A novel spider venom peptide from the predatory mite Neoseiulus barkeri shows lethal effect on phytophagous pests.

The long-term use of pesticides in the field, and the high fertility and adaptability of phytophagous mites have led to resistance problems; consequently, novel safe and efficient active substances are necessary to broaden the tools of pest mite control. Natural enemies of arthropods typically secrete substances with paralytic or lethal effects on their prey, and those substances are a resource for future biopesticides. In this study, two putative venom peptide genes were identified in a parasitic mite Neoseiulus barkeri transcriptome. Recombinant venom NbSP2 peptide injected into Tetranychus cinnabarinus mites was significantly more lethal than recombinant NBSP1. NbSP2 was also lethal to Spodoptera litura when injected but not when fed to third instar larvae. The interaction proteins of NbSP2 in T. cinnabarinus and S. litura were identified by affinity chromatography. Among these proteins, ATP synthase subunit ß (ATP SSß) was deduced as a potential target. Four binding sites were predicted between NBSP2 and ATP SSß of T. cinnabarinus and S. litura. In conclusion, we identified a venom peptide with activity against T. cinnabarinus and S. litura. This study provides a novel component for development of a new biological pesticide.

RNAi targeting ecdysone receptor blocks the larva to adult development of Tetranychus cinnabarinus.

RNA interference (RNAi) is a potentially useful pest control method because of its high specificity. Silencing the expression of important RNAi target genes of pests will block important biological processes and reduce pest damage. Ecdysone is a unique arthropod hormone and the ecdysone receptor (EcR) is a key factor in molting pathway. We investigated the possibility that dsRNA targeting of the EcR of Tetranychus cinnabarinus (TcEcR) could effectively block development from larvae to adults. The mRNA level of TcEcR was highest in the larva stage, and 73.1% of the mites failed to survive the larva stage when TcEcR expression was silenced. Only 11.7% of T. cinnabarinus ingesting dsRNA successfully developed into adults, while 86.7% in the control succeeded in molting across each stage. RNAi significantly increased the developmental intervals of T. cinnabarinus. Under the effects of dsRNA, development times for the larva and first nymph doubled. Phenotype of body size change and death were observed during the development of T. cinnabarinus ingesting dsRNA. These findings suggest that RNAi is a potential means for the control of T. cinnabarinus. Genes in hormone pathways such as EcR are possible RNAi targets.

Functional analysis of five trypsin-like protease genes in the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

Insect midgut proteases catalyze the release of free amino acids from dietary proteins and are essential for insect normal development. To date, digestive proteases as potential candidates have made great progress in pest control. To clarify the function of trypsin-like protease genes in the digestive system of Bactrocera dorsalis, a serious pest of a wide range of tropical and subtropical fruit and vegetable crops, five trypsin genes (BdTry1, BdTry2, BdTry3, BdTry4 and BdTry5) were identified from transcriptome dataset, and the effects of feeding condition on their expression levels were examined subsequently. RNA interference (RNAi) was applied to further explore their function on the growth of B. dorsalis. The results showed that all the BdTrys in starving midgut expressed at a minimal level but up-regulated upon feeding (except BdTry3). Besides, RNAi by feeding dsRNAs to larvae proved to be an effective method to cause gene silencing and the mixed dsRNAs of the five BdTrys slowed larvae growth of B. dorsalis. The current data suggest that trypsin genes are actively involved in digestion process of B. dorsalis larvae and thereafter play crucial roles in their development.

Reference Gene Validation for Quantitative PCR Under Various Biotic and Abiotic Stress Conditions in Toxoptera citricida (Hemiptera, Aphidiae).

The regulation of mRNA expression level is critical for gene expression studies. Currently, quantitative reverse transcription polymerase chain reaction (qRT-PCR) is commonly used to investigate mRNA expression level of genes under various experimental conditions. An important factor that determines the optimal quantification of qRT-PCR data is the choice of the reference gene for normalization. To advance gene expression studies in Toxoptera citricida (Kirkaldy), an important citrus pest and a main vector of the Citrus tristeza virus, we used five tools (GeNorm, NormFinder, BestKeeper, ΔCt methods, and RefFinder) to evaluate seven candidate reference genes (elongation factor-1 alpha [EF1α], beta tubulin [ß-TUB], 18S ribosomal RNA [18S], RNA polymerase II large subunit (RNAP II), beta actin (ß-ACT), alpha tubulin, and glyceraldhyde-3-phosphate dehydrogenase) under different biotic (developmental stages and wing dimorphism) and abiotic stress (thermal, starvation, and UV irradiation) conditions. The results showed that EF1α and 18S were the most stable genes under various biotic states, ß-ACT and ß-TUB during thermal stress, EF1α and RNAP II under starvation stress, and RNAP II, ß-ACT, and EF1α under UV irradiation stress conditions. This study provides useful resources for the transcriptional profiling of genes in T. citricida and closely related aphid species.

The Essential Role of Vitellogenin Receptor in Ovary Development and Vitellogenin Uptake in Bactrocera dorsalis (Hendel).

The vitellogenin receptor (VgR) functions as an essential component in uptaking and transporting vitellogenin (Vg) in female adults, which is involved in ovary development and oviposition. This study aimed to clarify the molecular characteristics and function of VgR in the oriental fruit fly Bactrocera dorsalis (Hendel). Here, we identified the full-length of BdVgR (GenBank Accession No. JX469118), encoding a 1925 residue (aa) protein with a 214.72 kDa molecular mass and several typical motifs of low-density lipoprotein receptor superfamily (LDLR). Phylogenic analysis suggested that BdVgR was evolutionary conserved with other Dipteran VgRs. The expression of BdVgR was exclusively detected in the ovaries rather than head, thorax or other tissues. The developmental expression patterns showed that the signal of BdVgR was detectable in very beginning of adult stage, and positively correlated with the growth rate of ovaries and the expression levels of its ligands. In addition, we also demonstrated that the expression level of BdVgR, and ovary development were significantly suppressed after being injected with BdVgR-targeted dsRNA. Together, all of these results indicated that BdVgR was critical for yolk protein absorption and ovary maturation in B. dorsalis, playing a vital role in female reproduction.

Inducible Expression of Mu-Class Glutathione S-Transferases Is Associated with Fenpropathrin Resistance in Tetranychus cinnabarinus.

The carmine spider mite, Tetranychus cinnabarinus (Boisduval), is a serious pest on a variety of economically important crops widely distributed in China, and its resistance to acaricides has quickly developed. In this study, we fully sequenced 13 GST genes of T. cinnabarinus (TcGSTs). The phylogenetic tree showed that five of them belonged to the delta class and the other eight belonged to the mu class. The alignment of gene sequences and comparison of gene expressions between a fenpropathrin-resistant strain (FR) and a susceptible strain (SS) showed that neither point mutation nor overexpression was detected in TcGSTs. However, when challenged by a sublethal dose of fenpropathrin, the mRNA levels of three GSTs from the mu class (TCGSTM2, TCGSTM3, and TCGSTM8) highly increased in FR, while in SS, the expression of these genes was still at the same level under the treatment. In conclusion, specific TcGSTs were identified that were inducible to stimulation by fenpropathrin, and proved that TcGSTs in FR were not constantly expressed at a high level, but could react much more quickly under the stress of fenpropathrin than SS.

Characterization of Bactrocera dorsalis serine proteases and evidence for their indirect role in insecticide tolerance.

The oriental fruit fly Bactrocera dorsalis (Hendel) causes devastating losses to agricultural crops world-wide and is considered to be an economically important pest. Little is known about the digestive enzymes such as serine proteases (SPs) in B. dorsalis, which are important both for energy supply and mitigation of fitness cost associated with insecticide tolerance. In this study, we identified five SP genes in the midgut of B. dorsalis, and the alignments of their deduced amino acid sequences revealed the presence of motifs conserved in the SP superfamily. Phylogenetic analyses with known SPs from other insect species suggested that three of them were trypsin-like proteases. Analyses of the expression profiles among the different developmental stages showed that all five genes were most abundant in larvae than in other stages. When larvae were continuously fed on diet containing 0.33 µg/g ß-Cypermethrin, expression of all five genes were upregulated in the midgut but the larval development was delayed. Biochemical assays were consistent with the increased protease activity exhibited by SPs in the midgut after treatment with ß-Cypermethrin. Taken together, these findings provide evidence for the hypothesis that enhanced SP activity may play an indirect role in relieving the toxicity stress of insecticide in B. dorsalis.

Retinoid X receptor 1 is a specific lethal RNAi target disturbing chitin metabolism during hatching of Tetranychus cinnabarinus.

RNA interference (RNAi) can be developed as an alternative method of chemical pesticides for pest control. In this study, we noticed a specifically expressed gene (retinoid X receptor 1, TcRXR1) in the egg stage of T. cinnabarinus. RNAi was applied to investigate the function of TcRXR1. Results showed that with continuous feeding of dsTcRXR1, the larvae of T. cinnabarinus could still successfully develop to adult, which was in accordance with the low expression of TcRXR1 out of egg stage. High mortality of eggs was observed after eggs were treated with dsTcRXR1. To investigate the downstream genes of TcRXR1, the RNA samples after successful RNAi of TcRXR1 were analyzed by transcriptome analysis. According to function annotation of differentially expressed genes, 6 genes were selected for their potential function with the phenotype of dsTcRXR1, and among them, a chitinase gene (TcCHT-E) attained a high expression level in the late stage of egg, peaking just after the expression peak of TcRXR1. Mortality of eggs was observed under the effect of dsTcCHT-E as well as dsTcRXR1. In conclusion, TcRXR1 is a specific RNAi target for control of T. cinnabarinus, and its lethal mechanism might be disturbing chitin metabolism hatching of egg.

Evaluation of suitable reference genes for quantitative RT-PCR during development and abiotic stress in Panonychus citri (McGregor) (Acari: Tetranychidae).

Quantitative real time reverse transcriptase polymerase chain reaction (RT-qPCR) is preferred for gene expression analysis in living organisms. Currently, it is a valuable tool for biological and ecological studies as it provides a relatively straightforward way to assess the relevance of transcriptional regulation under developmental and stress tolerance conditions. However, studies have shown that some commonly used reference genes varied among different experimental treatments, thus, systematic evaluation of reference genes is critical for gene expression profiling, which is often neglected in gene expression studies of arthropods. The aim of this study is to identify the suitable reference genes for RT-qPCR experiments involving various developmental stages and/or under abiotic stresses in citrus red mite Panonychus citri, a key pest in citrus orchards worldwide. GeNorm, NormFinder, and Bestkeeper software analysis indicates that elongation factor-1 alpha (ELF1A), RNA polymerase II largest subunit, alpha tublin, and glyceraldhyde-3-phosphate dehydrogenase (GAPDH) are the most stable reference genes in various developmental stages, meanwhile, ELF1A and GAPDH were the most stable reference genes under various abiotic stresses. Furthermore, this study will serve as a resource to screen reference genes for gene expression studies in any other spider mite species.

Molecular characterizations of two cytochrome P450 genes encoding CYP6A41 and CYP6EK1 from the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

Two P450 genes encoding CYP6A41 and CYP6EK1 were cloned from the oriental fruit fly using polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques. CYP6A41 and CYP6EK1 contained open reading frames of 1,530 and 1,524 nucleotides that encode 510 and 508 amino acid residues, respectively. The putative proteins shared 44% identity with each other. Phylogenetic analysis showed that CYP6A41 and CYP6EK1 were most closely related to Ceratitis capitata CYP6A10 and CYP6A subfamily. Expression patterns of the two genes in different geographical populations (Yunnan, Hainan, Dongguang, and Guangzhou), developmental stages (eggs, larvae, pupae, and adults), and tissues (midguts, fat bodies, and Malpighian tubules) were analyzed by real-time quantitative PCR (RT-qPCR) methods. The results showed that the expression levels of CYP6EK1 were significantly different among the four populations, but were not different for CYP6A41. Both the expressions of CYP6A41 and CYP6EK1 were development specific and had significantly higher levels in the larval stage. The expression of CYP6A41 did not vary among the midgut, fat body, or Malpighian tubules; however, CYP6EK1 expression was higher in the Malpighian tubules. The results suggest that CYP6A41 and CYP6EK1 might be involved in detoxification of xenobiotic compounds that were harmful to larval flies or development. Moreover, high expression of CYP6EK1 in the Malpighian tubules also implied participation in detoxification.

Transcription factors CncC and Maf connect the molecular network between pesticide resistance and resurgence of pest mites.

Pesticide resistance and resurgence are serious problems often occurring simultaneously in the field. In our long-term study of a fenpropathrin-resistant strain of Tetranychus cinnabaribus, enhancement of detoxification and modified fecundity mechanisms were both observed. Here we investigate the network across these two mechanisms and find a key node between resistance and resurgence. We show that the ecdysone pathway is involved in regulating the fecundity of T. cinnabaribus. The concentration change of ecdysone is consistent with the fecundity curve; the concentration of ecdysone is higher in the fenpropathrin-resistant strain which has stronger fecundity. The enhancement of ecdysone is due to overexpression of two P450 genes (CYP314A1 and CYP315A1) in the ecdysone synthesis pathway. Silencing expression of these CYP genes resulted in lower concentration of ecdysone, reduced expression of vitellogenin, and reduced fecundity of T. cinnabaribus. The expression of CYP315A1 is regulated by transcription factors Cap-n-collar isoform C (CncC) and Musculoaponeurotic fibrosarcoma protein (Maf), which are involved in regulating other P450 genes functioning in detoxification of fenpropathrin in T. cinnabaribus. A similar regulation is established in citrus pest mite Panonychus citri showing that the CncC pathway regulates expression of PcCYP315A1, which affects mite fecundity. Transcription factors are activated to upregulate detoxification genes facilitating pesticide resistance, while the "one to multiple" regulation mode of transcription factors simultaneously increases expression of metabolic enzyme genes in hormone pathways and alters the physiology of pests. This is an important response of arthropods to pesticides which leads to resistance and population resurgence.

Full length sequencing reveals novel transcripts of detoxification genes along with related alternative splicing events and lncRNAs in Phyllotreta striolata.

The striped flea beetle, Phyllotreta striolata (Fabricius), damages crops in the Brassicaceae. The genetic data for this pest are insufficient to reveal its insecticide resistance mechanisms or to develop molecular markers for resistance monitoring. We used PacBio Iso-Seq technology to sequence the full-length transcriptome of P. striolata. After isoform sequence clustering and removal of redundant transcripts, a total of 41,293 transcripts were obtained, and 35,640 of these were annotated in the database of gene products. Structure analysis uncovered 4,307 alternative splicing events, and 3,836 sequences were recognized as lncRNAs. Transcripts with the complete coding region of important detoxification enzymes were further classified. There were 57 transcripts of P450s distributed in CYP2, CYP3, CYP4, and Mito CYP clades, 29 transcripts of ESTs from 4 functional groups, 17 transcripts of GSTs classified into 5 families, 51 transcripts of ABCs distributed in 6 families, and 19 transcripts of UGTs. Twenty-five lncRNAs were predicted to be regulators of these detoxification genes. Full-length transcriptome sequencing is an efficient method for molecular study of P. striolata and it is also useful for gene function analysis.

Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae).

BACKGROUND: quantitative real-time reverse transcriptase PCR (RT-qPCR) has been widely used for quantification of mRNA as a way to determine key genes involved in different biological processes. For accurate gene quantification analysis, normalization of RT-qPCR data is absolutely essential. To date, normalization is most frequently achieved by the use of internal controls, often referred to as reference genes. However, several studies have shown that the reference genes used for the quantification of mRNA expression can be affected by the experimental set-up or cell type resulting in variation of the expression level of these key genes. Therefore, the evaluation of reference genes is critical for gene expression profiling, which is often neglected in gene expression studies of insects. For this purpose, ten candidate reference genes were investigated in three different tissues (midgut, Malpighian tubules, and fat body) of the oriental fruit fly, Bactrocera dorsalis (Hendel). RESULTS: two different programs, geNorm and Normfinder, were used to analyze the data. According to geNorm, α-TUB + ACT5 are the most appropriate reference genes for gene expression profiling across the three different tissues in the female flies, while ACT3 + α-TUB are considered as the best for males. Furthermore, we evaluated the stability of the candidate reference genes to determine the sexual differences in the same tissue. In the midgut and Malpighian tubules, ACT2 + α-TUB are the best choice for both males and females. However, α-TUB + ACT1 are the best pair for fat body. Meanwhile, the results calculated by Normfinder are quite the same as the results with geNorm; α-TUB is always one of the most stable genes in each sample validated by the two programs. CONCLUSIONS: in this study, we validated the suitable reference genes for gene expression profiling in different tissues of B. dorsalis. Moreover, appropriate reference genes were selected out for gene expression profiling of the same tissues taking the sexual differences into consideration. This work not only formed a solid basis for future gene expression study in B. dorsalis, but also will serve as a resource to screen reference genes for gene expression studies in any other insects.

Identification of Lysinibacillus sphaericus Binary toxin binding proteins in a malarial mosquito cell line by proteomics: A novel approach towards improving mosquito control.

Bacterial insecticidal proteins, such as the Bin toxin from Lysinibacillus sphaericus, could be used more extensively to control insecticide resistant mosquitoes. This study was aimed at identification of mosquito cell proteins binding Bin toxin. Results showed that purified toxin was toxic to Anopheles gambiae larvae and Ag55 cultured cells. Clathrin heavy chain (an endocytosis protein) and glycolytic enzymes such as pyruvate kinase, enolase and dihydrolipoamide dehydrogenase were identified as binders of Bin toxin. The viability of Ag55 cells in the presence of endocytosis inhibitor, pitstop2, was significantly decreased upon Bin treatment, while the inhibitor chlorpromazine did not affect Bin toxicity. Bin toxin treatment decreased ATP production and mitochondrial respiration in Ag55 cells, whereas non-mitochondrial oxygen consumption significantly increased after Bin toxin treatment. These findings are steps towards understanding how Bin toxin kills mosquitoes. SIGNIFICANCE: Mosquitoes are vectors of pathogens causing human diseases such as dengue fever, yellow fever, zika virus and malaria. An insecticidal toxin from Lysinibacillus sphaericus called Binary, or Bin, toxin could be used more extensively to control insecticide resistant mosquitoes. Bin toxin enter cells in susceptible mosquitoes and induces apoptosis or autophagy. In the current research, we used the malaria mosquito Anopheles gambiae Ag55 cell line as a model. A proteomic-based approach identified proteins that interact with Bin toxin. Interacting proteins include clathrin heavy chain (endocytosis protein) and glycolysis enzymes such as pyruvate kinase, enolase and dihydrolipoamide dehydrogenase. In Ag55 cell toxicity assays, an endocytosis inhibitor, pitstop2, increased Bin toxicity. Real time assays with a Seahorse™ flux analyzer showed that Bin significantly affects mitochondrial respiration, a result consistent with cell death via apoptosis or autophagy. These research findings add insights into how an unusual binary protein exploits cellular machinery to kill mosquitoes.

Functional analysis of UGT201D3 associated with abamectin resistance in Tetranychus cinnabarinus (Boisduval).

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are widely distributed within living organisms and share roles in biotransformation of various lipophilic endo- and xenobiotics with activated UDP sugars. In this study, it was found that the activity of UGTs in abamectin-resistant (AbR) strain was significantly higher (2.35-fold) than that in susceptible strain (SS) of Tetranychus cinnabarinus. Further analysis showed that 5-nitrouracil, the inhibitor of UGTs, could enhance the lethal effect of abamectin on mites. From the previous microarray results, we found an UGT gene (UGT201D3) overexpressed in AbR strain. Quantitative PCR analysis showed that UGT201D3 was highly expressed and more inducible with abamectin exposure in the AbR strain. After silencing the transcription of UGT201D3, the activity of UGTs was decreased and the susceptibility to abamectin was increased in AbR strain whereas it was not in SS. Furthermore, UGT201D3 gene was then successfully expressed in Escherichia coli. The recombinant UGT201D3 exhibited α-naphthol activity (2.81 ± 0.43 nmol/mg protein/min), and the enzyme activity could be inhibited by abamectin (inhibitory concentration at 50%: 57.50 ± 3.54 µmol/L). High-performance liquid chromatography analysis demonstrated that the recombinant UGT201D3 could effectively deplete abamectin (15.77% ± 3.72%) incubating with 150 µg protein for 6 h. These results provided direct evidence that UGT201D3 was involved in abamectin resistance in T. cinnabarinus.

The Transcription Factor MafB Regulates the Susceptibility of Bactrocera dorsalis to Abamectin via GSTz2.

Pesticide resistance is a serious problem that poses a major challenge to pest control. One of the most potent resistance mechanisms is the overexpression of genes coding for detoxification enzymes. The expression of detoxification genes is regulated by a series of transcription factors. Previous studies have revealed that the increased expression of detoxification genes contributes to the insecticide tolerance of Bactrocera dorsalis. Our objective was thus to identify the transcription factors involved in this process. Temporal expression profiles showed that the transcription factor MafB and detoxification genes were expressed highly in the fat body. Further analysis showed that the expression of MafB, GSTz2, and CYP473A3 was induced by abamectin. Disruption of the MafB transcription factor through RNA interference decreased the transcript levels of GSTz2 and CYP473A3 and increased the susceptibility to abamectin significantly. Direct silencing of the expression of GSTz2 also increased susceptibility to abamectin, while CYP473A3 did not. In conclusion, these results suggest that the expression of GSTz2 and CYP473A3 was regulated by the transcription factor MafB, and the up-regulation of GSTz2 via MafB decreased the susceptibility of B. dorsalis to abamectin.

Influence of various stressors on the expression of core genes of the small interfering RNA pathway in the oriental fruit fly, Bactrocera dorsalis.

RNA interference (RNAi)-based technology has emerged as a potential tool for controlling insect pests, however, previous studies found that the efficiency of RNAi in Bactrocera dorsalis was variable. In nature, insects often meet various challenges, such as pathogen infections, extreme temperatures, lack of nutrition and heavy metals. To better understand the association of the stressors with efficiency of RNAi, in the current study we tested the expression of three core genes, dicer2 (Bddcr2), r2d2 (Bdr2d2) and argonaute2 (Bdago2), of the small interfering RNA (siRNA) pathway of B. dorsalis upon various stressors. Our results showed that all three genes were upregulated by the infection of invertebrate iridescent virus 6, which suggested a function of the siRNA pathway against viral infection. The loading of FeCl3 could also increase the expression of Bddcr2. The treatments of Escherichia coli, extremely high (40°C) and low (0°C) temperatures, as well as starvation, could negatively influence the expression of Bddcr2 and/or Bdago2. In total, our results showed that various stressors could influence the expression of core components of B. dorsalis siRNA pathway. This highlights further speculation on the RNAi efficiency upon these stressors. Considering the complexity and variation of RNAi efficiency in different conditions, these results provide initial aspects in possible environmental stressors to influence the activity of the siRNA pathway, but the real impact of RNAi efficiency posed by these stressors requires further studies.

Transgenic cotton expressing CYP392A4 double-stranded RNA decreases the reproductive ability of Tetranychus cinnabarinus.

As a polyphagous pest, Tetranychus cinnabarinus has the ability to overcome the defense of various hosts, and causes severe losses to various economically important crops. Since the interaction between pest and host plants is a valuable clue to investigate potential ways for pest management, we intend to identify the key genes of T. cinnabarinus for its adaption on cotton, then, with RNA interference (RNAi) and transgenic technology, construct a transgenic cotton strain to interfere with this process, and evaluate the effect of this method on the management of the mites. The difference of gene expression of T. cinnabarinus was analyzed when it was transferred to a new host (from cowpea to cotton) through high-throughput sequencing, and a number of differentially expressed genes involved in detoxification, digestion and specific processes during the development were classified. From them, a P450 gene CYP392A4 with high abundance and prominent over-expression on the cotton was selected as a candidate. With transgenic technology, cotton plants expressing double-stranded RNA of CYP392A4 were constructed. Feeding experiments showed that it can decrease the expression of the target gene, result in the reduction of reproductive ability of the mites, and the population of T. cinnabarinus showed an apparent fitness cost on the transgenic cotton. These results provide a new approach to restrict the development of mite population on the host. It is also a useful attempt to control piercing sucking pests through RNAi and transgenic technology.

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

Phenoloxidases (POs) play a key role in melanin production, are involved in invertebrate immune mechanisms, and are considered important enzymes in the insect development process. In the present study, we report the developmental stage and tissue-specific expression patterns of BdPPO1 and PO activity from Bactrocera dorsalis. 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, particularly in the integument. Additionally, biochemical characterization showed that PO from different developmental stages and tissues all had maximum activity at pH 7.5 and 37°C. After feeding a metal ion-containing artificial diet, the activity of PO and expression of BdPPO1 were significantly increased, indicating 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 20-hydroxyecdysone (20E) after injection. Furthermore, injection of the double-stranded RNA of BdPPO1 into the 3rd instar larvae significantly reduced mRNA levels after 24 h and 48 h, and resulted in a lower pupation rate and abnormal phenotype. These results expand the understanding of the important role of PO and its zymogen in the growth of B. dorsalis.

Involvement of superoxide dismutase in oxidative stress in the oriental fruit fly, Bactrocera dorsalis: molecular cloning and expression profiles.

BACKGROUND: Bactrocera dorsalis, one of the most economically important fruit fly pests in East Asia, is well adapted to various environmental conditions. Pesticides, pathogens and other stresses can cause oxidative damage in most organisms. The superoxide dismutase (SOD) family contains some of the most important enzymes in the antioxidant protection system of the fruit fly and other organisms. RESULTS: Four full-length cDNA sequences encoding one MnSOD (BdSOD2-1) and three Cu-ZnSODs (BdSOD1-1, BdSOD1-2 and BdSOD1-3) were cloned. The expression profiles of these four genes under different stresses showed them to be involved in response to detrimental conditions including heavy metals, pesticides, extreme temperatures and lipopolysaccharide (LPS) stresses. More specifically, the expression levels of these genes were found to be depressed in the presence of copper, zinc and manganese. The expression of all four SOD genes increased upon exposure to lead, cadmium, low temperature (0 °C) and LPS stresses. Only BdSOD1-3 transcription increased significantly at high temperature (40 °C) exposure. The expressions levels of BdSOD1-2 and BdSOD1-3 increased significantly in the presence of ß-cypermethrin and malathion, but only the expression of BdSOD2-1 increased in the presence of avermectin treatment. CONCLUSION: These different expression profiles suggest that the four BdSODs play different roles and respond to different oxidative stresses in B. dorsalis. Some BdSODs undergo specific reaction in the response to specific oxidative stresses.