Bombyx mori P-element Somatic Inhibitor (BmPSI) Is a Key Auxiliary Factor for Silkworm Male Sex Determination

Manipulation of sex determination pathways in insects provides the basis for a wide spectrum of strategies to benefit agriculture and public health. Furthermore, insects display a remarkable diversity in the genetic pathways that lead to sex differentiation. The silkworm, Bombyx mori, has been cultivated by humans as a beneficial insect for over two millennia, and more recently as a model system for studying lepidopteran genetics and development. Previous studies have identified the B. mori Fem piRNA as the primary female determining factor and BmMasc as its downstream target, while the genetic scenario for male sex determination was still unclear. In the current study, we exploite the transgenic CRISPR/Cas9 system to generate a comprehensive set of knockout mutations in genes BmSxl, Bmtra2, BmImp, BmImpM, BmPSI and BmMasc, to investigate their roles in silkworm sex determination. Absence of Bmtra2 results in the complete depletion of Bmdsx transcripts, which is the conserved downstream factor in the sex determination pathway, and induces embryonic lethality. Loss of BmImp or BmImpM function does not affect the sexual differentiation. Mutations in BmPSI and BmMasc genes affect the splicing of Bmdsx and the female reproductive apparatus appeared in the male external genital. Intriguingly, we identify that BmPSI regulates expression of BmMasc, BmImpM and Bmdsx, supporting the conclusion that it acts as a key auxiliary factor in silkworm male sex determination.


Introduction
Genetic systems for sex determination in insects show high diversity in different species. Sex determination in the fruit fly, Drosophila melanogaster, is controlled hierarchically by X:A > Sxl > tra/tra2 > dsx and fru [1,2]. The X:A ratio of 1 promotes transcription of Sex-lethal (Sxl) and results in feminization, while 0.5 to Sxl suppression and male differentiation [3][4][5][6][7][8][9][10]. Sxl proteins control the splicing of female transformer (tra) mRNAs that give rise to functional proteins, while no functional Sxl proteins exist in the male [11,12]. The search of homolog genes in the sex determination of D. melanogaster has been found a conserved relationship among dsx/tra across dipterans [13,14] In another Diptera, Musca domestica, female employs a F D allele which is encoded by the tra gene [15]. The medfly, Ceratitis capitata, has an as yet unidentified dominant male-determining factor on the Y chromosome [16]. The sex determination factors (F or M) in these two insects control the downstream gene doublesex (dsx) to generate sex-specific splicing isoforms. In contrast to Drosophila, Cctra and Mdtra seem to initiate an autoregulatory mechanism in XX embryos that provides continuous tra female-specific function and acts as a cellular memory maintaining the female pathway [17][18][19]. Many other insect species also exploit tra as the sex determination factor. For example, the honeybee, Apis mellifera, uses the complementary sex determiner gene (csd) to regulate feminization, which activates the feminizer gene (fem) by directing splicing to form the female functional Fem protein [20]. This fem gene is considered an orthologue of Cctra gene [21]. The tra gene in the red flour beetle, Tribolium castaneum, controls female sex determination by regulating dsx sex-specific splicing [22]. Also, Lucilia cuprina and Nasonia vitripennis, are reported to use tra as a female-determining signal [23,24]. Recently, Hall et al. identified Nix a distant homolog of transformer2 (tra2) from Aedes aegypti as the male-determining factor [25]. These data shows that the dsx/tra axis is conserved in many insect species and tra is the key gene around which variation in sex determining mechanisms has evolved in all insect species with the exception of Aedes and Lepidopteran insects.
Species of lepidoptera exhibit markedly different sex determination pathways from those seen in the flies, bees and beetles [26]. In the silkworm Bombyx mori, females possess a femaledetermining W chromosome have heteromorphic sex chromosomes (ZW) and males are homomorphic (ZZ) [27]. No tra ortholog has been identified in this order, possibly as a result of highly divergent sequence [28]. Bioinformatic analyses fail to identify a tra ortholog in B. mori and no dsxRE (dsx cis-regulatory element) binding sites are found in the target gene ortholog, Bmdsx, resulting in the default mode of female-specific splicing of the latter [29,30]. BmPSI (P-element somatic inhibitor) and BmHrp28 (hnRNPA/B-like 28) were reported to regulate Bmdsx splicing through binding CE1 sequences of the female-specific exon 4 of Bmdsx pre-mRNA [30,31]. Another potential regulator, BmImp (IGF-II mRNA binding protein), enhances the male-specific splicing of Bmdsx pre-mRNA by increasing RNA binding activity of BmPSI [32]. The Z-linked BmImp binds to the A-rich sequences in its own pre-mRNA to induce the male-specific splicing of its pre-mRNA, and this splicing pattern is maintained by an autoregulatory mechanism, being BmImp M (the male-specific splicing form of BmImp) able to bind its corresponding pre-mRNA [33]. Since BmPSI or BmImp products do not exhibit any sequence similarities to known Ser/Arg (SR) proteins, such as Tra and Tra2, the regulatory mechanisms of sex-specific alternative splicing of Bmdsx that of exon skipping is distinct from that of Dmdsx that of 3' alternative splice [34]. Recently, the product of the W chromosomederived B. mori sex determination factor fem (female-enriched PIWI-interacting RNA) was identified to target the downstream gene BmMasc for controlling Bmdsx sex-specific splicing [35]. This remarkable finding reveals that fem is the primary female sex determinator in the silkworm. However, the genetic relationship among these genes in B. mori sex determination is still in mystery.
We use here a binary transgenic CRISPR/Cas9 system to generate somatic mutations in sex determination pathway genes in B. mori. Three genes, BmMasc, BmPSI and BmImp, are involved in sex regulation only in lepidopteran insects, and two, Bmtra2 and BmSxl, are structural orthologs of the key sex regulation factors in Drosophila. We focus on the sexually dimorphic traits of reproductive structures and sex-specific alternative splicing forms of Bmdsx, the bottom gene of the sex determination. The results show that BmPSI and BmMasc affect Bmdsx splicing and the male reproductive tissues, supporting the conclusion that they have roles in sex determination. Disruption of BmImp or Bmtra2 causes severe developmental defects in both sexes, supporting their critical roles other than sex determination. Furthermore, loss-offunction mutations of BmPSI altered transcriptional or post-transcriptional (splicing) of BmMasc, BmImp and Bmdsx in males. These data strongly support the conclusion that BmPSI plays a key auxiliary in male sex determination in B. mori.

CRISPR/Cas9-mediated mutagenesis of sex determination genes
We established a binary transgenic CRISPR/Cas9 system to make somatic mutagenesis targeting selected genes. This system contains two separated lines, one is to express Cas9 protein under the control of a B. mori nanos promoter (nos-Cas9) and another is to express sequencespecific sgRNAs under the control of a B. mori U6 promoter (U6-sgRNA). Two independent lines of nos-Cas9 and from three to 29 independent lines of each U6-sgRNA construct were obtained following piggyBac-mediated transgenesis (S1 Table). Genomic mutagenesis in F 0 animals was confirmed by genomic PCR, and subsequent physiological phenotypes were investigated (Fig 1). The results confirm that the transgenic CRISPR/Cas9 system works effectively (S1-S5 Figs).

BmPSI regulates transcription of BmMasc and BmImp M
Bmdsx is the conserved downstream component of the silkworm sex determination pathway and mutations in BmPSI and BmMasc had an effect on its splicing. Mutations in BmSxl, BmImp and BmImp M resulted in the appearance of a larger Bmdsx M (the male-specific splicing form of Bmdsx) mRNA isoform while the profile of Bmdsx F (the female-specific splicing form of Bmdsx) appeared unaltered (Fig 2, lanes 4, 10 and 12). Sequence analysis of the larger Bmdsx M isoform revealed an 81base-pairs (bp) length fragment insertion, creating a novel splice variant (S6 Fig). All Bmdsx isoforms were absent in both sexes in individuals carrying mutations in Bmtra2 (Fig 2, lanes 5 and 6). Mutations in BmMasc and BmPSI in males resulted in a decreased accumulation of Bmdsx M and the appearance of Bmdsx F (Fig 2, lanes 8 and 14), which was consistent with previous report when BmMasc expression was disrupted by using RNAi [35].
RT-PCR-based analysis revealed that mutations in BmSxl, BmMasc, BmImp and BmImp M had no or minor effect on other genes at transcriptional or post-transcriptional (splicing) levels except Bmdsx. The male-specific BmImp (BmImp M ) isoform appeared in female Bmtra2 mutants (Fig 2, lanes 5 and 6). This supports the conclusion that Bmtra2 is not only involved in regulating Bmdsx, but also has a role in splicing regulation of BmImp M . Interestingly, the abundance of BmMasc and BmImp M transcripts decreased in BmPSI mutant males (Fig 2, lane  14). Q-RT-PCR analysis showed that BmImp M and BmMasc mRNA levels decreased by 92% and 60%, respectively, in BmPSI mutant males (Fig 3). These results support the conclusion that BmPSI regulates BmMasc and BmImp M at the splicing level. In contrast to the results in males, with the exception of Bmtra2, no effects on transcript profiles were seen in any of the mutant females.

BmPSI and BmMasc control male sexual differentiation
The morphology of the external and internal genitalia provides a direct index of sexual differentiation in B. mori. Mutations in BmMasc result in males with degenerative testes similar to that observed with Bmdsx mutants (Fig 4A, lanes 2 and 4). The external genitalia of males exhibit characteristics of the copulatory organs of both males and females including the . The putative dsx target male-specific expression genes in the male olfactory system, pheromone binding protein 1 (BmPBP1), olfactory receptors (BmORs) BmOR1, BmOR3, were significantly down-regulated in the BmMasc and BmPSI male mutants (Fig 5A-5C). In contrast, the female-specific expression genes in the female oogenesis and olfactory system, vitellogenin (BmVg), BmOR19, BmOR30, were significantly up-regulated in the BmMasc and BmPSI male mutants (Fig 5D-5F). These morphological results and corresponding gene expression profiles provide additional support for the conclusion that BmPSI and BmMasc control male sexual differentiation.

Bmtra2 and BmImp have pleiotropic effects in development
Mutations in Bmtra2 result in lethality at later embryonic stages (Fig 6A and 6B). This phenotype is similar to that reported in the honeybee in which down-regulation of Amtra2 causes embryonic viability and affects the female-specific splicing of fem and Amdsx transcripts [36]. Also, in T. castaneum, only a few eggs could be produced by animals after the parental RNAi of Tctra-2 and these eggs ultimately failed to hatch [37]. This is different from Dipteran species, in which tra2 has no vital function in embryogenesis [38]. The similarity of these phenotypes supports the hypothesis that Bmtra2 and its orthologs have an essential, ancestrally-and evolutionarily-conserved function in embryogenesis that is not related to sex determination that predates the divergence of the Lepidoptera, Hymenoptera and Coleoptera.
Two distinct types of mutations were induced in BmImp, the first one targets all splice variants (BmImp), and the second one is only in the male-specific splice variant (BmImp M ) (S4 Fig). However, neither had an effect on the morphology of the genitalia despite the observed effect on Bmdsx F . While the growth indices of BmImp M mutant silkworms were normal, the body size and weight of the BmImp mutants was smaller than wild-type animals (Fig 6C and  6D). They failed to molt at each larval instar and the majority died at the later larval and prepupal stages (Fig 6E and 6F).

Discussion
We provide here genetic evidence for the proposed sex determination pathway in B. mori that emphasizes the key roles of the products of the BmPS1 and BmMasc genes in male determination and differentiation (Fig 7). B. mori has the ZZ/ZW sex chromosome system in which the female is ZW (heterogametic) and the male is ZZ (homogametic). The Fem piRNA gene is located on the W chromosome and maintains feminization through downregulating BmMasc expression. Without the BmMasc protein in ZW embryos, the default (full-length) splicing isoform of Bmdsx (Bmdsx F ) activates downstream gene expression and produces female-specific development. BmPSI is not involved in this pathway, since mutations in it have no observable effects on female differentiation. BmPSI protein in males interacts with the Bmdsx pre-mRNA and generates the male-specific Bmdsx splice variant (Bmdsx M ) [30]. The BmMasc product might play the role of a recruitment or splicing factor to participate in this event. BmMasc is expressed normally in males due to lack of Fem piRNA, and thus may be controlled by BmPSI.
Significant differences are evident in the B. mori sex-determination system when compared to D. melanogaster. Sxl has a major and early role in sex determination in the fruit fly. Its 'on/ off' status serves as the primary signal to trigger somatic sex differentiation by controlling its own splicing (autocatalytically) and of tra [38]. However, Sxl does not show sex-specific splicing and function in other dipterans [39]. Furthermore, the silkworm ortholog, BmSxl, has two splicing isoforms, neither of which is regulated in a sex-specific manner [40]. Depletion of Bmsxl induced a longer Bmdsx M sex-specific splicing form appeared, just as seen in BmImp or BmImp M disruption. Although the potential role of this splicing form was unclear, it did not bring any phenotypic consequence in male sexual dimorphism. The presence of a longer Bmdsx M splicing form might because the spliceosome is complex machinery containing up to 300 proteins and loss of additional auxiliary factors having little roles [41,42]. Dmtra2 is a co-binding protein of sex determination key gene Dmtra, and acts on the D. melanogaster sex-determination cascade to regulate both somatic sexual differentiation and male fertility [43]. However, the ortholog encoding the tra appears to not exist in the silkworm, and therefore Bmtra2 might have distinct roles. Bmtra2 could regulate female-specific splicing of Dmdsx and RNAi knockdown of Bmtra2 in the early embryo could cause abnormal testis formation in B. mori [44]. Our data support the conclusion that Bmtra2 regulates Bmdsx in both males and females, and has sex-independently essential roles during embryogenesis although the mechanism of embryonic lethality induced by Bmtra2 mutagenesis is not known. Another phenomena is BmImp M appeared in the Bmtra2 mutants, indicating that Bmtra2 is involved in regulating the upstream transcriptional factors of BmImp M . It is similar with Dmtra2 which regulates many downstream genes [45].
The BmImp gene was firstly identified in the silkworm as a co-binding protein with BmPSI [32]. BmImp produces two splice variants, one of which (BmImp M ) is expressed in various tissues only in males and is proposed to be an essential regulator in the B. mori sex determination cascade [33]. BmImp M could bind the Bmdsx pre-mRNA and knock-down of its product induced the female-specific Bmdsx splicing form in male cell lines and embryos [33,46]. We cannot account for the differences in our results, but speculate that they could be a consequence of different methods between RNAi and Cas9-mediated gene silencing, or different materials between cell lines and the intact animal. However, our phenotypic results are consistent with those seen in D. melanogaster and the mouse. In the fruit fly, loss-of-function Imp mutations were zygotic lethal which through imprecise P excision, or mutants die later as pharate adults by two loss-of-function alleles, H44 and H149 [47,48]. Imp1 mutations in mice produced animals that were~40% smaller than wild-type controls, and these exhibited high perinatal mortality [49]. Our experiments in which we mutate all isoforms of BmImp or only BmImp M did not result in any effects on sexual organs or sex determination genes. We conclude that BmImp and BmImp M are not essential for sex regulation but are needed for development.
BmMasc had been identified as a downstream target of Fem-piRNA which could induce Bmdsx F in male embryos after siRNA-mediated knocking down [35]. BmMasc also controls dosage compensation in male embryos, and male embryos injected with BmMasc siRNA did not hatch normally [35]. Kiuchi et al. [35] used two siRNAs to knockdown BmMasc and reported detecting Bmdsx F in male embryo and the down-regulation of BmImp M . They proposed that BmImp M and Bmdsx are located downstream of BmMasc in the sex determination cascade. In contrast, our genetic analyses show no evidence of an effect of BmMasc on BmImp M . We propose that BmMasc controls male sexual differentiation by regulating Bmdsx but has no regulatory effect on BmImp M . It is still unclear how BmMasc regulates Bmdsx. F or M factors have been identified as the tra or tra2 gene in several insect species, and these factors directly regulated the dsx gene [25,50]. Previous reports concluded that BmPSI could directly bind Bmdsx pre-mRNA and that the BmImp M product increased BmPSI RNA binding activity in vitro [32]. Mutations of DmPSI in D. melanogaster strongly affect 43 splicing events and DmImp is one of the downstream genes targeted by it [51,52]. We found that BmImp and BmImp M had minor effects on Bmdsx F splicing in our molecular genetic analyses. Furthermore, BmPSI did regulate expression of Bmdsx, BmImp M and BmMasc in vivo. It is unknown how BmPSI regulates the splicing of Bmdsx and other potential splicing factors involved in this process. Nonetheless, these data support the conclusion that BmPSI is at least a key auxiliary factor in the silkworm male sexual differentiation gene cascade, and provide the basis for the hypothesis that the BmPSI gene has a major initial role in the sex determination cascade.

Silkworm strains
Silkworms of the same genetic background (Nistari, a multivoltine, nondiapausing strain) were used in all experiments. Wild-type (WT) and mutant larvae were reared on fresh mulberry leaves under standard conditions [53].

Plasmid construction and germline transformation
A binary transgenic CRISPR/Cas9 system was established to target selected genes. The piggy-Bac-based plasmid, pBac[IE1-EGFP-nos-Cas9] (nos-Cas9), was constructed to express the Cas9 nuclease in germ-line cells under the control of the B. mori nanos (nos) promoter with the EGFP fluorescence marker gene under the control of the IE1 promoter. The plasmid pBac [IE1-DsRed2-U6-sgRNA] (U6-sgRNA) was constructed to express single guide RNAs (sgRNA) under the control of the silkworm U6 promoter and the DsRed fluorescence marker gene also under control of the IE1 promoter. The sgRNAs targeting sequences were designed by manually searching genomic regions that match the 5 0 -GG-N 18 -NGG-3 0 rule [54]. sgRNA sequences were checked bioinformatically for potential off-target binding using CRISPRdirect (http:// crispr.dbcls.jp/) by performing exhaustive searches against silkworm genomic sequences [55]. All sgRNA and oligonucleotide primer sequences for plasmid construction are listed in S2 Table. U6-dsx-sgRNA line from our previous report [56]. Each nos-cas9 or U6-sgRNA plasmid mixed with a piggyBac helper plasmid [53] was microinjected separately into fertilized eggs at the preblastoderm stage. G 0 adults were mated to WT moths, and resulting G 1 progeny scored for the presence of the marker gene product using fluorescence microscopy (Nikon AZ100) (S1 Table).

Genotyping and phenotypic analysis
Each U6-sgRNA transgenic line was mated individually with the nos-Cas9 line to derive mutated F 0 animals. Genomic DNA of mutated animals was extracted at the embryonic or larval stage using standard SDS lysis-phenol treatment after incubation with proteinase K, followed by RNase treatment and purification. The resulting individual DNA samples from mutant animals were separated by sex using gene amplification with primers specific to the W chromosome (S2 Table). For two sgRNA sites, mutation events were detected by amplification using gene-specific primers which set on the upstream or downstream of the each target (S2 Table). Amplified products were visualized by 2% agarose gel electrophoresis running 30 min at 100V. Amplicons were sub-cloned into the pJET-1.2 vector (Fermentas) and the positive clones of each we pick six were sequenced. For the one sgRNA, a restriction enzyme (HpyAV, New England Biolabs, Ipswich, MA, USA) cutting site adjacent to the AGG (PAM sequence) was selected to analyze the putative mutations by restriction enzyme digestion (RED) assay. The RED assay was carried out as previous report [57].
Phenotypic analysis focused light-microscope examination of the morphology of secondary sexual characteristics including internal and external genitalia. Photographs were taken with NRK-D90 (B) or DS-Ri1 (Nikon, Tokyo, Japan) digital cameras. Testes or ovaries were dissected from fourth-day, fifth-instar larvae and fixed overnight in Qurnah's solution (anhydrous ethanol: acetic acid: chloroform = 6:1:3v/v/v). Tissues were dehydrated, cleared three times using anhydrous ethanol and xylene, respectively, and embedded in the paraffin overnight. Tissue sections (8 μm) were cut (Leica; RM2235) and stained using a mixture of hematoxylin and eosin solution. All pictures were taken under a microscope (Olympus BX51) using differential interference contrast with the appropriate filter.

Qualitative and quantitative RT-PCR
Total RNA was extracted from silkworm at different stages using Trizol reagent (Invitrogen) and treated with RNase-free DNAse I (Ambion). cDNAs were synthesized using the Omniscript Reverse transcriptase kit (Qiagen) in a 20 μl reaction mixture containing 1 μg total RNA. RT-PCR reactions were carried out using KOD plus polymerase (Toyobo) with gene-specific primers (S2 Table). Amplification of the B. mori ribosomal protein 49 (Bmrp49) was used as a positive control.
Quantitative real-time RT-PCR (Q-RT-PCR) assays were performed using SYBR Green Realtime PCR Master Mix (Thermo Fisher Scientific) on an Eppendorf Real-time PCR System Mastercycler realplex (Eppendorf). Q-RT-PCR reactions were carried out with gene-specific primers (S2 Table). A 10-fold serial dilution of pooled cDNA was used as the template to make standard curves. Quantitative mRNA measurements were performed in three independent biological replicates and normalized to Bmrp49 mRNA.

Statistics
Experimental data were analyzed with the Student's t-test. t-test: Ã , p < 0.05, ÃÃ , p < 0.01, ÃÃÃ , p < 0.001. At least three independent replicates were used for each treatment and the error bars show means ± S.E.M.  BmPSI mutant males and females at the embryonic (144 h after post-oviposition), larval (third day of fifth larvae instar) and pupal (the third day) stages, respectively. The white bars indicate wild type females and the dot bars indicate wild type males. The purple bars indicate mutant females and the dot bars with purple indicate mutant males. Three individual biological replicates were performed in q-RT-PCR. Error bar: SD; Ã , ÃÃ and ÃÃÃ represented significant differences at the 0.05, 0.01, 0.001 level (t-test) compared with the control. (D) PCR-based gene amplification analyses for Bmdsx splicing in BmPSI mutant animals. Bmdsx F and Bmdsx M represent the female-and male-specific splicing isoforms of Bmdsx, respectively. The lower panel shows amplification of the rp49 transcript, which serves as an internal control for RNA extraction and RT-PCR. (TIF)