Agricultural Insect Pests Research Articles

Indigenous entomopathogenic nematode as biocontrol agents for insect pest management in hilly regions

The present investigation mainly emphasized on the development and use of entomopathogenic nematodes (EPNs) as a bio-insecticide. The success in controlling insect pests in the soil environments increased the production and use of the adapted indigenous EPNs species for insect management in the fields. EPNs as biocontrol agents were capable for high virulence, easy for application, safe for non-target animals and eco-friendly in nature. These nematodes have ubiquitous nature. These occur in low population in their natural habitat which was mass multiplied in the laboratory. In the present investigation, 5 concentrations (30IJs, 60IJs, 90IJs, 120IJs and 150IJs) of Heterorhabditis bacteriophora strain S15 were applied against the 3rd and 4th instar larvae of four major agricultural insect pests, namely Helicoverpa armigera, Spodoptera litura, Agrotis segetum and Mythimna separata under laboratory conditions at different time exposure (24, 48, 72 and 96 hr). It was observed that the 3rd and 4th larval instars of all 4 insects (H. armigera, S. litura, A. segetum and M. separata) were highly susceptible for the pathogenesis caused by H. bacteriophora Sirmaur isolates. Amongst all insects, both the larval instars of M. separata are highly susceptible for EPNs infection with highest 96% and 98% mortality in highest dose @150IJs. In 3rd instar larvae of other insects such as H. armigera, S. litura and A. segetum larval mortality ranges from 84%, 92% and 94% respectively. Among 4th instar larvae of H. armigera, S. litura and A. segetum the pathogenicity varies from 88%, 94% and 96% respectively. The recorded median lethal concentration (LC50) in 3rd instar larvae of H. armigera, S. litura, A. segetum and M. separata varies from 36.15, 30.05, 30.97 and 23.8. Similarly in 4th instar larvae of H. armigera, S. litura, A. segetum and M. separata, LC50 ranged from 31.41, 28.64, 26.92 and 20.64 respectively. Statistically significant variations were observed in the data recorded on the mortality, in all the treatments. EPNs are the best weapon to overcome insect resistance problems and must be employed to manage insect population.

Unraveling the Basis of Neonicotinoid Resistance in Whitefly Species Complex: Role of Endosymbiotic Bacteria and Insecticide Resistance Genes.

Bemisia tabaci (whitefly) is one of the most detrimental agricultural insect pests and vectors of many plant viruses distributed worldwide. Knowledge of the distribution patterns and insecticide resistance of this cryptic species is crucial for its management. In this study, genetic variation of mitochondrial cytochrome oxidase subunit 1 (MtCoI) gene of B. tabaci was analyzed followed by a study of the infection profile of various endosymbionts in 26 whitefly populations collected from West Bengal, India. Phylogenetic analysis revealed Asia I as the major cryptic species (65.38%), followed by Asia II 5, China 3, and Asia II 7, which were diversified into 20 different haplotypes. In addition to the primary endosymbiont (C. poriera), each of the four whitefly species showed a variable population of three secondary endosymbionts, majorly Arsenophonus with the highest infection rate (73.07%), followed by Wolbachia and Rickettsia. Further phylogenetic analyses revealed the presence of two subgroups of Arsenophonus, viz., A1 and A2, and one each in Wolbachia (W1) and Rickettsia (R3). Resistance to thiamethoxam, imidacloprid, and acetamiprid insecticides was analyzed for a clear picture of pesticide resistance status. The highest susceptibility was noted toward thiamethoxam (LC50 = 5.36 mg/L), followed by imidacloprid and acetamiprid. The whitefly population from Purulia and Hooghly districts bearing Asia II 7 and Asia II 5 cryptic species, respectively, shows maximum resistance. The differences in mean relative titer of four symbiotic bacteria among field populations varied considerably; however, a significant positive linear correlation was observed between the resistance level and relative titer of Arsenophonus and Wolbachia in the case of imidacloprid and thiamethoxam, while only Wolbachia was found in case of acetamiprid. Expression analysis demonstrated differential upregulation of insecticide resistance genes with Purulia and Hooghly populations showing maximally upregulated P450 genes. Moreover, thiamethoxam and imidacloprid resistance ratio (RR) showed a significant correlation with CYP6CM1, CYP6DZ7, and CYP4C64 genes, while acetamiprid RR correlated with CYP6CX1, CYP6DW2, CYP6DZ7, and CYP4C64 genes. Taken together, these findings suggested that P450 mono-oxygenase and symbiotic bacteria together affected whitefly resistance to neonicotinoids. Hence, a symbiont-oriented management programme could be a better alternative to control or delay resistance development in whitefly and can be used for pesticide clean-up in an agricultural field.

Identification and Detection of CYP4G68 Overexpression Associated With Cyantraniliprole Resistance in Bemisia tabaci From China

Bemisiatabaci, the tobacco whitefly, is one of the most notorious agricultural sucking insect pests that severely damage a series of crops worldwide. Throughout China, B. tabaci threatens agricultural production with increasing cases of resistance to commonly used insecticides, prompting the widespread use of cyantraniliprole as an alternative to control hemipteran pests. Here, we found overexpression of the CYP4G68 gene conferring cyantraniliprole resistance using quantitative real-time PCR (qPCR) and RNA interference (RNAi) in one lab-selected resistant strain CYAN-R (to about 80-fold higher than control). Furthermore, we measured levels of resistance to cyantraniliprole in whiteflies with 18 field-sampled populations across China and then confirmed that, among them, 14 field-sampled populations showed low-to-high resistance to cyantraniliprole compared with the susceptible strain. We measured CYP4G68 expression in the 14 field populations, and the results of qPCR and RNAi indicated that in two of these populations, Haikou and Wuhan, significant overexpression of CYP4G68 contributed to the development of field-evolved resistance to cyantraniliprole. These results indicate the need to facilitate strategies of management to delay the evolution of resistance to cyantraniliprole and control of whiteflies more sustainably, and to prevent overuse of insecticides in the environment through rational application practices.

Integrins in the Immunity of Insects: A Review.

Integrins are a large group of cell-surface proteins that are classified as transmembrane proteins. Integrins are classified into different types based on sequence variations, leading to structural and functional diversity. They are broadly distributed in animals and have a wide range of biological functions such as cell-to-cell communication, intracellular cytoskeleton organization, cellular signaling, immune responses, etc. Integrins are among the most abundant cell surface proteins in insects, exhibiting their indispensability in insect physiology. Because of their critical biological involvement in physiological processes, they appear to be a novel target for designing effective pest control strategies. In the current literature review, we first discuss the discovery and expression responses of integrins against various types of pathogens. Secondly, we examine the specific biological roles of integrins in controlling microbial pathogens, such as phagocytosis, encapsulation, nodulation, immune signaling, and so on. Finally, we describe the possible uses of integrins to control agricultural insect pests.

A high-quality de novo genome assembly based on nanopore sequencing of a wild-caught coconut rhinoceros beetle (Oryctes rhinoceros)

BackgroundAn optimal starting point for relating genome function to organismal biology is a high-quality nuclear genome assembly, and long-read sequencing is revolutionizing the production of this genomic resource in insects. Despite this, nuclear genome assemblies have been under-represented for agricultural insect pests, particularly from the order Coleoptera. Here we present a de novo genome assembly and structural annotation for the coconut rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae), based on Oxford Nanopore Technologies (ONT) long-read data generated from a wild-caught female, as well as the assembly process that also led to the recovery of the complete circular genome assemblies of the beetle’s mitochondrial genome and that of the biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). As an invasive pest of palm trees, O. rhinoceros is undergoing an expansion in its range across the Pacific Islands, requiring new approaches to management that may include strategies facilitated by genome assembly and annotation.ResultsHigh-quality DNA isolated from an adult female was used to create four ONT libraries that were sequenced using four MinION flow cells, producing a total of 27.2 Gb of high-quality long-read sequences. We employed an iterative assembly process and polishing with one lane of high-accuracy Illumina reads, obtaining a final size of the assembly of 377.36 Mb that had high contiguity (fragment N50 length = 12 Mb) and accuracy, as evidenced by the exceptionally high completeness of the benchmarked set of conserved single-copy orthologous genes (BUSCO completeness = 99.1%). These quality metrics place our assembly ahead of the published Coleopteran genomes, including that of an insect model, the red flour beetle (Tribolium castaneum). The structural annotation of the nuclear genome assembly contained a highly-accurate set of 16,371 protein-coding genes, with only 2.8% missing BUSCOs, and the expected number of non-coding RNAs. The number and structure of paralogous genes in a gene family like Sigma GST is lower than in another scarab beetle (Onthophagus taurus), but higher than in the red flour beetle (Tribolium castaneum), which suggests expansion of this GST class in Scarabaeidae. The quality of our gene models was also confirmed with the correct placement of O. rhinoceros among other members of the rhinoceros beetles (subfamily Dynastinae) in a phylogeny based on the sequences of 95 protein-coding genes in 373 beetle species from all major lineages of Coleoptera. Finally, we provide a list of 30 candidate dsRNA targets whose orthologs have been experimentally validated as highly effective targets for RNAi-based control of several beetles.ConclusionsThe genomic resources produced in this study form a foundation for further functional genetic research and management programs that may inform the control and surveillance of O. rhinoceros populations, and we demonstrate the efficacy of de novo genome assembly using long-read ONT data from a single field-caught insect.

Diet analysis of bats killed at wind turbines suggests large‐scale losses of trophic interactions

AbstractAgricultural practice has led to landscape simplification and biodiversity decline, yet recently, energy‐producing infrastructures, such as wind turbines, have been added to these simplified agroecosystems, turning them into multi‐functional energy‐agroecosystems. Here, we studied the trophic interactions of bats killed at wind turbines using a DNA metabarcoding approach to shed light on how turbine‐related bat fatalities may possibly affect local habitats. Specifically, we identified insect DNA in the stomachs of common noctule bats (Nyctalus noctula) killed by wind turbines in Germany to infer in which habitats these bats hunted. Common noctule bats consumed a wide variety of insects from different habitats, ranging from aquatic to terrestrial ecosystems (e.g., wetlands, farmland, forests, and grasslands). Agricultural and silvicultural pest insects made up about 20% of insect species consumed by the studied bats. Our study suggests that the potential damage of wind energy production goes beyond the loss of bats and the decline of bat populations. Bat fatalities at wind turbines may lead to the loss of trophic interactions and ecosystem services provided by bats, which may add to the functional simplification and impaired crop production, respectively, in multi‐functional ecosystems.

Insecticidal effect of entomopathogenic nematodes and the cell-free supernatants from their symbiotic bacteria against different larval instars of Agrotis segetum (Denis & Schiffermüller) (Lepidoptera: Noctuidae)

BackgroundEntomopathogenic nematodes (EPNs) (Steinernematidae and Heterorhabditidae) and their symbiotic bacteria are pathogenic for a wide range of insect pests and have been used successfully as a biological control agent. Although EPNs are well studied against many agricultural insect pests, the efficacy of their symbiotic bacteria still remains unclear for many insect pests of agricultural importance. In the present study, the virulence of native EPN isolates and their cell-free supernatants of symbiotic bacteria were tested against the 3rd and 4th larval instars of Agrotis segetum (Denis & Schiffermüller) (Lepidoptera: Noctuidae) under laboratory conditions (25 ± 1 °C and R.H. 60%).ResultsThe 4th instar larvae were more susceptible to infective juveniles (IJs) and mortalities over (95%) were achieved by all tested EPN isolates at the concentration of 100 IJs/cm2 after 72 hrs of exposure. The cell-free supernatants were more effective against the 3rd instar larvae and the highest mortalities were recorded as 42 and 60% in the contact and leaf disc bioassays, respectively.ConclusionThe results indicated that the cell-free supernatants can be an ideal application for young larval stages of A. segetum. However, further studies are required to test the effectiveness of both EPNs and the cell-free supernatants of their symbiotic bacteria in field conditions.

Selection and Evaluation of Reference Genes for miRNA Expression Analysis in Bemisia tabaci Under Insecticide Tolerance.

A growing number of studies have focused on the microRNA (miRNA) expression in Bemisia tabaci, one devastating agricultural insect pest of the tropical and subtropical areas for which the primary means of control are insecticides. In studying the genetic underpinnings of insecticide resistance, the choice of stable reference genes for normalizing data plays a key role to acquire unbiased expression profile results from quantitative real-time PCR (qPCR) analysis. Expression profiles of 11 selected reference genes were determined systematically in B. tabaci exposure to 11 insecticides. Furthermore, we assessed the stability of all the selected candidates in relation to other variables including sex, tissue type, and developmental stage. Candidate reference gene validation was conducted by analyzing the let-7-5p expression under various experimental treatments. Five programs BestKeeper, NormFinder, geNorm, △Ct, and RefFinder were applied to verify the accuracy of the selected candidates. Our results displayed that the best choices of the selected candidates for pymetrozine, sulfoxaflor, flonicamid, cyantraniliprole, afidopyropen, and deltamethrin treatment were miR-1-3p and miR-100-5p, U6 and miR-100-5p were best for chlorpyrifos and imidacloprid treatments, and U6 and miR-1-3p were best for flupyradifurone and β-cypermethrin treatments. The reference genes miR-624, miR-252, and miR-275 worked best in adult tissues, miR-100-5p and miR-1-3p worked best in either sex, and miR-624 and miR-11 were best to use across developmental stages. Not even one reference gene was found to be suitable for all experimental conditions. Our results contributed to the growing body of the literature on qPCR reference gene selection under various experimental conditions and facilitate further investigation on gene expression changes in B. tabaci, resulting from pesticide exposure.

Bacillus thuringiensis and Micromonospora echinospora ongrowth of Phaseolus vulgaris

The well-known bacteria genus called as Bacillus thuringiensis (Bt) is recognized for being used in the biological control of insect pests in agriculture. However, there is little information on the effect of Bt as a plant growth promoter and in interaction with Micromonospora echinopora, which, being an endophyte, could improve the response of Phaseolus vulgaris with NH4NO3 atreduced at 30%of common recommended without risk of affecting plant health or yield.The objective of this work was to analyze the response of P. vulgaris to Bt and M. echinospora with NH4NO3 at 30% of recommended dose. The experiment was conducted under a randomized block design, using the response variables: germination percentage, phenology and biomass. The experimental data were analyzed by ANOVA/Tukey HSD (P<0.05). In the results, it was found that B.thuringiensis induced in P. vulgaris a yield of 3.2Ton/Ha, while with M. echinospora 3.6Ton/Ha was reached, with NF reduced at 30%, while it was shown that B.thuringiensis improved the yield with M. echinospora with 4.0Ton /Ha, values with statistical difference compared to the 2.8 Ton/Ha of P. vulgaris without B.thuringiensis or M. echinospora used as relative control with 100% of the NF.Concluded that B. thuringiensis is not only useful in biological control but that it can be an excellent growth promoter that can be improved with M. echinospora, another option for the sustainable production of P. vulgaris.

Draft genome sequence of Bacillus thuringiensis strain S2195, an isolate toxic for lepidoptera / Projeto de sequência do genoma de Bacillus thuringiensis strain S2195, um tóxico isolado para lepidoptera

Bacillus thuringiensis is a Gram-positive bacterium which produces insecticidal proteins effective to control several medical and agriculture insect pests. These proteins belong to the Cry, Cyt, Vip and Sip families. This work describes the draft genome sequence of B. thuringiensis S2193, which contains genes encoding the parasporal crystal cry1Ad, cry1Ca, cry1Da, cry1Fa, cry1Ia, cry2A, cry9Ea and vip3Ca. Genes coding for plant growth promotion pathway were identified. And the operon which encodes the bacteriocin Thuricin 17 were annoted. We found the gene which encodes the toxin enhancin.

Management of Nuisance Macromoths in Expressways through Academic-Industrial Collaboration: Light Trap Designed on the Basis of Moths' Preferences for Light Attributes.

Aggregation of flying insects such as gypsy moths at commercial light sources in summer not only has an aesthetically negative impact on public facilities but also permits the establishment of new insect populations there from the next year. Although energy-efficient light traps equipped with light-emitting diodes (LEDs) have recently been used for controlling pest insects in agriculture, there are very few maintenance-free light traps that are available on the market. Based on the results of field surveys, we fabricated a prototype light trap in which the preferences of insects for light irradiation angle and wavelength are implemented. Field experiments revealed that flying moths were attracted more to light with a narrow irradiation angle than to light with a wide irradiation angle. Moreover, there was a tendency for fewer moths to be collected when fluorescent paint was applied to the surface of the flight-interception board, indicating that a high contrast made by illumination and the background is preferred by flying moths. Taken together with our previous results, we found that the moth catch was influenced more by modification of the light design than by change in visible light wavelengths. A semi-portable light trap, named the "Kurihara trap" after the primary contributor to its development, is made of light-weight plastic and is driven by solar power. This light trap is omnidirectional and maintenance-free and is therefore suitable for deployment in the backyards of rest areas as well as at houses for long-term macromoth sampling.

Genetic diversity and demographic history of the Old World Bollworm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), in Ethiopia inferred from mitochondrial gene sequences

The Old World bollworm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), is a globally distributed agricultural and horticultural insect pest. Despite the economic importance of this insect in Ethiopia, its genetic diversity and demographic history are poorly understood. We examined the nucleotide variation of the mitochondrial cytochrome c oxidase subunit I (COI) gene fragment of 74 H. armigera individuals from six collection sites in Ethiopia. We recorded 15 COI haplotypes in H. armigera, ten globally shared and five exclusive to Ethiopia (HaET15, HaET14, HaET10, HaET7, and HaET4). Haplotype HaET1 was the most widely geographically distributed and frequent (71.62%). Analysis of molecular variance (AMOVA) revealed a high and significant level of variation within H. armigera populations (θST = −0.0135). Negative values of the neutrality test and nonsignificant index of mismatch distribution supported the demographic expansion of H. armigera populations in Ethiopia; furthermore, this was also supported by the nonsignificant values of the sum of squared deviations (SSD) and raggedness index (r). The high genetic variation and population expansion of H. armigera have immense implications for devising locally adapted management strategies in area‐wide integrated pest management IPM programs. However, a comprehensive study of H. armigera genetic diversity and population structure using various molecular markers is needed for future confirmation.

Reactive Oxygen Species Initiate Defence Responses of Potato Photosystem II to Sap-Sucking Insect Feeding.

Simple SummaryPotato is one of the most universally cultivated horticultural crops and is vulnerable to a range of herbivorous insects. One of them is the brown marmorated stink bug, an invasive polyphagous sap-sucking agricultural insect pest that penetrates the phloem to sieve elements and removes sap via a specialized mouthpart, the stylet. By using the chlorophyll fluorescence imaging methodology, we examined potato photosystem II (PSII) photochemistry responses in the area of feeding on the whole leaf area. Highly increased reactive oxygen species (ROS) generation was observed as rapidly as 3 min after feeding to initiate defence responses and can be considered the primary plant defence response mechanism against herbivores. Our experimental results confirmed that chlorophyll fluorescence imaging methodology can detect spatial heterogeneity of PSII efficiency at the whole leaf surface and is a promising tool for investigating plant response mechanisms of sap-sucking insect herbivores. We suggest that PSII responses to insect feeding underlie ROS-dependent signalling. We conclude that the potato PSII response mechanism to sap-sucking insect herbivores is described by the induction of the defence response to reduce herbivory damage, instead of induction of tolerance, through a compensatory photosynthetic response mechanism that is observed after chewing insect feeding.Potato, Solanum tuberosum L., one of the most commonly cultivated horticultural crops throughout the world, is susceptible to a variety of herbivory insects. In the present study, we evaluated the consequence of feeding by the sap-sucking insect Halyomorpha halys on potato leaf photosynthetic efficiency. By using chlorophyll fluorescence imaging methodology, we examined photosystem II (PSII) photochemistry in terms of feeding and at the whole leaf area. The role of reactive oxygen species (ROS) in potato’s defence response mechanism immediately after feeding was also assessed. Even 3 min after feeding, increased ROS generation was observed to diffuse through the leaf central vein, probably to act as a long-distance signalling molecule. The proportion of absorbed energy being used in photochemistry (ΦPSII) at the whole leaf level, after 20 min of feeding, was reduced by 8% compared to before feeding due to the decreased number of open PSII reaction centres (qp). After 90 min of feeding, ΦPSII decreased by 46% at the whole leaf level. Meanwhile, at the feeding zones, which were located mainly in the proximity of the leaf midrib, ΦPSII was lower than 85%, with a concurrent increase in singlet-excited oxygen (1O2) generation, which is considered to be harmful. However, the photoprotective mechanism (ΦNPQ), which was highly induced 90 min after feeding, was efficient to compensate for the decrease in the quantum yield of PSII photochemistry (ΦPSII). Therefore, the quantum yield of non-regulated energy loss in PSII (ΦNO), which represents 1O2 generation, remained unaffected at the whole leaf level. We suggest that the potato PSII response to sap-sucking insect feeding underlies the ROS-dependent signalling that occurs immediately and initiates a photoprotective PSII defence response to reduce herbivory damage. A controlled ROS burst can be considered the primary plant defence response mechanism to herbivores.

Efficient control of western flower thrips by plastid-mediated RNA interference

Plastid-mediated RNA interference (PM-RNAi) has emerged as a promising strategy for pest control. Expression from the plastid genome of stable double-stranded RNAs (dsRNAs) targeted against essential insect genes can effectively control some herbivorous beetles, but little is known about the efficacy of the transplastomic approach in other groups of pest insects, especially nonchewing insects that do not consume large amounts of leaf material. Here we have investigated the susceptibility of the western flower thrip (WFT, Frankliniella occidentalis), a notorious pest in greenhouses and open fields, to PM-RNAi. We show that WFTs ingest chloroplasts and take up plastid-expressed dsRNAs. We generated a series of transplastomic tobacco plants expressing dsRNAs and hairpin RNAs (hpRNAs) targeted against four essential WFT genes. Unexpectedly, we discovered plastid genome instability in transplastomic plants expressing hpRNAs, suggesting that dsRNA cassettes are preferable over hpRNA cassettes when designing PM-RNAi strategies. Feeding studies revealed that, unlike nuclear transgenic plants, transplastomic plants induced a potent RNAi response in WFTs, causing efficient suppression of the targeted genes and high insect mortality. Our study extends the application range of PM-RNAi technology to an important group of nonchewing insects, reveals design principles for the construction of dsRNA-expressing transplastomic plants, and provides an efficient approach to control one of the toughest insect pests in agriculture and horticulture.

Functional characterization of two different decaprenyl diphosphate synthases in the vetch aphid Megoura viciae.

Long-chain polyprenyl diphosphate synthases play a critical role in the formation of the prenyl side-chain of ubiquinones, but up to date, their functions have scarcely been characterized in insects. Here, we first cloned the complementary DNAs encoding the subunits of decaprenyl diphosphate synthase (DPPS) in the vetch aphid Megoura viciae, an important agricultural pest insect. The results showed that there existed three DPPS subunits, designated as MvDPPS1, MvDPPS2a, and MvDPPS2b, with an open reading frame of 1218, 1275, and 1290 bp, and a theoretical isoelectric point of 7.91, 6.63, and 9.62, respectively. The sequences of MvDPPS1s from different aphid species were nearly identical, while the sequences of MvDPPS2a and MvDPPS2b shared only moderate sequence similarity. Phylogenetic analysis clearly separated MvDPPS2a and MvDPPS2b, indicating a functional differentiation between them. Functional coexpression analysis in Escherichia coli showed that MvDPPS1 plus MvDPPS2a and MvDPPS1 plus MvDPPS2b, respectively, catalyzed the formation of the prenyl side-chain of the ubiquinone coenzyme Q10 (CoQ10). Interestingly, MvDPPS1 plus MvDPPS2b catalyzed the formation of the prenyl side-chain of a ubiquinone other than CoQ10. RNA interference-mediated knockdown of MvDPPS2a imposed no significant effect on MvDPPS2b, and vice versa, suggesting no compensatory action between them. In the end, we detected the product CoQ10 in the aphid, the first identification of CoQ10 in an insect species. Taken together, we characterized two functional DPPSs in M. viciae, one of which might be multifunctional. Our study helps to understand the functional plasticity of the terpenoid backbone biosynthesis pathway in insects.

Culturing and Genetically Manipulating Entomopathogenic Nematodes.

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests.

Entomopathogenic fungi of the Metarhizium anisopliae species complex have gained importance as the biological control agents of agricultural insect pests. The increase in pest resistance to chemical insecticides, the growing concerns regarding the negative effects of insecticides on human health, and the environmental pollution from pesticides have led to a global drive to find novel sustainable strategies for crop protection and pest control. Previously, attempts to mass culture such entomopathogenic fungi (EPF) species as Beauveria bassiana have been conducted. However, only limited attempts have been conducted to mass culture Metarhizium robertsii and M. pinghaense for use against insect pests. This study aimed to mass-produce a sufficient number of resilient infective propagules of South African isolates of M. robertsii and M. pinghaense for commercial application. Three agricultural grain products, flaked oats, flaked barley, and rice, were used as the EPF solid fermentation substrates. Two inoculation methods, conidial suspensions and the liquid fungal culture of blastospores were used to inoculate the solid substrates. Inoculation using conidial suspensions was observed to be relatively less effective, as increased levels of contamination were observed on the solid substrates relative to when using the blastospore inoculation method. Flaked oats were found not to be a suitable growth substrate for both M. robertsii and M. pinghaense, as no dry conidia were harvested from the substrate. Flaked barley was found to favor the production of M. robertsii conidia over that of M. pinghaense, and an average of 1.83 g ± 1.47 g of dry M. robertsii conidia and zero grams of M. pinghaense conidia was harvested from the substrate. Rice grains were found to favor the conidial mass production of both M. pinghaense and M. robertsii isolates, with an average of 8.2 g ± 4.38 g and 6 g ± 2 g harvested from the substrate, respectively.

Impact of climate change on Helicoverpa armigera voltinism in different Agro-Climatic Zones of India

The Gram pod borer, Helicoverpa armigera (Noctuidae: Lepidoptera) is a cosmopolitan agricultural insect pest that prefers to feed on plant’s protein biomolecules. Out of different density-independent factors, surface air temperature majorly affects the incidence and damage of the H. armigera on the crops. Early prediction of H. armigera generations (voltinism) in future climate years perhaps prevent additional damage in various crops and improve the farmers preparedness. In this study, future climate data that is temperature obtained for eleven Agro-Climatic Zones (ACZs) of India under four Representative Concentration Pathways (RCPs) scenarios in different climate years (2010, 2030, 2050, 2070, 2090) using weather file generator MarkSim web application. The accumulation of Growing Degree-days (GDD) by H. armigera at eleven ACZs in each climate year under different RCP scenarios was estimated using temperature data. The mean surface air temperature is predicted to 0.51 °C, 1.03 °C, 1.57 °C and 2.1 °C in climate years 2030, 2050, 2070 and 2090, which escalated annual H. armigera Gen. to 12.88, 13.33, 13.79 and 14.23, respectively over the baseline climate year 2010. Likewise, under RCP 2.6, RCP 4.5, RCP 6.0 and RCP 8.5 scenarios H. armigera Gen. is predicted to 12.86, 13.29, 13.23 and 13.97 per annum with mean surface air temperatures 27.4 °C, 27.92 °C, 27.86 °C and 28.72 °C, respectively. The Eastern Coastal Plains and Hills Zone (ACZ 11) across climate years and RCPs has experienced a considerable increase in mean surface air temperature minimum (25.22 °C) and maximum (34.61 °C), which likely favor the GDD accumulation (6319.91) and the Genrations (14.97) in H. armigera. Therefore, the Eastern Coastal Plains and Hills Zone of India could be identified as H. armigera risk zone in near future. The present predictions in various ACZs of India may be significant in planning H. armigera management.

Preparation of Thymus vulgaris (L.) essential oil nanoemulsion and its chitosan encapsulation for controlling mosquito vectors

Here, we report a novel comparative assessment of preparation and characterization of thyme oil nanoemulsion and its chitosan encapsulation using high energy approach for the management of three major mosquito species viz., Anopheles stephensi (Liston,1901), Aedes aegypti (Linn., 1762) and Culex tritaeniorhynchus (Giles, 1901). The synthesized formulations were analysed for thermodynamic stability, indicating 1:0.5 (oil: surfactant) ratio to be the most stable of thyme oil nanoemulsion while 1:1 (nanoemulsion: chitosan solution) ratio of its chitosan encapsulation. These were further characterized by dynamic light scattering and transmission electron microscopy which revealed the size and morphology of the droplets which measured 52.18 ± 4.53 nm for thyme oil nanoemulsion and 50.18 ± 2.32 nm for its chitosan encapsulation. All the droplets were well dispersed with distinct flower-shaped nanoemulsion and somewhat mitochondria like chitosan encapsulation. In-vitro release study of thyme essential oil from its nanoemulsion and chitosan encapsulation showed that 91.68% and 73.41% of the total oil concentration in water was released respectively to the environment after 48 h clearly depicting controlled release in the encapsulation. Assessment of insecticidal potential against selected mosquito species revealed that both the nanoemulsion and its chitosan encapsulation were effective on the major mosquito species. Maximum activity of thyme oil nanoemulsion was noticed against C. tritaeniorhynchus (LC50—22.58 ppm) after 24 h of exposure while it was observed that its chitosan encapsulation was most effective on A. stephensi (LC50—18.88 ppm) after 24 h of exposure. Consistent morphological alterations could be noticed in the larvae of mosquito species. Hence, these nanoemulsions and encapsulations could be further tested for their applications against other insect pests in agriculture.

Methyl Benzoate as a Promising, Environmentally Safe Insecticide: Current Status and Future Perspectives

The widespread use of synthetic chemical pesticides beginning in the late 1930s has contributed to the development of insecticide resistance of many important species of pest insects and plants. Recent trends in pesticide development have emphasized the use of more environmentally benign control methods that take into consideration environmental, food safety, and human health. Biopesticides (e.g., naturally occurring pesticidal compounds) are alternative pest management tools that normally have no negative impact on human health or the environment. Here we review methyl benzoate, a relatively new botanical insecticide that occurs naturally as a metabolite in plants, and whose odor is an attractant to some insects. Since 2016, many studies have shown that methyl benzoate is an effective pesticide against a range of different agricultural, stored product, and urban insect pests. Methyl benzoate has several important modes of action, including as a contact toxicant, a fumigant, an ovicidal toxin, an oviposition deterrent, a repellent, and an attractant. In this review, we summarize various modes of action of methyl benzoate and its toxicity or control potential against various kinds of arthropods, including agricultural pests and their natural enemies, and pollinators. We conclude that methyl benzoate is a very promising candidate for use in integrated pest management under either greenhouse or field conditions.