CRISPR/Cas9 Mediated Disruption of Seminal Fluid Protein Sfp62 Induces Male Sterility in Bombyx mori

Simple Summary In gamogenetic animals, seminal fluid proteins are essential for male fertility. In this study, we investigated the function of the seminal fluid protein Sfp62 by using the CRISPR/Cas9 system in lepidopteran model insect Bombyx mori. Sfp62 mutation led to male sterility and can be inherited stably. The mutation did not affect growth and developmental nor female fertility. These data indicate that Sfp62 is an ideal target for sterile insect technology (SIT), in which genetically modified insects are released on a large scale to mate with wild-type insects in order to reduce or even eradicate the target pests. The determining factors for the effective implementation of SIT include the strong competitiveness of the modified individuals and multi-generational effects resulting from the mutation. Sfp62 meets these criteria and is therefore a promising target for biological pest control. Abstract Seminal fluid proteins provide factors necessary for development, storage, and activation of sperm. Altered expression of seminal fluid proteins can lead to defect in male infertility. We investigated the function of seminal fluid protein Sfp62 in the model lepidopteran insect Bombyx mori using CRISPR/Cas9 mediated mutagenesis. The knockout of BmSfp62 gene led to male sterility but has no effect on female fertility. The mutation did not affect growth and development of the silkworm of both sexes. Motility of sperm in male mutants was decreased and the mRNA expression levels of other genes encoding seminal fluid proteins were altered in BmSfp62 mutants compared to the wild-type controls. The male sterility caused by mutation of BmSfp62 was stably inherited. As the proteins encoded by Sfp62 genes are conserved among lepidopteran species, Sfp62 is a potential target for the biological management of lepidopteran pests.


Introduction
The males of gamogenetic animals produce seminal fluid proteins (SFPs) of various types that are critical for male fertility [1]. SFPs identified using transcriptome and proteome analyses include proteases, protease inhibitors, peptides, prohormones, lipases, trypsins, antioxidants, c-type lectins, and cysteine-rich secreted proteins [2][3][4][5]. SFPs provide a fluid environment for sperm and are necessary for development, storage, and activation of sperm [6,7]. Upon ejaculation, SFPs enter the genital tract of the female where these factors influence ovulation and fertilization [8,9].
In mammals, SFPs are biomarkers for male fertility [10]. About 50% of human infertility results from SFP dysfunction [11]. SFPs provide the main energy source for sperm and regulate sperm capacitation and acrosome reaction [12][13][14]. In Mus musculus, certain SFPs are essential for sperm-egg fusion including the membrane protein FIMP, SOF1; transmembrane protein TMEM95; the acrosome-associated protein SPACA6, DCST1, and DCST2 [15][16][17][18]. In fishes, SFPs sperm motility and fertilization are used as markers to evaluate the fish reproductive ability and to monitor sperm cryopreservation methods [19,20]. The ganglioside GM3 in SFPs is involved in the fertilization process in Oncorhynchus mykiss [21]. A high molecular weight glycosylated SFP is a sperm motility inhibitor in Oreochromis niloticus [22]. The lack of SPACA6 prevents sperm-egg fusion in Danio rerio [23].
In insects, SFPs not only regulate reproduction but also affect the lifespan of mating individuals and the numbers and survival rates of offspring [24][25][26][27][28][29]. In Drosophila melanogaster, Seminase is an essential factor for female oviposition induction, and its downregulation blocks sperm release, which in turn results in a significant reduction in post-copulation oviposition [30,31]. Acp70A (also known as sex peptide) and Acp26Aa (also known as Ovulin) regulate sperm storage and stimulate oviposition in Drosophila and also affect the survival of offspring [32][33][34]. In Culex pipiens, trypsin is essential for maintaining sperm motility and is necessary for maintenance of sperm viability in vitro [35]. In Anopheles, SFPs bind to symbiotic bacteria in males and transfer these bacteria to females, where they affect the female immune response [36]. In Ceratitis capitata, the SFPs enter the female and cause conformational changes in reproductive tract [37]. In Teleogryllus oceanicus, seven SFPs regulate sperm motility and enhance sperm competitiveness [38]. In Bombyx mori and Plutella xylostella, the serine protease Ser2 is an SFP necessary for male reproduction; its absence results in male sterility, but female mutants are fertile [39].
Sterile insect technology (SIT) is a new biological method for pest control [40]. The principle is that genetically modified to be sterile, are released into the field on a large scale to mate with wild-type insects to reduce or even eradicate target pests [41]. As SFPs are mainly secreted from insect gonads during the adult stage, loss of these factors does not usually impact growth and development [42]. Therefore, SFPs are potential targets for SIT [43].
In this study, we investigated the function of Sfp62 gene in B. mori, a model lepidopteran insect, by CRISPR/Cas9-induced mutation of the gene. This technique has been shown to result in stable inheritance of mutations [44]. Lepidoptera is the second largest order of insects and includes more than 70% of the agriculture pests [45]. We found that the deletion of BmSfp62 led to male sterility. Growth and development of the mutant insects was normal, and female fertility was not impacted by loss of BmSfp62. In females mated with male mutants, sperm motility in the bursa copulatrix was decreased. In addition, the mRNA levels of genes encoding other SFPs associated with sperm motility were altered in the BmSfp62 mutants. Changes in SFP composition and content caused abnormalities in the fluid environment of the sperm, which presumably results in male sterility. As Sfp62 is evolutionarily conserved among lepidopterans, the Sfp62 is a potential target gene for lepidopteran pest control.

Silkworm Strains and Rearing
A multivoltine and nondiapausing silkworm strain, Nistari, was used for the experiments and raised under standard conditions [46].

Evolutionary Analysis
The phylogenetic relationships of Sfp62 proteins in representative lepidopteran species was inferred using neighbor-joining method [47]. The evolutionary distances were computed using Poisson correction method [48]. Phylogenetic analyses were conducted in MEGA X [49]. The alignment of Sfp62 protein sequences was created with the ClustalX2 software and GENEDOC program.

Identification of mutations
TS-F ATGCAGAATGACCACGGTGGG TS-R CGTCGAGCCCTTCGTCTCAA

Plasmid Construction
Following the GGN 19 GG rule, small guide RNA (sgRNA) target sites were selected in the screened open reading frame of BmSfp62 [50][51][52]. Two target sites of BmSfp62 were identified. The activator was the plasmid pBac[IE1-EGFP-Nos-Cas9], which encodes Cas9 under the control of the Nos promoter and the enhanced green fluorescent protein (EGFP) marker under control of the IE1 promoter [53]. The effector plasmid was pBac[IE1-DsRed2-U6-sgRNAs], which encodes the sgRNAs and the discosoma red fluorescent protein (DsRed) driven by U6 and IE1 promoters, respectively. The primers are listed in Table 1.

Mutant Construction
The constructed plasmids were microinjected into embryos for germline transformation. The injected eggs were incubated and raised under standard conditions. When the mutant lines reached to adults, they were arranged to mate with wild-type (WT) moths. G1 progeny were screened under a fluorescence microscope (Nikon AZ100, Tokyo, Japan). The mutant germline (∆BmSfp62 individuals) with DsRed (red) and EGFP (green) fluorescence were obtained by hybridization of the Nos-Cas9 line with the U6-sgRNA line. Mutants with double-fluorescence were used in subsequent experiments.

Mutagenesis Analysis
The mutant genomic DNA was extracted by genomic DNA extraction kit (Thermo Fisher Scientific, USA). Genomic PCR was performed as follows: 94 • C for 2 min, 35 cycles of 94 • C for 15 s, 55 • C for 30 s, and 72 • C for 1 min, followed by a final extension period at 72 • C for 10 min. The PCR products were sub-cloned into pJET1.2 vectors (Thermo Fisher Scientific, USA) and sequenced. The primers are listed in Table 1.

Sperm Motility and Germline Transmission Assay
The bursa copulatrixes were dissected from control and ∆BmSfp62 females at the mated adult stage. Tissues were placed in physiological saline (Sangon, Shanghai, China) on glass slides. Photos were taken with a microscope (Olympus BX51, Tokyo, Japan).
The emerging moth was set in the center of a plastic container (30 × 18 × 4.5 cm 3 ). WT or ∆BmSfp62 moth of the opposite sex was placed 10 cm from the center of the container. Mutants that mated were recorded as responsive. The response index was calculated as responsive moth number divided by total test number multiplied by 100.

Expression Pattern of BmSfp62
The testis of silkworm in adult stage were nearly spherical rather than renal in the larval stage. Male gradually matured from the fifth larval instar to the adult stage. Therefore, we tested the mRNA expression of BmSfp62 in each representative stages. The expressions were first analyzed at day 3 of the fifth instar larvae (L5D3) and the wandering stage (W). In individuals from both developmental stages, BmSfp62 mRNA was much more highly expressed in the testis than head, epidermis, fat body, midgut, Malpighian tubules, anterior silk gland, middle silk gland, posterior silk gland, and ovary ( Figure 2A). We then quantified the expression in adult tissues. BmSfp62 mRNA was more highly expressed in the seminal vesicle and ejaculatory vesicle than male accessory gland, glandula prostatica, testis, female accessory gland, and bursa copulatrix in both virgin and mated adults ( Figure 2B). In the silkworm gonads, the mRNA expression of BmSfp62 increased significantly with development. These results suggest that the Sfp62 is important in male fertility. Biology 2022, 11, x 5 of 12

Expression Pattern of BmSfp62
The testis of silkworm in adult stage were nearly spherical rather than renal in the larval stage. Male gradually matured from the fifth larval instar to the adult stage. Therefore, we tested the mRNA expression of BmSfp62 in each representative stages. The expressions were first analyzed at day 3 of the fifth instar larvae (L5D3) and the wandering stage (W). In individuals from both developmental stages, BmSfp62 mRNA was much more highly expressed in the testis than head, epidermis, fat body, midgut, Malpighian tubules, anterior silk gland, middle silk gland, posterior silk gland, and ovary ( Figure 2A). We then quantified the expression in adult tissues. BmSfp62 mRNA was more highly expressed in the seminal vesicle and ejaculatory vesicle than male accessory gland, glandula prostatica, testis, female accessory gland, and bursa copulatrix in both virgin and mated adults ( Figure 2B). In the silkworm gonads, the mRNA expression of BmSfp62 increased significantly with development. These results suggest that the Sfp62 is important in male fertility.

Expression Pattern of BmSfp62
The testis of silkworm in adult stage were nearly spherical rather than renal in the larval stage. Male gradually matured from the fifth larval instar to the adult stage. Therefore, we tested the mRNA expression of BmSfp62 in each representative stages. The expressions were first analyzed at day 3 of the fifth instar larvae (L5D3) and the wandering stage (W). In individuals from both developmental stages, BmSfp62 mRNA was much more highly expressed in the testis than head, epidermis, fat body, midgut, Malpighian tubules, anterior silk gland, middle silk gland, posterior silk gland, and ovary ( Figure 2A). We then quantified the expression in adult tissues. BmSfp62 mRNA was more highly expressed in the seminal vesicle and ejaculatory vesicle than male accessory gland, glandula prostatica, testis, female accessory gland, and bursa copulatrix in both virgin and mated adults ( Figure 2B). In the silkworm gonads, the mRNA expression of BmSfp62 increased significantly with development. These results suggest that the Sfp62 is important in male fertility. . Asterisks indicate significant differences with a two-tailed t-test: *** p < 0.001.

CRISPR/Cas9-Mediated BmSfp62 Mutation Leads to Male Sterility
Two target sites were selected in exons of the BmSfp62 gene; the fragment containing the two sites was 994 bp ( Figure 3A). The activator line vector pBac[IE1-EGFP-Nos-Cas9] is designed to express the fluorescent marker EGFP and the effector line pBac[IE1-DsRed-U6-sgRNAs] is designed to express the fluorescent marker DsRed ( Figure 3B). Genomic DNA Biology 2022, 11, 561 6 of 12 was extracted from the mutants with EGFP and DsRed fluorescent markers to characterize deletions between the two target sites. Different numbers of bases were deleted in different mutant individuals ( Figure 3C). differences with a two-tailed t-test: *** p < 0.001.

CRISPR/Cas9-Mediated BmSfp62 Mutation Leads to Male Sterility
Two target sites were selected in exons of the BmSfp62 gene; the fragment containing the two sites was 994 bp ( Figure 3A). The activator line vector pBac[IE1-EGFP-Nos-Cas9] is designed to express the fluorescent marker EGFP and the effector line pBac[IE1-DsRed-U6-sgRNAs] is designed to express the fluorescent marker DsRed ( Figure 3B). Genomic DNA was extracted from the mutants with EGFP and DsRed fluorescent markers to characterize deletions between the two target sites. Different numbers of bases were deleted in different mutant individuals ( Figure 3C). We then mated WT males with both ΔBmSfp62 and WT females and ΔBmSfp62 males with both WT and ΔBmSfp62 females and examined numbers of eggs produced ( Figure  4A). All crosses resulted in similar numbers of eggs laid; however, eggs produced by females mated with ΔBmSfp62 males did not hatch. The control WT cross produced 363 ± 10 eggs; WT males mated with ΔBmSfp62 females produced 339 ± 10 eggs; ΔBmSfp62 males mated with WT females produced 329 ± 10 eggs; ΔBmSfp62 males mated with ΔBmSfp62 females produced 308 ± 10 eggs (n = 30/group). Almost all control eggs hatched (~95%, 346/363) as did eggs of WT males mated with ΔBmSfp62 females (~93%, 316/339), but no eggs of ΔBmSfp62 males mated with WT or ΔBmSfp62 females hatched (~0%, 0/329; ~0%, 0/308, respectively) ( Figure 4B). Subsequently, we tested the mRNA expression of BmSfp62 in adult tissues. BmSfp62 mRNA expression was significantly downregulated in ΔBmSfp62 individuals compared with WT individuals ( Figure 4C). We then mated WT males with both ∆BmSfp62 and WT females and ∆BmSfp62 males with both WT and ∆BmSfp62 females and examined numbers of eggs produced ( Figure 4A). All crosses resulted in similar numbers of eggs laid; however, eggs produced by females mated with ∆BmSfp62 males did not hatch. The control WT cross produced 363 ± 10 eggs; WT males mated with ∆BmSfp62 females produced 339 ± 10 eggs; ∆BmSfp62 males mated with WT females produced 329 ± 10 eggs; ∆BmSfp62 males mated with ∆BmSfp62 females produced 308 ± 10 eggs (n = 30/group). Almost all control eggs hatched (~95%, 346/363) as did eggs of WT males mated with ∆BmSfp62 females (~93%, 316/339), but no eggs of ∆BmSfp62 males mated with WT or ∆BmSfp62 females hatched (~0%, 0/329;~0%, 0/308, respectively) ( Figure 4B). Subsequently, we tested the mRNA expression of BmSfp62 in adult tissues. BmSfp62 mRNA expression was significantly downregulated in ∆BmSfp62 individuals compared with WT individuals ( Figure 4C).

∆BmSfp62 Sperm Motility Is Decreased and SPF Expression Is Altered
We next evaluated sperm motility in the bursa copulatrix of WT females after mating with WT or ∆BmSfp62 males. Normally, eupyrene sperm do not move on their own but are carried by the movement of apyrene sperm. Apyrene sperm motility was significantly decreased in the mutant compared to WT controls (Videos S1 and S2).
The mRNA levels of genes encoding sperm motility-related SFPs BmAtta, BmTry, BmPtp, and BmRca were significantly up-regulated in virgin adult gonads ∆BmSfp62 individuals compared to WT, whereas BmAmy and BmMlc were significantly downregulated in the mutant ( Figure 5). Thus, deletion of BmSfp62 leads to the altered expression of other genes encoding SFPs that are essential for fertility.

ΔBmSfp62 Sperm Motility Is Decreased and SPF Expression Is Altered
We next evaluated sperm motility in the bursa copulatrix of WT females after mating with WT or ΔBmSfp62 males. Normally, eupyrene sperm do not move on their own but are carried by the movement of apyrene sperm. Apyrene sperm motility was significantly decreased in the mutant compared to WT controls (Videos S1 and S2).
The mRNA levels of genes encoding sperm motility-related SFPs BmAtta, BmTry, BmPtp, and BmRca were significantly up-regulated in virgin adult gonads ΔBmSfp62 individuals compared to WT, whereas BmAmy and BmMlc were significantly downregulated in the mutant ( Figure 5). Thus, deletion of BmSfp62 leads to the altered expression of other genes encoding SFPs that are essential for fertility. . Asterisks indicate significant differences with a two-tailed t-test: *** p < 0.001.

Mutation of BmSfp62 Does Not Affect Adult Competitiveness and Is Stably Inherited
We used the percentage of successful mating as a response index to evaluate the adult competitiveness of the mutant females and males. There was no significant difference in competitiveness of WT and ΔBmSfp62 females (51.52% and 48.48%, respectively; n = 30/group) nor was there any significant difference in competitiveness of WT and WT is in blue, ∆BmSfp62 is in orange. mRNA expression level was normalized to Bmrp49. The data shown are means ± S.E.M. (n = 3). Asterisks indicate significant differences with a two-tailed t-test: *** p < 0.001.

Mutation of BmSfp62 Does Not Affect Adult Competitiveness and Is Stably Inherited
We used the percentage of successful mating as a response index to evaluate the adult competitiveness of the mutant females and males. There was no significant difference in competitiveness of WT and ∆BmSfp62 females (51.52% and 48.48%, respectively; n = 30/group) nor was there any significant difference in competitiveness of WT and ∆BmSfp62 males (51.09% and 48.91%, respectively; n = 30/group) ( Figure 6A, B). Heritability stability was analyzed by hybridization of the Nos-Cas9 and U6-sgRNAs lines. In successive generations, ∆BmSfp62 male sterility was observed ( Figure 6C). Thus, the BmSfp62 mutation did not affect competitiveness, and the resulting male sterility was stably inherited. . Asterisks indicate significant differences with a two-tailed t-test: *** p < 0.001.

Mutation of BmSfp62 Does Not Affect Adult Competitiveness and Is Stably Inherited
We used the percentage of successful mating as a response index to evaluate the adult competitiveness of the mutant females and males. There was no significant difference in competitiveness of WT and ΔBmSfp62 females (51.52% and 48.48%, respectively; n = 30/group) nor was there any significant difference in competitiveness of WT and ΔBmSfp62 males (51.09% and 48.91%, respectively; n = 30/group) ( Figure 6A, B). Heritability stability was analyzed by hybridization of the Nos-Cas9 and U6-sgRNAs lines. In successive generations, ΔBmSfp62 male sterility was observed ( Figure 6C). Thus, the BmSfp62 mutation did not affect competitiveness, and the resulting male sterility was stably inherited.

Discussion
Seminal fluid proteins are essential for male reproduction. There are many SFPs, and their characteristics and functions vary [54]. Here, we conducted a functional analysis of Sfp62 using the silkworm as a model. At the protein level, Sfp62 is highly conserved across lepidopteran species. BmSfp62 is highly expressed in male gonads; it was detected in other tissues but at low levels. We evaluated the physiological function of BmSfp62 gene in male reproduction by CRISPR/Cas9 technology. The technology enables accurate and efficient targeting of candidate genes [55,56]. The BmSfp62 mutation caused male sterility but did not decrease female fertility. Growth and reproductive behaviors of mutants were normal, but the sperm motility of the male BmSfp62 mutant was dramatically decreased compared to wild-type sperm, resulting in fertilization failure. Females mated with BmSfp62 mutant males laid normal numbers of eggs but did not hatch.
Mutation of BmSfp62 led to loss of BmSfp62 and alters expression of other genes that encode seminal fluid proteins including BmAtta, BmAmy, BmMlc, BmTry, BmPtp, and BmRca. In D. melanogaster, Atta, an immune defense gene, is highly expressed in virgin adults and regulates sperm competitiveness [57]. In D. melanogaster, Amy is expressed in the spermatid and mature sperm; the protein it encodes binds cAMP-dependent kinase to regulate spermatogenesis and sperm capacitation in human and mouse [58]. The Mlc gene encodes a motor protein that is required for sperm storage and release in D. melanogaster [59,60]. The hydrolase encoded by Try regulates the hydrolyzation of sex peptides that influence sperm motility and reproduction [61]. Ptp is expressed in lepidopteran testis, and its homolog in D. melanogaster is critical for efficient ATP synthesis necessary for sperm motility [62]. The SFP encoded by Rca regulates sperm quality and male fertility by influencing Ca + levels in seminal fluid in Rattus norvegicus [63]. The abnormal expression of SPFs resulting from BmSfp62 deficiency may contribute to the observed male sterility. Sperm motility and the levels of SFPs in the female's bursa copulatrix after mating influence the efficiency of sperm-egg fusion [64,65]. Loss of BmSfp62 gene function led to a decrease in sperm motility, which likely also impaired fertility.
In summary, our study demonstrated that the mutation of Sfp62 led to stably inherited male sterility by CRISPR/Cas9 system in B. mori. The BmSfp62 gene deletion caused male sterility, but females were fertile, and the mutation did not affect other growth and reproduction indicators. BmSfp62 mutant females released into the environment will mate with WT males and pass on effectively. The next generation of mutant males will be sterile but able to mate. Mating with mutants should eventually suppress the pest population. Further, BmSfp62 is evolutionarily conserved among lepidopterans, making it an ideal target for SIT-mediated control of lepidopteran pests.

Conclusions
We investigated the function of BmSfp62 in the lepidopteran model insect, B. mori. The BmSfp62 mutant males were sterile, but females were fertile. The mutation was stably inherited and did not affect other growth and reproductive indicators. BmSfp62 deletion decreased sperm motility and altered mRNA expression levels of other genes encoding seminal fluid proteins. Due to it evolutionary conservation in lepidopterans, BmSfp62 is a possible target for biological control of lepidopteran pests.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.