Abstract
Yellow genes are thought to be involved in the melanin biosynthetic pathway and play a crucial role in pigmentation reactions in insects. However, little research has been done on yellow genes in lepidopteran pests. To clarify the function of one of the yellow genes (yellow-y) in Spodoptera litura, we cloned the full-length of yellow-y, and investigated its spatial and temporal expression profiles by quantitative real-time PCR (qPCR). It revealed that yellow-y was highly expressed in larva of fourth, fifth, and sixth instars, as well as in epidermis (Ep), fat bodies (FB), Malpighian tubes (MT), and midguts (MG) of the larvae; whereas it was expressed in very low levels in different tissues of adults, and was almost undetected in pupa. This expression profile suggests an important role of yellow-y in larvae, minor role in adults, and no role in pupae. To confirm this, we disrupted yellow-y using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, and obtained G0 insects with mutation in yellow-y. The mutation in yellow-y clearly rendered the larvae body, a color yellower than that of wide type insects, and in addition, the mutation resulted in abnormal segmentation and molting for older larvae. The mutation of yellow-y also made various adult tissues (antennae, proboscis, legs, and wings) yellowish. However, the mutation had no effect on pigmentation of the pupal cuticle. Taken together, our study clearly demonstrated the role of yellow-y not only in the body pigmentation of larvae and adults, and but also in segmentation and molting of larvae, providing new insights into the physiology of larval development, as well as a useful marker gene for genome editing based studies.
Highlights
Coloration is deeply related to the adaptation and survival of insects, as it is involved in cuticle formation, crypsis, mimicry, aposematism, mating signals, and others (Tsuchida et al, 2010)
The amino acid sequences of these 11 proteins together with those yellow proteins from B. mori, T. castaneum, A. ipsilon, and D. melanogaster were subjected to phylogenetic analysis using MEGA6.0
The complete open reading frame (ORF) sequence of yellow-y consisted of 1,629 bp, encoding 542 amino acid residues
Summary
Coloration is deeply related to the adaptation and survival of insects, as it is involved in cuticle formation, crypsis, mimicry, aposematism, mating signals, and others (Tsuchida et al, 2010). In Tribolium castaneum, double-stranded RNA (dsRNA)-mediated transcript knockdown revealed that different yellow genes play different roles: yellow-y is required for melanin production in the hindwings, yellow-f is necessary for adult cuticle sclerotization but not for pigmentation (Arakane et al, 2010), and yellow-e gene is involved in body coloration and anti-dehydration (Noh et al, 2015). In another coleopteran insect Tenebrio molitor, knockdown of yellow-y gene by RNA interference (RNAi) leads to decrease of black pigmentation in the middle mesocuticle layer and hindwing (Mun et al, 2020). Considering the inefficiency of RNAi in lepidopteran insect species (Wang et al, 2016; Peng et al, 2020), a more effective gene knockdown or knockout technology is essential for the functional studies of yellow genes
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