| Phenoloxidases (POs) play a key role in insect development and metamorphosis. PO is uniquely associated with many important physiological and biochemical processes, such as cuticular sclerotization, melanization, and wound repair. Cuticular sclerotization and melanization are important for the insect integument, defensive encapsulation and melanization of foreign organisms. Therefore, PO and its zymogen are the hot spot in the research area of insect metamorphosis. Bactrocera dorsalis, the oriental fruit fly, is a destructive pest of many horticultural crops. Females typically oviposit in fruit, and the developing larvae tunnel through the fleshy mesocarp on which they feed, causing fruit damage and drop. Pupation and emergence are the important physiological and pathological events in insects, as well as B. dorsalis, and these physiological and biochemical processes are critical for the normal development and survival of insects in the environment. Current control techniques mainly rely on spraying chemical insecticides. However, there are some successive reports about the insecticides resistance from B. dorsalis. Hence, it is an urgent need to develop new pest management strategies. The current study aimed at the larval-pupal transition of B. dorsalis, and focused on PO and PPO, the main contents are as follows:1. The characterization of PO and the expression of its zymogen in different developmental stages and tissues from B. dorsalisSpecific PO activity was observed in all tested developmental stages with the substrate catechol. The 3rd instar larvae presented a significantly higher PO activity than the others, and the differences in the enzymatic activity among the different developmental stages were significant (P<0.05). In tested tissues, the highest PO activity was recovered in the integument followed by the midgut and fat body. PO activity in the integument was significantly higher than that in the midgut and fat body, whilst the enzymatic activity showed no significant difference between the midgut and fat bodyKinetic parameters for the activities determined for different developmental stages and tissues were analyzed with catechol and L-DOPA as substrates. For the catalytic activity of PO toward catechol, there was the significantly difference among Km values from the different developmental stages, and the Vmax value for the pupae was lower than those of the other developmental stages. However, a higher Vmax value was found for PO from the integument. The catalytic activity of PO toward L-DOPA showed a similar trend in its Km and Vmax values among different developmental stages and tissues. Additionally, biochemical characterization showed that PO from different developmental stages and tissues all had maximum activity at pH 7.5 and 37℃.The expression patterns of BdPPO1 were analyzed using qPCR with a-Tubulin as the reference gene, and the results indicited that the transcripts of BdPPO1 were expressed in all tested developmental stages, whilst transcripts of BdPPO1 were primarily identified in the late larval and pupal developmental stages, particularly during the larval-pupal transition. In different tissues, the expression results showed that BdPPO1 could be detected in all the examined tissues (integument, fat body, and midgut), and was mainly expressed in the integument. The results showed that the activity of PO and its zymogen expression were closely related to the development of B. dorsalis during the larval-pupal transition.2. Effect of Kojic Acid on PO and the development of B. dorsalisAs a specific inhibitor of PO, kojic acid (KA) is effectively capable of inhibiting PO. After feeding on a KA-containing artificial diet, the larval and pupal developmental periods were significantly prolonged. In addition, the larvae did not grow to normal size and rates of pupation and emergence were decreased when B. dorsalis larvae had been fed with KA-containing diet for 6 days. Compared to the control, PO activities from whole bodies of B. dorsalis were inhibited, so did in larval cuticles. Consistent with these, kinetic analysis showed that the catalytic capability of PO was significantly reduced. The present study indicated that KA is an effective inhibitor of PO activity in B. dorsalis. When PO was inhibited, the normal development of B. dorsalis was disrupted thus correlating the function of PO with larval development, pupation, and adult emergence.3. Effect of 20-hydroxyecdysone (20E) and metal ions on the expression of BdPPO1 in B. dorsalisThe relative expression levels of BdPPO1 were determined by qPCR after injection of 20E. The results showed that BdPPO1 was significantly up-regulated by 20E at 0.5μig/mL, whilst the expression of BdPPO1 was significantly down-regulated at 0.1 and 1μg/mL.PO activity increased in the presence of most concentrations of Zn2+, Mg2+, Ca2+ and Cu2+. PO activities from the larvae fed with a metal ion-containing diet for 6 days were determined using the L-DOPA substrate. The results showed that PO activity from Zn2+treatment was significantly higher than those of the other metal ions. The relative expression levels of BdPPO1 after metal ion treatments were determined by qPCR. The expression levels were significantly up-regulated by Zn2+, Mg2+, Ca2+, and Cu2+ treatments than the control. The results indicated that PO was a metalloprotein and it could be activated by Zn2+, Mg2+, Ca2+, and Cu2+. The functional analysis showed that the expression of BdPPO1 could be regulated by 20E after injection.4. Functional verification of BdPPO1 in B. dorsalisRNAi was applied to further explore the functions of BdPPO1 in B. dorsalis. injection of the double-stranded RNA of BdPPO1 into the 3rd instar larvae significantly reduced mRNA expression levels of BdPPO1 after 24 h and 48 h, and resulted in a lower pupation rate and abnormal phenotype. The present study demonstrated the potential implications for developing novel pest management strategies using BdPPO1 RNAi in the control of B. dorsalis and other insect pests. |
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