Shoaib Freed

Resistance selection, mechanism and stability of Spodoptera litura (Lepidoptera: Noctuidae) to methoxyfenozide

Methoxyfenozide belongs to a group of biorational insecticides known as insect growth regulators which is used in the control lepidopteran insect pests. Here we report a field collected population of Spodoptera litura selected with methoxyfenozide for thirteen consecutive generations resulted in the development of 83.24 and 2358.6-fold resistance to methoxyfenozide as compared to parental field population and susceptible laboratory population, respectively. The outcomes of synergism studies revealed methoxyfenozide resistance in S. litura to be monooxygenases (MO) mediated with high synergistic ratio (4.83) with piperonyl butoxide (PBO), while S,S,S-tributyl phosphorotrithioate (DEF) showed no synergism with methoxyfenozide (SR=1). This methoxyfenozide resistant strain showed a high cross resistance to deltamethrin (28.82), abamectin (12.87) and little to emamectin benzoate (2.36), however no cross resistance of methoxyfenozide and other tested insecticides was recorded. The results depicted the methoxyfenozide resistance in S. litura to be unstable with high reversion rate which decreased from 2358.6 to 163.9-fold (as compared to susceptible strain) when reared for five generations without any insecticidal exposure. The present research supports the significance of MO-mediated metabolism in resistance to methoxyfenozide, which demands some tactics to tackle this problem. The resistance against methoxyfenozide in S. litura can be overcome by switching off its use for few generations or insecticides rotation having different mode of action.

Cecropins from Plutella xylostella and Their Interaction with Metarhizium anisopliae.

Cecropins are the most potent induced peptides to resist invading microorganisms. In the present study, two full length cDNA encoding cecropin2 (Px-cec2) and cecropin3 (Px-cec3) were obtained from P. xylostella by integrated analysis of genome and transcriptome data. qRT-PCR analysis revealed the high levels of transcripts of Px-cecs (Px-cec1, Px-cec2 and Px-cec3) in epidermis, fat body and hemocytes after 24, 30 and 36 h induction of Metarhizium anisopliae, respectively. Silencing of Spätzle and Dorsal separately caused the low expression of cecropins in the fat body, epidermis and hemocytes, and made the P.xylostella larvae more susceptible to M. anisopliae. Antimicrobial assays demonstrated that the purified recombinant cecropins, i.e., Px-cec1, Px-cec2 and Px-cec3, exerted a broad spectrum of antimicrobial activity against fungi, as well as Gram-positive and Gram-negative bacteria. Especially, Px-cecs showed higher activity against M. anisopliae than another selected fungi isolates. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that cecropins exerted the vital morphological alterations to the spores of M. anisopliae. Based on our results, cecropins played an imperative role in resisting infection of M. anisopliae, which will provide the foundation of biological control of insect pests by using cecorpins as a target in the future.

Molecular cloning and characterization of a β-1,3-glucan recognition protein from Plutella xylostella (L.).

The β-glucan recognition proteins (βGRPs) play a significant role as important pattern recognition proteins (PRPs) for recognizing conserved surface determinants of pathogens and trigger complex signaling pathways in invertebrates. In the present study, a full-length cDNA 1793bp encoding 479 amino acids and βGRP1 was obtained from Plutella xylostella by reverse transcription PCR (RT-PCR) (designed as P×βGRP1) which showed significant similarities with other insects βGRPs. The transcription level was constitutively expressed and upregulated by microbial induction in all life stages of P. xylostella. Tissue distribution showed P×βGRP1 to be mainly expressed in fat body as detected by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. Subsequent to knock down the P×βGRP1 expression and the transcripts of Toll-like receptor, cecropin 1 and cecropin 2 decreased in P. xylostella. Meanwhile, the bacterial colonies increased and the expression of four AMP genes decreased on injection of anti-P×βGRP1 into Bombyx mori. The results demonstrated that P×βGRP1 can play a vital role in response to the expression of AMP genes in P. xylostella.

Infectivity of housefly, Musca domestica (Diptera: Muscidae) to different entomopathogenic fungi

The housefly Musca domestica is a worldwide insect pest that acts as a vector for many pathogenic diseases in both people and animals. The present study was conducted to evaluate the virulence of different local isolates of Beauveria bassiana, Metarhizium anisopliae and Isaria fumosorosea on M. domestica using two bioassay techniques: (1) adult immersion and (2) a bait method applied to both larvae and adults. The results showed evidence of a broad range of responses by both stages (larvae and adults) to the tested isolates of B. bassiana, M. anisopliae and I. fumosorosea. These responses were concentration-dependent, with mortality percentages ranging from 53.00% to 96.00%. Because it resulted in lower LC50 values and a shorter lethal time, B. bassiana (Bb-01) proved to be the most virulent isolate against both housefly larvae and adults. Sublethal doses of the tested isolates were also assessed to evaluate their effect on M. domestica fecundity and longevity. The fungal infections reduced housefly survival regardless of their sex and also decreased egg production in females.

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF TWO SERINE PROTEASES AND THEIR POTENTIAL INVOLVEMENT IN PROPHENOLOXIDASE ACTIVATION IN Plutella xylostella

The proteolytic activation of prophenoloxidase (proPO) is a humoral defense mechanism in insects and crustaceans. Phenoloxidase (PO) is produced as an inactive precursor namely, proPO and is activated via specific proteolytic cleavage by proPO-activating proteinase. The current research reports two novel serine proteinase genes (PxSP1-768 bp and PxSP2-816 bp) from Plutella xylostella, encoding 255 and 271 amino acid residues, respectively. Tissue distribution analyses by semiquantitative reverse transcription-PCR (RT-PCR) revealed the resultant genes to be primarily expressed in the hemocytes, while quantitative-RT-PCR (qRT-PCR) assay showed that transcription level of PxSP1 and PxSP2 increased significantly after injection of the fungal pathogen Beauveria bassiana. Purified recombinant fusion proteins of PxSP2 and PxSP1 were injected to New Zealand white rabbits and polyclonal antibodies were generated with the titers of 1:12,800. After silencing the expression of PxSP2 by RNAi, the PO activity decreased significantly. The results show that PxSP2 is involved in prophenoloxidase activation in P. xylostella.

Lethal and sub-lethal effects of spinosad on the life-history traits of army worm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), and its fitness cost of resistance

Spodoptera litura is one of the most destructive polyphagous insect pests, with more than 120 host‐plant species. In our present study, a field‐collected population of S. litura when selected with spinosad for 11 consecutive generations resulted in the development of 3921‐fold resistance to spinosad as compared to the susceptible strain. The spinosad‐resistant strain of S. litura had a relatively high fitness cost (0.17) as compared to the susceptible strain. Furthermore, the lethal and sub‐lethal effects of different concentrations of spinosad were checked on the susceptible strain at different levels; i.e., LC40, LC30, LC20 and LC10, which revealed that the impact of spinosad on the life‐history traits of S. litura increased with the increase in concentration of spinosad. A significant impact of spinosad was recorded on the larval duration, pre‐pupal weight, pupal duration, pupal weight, reproductive potential and adult emergence. The outcomes of the current research clearly indicate that fitness cost of spinosad and its sub‐lethal effects have a significant impact on population dynamics of S. litura, for which it can be incorporated in integrated pest management.

Patterns of genetic differentiation among populations of Amrasca biguttula biguttula (Shiraki) (Cicadellidae: Hemiptera)

Abstract Cotton leafhopper, Amrasca biguttula biguttula (Shiraki), a serious sucking insect pest of cotton and vegetables is present throughout South and Southeast Asia. Genetic differentiation within A. biguttula biguttula populations collected from 16 cotton growing areas of Punjab, Pakistan, was examined by sequencing the barcode region of the mitochondrial cytochrome oxidase subunit 1 (COI) gene. The dendrogram obtained by neighbour joining analysis of COI sequences confirmed the presence of single species of cotton leafhopper. The overall average pairwise divergence was 0.01. Very little variation was found among populations from cotton growing areas of Punjab, Pakistan and these were most similar to populations from North India. South Indian populations were grouped together and were generally more divergent. Extensive migration of this pest species among cotton-growing areas in the Indian subcontinent may hinder genetic diversification of cotton leafhopper. Four Pakistani samples of cotton leafhopper tested positive for Wolbachia infection but were not clearly differentiated from non-Wolbachia infected samples, suggesting that Wolbachia did not cause reproductive incompatibilities.

Mortality, Biological, and Biochemical Response of Musca domestica (Diptera: Muscidae) to Selected Insecticides1

Abstract  The concentration–mortality response of Musca domestica L. (Diptera: Muscidae) to nine insecticides, and the impacts of these insecticides on selected biological and biochemical parameters of the insect, were determined in laboratory assays. Adults displayed a concentration-dependent response for each insecticide. Median lethal concentration (LC50) values in baits were: acetamiprid (0.39 μg/ml), bifenthrin (0.22 μg/ml), chlorpyriphos (0.21 μg/ml), deltamethrin (0.41 μg/ml), emamectin benzoate (0.001 μg/ml), fipronil (0.002 μg/ml), imidacloprid (0.27 μg/ml), profenophos (0.63 μg/ml), and lufenuron (0.001 μg/ml). Based on 95% confidence intervals, fipronil proved to be the most lethal of the insecticides tested. LC10, LC30, and LC50 values of each of the insecticides were used to assess impacts on M. domestica longevity, fecundity, percentage eclosion, larval duration, percentage pupation, pupal weight, pupal duration, adult emergence, and sex ratio. In general, development parameters, with the exception of larval duration, were significantly (P > 0.05) altered in a concentration-dependent manner for each insecticide. Furthermore, enzymatic activity of total glutathione S-transferases, total esterases, acetylcholinesterase, and acid and alkaline phosphates was elevated at the LC10, LC30, and LC50 levels of the nine insecticides, which may contribute to development of resistance to these insecticides.

Activity of acetylcholinesterase and acid and alkaline phosphatases in different insecticide-treated Helicoverpa armigera (Hübner)

Helicoverpa armigera is a major devastating insect pest on a wide range of vegetables and cash crops. Insecticides are presently indispensable for its control in nearly all crops. H. armigera has acquired resistance against almost all insecticides because of the activity of detoxification enzymes used for the defensive mechanism. The current research was carried out to evaluate the activity of detoxification enzymes, i.e., acetylcholinesterase and alkaline and acid phosphatases in chlorpyrifos-, bifenthrin-, lufenuron-, lambda cyhalothrin-, and emamectin benzoate-treated larvae of H. armigera. The maximum AChE activity was recorded in emamectin benzoate-treated larval samples followed by chlorpyrifos, lufenuron, lambda cyhalothrin and bifenthrin, respectively, while the highest alkaline phosphatases’ activity was recorded in emamectin and the lowest in bifenthrin-treated H. armigera. As far as acid phosphatases’ activity is concerned, the highest activity was noted in lufenuron samples while the lowest in lambda cyhalothrin samples, respectively. Comparatively, activities of alkaline and acid phosphatases were higher than AChE. The elevated activities of detoxification enzymes can possibly lead to increase in resistance development against synthetic chemical insecticides.

Activity of glutathione S-transferase and esterase enzymes in Helicoverpa armigera (Hübner) after exposure to entomopathogenic fungi: Detoxification enzyme activity in H. armigera

Helicoverpa armigera, a polyphagous insect of crops and vegetables, is acquiring resistance against many commercial insecticides. The present study shows variations in the activity of two detoxification enzymes, namely esterase and glutathione S‐transferase (GST), in H. armigera after exposure to different isolates of entomopathogenic fungi. After treatment of larvae with the different isolates (Day 0), samples were collected on three days (Days 3, 5 and 7) for enzyme analysis. High GST activity was found in samples of hemolymph, intestine and fat bodies of H. armigera following treatment with Beauveria bassiana (isolate Bb‐08), Metarhizium anisopliae (isolates Ma‐11.1 and Ma‐4.1), and Isaria fumosorosea (isolates If‐02 and If‐2.3). High esterase activity was recorded in samples of the intestine and fat bodies on various days after treatment, whereas increased esterase activity in hemolymph was noted only in samples from Day 5 after treatment with M. anisopliae (Ma‐4.1). The detection of high GST and esterase activity demonstrates the possibility of the development of resistance against these microbial control agents in H. armigera.