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Transposon-based genetic transformation has facilitated insect functional genomics and new strategies of pest management. However, there is a need for alternative, site-specific approaches to overcome limitations of random integration (and associated position-effects) and potential instability of inserted transgenes. Here we describe a transposon-free, site-specific genetic transformation system mediated by transcription activator-like effector nucleases (TALENs) in the silkworm, Bombyx mori, a lepidopteran model insect. We successfully established a site-specific transgenic system with comparable transformation efficiency to transposon-based genetic transformation through microinjection of TALENs mRNA targeting the BmBLOS2 locus and a linearizable donor plasmid encoding an expression cassette of the DsRed2 red fluorescent protein. This system provides a valuable approach for insect transgenesis and will enable future functional gene analysis and generate novel applications in agricultural and medical insect pest-management technologies.

Insect development and metamorphosis are regulated by two major hormones, juvenile hormone and ecdysteroids. Despite being the key regulator of insect developmental transitions, the metabolic pathway of the primary steroid hormone, 20-hydroxyecdysone (20E), especially its inactivation pathway, is still not completely elucidated. A cytochrome P450 enzyme, CYP18A1, has been shown to play key roles in insect steroid hormone inactivation through 26-hydroxylation. Here, we identified two CYP18 (BmCYP18A1 and BmCYP18B1) orthologs in the lepidopteran model insect, Bombyx mori. Interestingly, BmCYP18A1 gene is predominantly expressed in the middle silk gland (MSG) while BmCYP18B1 expresses ubiquitously in B. mori. BmCYP18A1 is induced by 20E in vitro, suggesting its role in 20E metabolism. Using the binary Gal4/UAS transgenic system, we ectopically overexpressed BmCYP18A1 in a MSG-specific manner with a Sericin1-Gal4 (Ser-Gal4) driver or in a ubiquitous manner with an Actin3-Gal4 (A3-Gal4) driver. Ectopic overexpression of BmCYP18A1 in MSG or in all tissues resulted in developmental arrestment of transgenic animals during the final instar larval stage. The 20E titers in the transgenic animals expressing BmCYP18A1 were lower compared to the levels in the control animals. Although the biological significance of MSG-specific expression of BmCYP18A1 is unclear, our results provide the first evidence that BmCYP18A1, which is conserved in most arthropods, is involved in a tissue-specific steroid hormone inactivation in B. mori.

Understanding the mechanism of Bt resistance is one of the key elements of the effective application of Bt in pest control. The lepidopteran model insect, the silkworm, demonstrates qualities that make it an ideal species to use in achieving this understanding. We screened 45 strains of silkworm (Bombyx mori) using a Cry1Ab toxin variant. The sensitivity levels of the strains varied over a wide range. A resistant strain (P50) and a phylogenetically related susceptible strain (Dazao) were selected to profile the expressions of 12 Bt resistance-related genes. The SNPs in these genes were detected based on EST analysis and were validated by allelic-specific PCR. A comparison of allelic-specific expression between P50 and Dazao showed that the transcript levels of heterozygous genes containing two alleles rather than an imbalanced allelic expression contribute more to the resistance of P50 against Bt. The responses of the allelic-specific expression to Bt in hybrid larvae were then investigated. The results showed that the gene expression pattern of an ATP-binding cassette transporter C2 (ABCC2) and an aminopeptidase N (APN3), changed in an allelic-specific manner, with the increase of the resistant allele expression correlated with larval survival. The results suggest that a trans-regulatory mechanism in ABCC2 and APN3 allelic-specific expression is involved in the insect's response to the Bt toxin. The potential role of allelic-specific gene regulation in insect resistance to Bt toxins is discussed.