Biology subjects

Verhulst, E. C.

Publications and source records attributed to Verhulst, E. C..

11 recordsLinked to original sources

bioRxiv2025

SYNCAS-Mediated CRISPR-Cas9 Genome Editing in the Jewel Wasp, Nasonia vitripennis

Genetic engineering is a formidable approach to study biology. The development of CRISPR-Cas9 has allowed the genetic engineering of insect species from several orders, and in some species, this tool is used routinely for genetic research. However, insect gene editing often relies on the delivery of CRISPR-Cas9 components via embryo injection. This technique has a limitation: some species lay their eggs inside hard substrates or living hosts, making embryo collection impossible or labour-intensive. Recently, a variety of techniques that exploit maternal injection of nucleases have been developed to circumvent embryo injection. Yet, despite this variety of maternal delivery techniques, some insects remain refractory to gene editing. One of these is the parasitoid wasp, Nasonia vitripennis, an important hymenopteran model species. In this study, a recently developed method termed SYNCAS was used to perform knock-out (KO) of the cinnabar gene in this wasp, obtaining KO efficiencies up to ten times higher than reported for other maternal injection approaches. We found up to 2.73% of all offspring to display a KO phenotype, and we obtained up to 68 KO offspring per 100 injected mothers. The optimal timing of injection and provision of hosts for egg laying was determined. With this protocol, routine applications of CRISPR-Cas9 become feasible in this species, allowing reverse genetics studies of genes with unknown associated phenotypes and paving the way for more advanced editing techniques. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/643426v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@170bdd6org.highwire.dtl.DTLVardef@bf3cbaorg.highwire.dtl.DTLVardef@12be928org.highwire.dtl.DTLVardef@1d85e6a_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIMaternal injection of Cas9 RNP together with BAPC and saponins (SYNCAS) results in efficient gene editing of Nasonia vitripennis offspring C_LIO_LITiming of injection and egg collection is important for maximising efficiency and reducing screening effort C_LIO_LIThe efficient genetic engineering of Nasonia vitripennis suggests the applicability of SYNCAS to other parasitoid wasps, a category of insects particularly difficult to genetically engineer with embryonic Cas9 injections. C_LI

molecular biology↗

bioRxiv2025

A transcriptional control model for doublesex-dependent sex differentiation in Nasonia wasps

The transcription factor Doublesex (DSX) orchestrates insect sex differentiation. DSX affects Drosophila melanogaster male and female transcriptome, yet how DSX regulates gene expression in other species is poorly understood. We investigated sex-biased gene expression during the development of the parasitoid wasp Nasonia vitripennis, finding that more than three-quarters of its genes are sex-biased in at least one developmental point. Next, we transiently knocked down dsx expression to infer its role in sex-specific transcriptome regulation, revealing thousands of affected genes in males and a more subtle effect in females. Finally, we performed an in vitro DNA-protein interaction assay to identify DSX binding sites on the genome and primary DSX target genes. By integrating these three datasets, we defined DSXs regulatory function for all genes in N. vitripennis, revealing that DSX acts mainly in males as both an activator and a repressor. This male-centric model for DSX-mediated regulation is likely to apply to many other insect species.

molecular biology↗

bioRxiv2024

Insecticide resistance in Myzus persicae collected from sweet pepper

Populations of the green peach aphid, Myzus persicae, rapidly develop resistance to insecticides applied in agriculture, necessitating regular resistance monitoring of pest populations. Previous research identified two dominant multilocus genotypes (MLGs) in conventional (using insecticides + biological control agents) Dutch sweet pepper greenhouses: MLG-A under pymetrozine application and MLG-R under flonicamid application. This suggests positive selection for the genotypes by the application of these insecticides. However, no resistance of M. persicae to these insecticides has been reported yet. To investigate whether the insecticides were selectively driving the emergence of these specific MLGs, we compared the sensitivity of MLG-A and MLG-R to pymetrozine and flonicamid to that of six other genotypes from the same crop system. Additionally, we screened the M. persicae populations from Dutch sweet pepper greenhouses for known mutations conferring resistance to carbamates, pyrethroids, neonicotinoids, and tetronic and tetramic acid derivatives. Our results show that both MLG-A and MLG-R are less sensitive to pymetrozine compared to the other genotypes investigated. Furthermore, full mortality for MLG-R, the genotype least sensitive to flonicamid, was not achieved at the recommended field dose for this insecticide. Resistance mutations for carbamates and pyrethroids were prevalent among the MLGs, including MLG-A and MLG-R, while mutation A2226V, which is linked to resistance to tetronic and tetramic acid derivatives, was absent. Notably, the neonicotinoid resistance mutation R81T was found only in MLG-R, making this the northernmost detection of R81T in M. persicae to date. This study shows that various resistance mechanisms can accumulate in a single aphid genotype and that insecticides likely play a role in selecting for the dominant genotypes of M. persicae in conventional greenhouses.

genetics↗

bioRxiv2024

Population genetic structure of the aphid pest Myzus persicae in organic and conventional greenhouses

Aphids display remarkable adaptability to pest control strategies and their partheno-genetic reproduction results in rapid numerical increase of higher-fitness clones. Con-sequently, a high prevalence of only a few clonal lines could indicate positive selection for these genotypes, potentially reflecting adaptation to pest control methods. Here, we investigated the clonal diversity and population genetic structure of the green peach aphid Myzus persicae using microsatellite markers, in both organic and conventionally managed Dutch sweet pepper greenhouses over four consecutive years. In total, 26 distinct multilocus genotypes (MLG) were detected, with higher clonal diversity in organic than in conventional greenhouses. Strikingly, a single MLG dominated conventional greenhouses in 2019, only to be completely replaced by a new dominant MLG by 2022. Whole-genome sequencing of 15 sampled lines -- seven sharing the same MLG and eighth with unique MLGs -- revealed that all aphids with the dominant MLG of 2019, collected from various locations and over multiple years, originated from a single parthenogenetic ancestor. Our findings indicate that the population genetic structure of M. persicae differs between organically and conventionally managed sweet pepper greenhouses. The presence of dominant MLGs in conventional crop systems may suggest positive selection or evolutionary forces such as a founder effect. Understanding the forces driving these differences in population genetic structure and their impact on the efficacy of biocontrol agents will further help us improve control strategies for M. persicae in greenhouse crops.

genetics↗

bioRxiv2022

Evidence for an one-step mechanism of endosymbiont-induced thelytoky in the parasitoid wasp, Muscidifurax uniraptor

Wolbachia manipulates host reproduction in various haplodiploid insect species, in which fertilized eggs normally develop into diploid females while unfertilized eggs develop into haploid males. Females infected with a thelytoky-inducing Wolbachia produce diploid daughters from unfertilized eggs (thelytoky), but in some infected species diploid males spontaneously occur in low numbers. This suggests that diploidization and feminization are induced separately. In the Wolbachia-infected thelytokous parasitoid wasp, Muscidifurax uniraptor, occasional males have been found but with unknown ploidy. Therefore, we studied the mechanism of Wolbachia-induced thelytoky in M. uniraptor in the context of sex determination. We started by feeding different concentrations of tetracycline (antibiotic) to M. uniraptor females to gradually reduce the Wolbachia titre. We found that a decreased Wolbachia titre leads to an increased proportion of haploid male offspring, but we found no diploid males. Next, we studied the effect of Wolbachia infection on the expression and splicing of the sex determination genes transformer (Mutra) and transformer-2 (Mutra2) in female ovaries and conclude that Wolbachia does neither affect the expression nor the splicing of Mutra and Mutra2. We then measured Mutra and Mutra2 expression levels in developing zygotes at different time points and found a two-fold Mutra expression increase in Wol+ compared to Wol-. Finally, we used the sexually reproducing sister species, Muscidifurax raptorellus and artificially created triploid females to determine whether diploidization is sufficient for feminization. These triploid females, when virgin, produced haploid sons and diploid daughters, showing that in Muscidifurax feminization solely depends on ploidy level. This strongly suggests that Wolbachia only needs to induce diploidization and that bi-allelic Mutra expression is sufficient for female development.

genetics↗

bioRxiv2021

Silencing Doublesex expression triggers three-level pheromonal feminization in Nasonia males

The transcription factor Doublesex (Dsx) has a conserved function in controlling sexual morphological differences in insects, but our knowledge on its role in regulating sexual behavior is widely limited to Drosophila. Here, we show in the parasitoid wasp Nasonia vitripennis that males whose Dsx gene had been silenced by RNA interference (NvDsx-i) underwent a three-level pheromonal feminization: (1) NvDsx-i males were no longer able to attract females from a distance, owing to drastically reduced titers of the abdominal long-range sex pheromone. (2) NvDsx-i males were courted by wild-type males like females which correlated with a lower abundance of alkenes in their cuticular hydrocarbon (CHC) profiles. Supplementation of NvDsx-i male CHC profiles with realistic amounts of synthetic (Z)-9-hentriacontene (Z9C31), the most significantly reduced alkene in NvDsx-i males, interrupted courtship by wild-type conspecific males. Supplementation of female CHC profiles with Z9C31 reduced courtship and mating attempts by wild-type males. These results prove that Z9C31 is crucial for sex discrimination in Nasonia. (3) Nvdsx-i males were hampered in eliciting female receptivity during courtship and thus experienced severely reduced mating success, suggesting that they are unable to produce the hitherto unidentified oral aphrodisiac pheromone reported in N. vitripennis males. We conclude that Dsx is a multi-level key regulator of pheromone-mediated sexual communication in N. vitripennis. Silencing Dsx by RNA interference provides a new avenue for unraveling the molecular mechanisms underlying the pheromone-mediated sexual communication in insects.

molecular biology↗

bioRxiv2021

A conserved role of the duplicated Masculinizer gene in sex determination of the Mediterranean flour moth, Ephestia kuehniella

Sex determination in the silkworm, Bombyx mori, is based on Feminizer (Fem), a W-linked Fem piRNA that triggers female development in WZ individuals, and the Z-linked Masculinizer (Masc), which initiates male development and dosage compensation in ZZ individuals. While Fem piRNA is missing in a close relative of B. mori, Masc determines sex in several representatives of distant lepidopteran lineages. We studied the molecular mechanisms of sex determination in the Mediterranean flour moth, Ephestia kuehniella (Pyralidae). We identified an E. kuehniella Masc ortholog, EkMasc, and its paralog resulting from a recent duplication, EkMascB. Both genes are located on the Z chromosome and encode a similar Masc protein that contains two conserved domains but has lost the conserved double zinc finger domain. We developed PCR-based genetic sexing and demonstrated a peak in the expression of EkMasc and EkMascB genes only in early male embryos. Simultaneous knock-down experiments of both EkMasc and EkMascB using RNAi during early embryogenesis led to a shift from male- to female-specific splicing of the E. kuehniella doublesex gene (Ekdsx), their downstream effector, in ZZ embryos and resulted in a strong female-biased sex-ratio. Our results thus confirmed the conserved role of both EkMasc and EkMascB genes in masculinization. We suggest that the C-terminal proline-rich domain, we have identified in all functionally confirmed Masc proteins, in conjunction with the masculinizing domain, is important for transcriptional regulation of sex determination in Lepidoptera. The function of the Masc double zinc finger domain is still unknown, but appears to have been lost in E. kuehniella. Author summaryThe sex-determining cascade in the silkworm, Bombyx mori, differs greatly from those of other insects. In B. mori, female development is initiated by Fem piRNA expressed from the W chromosome during early embryogenesis. Fem piRNA silences Masculinizer (Masc) thereby blocking the male pathway resulting in female development. It is currently unknown whether this cascade is conserved across Lepidoptera. In the Mediterranean flour moth, Ephestia kuehniella, we identified an ortholog of Masc and discovered its functional duplication on the Z chromosome, which has not yet been found in any other lepidopteran species. We provide two lines of evidence that both the EkMasc and EkMascB genes play an essential role in masculinization: (i) they show a peak of expression during early embryogenesis in ZZ but not in WZ embryos and (ii) their silencing by RNAi results in female-specific splicing of the E. kuehniella doublesex gene (Ekdsx) in ZZ embryos and in a female-biased sex ratio. Our results suggest a conserved role of the duplicated Masc gene in sex determination of E. kuehniella.

genetics↗

bioRxiv2020

Braconidae revisited: Bracon brevicornis genome showcases the potential of linked-read sequencing in identifying a putative complementary sex determiner gene

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 genome of B. brevicornis, complete with an ab initio-derived annotation and protein comparisons with fellow braconids, Fopius arisanus and Diachasma alloem. 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).

genomics↗

bioRxiv2020

Sexually dimorphic traits and male-specific differentiation are actively regulated by Doublesex during specific developmental windows in Nasonia vitripennis

Sexually dimorphic traits in insects are rapidly evolving due to sexual selection which can ultimately lead to speciation. However, our knowledge of the underlying sex-specific molecular mechanisms is still scarce. Here we show that the highly conserved gene, Doublesex (Dsx), regulates rapidly diverging sexually dimorphic traits in the model parasitoid wasp Nasonia vitripennis (Hymenoptera: Pteromalidae). We present here the revised full Dsx gene structure with an alternative first exon, and two additional male NvDsx isoforms, which gives important insights into the evolution of the sex-specific oligomerization domains and C-termini. We show the sex-specific NvDsx expression throughout development, and demonstrate that transient NvDsx silencing in different male developmental stages dramatically shifts the morphology of two sexually dimorphic traits from male to female, with the effect being dependent on the timing of silencing. In addition, transient silencing of NvDsx in early male larvae affects male genitalia tissue growth but not morphology. This indicates that male NvDsx is actively required to suppress female-specific traits and to promote male-specific traits during specific developmental windows. These results also strongly suggest that in N. vitripennis most sex-specific tissues fully differentiate in the embryonic stage and only need the input of NvDsx for growth afterwards. This provides a first insight into the regulatory activity of Dsx in the Hymenoptera and will help to better understand the evolutionary and molecular mechanisms involved in sex-specific development in this parasitoid wasp, which can eventually lead to the development of new synthetic genetics-based tools for biological pest control by parasitoid wasps. Significance StatementIn insects, male and female differentiation is regulated by the highly conserved transcription factor Doublesex (Dsx). The role of Dsx in regulating rapidly evolving sexually dimorphic traits has received less attention, especially in wasps and bees. Here, we mainly focused on Dsx regulation of two sexually dimorphic traits and male genitalia morphology in the parasitoid wasp, Nasonia vitripennis. We demonstrate that Dsx actively regulates male-specific tissue growth and morphology during specific developmental windows. These findings will help to better understand the molecular mechanisms underlying the rapid evolution of sexual differentiation and sexually dimorphic traits in insects, but may also be the starting point for the development of new tools for biological control of pest insects by parasitoid wasps.

developmental biology↗

bioRxiv2020

Hybrid genome assembly and evidence-based annotation of the egg parasitoid and biological control agent Trichogramma brassicae

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.

genomics↗

bioRxiv2020

Jekyll or Hyde? The genome (and more) of Nesidiocoris tenuis, a zoophytophagous predatory bug that is both a biological control agent and a pest

Nesidiocoris tenuis (Reuter) is an efficient predatory biological control agent used throughout the Mediterranean Basin in tomato crops but regarded as a pest in northern European countries. Belonging to the family Miridae, it is an economically important insect yet very little is known in terms of genetic information - no published genome, population studies, or RNA transcripts. It is a relatively small and long-lived diploid insect, characteristics that complicate genome sequencing. Here, we circumvent these issues by using a linked-read sequencing strategy on a single female N. tenuis. From this, we assembled the 355 Mbp genome and delivered an ab initio, homology-based, and evidence-based annotation. Along the way, the bacterial "contamination" was removed from the assembly, which also revealed potential symbionts. Additionally, bacterial lateral gene transfer (LGT) candidates were detected in the N. tenuis genome. The complete gene set is composed of 24,688 genes; the associated proteins were compared to other hemipterans (Cimex lectularis, Halyomorpha halys, and Acyrthosiphon pisum), resulting in an initial assessment of unique and shared protein clusters. We visualised the genome using various cytogenetic techniques, such as karyotyping, CGH and GISH, indicating a karyotype of 2n=32 with a male-heterogametic XX/XY system. Additional analyses include the localization of 18S rDNA and unique satellite probes via FISH techniques. Finally, population genomics via pooled sequencing further showed the utility of this genome. This is one of the first mirid genomes to be released and the first of a mirid biological control agent, representing a step forward in integrating genome sequencing strategies with biological control research.

genomics↗