Assessment of a zinc finger protein gene (MPZC3H10) as potential RNAi target for green peach aphid Myzus persicae control.

BACKGROUND: RNA interference (RNAi) has potential application in pest control, and selection of the specific target gene is one of the key steps in RNAi. As an important effector, the zinc finger protein (ZFP) gene has high similarity among aphid species, and may have potential use in an RNAi-based pest control strategy. This study assessed the control efficiency of an RNAi target, MPZC3H10, a CCCH-type ZFP gene, against green peach aphid. RESULTS: ZC3H10 amino acid sequence similarity is more than 97.71% among the five tested aphid species: Myzus persicae, Aphis citricidus, Acyrthosiphon pisum, Diuraphis noxia and Rhopalosiphum maidis. However, no homologous sequence was found in the transcriptome of their ladybeetle predator, Propylaea japonica. Spatial expression patterns revealed that MPZC3H10 showed high expression in the muscle and fat body of M. persicae. The RNAi bioassay revealed that silencing of MPZC3H10 resulted in high mortality (53.33%) in M. persicae. By contrast, there were no observed negative effects on the growth and development of P. japonica when fed on aphids treated with double-stranded RNA (dsRNA) or injected with a "high dose" of dsRNA. CONCLUSION: Targeting MPZC3H10 showed promising efficiency for green peach aphid control via artificially designed dsRNA, and was safe for the predatory ladybeetle. © 2022 Society of Chemical Industry.

Characterization of carotenoid biosynthetic pathway genes in the pea aphid (Acyrthosiphon pisum) revealed by heterologous complementation and RNA interference assays.

Carotenoids are involved in many essential physiological functions and are produced from geranylgeranyl pyrophosphate through synthase, desaturase, and cyclase activities. In the pea aphid (Acyrthosiphon pisum), the duplication of carotenoid biosynthetic genes, including carotenoid synthases/cyclases (ApCscA-C) and desaturases (ApCdeA-D), through horizontal gene transfer from fungi has been detected, and ApCdeB has known dehydrogenation functions. However, whether other genes contribute to aphid carotenoid biosynthesis, and its specific regulatory pathway, remains unclear. In the current study, functional analyses of seven genes were performed using heterologous complementation and RNA interference assays. The bifunctional enzymes ApCscA-C were responsible for the synthase of phytoene, and ApCscC may also have a cyclase activity. ApCdeA, ApCdeC, and ApCdeD had diverse dehydrogenation functions. ApCdeA catalyzed the enzymatic conversion of phytoene to neurosporene (three-step product), ApCdeC catalyzed the enzymatic conversion of phytoene to ζ-carotene (two-step product), and ApCdeD catalyzed the enzymatic conversion of phytoene to lycopene (four-step product). Silencing of ApCscs reduced the expression levels of ApCdes, and silencing these carotenoid biosynthetic genes reduced the α-, ß-, and γ-carotene levels, as well as the total carotenoid level. The results suggest that these genes were activated and led to carotenoid biosynthesis in the pea aphid.

Prevalence of a Novel Bunyavirus in Tea Tussock Moth Euproctis pseudoconspersa (Lepidoptera: Lymantriidae).

Euproctis pseudoconspersa is a major pest of tea plants, and also causes a skin rash on workers in tea plantations. Research on virus could provide fundamental insights for classification, genetic diversity, evolution, and host-virus interaction mechanisms. Here, we identified a novel RNA virus, Euproctis pseudoconspersa bunyavirus (Phenuiviridae), and found that it is widely distributed in field populations of E. pseudoconspersa. The replication of virus in E. pseudoconspersa was indicated by Tag-PCR. These results contribute to the classification of bunyaviruses and provide insight into the diversity of commensal E. pseudoconspersa bunyavirus and the host.

Molecular characterization of ecdysis triggering hormone and its receptor in citrus red mite (Panonychus citri).

Neuropeptide ecdysis triggering hormone (ETH) plays crucial roles in invertebrates by activating a G protein-coupled receptor (GPCR), the ecdysis triggering hormone receptor (ETHR), which has been intensively investigated in Hexapoda. However, the molecular characterization of ETH and ETHR in Chelicerata remains unknown. In this study, we identified and characterized the full-length cDNA of ETH and ETHR in citrus red mite Panonychus citri (McGregor). Phylogenetic analysis indicated that PcETHR was closely related to the insect ETHR subtype B. A calcium mobilization-based functional assay showed that PcETH activated the PcETHR in a dose-dependent manner. Furthermore, a sharp upregulation of PcETH before ecdysis was observed by quantitative real-time PCR (qRT-PCR), and it revealed a tight correlation of ETH signaling and the molting process. These are the first insights into the molecular characterization of ETH and its receptor in mites as P. citri. Our work provides basic information of the ETH and ETHR in P. citri and lays a solid foundation for further physiological functions to better understand the ETH signaling system in mites.

Crustacean cardioactive peptide (CCAP) of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae): Molecular characterization, distribution and its potential roles in larva-pupa ecdysis.

Insects must undergo ecdysis for successful development and growth, and the crustacean cardioactive peptide (CCAP) is one of the most important hormone in this process. Here we reported a cDNA encoding for the CCAP precursor cloned from the oriental fruit fly, Bactrocera dorsalis, a most destructive insect pest of agriculture. The CCAP mature peptide (PFCNAFTGC-NH2) of B. dorsalis was generated by post-translational processing and found to be highly comparable with other insects. RT-qPCR showed that mRNA of CCAP in B. dorsalis (BdCCAP) was predominantly expressed in the central nervous system (CNS) and midgut of 3rd-instar larvae. By using immunohistochemical analysis, we also localized the endocrine cells that produce CCAP in the CNS, ring gland and midgut of 3rd-instar larvae of B. dorsalis. The synthetic CCAP mature peptide could induce the expression of mRNA of adipokinetic hormone (AKH), the metabolic neuropeptides in insects. The expression of BdCCAP mRNA in the CNS, but not in the midgut, could be upregulated in the response to the challenge of insect molting hormone, 20-hydroxyecdysone.

Tyrosine hydroxylase coordinates larval-pupal tanning and immunity in oriental fruit fly (Bactrocera dorsalis).

BACKGROUND: The oriental fruit fly Bactrocera dorsalis (Hendel), a notorious world pest infesting fruits and vegetables, has evolved a high level of resistance to many commonly used insecticides. In this study, we investigate whether tyrosine hydroxylase (TH) that is required for cuticle tanning (sclerotization and pigmentation) in many insects, could be a potential target in controlling B. dorsalis. RESULTS: We cloned TH cDNA (BdTH) of B. dorsalis. The complete open reading frame of BdTH (KY911196) was 1737 bp in length, encoding a protein of 578 amino acids. Quantitative real-time PCR confirmed that BdTH was highly expressed in the epidermis of 3rd instar larvae, and its expression increased prior to pupation, suggesting a role in larval-pupal cuticle tanning. When we injected dsBdTH or 3-iodo-tyrosine (3-IT) as a TH inhibitor or fed insect diet supplemented with 3-IT, there was significant impairment of larval-pupal cuticle tanning and a severe obstacle to eclosion in adults followed by death in most. Furthermore, injection of Escherichia coli into larvae fed 3-IT resulted in 92% mortality and the expressions of four antimicrobial peptide genes were significantly downregulated. CONCLUSION: These results suggest that BdTH might play a critical role in larval-pupal tanning and immunity of B. dorsalis, and could be used as a potential novel target for pest control. © 2017 Society of Chemical Industry.

Phytoecdysteroids from Ajuga iva act as potential antidiabetic agent against alloxan-induced diabetic male albino rats.

The present study investigated the protective effect of phytoecdysteroids extracted from the Ajuga iva plant on body weight changes, blood glucose, insulin total protein, blood urea nitrogen (BUN), creatinine, triglycerides (TG), cholesterol, lipid peroxidation, antioxidant enzymes, pancreatic histopathology and hexokinase-I expression in the alloxan-induced diabetic rats. Experimental diabetes was induced following 15day intraperitoneal administration of alloxan. The rats were divided into four groups. Group I served as a sham group, and group II served as the diabetic control. Group III served as a treatment for phytoecdysteroids (10mg/kg), and group IV served as a treatment for phytoecdysteroids (20mg/kg). Phytoecdysteroids restored body weight loss to its antihyperglycemic effect. Blood glucose was reduced 19.2 and 52.9% in group III and IV respectively. Blood insulin (54.9 and 105.88%) and total protein (25 and 72.2%) was increased in group III and IV respectively. BUN, creatinine, TG, cholesterol and lipid peroxidation was significantly reduced following treatment. Catalase, superoxide dismutase (SOD), and glutathione peroxidase activity were significantly increased following treatment. Islet ß-cells are lost in alloxan-induced diabetic rats. Regeneration of islets and reduced atrophy of acinar cells were noted. The number of insulin-secreting cells was tremendously reduced in alloxan-induced diabetic rats. Insulin-secreting cells were increased 48 and 61% in group III and IV respectively. Hexokinase-I mRNA (28.3 & 93.5%) and protein (27.9 and 55.3%) expression were significantly increased following treatment. Taking all these data together, it is suggested that the phytoecdysteroid could be a potential therapeutic agent against experimental diabetes.

Abamectin treatment affects glutamate decarboxylase expression and induces higher GABA levels in the citrus red mite, Panonychus citri.

The citrus red mite, Panonychus citri, is one of the most economically and globally destructive mite pests of citrus. Acaricide resistance has been a growing problem in controlling this pest. As the main inhibitory neurotransmitter in organisms, γ-aminobutyric acid (GABA) is synthesized from the amino acid glutamate by the action of glutamate decarboxylases (GADs). In the present study, one novel GAD gene, PcGAD, was identified and characterized from P. citri. The opening reading frame of PcGAD contained 1548 nucleotides that encode 515 amino acids. The subsequent spatiotemporal expression pattern by RT-qPCR revealed that the expression levels of PcGAD were significantly higher in larvae than in adults. Challenging with various concentrations of abamectin resulted in the upregulation of PcGAD transcript levels. Furthermore, biochemical characterization indicated that changes in GAD activity coincided with its mRNA levels. High-performance liquid chromatography confirmed that the GABA contents of P. citri increased upon abamectin treatment. The application of abamectin induces PcGAD expression and activates GAD activity, thereby resulting in an increase in GABA content in P. citri, which contributes to the adaptability of the mite to abamectin challenge.

Functional characterization of two acetylcholinesterase genes in the brown citrus aphid, Aphis (Toxoptera) citricidus (Kirkaldy), using heterologous expression and RNA interference.

Acetylcholinesterase (AChE) is the primary target of organophosphate- and carbamate-based insecticides. We sequenced the full-length cDNAs of two AChE genes from the brown citrus aphid Aphis (Toxoptera) citricidus (Kirkaldy). These two genes, Tcace1 and Tcace2, which encode TcAChE1 and TcAChE2, respectively, had a shared amino acid identity of 29% and were highly similar to other insect ace1 and ace2 genes, respectively, having specific functional motifs. Potential differences in enzymatic function were characterized by the heterologous expression of the two genes using a baculovirus system in Sf9 insect cells. Both of the recombinant AChEs had high specific activities for three typical substrates, acetylthiocholine iodide, butyrylthiocholine iodide, and propinylthiocholine iodide. TcAChE1 had a lower Michaelis-Menten constant value and a higher maximal reaction velocity than recombinant TcAChE2, indicating a higher affinity for substrates and greater catalytic efficiency, respectively. Bioassays showed a greater sensitivity of recombinant TcAChE1 to the 10 tested insecticides. Silencing of Tcace1 and Tcace2 by RNA interference significantly increased the susceptibility of A. citricidus to malathion and carbaryl; however, silencing Tcace1 resulted in a higher mortality rate than silencing Tcace2. Additionally, the specific enzyme activity decreased more after silencing Tcace1 than after silencing Tcace2. Thus, TcAChE1 plays a major role in postsynaptic neurotransmission in A. citricidus.

Regulation of three isoforms of SOD gene by environmental stresses in citrus red mite, Panonychus citri.

Superoxide dismutase (SOD) is a family of enzymes with multiple isoforms that possess antioxidative abilities in response to environmental stresses. Panonychus citri is one of the most important pest mites and has a global distribution. In this study, three distinct isoforms of SOD were cloned from P. citri and identified as cytoplasmic Cu-ZnSOD (PcSOD1), extracellular Cu-ZnSOD (PcSOD2), and mitochondrial MnSOD (PcSOD3). mRNA expression level analysis showed that all three isoforms were up-regulated significantly after exposure to the acaricide abamectin and to UV-B ultraviolet irradiation. In particular, PcSOD3 was up-regulated under almost all environmental stresses tested. The fold change of PcSOD3 expression was significantly higher than those of the two Cu-ZnSOD isoforms. Taken together, the results indicate that abamectin and UV-B can induce transcripts of all three SOD isoforms in P. citri. Furthermore, PcSOD3 seems to play a more important role in P. citri tolerance to oxidative stress.

Molecular characteristics, mRNA expression, and alternative splicing of a ryanodine receptor gene in the oriental fruit fly, Bactrocera dorsalis (Hendel).

Ryanodine receptors (RyRs) are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR) was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97%) to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis.

Effect of temperature on the occurrence and distribution of colorado potato beetle (Coleoptera: Chrysomelidae) in China.

Colorado potato beetle, Leptinotarsa decemlineata (Say), is the most destructive pest of potato in many countries of the world. It first invaded China from Kazakhstan in 1990s and now is a major pest of potato in many areas of Xinjiang Uygur Autonomous Region (Xinjiang). The objective of this study was to determine the effect of temperature on the spread of Colorado potato beetle in China after its invasion. Cold temperature in winter (December) and high temperature in summer (July) were analyzed in accordance with the absence and presence of Colorado potato beetle in Xinjiang. The boundary between the absence and presence of Colorado potato beetle in Xinjiang nearly coincided with the -8°C isotherm of monthly mean minimum temperature in winter. The stress of the low temperature in winter for Colorado potato beetle basically disappeared in the southeastern Hexi Corridor in Gansu Province of China, suggesting that the Hexi Corridor is the best channel to prevent any long-distance invasions of Colorado potato beetle into the Central Plains region. However, in Turpan City in northeastern Xinjiang, the extremely hot weather in the summer prevents the local colonization of Colorado potato beetle. Furthermore, according to our monitoring, high temperature in summer also limited Colorado potato beetle to diffuse eastward through Turpan. Results of this study suggest that it is essential to strengthen inspection and quarantine measures to prevent any artificial transmissions of Colorado potato beetle spreading eastward and thus to ensure the sustainable production of potato and other Solanaceae crops in northwest regions of China.

Measuring dynamics of caspase-3 activity in living cells using FRET technique during apoptosis induced by high fluence low-power laser irradiation.

BACKGROUND AND OBJECTIVES: Low-power laser irradiation (LPLI) has been used for therapies such as curing spinal cord injury, healing wound etc. Yet, the mechanism of LPLI remains unclear. In order to determine the effects of high fluence LPLI on cell growth and caspase-3 activity, we have measured the dynamics of caspase-3 activity during cell apoptosis induced by high fluence LPLI treatment. STUDY DESIGN/MATERIALS AND METHODS: He-Ne laser was used to irradiate human lung adenocarcinoma cells (ASTC-a-1). Cell Counting Kit-8 was used for cytotoxicity assay. A fluorescent microscope was used to perform fluorescence resonance energy transfer (FRET) imaging. A luminescence spectrometer was used to acquire the fluorescent emission spectrum. Statistical analysis was performed with Student's paired t-test. RESULTS: Cytotoxicity assay showed that when light irradiation fluence exceeded 60 J/cm2, LPLI treatment induced ASTC-a-1 cell apoptosis in a fluence-dependent manner. FRET imaging and spectrofluorometric analysis demonstrated that caspase-3 was activated during high fluence LPLI-induced cell apoptosis. CONCLUSIONS: Using FRET technique, we have reported that high fluence LPLI can induce human lung adenocarcinoma cells (ASTC-a-1) apoptosis. The activation of caspase-3 plays an important role in the apoptotic process.