Genetic control of Plutella xylostella in omics era

Diamondback moth, Plutella xylostella (L.), is a specialist pest on cruciferous crops of economic importance. The large‐scale use of chemical insecticides for the control of this insect pest has caused a number of challenges to agro‐ecosystems. With the advent of the omics era, genetic pest management strategies are becoming increasingly feasible and show a powerful potential for pest control. Here, we review strategies for using transgenic plants and sterile insect techniques for genetic pest management and introduce the major advances in the control of P. xylostella using a female‐specific RIDL (release of insects carrying a dominant lethal gene) strategy. Further, the advantages of gene drive developed in combination with sex determination and CRISPR/Cas9 systems are addressed, and the corresponding prospects and implementation issues are discussed. It is predictable that under the policy and regulation of professional committees, the genetic pest control strategy, especially for gene drive, will open a new avenue to sustainable pest management not only for P. xylostella but also for other insect pests.     

Functional characterization of SlitPBP3 in Spodoptera litura by CRISPR/Cas9 mediated genome editing

Functional gene analysis by using genome editing techniques is limited only in few model insects. Here, we reported an efficient and heritable gene mutagenesis analysis in an important lepidopteran pest, Spodoptera litura, using the CRISPR/Cas9 system. By using this system, we successfully obtained the homozygous S. litura strain by targeting the pheromone binding protein 3 gene (SlitPBP3), which allowed us to elucidate the role of this gene in the olfaction of the female sex pheromones. By co-injection of Cas9 mRNA and sgRNA into S. litura eggs, highly efficient chimera mutation in SlitPBP3 loci was detected both in injected eggs (39.1%) and in the resulting individual moths (87.5%). We used the mutant moths as parents to obtain the G1 offspring and the homozygous mutant strain in G2. The function of SlitPBP3 was explored by Electroantennogram (EAG) recordings with a homozygous mutant strain. The result showed that the EAG responses were significantly decreased in mutant males than in control males when treated with the major sex pheromone component (Z9,E11-14:Ac) and a minor component (Z9-14:Ac) at higher dosages. The results demonstrate that s SlitPBP3 gene plays a minor role in the perception of the female sex pheromones. Furthermore, our study provides a useful methodology with the CRISPR/Cas9 system for gene in vivo functional study, particular for lepidopteran species in which the RNAi approach is not efficient.     

CRISPR/Cas9‐mediated ebony knockout results in puparium melanism in Spodoptera litura

Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.     

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 characterization of sex‐identification markers in Sebastes carnatus and Sebastes chrysomelas rockfishes

Fish have evolved a variety of sex‐determining (SD) systems including male heterogamy (XY), female heterogamy (ZW) and environmental SD. Little is known about SD mechanisms of Sebastes rockfishes, a highly speciose genus of importance to evolutionary and conservation biology. Here, we characterize the sex determination system in the sympatrically distributed sister species Sebastes chrysomelas and Sebastes carnatus. To identify sex‐specific genotypic markers, double digest restriction site – associated DNA sequencing (ddRAD‐seq) of genomic DNA from 40 sexed individuals of both species was performed. Loci were filtered for presence in all of the individuals of one sex, absence in the other sex and no heterozygosity. Of the 74 965 loci present in all males, 33 male‐specific loci met the criteria in at least one species and 17 in both. Conversely, no female‐specific loci were detected, together providing evidence of an XY sex determination system in both species. When aligned to a draft reference genome from Sebastes aleutianus, 26 sex‐specific loci were interspersed among 1168 loci that were identical between sexes. The nascent Y chromosome averaged 5% divergence from the X chromosome and mapped to reference Sebastes genome scaffolds totalling 6.9Mbp in length. These scaffolds aligned to a single chromosome in three model fish genomes. Read coverage differences were also detected between sex‐specific and autosomal loci. A PCR‐RFLP assay validated the bioinformatic results and correctly identified sex of five additional individuals of known sex. A sex‐determining gene in other teleosts gonadal soma‐derived factor (gsdf) was present in the model fish chromosomes that spanned our sex‐specific markers.     

Characterization of a Y‐specific duplication/insertion of the anti‐Mullerian hormone type II receptor gene based on a chromosome‐scale genome assembly of yellow perch,Perca flavescens

Yellow perch, Perca flavescens, is an ecologically and economically important species native to a large portion of the northern United States and southern Canada and is also a promising candidate species for aquaculture. However, no yellow perch reference genome has been available to facilitate improvements in both fisheries and aquaculture management practices. By combining Oxford Nanopore Technologies long‐reads, 10X Genomics Illumina short linked reads and a chromosome contact map produced with Hi‐C, we generated a high‐continuity chromosome‐scale yellow perch genome assembly of 877.4 Mb. It contains, in agreement with the known diploid chromosome yellow perch count, 24 chromosome‐size scaffolds covering 98.8% of the complete assembly (N50 = 37.4 Mb, L50 = 11). We also provide a first characterization of the yellow perch sex determination locus that contains a male‐specific duplicate of the anti‐Mullerian hormone type II receptor gene (amhr2by) inserted at the proximal end of the Y chromosome (chromosome 9). Using this sex‐specific information, we developed a simple PCR genotyping assay which accurately differentiates XY genetic males (amhr2by⁺) from XX genetic females (amhr2by^?). Our high‐quality genome assembly is an important genomic resource for future studies on yellow perch ecology, toxicology, fisheries and aquaculture research. In addition, characterization of the amhr2by gene as a candidate sex‐determining gene in yellow perch provides a new example of the recurrent implication of the transforming growth factor beta pathway in fish sex determination, and highlights gene duplication as an important genomic mechanism for the emergence of new master sex determination genes.     

A biotechnology‐based male‐sterility system for hybrid seed production in tomato

Incorporating male sterility into hybrid seed production reduces its cost and ensures high varietal purity. Despite these advantages, male‐sterile lines have not been widely used to produce tomato (Solanum lycopersicum) hybrid seeds. We describe the development of a biotechnology‐based breeding platform that utilized genic male sterility to produce hybrid seeds. In this platform, we generated a novel male‐sterile tomato line by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 (Cas9)‐mediated mutagenesis of a stamen‐specific gene SlSTR1 and devised a transgenic maintainer by transforming male‐sterile plants with a fertility‐restoration gene linked to a seedling‐colour gene. Offspring of crosses between a hemizygous maintainer and the homozygous male‐sterile plant segregated into 50% non‐transgenic male‐sterile plants and 50% male‐fertile maintainer plants, which could be easily distinguished by seedling colour. This system has great practical potential for hybrid seed breeding and production as it overcomes the problems intrinsic to other male‐sterility systems and can be easily adapted for a range of tomato cultivars and diverse vegetable crops.     

Identification of sex‐specific molecular markers using restriction site‐associated DNA sequencing

A major barrier to evolutionary studies of sex determination and sex chromosomes has been a lack of information on the types of sex‐determining mechanisms that occur among different species. This is particularly problematic in groups where most species lack visually heteromorphic sex chromosomes, such as fish, amphibians and reptiles, because cytogenetic analyses will fail to identify the sex chromosomes in these species. We describe the use of restriction site‐associated DNA (RAD) sequencing, or RAD‐seq, to identify sex‐specific molecular markers and subsequently determine whether a species has male or female heterogamety. To test the accuracy of this technique, we examined the lizard Anolis carolinensis. We performed RAD‐seq on seven male and ten female A. carolinensis and found one male‐specific molecular marker. Anolis carolinensis has previously been shown to possess male heterogamety and the recently published A. carolinensis genome facilitated the characterization of the sex‐specific RAD‐seq marker. We validated the male specificity of the new marker using PCR on additional individuals and also found that it is conserved in some other Anolis species. We discuss the utility of using RAD‐seq to identify sex‐determining mechanisms in other species with cryptic or homomorphic sex chromosomes and the implications for the evolution of male heterogamety in Anolis.     

Identification and functional characterization of doublesex gene in the testis of Spodoptera litura

Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at Go generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.     

Multiple molecular approaches yield no evidence for sex‐determining genes in lake sturgeon (Acipenser fulvescens)

Common DNA‐based sexing assays have been widely used for the conservation and management of mammals and birds. However, many fishes do not have genetic sex determination and in those that do, the plasticity of the genes involved means that species‐specific assays are normally required. Such DNA‐sexing markers would be especially valuable in lake sturgeon (Acipenser fulvescens) because of their sexual monomorphism, delayed sexual maturity, and conservation status. We tried to identify genetic differences between male and female lake sturgeon using several different molecular genetic methods, including randomly amplified polymorphic DNA, representational difference analyses, subtractive hybridization, and a candidate gene approach. Ultimately, a number of genes were identified but none was sex‐specific. Although the ultimate mechanism of sex determination is yet unknown, it is possible that sex determination is environmental in lake sturgeon, especially since recent studies have also failed to identify sex determination genes in other sturgeon species.