Cooperative herbivory between two important pests of rice.

Normally, when different species of herbivorous arthropods feed on the same plant this leads to fitness-reducing competition. We found this to be different for two of Asia's most destructive rice pests, the brown planthopper and the rice striped stem borer. Both insects directly and indirectly benefit from jointly attacking the same host plant. Double infestation improved host plant quality, particularly for the stemborer because the planthopper fully suppresses caterpillar-induced production of proteinase inhibitors. It also reduced the risk of egg parasitism, due to diminished parasitoid attraction. Females of both pests have adapted their oviposition behaviour accordingly. Their strong preference for plants infested by the other species even overrides their avoidance of plants already attacked by conspecifics. This cooperation between herbivores is telling of adaptations resulting from the evolution of plant-insect interactions, and points out mechanistic vulnerabilities that can be targeted to control these major pests.

Screening Potential Reference Genes in Tuta absoluta with Real-Time Quantitative PCR Analysis under Different Experimental Conditions.

Tuta absoluta is one of the most significant invasive pests affecting tomato plants worldwide. RT-qPCR has emerged as one of the most sensitive and accurate methods for detecting gene expression data. The screening of stable internal reference genes is the most critical step for studying the molecular mechanisms of environmental adaptability. The stable reference genes expressed in T. absoluta under specific experimental conditions have not yet been clarified. In this study, seven candidate reference genes (RPL27, RPS13, RPS15, EF1-α, TUB, TBP, and ß-actin) and their optimal numbers were evaluated under biotic (developmental stages and adult tissues) and abiotic (insecticide, temperature, and plant VOC) conditions using four software programs. Our results identified the following reference genes and numbers as optimal: three genes (EF1-α, RPS13, and RPL27) for different developmental stages (egg, larva, pupa, unmated adult), two genes (RPS13 and TBP) for adult tissues (antenna, head, thorax, abdomen, leg), two genes (TBP and RPS13) for insecticides (Bacillus thuringiensis, chlorpyrifos, abamectin-aminomethyl, and chlorantraniliprole), two genes (RPL27 and TUB) for temperature-induced stresses (0, 25, and 40 °C), and two genes (RPS13 and TUB) for VOC-induced stresses (nonanal, α-phellandrene, and tomato leaves). Our results provide a reference for selecting appropriate reference genes for further study of the functional genes of T. absoluta under different experimental conditions.

Induction of cell wall phenolic monomers as part of direct defense response in maize to pink stem borer (Sesamia inferens Walker) and non-insect interactions.

Pink stem borer (PSB) causes considerable yield losses to maize. Plant-insect interactions have significant implications for sustainable pest management. The present study demonstrated that PSB feeding, mechanical wounding, a combination of mechanical wounding and PSB regurgitation and exogenous application of methyl jasmonate have induced phenolic compound mediated defense responses both at short term (within 2 days of treatment) and long term (in 15 days of treatment) in leaf and stalk tissues of maize. The quantification of two major defense related phenolic compounds namely p-Coumaric acid (p-CA) and ferulic acid (FA) was carried out through ultra-fast liquid chromatography (UFLC) at 2 and 15 days after imposing the above treatments. The p-CA content induced in leaf tissues of maize genotypes were intrinsically higher when challenged by PSB attack at V3 and V6 stages in short- and long-term responses. Higher p-CA content was observed in stalk tissues upon wounding and regurgitation in short- and long-term responses at V3 and V6 stages. Significant accumulation of FA content was also observed in leaf tissues in response to PSB feeding at V3 stage in long-term response while at V6 stage it was observed both in short- and long-term responses. In stalk tissues, methyl jasmonate induced higher FA content in short-term response at V3 stage. However, at V6 stage PSB feeding induced FA accumulation in the short-term while, wounding and regurgitation treatment-induced defense responses in the long-term. In general, the resistant (DMRE 63, CM 500) and moderately resistant genotypes (WNZ ExoticPool) accumulated significantly higher contents of p-CA and FA content than susceptible ones (CM 202, BML 6) in most of the cases. The study indicates that phenolic mediated defense responses in maize are induced by PSB attack followed by wounding and regurgitation compared to the other induced treatments. Furthermore, the study confirmed that induced defense responses vary with plant genotype, stage of crop growth, plant tissue and short and long-term responses. The results of the study suggested that the Phenolic acids i.e. p-CA and FA may contribute to maize resistance mechanisms in the maize-PSB interaction system.

CRISPR-mediated mutations in the ABC transporter gene ABCA2 confer pink bollworm resistance to Bt toxin Cry2Ab.

Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) have many benefits and are important globally for managing insect pests. However, the evolution of pest resistance to Bt crops reduces their benefits. Understanding the genetic basis of such resistance is needed to better monitor, manage, and counter pest resistance to Bt crops. Previous work shows that resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2 in lab- and field-selected populations of the pink bollworm (Pectinophora gossypiella), one of the world's most destructive pests of cotton. Here we used CRISPR/Cas9 gene editing to test the hypothesis that mutations in the pink bollworm gene encoding ABCA2 (PgABCA2) can cause resistance to Cry2Ab. Consistent with this hypothesis, introduction of disruptive mutations in PgABCA2 in a susceptible strain of pink bollworm increased the frequency of resistance to Cry2Ab and facilitated creation of a Cry2Ab-resistant strain. All Cry2Ab-resistant individuals tested in this study had disruptive mutations in PgABCA2. Overall, we found 17 different disruptive mutations in PgABCA2 gDNA and 26 in PgABCA2 cDNA, including novel mutations corresponding precisely to single-guide (sgRNA) sites used for CRISPR/Cas9. Together with previous results, these findings provide the first case of practical resistance to Cry2Ab where evidence identifies a specific gene in which disruptive mutations can cause resistance and are associated with resistance in field-selected populations.

Pararamosis: Disease of the Rubber Plantations.

Pararamosis is a medical condition, described in the latex extracting areas of the Amazon (rubber tree regions), resulting from contact with the caterpillar of the Premolis semirufa moth. The disease can present itself in an acute form-similarly to other erucisms (injuries caused by moth larvae in humans)-or in a chronic form, typically characterized by the occurrence of changes in the joints of the hands. Because of its importance, in the context of tropical diseases, the objective of this article was to review the main facets of the disease, emphasizing the different pathogenic aspects of the interaction between the arthropod and man.

Infectivity of entomopathogenic nematodes against the legume pod-borer, Maruca vitrata Fabricius, infesting pigeon pea.

The legume pod-borer, Maruca vitrata Fabricius (Lepidoptera: Crambidae) (LPB), is an important insect pest of pigeon pea. Chemical pesticides are generally employed to manage this pest, but because of the soil residue issues and other environmental hazards associated with their use, biopesticides are also in demand. Another benign alternative is to use entomopathogenic nematodes (EPNs) to manage this vital pest. In the present study, the infectivity of ten native EPNs was evaluated against LPB by assessing their penetration and production in the LPB. The effectiveness of the promising EPNs against second-, third- and fourth-instar LPB larvae was also studied. Heterorhabditis sp. (Indian Agricultural Research Institute-Entomopathogenic Nematodes Rashid Pervez (IARI-EPN RP) 06) and Oscheius sp. (IARI-EPN RP 08) were found to be most pathogenic to LPB, resulting in about 100% mortality within 72 h, followed by Steinernema sp. (IARI-EPN RP 03 and 09). Oscheius sp. (IARI-EPN RP 04) was found to be the least pathogenic to LPB larva with 67% mortality. Maximum penetration was exhibited by Heterorhabditis sp. (IARI-EPN RP 06) followed by Oscheius sp. (IARI-EPN RP 08), whereas the lowest rate of penetration was exhibited by Oscheius sp. (IARI-EPN RP 01). The highest rate of production was observed with Oscheius sp. (IARI-EPN RP 08), followed by Oscheius sp. (IARI-EPN RP 04 and 10). Among the tested instars of the LPB larvae, second-instar larvae were more susceptible to EPNs, followed by third- and fourth-instar larvae. The results indicate that Heterorhabditis sp. (IARI-EPN RP 06) and Oscheius sp. (IARI-EPN RP 08) have a good potential to the manage LPB.

Transgenic cotton and sterile insect releases synergize eradication of pink bollworm a century after it invaded the United States.

Invasive organisms pose a global threat and are exceptionally difficult to eradicate after they become abundant in their new habitats. We report a successful multitactic strategy for combating the pink bollworm (Pectinophora gossypiella), one of the world's most invasive pests. A coordinated program in the southwestern United States and northern Mexico included releases of billions of sterile pink bollworm moths from airplanes and planting of cotton engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). An analysis of computer simulations and 21 y of field data from Arizona demonstrate that the transgenic Bt cotton and sterile insect releases interacted synergistically to reduce the pest's population size. In Arizona, the program started in 2006 and decreased the pest's estimated statewide population size from over 2 billion in 2005 to zero in 2013. Complementary regional efforts eradicated this pest throughout the cotton-growing areas of the continental United States and northern Mexico a century after it had invaded both countries. The removal of this pest saved farmers in the United States $192 million from 2014 to 2019. It also eliminated the environmental and safety hazards associated with insecticide sprays that had previously targeted the pink bollworm and facilitated an 82% reduction in insecticides used against all cotton pests in Arizona. The economic and social benefits achieved demonstrate the advantages of using agricultural biotechnology in concert with classical pest control tactics.

Synergistic Effect of Beauveria bassiana and Trichoderma asperellum to Induce Maize (Zea mays L.) Defense against the Asian Corn Borer, Ostrinia furnacalis (Lepidoptera, Crambidae) and Larval Immune Response.

Ostrinia furnacalis, is the major pest of maize causing significant yield losses. So far, many approaches have been used to increase the virulence of entomopathogenic fungal isolates. The current study is an attempt to estimate synergistic effect of Beauveria bassiana and Trichoderma asperellum in order to explore larval immune response through RNA sequencing and differentially expression analysis. In vivo synergism was examined in seven proportions (B. bassiana: T. asperellum = 1:1, 1:2, 1:3, 1:4, 4:1, 3:1, 2:1) and in the in vitro case, two inoculation methods were applied: seed coating and soil drenching. Results revealed significant decrease in plant damage and high larval mortality in fungal treatments. Fungal isolates mediated the plant defense by increasing proline, superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO) and protease activities. Seed coating method was proved to be the most effective in case of maize endophytic colonization. In total, 59 immune-related differentially expressed genes DEGs were identified including, cytochrome P450, heat shock protein, ABC transporter, cadherin, peptidoglycan recognition protein (PGRP), cuticlular protein, etc. Further, transcriptomic response was confirmed by qRT-PCR. Our results concluded that, coculture of B. bassiana and T. asperellum has the synergistic potential to suppress the immune response of O. furnacalis and can be used as sustainable approach to induce plant resistance through activation of defense-related enzymes.

Large-scale genetic admixture suggests high dispersal in an insect pest, the apple fruit moth.

Knowledge about population genetic structure and dispersal capabilities is important for the development of targeted management strategies for agricultural pest species. The apple fruit moth, Argyresthia conjugella (Lepidoptera, Yponomeutidae), is a pre-dispersal seed predator. Larvae feed on rowanberries (Sorbus aucuparia), and when rowanberry seed production is low (i.e., inter-masting), the moth switches from laying eggs in rowanberries to apples (Malus domestica), resulting in devastating losses in apple crops. Using genetic methods, we investigated if this small moth expresses any local genetic structure, or alternatively if gene flow may be high within the Scandinavian Peninsula (~850.000 km2, 55o - 69o N). Genetic diversity was found to be high (n = 669, mean He = 0.71). For three out of ten tetranucleotide STRs, we detected heterozygote deficiency caused by null alleles, but tests showed little impact on the overall results. Genetic differentiation between the 28 sampling locations was very low (average FST = 0.016, P < 0.000). Surprisingly, we found that all individuals could be assigned to one of two non-geographic genetic clusters, and that a third, geographic cluster was found to be associated with 30% of the sampling locations, with weak but significant signals of isolation-by-distance. Conclusively, our findings suggest wind-aided dispersal and spatial synchrony of both sexes of the apple fruit moth over large areas and across very different climatic zones. We speculate that the species may recently have had two separate genetic origins caused by a genetic bottleneck after inter-masting, followed by rapid dispersal and homogenization of the gene pool across the landscape. We suggest further investigations of spatial genetic similarities and differences of the apple fruit moth at larger geographical scales, through life-stages, across inter-masting, and during attacks by the parasitoid wasp (Microgaster politus).

Population dynamics of Hippophae rhamnoides shrub in response of sea-level rise and insect outbreaks.

The coastal vegetation of islands is expected to be affected by future sea-level rise and other anthropogenic impacts. The biodiverse coastal vegetation on the eastern part of the Dutch Wadden Island of Ameland has experienced land subsidence caused by gas extraction since 1986. This subsidence mimics future sea-level rising through increased flooding and raising groundwater levels. We studied the effects of this relative sea-level rise and other environmental factors (i.e. insect outbreaks, temperature and precipitation) on the population dynamics (i.e. cover and age structure and annual growth) of the shrub sea-buckthorn (Hippophae rhamnoides L.) in young (formed after 1950) and old (formed before 1950) dune areas over a period of 56 years (1959-2015). We found an increase in sea-buckthorn cover in the young dune areas since 1959, while over time the population in the old dunes decreased due to successional replacement by other species. With the increasing age of the young dunes, we found also a decrease in sea-buckthorn after 2009. However the sharp decrease indicated that other environmental factors were also involved. The most important determinant of annual shrub growth appeared to be five outbreaks of the brown-tail moth (Euproctis chrysorrhoea L.), in the last decade. Relative sea-level rise caused more frequent flooding and reduced growth at lower elevations due to inundation or soil water saturation. This study clearly indicates that sea-buckthorn is affected by relative sea-level rise, but that other ecological events better explain its variation in growth. Although shrub distribution and growth can be monitored with robust methods, future predictions of vegetation dynamics are complicated by unpredictable extreme events caused by (a)biotic stressors such as insect outbreaks.

Distinct defensive activity of phenolics and phenylpropanoid pathway genes in different cotton varieties toward chewing pests.

Identifying the maximum level of inherent defense against harmful insects in natural variation among wild lineages of crop plants may result in high yield tolerant varieties and reducing use of chemical insecticides. However, knowledge of natural cotton genotypes with high insect-resistance is still indistinguishable at the biochemical or molecular level. In the present study, different cultivated Gossypium hirsutum varieties were evaluated for their inherent insect-tolerance against two major cottons chewing pests. The insect bio-assay identified two tolerant and one susceptible cotton varieties. The study demonstrates difference in phenolic acids, proanthocyanidin and tannin accumulation in tolerant and susceptible varieties. The post-infestation of chewing pests increases transcript level of the phenylpropanoid pathway genes were detected in tolerant varieties as compared to the susceptible varieties. Altogether, chewing pest-tolerance level in cotton varieties is the cumulative effect of enhanced phenylpropanoid pathway genes expression and secondary metabolite leading to defense responses to conventional host plant.

Characterization of the complete mitochondrial genome of Orthaga olivacea Warre (Lepidoptera Pyralidae) and comparison with other Lepidopteran insects.

Orthaga olivacea Warre (Lepidoptera: Pyralidae) is an important agricultural pest of camphor trees (Cinnamomum camphora). To further supplement the known genome-level features of related species, the complete mitochondrial genome of Orthaga olivacea is amplified, sequenced, annotated, analyzed, and compared with 58 other species of Lepidopteran. The complete sequence is 15,174 bp, containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a putative control region. Base composition is biased toward adenine and thymine (79.02% A+T) and A+T skew are slightly negative. Twelve of the 13 PCGs use typical ATN start codons. The exception is cytochrome oxidase 1 (cox1) that utilizes a CGA initiation codon. Nine PCGs have standard termination codon (TAA); others have incomplete stop codons, a single T or TA nucleotide. All the tRNA genes have the typical clover-leaf secondary structure, except for trnS(AGN), in which dihydrouridine (DHU) arm fails to form a stable stem-loop structure. The A+T-rich region (293 bp) contains a typical Lepidopter motifs 'ATAGA' followed by a 17 bp poly-T stretch, and a microsatellite-like (AT)13 repeat. Codon usage analysis revealed that Asn, Ile, Leu2, Lys, Tyr and Phe were the most frequently used amino acids, while Cys was the least utilized. Phylogenetic analysis suggested that among sequenced lepidopteran mitochondrial genomes, Orthaga olivacea Warre was most closely related to Hypsopygia regina, and confirmed that Orthaga olivacea Warre belongs to the Pyralidae family.

Resistance to Chilo infuscatellus (Lepidoptera: Pyraloidea) in transgenic lines of sugarcane expressing Bacillus thuringiensis derived Vip3A protein.

Sustainable agriculture requires management of insect pests through resistance development. The biological potential of Cry toxins and Vip protein, derived from Bacillus species, is widely recognized in this context. The identification, evaluation of new insecticidal protein genes with different mode of action and entomotoxicity against sugarcane stem borer (Chilo infuscatellus) is important to overcome evolved insect resistance. In this study, we reported the generation of transgenic sugarcane lines expressing Vip3A toxin driven by polyubiquitin promoter for resistance against sugarcane stem borer. The V0 transgenic sugarcane plants were initially characterized by GUS histochemical staining, PCR and Southern blot assays that confirmed genetic transformation of twelve independent sugarcane lines. Variable transgene expression was found among transgenic sugarcane lines when revealed through Realtime quantitative PCR (RT-qPCR) with highest in S10 line while minimum was observed in V5 line. A similar expression pattern was observed in transgenic sugarcane lines for Vip3A protein concentration which ranged from 5.35 to 8.89 µg/mL. A direct correlation was observed between the Vip3A protein and Vip3A transgene expression in the transgenic sugarcane lines. In in-vitro insect bioassay on V1, Vip3A transgenic sugarcane lines exhibited high resistance to C. infuscatellus with upto 100% mortality compared to the control sugarcane line. Our findings suggest that a single copy insertion of Vip3A gene in transgenic sugarcane lines render them resistant to borer and these lines can be potentially used for generation of insect resistant transgenic sugarcane and could also be employed in gene pyramiding with Bt toxin to prolong resistance.

JA-Ile-macrolactone 5b Induces Tea Plant (Camellia sinensis) Resistance to Both Herbivore Ectropis obliqua and Pathogen Colletotrichum camelliae.

Jasmonates (JAs), the group of lipid-derived hormones, were found to control the defense responses in a myriad of plants. Meaningfully, the macrolactones of 12-hydroxy jasmonate isoleucine (12OH-JA-Ile) were reported to induce the defensive response of wild tobacco. However, little to nothing has been known about the elicitation effect of JA-Ile-macrolactones on woody plants to harmful organisms, let alone its underlying mechanisms. Here, we first optimized the synthetic routine using mild toxic reagent isobutyl chloroformate instead of ethyl chloroformate for conjugation, and we used acetonitrile (MeCN) instead of ethyl alcohol for the better dissolution of p-toluenesulfonic acid (p-TsOH) to gain JA-Ile-macrolactones. JA-Ile-macrolactone 5b-treated tea plants significantly inhibited the larvae weight gain of Ectropis obliqua larvae and the lesions caused by Colletotrichum camelliae. Furthermore, the expression level of CsOPR3 was significantly upregulated in 5b-treated leaves. Meanwhile, 5b reduced the accumulation of eriodictyol 7-O-glucuronide (EDG) in tea plants, which was confirmed to promote the growth rate of E. obliqua larvae by artificial diet assay. In conclusion, our study proved that the exogenous application of 5b could induce the tea plant resistance both to herbivore E. obliqua and pathogen C. camelliae, and EDG was identified as one of the secondary metabolites that could influence the growth rate of E. obliqua, but it did not directly influence the infection of C. camelliae in vitro. Further research should be carried out to clarify the mechanism through which 5b induces tea plant resistance to C. camelliae.

Introgression of a cry1Ab transgene into open pollinated maize and its effect on Cry protein concentration and target pest survival.

In Africa, the target pests of genetically modified Bt maize are lepidopteran stem borers, notably Busseola fusca (Lepidoptera: Noctuidae). Gene flow between Bt maize hybrids and open pollinated varieties (OPVs) that do not contain the Bt trait is highly likely in areas where both types of maize are cultivated. Consequently, introgression of the cry1Ab transgene into local OPVs will result in unknown patterns of Cry1Ab protein expression in plants during follow-up seasons when recycled seed of OPVs is planted. Too low concentrations of Cry protein in such plants may result in selection for resistant alleles and accelerate resistance evolution. The aim of this study was to determine the effects of introgression of the cry1Ab transgene into an OPV, on Cry protein concentration levels and pest survival. Bt transgene introgression was done by crossing a transgenic donor hybrid containing the cry1Ab gene with a non-Bt OPV as well as with a non-Bt near-isogenic hybrid. F1 and F2 crosses as well as back crosses were done yielding 11 genotypes (treatments). Cry1Ab protein concentrations in leaf tissue of these crosses were determined by means of ELISAs. All crosses that contained the transgene had similar or higher Cry1Ab concentrations when compared to the Bt parental hybrid, except for the Bt x OPV F1-cross that had a significantly lower Cry1Ab concentration. Survival B. fusca larvae were evaluated in assays in which larvae were reared for 14 days on whorl leaf tissue of the different treatments. Larval survival did not differ between any of the maize plant treatments which contained the Bt gene. Results suggest that Bt transgene introgression into OPVs may produce plant progenies that express Cry1Ab protein at sufficient concentrations, at last up to the F2 seed, to control B. fusca larvae. Resistance evolution is however not only influenced by the frequency of pest individuals that survive exposure to the Cry proteins but also by factors such as genetics of the pest and recipient OPV, pest biology and migration behaviour.

Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests.

Cotton is the main fiber producing crop globally, with a significant impact on the economy of Pakistan. Bt cotton expressing a Cry1Ac gene is grown over a large area in Pakistan, however, there is a major concern that bollworms may develop resistance. Here we have used a durable resistance strategy against bollworms by developing a double gene construct containing Cry1Ac and Cry2Ab (pGA482-12R) for cotton transformation. Both Cry toxin genes have been cloned in the same T-DNA borders and transferred successfully into cotton via Agrobacterium-mediated transformation. Both genes are expressed in transgenic cotton plants and is likely to help breeders in developing new cotton cultivars by incorporating these genes in cotton lines having no Bt genes or expressing Cry1Ac gene (Mon 531). Positive transgenic cotton was identified by PCR using specific primers for the amplification of both Cry1Ac and Cry2Ab genes. Cry1Ac and Cry2Ab expression was confirmed with an immunostrip test and quantified using ELISA that showed significant spatio-temporal expression of Cry2Ab ranging from 3.28 to 7.72 µg/g of the tissue leaf. Insect bioassay with army worm (Spodoptera litura) was performed to check the efficacy of NIBGE (National Institute for Biotechnology and Genetic Engineering) double gene transgenic cotton plants and up to 93% insect mortality was observed.

Genetic characterization of fall armyworm infesting South Africa and India indicate recent introduction from a common source population.

The invasion of the Western Hemisphere native fall armyworm (Spodoptera frugiperda; J. E. Smith) (Lepidoptera: Noctuidae) into the Eastern Hemisphere has been notable for the rapidity and geographical breadth of new detections. In the year following the first discovery in western sub-Saharan Africa in 2016, infestations have been documented in most sub-Saharan maize growing regions and has now expanded beyond Africa with populations recently reported in India. These observations could indicate a remarkable capacity for rapid establishment and long-distance dissemination. However, while fall armyworm does exhibit extended migration in North America where it annually traverses thousands of kilometers, this behavior is known to be dependent on highly favorable wind patterns and so can't be assumed to occur in all locations. An alternative possibility is that the species has long been present in Africa, and perhaps the rest of the hemisphere, but was undetected until the enhanced monitoring that resulted after its initial discovery. Determining whether the fall armyworm in the Eastern Hemisphere is newly arrived or long pre-existing is important for assessing the risks of significant economic impacts, as the former indicates a change in pest composition while the latter does not. This study examined this issue by comparing collections from two geographically distant locations, South Africa and India. Sequence comparisons were used to quantify differences between the South Africa and India collections, assess the likelihood of their sharing a common source population, and their possible relationship with previously characterized fall armyworm from other regions of Africa. The results indicate genetic homogeneity between the South African and Indian fall armyworm populations tested and substantial similarities between these and collections from eastern Africa. The implications of these findings on fall armyworm population behavior and composition are discussed.

Examining population structure of a bertha armyworm, Mamestra configurata (Lepidoptera: Noctuidae), outbreak in western North America: Implications for gene flow and dispersal.

The bertha armyworm (BAW), Mamestra configurata, is a significant pest of canola (Brassica napus L. and B. rapa L.) in western North America that undergoes cyclical outbreaks every 6-8 years. During peak outbreaks millions of dollars are spent on insecticidal control and, even with control efforts, subsequent damage can result in losses worth millions of dollars. Despite the importance of this pest insect, information is lacking on the dispersal ability of BAW and the genetic variation of populations from across its geographic range which may underlie potential differences in their susceptibility to insecticides or pathogens. Here, we examined the genetic diversity of BAW populations during an outbreak across its geographic range in western North America. First, mitochondrial cytochrome oxidase 1 (CO1) barcode sequences were used to confirm species identification of insects captured in a network of pheromone traps across the range, followed by haplotype analyses. We then sequenced the BAW genome and used double-digest restriction site associated DNA sequencing, mapped to the genome, to identify 1000s of single nucleotide polymorphisms (SNP) markers. CO1 haplotype analysis identified 9 haplotypes distributed across 28 sample locations and three laboratory-reared colonies. Analysis of genotypic data from both the CO1 and SNP markers revealed little population structure across BAW's vast range. The CO1 haplotype pattern showed a star-like phylogeny which is often associated with species whose population abundance and range has recently expanded and combined with pheromone trap data, indicates the outbreak may have originated from a single focal point in central Saskatchewan. The relatively recent introduction of canola and rapid expansion of the canola growing region across western North America, combined with the cyclical outbreaks of BAW caused by precipitous population crashes, has likely selected for a genetically homogenous BAW population adapted to this crop.

A Combinational Approach of Enhanced Methanol Production and Double Bt Genes for Broad Spectrum Insect Resistance in Transgenic Cotton.

The prevalence of insect resistance against Bt toxins has led to the idea of enhancing demethylation from cell wall pectin by pectin methylesterase enzyme for overproduction of methanol which is toxic to insects pests. The AtPME and AnPME fragments ligated into pCAMBIA1301 vector were confirmed through restriction digestion with EcoR1 and BamH1. Excision of 3363 bp fragment from 11,850 bp vector confirmed the ligation of both fragments into pCAMBIA1301 vector. Transformation of pectin methylesterase-producing genes, i.e., AtPME and AnPME from Arabidopsis thaliana and Aspergillus niger cloned in plant expression vector pCAMBIA1301 under 35S promoter into cotton variety CEMB-33 harboring two Bt genes Cry1Ac and Cry2A, respectively, was done by using shoot apex-cut Agrobacterium-mediated transformation method. The plantlets were screened on MS medium supplemented with hygromycin on initial basis. Amplification of 412 and 543 bp, respectively, through gene-specific primer has been obtained which confirmed the successful introduction of pCAMBIA AtPME and AnPME genes into cotton variety CEMB 33. Relative expression of AtPME and AnPME genes through real-time PCR determined the expression level of both gene ranges between 3- and 3.5-fold in different transgenic cotton lines along with quantity of methanol ranging from 0.8 to 0.9% of maximum while 0.5% to 0.6% of minimum but no expression was obtained in negative non-transgenic control cotton plant with least quantity of methanol, i.e., 0.1%. Almost 100% mortality was observed in insect bioassay for Helicoverpa armigera on detached leaves bioassay and 63% for Pink Bollworm (Pectinophora gossypiella) on growing transgenic cotton bolls as compared to positive control transgenic cotton with double Bt genes where mortality was found to be 82% for H. armigera and 50% for P. gossypiella while 0% in negative control non-transgenic plants.

An effector from cotton bollworm oral secretion impairs host plant defense signaling.

Insects have evolved effectors to conquer plant defense. Most known insect effectors are isolated from sucking insects, and examples from chewing insects are limited. Moreover, the targets of insect effectors in host plants remain unknown. Here, we address a chewing insect effector and its working mechanism. Cotton bollworm (Helicoverpa armigera) is a lepidopteran insect widely existing in nature and severely affecting crop productivity. We isolated an effector named HARP1 from H. armigera oral secretion (OS). HARP1 was released from larvae to plant leaves during feeding and entered into the plant cells through wounding sites. Expression of HARP1 in Arabidopsis mitigated the global expression of wounding and jasmonate (JA) responsive genes and rendered the plants more susceptible to insect feeding. HARP1 directly interacted with JASMONATE-ZIM-domain (JAZ) repressors to prevent the COI1-mediated JAZ degradation, thus blocking JA signaling transduction. HARP1-like proteins have conserved function as effectors in noctuidae, and these types of effectors might contribute to insect adaptation to host plants during coevolution.