Study On Important Genes In The Biosynthetic And Metabolic Pathways Of Chitin And Juvenile Hormone In Bactrocera Dorsalis

Chitin and juvenile hormone (JH) play key roles in insect development and metamorphosis. Chitin is the main component of integument, trachea, and peritrophic matrix of midgut. The growth and development of insect will reliance on rigorously controlling the synthesis and degradation of chitin. The physiological balance of JH in insects depends on its biosynthesis and degradation pathway. The regulation of JH titer is crucial to normal development in insects. Chitin and JH biosynthetic and metabolic pathway is not only a hot spot in the area of insect metamorphosis, but also an important research field in exploring the new strategies for controlling pests. The polyphagous tephritid fruit fly Bactrocera dorsalis is an important pest in the tropical and subtropical areas, damaging more than250plant species, including numerous fruits and vegetables. Females typically oviposit in fruit, and the developing larvae tunnel through the fleshy mesocarp on which they feed, causing fruit damage and drop. Current control techniques mainly rely on spraying chemical insecticides, but there are some successive reports about the insecticides resistance from B. dorsalis. Therefore, it is an urgent need to develop new pest management strategies. Based on the B. dorsalis transcriptomic database, the molecular characteristics and functions of the important genes in the biosynthetic and metabolic pathways of chitin and JH from B. dorsalis were analyzed comprehensively. The main contents are as follows:1. Molecular characterization and functional analysis of chitin synthase1gene and its two alternative splicing variants in B. dorsalisUsing RT-PCR and RACE techniques, the full-length cDNA sequence of chitin synthase1gene(BdCHSl) were cloned and characterized from B. dorsalis. Two alternative splicing variants of BdCHSl were identified, and termed as BdCHSl a and BdCHSlb, respectively. The cDNA of both variants consisted of5552bp, with an open reading frame (ORF) of4776bp, encoding a protein of1592amino acid residues, plus685bp and88bp of5’-and3’-noncoding regions, respectively. The alternative splicing site was located between positions3784-3960and formed a pair of mutually exclusive exons (a/b) that were same in size (177bp), but showed only65%identity at the nucleotide level. During B. dorsalis growth and development, BdCHSl and BdCHSla were both mainly expressed during the larval-pupal and pupal-adult transitions, while BdCHSlb was mainly expressed during pupal-adult metamorphosis and in the middle of the pupal stage. BdCHSla was predominately expressed in the integument whereas BdCHSlb was mainly expressed in the trachea, and lowly expressed in fat body, midgut and Malpighian tubules. The20-hydroxyecdysone (20E) induced the expression of BdCHSl and its variants. Both BdCHSl and BdCHSla were up-regulated dramatically at8h post-injection compared with control insects, whereas the expression of BdCHSlb was up-regulated as early as at1h post-injection, indicating that BdCHSlb quickly responded to20E. The results showed the expressions of BdCHSl and its two variants in the larvae of B. dorsalis exprosed to diflubenzuron increased significantly compared to the control. Increased expression of these genes mRNA may be due to compensation response of the BdCHSl, BdCHSla, and BdCHSlb gene at the transcritional level that is caused by the retarded chitin synthesis, indicating that chitin synthase may be one of targets of diflubenzuron. Injection of dsRNA of BdCHSl, BdCHSla, and BdCHSlb 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 BdCHSl and BdCHSla had a similar result in that the larva was trapped in old cuticle and died without tanning completely, while silencing of BdCHSlb has no effect on insect morphology. These results demonstrated that BdCHSl plays an important role in the larval-pupal transition and the expression of BdCHSl in B. dorsalis is regulated by20E.2. Molecular characterization and functional analysis of two chitinase genes in B. dorsalisIn this study, we identified and characterized two full-length cDNAs of chitinase genes in B. dorsalis. The chitinase genes were named BdCht2and BdCht5, respectively. The cDNA of BdCht2contains an open reading frame (ORF) of1449bp that encodes483amino acid residues, and126-and296-bp non-coding regions at the5’-and3’-ends, respectively. The BdCht2genome has four exons and three introns. The cDNA of BdCht5contains an ORF of1785bp that encodes595amino acid residues. Phylogenetic analysis with other insect chitinases suggested that the two chitinases were grouped into two classes, Group Ⅶ and Group Ⅰ, respectively. The predicted molecular mass of BdCht2and BdCht5proteins are approximately54.3kDa and67.5kDa and isoelectric point are5.97and5.70, respectively. The977bp5’flanking region of BdCht2was identified, and the transcription factor binding sites were predicted.The qPCR analysis showed that BdCht2and BdCht5were both mainly expressed during the larval-pupal and pupal-adult transitions, which suggested that two genes might play important roles in development and metamorphosis. BdCht2and BdCht5were all expressed in five tissues, with its higher expression occurring in the integument and trachea, followed by the fat body, and other tissues. BdCht2was predominately expressed in the integument whereas BdCht5was mainly expressed in the trachea. Moreover, the expressions of BdCht2and BdCht5were up-regulated significantly upon20E at different doses injection, compared to that of the control after8h and12h of20E treatment, respectively. In B. dorsalis, starvation can shorten the growth duration of third instar larvae, and lead to precocious metamorphosis. Starvation also increased the expression of BdCht2and BdCht5, and was suppressed again by re-feeding the insects. Although we performed RNAi for two chitinase genes by injection of dsRNA into the third-instar larvae, no phenotypic abnormalities were observed in the treated larvae. The knockdown efficiency of dsRNA for BdCht5was highly, whereas BdCht2was a limited RNAi response.3. Molecular characterization and expression analysis of JH biosynthetic pathway genes in B. dorsalisTo data, six JH biosynthesis pathway genes were identified, of which, four genes, including BdHMGS, BdHMGR, BdIPPI and BdFPS, involve in upstream mevalonic acid pathway (MVA) of JH biosynthesis and catalyze acetyl-CoA to generate isopentenyl diphosphate. BdSDR and BdJHAMT-like participate in downstream of JH biosyhthesis pathway. The ORF of BdHMGS was1383bp in length encoded460amino acids while the full-length cDNA of BdHMGR was a3157bp sequence with an ORF encoded928amino acids. Furthermore, BdIPPI had an ORF encoding245amino acids, while the1434bp cDNA of BdFPS had an ORF encoding408amino acids. Moreover, the920bp cDNA of BdSDR had an ORF encoding248amino acids, while BdJHAMT-like possessed an ORF of276amino acids. It is found that the domain regions of encoded proteins were highly conserved.The qPCR was used to analyze the mRNA expression of important genes involved in JH biosynthesis in different developmental stages and tissues of B. dorsalis. During the third-instar larval stage, the highest expressions of BdFPS and BdSDR were observed, but low or no expressions of other genes were observed. During the pupal stage, the expression of these genes was abundantly observed except BdSDR. The JH biosynthetic pathway genes have sex-specific expression patterns, and most genes were higher expressed in females. These genes were highly expressed in the adult fat body and head. Corpora allata is mainly localized in the head, and fat body is an important organ that plays important roles in sexual maturity. After adding JH analog to the incubated fat body in vitro, the expression levels of most JH biosynthetic pathway genes were inhibited, and20E also could inhibited the expressions of BdSDR and BdJHAMT-like. The results suggested that exogenous hormones might regulate the expression of these genes involved in JH biosynthesis. Furthermore, the expressions of BdHMGS, BdIPPI and BdJHAMT-like were significantly decreased after48h starvation compared to the fed larvae, and the rate of JH biosynthesis was effectively reduced. The JH titer was significantly decreased, leading to precocious metamorphosis in the larvae. However, the expression of BdHMGR was up-regulated, no significant change of BdSDR and BdFDR was detected after starvation. Based on these results, it is deduced that the regulation mechanism of important genes involved in JH biosynthesis may be different in response to hormone and starvation treatment.4. Functional analysis of BdHMGR gene in B. dorsalisRNAi was applied to further explore the functions of BdHMGR in B. dorsalis. Injection dsRNA of BdHMGR into day-5female adult significantly inhibited the expression level of the corresponding gene compared to control groups injected with dsGFP, significantly reduced BdVg2mRNA levels in ovaries, inhibited the development of ovaries. Moreover, the diameter of ovaries of the females was significantly lower than that in control groups, leading to effectively inhibit oviposition. The present study demonstrated the potential implications for developing novel pest management strategies using BdHMGR RNAi in the control of B. dorsalis and other insect pests.5. Molecular characterization and expression analysis of JH metabolic enzyme genes in B. dorsalisThree key enzymes, namely JH esterase (JHE), JH epoxide hydrolase (JHEH), and JH diol kinase (JHDK) are required for JH degradation in insects. In this study, we identified four JH metabolism-related genes, including BdJHE, BdJHEH2, BdJHEH3and BdJHDK. The cDNA of BdJHE contains an ORF of1788bp that encodes596amino acid residues, and52-and443-bp non-coding regions at the5’-and3’-ends, respectively. The ORF of BdJHEH2was1395bp in length encoded465amino acids while the full-length cDNA of BdJHEH3was a1651bp sequence with an ORF encoded459amino acids. The full-length cDNA of BdJHDK was a1065bp sequence with an ORF encoded184amino acids. It is found that the domain regions of encoded proteins were highly conserved. Phylogenetic analyses showed that separated clusters of each gene and the evolutional conservation in insects with a high similarity in Diptera.BdJHE, BdJHEH2and BdJHDK were mainly expressed during the larval-pupal transition, suggesting that these genes play important roles in the metamorphosis. However, BdJHEH3were predominately expressed in the early third-instar larvae, and lowly expressed during insect metamorphosis, indicating that this gene might be not JH metabolism enzyme. Furthermore, these genes were highly expressed on day-7and day-10adult, and have sex-specific expression patterns. The relative expression levels of BdJHE and BdJHEH2are substantially higher in females than males, while BdJHDK showed higher expression in males. Furthermore, BdJHE and BdJHEH2were mainly expressed in fat body, whereas BdJHEH3and BdJHDK were highly expressed in midgut and Malpighian tubules, respectively. The expressions levels of JH metabolism enzyme genes in third instar larvae were remarkably changed except BdJHE gene by20E injection. Specifically, At1h after20E injection, the expression levels of BdJHEH2and BdJHEH3were increased significantly. However, the expression level of BdJHDK was significantly decreased compared with control larvae. At4h and8h after20E application, the expression levels of BdJHEH2and BdJHEH3were increased remarkably, respectively. Expression level of BdJHDK was increased significantly at12h after20E injection. The results suggested that different juvenile hormone metabolism enzyme genes play various roles, and the induced expressions of JHEH and JHDK were mainly response to20E. Experiment was performed to analyze whether or not the JH metabolism enzymes expressions were regulated by starvation, the results showed that the expressions of BdJHE and BdJHDK were significantly increased after starvation treatment compared to the fed larvae, however, the expression of BdJHEH2and BdJHEH3were decreased, no significant change was observed after re-feeding. The changes in expression levels of these genes in response to food suggest that they may be have different roles in regulating growth and development of larvae during feeding.In summary, thirteen full-length cDNAs of genes involved in chitin and JH biosynthetic and metabolic pathway were cloned based on the B. dorsalis transcriptome database, and the sequences were analyzed using bioinformatics methods. The qPCR was applied to analyze the expression patterns of these genes in different developmental stages and tissues, but also evaluate the effects of hormone and starvation treatment in molecular responses under stress. Furthermore, RNAi was performed to investigate the functions of important genes. Results of our investigation provide foundational information for understanding the physiological and biological functions of these genes, and for facilitating the development of new strategies for pest control.