Compatibility and synergistic interactions of fungi, Metarhizium anisopliae, and insecticide combinations against the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae).

Aphids are major pests affecting cereals, vegetables, fruit, forestry and horticultural produce. A multimodal approach may be an effective route to controlling this prolific pest. We assessed the individual and combined effect of eight insecticides and the entomopathogenic fungi, Metarhizium anisopliae (Metschin.) against the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), under laboratory conditions. Six of the insecticides tested were found to be highly compatible (flonicamid, imidacloprid, nitenpyram, dinotefuran, pyriproxyfen and spirotetramat), showing positive integration with the fungus and were selected for bioassays. The combination mixtures (1:1 ratio of M. anisopliae: insecticide) were significantly more toxic to A. gossypii than individual treatments. Maximum mortality (91.68%) of A. gossypii was recorded with combination of flonicamid and M. anisopliae (2.4 × 106 cfu/ml) 72 h after application. While minimum mortality (17.08%) was observed with the individual treatment of M. anisopliae (2.4 × 106 cfu/ml). The insecticides revealed toxicity consistent with their compatibility with M. anisopliae, ranking for efficacy exactly as they did for compatibility. In addition, the synergy factor (SF) and co-toxicity coefficient (CTC) values indicated synergistic interactions at different time intervals. The synergistic efficacy revealed the potential of fungus-insecticide integration against sucking insect pests.

Characterization of Ocimum basilicum synthesized silver nanoparticles and its relative toxicity to some insecticides against tobacco cutworm, Spodoptera litura Feb. (Lepidoptera; Noctuidae).

Tobacco cutworm, Spodoptera litura Feb. (Lepidoptera; Noctuidae) is a notorious cosmopolitan pest of numerous crops. Frequent evolution of insecticide resistance and polyphagous nature favors the survival of this voracious pest. Nanotechnology offers an alternative technique to overcome the limitations of existing pest management strategies. In the present study, the silver nanoparticles were synthesized from Ocimum basilicum leaf extract and UV-spectra at 464.17 nm confirmed their synthesis while crystalline nature of nanoparticles was evaluated by X-rays diffraction (XRD) pattern and scanning electron microscopy (SEM) revealed their octagonal to spherical shape. Insecticidal potential of O. basilicum synthesized silver nanoparticles (ObAgNPs) was evaluated in comparison with some synthetic insecticides owning different mode of actions, including Coragen®, Proclaim®, Tracer® and Talstar® against S. litura. Probit analysis of 2nd, 3rd, 4th and 5th instars of S. litura showed that lethal concentrations (LCs) of tested nanoparticles and pesticides were positively correlated with older instars. All values of LC50, LC90 and LC95 were statistically different from each other with maximum on the 4th instar and minimum on 2nd instar of S. litura. Tukey's HSD test revealed that ObAgNPs caused maximum mortality (21.67-96.67%) in 2nd instar larvae at the rate of 100-1500 mg/L followed by Coragen® (18.33-91.67%), Proclaim® (13.33-78.33%) and Talstar® (13.33-68.33%), while Tracer indicated the lowest larval mortality (11.67-66.67%) at the rate of 60-120 mg/L and a similar trend was assessed for all the successive instars of S. litura. Higher activity of carboxyl esterase and glutathione-s-transferases confirmed the development of insecticide resistance against the synthetic chemicals. Conclusively, ObAgNPs were found to be eco-friendly but the most effective biogenic compounds for the suppression of S. litura population as compared to the selected synthetic chemicals which may be hazardous for the environment as well as human health. This study will assist the pesticide industries to re-standardize the dose rates against Spodoptera litura.

Efficacy of biorational insecticides against Bemisia tabaci (Genn.) and their selectivity for its parasitoid Encarsia formosa Gahan on Bt cotton.

The toxicity of seven biorational insecticides [five insect growth regulators (Buprofezin, Fenoxycarb, Pyriproxyfen, Methoxyfenozide, and Tebufenozide) and two oil-extracts of neem and bitter gourd seeds] against Bemisia tabaci and their selectivity for its parasitoid, Encarsia formosa were evaluated in laboratory and field conditions for 2 years (2018-2019) in Pakistan. Toxicity results demonstrate that Pyriproxyfen, Buprofezin, and Fenoxycarb proved to be effective (80-91% mortality and 66.3-84.2% population-reduction) against B. tabaci followed by Methoxyfenozide, Tebufenozide (50-75% mortality and 47.8-52.4% population-reduction), and then oil-extracts of neem and bitter gourd (25-50% mortality and 36.5-39.8% population-reduction) in the laboratory [72 h post-application exposure interval (PAEI)] and field trails (168 h PAEI), respectively. All tested biorationals, except Methoxyfenozide [(slightly-harmful/Class-II), i.e., causing mortality of parasitoids between a range of 25-50%] and Tebufenozide [(moderately-harmful/Class-III), i.e., causing mortality of parasitoids between the ranges of 51-75%], proved harmless/Class-I biorationals at PAEI of 7-days in the field (parasitism-reduction < 25%) and 3-days in the lab (effect < 30%). In laboratory bioassays, exposure of parasitized-pseudopupae and adult-parasitoids to neem and bitter gourd oils demonstrated that these compounds proved harmless/Class-I biorationals (< 30% mortality). Alternatively, Pyriproxyfen, Buprofezin, Fenoxycarb, Methoxyfenozide, and Tebufenozide were slightly-harmful biorationals (30-79% mortality) against the respective stages of E. formosa. We conclude that most of the tested biorationals proved harmless or slightly harmful to E. formosa, except tebufenozide after PAEI of 7-days (168 h) in the field and, therefore, may be used strategically in Integrated Pest Management (IPM) of B. tabaci.

Comparative bio-efficacy of nuclear polyhedrosis virus (NPV) and Spinosad against American bollwormm, Helicoverpa armigera (Hubner)

ABSTRACT American bollworm (ABW), Helicoverpa armigera (Hubner), is considered as a major pest of cotton, Gossypium hirsutum, all over the globe. Due to its destructive feeding nature and continuous consumption of the same chemicals, it devolved resistant against many insecticides. Therefore, a combined application of bio- and synthetic-pesticide need to evaluate against this pest. The entomopathogenic viruses like nuclear polyhedrosis virus (NPV), a member of baculoviruses, can be the potential candidates for better control against ABW. The present study was conducted to assess the comparative efficacy of NPV and Spinosad 240SC (with the concentration of 250 mL · ha-1) against ABW in the controlled environment. The ABW was treated with different concentrations of NPV and Spinosad separately and in a combination of NPV with 0.1% Spinosad. The results revealed that highest concentrations showed highest mortality (95%) followed by 95%, 92%, 84%, 82% and 78% mortality at 1 × 109, 1 × 108, 1 × 107, 1 × 106 and 1 × 105 POBs, respectively. Spinosad when mixed in diet give 100% mortality at 0.8% followed by 50.87%, 42.10%, 29.82%, 26.31% and 22.80% mortality at 0.4%, 0.2%, 0.1%, 0.5% and 0.025% respectively. The results of this study revealed that microbial control of ABW through NPV is an effective tool. The repeated use of synthetic pesticides caused the resurgence of many insect pests, and this study results would provide useful insight to build a framework for future investigations for the management of many major insect pests.

De novo sequencing and transcriptome analysis of venom glands of endoparasitoid Aenasius arizonensis (Girault) (=Aenasius bambawalei Hayat) (Hymenoptera, Encyrtidae).

Aenasius bambawalei Hayat (Encyrtidae: Hymenoptera) has been synonymized with Aenasius arizonensis (Girault) is a small, newly discovered endoparasitoid of the cotton mealybug Phenacoccuss solenopsis Tinsley (Pseudococcidae: Hemiptera), which completes its life cycle inside the body of its host and it is a potential insect control tool. Despite the acquired knowledge regarding host-parasitoid interaction, little information is available on the factors of parasitoid origin able to modulate mealybug physiology. The components of A. arizonensis venom have not been well studied but venom from other parasitoids and wasps contain biologically active proteins that have potential applications in pest management or may be of medicinal importance. To provide an insight into the transcripts expressed in the venom gland of A. arizonensis, a transcriptomic database was developed utilizing high throughput RNA sequencing approaches to analyze the genes expressed in venom glands of this endoparasitic wasp. The resulting A. arizonensis RNA sequences were assembled de-novo with contigs then blasted against the NCBI non-redundant sequence database. Contigs which matched database sequences were mostly homologous to genes from hymenopteran parasitoids such as Nasonia vitripennis, Copidosoma floridanum, Fopius arsenus and Pteromalas puparium. Further analysis of the A. arizonensis database was then performed which focused on selected genes encoding proteins potentially involved in host developmental arrest, disrupting the host immune system, host paralysis, and transcripts that support these functions. Sequenced mRNAS predicted to encode full length ORFs of Calreticulin, Serine Protease Precursor and Arginine kinase proteins were identified and the tissue specific expression of these putative venom genes was analyzed by RT-PCR. In addition, results also demonstrate that de novo transcriptome assembly allows useful venom gene expression analysis in a species lacking a genome sequence database and may provide useful information for devising control tools for insect pests and other applications.

De novo sequencing and transcriptome analysis of female venom glands of ectoparasitoid Bracon hebetor (Say.) (Hymenoptera: Braconidae).

Venom is a key-factor in the regulation of host physiology by parasitic Hymenoptera and a potentially rich source of novel bioactive substances for biotechnological applications. The limited study of venom from the ectoparasitoid Bracon hebetor, a tiny wasp that attacks larval pest insects of field and stored products and is thus a potential insect control agent, has not described the full complement and composition of these biomolecules. To have a comprehensive picture of genes expressed in the venom glands of B. hebetor, a venom gland transcriptome was assembled by using next generation sequencing technologies followed by de novo assemblies of the 10.81 M sequence reads yielded 22,425 contigs, of which 10,581 had significant BLASTx hits to know genes. The majority of hits were to Diachasma alloeum, an ectoparasitoid from same taxonomic family, as well as other wasps. Gene ontology grouped the sequences into molecular functions in which catalytic activity with 42.2% was maximum, cellular components in which cells with 33.8% and biological processes among which metabolic process with 30% had the most representatives. In this study, we highlight the most abundant sequences, and those that are likely to be functional components of the venom for parasitization. Full length ORFs of Calreticulin, Venom Acid Phosphatase Acph-1 like protein and arginine kinase proteins were isolated and their tissue specific expression was studied by RT-PCR. Our report is the first to characterize components of the B. hebetor venom glands that may be useful for developing control tools for insect pests and other applications.

Multiple Resistances Against Formulated Organophosphates, Pyrethroids, and Newer-Chemistry Insecticides in Populations of Helicoverpa armigera (Lepidoptera: Noctuidae) from Pakistan.

Field populations of Helicoverpa armigera Hübner from 15 localities across the Punjab, Pakistan, were assessed by the leaf dip method for resistance against formulated organophosphates, pyrethroids, and newer insecticide groups. Resistance levels in H. armigera have been incrementally increasing for organophosphate and pyrethroid insecticides after decades of use in Pakistan. Resistance ratios (RRs) documented for organophosphates were 24- to 116-fold for profenofos and 22- to 87-fold for chlorpyrifos. For pyrethroids, RRs were 3- to 69-fold for cypermethrin and 3- to 27-fold for deltamethrin. Resistance levels against newer chemistries were 2- to 24-fold for chlorfenapyr, 1- to 22-fold for spinosad, 1- to 20-fold for indoxacarb, 1- to 18-fold for abamectin, and 1- to 16-fold for emamectin benzoate. Resistant populations of H. armigera were mainly in the southern part of the Punjab, Pakistan. The most resistant populations were collected from Pakpattan, Multan, and Muzzafargarh. Of the nine insecticides tested, LC50 and LC90 values were lower for newer insecticide groups; resistance levels were moderate to very high against organophosphates, very low to high against pyrethroids, and very low to low against the newer-chemistry insecticides. These findings suggest that the newer-chemistry insecticides with different modes of action could be included in insecticide rotations or replace the older insecticides. Supplementing the use of synthetic insecticides with safer alternatives could help to successfully lower the farmer's reliance on insecticides and the incidence of resistance due to repeated use of insecticides against major insect pests.

Phenology of Asian citrus psyllid (Hemiptera: Liviidae) and associated parasitoids on two species of Citrus, kinnow mandarin and sweet orange, in Punjab Pakistan.

The population phenology of Asian citrus psyllid, Diaphorina citri Kuwayama, was monitored weekly for 110 wk on two species of Citrus, kinnow mandarin and sweet orange, at two different research sites in Faisalabad, Punjab Pakistan. Citrus flush growth patterns were monitored and natural enemy surveys were conducted weekly. Flush patterns were similar for kinnow and sweet orange. However, flush on sweet orange was consistently more heavily infested with Asian citrus psyllid than kinnow flush; densities of Asian citrus psyllid eggs, nymphs, and adults were higher on sweet orange when compared with kinnow. When measured in terms of mean cumulative insect or Asian citrus psyllid days, eggs, nymphs, and adults were significantly higher on sweet orange than kinnow. Two parasitoids were recorded attacking Asian citrus psyllid nymphs, Tamarixia radiata (Waterston) and Diaphorencyrtus aligarhensis (Shafee, Alam and Agarwal). The dominant parasitoid species attacking Asian citrus psyllid nymphs on kinnow and sweet orange was T. radiata, with parasitism averaging 26%. D. aligarhensis parasitism averaged 17%. Generalist predators such as coccinellids and chrysopids were collected infrequently and were likely not important natural enemies at these study sites. Immature spiders, in particular, salticids and yellow sac spiders, were common and may be important predators of all Asian citrus psyllid life stages. Low year round Asian citrus psyllid densities on kinnow and possibly high summer temperatures, may, in part, contribute to the success of this cultivar in Punjab where Candidatus Liberibacter asiaticus, the putative causative agent of huanglongbing, a debilitating citrus disease, is widespread and vectored by Asian citrus psyllid.