Identification and characterization of the masculinizing function of the Helicoverpa armigera masc gene

Authors: DENG, ZHONGYUAN - Zhengzhou University; LEYAO, LI - Zhengzhou University; ZHANG, YUTING - Zhengzhou University; ZHANG, YAKUN - Chinese Academy Of Agricultural Sciences; XIE, XINGCHENG - Chinese Academy Of Agricultural Sciences; ZHANG, MIN - Zhengzhou University; HUANG, JINYONG - Zhengzhou University; Ni, Xinzhi; LI, XIANCHUN - University Of Arizona

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/5/2021
Publication Date: 8/11/2021
Citation: Deng, Z., Leyao, L., Zhang, Y., Zhang, Y., Xie, X., Zhang, M., Huang, J., Ni, X., Li, X. 2021. Identification and characterization of the masculinizing function of the Helicoverpa armigera masc gene. International Journal of Molecular Sciences. 22(16):8650. https://doi.org/10.3390/ijms22168650.
DOI: https://doi.org/10.3390/ijms22168650

Interpretive Summary: Sex of an individual insect has been known to be determined genetically upon fertilization, and then developmentally upon embryogenesis. Developmental determination of sex is regulated by a cascade of genes that act upon one another via alternatively splicing to transduce primary or initial signal, which is derived from the chromosomal makeup of a fertilized egg, to two terminal double-switch genes. Although manipulation of sex ratio (with low number of females for reproduction) could be a good pest management tool, relatively much less is known about the sex determination pathway in moths, except sex determination studies in the silkworm. In moths, males are homogametic (with ZZ chromosomes) and females are heterogametic (with ZW chromosomes). The question addressed in this study was if a homologue of silkworm genes exists and plays the same role in the distantly-related corn earworm, a key insect pest. We initiated this study by examining a corn earworm larva transcriptome, followed by a series of genomic studies on the corn earworm. The findings from this study demonstrated that the gene (HaMasc), like BmMasc, is a Z chromosome-linked male determinant required for the regulated normal development of testes in male insects. The study established baseline information for potentially manipulating sex ratio of a pest moth population to reduce offspring population and its damage on crops.

Technical Abstract: Masculinizer (Masc) gene has been known to control sex development and dosage compensation in Bombyx mori. However, it remains unclear whether its orthologue also exists and plays the same roles in distantly-related lepidopterans such as, the corn earworm, Helicoverpa armigera, which is also known as the old world cotton bollworm. To address this question, we BLAST-searched the publicly-available transcriptome datasets of H. armigera using B. mori Masc protein sequence as a query and retrieved a transcript (asmbl_65189) representing a partial sequence of Masc from H. armigera (HaMasc). Subsequent 5’/3’ RACE, full-length RT-PCR cloning and BLAST search against the unpublished scaffolds of the male H. armigera genome showed that HaMasc contains 12 exons and transcribes into one constitutively-spliced transcript and another alternative transcript retaining its 1st intron. The two transcripts shared an identical open reading frame (ORF) of 1896 bp encoding a protein of 632 amino acids, which contained all essential functional domains of BmMasc, albeit with less than 30% amino acid sequence identity with BmMasc. Genomic PCR and qPCR showed that HaMasc genomic copy number was 2-fold greater in males than in females. RT-PCR and RT-qPCR analyses revealed that HaMasc transcripts were significantly more abundant in males than in females; in embryonic stage than other developmental stages; and in head tissue than in the tissues of other body parts. Transfection of a mixture of 3 siRNAs of HaMasc into a male embryonic cell line of H. armigera led to appearance of female-specific isoforms of H. armigera doublesex (Hadsx) mRNAs. Knockdown of HaMasc starting from the 3rd instar larvae resulted in smaller male pupae and testes, fewer but larger/longer spermatocytes and sperm bundles, delayed pupation and internal fusion of the testes and follicles. These data demonstrate that HaMasc functions as a male determinant targeted by an unidentified female-specific primary signal in the H. armigera sex determination cascade.