Two Leptinotarsa uridine diphosphate N-acetylglucosamine pyrophosphorylases are specialized for chitin synthesis in larval epidermal cuticle and midgut peritrophic matrix

Shi, Ji-Feng; Jia, Fu; Mu, Lili; Guo, Wen‐Chao; Li, Guoqing

doi:10.1016/j.ibmb.2015.11.005

Cited by 51 publications

(65 citation statements)

References 44 publications

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“…A). Consistently, the mRNA levels of five transcripts ( LdUAP1 , LdUAP2 , LdChSAa , LdChSAb , and LdChSB ) encoding two chitin biosynthesis enzymes, uridine diphosphate N ‐acetylglucosamine pyrophosphorylase and chitin synthase (Shi et al ., ; ), were significantly declined in the LdCDA2 and LdCDA2b ‐depleted larvae (Fig. B–F).…”

Section: Resultsmentioning

confidence: 78%

“…Our previous results showed that 20E signal in the late stage of each L. decemlineata larval instar stimulated the expression of two chitin synthase ( ChS ) genes, two uridine diphosphate‐ N ‐acetylglucosamine‐pyrophosphorylase genes, three trehalase genes, and a trehalose biosynthesis gene (trehalose‐6‐phosphate synthase) (Shi et al ., ; ; ; Xu et al ., ). Similarly, in vivo tests revealed that 20E upregulated ChS s in three insect species: B. mori (Zhuo et al ., ; ), Spodoptera exigua (Yao et al ., ), and Bactrocera dorsalis (Yang et al ., ).…”

Section: Discussionmentioning

confidence: 99%

“…The defects in LdCDA2 RNAi larvae are reminiscent of the lethal phenotypes in chitin‐biosynthesis‐gene‐depleted ( LdUAP or LdChS ) L. decemlineata beetles, which consumed less food, grew slowly, and failed to molt (Shi et al ., ; ). Accordingly, our results in this paper revealed that chitin quantity was significantly reduced in the LdCDA2 RNAi larvae.…”

Section: Discussionmentioning

confidence: 99%

“…Fortunately, dietary introduction of bacterially expressed double‐stranded RNAs (dsRNAs) is able to effectively knock down target genes in L. decemlineata (Sun et al ., ; Meng et al ., ; ; Xu et al ., ). Moreover, chitin is absent in vertebrates, and chitin biosynthesis enzymes have been confirmed to be potential targets for novel insecticide development (Shi et al ., ; ). These prompt us to carry out a functional analysis of CDA2 in L. decemlineata larvae and to estimate its potential effect on the larvae.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Silencing chitin deacetylase 2 impairs larval–pupal and pupal–adult molts in Leptinotarsa decemlineata

Chen

Wang

et al. 2018

Insect Molecular Biology

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Insect chitin deacetylases (CDAs) are carbohydrate esterases that catalyze N-deacetylation of chitin to generate chitosan, a process essential for chitin organization and compactness during the formation of extracellular chitinous structure. Here we identified two CDA2 splice variants (LdCDA2a and LdCDA2b) in Leptinotarsa decemlineata. Both splices were abundantly expressed in larval foregut, rectum, and epidermis; their levels peaked immediately before ecdysis within each instar. In vivo results revealed that the two isoforms transcriptionally responded, positively and negatively respectively, to 20-hydroxyecdysone and juvenile hormone signaling pathways. RNA interference (RNAi)-aided knockdown of the two LdCDA2 variants (hereafter LdCDA2) or LdCDA2b, rather than LdCDA2a, resulted in three negative effects. First, foliage consumption was significantly reduced, larval developing period was lengthened, and larval growth was retarded. Second, chitin contents were reduced, whereas glucose, trehalose, and glycogen contents were increased in the LdCDA2 and LdCDA2b RNAi larvae. Third, approximately 20% of LdCDA2 and LdCDA2b RNAi larvae were trapped within the exuviae and finally died. About 60% of the abnormal pupae died as pharate adults. Around 20% of the RNAi pupae emerged as deformed adults, with small size and wrinkled wings. These adults eventually died within 1 week after molting. Our results reveal that knockdown of CDA2 affects chitin accumulation. Consequently, LdCDA2 may be a potential target for control of L. decemlineata larvae.

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Section: Resultsmentioning

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Silencing chitin deacetylase 2 impairs larval–pupal and pupal–adult molts in Leptinotarsa decemlineata

Chen

Wang

et al. 2018

Insect Molecular Biology

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“…Guts and tracheae were observed under a light microscope for retention/loss of structural integrity of the chitin-containing structures. Moreover, the tracheae were incubated with 10 M of NaOH at 95 °C for 2 h to solubilize the cuticular proteins 30, and the treated tracheae were then seen under the light microscope.…”

Section: Methodsmentioning

confidence: 99%

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

Shi

Chen

et al. 2016

Int. J. Biol. Sci.

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Dietary introduction of bacterially expressed double-stranded RNA (dsRNA) has great potential for management of Leptinotarsa decemlineata. Identification of the most attractive candidate genes for RNA interference (RNAi) is the first step. In the present paper, three complete chitin synthase cDNA sequences (LdChSAa, LdChSAb and LdChSB) were cloned. LdChSAa and LdChSAb, two splicing variants of LdChSA gene, were highly expressed in ectodermally-derived epidermal cells forming epidermis, trachea, foregut and hindgut, whereas LdChSB was mainly transcribed in midgut cells. Feeding bacterially expressed dsChSA (derived from a common fragment of LdChSAa and LdChSAb), dsChSAa, dsChSAb and dsChSB in the second- and fourth-instar larvae specifically knocked down their target mRNAs. RNAi of LdChSAa+LdChSAb and LdChSAa lowered chitin contents in whole body and integument samples, and thinned tracheal taenidia. The resulting larvae failed to ecdyse, pupate, or emerge as adults. Comparably, knockdown of LdChSAb mainly affected pupal-adult molting. The LdChSAb RNAi pupae did not completely shed the old larval exuviae, which caused failure of adult emergence. In contrast, silencing of LdChSB significantly reduced foliage consumption, decreased chitin content in midgut sample, damaged midgut peritrophic matrix, and retarded larval growth. As a result, the development of the LdChSB RNAi hypomorphs was arrested. Our data reveal that these LdChSs are among the effective candidate genes for an RNAi-based control strategy against L. decemlineata.

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Molecular cloning, gene expression analysis, and in silico characterization of UDP‐N‐acetylglucosamine pyrophosphorylase from Bombyx mori

Palaka

Ilavarasi

Sapam

et al. 2019

Biotech and App Biochem

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The present study was aimed to explore the molecular and structural features of UDP‐N‐acetylglucosamine pyrophosphorylase of Bombyx mori (BmUAP), an essential enzyme for chitin synthesis in insects. The BmUAP cDNA sequence was cloned and expression profiles were monitored during the molting and feeding stages of silkworm larvae. The effect of 20‐hydroxyecdysone (20E) on BmUAP expression, and on silkworm molting was studied, which revealed that 20E regulates its expression. Multiple sequence alignment of various pyrophosphorylases revealed that the residues N223, G290, N327, and K407 of human UAP (PDB ID: 1JV1) were found to be highly conserved in BmUAP and all other eukaryotic UAPs considered for the study. Phylogenetic analysis inferred that the UAPs possess discrete variations in primary structure among different insect Orders while sharing good identity between species of the Order. The structure of BmUAP was predicted and its interactions with uridine triphosphate, N‐acetylglucosamine‐1‐phosphate, and UDP‐N‐acetylglucosamine were analyzed. Virtual screening with a library of natural compounds resulted in five potential hits with good binding affinities. On further analysis, these five hits were found to be mimicking substrate and product, in inducing conformational changes in the active site. This work provides crucial information on molecular interactions and structural dynamics of insect UAPs.

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Two Leptinotarsa uridine diphosphate N-acetylglucosamine pyrophosphorylases are specialized for chitin synthesis in larval epidermal cuticle and midgut peritrophic matrix

Cited by 51 publications

References 44 publications

Silencing chitin deacetylase 2 impairs larval–pupal and pupal–adult molts in Leptinotarsa decemlineata

Silencing chitin deacetylase 2 impairs larval–pupal and pupal–adult molts in Leptinotarsa decemlineata

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

Molecular cloning, gene expression analysis, and in silico characterization of UDP‐N‐acetylglucosamine pyrophosphorylase from Bombyx mori

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