Genome-wide identification of yellow gene family in Hermetia illucens and functional analysis of yellow-y by CRISPR/Cas9.

The yellow gene family plays a crucial role in insect pigmentation. It has potential for use as a visible marker gene in genetic manipulation and transgenic engineering in several model and non-model insects. Sadly, yellow genes have rarely been identified in Stratiomyidae species and the functions of yellow genes are relatively unknown. In the present study, we first manually annotated and curated 10 yellow genes in the black soldier fly (BSF), Hermetia illucens (Stratiomyidae). Then, the conserved amino acids in the major royal jelly proteins (MRJPs) domain, structural architecture and phylogenetic relationship of yellow genes in BSF were analyzed. We found that the BSF yellow-y, yellow-c and yellow-f genes are expressed at all developmental stages, especially in the prepupal stage. Using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we successfully disrupted yellow-y, yellow-c and yellow-f in the BSF. Consequently, the mutation of yellow-y clearly resulted in a pale-yellow body color in prepupae, pupae and adults, instead of the typical black body color of the wild type. However, the mutation of yellow-c or yellow-f genes did not result in any change in color of the insects, when compared with the wild type. Our study indicates that the BSF yellow-y gene plays a role in body pigmentation, providing an optimal marker gene for the genetic manipulation of BSF.

Revision of the genus Nephelobotys Munroe & Mutuura, 1970 (Lepidoptera, Crambidae, Pyraustinae).

The genus Nephelobotys Munroe & Mutuura, 1970 is revised to include eight species. Three species, N. apiculata sp. nov., N. semicircularis sp. nov. and N. brevis sp. nov. are described as new to science. Two new combinations, N. habisalis (Walker, 1859) comb. nov. and N. flavicilialis (Snellen, 1890) comb. nov., are proposed. N. forcipatusKo & Bae, 2022 syn. nov. is found to be identical with N. habisalis (Walker, 1859). Diagnoses for all species are provided, with illustrations of external features and genitalia.

Establishment of highly efficient transgenic system for black soldier fly (Hermetia illucens).

The black soldier fly (BSF), Hermetia illucens, is a promising insect for mitigating solid waste problems as its larvae are able to bioconvert organic waste into valuable biomass. We recently reported a high-quality genome assembly of the BSF; analysis of this genome sequence will further the understanding of insect biology and identify genes that can be manipulated to improve efficiency of bioconversion. To enable genetic manipulation of the BSF, we have established the first transgenic methods for this economically important insect. We cloned and identified the ubiquitous actin5C promoter (Hiactin5C-p3k) and 3 endogenous U6 promoters (HiU6:1, HiU6:2, and HiU6:3). The Hiactin5C promoter was used to drive expression of a hyperactive variant of the piggyBac transposase, which exhibited up to 6-fold improvement in transformation rate when compared to the wild-type transposase. Furthermore, we evaluated the 3 HiU6 promoters using this transgenic system. HiU6:1 and HiU6:2 promoters provided the highest knockdown efficiency with RNAi and are thus promising candidates for future Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) development. Overall, our findings provide valuable genetic engineering toolkits for basic research and genetic manipulation of the BSF.

Intersex is required for female sexual development in Hermetia illucens.

Sex-determination pathways are extremely diverse. Understanding the mechanism of sex determination in insects is important for genetic manipulation of the pest population and for breeding of economically valuable insects. Although sex determination has been well characterized in the model species Drosophila melanogaster, little is known about this pathway in Stratiomyidae. In the present study, we first identified the Drosophila intersex (ix) homolog in Hermetia illucens, also known as the black soldier fly, which belongs to the Stratiomyidae family and which is an important insect for the conversion of various organic wastes. Phylogenetic analyses and multiple sequence alignment revealed that Hiix is conserved compared with Drosophila. We showed that Hiix is highly expressed in internal genitalia. Disruption of the Hiix gene using CRISPR/Cas9 resulted in female-specific defects in external genitalia and abnormal and undersized ovaries. Taken together, our study furthers our understanding of sex determination in insects and could facilitate breeding of H. illucens.

Genomic landscape and genetic manipulation of the black soldier fly Hermetia illucens, a natural waste recycler.

The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is renowned for its bioconversion of organic waste into a sustainable source of animal feed. We report a high-quality genome of 1.1 Gb and a consensus set of 16,770 gene models for this beneficial species. Compared to those of other dipteran species, the BSF genome has undergone a substantial expansion in functional modules related to septic adaptation, including immune system factors, olfactory receptors, and cytochrome P450s. We further profiled midgut transcriptomes and associated microbiomes of BSF larvae fed with representative types of organic waste. We find that the pathways related to digestive system and fighting infection are commonly enriched and that Firmicutes bacteria dominate the microbial community in BSF across all diets. To extend its potential practical applications, we further developed an efficient CRISPR/Cas9-based gene editing approach and implemented this to yield flightless and enhanced feeding capacity phenotypes, both of which could expand BSF production capabilities. Our study provides valuable genomic and technical resources for optimizing BSF lines for industrialization.