CRISPR/Cas9‐based functional analysis of yellow gene in the diamondback moth, Plutella xylostella

Abstract

The diamondback moth, Plutella xylostella (L.), is an economically important pest of cruciferous crops worldwide. This pest is notorious for rapid evolution of the resistance to different classes of insecticides, making it increasingly difficult to control. Genetics‐based control approaches, through manipulation of target genes, have been reported as promising supplements or alternatives to traditional methods of pest management. Here we identified a gene of pigmentation (yellow) in P. xylostella, Pxyellow, which encodes 1674 bp complementary DNA sequence with four exons and three introns. Using the clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR‐associated protein 9 system, we knocked out Pxyellow, targeting two sites in Exon III, to generate 272 chimeric mutants (57% of the CRISPR‐treated individuals) with color‐changed phenotypes of the 1st to 3rd instar larvae, pupae, and adults, indicating that Pxyellow plays an essential role in the body pigmentation of P. xylostella. Fitness analysis revealed no significant difference in the oviposition of adults, the hatchability of eggs, and the weight of pupae between homozygous mutants and wildtypes, suggesting that Pxyellow is not directly involved in regulation of growth, development, or reproduction. This work advances our understanding of the genetic and insect science molecular basis for body pigmentation of P. xylostella, and opens a wide avenue for development of the genetically based pest control techniques using Pxyellow as a screening marker.

Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Protein 9 Mediated Knockout Reveals Functions of the yellow-y Gene in Spodoptera litura

“…The yellow-y gene also plays a role in cuticle pigmentation in adults. In the present study, CRISPR/Cas9-treated adults exhibited a yellow color in body, wings, antennae, proboscis, and legs of adult S. litura , similar as the findings in D. melanogaster ( Wittkopp et al, 2002a , b , 2009 ), A. ipsilon ( Chen et al, 2018 ), and P. xylostella ( Wang et al, 2020 ). However, there were also studies with coleopteran species showed different results.…”

“…Our observation of phenotypes with yellow-y knockout individuals support the hypothesis that yellow-y is involved in the cuticle pigmentation in larvae but not in pupae, which accords with the findings in A. ipsilon mutants ( Chen et al, 2018 ). However, CRISPR/Cas9-treated pupae exhibited a yellow color in P. xylostella ( Wang et al, 2020 ). Besides, some larvae showed deformed body segmentation at fifth instar and abnormal molting at sixth instar in S. litura , which was not showed in B. mori ( Futahashi et al, 2008 ), A. ipsilon ( Chen et al, 2018 ), and P. xylostella ( Wang et al, 2020 ).…”

“…However, CRISPR/Cas9-treated pupae exhibited a yellow color in P. xylostella ( Wang et al, 2020 ). Besides, some larvae showed deformed body segmentation at fifth instar and abnormal molting at sixth instar in S. litura , which was not showed in B. mori ( Futahashi et al, 2008 ), A. ipsilon ( Chen et al, 2018 ), and P. xylostella ( Wang et al, 2020 ). Similarly, in T. castaneum , knockdown of yellow-y expression by RNAi made the adults unable to shed the pupal cuticle properly and died entrapped in their pupal cuticle ( Arakane et al, 2010 ).…”

“…The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is a newly developed and an effective genome editing technology, which has been used in gene function study in some lepidopterans, such as Helicoverpa armigera ( Chang et al, 2017 ; Ye et al, 2017 ; Wang et al, 2018 ), Plutella xylostella ( Liu et al, 2020 ), and Manduca sexta ( Fandino et al, 2019 ). By using CRISPR/Cas9 system, the yellow-y gene has been recently identified to be involved in synthesizing melanin in Bicyclus anynana ( Matsuoka and Monteiro, 2018 ), Agrotis ipsilon ( Chen et al, 2018 ), and P. xylostella ( Wang et al, 2020 ), but phenotypes (observable morphological, developmental, and behavioral properties) induced by the knockout of yellow-y were not very consistent among these species. Thus, more studies are undoubtedly needed to further refine the link between the phenotypes and the yellow genes.…”

CRISPR/Cas9‐based functional characterization of the pigmentation gene ebony in Plutella xylostella

“…However, further investigations (e.g., with mathematical modelling) would be needed to analyse such possibilities in detail for specific gene drive designs. In contrast, previous report of another pigmentation gene Pxyellow showed no significant impacts on moth fitness caused by Pxyellow defect (Wang et al ., 2020), making it an ideal neutral marker for testing drive systems but without the additional limiting function shown in Pxebony . Therefore, our research on Pxebony provides more options and expands the genetic tool box for developing different gene drives in DBM.…”