Identification, mRNA Expression, and Functional Analysis of Chitin Synthase 1 Gene and Its Two Alternative Splicing Variants in Oriental Fruit Fly, Bactrocera dorsalis

Abstract

Two alternative splicing variants of chitin synthase 1 gene (BdCHS1) were cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel). The cDNA of both variants (BdCHS1a and BdCHS1b) consisted of 5,552 nucleotides (nt), with an open reading frame (ORF) of 4,776 nt, encoding a protein of 1,592 amino acid residues, plus 685- and 88-nt of 5′- and 3′-noncoding regions, respectively. The alternative splicing site was located between positions 3,784-3,960 and formed a pair of mutually exclusive exons (a/b) that were same in size (177 nt), but showed only 65% identity at the nucleotide level. During B. dorsalis growth and development, BdCHS1 and BdCHS1a were both mainly expressed during the larval-pupal and pupal-adult transitions, while BdCHS1b was mainly expressed during pupal-adult metamorphosis and in the middle of the pupal stage. BdCHS1a was predominately expressed in the integument whereas BdCHS1b was mainly expressed in the trachea. The 20-hydroxyecdysone (20E) induced the expression of BdCHS1 and its variants. Injection of dsRNA of BdCHS1, BdCHS1a, and BdCHS1b into third-instar larvae significantly reduced the expression levels of the corresponding variants, generated phenotypic defects, and killed most of the treated larvae. Furthermore, silencing of BdCHS1 and BdCHS1a had a similar result in that the larva was trapped in old cuticle and died without tanning completely, while silencing of BdCHS1b has no effect on insect morphology. These results demonstrated that BdCHS1 plays an important role in the larval-pupal transition and the expression of BdCHS1 in B. dorsalis is regulated by 20E.

RNA interference of chitin synthase genes inhibits chitin biosynthesis and affects larval performance in Leptinotarsa decemlineata (Say)

“…In the present paper, we found that LdChSAa and LdChSAb were highly expressed in ectodermally-derived epidermal cells forming epidermis, trachea, foregut and hindgut, whereas LdChSB was mainly transcribed in midgut cells in L. decemlineata . Similar tissue-biased expression profiles have been documented in other insect species 24, 33, 34. Therefore, we compared the RNAi efficiency in the epidermis and in the midgut by ingestion of dsRNAs respectively targeting LdChSA and LdChSB in L. decemlineata larvae.…”

“…In T. castaneum , ds TcChSAa disrupted larval-larval, larval-pupal, and pupal-adult ecdysis, while ds TcChSAb only influenced pupal-adult molting 2, 7. Knockdown of BdChSAa + BdChSAb or only BdChSAa in B. dorsalis had a similar result in that the larvae were trapped in old cuticles and died without completely tanning, whereas injection of ds BdChSAb into B. dorsalis third (final) instar larvae caused no visible abnormal morphological changes, and no influence on pupation 24. In L. migratoria , ds LmChSAa resulted in three different lethal phenotypes, i.e., translucent new cuticle, trouble shedding old cuticle, and stunted development, whereas ds LmChSAb only caused crumpled cuticle 17.…”

“…Injection of ds ChS s at an amount from 200 to 3000 ng per individual caused larval lethality, impaired ecdysis, decreased adult fecundity and/or affected egg hatching in Orthopteran Locusta migratoria 17, 22, Hemipteran Rhodnius prolixus 23 and Nilaparvata lugens 45, Coleopteran T. castaneum 7, 8, 21, Lepidopteran Spodoptera exigua 19, Ostrinia furnacalis 14 and Bombyx mori 46, 47, and Dipteran Drosophila melanogaster 48, 49 and Bactrocera dorsalis 24. These data demonstrate that ChS s are the potential candidate genes for an RNAi-based pest control strategy.…”

“…Moreover, in most insect species ChSA contains alternative exons which lead to the production of two splicing mRNA variants, ChSAa and ChSAb 7, 10, 12-17. Functional analyses using RNAi in different insect species show that ChSs are required for survival, ecdysis, oviposition and egg hatching 7, 8, 17-24. Furthermore, chitin is absent in plants and vertebrates.…”

Phenoloxidase and its zymogen are required for the larval–pupal transition in Bactrocera dorsalis (Diptera: Tephritidae)

“…The final concentration of dsRNA was 1.5 lg/lL. According to the method described by Yang et al (2013) with modifications, approximately 1 lg of dsRNA was slowly injected into the antepenultimate abdominal segments of 3-day-old 3rd instar larvae with a Nanoject II Auto-Nanoliter Injector (Drummond Scientific, Broomall, PA, USA). Control larvae were injected with equivalent volumes of dsGFP.…”