Kang-Kang Xu

Identification, mRNA expression, and functional analysis of chitin synthase 1 gene and its two alternative splicing variants in oriental fruit fly, Bactrocera dorsalis.

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.

Transcriptional Regulation of a Chitinase Gene by 20-Hydroxyecdysone and Starvation in the Oriental Fruit Fly, Bactrocera dorsalis

Insect chitinases are hydrolytic enzymes that are required for the degradation of glycosidic bonds of chitin. In this study, we identified and characterized a full-length cDNA of the chitinase gene (BdCht2) in the oriental fruit fly, Bactrocera dorsalis. The cDNA contains an open reading frame (ORF) of 1449 bp that encodes 483 amino acid residues and 126- and 296-bp non-coding regions at the 5′- and 3′-ends, respectively. The BdCht2 genome has four exons and three introns. The predicted molecular mass of the deduced BdCht2 is approximately 54.3 kDa, with an isoelectric point of 5.97. The 977 bp 5′ flanking region was identified and the transcription factor binding sites were predicted. Bioinformatic analyses showed that the deduced amino acid sequence of BdCht2 had 34%–66% identity to that of chitinases identified in other insect species. Quantitative real-time PCR (qPCR) analyses indicated that BdCht2 was mainly expressed during the larval-pupal and pupal-adult transitions. The tissue-specific expression showed that the highest expression was in the integument, followed by the fat body and other tissues. Moreover, the expression of BdCht2 was upregulated significantly upon 20-hydroxyecdysone (20E) at different dose injections after 8 h compared to that of the control. Starvation also increased the expression of BdCht2 in the third-instar larvae and was suppressed again by re-feeding the insects. These results suggest that BdCht2 plays an important role in the molting process of B. dorsalis larvae and can be regulated by 20E.

Two Chitin Biosynthesis Pathway Genes in Bactrocera dorsalis (Diptera: Tephritidae): Molecular Characteristics, Expression Patterns, and Roles in Larval—Pupal Transition

ABSTRACT Glucose-6-phosphate isomerase (G6PI) and UDP-N-acetylglucosamine pyrophosphorylase (UAP), two key components in the chitin biosynthesis pathway, are critical for insect growth and metamorphosis. In this study, we identified the genes BdG6PI and BdUAP from the oriental fruit fly, Bactrocera dorsalis (Hendel). The open reading frames (ORFs) of BdG6PI (1,491 bp) and BdUAP (1,677 bp) encoded 496 and 558 amino acid residues, respectively. Multiple sequence alignments showed that BdG6PI and BdUAP had high amino acid sequence identity with other insect homologues. Quantitative real-time polymerase chain reaction (qPCR) analysis indicated that BdG6PI was mainly expressed in the early stages of third-instar larvae and adults, while significantly higher expression of BdUAP was observed in adults. Both transcripts were expressed highly in the Malpighian tubules, but only slightly in the tracheae. The expression of both BdG6PI and BdUAP was significantly up-regulated by 20-hydroxyecdysone exposure and down-regulated by starvation. Moreover, injection of double-stranded RNAs of BdG6PI and BdUAP into third-instar larvae significantly reduced the corresponding gene expressions. Additionally, silencing of BdUAP resulted in 65% death and abnormal phenotypes of larvae, while silencing of BdG6PI had a slight effect on insect molting. These findings provide some data on the roles of BdG6PI and BdUAP in B. dorsalis and demonstrate the potential role for BdUAP in larval—pupal transition.

Insulin signaling pathway in the oriental fruit fly: The role of insulin receptor substrate in ovarian development

Insulin signaling pathways have integral roles in regulating organ growth and body size of insects. Here, we identified and characterized six insulin signaling pathway components-InR, IRS, PI3K92E, PI3K21B, Akt, and PDK-from Bactrocera dorsalis. Quantitative real-time polymerase chain reaction was used to establish gene expression profiles for the insulin signaling pathway components for different developmental stages and tissues, and in response to 20-hydroxyecdysone (20E) and starvation. IRS, PI3K92E, and PI3K21B were highly expressed in the head, while InR, Akt, and PDK were most abundant in Malpighian tubules. Both IRS and PI3K92E were highly expressed during the larval-pupal and pupal-adult transition, while the remaining four genes were highly expressed only during the pupal-adult transition. Following initial exposure to 20E, the expression levels of most genes were significantly decreased. However, the expression levels of IRS, PI3K92E, and PI3K21B were significantly increased at 8 and 12h post-treatment compared with the control. Moreover, we found that most insulin signaling pathway genes in B. dorsalis were up-regulated in response to starvation, but decreased when re-fed. On the contrary, transcript levels of PI3K21B decreased significantly during starvation. Furthermore, injection of IRS dsRNA into adult females significantly reduced IRS transcript levels. Suppression of IRS expression inhibited ovarian development, and the average ovary size was reduced by 33% compared with the control. This study provides new insight into the roles of insulin signaling pathway components in B. dorsalis, and demonstrates an important role for IRS in ovarian development.

Molecular Cloning, Characterization and mRNA Expression of a Chitin Synthase 2 Gene from the Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae)

Chitin synthase (CHS), a potential target for eco-friendly insecticides, plays an essential role in chitin formation in insects. In this study, a full-length cDNA encoding chitin synthase 2 (BdCHS2) was cloned and characterized in the oriental fruit fly, Bactrocera dorsalis. The BdCHS2 cDNA had 4417 nucleotides, containing an open reading frame of 4122 nucleotides, which encoded 1373 amino acid residues with a predicted molecular weight of 158.5 kDa. Phylogenetic analysis with other insect CHSs suggested that BdCHS2 belongs to insect CHS2. The BdCHS2 transcript was predominately found in midgut but was detected at low levels in fat body, Malpighian tubules, integument, and trachea. Moreover, BdCHS2 was expressed in all developmental stages, and highly expressed in the feeding stages. There was a positive relationship between BdCHS2 expression and total chitin content during development. Furthermore, both the gene expression and chitin content in midgut decreased when the insect was fed for 24 h, then starved for 24 h, while they increased dramatically and rapidly under the condition of starvation for 24 h then feeding for 24 h. These results suggest that BdCHS2 may play an important role in regulating chitin content of the midgut, and subsequently affect the growth and development of B. dorsalis.

Molecular characterization and functional analysis of BdFoxO gene in the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

The forkhead box O transcription factor (FoxO) is an important downstream transcription factor in the well-conserved insulin signaling pathway, which regulates the body size and development of insects. In this study, the FoxO gene (BdFoxO) was identified from the oriental fruit fly, Bactrocera dorsalis (Hendel). The open reading frame of BdFoxO (2732 bp) encoded a 910 amino acid protein, and the sequence was well conserved with other insect species. The BdFoxO was highly expressed in larvae and pupae among different development stages, and the highest tissue-specific expression level was found in the fat bodies compared to the testis, ovary, head, thorax, midgut, and Malpighian tubules of adults. Interestingly, we found BdFoxO expression was also up-regulated by starvation, but down-regulated when re-fed. Moreover, the injection of BdFoxO double-stranded RNAs into third-instar larvae significantly reduced BdFoxO transcript levels, which in turn down-regulated the expression of other four genes in the insulin signaling pathway. The silencing of BdFoxO resulted in delayed pupation, and the insect body weight increased significantly compared with that of the control. These results suggested that BdFoxO plays an important role in body size and development in B. dorsalis.

Identification and characterization of three juvenile hormone genes from Bactrocera dorsalis (Diptera: Tephritidae)

Abstract Juvenile hormone (JH) plays an important role in regulating growth, development, and reproduction of insects. Three key enzymes, namely, JH esterase (JHE), JH epoxide hydrolase (JHEH), and JH diol kinase (JHDK), are involved in JH degradation. In this study, we identified the full-length cDNAs of the 3 genes BdJHEH2, BdJHEH3, and BdJHDK encoding JHEH and JHDK from the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). We used quantitative real-time polymerase chain reaction to investigate mRNA expression profiles of these 3 genes in various development stages and tissues, and in response to both 20-hydroxyecdysone (20E) and starvation. Both BdJHEH2 and BdJHDK were highly expressed during the larval—pupal transition, whereas BdJHEH3 was mainly expressed in the early 3rd instars. All 3 genes were highly expressed in 7-d-old and 10-d-old adults, but exhibited sex-specific expression patterns. BdJHEH2 was highly expressed in fat body, whereas BdJHEH3 and BdJHDK were most abundant in Malpighian tubules. In response to 20E exposure, the 3 genes were significantly up-regulated at various time points compared with the control. However, the transcript levels of BdJHDK decreased significantly during the initial exposure to 20E. After starvation treatment, expression of BdJHEH2 and BdJHEH3 significantly decreased, whereas BdJHDK was up-regulated. No significant change was observed after feeding resumption. These 3 genes have distinct roles in regulating growth and development of B. dorsalis.

The complete mitochondrial genome of Stegobium paniceum (Coleoptera: Anobiidae)

Abstract The complete mitochondrial genome of Stegobium paniceum (Coleoptera: Anobiidae) is a circular DNA molecule of 15,271 bp (GenBank accession number XK819317), and its nucleotide composition is biased towards A + T nucleotides (78.32%). This genome comprises 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA (tRNA) genes, and an A + T-rich region. The gene order of S. paniceum was similar to those found in other known Coleoptera species. Sixteen reading frame overlaps and six intergenic regions were found in the mitochondrial genome of S. paniceum. All 22 tRNA genes have the typical cloverleaf secondary structure, with an exception for trnS1 (AGN). The phylogenetic relationships based on neighbour-joining method revealed that S. paniceum is closely related to Apatides fortis, which is consistent with the traditional morphological classification.

Complete mitochondrial genome of the bamboo snout beetle, Cyrotrachelus buqueti (Coleoptera: Curculionidae)

Abstract The bamboo snout beetle Cyrotrachelus buqueti (Coleoptera: Curculionidae) is a destructive forest pest and distributed widely in Southeast Asia. The 15,035 bp complete mitochondrial genome of the species consists of 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 21 transfer RNA genes (tRNAs) and a control region (GenBank accession no. MG674390). The trnl gene was not found in the C. buqueti mitogenome. The gene order and the orientation of C. buqueti were similar to those found in other Coleoptera species. The nucleotide composition was significantly biased (A, G, C, and T was 38.18%, 10.10%, 16.16%, and 35.56%, respectively) with A + T contents of 73.74%. ATG, ATA, ATT, AAT, and TTG were initiation codons and TAA, TAG, and T were termination codons. All the 21 tRNAs displayed a typical cloverleaf secondary structure, except for trnS1 which lacked the dihydrouridine arm. Phylogenetic analysis was performed using 13 PCGs with 14 other beetles showed that C. buqueti is closely related to Eucryptorhynchus brandti, which agree with the traditional classification.

Fabrication and characterization of Chinese giant salamander skin composite collagen sponge as a high-strength rapid hemostatic material

Abstract Chinese giant salamander (CGS) has high medicinal value and long history of clinical use in ancient China. In this study, CGS skin (CGSS) collagen was extracted and purified to prepare collagen sponge by freeze-drying. TEMPO oxidized microfibrillated cellulose (TEMPO-MFC) and genipin were adopted to improve the mechanical properties of collagen sponge. The hygroscopicity, porosity, mechanical properties, hemostatic performance, morphology, and biodegradability of the resultant sponges were investigated in detail. The results indicated that CGSS collagen was type I collagen with intact triple-helical structure, and the prepared sponge had porous structure and excellent hemostatic performance with procoagulant ratio of 53.28%. However, the CGSS collagen sponge showed low tensile strength (TS) of 98.80 KPa, compression strength (CS) of 1.48 KPa, and elongation at break (E) of 4.72%. Incorporating 2.5% TEMPO-MFC into the native CGSS collagen sponge resulted in an increase of 188.26% in TS to 284.80 KPa, 166.89% in CS to 3.95 KPa, and 73.52% in E to 8.19%. The improvements were attributed to the physical filling of TEMPO-MFC in cavity and cavity wall of collagen sponge and the stable chemical linkage between carboxyl of TEMPO-MFC and amino group of collagen which effectively improved the toughening of sponge and formed good interface bonding, respectively. Subsequent 0.3% genipin treatment further improved the TS to 605.00 KPa and the CS to 8.66 KPa as a result of crosslinking reaction. Moreover, the composite reinforcing also improved the anti-degradation ability and procoagulant ratio of collagen sponge. All results suggested that the TEMPO-MFC toughened and genipin crosslinked CGSS composite collagen sponge is a promising rapid hemostatic material with high-strength and can be applicated in biomedical field.