Biochemical characterization of Aedes aegypti (Linnaeus) (Diptera: Culicidae) resistance to deltamethrin, fipronil, and imidacloprid.

Aedes aegypti is an important vector of dengue fever, dengue hemorrhagic fever and yellow fever, chikungunya, and Zika virus. The objective was to evaluate the resistance of A. aegypti exposed to insecticides with different action modes (deltamethrin, imidacloprid, and fipronil) under intense selection pressure for 10 generations in laboratory. Bioassays were conducted according to World Health Organization. Biochemical assay performed after selection with deltamethrin (Delta-SEL), fipronil (Fipro-SEL), and imidacloprid (Imida-SEL) from G1 to G10 was used for the assessment of detoxification enzymes (esterase (EST), acetylcholinesterase (AChE), glutathione S-transferases (GST), and acid and alkaline phosphatases (ACP and ALP)). The Fipro-SEL (G10) had high resistance (77-fold), whereas Delta-SEL and Imida-SEL populations presented very high resistance with 118 and 372-fold, respectively, in comparison with unselected (UNSEL). The levels of EST, AChE, GST, ACP, and ALP enzymes amplified on application from G1 to G10. The enzymes contributing in resistance development of insecticides were as follows: GST (20.7 µmol/min/mg of protein) in Delta-SEL (G10), while AChE 9.71 µmol/min/mg of protein in Imida-SEL (G10) and the peak ACP and ALP enzyme activities 13.32 and 12.93 µmol/min/mg of protein, respectively, in Fipro-SEL (G10). The results showed that detoxification enzymes trigger insecticide resistance in A. aegypti and their suppression may aid in the resistance breakage.

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.

Molecular cloning and characterization of gloverin from the diamondback moth, Plutella xylostella L. and its interaction with bacterial membrane.

Gloverin restricted to Lepidoptera is known to be a glycine-rich and heat stable antimicrobial protein. The current research reports a 650 bp full-length cDNA encoding gloverin from Plutella xylostella (PxGlo) by reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. PxGlo transcript was detected in both developmental stages and several tissues of 4th instar naïve larvae of P. xylostella with higher levels in the fat bodies. The mRNA levels of PxGlo increased appreciably in fat bodies after injection of Escherichia coli K12. The recombinant PxGlo expressed in S2 cells was purified by Anti-V5 M2 agarose beads which showed high activity against E. coli K12, while low activity against Bacillus thuringiensis, Staphylococcus aureus and E. coli D31. The analysis of transmission electron microscope and scan electron microscope showed PxGlo to cause significant morphological alteration in the E. coli K12 cell surface. Knockdown of PxGlo expression by RNAi increased the larval susceptibility towards the pathogenic bacteria i.e., Serratia marcescens and B. thuringiensis. Our results showed that PxGlo is an inducible antibacterial peptide which exhibits high activity mainly against E. coli K12, and PxGlo performs vital roles against the infection of pathogenic bacteria.

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.

Effect of temperature on development and reproduction of Proprioseiopsis asetus (Acari: Phytoseiidae) fed on asparagus thrips, Thrips tabaci.

Thrips tabaci Lindeman (Thysanoptera: Thripidae) is one of the most important pests of asparagus in China. In this study the effects of five constant temperatures (15, 20, 25, 30 and 35 °C) on the growth, survivorship and reproduction of Proprioseiopsis asetus (Chant) (Acari: Phytoseiidae) fed on T. tabaci was examined under laboratory conditions. Development time of immatures decreased with increasing temperature. The lower egg-to-adult developmental threshold (T 0) and thermal constant (K) of P. asetus were estimated at 15.2 °C and 75.8 degree days by means of a linear model. Fertilized females fed on T. tabaci produced offspring of both sexes, whereas the offspring sex ratio [♀/(♀ + â™‚)] of P. asetus at 20-35 °C was female-biased (0.68-0.78) and not significantly influenced by temperature. Survivorship during immature development was significantly influenced by temperature, and was especially low at 15 °C. Pre- and post-oviposition periods of fertilized females shortened with the increase in temperature. The longest oviposition period was 20.4 days, at 25 °C, whereas at 15 °C the mites did not reproduce. Maximum average life time fecundity and mean daily fecundity was recorded at 25 and 35 °C, respectively; the intrinsic rate of increase ranged from 0.05 (20 °C) to 0.17 (35 °C). The results indicate the capability of P. asetus to develop and reproduce at a broad range of temperatures, especially above 25 °C, which can be used for better management of T. tabaci in asparagus.

Molecular characterization of a short peptidoglycan recognition protein (PGRP-S) from Asian corn borer (Ostrinia furnacalis) and its role in triggering proPO activity.

Peptidoglycan recognition proteins (PGRPs) are non-specific immune molecules of insects, and vertebrates etc., but are not present in plants and nematodes. In the current experiment, a PGRP DNA sequence (2,910 bp containing four exons) was identified from genomic DNA library of Asian corn borer, Ostrinia furnacalis, and a full-length cDNA programming PGRP was cloned (designed as OfPGRP-S) with an open reading frame of 579 bp, having 192 amino acid. This inferred amino acid sequence showed maximum similarity to known lepidopteran PGRPs. Quantitative real-time PCR investigation disclosed the level of mRNA of OfPGRP-S to be constitutively expressed in the whole developmental stages and with higher expression in the mature larvae. Even more the OfPGRP-S was mainly expressed in immune capable organs i.e., fat body and midgut, and was strongly induced by injecting gram-positive bacteria i.e., Staphylococus aureus. Recombinant protein OfPGRP-S could bind to S. aureus and Bacillus thuringiensis which enhance proPO activation in the presence of these microbes. The results indicated that OfPGRP-S is an inducible protein acting as a receptor-type PGRP for enhancing the proPO activation on exposure to bacteria.

Virulence and transgenerational effects of Metarhizium anisopliae on Oxycarenus hyalinipennis.

BACKGROUND: Insect pests cause major yield losses to Gossypium hirsutum, often requiring the use of chemical insecticides. To avoid human health, environmental and resistance problems, entomopathogenic fungi (EPF) can be used to control insect pests. In our study, the pathogenicity of Metarhizium anisopliae to Oxycarenus hyalinipennis was determined by the immersion method. Furthermore, the sublethal and lethal effects of M. anisopliae on the biological parameters of O. hyalinipennis were investigated by age-stage, two-sex life table software. RESULTS: M. anisopliae infection was lethal to the fourth instar of O. hyalinipennis with LC50 values of 8.84 × 104 spores mL-1 . The sublethal and lethal concentrations of M. anisopliae not only affected the parental generation (F0 ) but also the demographic parameters of the offspring of the filial generation (F1 ). Transgenerational results of F1 infected with M. anisopliae showed decreased net reproductive rate (R0 ), intrinsic rate of increase (r) and mean generation time (T) compared to those of the control group. The larval developmental duration significantly decreased to 15.52 and 19.02 days in the LC50 and LC20 groups, respectively, compared to 21.08 days in the control group. There was a noteworthy decline in mean fecundity in the LC50 and LC20 groups, i.e., 16.0 and 20.96 eggs, compared to 33.26 eggs in the control group. Adult longevity was likewise considerably reduced in the LC50 and LC20 treated groups. CONCLUSION: The study showed that M. anisopliae can have an enduring impact on the biological parameters of O. hyalinipennis, which may enhance its use in eco-friendly management programs. © 2023 Society of Chemical Industry.

Botanical biopesticides have an influence on tomato quality through pest control and are cost-effective for farmers in developing countries.

Synthetic insecticides heavily applied to manage agricultural pests are highly hazardous to the environment and non-target organisms. Their overuse through repeated treatments in smallholder farming communities is frequent. Botanical biopesticides are ideal for sustainable pest management in agricultural environments by keeping synthetic insecticide use at a minimum. Here we evaluated a locally prepared neem seed extract (NSE) alongside emamectin benzoate against both lepidopteran pests Helicoverpa armigera (Hübner) and Spodoptera exigua (Hübner) on tomato Lycopersicon esculentum Mill under natural field conditions in Pakistan. We compared pest severity, fruit injury, quality, marketability, and cost:benefit ratio (CBR) between treatments. The concentration of azadirachtin A in the NSE was 26.5 ppm. NSE at 2% (20 mL/L) and the emamectin benzoate at the recommended field rate in Pakistan were sprayed weekly throughout the fruiting stage. The pest larvae were significantly more abundant on fruits than on flowers and leaves. Fruit injury and losses were significantly more important in untreated control compared to NSE and emamectin benzoate treatments. NSE efficacy varied with respect to the cultivars used and the seasons. Cultivar Eden harboured more pests than Adventa, and emamectin benzoate suppressed more pest individuals than NSE. Both the insecticidal treatments were comparable in terms of marketable yield productions as well as unmarketable, uninjured, and recovered fruit yields. NSE generated a higher CBR (1: 9.26) than emamectin benzoate (1: 3.23). NSE suppressed pests by acting as an antifeedant, similar to its synthetic counterpart. Smallholder growers can thus use NSE as a cost-effective solution in tomato pest management in Pakistan.

Spatio-Temporal Profiling of Metarhizium anisopliae-Responsive microRNAs Involved in Modulation of Plutella xylostella Immunity and Development.

Metarhizium anisopliae, a ubiquitous pathogenic fungus, regulates a wide array of the insect pest population. The fungus has been employed to control Plutella xylostella, an insecticide-resistant destructive lepidopteran pest, which causes substantial economic losses in crops worldwide. Integration of modern gene-silencing technologies in pest control strategies has become more crucial to counter pesticide-resistant insects. MicroRNAs (miRNA) play essential roles in the various biological process via post-transcriptional gene regulation. In the present study, RNA-seq analysis of control (CK36h, CK72h) and fungal-infected (T36h, T72h) midguts was performed to reveal underlying molecular mechanisms occurring in larval midgut at different time courses. We aimed at exploring M. anisopliae-responsive miRNAs and their target genes involved in development and immunity. After data filtration, a combined set of 170 miRNAs were identified from all libraries. Interestingly, miR-281, miR-263, miR-1, miR-6094 and miR-8 were listed among the most abundantly expressed conserved miRNAs. Furthermore, we experimentally studied the role of differentially expressed miR-11912-5p in regulating corresponding target trypsin-like serine proteinase (Px_TLSP). The luciferase assay (in vitro) revealed that miRNA-11912-5p significantly downregulated its target gene, suggesting it might play a crucial role in defense mechanism of P. xylostella against M.+ anisopliae infection. We used synthetic miRNA mimic/inhibitor (in vivo), to overexpress/silence miRNA, which showed harmful effects on larval duration, survival and adult fecundity. Additionally, fungal application in the presence of mimics revealed enhanced sensitivity of P. xylostella to infection. Our finding provides an insight into the relatively obscure molecular mechanisms involved in insect midgut during the fungal infection.