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...

SignificanceThe use of heterologous systems to express spider silk has become an attractive method. However, achieving cost-effective production and high yields is still challenging. Here, we describe the establishment of a targeted gene replacement system in Bombyx mori to express the major ampullate spidroin-1 gene (MaSp1) from the spider Nephila clavipes. With the aid of transcription activator-like effector nuclease-mediated homology-directed repair, we genetically replaced the silkworm fibroin heavy chain gene with MaSp1 with considerable transformation efficiency, and the chimeric MaSp1 yields reached up to 35.2% wt/wt of cocoon shells in transformed silkworms. The genetically modified silk fiber had significant changes in mechanical properties, with improved extensibility. This system will shed light on the future mass production of new biomaterials, including spider silk.

Feeding preference is critical for insect adaptation and survival. However, little is known regarding the determination of insect feeding preference, and the genetic basis is poorly understood. As a model lepidopteran insect with economic importance, the domesticated silkworm, Bombyx mori, is a well-known monophagous insect that predominantly feeds on fresh mulberry leaves. This species-specific feeding preference provides an excellent model for investigation of host-plant selection of insects, although the molecular mechanism underlying this phenomenon remains unknown. Here, we describe the gene GR66, which encodes a putative bitter gustatory receptor (GR) that is responsible for the mulberry-specific feeding preference of B. mori. With the aid of a transposon-based, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system, the GR66 locus was genetically mutated, and homozygous mutant silkworm strains with truncated gustatory receptor 66 (GR66) proteins were established. GR66 mutant larvae acquired new feeding activity, exhibiting the ability to feed on a number of plant species in addition to mulberry leaves, including fresh fruits and grain seeds that are not normally consumed by wild-type (WT) silkworms. Furthermore, a feeding choice assay revealed that the mutant larvae lost their specificity for mulberry. Overall, our findings provide the first genetic and phenotypic evidences that a single bitter GR is a major factor affecting the insect feeding preference.

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Laccase, a member of a group of proteins collectively known as multicopper oxidases, is hypothesized to play an important role in insect cuticle sclerotization by oxidizing catechols in the cuticle to their corresponding quinones, which then catalyze protein cross-linking reactions. Laccase 2 has been proved as the gene required for beetle cuticle tanning through RNA interference (RNAi) experiments on red flour beetle Tribolium castaneum. The pine sawyer beetle, Monochamus alternatus (Coleoptero: Cerambycidae) is the insect serving as a major vector of the pinewood nematode, Bursaphelenchus xylophilus, which is the causative agent for pine wilt disease. The cDNA of MaLac2 was cloned from the insect in this study. The conceptual amino-acid sequence deduced was much conserved with other known insect laccases, particularly with the enzyme of Tribolium castaneum. Injection in hemolymph of pine sawyer larva of dsRNA targeting the laccase 2 mRNA leads to important alterations of the tanning, hardening and sclerotization of the pupal and adult cuticles. Defaults appear in a dose-dependent manner and high loads of dsRNA are lethal. The decrease of the endogenous laccase 2 mRNA affects the procuticle which is thinner and without the characteristic piling up of successive layers. The observations reinforce the role of laccase 2 as an essential phenoloxidase for making cuticle.