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

Abstract

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.

Fig 1. Loss-of-function analysis of B.mori sex determination genes.

(A) Schematic representation of functional analysis using the transgenic CRISPR/Cas9 system. nos-Cas9 (green) transgenic moths were crossed with U6-sgRNA (red) transgenic moths and the F₀ heterozygotes were analyzed as for genotypic and phenotypic effects. (B) Genes affected and major phenotypic effects observed in mutants.

Fig 2. PCR-based gene amplification analyses for targeted genes in wild type (WT) and mutant animals.

Six genes, BmSxl, Bmtra2, BmMasc, BmImp, BmImp^M, and BmPSI, were mutated and corresponding expression files were listed. Expression of Bmdsx was investigated as a general indicator. Bmdsx^F and Bmdsx^M represent the female- and male-specific splicing isoforms of Bmdsx, respectively. BmSxl-L and BmSxl-S represent two different isoforms of the BmSxl gene. The lower panel shows amplification of the rp49 transcript, which serves as an internal control for RNA extraction and RT-PCR.

Fig 3. Q-RT-PCR analysis for investigation of BmMasc, BmImp^M, BmImp and BmPSI transcript abundance in corresponding mutants.

(A-D) Relative mRNA expression levels of BmMasc, BmPSI, BmImp and BmImp^M in mutant males and females, respectively. Three individual biological replicates were performed in q-RT-PCR. Error bar: SD; *, ** and *** represented significant differences at the 0.05, 0.01, 0.001 level (t-test) compared with the control.

Fig 4. Morphological changes in sexually-dimorphic reproductive system structures in wild-type (WT) and mutant silkworms.

(A) Morphology of internal gonad structure in paraffin sections stained with hematoxylin and eosin. Gonads are dissected from wild-type and mutant males and females on the fourth day of the fifth-instar. White arrows show the separation of the testis envelope from the testicular lobe. Scale bars: 250μm. (B) Gross morphology of external genitalia of control and mutant adults. Key to arrows: red, ventral chitin plate; blue, genital papillae; white, penis; green, clasper; and purple, ventral plates. Scale bars: 1mm.

Fig 5. Q-RT-PCR analysis of the putative downstream genes of Bmdsx in the BmMasc and BmPSI mutants.

(A-F) Relative mRNA expression levels of BmPBP1, BmOR1, BmOR3, BmVg, BmOR19 and BmOR30 in mutant males. Three individual biological replicates were performed in q-RT-PCR. Error bar: SD; ** and *** represented significant differences at the 0.01, 0.001 level (t-test) compared with the control.

Fig 6. Pleiotropic effects of mutations in the Bmtra2 and BmImp genes.

(A and B) Embryonic lethality in progeny of the mating of nos-Cas9 and U6-Bmtra2-sgRNA: F₀ individuals complete embryogenesis (A) but fail to hatch (B). Embryos appear intact at eight days after oviposition and died on the twelfth day. Red arrows indicate control and black arrows indicate mutants. (C-F) Loss of BmImp function affects growth and metamorphic development. (C and D) Phenotypes of larvae resulted from crosses between nos-Cas9 and U6-BmImp-sgRNA at the fifth day of the final larval instar. Smaller larvae are from mutant insects and larger larvae were from controls as showed in C. Scale bar: 1 cm. Data are from the crosses of ΔBmImp (nos-Cas9/U6-sgRNA), nos-Cas9 (nos-Cas9/-), U6-sgRNA (-/U6-sgRNA) and WT (wild-type) as shown in D. E, Most larvae do not complete metamorphosis and die at the prepupal stage. Morphology of fifth instar larvae (ΔBmImp) compared to control. F, percent of wild-type (control, blue line) and mutant (ΔBmImp, red line) silkworms progressed through development. Key: L1-5, last day of the first, second, third and fourth larval instars, respectively. Data are from ΔBmImp (n = 105) and control (n = 100) animals.

Fig 7. Proposed genetic cascade of sex determination in B. mori.

The Fem piRNA derived from female W chromosome downregulates Masc expression (yellow panel) [27]. Absence of Masc products and inactive BmPSI result in splicing that produces the female-specific isoform of Bmdsx (Bmdsx^F), which contain all of the exons of the gene. Bmdsx^F products induce female determination and differentiation of morphological sex characteristics. Thus, female development is the default sex determination pathway. Masc products are expressed normally in the male due to the lack of Fem and presence of active BmPSI, and this results in splicing of the male-specific isoform of Bmdsx (Bmdsx^M, blue panel), which contain exons of 1, 2, 5 of the Bmdsx pre-mRNA. Bmdsx^M products promote male development. The tra2 product also is involved in the regulation of Bmdsx.