on the antennae. The results show a similar level of complexity of the olfactory system morphology of small and large wasps. Wasps with a similar genotype but very different brain and body size have similarly sized olfactory sensilla and most of them occur in equal numbers on the antennae. Small and large wasps also have a similar number of glomeruli in the antennal lobe. Glomeruli in small brains are, however, smaller in both absolute and relative volume. These similarities between small and large wasps may indicate that plasticity in brain size does not require plasticity in the gross morphology of the olfactory system. It may be vital for wasps of all sizes to have a large number of olfactory receptor types, to maintain olfactory precision in their search for suitable hosts, and consequently maintain their reproductive success and Darwinian fitness.

Differentially expressed genes linked to natural variation in long-term memory formation in Cotesia parasitic wasps.

Even though learning and memory are universal traits in the Animal Kingdom, closely related species reveal substantial variation in learning rate and memory dynamics. To determine the genetic background of this natural variation, we studied two congeneric parasitic wasp species, Cotesia glomerata and C. rubecula, which lay their eggs in caterpillars of the large and small cabbage white butterfly. A successful egg laying event serves as an unconditioned stimulus (US) in a classical conditioning paradigm, where plant odors become associated with the encounter of a suitable host caterpillar. Depending on the host species, the number of conditioning trials and the parasitic wasp species, three different types of transcription-dependent long-term memory (LTM) and one type of transcription-independent, anesthesia-resistant memory (ARM) can be distinguished. To identify transcripts underlying these differences in memory formation, we isolated mRNA from parasitic wasp heads at three different time points between induction and consolidation of each of the four memory types, and for each sample three biological replicates, where after strand-specific paired-end 100 bp deep sequencing. Transcriptomes were assembled de novo and differential expression was determined for each memory type and time point after conditioning, compared to unconditioned wasps. Most differentially expressed (DE) genes and antisense transcripts were only DE in one of the LTM types. Among the DE genes that were DE in two or more LTM types, were many protein kinases and phosphatases, small GTPases, receptors and ion channels. Some genes were DE in opposing directions between any of the LTM memory types and ARM, suggesting that ARM in Cotesia requires the transcription of genes inhibiting LTM or vice versa. We discuss our findings in the context of neuronal functioning, including RNA splicing and transport, epigenetic regulation, neurotransmitter/peptide synthesis and antisense transcription. In conclusion, these brain transcriptomes provide candidate genes that may be involved in the observed natural variation in LTM in closely related Cotesia parasitic wasp species.

Insects Can Count: Sensory Basis of Host Discrimination in Parasitoid Wasps Revealed.

The solitary parasitoid Leptopilina heterotoma is one of the best studied organisms concerning the ecology, behaviour and physiology of host discrimination. Behavioural evidence shows that L. heterotoma uses its ovipositor to discriminate not only between parasitized and unparasitized Drosophila melanogaster larvae, but also to discriminate between hosts with different numbers of parasitoid eggs. The existing knowledge about how and when the parasitoid marks the host motivated us to unravel the chemosensory basis of host discrimination by L. heterotoma that allows it to choose the "best" host available. In this paper we report on electrophysiological recordings of multi-neural responses from the single taste sensillum on the tip of the unpaired ovipositor valve. We stimulated this sensillum with haemolymph of unparasitized, one-time-parasitized and two-times-parasitized Drosophila larvae. We demonstrate for the first time that quantitative characteristics of the neural responses to these haemolymph samples differed significantly, implying that host discrimination is encoded by taste receptor neurons in the multi-neuron coeloconic ovipositor sensillum. The activity of three of the six neurons present in the sensillum suffices for host discrimination and support the hypothesis that L. heterotoma females employ an ensemble code of parasitization status of the host.

Isoprene emission by poplar is not important for the feeding behaviour of poplar leaf beetles.

BACKGROUND: Chrysomela populi (poplar leaf beetle) is a common herbivore in poplar plantations whose infestation causes major economic losses. Because plant volatiles act as infochemicals, we tested whether isoprene, the main volatile organic compound (VOC) produced by poplars (Populus x canescens), affects the performance of C. populi employing isoprene emitting (IE) and transgenic isoprene non-emitting (NE) plants. Our hypothesis was that isoprene is sensed and affects beetle orientation or that the lack of isoprene affects plant VOC profiles and metabolome with consequences for C. populi feeding. RESULTS: Electroantennographic analysis revealed that C. populi can detect higher terpenes, but not isoprene. In accordance to the inability to detect isoprene, C. populi showed no clear preference for IE or NE poplar genotypes in the choice experiments, however, the beetles consumed a little bit less leaf mass and laid fewer eggs on NE poplar trees in field experiments. Slight differences in the profiles of volatile terpenoids between IE and NE genotypes were detected by gas chromatography - mass spectrometry. Non-targeted metabolomics analysis by Fourier Transform Ion Cyclotron Resonance Mass Spectrometer revealed genotype-, time- and herbivore feeding-dependent metabolic changes both in the infested and adjacent undamaged leaves under field conditions. CONCLUSIONS: We show for the first time that C. populi is unable to sense isoprene. The detected minor differences in insect feeding in choice experiments and field bioassays may be related to the revealed changes in leaf volatile emission and metabolite composition between the IE and NE poplars. Overall our results indicate that lacking isoprene emission is of minor importance for C. populi herbivory under natural conditions, and that the lack of isoprene is not expected to change the economic losses in poplar plantations caused by C. populi infestation.