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The sweat bee Halictus rubicundus is an important pollinator with a large latitudinal range and many potential barriers to gene flow. Alongside typical physical barriers, including mountain ranges and oceans, the climate may also impose restrictions on gene flow in this species. The climate influences voltinism and sociality in H. rubicundus, which is bivoltine and can nest socially at warmer lower latitudes but tends to be univoltine and solitary in the cooler north. Variation in voltinism could result in phenological differences, potentially limiting gene flow, but a previous study found no evidence for this in H. rubicundus populations in mainland Britain. Here we extend the previous study to consider populations of H. rubicundus at extreme northern and southern latitudes in the UK. We found that bees from a population in the far north of Scotland were genetically differentiated from bees collected in Cornwall in the south-west of England. In contrast, bees collected across the Irish Sea in Northern Ireland showed slight genetic overlap with both the Scottish and Cornish bees. Our results suggest that when populations at extreme latitudes are considered, phenology and the climate may act alongside physical barriers such as the Scottish Highlands and the Irish Sea to restrict gene flow in H. rubicundus. We discuss the implications of our results for local adaptation in the face of rapidly changing selection pressures which are likely under climate change.

The occurrence of facultative endosymbionts has been studied in many commercially important crop pest aphids, but their occurrence and effects in non-commercial aphid species in natural populations have received less attention. We screened 437 aphid samples belonging to 106 aphid species for the eight most common facultative aphid endosymbionts. We found one or more facultative endosymbionts in 53% (56 of 106) of the species investigated. This likely underestimates the situation in the field because facultative endosymbionts are often present in only some colonies of an aphid species. Oligophagous aphid species carried facultative endosymbionts significantly more often than monophagous species. We did not find a significant correlation between ant tending and facultative endosymbiont presence. In conclusion, we found that facultative endosymbionts are common among aphid populations. This study is, to our knowledge, the first of its kind in the Netherlands and provides a basis for future research in this field. For instance, it is still unknown in what way many of these endosymbionts affect their hosts, which is important for determining the importance of facultative endosymbionts to community dynamics.

Aphids are major agricultural pests and laboratory studies have shown that heritable bacterial endosymbionts can protect aphids against biocontrol parasitoids, but results from agricultural crop systems are lacking. Here we assess (1) which aphid species are present in Dutch strawberry crops, (2) which facultative endosymbionts they carry, and (3) how such facultative endosymbionts affect parasitism success of the biocontrol parasitoid wasps Aphidius ervi and Praon volucre on the most prevalent aphid species Acyrthosiphon malvae. By curing endosymbiont infections, we were able to determine the effects of endosymbionts and aphid genotypes on the parasitism success of both parasitoid species. We show that aphid species composition on strawberry crops is dynamic across the season and that facultative endosymbiont (co-)infections are common. Infection with facultative endosymbionts protected A. malvae against A. ervi, but not against P. volucre. Finally, we also found variation in levels of endogenous resistance among A. malvae lines against A. ervi. Our study is the first to show that a common aphid pest on agricultural farms can be protected against biocontrol parasitoids by both facultative endosymbionts and endogenous resistance.

Ecotypes, subpopulations or strains of a single species locally adapted to divergent ecological conditions within the same habitat are often considered to be the first steps in sympatric speciation. It has been suggested that two ecotypes are distinguishable in Nasonia vitripennis, a prominent model organism for parasitic Hymenoptera, with one ecotype parasitizing fly pupae in bird nests, and the other one parasitizing fly pupae on carrion. This differentiation into two ecotypes has been hypothesized to indicate incipient sympatric speciation in populations of this globally distributed species. In the present study, we investigated the differentiation into these two distinct ecotypes focusing on chemical profiles and the population genetic divergence in a wild N. vitripennis population from the Netherlands. Isofemale lines were obtained from bird nest boxes and from deer carrion, respectively, representing both microhabitats. To test for phenotypic differentiation, we determined the surface cuticular hydrocarbon (CHC) profiles from wasps of both host patches. Using a panel of 14 microsatellites, we concordantly determined the population genetic structure and tested for genetic differentiation between foundresses obtained from both microhabitats. Both the phenotypic as well as the genetic datasets show no evidence for any kind of separation based on the postulated two ecotypes, but rather suggest free interbreeding with no gene flow interruption between the two distinct host patches. Our findings challenge previous assumptions on clearly distinguishable ecotypes in N. vitripennis, and demonstrate how a chemical ecological assessment coupled with population genetics can be instrumental in re-evaluating the potential of ecological differentiation and incipient speciation mechanisms in parasitoid wasps.

Ecotypes, strains locally adapted to divergent ecological conditions, are often considered to be the first steps in sympatric speciation. It has been suggested that two distinguishable ecotypes hint at incipient sympatric speciation in Nasonia vitripennis, the prominent model organism for parasitoid wasps, with one ecotype parasitizing fly pupae in bird nests, and the other parasitizing fly pupae on carrion. In the present study, we investigated the differentiation into these two distinct ecotypes on a population genetic and on a phenotypic level in a wild N. vitripennis population in the Netherlands. Isofemale lines were obtained from bird nest boxes and from deer carrion, respectively, representing both microhabitats. Using a panel of 14 microsatellites, we determined the population genetic structure and tested for genetic differentiation between the foundresses obtained from both microhabitats. To test for phenotypic differentiation, we determined the cuticular hydrocarbon (CHC) profiles from wasps of both microhabitats. Both the genetic and the phenotypic datasets show no evidence for any kind of separation based on the postulated two ecotypes, but rather suggest free interbreeding with no gene flow interruption between the two distinct host patches. This challenges previous assumptions on distinguishable ecotypes in N. vitripennis, and invites a re-evaluation of potential ecological speciation mechanisms in parasitoid wasps. This dataset contains: 1) microsatellite data for 14 loci 25 N. vitripennis females collected in 2018 in the Hoge Veluwe in The Netherlands (HV2018_MicrosatelliteDataset.csv) 2) Raw data of GC-MS profiles of cuticular hydrocarbon (CHC) of N.vitripennis males and females from iso-female lines collected in 2018 in the Hoge Veluwe in The Netherlands (HV2018_CHC_RawData.tar) and an overview (HV2018_GCMS_Data.csv).

Endosymbionts are maternally transmitted, and therefore benefit from maximizing female offspring numbers. Parthenogenesis-induction (PI) is the most effective type of manipulation for transmission, but has solely been detected in haplodiploid species, whereas cytoplasmic incompatibility (CI) is detected frequently across the arthropod phylum, including haplodiploids. This puzzling observation led us to hypothesize that the molecular sex-determination mechanism of the haplodiploid host may be a constraining factor in the ability of endosymbionts to induce parthenogenesis. Recent insights indicate that PI-endosymbionts may be able to directly manipulate sex-determination genes to induce the necessary steps required for PI in haplodiploids. However, sex-determination cascades vary extensively, so PI-induction would require a specialized and host-dependent tool set. Contrastingly, CI-related genes target conserved cell-cycle mechanisms, are located on mobile elements, and spread easily. Finally, endosymbiont-manipulations may have a strong impact on the effectiveness of haplodiploid biocontrol agents, but can also be used to enhance their efficacy.

Investigate the role of oviposition- (OIPVs) and herbivore-induced plant volatiles (HIPVs) emitted by olive trees upon infestation by Bactrocera oleae as well as cues emitted by the insect host B. oleae.

We sequenced the microbiomes of aphids collected from Dutch bell pepper greenhouses in 2019 to study the prevalence of secondary endosymbionts., We sequenced the microbiomes of aphids collected from Dutch bell pepper greenhouses in 2019 to study the prevalence of secondary endosymbionts.

Biological control (biocontrol) of crop pests is a sustainable alternative to the use of biodiversity and organismal health-harming chemical pesticides. Aphids can be biologically controlled with parasitoid wasps; however, variable results of parasitoid-based aphid biocontrol in greenhouses are reported. Aphids may display genetically encoded (endogenous) defences that increase aphid resistance against parasitoids as under high parasitoid pressure there will be selection for parasitoid-resistant aphids, potentially affecting the success of parasitoid-based aphid biocontrol in greenhouses. Additionally, aphids may carry secondary bacterial endosymbionts that protect them against parasitoids. We studied whether there is variation in either of these heritable elements in aphids in greenhouses of sweet pepper, an agro-economically important crop in the Netherlands that is prone to aphid pests and where pest management heavily relies on biocontrol. We sampled aphid populations in organic (biocontrol only) and conventional (biocontrol and pesticides) sweet pepper greenhouses in the Netherlands during the 2019 crop growth season. We assessed the aphid microbiome through both diagnostic PCR and 16S rRNA sequencing and did not detect any secondary endosymbionts in the two most encountered aphid species, Myzus persicae and Aulacorthum solani. We also compared multiple aphid lines collected from different greenhouses for variation in levels of endogenous-based resistance against the parasitoids commonly used as biocontrol agents. We found no differences in the levels of endogenous-based resistance between different aphid lines. This study does not support the hypothesis that protective endosymbionts or the presence of endogenous resistant aphid lines affects the success of parasitoid-based biocontrol of aphids in Dutch greenhouses. Future investigations will need to address what is causing the variable successes of aphid biocontrol and what (biological and management-related)

Insects have a wide variety of mating systems. For parasitic wasps, mating sites are often confined to where their hosts occur and this mutual interaction therefore greatly affects the evolution of alternative mating strategies. Parasitoids of the genus Nasonia (Hymenoptera: Pteromalidae) – comprising the species Nasonia vitripennis (Walker), Nasonia longicornis Darling, Nasonia giraulti Darling, and Nasonia oneida Raychouhury & Desjardins – parasitize blowfly pupae and typically mate immediately after emergence of the females from the host puparium. However, in one Nasonia species, N. giraulti, mating predominantly occurs within the host. Here, we re-assess within-host mating (WHM) rates within the Nasonia genus, using laboratory lines and field-collected hosts, including N. oneida for which no data were available yet. We confirm that WHM rates are low in N. vitripennis and N. longicornis, but high in N. giraulti. WHM is rare in N. oneida, despite its sympatry and close phylogenetic relationship with N. giraulti. Multiparasitization experiments with N. vitripennis and N. giraulti resulted in lower WHM proportions of N. giraulti. To evaluate whether this was due to an exit hole in the host pupa created by N. vitripennis males – where an exit hole may result in emergence of N. giraulti females and possibly males prior to mating – we artificially created exit holes in the host parasitized by N. giraulti only. This also yielded a lower WHM proportion. Progeny analysis of interspecific crosses of N. longicornis and N. oneida with N. giraulti, generating hybrid female but non-hybrid male offspring, revealed that WHM is largely determined by the maternal species and, hence, by the species to which the male offspring belong. We conclude that WHM is predominantly a male-mediated trait in N. giraulti, resulting from males refraining from making exit holes and mating with females inside the host. We discuss how these findings can be used to evaluate whether WHM contributes to

During the transition from sexual to asexual reproduction, a suite of reproduction-related sexual traits become superfluous, and may be selected against if costly. Female functional virginity refers to asexual females resisting to mate or not fertilizing eggs after mating. These traits appear to be among the first that evolve during transitions from sexual to asexual reproduction. The genetic basis of female functional virginity remains elusive. Previously, we reported that female functional virginity segregates as expected for a single recessive locus in the asexual parasitoid wasp Asobara japonica. Here, we investigate the genetic basis of this trait by quantitative trait loci (QTL) mapping and candidate gene analyses. Consistent with the segregation of phenotypes, we found a single QTL of large effect, spanning over 4.23 Mb and comprising at least 131 protein-coding genes, of which 15 featured sex-biased expression in the related sexual species Asobara tabida. Two of the 15 sex-biased genes were previously identified to differ between related sexual and asexual population/species: CD151 antigen and nuclear pore complex protein Nup50. A third gene, hormone receptor 4, is involved in steroid hormone mediated mating behaviour. Overall, our results are consistent with a single locus, or a cluster of closely linked loci, underlying rapid evolution of female functional virginity in the transition to asexuality. Once this variant, causing rejection to mate, has swept through a population, the flanking region does not get smaller owing to lack of recombination in asexuals.

Biological control is widely successful for controlling pests, but effective biocontrol agents are now more difficult to obtain due to more restrictive international trade laws. Coupled with increasing demand, the efficacy of existing and new biocontrol agents needs to be improved with genetic and genomic approaches. Although they have been underutilised in the past, applying genetic and genomic techniques is becoming more feasible from both technological and economic perspectives. We review current methods and provide a framework for using them, incorporating evolutionary and ecological principles. First, it is necessary to identify which biocontrol trait to select and in what direction. Next, the genes or markers linked to these traits need be determined to better target their selection, followed by how to implement this information into a breeding program. Choosing a trait can be assisted by modelling to account for the proper agro-ecological context, and by knowing which traits have sufficiently high heritability values. We provide guidelines for designing genomic strategies in biocontrol programs, which depends on the organism, budget, and desired objective. Genomic approaches start with genome sequencing and assembly. We provide a guide for deciding the most successful sequencing strategy for biocontrol agents. Gene discovery involves quantitative trait loci (QTL) analyses, transcriptomic and proteomic studies, and gene editing. Improving biocontrol practices include marker-assisted selection, genomic selection and microbiome manipulation of biocontrol agents, and monitoring for genetic variation during rearing and post-release. We conclude by identifying the most promising applications of genetic and genomic methods to improve biological control efficacy.

This project aims to provide a decent quality of genome assembly with PacBio long reads, also generates genome-wide SNP markers with Illuminar short reads, and gene expression data with RNAseq reads.

In coordination with BINGO project (see other work under BioProject PRJNA433466), a single female Nesidiocoris tenuis, a zoophytophagous mirid and biocontrol agent, was sequenced using the 10X Genomics platform and assembled using Supernova version 2. Also provided is an ab initio-, homology-, and evidence-based annotation.

R-scripts and corresponding .csv data files for statistical analyses. Abbreviations used in the csv. files: WM: Winter Morph, SM: Summer Morph, EM: Pre-cold mated, LM: Post-cold mated, V: Virgin, F: Female, M: Male, R-scripts and corresponding .csv data files for statistical analyses. Abbreviations used in the csv. files: WM:Winter Morph SM:Summer Morph EM:Pre-cold mated LM:Post-cold mated V:Virgin F:Female M:Male

Bracon brevicornis is an ectoparasitoid of a wide range of larval-stage Lepidopterans, including several pests of important crops, such as the corn borer, Ostrinia nubilalis. It is also one of the earliest documented cases of complementary sex determination in Hymenoptera. Here, we present the linked-read-based genome of B. brevicornis, complete with an ab initio-derived annotation and protein comparisons with fellow braconids, Fopius arisanus and Diachasma alloeum. We demonstrate the potential of linked-read assemblies in exploring regions of heterozygosity and search for structural and homology-derived evidence of the complementary sex determiner gene (csd).

Collection of 34 inbred lines, derived from laboratory outbred Nasonia vitripennis HVRx population

Trichogramma brassicae (Bezdenko) are egg parasitoids that are used throughout the world as biological control agents and in laboratories as model species. Despite this ubiquity, few genetic resources exist beyond COI, ITS2, and RAPD markers. Aided by a Wolbachia infection, a wild-caught strain from Germany was reared for low heterozygosity and sequenced in a hybrid de novo strategy, after which several assembling strategies were evaluated. The best assembly, derived from a DBG2OLC-based pipeline, yielded a genome of 235 Mbp made up of 1,572 contigs with an N50 of 556,663 bp. Following a rigorous ab initio-, homology-, and evidence-based annotation, 16,905 genes were annotated and functionally described. As an example of the utility of the genome, a simple ortholog cluster analysis was performed with sister species T. pretiosum, revealing over 6000 shared clusters and under 400 clusters unique to each species. The genome and transcriptome presented here provides an essential resource for comparative genomics of the commercially relevant genus Trichogramma, but also for research into molecular evolution, ecology, and breeding of T. brassicae.

Biological control is widely successful at controlling pests, but effective biocontrol agents are now more difficult to import from countries of origin due to more restrictive international trade laws (the Nagoya Protocol). Coupled with increasing demand, the efficacy of existing and new biocontrol agents needs to be improved with genetic and genomic approaches. Although they have been underutilised in the past, application of genetic and genomic techniques is becoming more feasible from both technological and economic perspectives. We review current methods and provide a framework for using them. First, it is necessary to identify which biocontrol trait to select and in what direction. Next, the genes or markers linked to these traits need be determined, including how to implement this information into a selective breeding program. Choosing a trait can be assisted by modelling to account for the proper agro-ecological context, and by knowing which traits have sufficiently high heritability values. We provide guidelines for designing genomic strategies in biocontrol programs, which depend on the organism, budget, and desired objective. Genomic approaches start with genome sequencing and assembly. We provide a guide for deciding the most successful sequencing strategy for biocontrol agents. Gene discovery involves quantitative trait loci analyses, transcriptomic and proteomic studies, and gene editing. Improving biocontrol practices includes marker-assisted selection, genomic selection and microbiome manipulation of biocontrol agents, and monitoring for genetic variation during rearing and post-release. We conclude by identifying the most promising applications of genetic and genomic methods to improve biological control efficacy.

Seasonal polyphenism in Drosophila suzukii manifests itself in two discrete adult morphotypes, the “winter morph” (WM) and the “summer morph” (SM). These morphotypes are known to differ in thermal stress tolerance, and they co-occur during parts of the year. In this study, we aimed to estimate morph-specific survival and fecundity in laboratory settings simulating field conditions. We specifically analyzed how WM and SM D. suzukii differed in mortality and reproduction during and after a period of cold exposure resembling winter and spring conditions in temperate climates. The median lifespan of D. suzukii varied around 5 months for the WM flies and around 7 months for the SM flies. WM flies showed higher survival during the cold-exposure period compared with SM flies, and especially SM males suffered high mortality under these conditions. In contrast, SM flies had lower mortality rates than WM flies under spring-like conditions. Intriguingly, reproductive status (virgin or mated) did not impact the fly survival, either during the cold exposure or during spring-like conditions. Even though the reproductive potential of WM flies was greatly reduced compared with SM flies, both WM and SM females that had mated before the cold exposure were able to continuously produce viable offspring for 5 months under spring-like conditions. Finally, the fertility of the overwintered WM males was almost zero, while the surviving SM males did not suffer reduced fertility. Combined with other studies on D. suzukii monitoring and overwintering behavior, these results suggest that overwintered flies of both morphotypes could live long enough to infest the first commercial crops of the season. The high mortality of SM males and the low fertility of WM males after prolonged cold exposure also highlight the necessity for females to store sperm over winter to be able to start reproducing early in the following spring.

Zoophytophagous predators provide biocontrol services in various major crops of modern horticulture due to the combination of its predatory capacity and the induction of plant defenses derived from its phytophagy. However, under certain conditions of prey scarcity, these natural enemies can inflict plant damage. Exploitation of genetic variation and subsequent selective breeding on foraging traits is a potential alternative to overcome this inconvenience. In this study, we quantified the genetic variation of phytophagy and zoophagy of Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae), a zoophytophagous predator widely used in tomato crops to suppress key pests. We compared nine isofemale lines on their capacity to produce necrotic rings and wilting on tomato plants as a proxy for phytophagy, as well as their efficacy to prey on Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) eggs, as a proxy for zoophagy. Differences between isofemale lines in phytophagy and zoophagy indicated a genetic basis. Variation found in the zoophagy levels was larger than that in phytophagy levels. Our results showed that there is a genetic basis for the variation observed in the feeding behavior of isofemale lines of N. tenuis, highlighting the potential importance of selective breeding for such traits of biocontrol interest.