Muhammad Rizwan-ul-Haq

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.

Cloning and characterization of three chemosensory proteins from Spodoptera exigua and effects of gene silencing on female survival and reproduction.

Insect chemosensory proteins (CSPs) are supposed to transport hydrophobic chemicals to receptors on sensory neurons. However, CSPs are broadly expressed in various insect tissues, suggesting their involvement in the physiological processes beyond chemoreception. So, the exact physiological roles of CSPs in insects still need to be unraveled. In this study, three full-length of CSP genes from Spodoptera exigua have been cloned and characterized. The deduced amino acid sequences of SexiCSP1, SexiCSP2 and SexiCSP3 revealed open reading frames of 128, 128 and 126 amino acids, respectively, with four conserved cysteine residues. The expression patterns of the three SexiCSPs were further investigated by real-time PCR. Three SexiCSPs were expressed in antennae, heads, legs, wings, thoraxes, abdomens, testes and ovaries, with the highest expression level in female and male antennae. Furthermore, all three SexiCSPs mRNA were distributed extensively in the tested development stages with the highest expression level in pupae. RNAi-based gene silencing study resulted in a dramatic reduction of corresponding mRNA in female S. exigua after injection with dsRNA of all three SexiCSPs. Consequentially, 42.5% of mortalities, 68.3% (compare to DEPC water injected control) and 71.4% (compare to uninjected control) oviposition inhibition, and significantly effected egg hatching were observed in the female S. exigua injected with dsSexiCSP3 as compared to control treatments. On the other hand, dsSexiCSP1 and dsSexiCSP2 injected female adults did not show effects on survival and reproduction. Our study confirms the utility of RNAi approach to functional characterization of CSP genes in S. exigua and provides a starting point for further studies on female survival and reproduction in this insect. It also reveals the potential pest controlling method, as insect behavior regulation agent that disrupts the expression of chemosensory proteins.

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.

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.

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. Copyright

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.

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.