Haplodiploid Sex Determination Research Articles

Identification of Parthenogenesis-Inducing Effector Proteins in Wolbachia.

Bacteria in the genus Wolbachia have evolved numerous strategies to manipulate arthropod sex, including the conversion of would-be male offspring to asexually reproducing females. This so-called "parthenogenesis induction" phenotype can be found in a number of Wolbachia strains that infect arthropods with haplodiploid sex determination systems, including parasitoid wasps. Despite the discovery of microbe-mediated parthenogenesis more than 30 yr ago, the underlying genetic mechanisms have remained elusive. We used a suite of genomic, computational, and molecular tools to identify and characterize two proteins that are uniquely found in parthenogenesis-inducing Wolbachia and have strong signatures of host-associated bacterial effector proteins. These putative parthenogenesis-inducing proteins have structural homology to eukaryotic protein domains including nucleoporins, the key insect sex determining factor Transformer, and a eukaryotic-like serine-threonine kinase with leucine-rich repeats. Furthermore, these proteins significantly impact eukaryotic cell biology in the model Saccharomyces cerevisiae. We suggest that these proteins are parthenogenesis-inducing factors and our results indicate that this would be made possible by a novel mechanism of bacterial-host interaction.

Testing the local mate competition rule in a quasi-gregarious parasitoid with facultative superparasitism

Abstract As an important domain of evolutionary ecology, sex allocation theory well explains the evolution of investment into female versus male offspring. Local mate competition (LMC) is a good predictor of sex allocation, where the optimal sex ratio becomes less female-biased and asymptotically approaches 0.5 as the number of foundresses increases. Parasitoid wasps, with haplodiploid sex determination, offer excellent opportunities to test how organisms manipulate their offspring sex ratio in response to environmental variation, and many species have been proved to allocate sex according to predictions under LMC. When hosts are spatially clustered, as in gregarious species, the mating systems of quasi-gregarious parasitoids meet the essential assumptions (female mating before dispersal) of LMC. However, inconsistent with predictions, in the quasi-gregarious species Anastatus disparis (Hymenoptera: Eupelmidae), a strongly female-biased eclosion sex ratio (0.156 ± 0.018 to 0.185 ± 0.016) was observed as the number of females laying eggs in a patch increased. Superparasitism, in which 44.7% of parasitized hosts contained more than one egg but only one adult emerged from each host, was common in A. disparis. However, the egg sex ratio was determined by microsatellites and likely fit the predictions of LMC theory. Male-biased offspring mortality arising from superparasitism during development likely contributes to the shift from the primary sex ratio predicted under LMC to the observed female-biased eclosion sex ratio. Inconsistent with results in gregarious parasitoids, the role of superparasitism in driving sex ratio shifts in quasi-gregarious parasitoids should be incorporated into LMC-based predictions of sex ratios.

A One-Step Multiplex PCR Method to Rapidly Distinguish Two Strains of Diglyphus wani (Hymenoptera: Eulophidae) Against Agromyzid Leafminers.

Hymenopteran parasitoids generally show a haplo-diploid sex determination system. Haploid males are produced from unfertilized eggs, whereas diploid females develop from fertilized eggs (arrhenotokous). In some cases, diploid females develop from unfertilized eggs (thelytokous). Diglyphus wani (Hymenoptera: Eulophidae) is a biological control agent for agromyzid leafminers and have arrhenotokous and thelytokous strains. However, the morphological characteristics of two strains of D. wani are so similar that it is difficult to accurately distinguish them based on morphology. Here, a rapid molecular identification method was developed based on the mitochondrial gene cytochrome c oxidase I (COI) and one-step multiplex PCR. Two primer combinations, PC1 (Ar-F1/Th-F1/WR2) and PC2 (Ar-F1/Th-F4/WR2), were designed and repeatedly screened to distinguish two strains simultaneously, of which two special forward primers Th-F1/Th-F4 were used for the thelytokous strain and one special forward primer Ar-F1 was used for the arrhenotokous strain. In addition, a common reverse primer, WR2, was used for both strains. The PC1 and PC2 PCR assays were effective in distinguishing the two strains at different developmental stages and field colonies. This method provides a reliable, highly sensitive, and cost-effective tool for the rapid identification of the two strains of D. wani.

Revisiting the hymenopteran diploid male vortex: a review of avoidance mechanisms and incidence

AbstractAll hymenopterans (bees, ants, wasps, and sawflies) have haplodiploid sex determination. Generally, this involves haploid males developing from unfertilized eggs and diploid females developing from fertilized eggs, but diploid male production (DMP) frequently occurs. Some species have complementary sex determination (CSD), in which heterozygotes of a csd locus or loci are diploid females, hemizygotes are haploid males, and homozygotes are typically sterile diploid males. These diploid males underlie the diploid male vortex (DMV), the phenomenon of inbreeding reducing csd allelic diversity and causing increasingly higher production of sterile diploid males until the population dies out. The DMV has been cited as an extinction risk for many species across the order and a danger to both human‐controlled and natural hymenopteran populations important in biological control, pollination, and endangered species conservation. However, it has been unclear how frequently it occurs. We review the known mechanisms for DMV avoidance. Many of these mechanisms are linked to lifestyle, and so we structure our investigation around eusocial lifestyle and non‐eusocial (solitary or gregarious) species. We also review documented DMV incidence to make an inference about its prevalence. There are many means to avoid inbreeding or diploid male production, including eusocial‐exclusive polygyny, polyandry, and diploid queen execution. For both eusocial and non‐eusocial species these include biological mechanisms such as multi‐locus CSD or non‐CSD sex determination, diploid male inviability or brood removal, partial diploid male fecundity, and mating avoidance of kin or diploid males; or population genetic mechanisms such as gene flow and dispersal, balancing selection of csd alleles (with some being more prominent in eusocial vs. non‐eusocial species). Documented cases of DMV are uncommon, and incidence is often tied to exacerbatory conditions such as habitat fragmentation and host declines. With this review we suggest that due to numerous avoidance mechanisms, DMV risk may not be as high as previously believed, and requires specific circumstances. In doing so we aim to better inform hymenopteran breeding and conservation efforts.

Breeding for hygienic behavior in honey bees (Apis mellifera): a strong paternal effect

The haplodiploid sex determination of honey bees and the multiple mating of queens pose challenges in determining the genetic contribution of drones (male honey bees). This is especially important for breeding programs as, for example, when attempting to reinforce traits governing social immunity against pests and diseases. Here, we focused on breeding aiming at enhancing hygienic behavior, a trait that is known to reduce parasite load in honey bee colonies. To evaluate the contribution of drones versus queens to this trait, we conducted a two-step bidirectional selection program. First, we selected colonies with consistent phenotypes for low- or high-hygienic behavior (generation P). From those, we generated two types of daughter colonies (F1). One type originated from queens that had been artificially inseminated with selected drones that originated from queens of either low- or high-hygienic phenotype. The other type of colonies was set from naturally mated queens. We then compared the hygienic performance of the progeny colonies. In the next step, we used the F1 colonies (from either artificially inseminated or naturally mated queens) to produce naturally mated queens, which subsequently generated F2 colonies. These were then examined for the level of hygienic behavior. The results demonstrate the significant contribution of both parents to the phenotype of offspring. In particular, drones had a consistent and significant influence on the hygienic performance of the progeny throughout generations. These findings emphasize the great potential to propagate the hygienic trait in local populations by selecting lines for drones that carry the high-hygienic trait.

In vitro larval rearing method of eusocial bumblebee Bombus terrestris for toxicity test

Bumblebees are important pollinators of wild and agricultural plants but recently have been declining due to various stressors, such as pesticides and diseases. Because of the haplo-diploid sex determination system in hymenopterans, experiments using micro-colonies (small sub colonies without a queen) to identify risks to bumblebee health are limited as they are only able to produce males. Therefore, an experimental protocol for rearing bumblebee larvae in vitro is needed to better understand effects on worker larvae. Here, we aimed to establish a rearing method for larvae of Bombus terrestris for use in risk assessment assays. To confirm the validity of our rearing method, we tested two insecticides used for tomato cultivation, chlorfenapyr and dinotefuran. Bombus terrestris larvae fed with a high nutrient quantity and quality diet increased growth per day. All chlorfenapyr-exposed individuals died within 10 days at 2000-fold dilution, an application dose used for tomatoes. There were significant differences in adult emergence rate among almost all chlorfenapyr treatments. On the other hand, sublethal dinotefuran-exposure did not affect rates of pupation and adult emergence, growth, or larval and pupal periods. Although larvae were smaller than in the natural colony, this rearing method for B. terrestris larvae proved to be effective at evaluating realistic sub-colonies to pesticide exposures.

INSIGHTS OF CASTE DETERMINATIONS IN SOCIAL INSECTS

Eusocial insects display caste structures in which reproductive ability is possessed by a single or a few queens while all other colony members act as workers. In social insects like ants, bees, and termites, vital physiological processes are regulated at the colony scale. Females in social insects have at least one reproductive caste and one nonreproductive caste; many termites have at least two male castes. The castes have considerable anatomical, physiological, and behavioural differences in higher social insects. Organismal systems, such as pheromone sensing, hormone signaling, and brain signaling pathways, are deployed in novel circumstances to impact nestmate and colony behaviours due to physiological decentralization over evolutionary time. Significant morphogenesis with region-specific cellular proliferation and degradation occurs during soldier development through two moulting via a presoldier stage in termite. JH action has been developed, in which a high JH titer causes soldier differentiation and a low JH titer causes alate differentiation. A monogamous pair of primary reproductives (one king and one queen) generated from alates normally establishes termite colonies (winged adults). The nymph-alate pathway (sexual pathway) or the worker pathway differentiates larvae in Reticulitermes termites (neuter pathway). Haplo-diploid sex determination controls the first developmental transition, in which unfertilized (haploid) embryos become males and fertilized (diploid) embryos become females in the case of Cataglyphis ant genus. The queen’s mandibular gland secretion, a mix of fatty acids and aromatic chemicals, is critical for maintaining the reproductive division of labour in honeybees (Apis mellifera), suppressing ovary growth in workers. Besides this, the brood produced by the queen also inhibits ovary development in workers by emitting two pheromones: the brood pheromone (BP), mainly composed of esters, and the highly volatile E-b-ocimene.

Life History and the Transitions to Eusociality in the Hymenoptera

Although indirect selection through relatives (kin selection) can explain the evolution of effectively sterile offspring that act as helpers at the nest (eusociality) in the ants, bees, and stinging wasps (aculeate Hymenoptera), the genetic, ecological, and life history conditions that favor transitions to eusociality are poorly understood. In this study, ancestral state reconstruction on recently published phylogenies was used to identify the independent transitions to eusociality in each of the taxonomic families that exhibit eusociality. Semisociality, in which a single nest co-foundress monopolizes reproduction, often precedes eusociality outside the vespid wasps. Such a route to eusociality, which is consistent with groups consisting of a mother and her daughters (subsocial) at some stage and ancestral monogamy, is favored by the haplodiploid genetic sex determination of the Hymenoptera (diploid females and haploid males) and thus may explain why eusociality is common in the Hymenoptera. Ancestral states were also reconstructed for life history characters that have been implicated in the origins of eusociality. A loss of larval diapause during unfavorable seasons or conditions precedes, or coincides with, all but one transition to eusociality. This pattern is confirmed using phylogenetic tests of associations between state transition rates for sweat bees and apid bees. A loss of larval diapause may simply reflect the subsocial route to eusociality since subsociality is defined as females interacting with their adult daughters. A loss of larval diapause and a gain of subsociality may be associated with an extended breeding season that permits the production of at least two broods, which is necessary for helpers to evolve. Adult diapause may also lower the selective barrier to a first-brood daughter becoming a helper. Obligate eusociality meets the definition of a major evolutionary transition, and such transitions have occurred five times in the Hymenoptera.

Estimating heritability in honeybees: Comparison of three major methods based on empirical and simulated datasets

AbstractThe genetic contribution to phenotypic variation (namely the heritability) affects the response to selection. In honeybee, the haplodiploid sex determination does not allow the straightforward use of classical quantitative genetics methods to estimate heritability and genetic correlation. Nevertheless, specific methods have been developed for about 40 years. In particular, sibling analyses are frequently used with three main methods: an historical model using the average colony relatedness, a half‐sib/full‐sib model, and the more recent animal model. We compared those three methods using experimental and simulated datasets to see which performs the best. Our experimental dataset is composed of 10 colonies with a total sample of 853 workers. All individuals were genotyped to reconstitute the pedigree, and phenotypic traits were measured: labial palpus and wing cubital veins lengths. We also simulated phenotypic datasets with varying levels of heritability, common environment effect, and genetic correlation between traits. The simulation approach showed that the average colony relatedness was highly biased in presence of common environment effect whereas the half‐sib/full‐sib and the animal model gave reliable estimates of heritability. The animal model provided the greatest precision in genetic correlations. Using this latter method, we found that wing vein lengths had high heritabilities whereas the palpus length had lower heritability due to larger environmental variance and/or measurement error. Finally, significant genetic correlations among measured traits indicate that they do not evolve independently.

Lack of parent-of-origin effects in Nasonia jewel wasp: A replication and extension study

In diploid cells, the paternal and maternal alleles are, on average, equally expressed. There are exceptions from this: a small number of genes express the maternal or paternal allele copy exclusively. This phenomenon, known as genomic imprinting, is common among eutherian mammals and some plant species; however, genomic imprinting in species with haplodiploid sex determination is not well characterized. Previous work reported no parent-of-origin effects in the hybrids of closely related haplodiploid Nasonia vitripennis and Nasonia giraulti jewel wasps, suggesting a lack of epigenetic reprogramming during embryogenesis in these species. Here, we replicate the gene expression dataset and observations using different individuals and sequencing technology, as well as reproduce these findings using the previously published RNA sequence data following our data analysis strategy. The major difference from the previous dataset is that they used an introgression strain as one of the parents and we found several loci that resisted introgression in that strain. Our results from both datasets demonstrate a species-of-origin effect, rather than a parent-of-origin effect. We present a reproducible workflow that others may use for replicating the results. Overall, we reproduced the original report of no parent-of-origin effects in the haplodiploid Nasonia using the original data with our new processing and analysis pipeline and replicated these results with our newly generated data.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in <em>Nasonia vitripennis</em>

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

A novel method for using RNA‐seq data to identify imprinted genes in social Hymenoptera with multiply mated queens

Genomic imprinting results in parent-of-origin-dependent gene expression biased towards either the maternally or paternally derived allele at the imprinted locus. The kinship theory of genomic imprinting argues that this unusual expression pattern can be a manifestation of intra-genomic conflict between the maternally and paternally derived halves of the genome that arises because they are not equally related to the genomes of social partners. The theory thus predicts that imprinting may evolve wherever there are close interactions among asymmetrically related kin. The social Hymenoptera with permanent caste differentiation are suitable candidates for testing the kinship theory because haplodiploid sex determination creates strong relatedness asymmetries and nursing workers interact closely with kin. However, progress in the search for imprinted genes in the social Hymenoptera has been slow, in part because tests for imprinting rely on reciprocal crosses that are impossible in most species. Here, we develop a method to systematically search for imprinting in haplodiploid social insects without crosses, using instead samples of pooled individuals collected from natural colonies. We tested this protocol using data available for the leaf-cutting ant Acromyrmex echinatior, providing the first genome-wide search for imprinting in any ant. Although we identified several genes as potentially imprinted, none of the four genes tested could be verified as imprinted using digital droplet PCR, highlighting the need for higher quality genomic assemblies that accurately map duplicated genes.

The effects of diploid male production on honey bee colony evolution and survival

The order Hymenoptera includes most of the eusocial species on the planet. Correlated is the fact that many of the social species within the order are haplodiploid and use complementary sex determination (CSD) to determine the sex of offspring. CSD is the mechanism by why single sex alleles within an organism result in male development (haploid) and mismatched sex alleles develop into females (diploids). Related to this is the production of diploid males: fertilized eggs with matched sex alleles which develop as male instead of female. Honey bees are no exception to this, and as their numbers continue to suffer globally and their genetic diversity lowers, the effects of diploid male production (DMP) may pose an increased risk to the survival of bee colonies. In the present study, we develop a model for diploid male production in a honey bee colony and show that with ample resources, this phenomena has little effect on a colony’s health, but there is a limit to the sustainability of a colony suffering from diploid male production. We use our model to show that there were likely no great evolutionary pressures against CSD and DMP in wild honey bees as its effects on colony health in the wild would have been negligible but increased environmental hazards such as pesticides and monoculture crops increase the effects of DMP on colony health.

A comparative analysis of methods for de novo assembly of hymenopteran genomes using either haploid or diploid samples

Diverse invertebrate taxa including all 200,000 species of Hymenoptera (ants, bees, wasps and sawflies) have a haplodiploid sex determination system, where females are diploid and males are haploid. Thus, hymenopteran genome projects can make use of DNA from a single haploid male sample, which is assumed advantageous for genome assembly. For the purpose of gene annotation, transcriptome sequencing is usually conducted using RNA from a pool of individuals. We conducted a comparative analysis of genome and transcriptome assembly and annotation methods, using genetic sources of different ploidy: (1) DNA from a haploid male or a diploid female (2) RNA from the same haploid male or a pool of individuals. We predicted that the use of a haploid male as opposed to a diploid female will simplify the genome assembly and gene annotation thanks to the lack of heterozygosity. Using DNA and RNA from the same haploid individual is expected to provide better confidence in transcript-to-genome alignment, and improve the annotation of gene structure in terms of the exon/intron boundaries. The haploid genome assemblies proved to be more contiguous, with both contig and scaffold N50 size at least threefold greater than their diploid counterparts. Completeness evaluation showed mixed results. The SOAPdenovo2 diploid assembly was missing more genes than the haploid assembly. The SPAdes diploid assembly had more complete genes, but a higher level of duplicates, and a greatly overestimated genome size. When aligning the two transcriptomes against the male genome, the male transcriptome gave 2–3% more complete transcripts than the pool transcriptome for genes with comparable expression levels in both transcriptomes. However, this advantage disappears in the final results of the gene annotation pipeline that incorporates evidence from homologous proteins. The RNA pool is still required to obtain the full transcriptome with genes that are expressed in other life stages and castes. In conclusion, the use of a haploid source material for a de novo genome project provides a substantial advantage to the quality of the genome draft and the use of RNA from the same haploid individual for transcriptome to genome alignment provides a minor advantage for genes that are expressed in the adult male.

Sperm-limited males continue to mate, but females cannot detect the male state in a parasitoid wasp

Female mating frequency and male ejaculate allocation are likely to interact. Females may adjust their propensity for remating based on the amount of provided sperm to ensure a sufficient sperm supply, and males may determine sperm allocation based on female availability and female mating frequency. In this study, I investigated male and female mating behaviors in the parasitoid wasp Anisopteromalus calandrae. The wasp exhibits the haplo-diploid sex determination, in which sperm-depleted females are constrained to produce only sons by laying unfertilized eggs. The first experiment showed that a rapid succession of male mating decreased the production of daughters (fertilized eggs) by the inseminated females, suggesting that sperm-limited males provided an insufficient amount of sperm to the females. Although the males appeared to replenish their sperm store after 6 h, they mated upon encountering females despite their sperm shortage. The second experiment showed that copulation reduced the subsequent mating receptivity of the inseminated females irrespective of whether the females received a sufficient amount of sperm. Moreover, although approximately 26% of females accepted a second mating and recovered a certain degree of daughter production, remating was independent of the mating status of their first mating partner or the social environment. These results suggest that sperm-limited males may benefit from continuing to mate because their copulation prevents competing males from reproducing with their mates. Females incur a cost from not remating depending on the amount of sperm provided, which may result from weak environmental selection pressure or manipulation by the initial mate.Significance statementMale and female mating strategies are likely to evolve interdependently. Specifically, in species that produce limited numbers of sperm, the mating behavior of males and females is likely to be influenced by sperm transfer. The results of the present study suggest that males of the parasitoid wasp Anisopteromalus calandrae can replenish their sperm stores over time. However, sperm-limited males that encounter a potential mating partner mate rather than wait for sperm recovery. Females do not discriminate the male state before copulation, and those that mate with sperm-limited males suffer from sperm shortage. Although females can augment their sperm supply by remating, only a portion of females do so, regardless of sperm supply. The lack of a female facultative response suggests weak environmental selection on the species or the existence of sexual conflict over sperm transfer.

The role of epistatic interactions underpinning resistance to parasitic Varroa mites in haploid honey bee (Apis mellifera) drones.

The Red Queen hypothesis predicts that host-parasite coevolutionary dynamics can select for host resistance through increased genetic diversity, recombination and evolutionary rates. However, in haplodiploid organisms such as the honeybee (Apis mellifera), models suggest the selective pressure is weaker than in diploids. Haplodiploid sex determination, found in A.mellifera, can allow deleterious recessive alleles to persist in the population through the diploid sex with negative effects predominantly expressed in the haploid sex. To overcome these negative effects in haploid genomes, epistatic interactions have been hypothesized to play an important role. Here, we use the interaction between A.mellifera and the parasitic mite Varroa destructor to test epistasis in the expression of resistance, through the inhibition of parasite reproduction, in haploid drones. We find novel loci on three chromosomes which explain over 45% of the resistance phenotype. Two of these loci interact only additively, suggesting their expression is independent of each other, but both loci interact epistatically with the third locus. With drone offspring inheriting only one copy of the queen's chromosomes, the drones will only possess one of two queen alleles throughout the years-long lifetime of the honeybee colony. Varroa, in comparison, completes its highly inbred reproductive cycle in a matter of weeks, allowing it to rapidly evolve resistance. Faced with the rapidly evolving Varroa, a diversity of pathways and epistatic interactions for the inhibition of Varroa reproduction could therefore provide a selective advantage to the high levels of recombination seen in A.mellifera. This allows for the remixing of phenotypes despite a fixed queen genotype.

Can Plant Defence Mechanisms Provide New Approaches for the Sustainable Control of the Two-Spotted Spider Mite Tetranychus urticae?

Tetranychus urticae (T. urticae) Koch is a cosmopolitan, polyphagous mite which causes economic losses in both agricultural and ornamental plants. Some traits of T. urticae hamper its management, including a short life cycle, arrhenotokous parthenogenesis, its haplodiploid sex determination system, and its extraordinary ability to adapt to different hosts and environmental conditions. Currently, the use of chemical and biological control are the major control methods used against this mite. In recent years, some studies have focused on plant defence mechanisms against herbivores. Various families of plant compounds (such as flavonoids, glucosinolates, or acyl sugars) have been shown to behave as acaricides. Plants can be induced upon appropriate stimuli to increase their resistance against spider mites. This knowledge, together with the understanding of mechanisms by which T. urticae detoxifies and adapts to pesticides, may complement the control of this pest. Herein, we describe plant volatile compounds (VOCs) with repellent activity, and new findings about defence priming against spider mites, which interfere with the T. urticae performance. The use of VOCs and defence priming can be integrated into current management practices and reduce the damage caused by T. urticae in the field by implementing new, more sustainable crop management tools.

Embryo Microinjection and Transplantation Technique for <em>Nasonia vitripennis</em> Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation.

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Generation of heritable germline mutations in the jewel wasp Nasonia vitripennis using CRISPR/Cas9

The revolutionary RNA-guided endonuclease CRISPR/Cas9 system has proven to be a powerful tool for gene editing in a plethora of organisms. Here, utilizing this system we developed an efficient protocol for the generation of heritable germline mutations in the parasitoid jewel wasp, Nasonia vitripennis, a rising insect model organism for the study of evolution, development of axis pattern formation, venom production, haplo-diploid sex determination, and host–symbiont interactions. To establish CRISPR-directed gene editing in N. vitripennis, we targeted a conserved eye pigmentation gene cinnabar, generating several independent heritable germline mutations in this gene. Briefly, to generate these mutants, we developed a protocol to efficiently collect N. vitripennis eggs from a parasitized flesh fly pupa, Sarcophaga bullata, inject these eggs with Cas9/guide RNA mixtures, and transfer injected eggs back into the host to continue development. We also describe a flow for screening mutants and establishing stable mutant strains through genetic crosses. Overall, our results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in N. vitripennis, with strong potential for expansion to target critical genes, thus allowing for the investigation of several important biological phenomena in this organism.