Lethality of Sesquiterpenes Reprogramming Red Palm Weevil Detoxification Mechanism for Natural Novel Biopesticide Development.

Natural biopesticide development for invasive populations of red palm weevils is mainly responsible for the destruction of date palms and demands an extensive screening program of plant secondary metabolites. In the current study, the pesticidal potential of sesquiterpenes (C15 H24), an important class of plant secondary metabolites primarily composed of three isoprene units, was evaluated by laboratory toxicity, feeding performance bioassays, and host detoxification gene expression patterns. Dose-mortality response bioassays performed against mid-aged eighth-instar red palm weevil larvae revealed dose-dependent mortality. Only three sesquiterpenes, including Farnesol (LD50 = 6559 ppm) and Farnesyl acetate (LD50 = 7867 ppm), are considered to have significant toxicity, with Picrotoxin (LD50 = 317 ppm) being the most toxic. Furthermore, highly toxic sesquiterpene (Picrotoxin) established in the current study tremendously reduced the feeding performance indices, including the efficacy of conversion of digested food (ECD) (81.74%) and the efficacy of conversion of ingested food (ECI) (73.62%). The least toxic sesquiterpenes, including ß-Caryophyllene, (+)-Cedrol, Nerolidol, (+)-Nootkatone, and Parthenolide, observed in the current study failed to impart significant reductions of ECI and ECD indices. Lethality of the least toxic sesquiterpenes was overcome by greatly inducing gene expressions of Glutathione S transferase (GST) and Cytochrome P450. These encouraging results enabled us to suggest Picrotoxin as a promising biopesticide for the control of red palm weevil infestations.

Toxin-Pathogen Synergy Reshaping Detoxification and Antioxidant Defense Mechanism of Oligonychus afrasiaticus (McGregor).

Current study reveals the likelihood to use pathogen and toxin mutually as an effective and eco-friendly strategy for Oligonychus afrasiaticus (McGregor) management, which could reduce toxicant dose and host killing time. Therefore, phytol and Beauveria bassiana in different proportions were evaluated to determine their effectiveness. Prior to ascertaining host mortality and defense mechanisms, we have recorded in vitro action of phytol using different concentrations (0.70, 1.40, 2.10, 2.80, and 3.50 mg/mL) against B. bassiana suspension. In vitro compatibility assays revealed that growth parameters (vegetative growth, sporulation, and viability) of B. bassiana were least affected by the action of phytol at all tested concentrations. Biological Index of B. bassiana exhibited compatibility with phytol allowed us to conduct Joint toxicity bioassays in which phytol and spores mixed in different proportions in order to attain maximum treatment effect in terms of high mortality at low concentration under short time. Results revealed that joint-application exhibited both synergistic (treatments with higher proportions of phytol), and antagonistic interaction (treatments with higher proportions of spores) interactions. Biochemical mechanisms involved in host antioxidant and detoxification response were explored by quantifying their respective enzymatic activities. Lethality of different treatments induced different patterns of detoxification enzymes including glutathione S-transferase (GST) and acetylcholinesterase (AchE). Overall, the least potent treatments (20% phytol:80% spores, and 40% phytol:60% spores) established in the current study induced relatively higher GST and AchE activities. On the other hand, the most potent treatment (80% phytol:20% spores) at its maximum concentration exhibited negligible relative GST and AchE activities. Antioxidant enzyme activities of CAT and SOD measured in the current study showed moderate to complex interaction might because of toxin-pathogen remarkable synergy. This study suggested that joint application of phytol with B. bassiana spores have shown tremendous acaricidal potential and found to be promising new strategy for controlling old world date mites.

Toxicity of Plant Secondary Metabolites Modulating Detoxification Genes Expression for Natural Red Palm Weevil Pesticide Development.

This study aimed to explore the larvicidal and growth-inhibiting activities, and underlying detoxification mechanism of red palm weevil against phenylpropanoids, an important class of plant secondary metabolites. Toxicity of α-asarone, eugenol, isoeugenol, methyl eugenol, methyl isoeugenol, coumarin, coumarin 6, coniferyl aldehyde, diniconazole, ethyl cinnamate, and rosmarinic acid was evaluated by incorporation into the artificial diet. All of the phenylpropanoids exhibited dose- and time-dependent insecticidal activity. Among all the tested phenylpropanoids, coumarin exhibited the highest toxicity by revealing the least LD50 value (0.672 g/L). In addition, the most toxic compound (coumarin) observed in the current study, deteriorated the growth resulting tremendous reduction (78.39%) in efficacy of conversion of digested food (ECD), and (ECI) efficacy of conversion of ingested food (70.04%) of tenth-instar red palm weevil larvae. The energy-deficient red palm weevil larvae through their intrinsic abilities showed enhanced response to their digestibility resulting 27.78% increase in approximate digestibility (AD) compared to control larvae. The detoxification response of Rhynchophorus ferrugineus larvae determined by the quantitative expression of cytochrome P450, esterases, and glutathione S-transferase revealed enhanced expression among moderately toxic and ineffective compounds. These genes especially cytochrome P450 and GST detoxify the target compounds by enhancing their solubility that leads rapid excretion and degradation resulting low toxicity towards red palm weevil larvae. On the other hand, the most toxic (coumarin) silenced the genes involved in the red palm weevil detoxification mechanism. Based on the toxicity, growth retarding, and masking detoxification activities, coumarin could be a useful future natural red palm weevil-controlling agent.

Susceptibility and Immune Defence Mechanisms of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) against Entomopathogenic Fungal Infections.

Insects infected with entomopathogenic fungi, experience physiological changes that influence their growth and immune defence. The potential of nine isolates of entomopathogenic fungi was evaluated after determining percent germination and relative conidial hydrophobicity. However, nutritional indices were evaluated after immersing eighth-instar Rhynchophorus ferrugineus larvae into each isolate suspension (1 × 107 conidia/mL). The results showed that isolates B6884 and M9374 had 44.51% and 39.02% higher conidial hydrophobicity compared with isolate I03011 (least virulent). The results of nutritional index assays revealed a significant reduction in growth indices after infection with different isolates. Compared with control, B6884 and M9374 greatly decreased larval growth by reducing the efficacy of conversion of ingested food (36%-47%) and Efficacy of conversion of digested food (50%-63%). Furthermore, only isolate B6884 induced 100% mortality within 12 days. Compared with control, isolate I03011, possessing the lowest conidial hydrophobicity, only reduced 0.29% of the efficacy of conversion of ingested food (ECI) and 0.48% of the efficacy of conversion of digested food (ECD). Similarly, transcriptomic analysis of genes related to the Red palm weevil (RPW) immune response, including pathogen recognition receptors (C-type lectin and endo-beta-1,4-glucanse), signal modulator (Serine protease-like protein), signal transductors (Calmodulin-like protein and EF-hand domain containing protein) and effectors (C-type lysozyme, Cathepsin L., Defensin-like protein, Serine carboxypeptidase, and Thaumatin-like protein), was significantly increased in larval samples infected with B6884 and M9374. These results suggest that for an isolate to be virulent, conidial hydrophobicity and germination should also be considered during pathogen selection, as these factors could significantly impact host growth and immune defence mechanisms.

Insecticidal potency of RNAi-based catalase knockdown in Rhynchophorus ferrugineus (Oliver) (Coleoptera: Curculionidae).

BACKGROUND: Palm trees around the world are prone to notorious Rhynchophorus ferrugineus, which causes heavy losses of palm plantations. In Middle Eastern countries, this pest is a major threat to date palm orchards. Conventional pest control measures with the major share of synthetic insecticides have resulted in insect resistance and environmental issues. Therefore, in order to explore better alternatives, the RNAi approach was employed to knock down the catalase gene in fifth and tenth larval instars with different dsRNA application methods, and their insecticidal potency was studied. RESULTS: dsRNA of 444 bp was prepared to knock down catalase in R. ferrugineus. Out of the three dsRNA application methods, dsRNA injection into larvae was the most effective, followed by dsRNA application by artificial feeding. Both methods resulted in significant catalase knockdown in various tissues, especially the midgut. As a result, the highest growth inhibition of 123.49 and 103.47% and larval mortality of 80 and 40% were observed in fifth-instar larvae, whereas larval growth inhibition remained at 86.83 and 69.08% with larval mortality at 30 and 10% in tenth-instar larvae after dsRNA injection and artificial diet treatment. The topical application method was the least efficient, with the lowest larval growth inhibition of 57.23 and 45.61% and 0% mortality in fifth- and tenth-instar larvae. Generally, better results were noted at the high dsRNA dose of 5 µL. CONCLUSION: Catalase enzyme is found in most insect body tissues, and thus its dsRNA can cause broad-scale gene knockdown within the insect body, depending upon the application method. Significant larval mortality and growth inhibition after catalase knockdown in R. ferrugineus confirms its insecticidal potency and suggests a bright future for RNAi-based bioinsecticides in pest control. © 2016 Society of Chemical Industry.

Rhynchophorus ferrugineus midgut cell line to evaluate insecticidal potency of different plant essential oils.

Cell cultures can be a potent and strong tool to evaluate the insecticidal efficiency of natural products. Plant essential oils have long been used as the fragrance or curative products around the world which means that they are safer to be used in close proximity of humans and mammals. In this study, a midgut cell line, developed from Rhynchophorus ferrugineus (RPW-1), was used for screening essential oils from nine different plants. Assays revealed that higher cell mortality was observed at 500 ppm which reached to 86, 65, 60, 59, 56, 54, 54, 53, and 53%, whereas lowest cell mortality at 1 ppm remained at 41, 23, 20, 17, 16, 15, 14, 13, and 10%, for Azadirachta indica, Piper nigrum, Mentha spicata, Cammiphora myrrha, Elettaria cardamomum, Zingiber officinale, Curcuma longa, Schinus molle, and Rosmarinus officinalis, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay revealed the percentage of cell growth inhibition was highest at 500 ppm and remained at 48, 45, 42, 37, 34, 29, 24, 22, and 18% against A. indica, P. nigrum, M. spicata, C. myrrha, E. cardamomum, Z. officinale, C. longa, S. molle, and R. officinalis, respectively. Lowest LC50 value (7.98 ppm) was found for A. indica, whereas the highest LC50 (483.11 ppm) was against R. officinalis. Thus, in this study, essential oils of A. indica exhibited the highest levels of toxicity, whereas those from R. officinalis exhibited the lowest levels of toxicity toward RPW-1 cells.

Mycoinsecticides: potential and future perspective.

Crop damage and consequent loss in productivity associated with arthropod pests represent the most serious threats to the world economy. Different methods have been used in the past to control arthropod pests; however, the increase in incidences of resistance to pesticides, food safety concerns, and concerns related to environmental impact associated with the use of chemical pesticides have led to the recent expansion of biological control agents. Entomopathogenic fungi are important and promising bio-control agents for controlling arthropod pests. This review compiles recent information regarding the potential of entomopathogenic fungi used against arthropod pests, traits responsible for mycoinsecticide virulence, and possible ways to enhance the virulence of entomopathogenic fungi, in order to pave the way for the development of bio-rational and eco-friendly arthropod pest management strategies.

Establishing midgut cell culture from Rhynchophorus ferrugineus (Olivier) and toxicity assessment against ten different insecticides.

Midgut epithelial cell culture was successfully developed from red palm weevil (Rhynchophorus ferrugineus) during this study and named as RPW-1. Optimum conditions for four different commercial media were also worked out to successfully maintain the culture. Grace's medium was found to be the most effective for RPW-1 culturing which resulted in the highest cell density of 7.5 × 10(6) cells/ml after 72 h of cell seeding with 96% cell viability. It was followed by Schneider's medium and TNM-FH medium where cell densities reached up to 7.4 × 10(6) and 5.9 × 10(6) cells/ml, respectively, after 72 h having 91 and 89% cell viability. Comparatively, Media-199 was least effective for RPW-1 cell culturing. As a whole, temperature at 27°C and pH 6.3 were the best for RPW-1 culturing where the highest cell density and maximum cell viability were noted. Individually, Grace's medium, Schneider's medium, TNM-FH medium, and Media-199 produced better results at 27°C, 27°C, 24°C, and 21°C and pH 6.3, 6.4, 5.3, and 7.1, respectively. The toxicity assay and MTT cell proliferation assay revealed that, out of the ten insecticides used in this study, emamectin benzoate was the most toxic insecticide to RPW-1 cells resulting in 92% cell mortality and 74% cell growth inhibition. Dieldrin was the least potent, causing only 19% cell mortality and 18% cell growth inhibition.

Identification of a novel interacting partner of the chemosensory protein 1 from Plutella xylostella L.

Chemosensory proteins (CSPs) are small soluble proteins endowed with heterogeneous functions. The information so far available for CSPs suggested these well-defined and conserved proteins were involved in diverse activities, including chemical communication, feeding, development, mating, immune regulation, as well as circadian rhythms. However, the detailed mechanisms of these physiological functions remain elusive. To explore the underlying mechanisms of CSPs and their interaction partners, a cDNA library from the head of Plutella xylostella was screened against CSP1 to identify proteins involved in the PxylCSP1-related physiological activities. Protein kinase C (PKC) was screened out as a putative interacting protein of PxylCSP1. The full length of PxylPKC cDNA was obtained, and the results of semi-quantitative real-time PCR and quantitative real-time PCR revealed that PxylPKC showed similar expression pattern as PxylCSP1. In vivo and in vitro interactions between PxylCSP1 and PxylPKC were further confirmed by co-immunoprecipitation and GST pull-down assays, respectively. These findings extended our knowledge on the mechanisms of CSP-regulated functions, and providing new target proteins to facilitate the design of novel intervention strategies against the pest.

Evaluation of glutathione s-transferase as toxicity indicator for roxarsone and arsanilic acid in Eisenia fetida.

Different compounds can induce stress response by targeting specific genes. Studies related to elucidating the detoxification and adaptive responses of proteins like glutathione-s-transferase (GST) can be helpful in better understanding toxicity. Roxarsone and arsanilic acid, which have been exhaustively used as animal and poultry feed additives, pose a threat to the environment and human health. GST enzyme bioassay revealed fluctuations in response to different concentrations of roxarsone and arsanilic acid at different time intervals. The highest GST enzyme activity (40.51%) was observed on day 15 of treatment with roxarsone. On the other hand, arsanilic acid caused the maximum enzyme activity (52.11%) on day 10 of treatment. During this study, the full-length gene sequence of GST, having the size 984 bp (Genbankno. HQ693699), was achieved from Eisenia fetida and established as a biomarker to assess the toxicity of roxarsone and arsanilic acid. The deduced protein has a computed molecular mass of 23.56 kDa and a predicted isoelectric point of 9.92. Quantitative real-time PCR revealed significant differential gene expression in response to roxarsone and arsanilic acid treatment as compared with control treatment. Roxarsone caused the highest gene expression of 7.0-fold increase over control on day 15 of treatment, whereas arsanilic acid resulted in the highest gene expression reaching to 14.56-fold as compared with control. This study is helpful in understanding the role of GST as a potential biomarker for chemicals like roxarsone and arsanilic acid, which can pollute the food chain.

Combination of Kluyveromyces marxianus and sodium bicarbonate for controlling green mold of citrus fruit.

Biocontrol efficacy of an antagonistic yeast Kluyveromyces marxianus was evaluated individually or in combination with sodium bicarbonate (SBC) against green mold of citrus fruit caused by Penicillium digitatum. Their effects on postharvest quality of citrus fruit were also investigated. The results indicated that the antagonistic activity of K. marxianus at 1×108 CFU/mL on green mold of citrus fruit was enhanced by 2% SBC treatment. In artificial inoculation trials, disease control after 3 and 6 days, respectively, with the mixture of K. marxianus and 2% SBC (18.33%, 58.33%) was significantly improved over that obtained with K. marxianus (41.67%, 70.00%) or SBC (43.33%, 81.67%) alone. The combination of K. marxianus with SBC was as effective as the imazalil treatment in natural infection trials, which gave about 90% control of green mold. Addition of 2% SBC significantly stimulated the growth of K. marxianus in citrus fruit wounds after 72 h. Moreover, K. marxianus, SBC and their combination did not impair quality parameters including weight loss, fruit firmness, total soluble solids, titratable acidity and ascorbic acid at 4 °C for 30 days followed by 20 °C for 15 days. These results suggested that the use of SBC is a useful approach to improve the efficacy of K. marxianus for the postharvest green mold of citrus fruit.

Apolipophorin III and transmission electron microscopy as toxicity indicators for harmaline and tea saponin in Spodoptera exigua (Noctuidae: Lepidoptera).

Apolipophorin III, traditionally known for lipid transport in insects is fairly established as toxicity indicator against harmaline and tea saponin during this study. Apolipophorin III expressed in the hemolymph and midgut tissues of 3rd, 4th, 5th larval instars and pupae of Spodoptera exigua. Apolipophorin III presence was further confirmed by achieving its partial cDNA (Genbank accession no. FJ606822) of 448bp. qRT PCR revealed that tea saponin resulted in significant reduction of gene expression in 3rd and 4th larval instars but increased in 5th instar as compared to control. Harmaline caused gradual increase of gene expression in 3rd, 4th and 5th instars after feeding on the treated diet. Fifth instar larvae synonymously resulted in the highest gene expressions against both the biochemicals. After the injection of harmaline and tea saponin abrupt increase in gene expression of 4th, 5th larval instar and pupae was observed as compared to control treatment. Transmission electron microscopy of midgut epithelium after being fed with harmaline and tea saponin depicted certain cytological changes. Harmaline treatment lead to cytoplasm vacuolization, mitochondrial disruption, spherocrystals with concentric layers, irregular nucleus and floating nuclei in cytoplasm. Tea saponin treatment resulted in denser cytoplasm, higher intracellular osmotic concentration and reduced complement of apical microvilli. Cells were found to have only a few mitochondria and glycogen deposits in comparison to control treatment.

Biodegradation of beta-cypermethrin and 3-phenoxybenzoic acid by a novel Ochrobactrum lupini DG-S-01.

A newly isolated bacterium DG-S-01 from activated sludge utilized beta-cypermethrin (beta-CP) and its major metabolite 3-phenoxybenzoic acid (3-PBA) as sole carbon and energy source for growth in mineral salt medium (MSM). Based on the morphology, physio-biochemical characteristics, and 16S rDNA sequence analysis, DG-S-01 was identified as Ochrobactrum lupini. DG-S-01 effectively degraded beta-CP with total inocula biomass A(590 nm) = 0.1-0.8, at 20-40 °C, pH 5-9, initial beta-CP 50-400 mg L(-1) and metabolized to yield 3-PBA leading to complete degradation. Andrews equation was used to describe the special degradation rate at different initial concentrations. Degradation rate parameters q(max), K(s) and K(i) were determined to be 1.14 d(-1), 52.06 mg L(-1) and 142.80 mg L(-1), respectively. Maximum degradation was observed at 30 °C and pH 7.0. Degradation of beta-CP was accelerated when MSM was supplemented with glucose, beef extract and yeast extract. Studies on biodegradation in liquid medium showed that over 90% of the initial dose of beta-CP (50 mg L(-1)) was degraded under the optimal conditions within 5d. Moreover, the strain also degraded beta-cyfluthrin, fenpropathrin, cyhalothrin and deltamethrin. These results reveal that DG-S-01 may possess potential to be used in bioremediation of pyrethroid-contaminated environment.

Screening of T7 phage displayed Bactrocera dorsalis (Hendel) antenna cDNA library against chemosensory protein.

Recent studies have shown that chemosensory proteins (CSPs) were involved in diverse life activities such as insect feeding, development, mating, immune regulation, as well as other important circadian rhythms, etc. To screen the proteins involved in the BdorCSP-related physiological activity, a cDNA library of the Bactrocera dorsalis (Hendel) antenna expressed on the surface of T7 phage was screened against BdorCSP. After four rounds of screening, ELISA-positive samples of selected phages were sequenced and identified as protein disulfide isomerase (PDI), trypsin-like serine protease (Ser), TakeOut (TO), and a new protein by GenBank blast, respectively. Real-time quantitative PCR results showed that the expression levels of Ser, TO, and the new protein were the highest in antenna, sharing similar expression pattern with BdorCSP. These results reveal that these proteins might be involved in the BdorCSP-related physiological or metabolic activities. This work paves a new way for exploring the function of CSPs.

Molecular characterization and expression pattern of four chemosensory proteins from diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

Some chemosensory proteins (CSPs) expressed in insect sensory appendages are thought to be involved in chemical signaling in moths. We cloned and characterized four CSP genes from Plutella xylostella. The deduced amino acid sequences of PxylCSP1, PxylCSP2, PxylCSP3 and PxylCSP4 revealed open reading frames of 152, 128, 126 and 126 amino acids, respectively, with four conserved cysteine residues. The expression patterns of the four PxylCSP genes were further investigated by reverse transcription (RT) PCR and real-time PCR. PxylCSP1 and PxylCSP2 genes were expressed in all the tested tissues with the highest expression level in the antennae and heads (without antennae) whereas PxylCSP3 and PxylCSP4 mRNA were distributed extensively in all the tested tissues without apparent quantitative differences. The transcription levels of these CSP genes depended on sex, age, mating and the genes. Fluorescence quenching with Rhodojaponin-III (R-III) and homology modelling studies indicated that PxylCSP1 was able to bind non-volatile oviposition deterrents, such as R-III. These ubiquitous proteins might have the role of extracting non-volatile compounds (oviposition deterrents or antifeedants) dispersed in the environment and transporting them to their receptor.