Direct Parental (DIPA) CRISPR in the jewel wasp, Nasonia vitripennis.

While CRISPR-Cas9 technology has demonstrated remarkable promise as a gene editing tool, its application in certain insects, such as the jewel wasp, Nasonia vitripennis, has been hindered by a lack of a tractable method for reagent delivery. Direct Parental-CRISPR (DIPA-CRISPR) recently emerged as a facile way to induce gene lesions because it involves adult injection with commercially available Cas9-sgRNA with no helper reagent. However, DIPA-CRISPR has so far been tested in only a few insects. Here, we have assessed the viability of DIPA-CRISPR in N. vitripennis by targeting two eye-pigmentation genes, cinnabar and vermilion, which function in the ommochrome pathway. Successful generation of lesions in both genes demonstrated the functionality of DIPA-CRISPR in N. vitripennis and its potential application to other genes, thereby expanding the range of insects suitable for this method. We varied two parameters, Cas9-sgRNA concentration and injection volume, to determine optimal injection conditions. We found that the larger injection volume coupled with either higher or lower concentration was needed for consistent mutation production. However, DIPA-CRISPR yields an overall low mutation rate in N. vitripennis when compared to other tested insects, a characteristic that may be attributed to a proportionally low vitellogenic import efficiency in the jewel wasp. We discuss different factors that may be considered in determining when DIPA-CRISPR may be preferable over other reagent delivery methods.

What is a B chromosome? Early definitions revisited.

Since the discovery of B chromosomes, multiple different definitions of these selfish genetic elements have been put forth. We reconsidered early definitions in light of recently published studies. While there are many characteristics that vary among different B chromosomes, such as their evolutionary origins, size, segregation behaviors, gene content, and function, there is one defining trait of all B chromosomes: they are nonessential for the organism. The points raised here may be useful for framing future B chromosome studies and help guide the categorization of new chromosomal elements that are uncovered in genomic studies.

PSRs: Selfish chromosomes that manipulate reproductive development.

B chromosomes are intriguing "selfish" genetic elements, many of which exhibit higher-than-Mendelian transmission. This perspective highlights a group of B chromosomes known as Paternal Sex Ratio chromosomes (PSRs), which are found in several insects with haplo-diploid reproduction. PSRs harshly alter the organism's reproduction to facilitate their own inheritance. A manifestation of this effect is the conversion of female destined individuals into males. Key to this conversion is the mysterious ability of PSRs to cause elimination of the sperm-inherited half of the genome during zygote formation. Here we discuss how PSRs were discovered, what is known about how they alter paternal chromatin dynamics to cause sex conversion, and how PSR-induced genome elimination is different from other forms of programmed genome elimination in different insects. PSRs also stand out because their DNA sequence compositions differ in remarkable ways from their insect's essential chromosomes, a characteristic suggestive of interspecies origins. Broadly, we also highlight poorly understood aspects of PSR dynamics that need to be investigated.

Ability of a selfish B chromosome to evade genome elimination in the jewel wasp, Nasonia vitripennis.

B chromosomes are non-essential, extra chromosomes that can exhibit transmission-enhancing behaviors, including meiotic drive, mitotic drive, and induction of genome elimination, in plants and animals. A fundamental but poorly understood question is what characteristics allow B chromosomes to exhibit these extraordinary behaviors. The jewel wasp, Nasonia vitripennis, harbors a heterochromatic, paternally transmitted B chromosome known as paternal sex ratio (PSR), which causes complete elimination of the sperm-contributed half of the genome during the first mitotic division of fertilized embryos. This genome elimination event may result from specific, previously observed alterations of the paternal chromatin. Due to the haplo-diploid reproduction of the wasp, genome elimination by PSR causes female-destined embryos to develop as haploid males that transmit PSR. PSR does not undergo self-elimination despite its presence with the paternal chromatin until the elimination event. Here we performed fluorescence microscopic analyses aimed at understanding this unexplained property. Our results show that PSR, like the rest of the genome, participates in the histone-to-protamine transition, arguing that PSR does not avoid this transition to escape self-elimination. In addition, PSR partially escapes the chromatin-altering activity of the intracellular bacterium, Wolbachia, demonstrating that this ability to evade chromatin alteration is not limited to PSR's own activity. Finally, we observed that the rDNA locus and other unidentified heterochromatic regions of the wasp's genome also seem to evade chromatin disruption by PSR, suggesting that PSR's genome-eliminating activity does not affect heterochromatin. Thus, PSR may target an aspect of euchromatin to cause genome elimination.

Mechanistically comparing reproductive manipulations caused by selfish chromosomes and bacterial symbionts.

Insects naturally harbor a broad range of selfish agents that can manipulate their reproduction and development, often leading to host sex ratio distortion. Such effects directly benefit the spread of the selfish agents. These agents include two broad groups: bacterial symbionts and selfish chromosomes. Recent studies have made steady progress in uncovering the cellular targets of these agents and their effector genes. Here we highlight what is known about the targeted developmental processes, developmental timing, and effector genes expressed by several selfish agents. It is now becoming apparent that: (1) the genetic toolkits used by these agents to induce a given reproductive manipulation are simple, (2) these agents target sex-specific cellular processes very early in development, and (3) in some cases, similar processes are targeted. Knowledge of the molecular underpinnings of these systems will help to solve long-standing puzzles and provide new tools for controlling insect pests.

A novel pattern of germ cell divisions in the production of hymenopteran insect eggs.

Egg development is a defining process of reproduction in higher eukaryotes. In the fruit fly, Drosophila melanogaster, this process begins with four mitotic divisions starting from a single germ cell, producing a cyst of 16 cystocytes; one of these cells will become the oocyte and the others supporting nurse cells. These mitotic divisions are exceptional because cytokinesis is incomplete, resulting in the formation of cytoplasmic bridges known as ring canals that interconnect the cystocytes. This organization allows all cystocytes to divide synchronously during each mitotic round, resulting in a final, power-of-2 number of germ cells. Given that numerous insects obey this power-of-2 rule, we investigated if strict cell doubling is a universal, underlying cause. Using confocal microscopy, we found striking departures from this paradigm in three different power-of-2 insects belonging to the Apocrita suborder (ants, bees and wasps). In these insects, the earliest-formed cystocytes cease to divide during the latter mitotic cycles while their descendants undergo further division, thereby producing a 'radial' direction of division activity. Such cystocyte division patterns that depart from strict cell doubling may be 'fine-tuned' in order to maintain a final, power-of-2 germ cell number.

Genome elimination mediated by gene expression from a selfish chromosome.

Numerous plants and animals harbor selfish B chromosomes that "drive" or transmit themselves at super-Mendelian frequencies, despite long-term fitness costs to the organism. Currently, it is unknown how B chromosome drive is mediated, and whether B-gene expression plays a role. We used modern sequencing technologies to analyze the fine-scale sequence composition and expression of paternal sex ratio (PSR), a B chromosome in the jewel wasp Nasonia vitripennis. PSR causes female-to-male conversion by destroying the sperm's hereditary material in young embryos to drive. Using RNA interference, we demonstrate that testis-specific expression of a PSR-linked gene, named haploidizer, facilitates this genome elimination-and-sex conversion effect. haploidizer encodes a putative protein with a DNA binding domain, suggesting a functional link with the sperm-derived chromatin.

Spermatogenesis in haploid males of the jewel wasp Nasonia vitripennis.

Males of hymenopteran insects, which include ants, bees and wasps, develop as haploids from unfertilized eggs. In order to accommodate their lack of homologous chromosome pairs, some hymenopterans such as the honeybee have been shown to produce haploid sperm through an abortive meiosis. We employed microscopic approaches to visualize landmark aspects of spermatogenesis in the jewel wasp Nasonia vitripennis, a model for hymenopteran reproduction and development. Our work demonstrates that N. vitripennis, like other examined hymenopterans, exhibits characteristics indicative of an abortive meiosis, including slight enlargement of spermatocytes preceding meiotic initiation. However, we saw no evidence of cytoplasmic buds containing centrioles that are produced from the first abortive meiotic division, which occurs in the honeybee. In contrast to other previously studied hymenopterans, N. vitripennis males produce sperm in bundles that vary widely from 16 to over 200, thus reflecting a range of cellular divisions. Our results highlight interesting variations in spermatogenesis among the hymenopteran insects, and together with previous studies, they suggest a pattern of progression from meiosis to a more mitotic state in producing sperm.

Sequence Expression of Supernumerary B Chromosomes: Function or Fluff?

B chromosomes are enigmatic heritable elements found in the genomes of numerous plant and animal species. Contrary to their broad distribution, most B chromosomes are non-essential. For this reason, they are regarded as genome parasites. In order to be stably transmitted through generations, many B chromosomes exhibit the ability to "drive", i.e., they transmit themselves at super-Mendelian frequencies to progeny through directed interactions with the cell division apparatus. To date, very little is understood mechanistically about how B chromosomes drive, although a likely scenario is that expression of B chromosome sequences plays a role. Here, we highlight a handful of previously identified B chromosome sequences, many of which are repetitive and non-coding in nature, that have been shown to be expressed at the transcriptional level. We speculate on how each type of expressed sequence could participate in B chromosome drive based on known functions of RNA in general chromatin- and chromosome-related processes. We also raise some challenges to functionally testing these possible roles, a goal that will be required to more fully understand whether and how B chromosomes interact with components of the cell for drive and transmission.

Cytological analysis of cytoplasmic incompatibility induced by Cardinium suggests convergent evolution with its distant cousin Wolbachia.

Cytoplasmic incompatibility (CI) is a conditional sterility in numerous arthropods that is caused by inherited, intracellular bacteria such as Wolbachia Matings between males carrying CI-inducing Wolbachia and uninfected females, or between males and females infected with different Wolbachia strains, result in progeny that die during very early embryogenesis. Multiple studies in diploid (Drosophila) and haplodiploid (Nasonia) insects have shown that CI-Wolbachia cause a failure of the paternally derived chromatin from resolving into distinct chromosomes. This leads to the formation of chromatin bridges and other mitotic defects as early as the first mitotic division, and to early mitotic arrest. It is currently unknown if CI-inducing symbionts other than Wolbachia affect similar cellular processes. Here, we investigated CI caused by an unrelated bacterium, Cardinium, which naturally infects a parasitic wasp, Encarsia suzannae CI crosses in this host-symbiont system resulted in early mitotic defects including asynchrony of paternal and maternal chromosome sets as they enter mitosis, chromatin bridges and improper chromosome segregation that spanned across multiple mitotic divisions, triggering embryonic death through accumulated aneuploidy. We highlight small differences with CI-Wolbachia, which could be due to the underlying CI mechanism or host-specific effects. Our results suggest a convergence of CI-related cellular phenotypes between these two unrelated symbionts.

Unique sequence organization and small RNA expression of a "selfish" B chromosome.

B chromosomes are found in numerous plants and animals. These nonessential, supernumerary chromosomes are often composed primarily of noncoding DNA repeats similar to those found within transcriptionally "silenced" heterochromatin. In order to persist within their resident genomes, many B chromosomes exhibit exceptional cellular behaviors, including asymmetric segregation into gametes and induction of genome elimination during early development. An important goal in understanding these behaviors is to identify unique B chromosome sequences and characterize their transcriptional contributions. We investigated these properties by examining a paternally transmitted B chromosome known as paternal sex ratio (PSR), which is present in natural populations of the jewel wasp Nasonia vitripennis. To facilitate its own transmission, PSR severely biases the sex ratio by disrupting early chromatin remodeling processes. Through cytological mapping and other approaches, we identified multiple DNA repeats unique to PSR, as well as those found on the A chromosomes, suggesting that PSR arose through a merger of sequences from both within and outside the N. vitripennis genome. The majority of PSR-specific repeats are interspersed among each other across PSR's long arm, in contrast with the distinct "blocks" observed in other organisms' heterochromatin. Through transcriptional profiling, we identified a subset of repeat-associated, small RNAs expressed by PSR, most of which map to a single PSR-specific repeat. These RNAs are expressed at much higher levels than those arising from A chromosome-linked repeats, suggesting that in addition to its sequence organization, PSR's transcriptional properties differ substantially from the pericentromeric regions of the normal chromosomes.

Sex Differences: Satellite DNA Directs Male-Specific Gene Expression.

Dosage compensation in some animals involves up-regulation of genes on the male's X chromosome. A study in the fruit fly Drosophila melanogaster shows that satellite DNA, and corresponding small non-coding RNA, helps the dosage compensation machinery preferentially find X sequences.

Genome Silencing and Elimination: Insights from a "Selfish" B Chromosome.

B chromosomes are non-essential components of numerous plant and animal genomes. Because many of these "extra" chromosomes enhance their own transmission in ways that are detrimental to the rest of the genome, they can be thought of as genome parasites. An extreme example is a paternally inherited B chromosome known as paternal sex ratio (PSR), which is found in natural populations of the jewel wasp Nasonia vitripennis. In order to ensure its own propagation, PSR severely biases the wasp sex ratio by converting diploid female-destined embryos into transmitting haploid males. This action occurs at the expense of the other paternally inherited chromosomes, which fail to resolve during the first round of division and are thus eliminated. Recent work has revealed that paternal genome elimination by PSR occurs through the disruption of a number of specific histone post-translational modifications, suggesting a central role for chromatin regulation in this phenomenon. In this review, we describe these recent advances in the light of older ones and in the context of what is currently understood about the molecular mechanisms of targeted genome silencing and elimination in other systems.

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.

A 'selfish' B chromosome induces genome elimination by disrupting the histone code in the jewel wasp Nasonia vitripennis.

Intragenomic conflict describes a phenomenon in which genetic elements act 'selfishly' to gain a transmission advantage at the expense of the whole genome. A non-essential, selfish B chromosome known as Paternal Sex Ratio (PSR) induces complete elimination of the sperm-derived hereditary material in the jewel wasp Nasonia vitripennis. PSR prevents the paternal chromatin from forming chromosomes during the first embryonic mitosis, leading to its loss. Although paternally transmitted, PSR evades self-elimination in order to be inherited. We examined important post-translational modifications to the DNA packaging histones on the normal genome and the PSR chromosome in the fertilized embryo. Three histone marks - H3K9me2,3, H3K27me1, and H4K20me1 - became abnormally enriched and spread to ectopic positions on the sperm's chromatin before entry into mitosis. In contrast, other histone marks and DNA methylation were not affected by PSR, suggesting that its effect on the paternal genome is specific to a subset of histone marks. Contrary to the paternally derived genome, the PSR chromosome was visibly devoid of the H3K27me1 and H4K20me1 marks. These findings strongly suggest that PSR causes paternal genome elimination by disrupting at least three histone marks following fertilization, while PSR avoids self-elimination by evading two of these marks.

Male-Killing Spiroplasma Alters Behavior of the Dosage Compensation Complex during Drosophila melanogaster Embryogenesis.

Numerous arthropods harbor maternally transmitted bacteria that induce the preferential death of males [1-7]. This sex-specific lethality benefits the bacteria because males are "dead ends" regarding bacterial transmission, and their absence may result in additional resources for their viable female siblings who can thereby more successfully transmit the bacteria [5]. Although these symbionts disrupt a range of developmental processes [8-10], the underlying cellular mechanisms are largely unknown. It was previously shown that mutations in genes of the dosage compensation pathway of Drosophila melanogaster suppressed male killing caused by the bacterium, Spiroplasma [10]. This result suggested that dosage compensation is a target of Spiroplasma. However, it remains unclear how this pathway is affected, and whether the underlying interactions require the male-specific cellular environment. Here, we investigated the cellular basis of male embryonic lethality in D. melanogaster induced by Spiroplasma. We found that the dosage compensation complex (DCC), which acetylates X chromatin in males [11], becomes mis-localized to ectopic regions of the nucleus immediately prior to the killing phase. This effect was accompanied by inappropriate histone acetylation and genome-wide mis-regulation of gene expression. Artificially induced formation of the DCC in infected females, through transgenic expression of the DCC-specific gene msl-2, resulted in mis-localization of this complex to non-X regions and early Spiroplasma-induced death, mirroring the killing effects in males. These findings strongly suggest that Spiroplasma initiates male killing by targeting the dosage compensation machinery directly and independently of other cellular features characteristic of the male sex.

Identification of Genes Uniquely Expressed in the Germ-Line Tissues of the Jewel Wasp Nasonia vitripennis.

The jewel wasp Nasonia vitripennis is a rising model organism for the study of haplo-diploid reproduction characteristic of hymenopteran insects, which include all wasps, bees, and ants. We performed transcriptional profiling of the ovary, the female soma, and the male soma of N. vitripennis to complement a previously existing transcriptome of the wasp testis. These data were deposited into an open-access genome browser for visualization of transcripts relative to their gene models. We used these data to identify the assemblies of genes uniquely expressed in the germ-line tissues. We found that 156 protein-coding genes are expressed exclusively in the wasp testis compared with only 22 in the ovary. Of the testis-specific genes, eight are candidates for male-specific DNA packaging proteins known as protamines. We found very similar expression patterns of centrosome associated genes in the testis and ovary, arguing that de novo centrosome formation, a key process for development of unfertilized eggs into males, likely does not rely on large-scale transcriptional differences between these tissues. In contrast, a number of meiosis-related genes show a bias toward testis-specific expression, despite the lack of true meiosis in N. vitripennis males. These patterns may reflect an unexpected complexity of male gamete production in the haploid males of this organism. Broadly, these data add to the growing number of genomic and genetic tools available in N. vitripennis for addressing important biological questions in this rising insect model organism.

Simple method for fluorescence DNA in situ hybridization to squashed chromosomes.

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.