Role of CYP6MS subfamily enzymes in detoxification of Sitophilus zeamais after exposure to terpinen-4-ol and limonene.

Food security is an important basis and guarantee to national safety, the loss caused by storage pests was a serious problem which affects the food security widely. Frequent application of chemical pesticides caused several critical crises including the development of resistance, pesticide residues, environmental pollution, and exposure risk to human or non-target organisms. The utilization of volatile components acts as a natural alternative for controlling storage pests has aroused extensive interest in recent years. It has been reported that terpinene-4-ol and limonene showed significant insecticidal activity against Sitophilus zeamais in our previous studies, which was evaluated to have strong influences to CYP450 genes. To determine the links and roles of related genes, we identified the SzCYP6MS subfamily genes which encoded a putative protein of 493 or 494 amino acids. Then, the expression of four CYP6MS subfamily genes were increased significantly under the fumigation stress by terpinen-4-ol and limonene, which was determined by the RT-qPCR analysis compared with non-fumigated colonies. In addition, we determined that RNAi-mediated CYP6MS genes knockdown significantly increased the sensitivity of S. zeamais to terpinen-4-ol and limonene, the mortality rates of insects with knocked down CYP6MS1, CYP6MS5, CYP6MS6, CYP6MS8, and CYP6MS9 genes increased by 25%, 25%, 16%, 17%, and 4% in terpinen-4-ol treatment groups and by 29%, 25%, 15%, 22%, and 3% in limonene treatment groups compared with that in the control groups, respectively. Finally, it was validated that CYP6MS5 exhibited the most stable binding with terpinen-4-ol that was similar to the result between CYP6MS8 and limonene which were verified by molecular docking analysis. In together, this study demonstrates the potential of terpinen-4-ol and limonene used as novel botanical pesticides to control storage pests, thereby reducing application of chemical pesticides and postponing resistance development.

Cloning and expression of the mitochondrial cytochrome c oxidase subunit II gene in Sitophilus zeamais and interaction mechanism with allyl isothiocyanate.

In the United States, allyl isothiocyanate (AITC) has been registered as an insecticide, bactericide, and nematicide. And it has been confirmed that AITC has significant insecticidal activities against four stored product pests including Sitophilus zeamais Mostchulky (Coleoptera: Curculionidae). This study aimed to verify the mechanism of action of AITC on cytochrome c oxidase core subunits II in S. zeamais. Enzyme - catalyzed reactions and Fourier transform infrared spectrometer (FTIR) analysis revealed that the expressed COX II proteins could competitively bind and inhibit the activity of COX II. Furthermore, molecular docking results showed that a sulfur atom of AITC could form a 2.9 Å hydrogen bond with Ile-30, having a binding energy of -2.46 kcal/mol.

Assessment of Colocasia esculenta leaf extract as a natural alternative for Sitophilus zeamais control: Toxicological, biochemical, and mechanistic insights.

The present study assessed the toxicological, biochemical, and mechanism of action of Colocasia esculenta leaf extract (CELE) on Wistar albino rat and on cholinergic, antioxidant, and antiinflammatory enzymes in Sitophilus zeamais. This was with a view to assessing the potential benefits and safety profile of CELE as a natural alternative for insect control. The bioactivity of the fraction was evaluated using insecticidal and repellent activities against colonies of Sitophilus zeamais to obtain a VLC-chromatographed fraction which was spectroscopically characterized and investigated for enzyme inhibition. The results revealed the ethyl acetate fraction (EAF) as the most potent one with LC50 6.198 µg/ml and 6.6 ± 0.5 repellency. The EAF had an LD50 > 5000 mg/kg but repeated dose >800 mg/kgbw po administration caused significant (p < 0.05) increase in liver and kidney function biomarkers accompanied with elevated atherogenic and coronary indices. Also, renal and hepatomorphological lesions increased in a dose-dependent manner. The High-Performance Liquid Chromatography analysis profiled 7 unknown compounds while the GC-qMS revealed 103 compounds in the CC6 fraction allowing for their identification, quantification, and providing insights into the biological activities and its potentials application. The CC6 fraction inhibited glutathione S-transferase (IC50 = 2265.260.60 mg/ml), superoxide dismutase (IC50 = 1485.300.78 mg/ml), catalase (IC50 = 574.471.57 mg/ml), acetyl cholinesterase (IC50 = 838.280.51 mg/ml), butyryl cholinesterase (IC50 = 1641.76 ± 1.14 mg/ml) and upregulated cyclooxygenase-2 (IC50 = 37.89 ± 0.15 mg/ml). Based on the result of the study, it could be inferred that the unidentified compounds present in the EAF exhibit strong insecticidal properties. The study concluded that the acute toxicity of the potent fraction showed no abnormal clinical toxic symptoms while a repeated dose of the extract in sub-acute studies showed a toxic effect that is dose-dependent. The mechanism of action of the purified fraction could be said to be by inhibition of cholinergic and antioxidant enzymes. However, the potent fraction also upregulated the activity of anti-inflammatory enzymes. Hence, regulated amount of CELE at a repeated dose <800 mg/kgbw could be considered for use as an anti-pest agent in Integrated Pest Management of Sitophilus zeamais.

Effiiency of the Essential Oil of Porophyllum linaria (Asteraceae) a Mexican Endemic Plant Against Sitophilus zeamais (Coleoptera: Curculionidae).

The insecticidal and repellent effect of essential oil isolated from fresh leaves of Porophyllum linaria on maize weevil was evaluated, as well as the effect on the grain germination after treated. In total, 28 constituents were identified by gas chromatography coupled with mass spectrometry accounting for 99.86% of whole essential oil. The main majority compounds were ß-myrcene (41.94%), D-limonene (20.29%), and estragole (20.03%). Contact toxicity significantly increased with dose and time after treatment. With the 800 ppm (highest concentration), the mortality (%) obtained for the tenth and fifteenth day was 43 and 82%, respectively, whereas with 50 ppm (lowest concentration) 30% mortality was obtained at the end of the experiment (fifteenth day). At 15 d (end of the experiment), the LC50 y LC90 were obtained with values of 329.01 ± 44.35 y 1058.86 ± 117.76 ppm, respectively. For a concentration of 800 ppm, a selection index of zero was obtained, indicating the preference of the pest to the untreated maize (control). The maize grains germination test showed a significant reduction both in the length of hypocotyl and radicle of maize grain. So, in the highest dose, the hypocotyl and radicle length was 1.40 ± 0.34 and 9.14 ± 0.55 cm, respectively, whereas the control group registered 3.28 ± 0.39 and 13.02 ± 0.97 cm, respectively. This finding is promising since as it could result in the identification of botanical substances capable of suppressing maize weevil, Sitophilus zeamais development.

Low-pressure ozone injection system: relationship between reaction kinetics and physical properties of grains.

BACKGROUND: The ozonation of grains in a closed system at low pressure is a strategy with the potential for treating packaged products. Research is necessary to characterize the reaction kinetics of ozone in this type of injection system so that it is possible to design chambers and determine the ozone concentrations suitable for commercial-scale applications. The objective of this study was therefore to characterize the low-pressure ozone injection system in relation to the physical properties of the grains and determine possible changes in their quality. Samples (5 kg each) of common beans, cowpea beans, corn, popcorn kernels, paddy rice, and polished rice were exposed to ozone in a 70 L hypobaric chamber. Initially, the internal pressure of the chamber was reduced to 500 hPa. Then, ozone was injected at a concentration of 32.10 g m-3 at a volumetric flow rate of 1 L min-1 until reaching a pressure of 1000 hPa. To relate the decomposition of ozone to the grains that were being evaluated, different physical properties were determined, and quality analysis was conducted. RESULTS: Ozone gas half-life outside and inside the package depended on the grain type. Ozone decomposition was quickest in polished rice and slowest in common beans. The half-life of the different grains ranged from 17.8 to 52.9 and 16.4 to 52.9 min, outside and inside the package, respectively. Considering the physical properties, specific surface (Ss), surface area (SA), and sphericity (φ) exhibited a significant correlation with the decomposition rate constant (k) of ozone. However, the variables volume (V), permeability (K), porosity (ε), and specific mass (ρ) showed no correlation with k. CONCLUSION: The physical properties of grain influenced the reaction kinetics of ozone gas during the low-pressure injection process. Ozone gas injection at low pressures did not alter the quality attributes of the grains under study. © 2022 Society of Chemical Industry.

Efficacy of different hermetic bag storage technologies against insect pests and aflatoxin incidence in stored maize grain.

The performance of six grain storage technologies for the control of insect pests in maize was evaluated over a 36-week (9-month) storage period. The six technologies used were: two ZeroFly® hermetic bag brands (laminated and non-laminated); Purdue Improved Crop Storage (PICS) bag; non-hermetic ZeroFly® bag; woven polypropylene (PP) bag containing maize grain treated with Actellic Gold® Dust (pirimiphos-methyl 1.6% + thiamethoxam 0.3%) and woven PP bag containing untreated grain. Each bag was filled with 50 kg maize grain and four replicates of each were set up. With the exception of the non-hermetic ZeroFly® bag, 50 live adults of the larger grain borer Prostephanus truncatus and of the maize weevil Sitophilus zeamais, were introduced into all the bags. Insects were not introduced into the non-hermetic ZeroFly® bag to assess its effectiveness in repelling infestation from outside. Parameters recorded were gas composition (oxygen and carbon dioxide) levels inside the bags; weight of flour generated by insect feeding activities; grain moisture level; live adult insect counts; grain damage and weight loss; grain germination rate and aflatoxin level. At termination, the plastic liners of the hermetic bags were examined for perforations. Results show that oxygen depletion and carbon dioxide evolution were faster in ZeroFly® hermetic compared to PICS bags. Throughout the 36-week storage trial, grain damage remained below 4% and weight loss below 3% in all the treatments except in the untreated PP bags in which it increased to 81.1 and 25.5%, respectively. The hermetic PICS, ZeroFly® and Actellic Gold dust-treated PP bags maintained grain germination at 60%, which was lower than the initial 90%, while in untreated control, it reduced to 4.7%. The mean aflatoxin levels fluctuated between 0.39 and 3.56 parts per billion (ppb) during 24 weeks of storage in all the technologies tested, which is below the acceptable maximum level of 10 ppb in maize. Based on the evaluation results, it can be concluded that hermetic PICS and ZeroFly® bags and woven PP bag with Actellic Gold dust-treated grain effectively protected stored maize grain from insect attack and weight losses. Appropriate strategies and mechanisms for the effective and efficient adoption of hermetic storage bag technology at scale would contribute towards global food security.

Function of four mitochondrial genes in fumigation lethal mechanisms of Allyl Isothiocyanate against Sitophilus zeamais adults.

Allyl isothiocyanate (AITC) is a promising alternative to chemical fumigants, and mitochondrial dysfunction has been proposed to play a crucial role in its lethal mechanisms; however, the specific lethal mechanisms of AITC remain unknown. Four mitochondrial electron transport chain genes, nd5, nd6, cox1, and cox5, were selected from adult Sitophilus zeamais and processed with RNA interference experiments. Then, the biochemical and biophysical effects were compared between double-stranded RNA (dsRNA)-mediated insects and wild-type insects after AITC fumigation at the concentration of LC50 values. The bioactivity of AITC against dsnd6-mediated insects increased, while the bioactivity against dcox1-mediated insects decreased. Compared with the wild-type insects, the increase of reactive oxygen species (ROS) levels by AITC in mitochondria from dsnd6-mediated insects increased by 18.95%, while that of dscox1-mediated insects decreased by 27.45%. The effects of AITC on mRNA expression levels of detoxifying enzymes including CAT (down-regulation effect) and CuZnSOD (overexpression effect) partly recovered in the dsnd5-mediated insects, while a greater effect was observed for dscox1-mediated insects. Molecular docking results indicated that ASN511 at the cox1 subunit was the binding site of AITC by one hydrogen bond, with a bond distance of 2.1 Å. These findings provide insight for further applications of AITC and could provide a novel strategy to investigate lethal mechanisms of insecticides.

Bacterial chitinase biochemical properties, immobilization on zinc oxide (ZnO) nanoparticle and its effect on Sitophilus zeamais as a potential insecticide.

It has been planned to minimize the yield and quality impairment of the seed corn, which is strategically important in the world, by pests under storage conditions with a biological product produced with a biotechnological approach. In this context, the present study aimed to control the maize weevil Sitophilus zeamais, known as a warehouse pest, using a nanoformulation. In the study, the chitinase enzyme from Lactobacillus coryniformis was purified first using ammonium sulfate precipitation and then by using the HiTrap Capto DEAE column, and the molecular mass of the purified enzyme was determined to be ~ 33 kDa, and the optimum pH and the values as pH 6.0 and 65-75 °C, respectively. Five different doses of nanoformulation (2, 4, 6, 8 and 10 mg/L) were applied to corn grains by the spraying method with three repetitions so that the insect can ingest the formulation through feeding. The effects of the applications on the death rate and mean time of death of Sitophilus zeamais were determined. According to these findings, it was concluded that the best practice was nanoformulation with 6 mg/L, considering both the mortality rate (100%) and the average death time (2.4 days). Chitinase from L. coryniformis is a promising candidate for corn lice control and management.

The Role of Fumonisins in the Biological Interaction between Fusarium verticillioides and Sitophilus zeamais.

The aim of the current study was to investigate the entomopathogenic capacity of the mold Fusarium verticillioides and the effect of its mycotoxins fumonisins, on the grain beetle Sitophilus zeamais. We evaluated the capacity of this fungus to infect live insects, the antifungal activity of constituents of the insect's epicuticle, and the effect of a fumonisin extract on the fitness of the insects. We found that F. verticillioides could not penetrate the cuticle of S. zeamais and that the fumonisin extract had no negative effects on the fitness of the insects. However, the progeny of the insects increased, and the fumonisin extract had repellent effects. This is the first report about the effects of fumonisins on the relationship between F. verticillioides and S. zeamais, which may provide useful information about interactions between pathogenic microorganisms and insects, especially on stored product pests.

Knockdown of NADPH-cytochrome P450 reductase and CYP6MS1 increases the susceptibility of Sitophilus zeamais to terpinen-4-ol.

Terpinen-4-ol showed highly insecticidal activity to stored-grain pest Sitophilus zeamais, and cytochrome P450s were strongly induced in response to terpinen-4-ol fumigation. Understanding of the function of P450 enzyme system in the susceptibility to terpinen-4-ol in S. zeamais will benefit the potential application of terpinen-4-ol in controlling stored-grain pests. In the present study, the synergist piperonyl butoxide increased the toxicity of terpinen-4-ol to S. zeamais, with a synergism ratio of 3.5-fold. Two isoforms of NADPH-cytochrome P450 reductase (SzCPR) were identified, with the difference at the N-terminal. SzCPR contained an N-terminal membrane anchor, FMN, FAD, and NADP binding domains. Expression levels of SzCPR were upregulated by tea tree oil (TTO) and its main constituent terpinen-4-ol under different concentrations and time periods. RNAi was generated for S. zeamais by feeding adults dsRNA and the knockdown of SzCPR increased the susceptibility of S. zeamais to terpinen-4-ol, with higher mortality of adults than control under terpinen-4-ol fumigation. Further RNAi analysis showed that P450 gene CYP6MS1 mediated the susceptibility of S. zeamais to terpinen-4-ol. These results revealed that cytochrome P450 enzyme system, especially CYP6MS1 participated in the susceptibility of S. zeamais to terpinen-4-ol. The findings provided a foundation to clarify the metabolic mechanisms of terpinen-4-ol in stored-grain pests.

Optimal Extraction of Ocimum basilicum Essential Oil by Association of Ultrasound and Hydrodistillation and Its Potential as a Biopesticide against a Major Stored Grains Pest.

The essential oil of basil (Ocimum basilicum) has significant biological activity against insect pests and can be extracted through various techniques. This work aimed to optimize and validate the extraction process of the essential oil of O. basilicum submitted to different drying temperatures of the leaves and extracted by the combination of a Clevenger method and ultrasound. The biological activity of the extracted oil under different conditions was evaluated for potential control of Sitophilus zeamais. The extraction method was optimized according to the sonication time by ultrasound (0, 8, 19, 31 and 38 min) and hydrodistillation (20, 30, 45, 60 and 70 min) and drying temperature (20, 30, 45, 60 and 70 °C). The bioactivity of the essential oil was assessed against adults of S. zeamais and the effects of each variable and its interactions on the mortality of the insects were evaluated. The best yield of essential oil was obtained with the longest sonication and hydrodistillation times and the lowest drying temperature of leaves. Higher toxicity of the essential oil against S. zeamais was obtained by the use of ultrasound for its extraction. The identification and the relative percentage of the compounds of the essential oil were performed with a gas chromatograph coupled to a mass selective detector. The performance of the method was assessed by studying selectivity, linearity, limits of detection (LOD) and quantification (LOQ), precision and accuracy. The LOD and LOQ values for linalool were 2.19 and 6.62 µg mL-1 and for estragole 2.001 and 6.063 µg mL-1, respectively. The coefficients of determination (R2) were >0.99. The average recoveries ranged between 71 and 106%, with coefficient of variation ≤6.4%.

Overexpression of the ZmDEF1 gene increases the resistance to weevil larvae in transgenic maize seeds.

Plant defensins are divided into 18 groups and are multifunctional proteins. The Zea mays defensin 1 (ZmDEF1) gene encodes the defensin 1 protein, which can inhibit alpha-amylase in the insect gut. In this study, the ZmDEF1 gene was transferred into two maize cultivars, LC1 and LVN99, to improve weevil resistance in maize. The recombinant ZmDEF1 protein was assessed for its ability to inhibit alpha-amylase in the gut of the larvae of the maize weevil (Sitophilus zeamais Motsch.). ZmDEF1 was cloned into a pBetaPhaso-dest vector, which harbours phaseolin, a seed-specific promoter, and the Agrobacterium tumefaciens strain C58 harbouring the pBetaPhaso-ZmDEF1 vector was used to transfer the ZmDEF1 gene into two maize cultivars using immature embryos. Transformed calluses were selected on selection media containing kanamycin. The stable integration of the ZmDEF1 transgene into the transgenic maize plant genome was confirmed using Southern blotting. The recombinant ZmDEF1 protein of approximately 10 kDa was expressed in three transgenic maize lines from the LC1 cultivar (C1, C3, and C5) and two transgenic maize lines from the LVN99 cultivar (L1 and L3). The ZmDEF1 transgenic efficiency based on the results of PCR, as well as Southern and Western blotting, was 1.32% and 0.82%, respectively, which depends on the genotypes of LC1 and LVN99. The recombinant ZmDEF1 protein inhibited the alpha-amylase activity of the maize weevil larvae, and its ability to inhibit alpha-amylase is 54.52-63.09% greater than the ZmDEF1 protein extracted from non-transgenic plants.

A time-saving method for sealing Purdue Improved Crop Storage (PICS) bags.

Purdue Improved Crop Storage (PICS) bags were designed to reduce grain storage losses on smallholder farms. The bag consists of three layers: two high-density polyethylene liners fitted inside a woven polypropylene bag. Recently, farmer groups, development relief programs, and government food security agencies have shown interest in PICS bags for large-scale use. PICS bags are conventionally closed by a twist-tie (TT) method, which involves twisting, folding, and tying the lip of each layer individually with a cord. This is not only time and labor intensive, but also may affect the integrity of the liners. We evaluated three new bag closure methods: i) inner liner rolled onto itself and middle liner fold-tied (IR), ii) both liners folded together and tied (FT), and iii) both liners folded and tied separately (FS), along with the conventional twist tie (TT) method. The time to close partially or fully filled 50 kg-capacity PICS bags filled with maize grain was assessed. Results showed that FT was the most time-saving method, reducing bag sealing time by >34% versus the usual TT method. The average internal oxygen levels reached <2% within a week in bags containing grain highly infested with Sitophilus zeamais, while it remained >5% levels for less-infested bags. In both cases, insect population growth was suppressed. Oxygen depletion rates among tying methods remained the same regardless of the closure method used. When large numbers of bags need to be closed, the time-saving FT method is a good alternative PICS sealing method over the conventional twist-tie approach.

Identification of Insecticidal Constituents from the Essential Oil from the Aerial Parts Stachys riederi var. japonica.

The essential oil of Stachys riederi var. japonica (Family: Lamiaceae) was extracted by hydrodistillation and determined by GC and GC-MS. A total of 40 components were identified, representing 96.01% of the total oil composition. The major compounds in the essential oil were acetanisole (15.43%), anisole (9.43%), 1,8-cineole (8.07%), geraniol (7.89%), eugenol (4.54%), caryophyllene oxide (4.47%), caryophyllene (4.21%) and linalool (4.07%). Five active constituents (acetanisole, anisole, 1,8-cineole, eugenol and geraniol) were identified by bioactivity-directed fractionation. The essential oil possessed fumigant toxicity against maize weevils (Sitophilus zeamais) and booklice (Liposcelis bostrychophila), with LC50 values of 15.0 mg/L and 0.7 mg/L, respectively. Eugenol and anisole exhibited stronger fumigant toxicity than the oil against booklice. 1,8-Cineole showed stronger toxicity, and anisole as well as eugenol exhibited the same level of fumigant toxicity as the essential oil against maize weevils. The essential oil also exhibited contact toxicity against S. zeamais adults and L. bostrychophila, with LC50 values of 21.8 µg/adult and 287.0 µg/cm², respectively. The results indicated that the essential oil of S. riederi var. japonica and its isolates show potential as fumigants, and for their contact toxicity against grain storage insects.

Cinnamaldehyde and related phenylpropanoids, natural repellents, and insecticides against Sitophilus zeamais (Motsch.). A chemical structure-bioactivity relationship.

BACKGROUND: The insecticidal and repellent effects on adult Sitophilus zeamais of 12 cinnamaldehyde-related compounds was evaluated by contact toxicity bioassays and a two-choice olfactometer. To determine non-toxicity in mammals, body weight, serum biochemical profiles, liver weight, physiological parameters, sperm motility, and histopathological data were obtained as complementary information in C57BL/6 mice treated with the best natural compound. RESULTS: Based on 24 h LC95 and LC50 values, α-methyl-cinnamaldehyde and cinnamaldehyde exhibited better insecticidal action than the other compounds. The best repellent effect was observed with α-bromo-cinnamaldehyde, which even repelled at the lowest concentration studied (0.28 µmol L-1 ). The evaluation of a quantitative structure-activity relationship found a linear relationship between the LC50 values for adult weevil toxicity and dipolo with Q values (giving the difference between orbital electronegativity carbon 1 and orbital electronegativity carbon 3 of the molecule) in cinnamaldehyde-related compounds. The polar surface and Log P descriptors also revealed a linear relationship with the S. zeamais repellent effect for cinnamaldehyde analogues. Cinnamaldehyde did not show toxicity in the parameters evaluated in mice. CONCLUSION: From the phenylpropanoid components studied, the natural compound that had the best insecticidal and repellent action against S. zeamais was cinnamaldehyde. It presented no mammalian toxicity. © 2018 Society of Chemical Industry.

Transcriptomic alterations in Sitophilus zeamais in response to allyl isothiocyanate fumigation.

To study the fumigation mechanisms of Allyl isothiocyanate (AITC) a promising biorational alternative to present fumigants (phosphine and methyl bromide), and provide theoretical basis for its further development in the control of stored grain pests, this research presents a transcriptome analysis of Sitophilus zeamais fumigated with AITC at the concentration of LC50 (5.69µg/mL) and control over 8h. 21,869,022 and 23,873,110 clean reads in insects fumigated with AITC and control were gained, respectively. The results of RNA-seq were confirmed by qRT-PCR determination of the expression levels of NADH dehydrogenase subunit 6 and Vacuolar ATP synthase subunit B in the insects fumigated with AITC at different concentrations. After enrichment analysis of differentially expressed genes, 117 over-expressed and 271 down-regulated transcripts were gained. Following GO enrichment, these transcripts were classified into 38 GO subgroups (at level 2), and the majority enriched GO terms were "Binding" "Cell process" and "metabolic". KEGG enrichment analysis showed that the majority enriched pathway were "Folding, sorting and degradation", "Transport and catabolism", "Energy metabolism", and "Carbohydrate metabolism". Connected with previous researches on mechanisms of isothiocyanates, cytoskeleton collapse and mitochondria dysfunction are proposed to be significant lethal mechanisms of AITC.

Molecular cloning and expression analysis of cytochrome c oxidase subunit II from Sitophilus zeamais.

Cytochrome c oxidase subunit II (COX II) containing a dual core CuA active site is one of the core subunits of mitochondrial Cytochrome c oxidase (Cco), which plays a significant role in the physiological process. In this report, the full-length cDNA of COXII gene was cloned from Sitophilus zeamais, which had an open reading frame (ORF) of 684 bp encoding 227 amino acids residues. The predicted COXII protein had a molecular mass of 26.2 kDa with pI value of 6.37. multiple sequence alignment and phylogenetic analysis indicated that Sitophilus zeamais COXII had high sequence identity with the COXII of other insect species. The gene was subcloned into the expression vector pET-32a, and induced by isopropyl ß-d-thiogalactopyranoside (IPTG) in E. coli Transetta (DE3) expression system. Finally the recombinant COXII with 6-His tag was purified using affinity chromatography with Ni(2+)-NTA agarose. Western Blotting (WB) showed the recombinant protein was about 44 kD, and the concentration of fusion protein was 50 µg/mL. UV-spectrophotometer and infrared spectrometer analysis showed that recombinant COXII could catalyze the oxidation of substrate Cytochrome C (Cyt c), and influenced by allyl isothiocyanate (AITC). By using molecular docking method, It was found that a sulfur atom of AITC structure could form a length of 2.9 Å hydrogen bond with Leu-31. These results suggested that tag-free COXII was functional and one of the action sites of AITC, which will be helpful to carry out a point mutation in binding sites for the future research.

Comparative studies on mitochondrial electron transport chain complexes of Sitophilus zeamais treated with allyl isothiocyanate and calcium phosphide.

With Sitophilus zeamais as the target organism, the present study for the first time attempted to elucidate the comparative effects between allyl isothiocyanate (AITC) and calcium phosphide (Ca3P2), exposure on mitochondrial electron transport chain (ETC.) complex I & IV and their downstream effects on enzymes relevant to reactive oxygen species (ROS). In vivo, both AITC and Ca3P2 inhibited complex I and IV with similar downstream effects. In contrast with Ca3P2, the inhibition of complex I caused by AITC was dependent on time and dose. In vitro, AITC inhibited complex IV more significantly than complex I. These results indicate that mitochondrial complex IV is the primary target of AITC, and that complex I is another potential target.

Resistance of Rice Varieties to the Stored-Product Insect, Sitophilus zeamais (Coleoptera: Curculionidae).

Four common Portuguese rice varieties--Thaibonnet, Gladio, Albatros, and Eurosis--were tested for their relative susceptibility to Sitophilus zeamais Motschulsky, a common pest of stored rice in Portugal and in tropical countries. Physical (moisture content, hardness, length, and width) and chemical (by attenuated total reflection-Fourier transform infrared spectroscopy) properties of rice kernels were measured. Insect bioassays measured median developmental time, Dobie's index of susceptibility, percentage of damaged grains and weight loss, and progeny developed. This was done for paddy, brown rice, and polished rice for each variety. There were small, but significant, differences in insect resistance among the varieties. However, it was different for paddy and polished rice. In paddy, these differences were correlated with hull damage, and Eurosis was the most susceptible variety. In polished rice, resistance was correlated with hardness, and Thaibonnet was the most susceptible variety. In general, paddy rice was more resistant to insect attack, followed by polished rice and then brown rice. Paddy kernels selected with undamaged hull were completely resistant to attack. Implications for IPM and breeding for resistant varieties are discussed.

Typical Monoterpenes as Insecticides and Repellents against Stored Grain Pests.

Five monoterpenes naturally occurring in essential oils were tested for their insecticidal and repellent activities against the bruchid beetle Callosobruchus maculatus and the maize weevil Sitophilus zeamais. The monoterpenes were highly efficient as inducers of mortality or repellency against both insect species. They were more efficient in their fumigant activity against C. maculatus than against S. zeamais, while this profile of action was inverted when considering the repellent activities. Eugenol was one the most effective fumigants against both insects and one the most effective repellent against C. maculatus, while citronellal and geranial were one the most effective repellents against S. zeamais. Functional and positional isomerism of the monoterpenes pairs appears to exert little or no influence on theirs effects, especially in case of repellency. The validation of the insecticidal/repellent efficacy of isolated monoterpenes may permit a more advantageous, rapid, economic and optimized approach to the identification of promising oils for commercial formulations when combined with ethnobotanical strategies.