Transgene-based, female-specific lethality system for genetic sexing of the silkworm, Bombyx mori

Abstract

Transgene-based genetic sexing methods are being developed for insects of agricultural and public health importance. Male-only rearing has long been sought in sericulture because males show superior economic characteristics, such as better fitness, lower food consumption, and higher silk yield. Here we report the establishment of a transgene-based genetic sexing system for the silkworm, Bombyx mori. We developed a construct in which a positive feedback loop regulated by sex-specific alternative splicing leads to high-level expression of the tetracycline-repressible transactivator in females only. Transgenic animals show female-specific lethality during embryonic and early larval stages, leading to male-only cocoons. This transgene-based female-specific lethal system not only has wide application in sericulture, but also has great potential in lepidopteran pest control.Lepidoptera | doublesex T he mulberry silkworm, Bombyx mori, is a completely domesticated insect and is the foundation of sericulture, an endeavor of great economic importance. It is believed that sericulture originated in China and has been conducted there for more than 5,000 y (1). Male-only rearing techniques for B. mori are desirable because males show higher resistance to disease, lower food consumption, and better silk quality (2). To this end, several B. mori strains for male-only rearing have been developed by classical genetics. In the last century, Strunnikov (3) established a sex-linked, balanced-lethal system using radiation-induced chromosome translocations. However, conventional approaches involving selective breeding or irradiation for developing male-only silkworm strains are time and labor consuming. Thus, novel approaches are desired to improve modern silkworm breeding. In recent years, advances in B. mori genetic manipulation, notably genetic transformation, have been successfully established and applied extensively in gene function analysis and the production of bioreactors (1, 4-7). These technologies provide a potential basis for improvements in sericulture including the development of a male-only rearing system.Transgene-based genetic sexing systems have been developed in Drosophila melanogaster (8, 9) and several medically and agriculturally important insect species (10-13) as part of a series of genetics-based improvements and alternatives to the sterile insect technique (14). Systems based on sex-specific lethality for improving silk production are preferable to those using differential expression of a marker gene, for example fluorescence, which then would require manual or automated examination of each individual as part of the sorting process. Molecular designs developed for Diptera should be able to provide genetic sexing in B. mori, as the ability to transfer systems from one species to another is a key advantage of transgenic approaches over classical genetic methods (14-16). Furthermore, despite early reports to the contrary, more recent work has established clearly that transgenic strains can be developed with good...

Transgenic Clustered Regularly Interspaced Short Palindromic Repeat/Cas9-Mediated Viral Gene Targeting for Antiviral Therapy of Bombyx mori Nucleopolyhedrovirus

“…1B). The generation of TG silkworms has been described previously (23). We obtained three TG lines (TG-A, TG-B, and TG-C), and the transgene locations in the genome were identified by inverse PCR and sequencing (Fig.…”

MiR-2 family targetsawdandfngto regulate wing morphogenesis inBombyx mori

“…Inverse PCR was carried out as described previously 25,47 to investigate genomic insertion loci of transgenes. Both lines crossed with pBacA3-Gal4 animals respectively, and their transformed G2 offspring showed the identical phenotype thus we The transcript level of BmWnt-1 is down-regulated significantly in 4A (89%) and 4F (57%).…”

Genetics‐based methods for agricultural insect pest management

“…One important outcome is dominant female‐specific lethality, alternatively described as male‐selecting constructs (Heinrich & Scott, 2000; Thomas et al ., 2000; Fu et al ., 2007, 2010; Wise de Valdez et al ., 2011; Ant et al ., 2012; Labbé et al ., 2012; Jin et al ., 2013; Tan et al ., 2013) (Fig. 1B).…”