Patrick M. Ferree

The DNA of sperm is uniquely packaged into an exceptionally condensed chromatin state with sperm nuclear basic proteins (SNBPs). These proteins are diverse, including protamines, protamine-like (PL) proteins, testis-specific (TS) histones, and HMG-box proteins. Usage of SNBP type varies widely among and within phyla. Moreover, SNBPs can be evolutionarily dynamic in copy number and amino acid composition, even among closely related species. Despite the diversity of insects, little is known about SNBP usage across members of this taxonomic group. A previous study biochemically identified SNBP candidates from the male reproductive tissue of the jewel wasp, Nasonia vitripennis, in addition to other insects. Here, we computationally examined existing N. vitripennis tissue transcriptomes to independently identify SNBP candidates. Our analyses uncovered 13 genes encoding proteins with SNBP characteristics, including exclusive gene expression in the testis, short protein length, and a high proportion of basic residues. The two highest-expressed of these genes encode PL proteins, which are evolutionary derivatives of the histone H1 family. Targeted degradation of the transcripts produced by the highest-expressed PL gene resulted in partial male sterility and defective sperm nuclear morphology in fertilized embryos, while RNAi treatment of the other PL gene yielded a subtle fertility effect. Both genes are present across hymenopteran insects but not outside this insect order. Moreover, they are not evolutionarily dynamic like the testis-specific HMG-box SNBPs in Drosophila species, which may reflect the lack of sex chromosomes, and resulting conflict between them, in hymenopteran insects.

Many organisms carry extra, non-essential chromosomes known as B chromosomes (Bs), which are selfishly transmitted at super-mendelian levels to offspring. This heightened transmission, termed drive, occurs during gametogenesis, usually in one of the two parents. In some cases, Bs can experience an opposing process, drag, which reduces their transmission. If these processes occur together in the same organism, one in each parental sex, then they may facilitate the spread of Bs while countering their accumulation in the genome to harmful levels. While previous studies have elucidated mechanistic aspects of B drive, little is known about drag or other factors that govern the inheritance of these selfish genetic elements. Here, we examined the inheritance of Paternal Sex Ratio (PSR), a single-copy B in the jewel wasp, Nasonia vitripennis , which is transmitted paternally to offspring. PSR drives by converting female-destined embryos into PSR-transmitting males. Using genetic manipulation, we produced exceptional PSR-carrying females, which were used to assess B transmission potential. We found that females transmit PSR at an unexpectedly low level compared to univalent chromosomes in other organisms. This reduced transmission stems from remarkable loss of PSR from the egg’s nucleus upon entry into meiosis, an effect that may be caused by an absence of microtubule-based spindle fibers in meiosis I-arrested wasp eggs. We also found that PSR is strictly limited to a single copy per genome, likely because wasps having two PSR copies die during development. Our findings reveal the successful inheritance of this selfish B chromosome involves its restriction to a single copy and hidden female meiotic drag in addition to strong paternal drive.

Many organisms carry extra, non-essential chromosomes known as B chromosomes (Bs), which are selfishly transmitted at super-Mendelian levels to offspring. This heightened transmission, termed drive, occurs during gametogenesis, usually in one of the two parents. In some cases, Bs can experience an opposing process, drag, which reduces their transmission. If these processes occur together in the same organism, one in each parental sex, then they may facilitate the spread of Bs while countering their accumulation in the genome to harmful levels. While previous studies have elucidated mechanistic aspects of B drive, little is known about drag or other factors that govern the inheritance of these selfish genetic elements. Here we examined the inheritance of Paternal Sex Ratio (PSR), a single-copy B in the jewel wasp, Nasonia vitripennis , which is transmitted paternally to offspring. PSR drives by converting female-destined embryos into PSR-transmitting males. Using genetic manipulation, we produced exceptional PSR-carrying females, which were used to assess B transmission potential. We found that females transmit PSR at unexpectedly low levels compared to univalent chromosomes in other organisms. This reduced transmission stems from remarkable loss of PSR from the egg’s nucleus upon entry into meiosis, an effect that may be caused by an absence of microtubule-based spindle fibers in meiosis I-arrested wasp eggs. We also found that PSR is strictly limited to a single copy per genome, suggesting that two PSR copies are lethal during development. Our findings reveal the successful inheritance of this selfish B chromosome involves a restriction to a single copy and hidden female meiotic drag in addition to its strong paternal drive.

Insects are the largest group of animals when it comes to the number and diversity of species. Yet, with the exception of Drosophila, no information is currently available on the primary structure of their sperm nuclear basic proteins (SNBPs). This paper represents the first attempt in this regard and provides information about six species of Neoptera: Poecillimon thessalicus, Graptosaltria nigrofuscata, Apis mellifera, Nasonia vitripennis, Parachauliodes continentalis, and Tribolium castaneum. The SNBPs of these species were characterized by acetic acid urea gel electrophoresis (AU-PAGE) and high-performance liquid chromatography fractionated. Protein sequencing was obtained using a combination of mass spectrometry sequencing, Edman N-terminal degradation sequencing and genome mining. While the SNBPs of several of these species exhibit a canonical arginine-rich protamine nature, a few of them exhibit a protamine-like composition. They appear to be the products of extensive cleavage processing from a precursor protein which are sometimes further processed by other post-translational modifications that are likely involved in the chromatin transitions observed during spermiogenesis in these organisms.

While clustered regularly interspaced short palindromic repeats (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 (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 amenability 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 reagent 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.

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.

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.