Search

A key component in understanding plant-insect interactions is the nature of host defenses. Research on defense traits among Pinus species has focused on specialized metabolites and axial resin ducts, but the role of lignin in defense within diverse systems is unclear. We investigated lignin levels in the outer bark and phloem of P. longaeva, P. balfouriana, and P. flexilis; tree species growing at high elevations in the western United States known to differ in susceptibility to mountain pine beetle (Dendroctonus ponderosae; MPB). Pinus longaeva and P. balfouriana are attacked by MPB less frequently than P. flexilis, and MPB brood production in P. longaeva is limited. Because greater lignification of feeding tissues has been shown to provide defense against bark beetles in related genera, such as Picea, we hypothesized that P. longaeva and P. balfouriana would have greater lignin concentrations than P. flexilis. Contrary to expectations, we found that the more MPB-susceptible P. flexilis had greater phloem lignin levels than the less susceptible P. longaeva and P. balfouriana. No differences in outer bark lignin levels among the species were found. We conclude that lignification in Pinus phloem and outer bark is likely not adaptive as a physical defense against MPB., Competing Interests: The authors have declared that no competing interests exist.

Insecticidal double-stranded RNAs (dsRNAs) silence expression of vital genes by activating the RNA interference (RNAi) mechanism in insect cells. Despite high commercial interest in insecticidal dsRNA, information on resistance to dsRNA is scarce, particularly for dsRNA products with non-transgenic delivery (ex. foliar/topical application) nearing regulatory review. We report the development of the CEAS 300 population of Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) with > 11,100-fold resistance to a dsRNA targeting the V-ATPase subunit A gene after nine episodes of selection using non-transgenic delivery by foliar coating. Resistance was associated with lack of target gene down-regulation in CEAS 300 larvae and cross-resistance to another dsRNA target (COPI β; Coatomer subunit beta). In contrast, CEAS 300 larvae showed very low (~ 4-fold) reduced susceptibility to the Cry3Aa insecticidal protein from Bacillus thuringiensis. Resistance to dsRNA in CEAS 300 is transmitted as an autosomal recessive trait and is polygenic. These data represent the first documented case of resistance in an insect pest with high pesticide resistance potential using dsRNA delivered through non-transgenic techniques. Information on the genetics of resistance and availability of dsRNA-resistant L. decemlineata guide the design of resistance management tools and allow research to identify resistance alleles and estimate resistance risks.

Factors influencing the efficacy of insectivorous vertebrates in providing natural pest control services inside crops at increasing distances from the crop edge are poorly understood. We investigated the identity of vertebrate predators (birds and bats) and removal of sentinel prey (mealworms and beetles) from experimental feeding trays in cotton crops using prey removal trials, camera traps and observations. More prey was removed during the day than at night, but prey removal was variable at the crop edge and dependent on the month (reflecting crop growth and cover) and time of day. Overall, the predation of mealworms and beetles was 1-times and 13-times greater during the day than night, respectively, with predation on mealworms 3-5 times greater during the day than night at the crop edge compared to 95 m inside the crop. Camera traps identified many insectivorous birds and bats over crops near the feeding trays, but there was no evidence of bats or small passerines removing experimental prey. A predation gradient from the crop edge was evident, but only in some months. This corresponded to the foraging preferences of open-space generalist predators (magpies) in low crop cover versus the shrubby habitat preferred by small passerines, likely facilitating foraging away from the crop edge later in the season. Our results are in line with Optimal Foraging Theory and suggest that predators trade-off foraging behaviour with predation risk at different distances from the crop edge and levels of crop cover. Understanding the optimal farm configuration to support insectivorous bird and bat populations can assist farmers to make informed decisions regarding in-crop natural pest control and maximise the predation services provided by farm biodiversity.

The Western corn rootworm (WCR) Diabrotica virgifera virgifera LeConte is one of the most economically important insect pests in North America. Since 2003, transgenic maize expressing WCR-active proteins from Bacillus thuringiensis (Bt) have been widely adopted as the main approach to controlling WCR in the U.S. However, the emergence of field resistance to the Bt proteins in current commercial products has been documented in recent years, highlighting the need to develop additional tools for controlling this devasting pest. Here we report the discovery of Vpb4Da2 (initially assigned as Vip4Da2), a new insecticidal protein highly selective against WCR, through high-throughput genome sequencing of a Bt strain sourced from grain dust samples collected in the eastern and central regions of the US. Vpb4Da2 contains a sequence and domain signature distinct from families of other WCR-active proteins. Under field conditions, transgenic maize expressing Vpb4Da2 demonstrates commercial-level (at or below NIS 0.25) root protection against WCR, and reduces WCR beetle emergence by ≥ 97%. Our studies also conclude that Vpb4Da2 controls WCR populations that are resistant to WCR-active transgenic maize expressing Cry3Bb1, Cry34Ab1/Cry35Ab1 (reassigned as Gpp34Ab1/Tpp35Ab1), or DvSnf7 RNA. Based on these findings, Vpb4Da2 represents a valuable new tool for protecting maize against WCR., Competing Interests: Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Author’s employment by Bayer does not alter our adherence to PLOS ONE policies on sharing data and materials.

Quantitative real-time polymerase chain reaction (qPT-PCR) is commonly used to analyze gene expression, however, the accuracy of the normalized results is affected by the expression stability of reference genes. Holotrichia oblita (Coleoptera: Scarabaeidae) causes serious damage to crops. Reliable reference genes in H. oblita are needed for qRT-PCR analysis. Therefore, we evaluated 13 reference genes under biotic and abiotic conditions. RefFinder provided a comprehensive stability ranking, and geNorm suggested the optimal number of reference genes for normalization. RPL13a and RPL18 were the most suitable reference genes for developmental stages, tissues, and temperature treatments; RPL13a and RPS3 were the most suitable for pesticide and photoperiod treatments; RPS18 and RPL18 were the most suitable for the two sexes. We validated the normalized results using odorant-binding protein genes as target genes in different tissues. Compared with the selected suitable reference genes, the expression of OBP1 in antennae, abdomen, and wings, and OBP2 in antennae and wings were overestimated due to the instability of ACTb. These results identified several reliable reference genes in H. oblita for normalization, and are valuable for future molecular studies., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: Competing interests: None declared.

Hippodamia variegata is one of the most commercialized ladybirds used for the biological control of aphid pest species in many economically important crops. This species is the first Coccinellidae whose satellitome has been studied by applying new sequencing technologies and bioinformatics tools. We found that 47% of the H. variegata genome is composed of repeated sequences. We identified 30 satellite DNA (satDNA) families with a median intragenomic divergence of 5.75% and A+T content between 45.6% and 74.7%. This species shows satDNA families with highly variable sizes although the most common size is 100-200 bp. However, we highlight the existence of a satDNA family with a repeat unit of 2 kb, the largest repeat unit described in Coleoptera. PCR amplifications for fluorescence in situ hybridization (FISH) probe generation were performed for the four most abundant satDNA families. FISH with the most abundant satDNA family as a probe shows its pericentromeric location on all chromosomes. This location is coincident with the heterochromatin revealed by C-banding and DAPI staining, also analyzed in this work. Hybridization signals for other satDNA families were located only on certain bivalents and the X chromosome. These satDNAs could be very useful as chromosomal markers due to their reduced location.

Maize (Zea mays L) is one of main nutrients sources for humans and animals worldwide. In Africa, storage of maize ensures food resources availability throughout the year. However, it often suffers losses exceeding 20% due to insects such as the larger grain borer, Prostephanus truncatus (Horn) (Coleoptera; Bostrichidae), major pest of stored maize in the tropical countries. This study aims to select resistant varieties to reduce maize storage losses and explain the physicochemical parameters role in grains susceptibility. In the first study, maize grains were artificially infested under no-choice method with insects. Susceptibility parameters such as weight loss, grain damage, number of emerged insects, median development time and susceptibility index varied significantly through maize varieties. Dobie susceptibility index (SI) was assessed as a major indicator of resistance. The most resistant varieties were Early-Thaï, DMR-ES and Tzee-Yellow. Conversely, Synth-9243, Obatampa and Synth-C varieties were susceptible. SWAN, Across-Pool and Tzee-White were classified as moderately resistant varieties. The insect reproductive potential was significantly different in the nine maize varieties and Early-Thaï, DMR-ES and Tzee-Yellow varieties were the least favourable host. To assess the relationship between grains physicochemical characteristics and varietal susceptibility, moisture, total phenolics, palmitic acid, proteins, amylose, density and grain hardness were evaluated according to standardized methods. Palmitic acid, SI, insects emerged and grain damage were significantly and positively correlated with each other, and negatively correlated with grains hardness, phenolics and amylose contents. Maize susceptibility index was significantly and negatively correlated to amylose, and phenolics contents and positively correlated to palmitic acid content. This study identified three resistant maize varieties to P. tuncatus and revealed that the major factors involved in this resistance were hardness, phenolic and amylose contents of grains., Competing Interests: The authors have declared that no competing interests exist.

Genes that affect adaptive traits have been identified, but our knowledge of the genetic basis of adaptation in a more general sense (across multiple traits) remains limited. We combined population-genomic analyses of evolve-and-resequence experiments, genome-wide association mapping of performance traits, and analyses of gene expression to fill this knowledge gap and shed light on the genomics of adaptation to a marginal host (lentil) by the seed beetle Callosobruchus maculatus . Using population-genomic approaches, we detected modest parallelism in allele frequency change across replicate lines during adaptation to lentil. Mapping populations derived from each lentil-adapted line revealed a polygenic basis for two host-specific performance traits (weight and development time), which had low to modest heritabilities. We found less evidence of parallelism in genotype-phenotype associations across these lines than in allele frequency changes during the experiments. Differential gene expression caused by differences in recent evolutionary history exceeded that caused by immediate rearing host. Together, the three genomic datasets suggest that genes affecting traits other than weight and development time are likely to be the main causes of parallel evolution and that detoxification genes (especially cytochrome P450s and beta-glucosidase) could be especially important for colonization of lentil by C. maculatus .

The African coffee white stem borer Monochamus leuconotus (Pascoe) (Coleoptera: Cerambycidae) is a destructive insect pest of Arabica coffee trees in African highlands. Our study aims to provide information on the pest biology as influenced by temperature, determine thermal thresholds, and provide life table parameters for M. leuconotus reared in the laboratory. The life cycle of M. leuconotus was studied at seven constant temperatures in the range 15-35 °C, with 80 ± 5% RH and a photoperiod of L:D 12:12. Linear and nonlinear models were fitted to laboratory data to describe the impact of temperature on M. leuconotus development, mortality, fecundity and senescence. The complete life cycle was obtained between 18 and 30 °C, with the egg incubation period ranging 10.8-29.2 days. The development time was longest for the larva, with 194.2 days at 30 °C and 543.1 days at 18 °C. The minimum temperature threshold (T min ) was estimated at 10.7, 10.0 and 11.5 °C, for egg, larva and pupa, respectively. The maximum temperature threshold (T max ) was estimated at 37.4, 40.6 and 40.0 °C for egg, larva and pupa, respectively. The optimum temperature for immature stage survival was estimated between 23.0 and 23.9 °C. The highest fecundity was 97.8 eggs per female at 23 °C. Simulated life table parameters showed the highest net reproductive rate (R o ) of 11.8 daughters per female at 26 °C and maximal intrinsic rate of increase (r m ) between 26 and 28 °C, with a value of 0.008. Our results will help understanding M. leuconotus biology as influenced by temperature and may be used to predict the distribution and infestation risk under climate warming for this critical coffee pest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Zygogramma bicolorata Pallister (Coleoptera: Chrysomelidae) is an effective biocontrol agent of Parthenium hysterophorus L. which is an alien invasive herbaceous weed with a pan-tropical distribution. The present study aimed to assess the effects of temperature and altitude on feeding attributes (consumption rate, conversion efficiency and growth rate) of adults from the wild populations of Z. bicolorata inhabiting India and Nepal. Results revealed that adults inhabiting areas of low temperature (24°C ‒ 25°C) and high altitude (415 m ‒1400 m) were large and had higher food consumption rates. In contrast, those inhabiting areas of high temperature (34°C ‒ 36°C) and low altitude (81 m ‒ 229 m) were smaller and had higher food utilization efficiencies. In all the eco-climatic regions, females were larger than males and had higher feeding attributes than their counterparts. Temperature between 27°C and 30°C was found optimal for Z. bicolorata adults to convert and utilize the food biomass to body mass. Above the optimal temperature the feeding attributes decreased. Present results suggest that there exists a possibility for decrease in body size, and thereby weed biocontrol efficiency of Z. bicolorata adults with an increase in temperature due to global climate change., Competing Interests: Declaration of competing interest NONE., (Copyright © 2020. Published by Elsevier Ltd.)

Two bruchid species, azuki bean weevil (Callosobruchus chinensis L.) and cowpea weevil (Callosobruchus maculatus F.), are the most important insect pests of mungbean [Vigna radiata (L.) Wilczek] after harvest. Improving bruchid resistance is a major goal for mungbean breeders. Bruchid resistance in mungbean is controlled by a single major locus, Br. The tightly linked VrPGIP1 and VrPGIP2, which encode polygalacturonase-inhibiting proteins (PGIPs), are the candidate genes at the Br locus associated with bruchid resistance. One VrPGIP1 resistance allele and two VrPGIP2 resistance alleles have been identified. In this study, we fine-mapped the bruchid-resistance genes in wild mungbean (V. radiata var. sublobata) accession ACC41 using the F 2 population (574 individuals) derived from the 'Kamphaeng Saen 2' (susceptible) × ACC41 (resistant) cross. A QTL analysis indicated that the resistance to the azuki bean weevil and cowpea weevil in ACC41 is controlled by a major QTL (qBr5.1) and a minor QTL (qBr5.2), which are only 0.3 cM apart. qBr5.1 and qBr5.2 accounted for about 82% and 2% of the resistance variation in the F 2 population, respectively. qBr5.1 was mapped to a 237.35-kb region on mungbean chromosome 5 containing eight annotated genes, including VrPGIP1 and VrPGIP2. An examination of the ACC41 VrPGIP1 and VrPGIP2 sequences revealed a new allele for VrPGIP1 (i.e., VrPGIP1-2). Compared with the wild-type sequence, VrPGIP1-2 has five SNPs, of which four cause amino acid changes (residues 125, 129, 188, and 336). A protein sequence analysis indicated that residues 125 and 129 in VrPGIP1-2 are in a β-sheet B1 region, whereas residues 188 and 336 are in a C 10 -helix region and at the end of the C-terminal region, respectively. Because the β-sheet B1 region is important for interactions with polygalacturonase (PG), residues 125 and 129 in VrPGIP1-2 likely contribute to bruchid resistance by inhibiting PG. Our results imply that VrPGIP1-2 is associated with the bruchid resistance of wild mungbean accession ACC41. This new resistance allele may be useful for breeding mungbean varieties exhibiting durable bruchid resistance.

Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae and Platypodinae) bore into tree xylem to complete their life cycle, feeding on symbiotic fungi. Ambrosia beetles are a threat to avocado where they have been found to vector a symbiotic fungus, Raffaelea lauricola, the causal agent of the laurel wilt disease. We assessed the repellency of methyl salicylate and verbenone to two putative laurel wilt vectors in avocado, Xyleborus volvulus (Fabricius) and Xyleborus bispinatus (Eichhoff), under laboratory conditions. Then, we tested the same two chemicals released from SPLAT flowable matrix with and without low-dose ethanol dispensers for manipulation of ambrosia beetle populations occurring in commercial avocado. The potential active space of repellents was assessed by quantifying beetle catch on traps placed 'close' (~5-10 cm) and 'far' (~1-1.5 m) away from repellent dispensers. Ambrosia beetles collected on traps associated with all in-field treatments were identified to species to assess beetle diversity and community variation. Xyleborus volvulus was not repelled by methyl salicylate (MeSA) or verbenone in laboratory assays, while X. bispinatus was repelled by MeSA but not verbenone. Ambrosia beetle trap catches were reduced in the field more when plots were treated with verbenone dispensers (SPLAT) co-deployed with low-dose ethanol dispensers than when treated with verbenone alone. Beetle diversity was highest on traps deployed with low-dose ethanol lures. The repellent treatments and ethanol lures significantly altered the species composition of beetles captured in experiment plots. Our results indicate that verbenone co-deployed with ethanol lures holds potential for manipulating ambrosia beetle vectors via push-pull management in avocado. This tactic could discourage immigration and/or population establishment of ambrosia beetles in commercial avocado and function as an additional tool for management programs of laurel wilt.

Despite its high ecological importance, the commensal interactions at community level are poorly studied. In tropical dry forests (TDF) there is a great diversity of species adapted to the high seasonality that characterizes them; however, little is known regarding how the spatial and temporal availability of resources generates changes in the pattern of commensal interactions. We experimentally studied changes in the diversity, composition, and pattern of interactions in spatio-temporal associations between the saproxylophagous beetles and their host trees in a TDF in Morelos, Mexico. A total of 65 host tree species were selected, from which 16 wood sections were obtained per species. These sections were exposed in the field to allow oviposition by the cerambycids under four different (spatio-temporal) treatments. We analyzed the network structure and generated indices at species level (i.e., specialization, species strength, and effective partners) and those related to physical characteristics of the wood (hardness and degradation rate) and the cerambycids (body size). In total, 1,323 individuals of 57 species of cerambycids emerged. Our results showed that, independently of the space and time, the network presented a nested and modular structure, with a high specialization degree and a high turnover of cerambycid species and their interactions. In general, we found that the cerambycids are mostly associated with softwood species with a lower decomposition rate of wood, as well as with the most abundant host species. The commensalistic interactions between the cerambycids and their host trees are highly specialized but are not spatio-temporally static. The high turnover in the interactions is caused by the emergence patterns of cerambycids, which seem to restrict their use to certain species. The knowledge of the spatio-temporal variation in Cerambycidae-host tree interactions allows us to predict how environmental and structural changes in the habitat can modify the species ensemble, and therefore its interactions., Competing Interests: The authors have declared that no competing interests exist.

The manipulation of animal behavior by parasitic organisms is one of the most complex adaptations to have arisen via natural selection. Among the most impressive examples of behavioral manipulation are the zombie-ant fungi [1]. In this association, ants are controlled to leave the colony and perform a stereotyped death grip behavior, where they bite onto vegetation over foraging trails, before being killed for the post mortem fungal growth. Manipulation functions to provide a platform outside the nest, from which fungal parasites actively shoot out spores, targeting foraging ants because within colony transmission is prevented by strong social immunity exhibited by social insect societies [2, 3]. It is not clear how such complex examples of host manipulation arose. To address this, we performed a broad-scale phylogenetic reconstruction of the order Hypocreales, to which the zombie-ant fungi, Ophiocordyceps, belong. In order to understand the patterns of host association and host switching along the evolution of Ophiocordyceps, we performed ancestral character state reconstruction analysis. We found that zombie-ant fungi likely arose from an ancestor that infected beetle larvae residing in soil or decaying wood, similar to extant beetle-infecting Ophiocordyceps species. Surprisingly, the jump led to an extensive species radiation observed after the development of behavioral manipulation. We suggest that the jump from solitary beetle larva to ants within a colony exposed the fungus to the robust social immunity of ant societies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Cry8-like from Bacillus thuringiensis (Bt) encodes an insecticidal crystal (Cry) protein. Holotrichia parallela (Coleoptera: Scarabaeoidae), commonly known as the dark black chafer, is a troublesome pest of soybean (Glycine max). To test whether cry8-like can confer resistance against H. parallela to soybean, we introduced cry8-like from the Bt strain HBF-18 into soybean cultivar Jinong 28., Results: Quantitative reverse transcription-PCR analysis demonstrated that cry8-like was expressed most highly in soybean leaves. In addition, Southern blot assays revealed that one copy of the integrated fragment was present in the transformed plants. Eight independent cry8-like transgenic lines were subsequently fed on by H. parallela. Under H. parallela feeding stress, the survival rates of the non-transgenic plants were 92% lower than those of the transgenic plants. The mortality rate of H. parallela increased when the larvae fed on the roots of T 1 transgenic soybean plants. Moreover, the surviving larvae were deformed, and their growth was inhibited., Conclusions: Collectively, our data suggest that transgenic soybean plants expressing the cry8-like gene are more resistant to H. parallela than non-transgenic plants and that transgenic expression of the cry8-like gene may represent a promising strategy for engineering pest tolerance. The events generated in this study could thus be utilized in soybean breeding programs.

The cabbage stem flea beetle (CSFB), Psylliodes chrysocephala (Coleoptera: Chrysomelidae), has recently become a major pest species in winter oilseed rape in the Czech Republic. The susceptibility of CSFB populations from two localities to six pyrethroids, two neonicotinoids, one organophosphate and one oxadiazine was evaluated in 2015-2018 in glass vial experiments. The susceptibility of CSFB to thiacloprid and thiamethoxam was evaluated in feeding experiment in 2017 and 2018. High susceptibility of CSFB populations to lambda-cyhalothrin, cypermethrin, esfenvalerate, tau-fluvalinate, etofenprox, deltamethrin, chlorpyrifos, indoxacarb and acetamiprid was observed in the glass vial experiments. The LC50 and LC90 data obtained for pyrethroids in these experiments in 2015 represent baseline for CSFB resistance monitoring to pyrethroids in the Czech Republic. High tolerance of CSFB to thiacloprid of CSFB was demonstrated in glass vial and the feeding experiment, too., Competing Interests: The authors have declared that no competing interests exist.

We studied seeds from a set of plant species from the Convolvulaceae family. Seeds collected from natural populations and infested with beetles of genus Megacerus were monitored until the beetle emergence. We analyze the relationship between body weight of beetles and seed weight of host plants, and its connection with between-species differences and sexual dimorphism. The results show that differences in the scaling of body weight of beetles are associated with sexual dimorphism. For the same species of beetle, the females tend to have heavier bodies than the males. Differences between host plants species in the weight of seeds are related to differences in the body weight Megacerus species, resulting in a distinctive pattern of seed infestation across hosts. Small-sized (lighter) species of beetles tended to infest small-sized (lighter) seed species and, correspondingly, heavier beetles species tended to do it in heavier seed species. Mechanisms of female oviposition preferences may be involved to generate that pattern. In general, the beetle weight showed an asymptotic relation with the host seed weight. The greater the weight of the seed, the greater the weight of adult beetle was. However, the proportion in weights reaches an asymptotic value probably because beetles reached the maximum possible weight for their species. We conclude that the process of specialization in the seed-beetle assemblage studied is influenced by intrinsic traits of the species involved in the interaction (beetles and seeds) and by differences between sexes in their sexual-allocation paths.

The mountain pine beetle (MPB; Dendroctonus ponderosae) is a forest insect pest that attacks several different pine (Pinus) species in its native range of distribution in western North America. MPB are exposed for most of their life cycle to the chemical defenses of their hosts. These defenses are dominated by oleoresin secretions containing mostly various monoterpenes and diterpene resin acids (DRAs). Cytochrome P450 enzymes (P450s) of the MPB are thought to be involved in the metabolism of at least some of these defense compounds. Here we describe the cloning and characterization of three MPB P450s, CYP6DJ1, CYP6BW1 and CYP6BW3, and their functions in the oxidation of various monoterpenes and diterpene resin acids. CYP6DJ1 oxidizes the monoterpenes (+)-(4R)-limonene, (-)-(4S)-limonene and terpinolene and produces (4R,8R)-limonene-8,9-epoxide, (4R,8S)-limonene-8,9-epoxide, (4S,8S)-limonene-8,9-epoxide, (4S,8R)-limonene-8,9-epoxide, perilla alcohol and several unidentified oxidized compounds. These products of CYP6DJ1 were also identified in extracts of MPB treated with the same monoterpenes. CYP6BW1 and CYP6BW3 both oxidize the DRAs abietic acid, dehydroabietic acid, neoabietic acid, levopimaric acid, palustric acid, and isopimaric acid, producing hydroxylated and epoxidized DRAs. CYP6DJ1, CYP6BW1 and CYP6BW3 appear to contribute to the metabolism of oleoresin terpenes as part of the MPB's ability to cope with host defenses., Competing Interests: The authors have declared that no competing interests exist.

Vigna unguiculata, one of the most important legumes, mainly in underdeveloped countries, is susceptible to post-harvest losses in storage by Callosobruchus maculatus (Fabricius, 1775) (Coleoptera: Chrysomelidae). The work evaluated the toxicity, inhibition of oviposition, instantaneous rate of population growth (r i ) and the development of fumigated C. maculatus with the essential oil of Vanillosmopsis arborea and its major constituent, α-bisabolol. The experimental units consisted of 0.8 L flasks treated with concentrations of 1.2-11.2 μL L -1 of air of the essential oil of V. arborea or its major constituent applied to disks of filter paper. α-Bisabolol was quantified as 409.33 mL L -1 of the essential oil. The development rate of C. maculatus was evaluated by daily adult counts. Oviposition was evaluated at lethal concentrations (LC 50 , LC 25 , LC 10 and LC 1 ). The LC 50 and LC 95 of the essential oil of V. arborea and α-bisabolol were 5.23 and 12.97 μL L -1 of air and 2.47 and 8.82 μL L -1 of air, respectively. At some concentrations, the α-bisabolol was more toxic to males than to females of the insect. Increased concentrations of the essential oil reduced the r i , rate of development, oviposition, and number of eggs of C. maculatus and therefore have potential for pest control.

Most of the commercialized insect resistant transgenic crops express cry gene(s) isolated from Bacillus thuringiensis; however, intensive cultivation of Bt crops over almost two decades has been questioned regarding its sustainability and durability in pest management. The present study focused on silencing of highly specific molting-associated Ecdysone receptor (EcR) gene of Colorado potato beetle (CPB) using RNA interference (RNAi) approach. The partial cDNA of EcR gene of CPB was amplified using specific primers in sense and anti-sense orientations, and cloned in pRNAi-GG vector flanked by an intronic sequence (pdk). Leaf and internodal explants of Agria and Lady Olympia potato cultivars were infected with Agrobacterium strain LBA4404 harboring constructs under the control of CaMV 35S promoter. Standard molecular analysis of primary transformants showed proper integration of T-DNA in plant genome. The transgenic plants of both cultivars were evaluated for their efficacy against first, second and third instar CPB larvae. The leaf biotoxicity assays revealed 15-80% of CPB mortality. A significantly lower fold-change (0.87-4.14×) in larval weight was observed in insects fed on transgenic plants compared to the ones fed on control plants (1.87-6.53×). Furthermore, CPB larvae fed on transgenic plants exhibited reduced EcR transcripts, indicating the functionality of dsRNA EcR in silencing EcR gene expression. This study is an excellent example of the integration of an alternative, effective and reliable method to cope with potato insect pests that incur significant losses to potato production in the world.

Volatiles are often released upon herbivory as plant defense compounds. While the formation of volatiles above-ground has been intensively studied, little is known about herbivore-induced root volatiles. Here, we show that cockchafer larvae-damaged roots of Populus trichocarpa and P. nigra release a mixture of monoterpenes, including (-)-α-pinene, (-)-camphene, (-)-β-pinene, p-cymene, and 1,8-cineole. Three terpene synthases, PtTPS16 and PtTPS21 from P. trichocarpa and PnTPS4 from P. nigra, could be identified and characterized in vitro. PnTPS4 was found to produce 1,8-cineole as sole product. PtTPS16 and PtTPS21, although highly similar to each other, showed different product specificities and produced γ-terpinene and a mixture of (-)-camphene, (-)-α-pinene, (-)-β-pinene, and (-)-limonene, respectively. Four active site residues were found to determine the different product specificities of the two enzymes. The expression profiles of PtTPS16, PtTPS21, and PnTPS4 in undamaged and herbivore-damaged poplar roots generally matched the emission pattern of monoterpenes, indicating that monoterpene emission in roots is mainly determined at the gene transcript level. Bioassays with Phytophtora cactorum (Oomycetes) revealed inhibitory effects of vapor-phase 1,8-cineole and (-)-β-pinene on the growth of this important plant pathogen. Thus herbivore-induced volatile monoterpenes may have a role in defense against pathogens that cause secondary infections after root wounding.

The premises of stored agricultural products and food consists of a complex ecosystem in which several pests can seriously affect the quality and quantity of the products. In this study we utilize a 4-level hierarchical linear multilevel model in order to assess the effect of temperature, relative humidity (RH) and interspecific competition on the population size and damage potential of the larger grain borer, Prostephanus truncatus (Horn) (Coleoptera: Bostrychidae) and the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae). As RH was increased, we observed higher percentage of live insects, while increased levels of temperature significantly decreased the percentage of live insects. The combination of R. dominica and P. truncatus lead to reduction of the percentages of live insects in comparison to single species treatments. However, P. truncatus is more damaging than R. dominica in maize, based on the proportion of damaged kernels which were infested by each insect species. We expect our results to have bearing in the management of these species., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Vicilins are 7S globulins which constitute the major seed storage proteins in leguminous species. Variant vicilins showing differential binding affinities for chitin have been implicated in the resistance and susceptibility of cowpea to the bruchid Callosobruchus maculatus. These proteins are members of the cupin superfamily, which includes a wide variety of enzymes and non-catalytic seed storage proteins. The cupin fold does not share similarity with any known chitin-biding domain. Therefore, it is poorly understood how these storage proteins bind to chitin. In this work, partial cDNA sequences encoding β-vignin, the major component of cowpea vicilins, were obtained from developing seeds. Three-dimensional molecular models of β-vignin showed the characteristic cupin fold and computational simulations revealed that each vicilin trimer contained 3 chitin-binding sites. Interaction models showed that chito-oligosaccharides bound to β-vignin were stabilized mainly by hydrogen bonds, a common structural feature of typical carbohydrate-binding proteins. Furthermore, many of the residues involved in the chitin-binding sites of β-vignin are conserved in other 7S globulins. These results support previous experimental evidences on the ability of vicilin-like proteins from cowpea and other leguminous species to bind in vitro to chitin as well as in vivo to chitinous structures of larval C. maculatus midgut., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Insect pests are one of the factors that most impact plant yield. The magnitude of the losses and the spatiotemporal pest distribution in crops is a result of their interactions with the environment. Therefore, the understanding of the causes of production losses and the pest spatial patterns is important for the development of suitable sampling plans and pest management programs. Thus, this study aimed to quantify grain losses caused by insects and to determine the spatial distribution pattern of arthropod pest species in Bt and non-Bt corn. The prevailing insect pests in the corn ears were the earworm and fall armyworm caterpillars (Helicoverpa spp. and Spodoptera frugiperda), the cornsilk fly (Euxesta spp.), the maize weevil (Sitophilus zeamais), and the square-necked grain beetle (Cathartus quadricollis). The non-Bt corn was more attacked by the caterpillars and the weevil, while Bt corn was more affected by the cornsilk fly Euxesta spp. Spatial dependence was significant for the damage caused by the caterpillars, the grain beetle and the maize weevil in both the Bt and non-Bt corn genotypes. The range of the damage caused by the insects was between 9.0-9.7 m for the caterpillars, 6.9-12.20 m for the cornsilk fly, 10.7-80.4 m for the square-necked grain beetle, and 51.9-170.7 m for the maize weevil. The pattern of the spatial distribution of pest damage in both corn genotypes (i.e., Bt and non-Bt corn) was similar with a prevalence of moderate to strong spatial dependence and aggregate damage distribution. The plants near to the sampling points exhibited injury and infestation levels similar to those of the sampled plants., Competing Interests: The authors have declared that no competing interests exist.

In this paper, we present a new method for the prediction and uncertainty quantification of data-driven multivariate systems. Traditionally, either mechanistic or non-mechanistic modeling methodologies have been used for prediction; however, it is uncommon for the two to be incorporated together. We compare the forecast accuracy of mechanistic modeling, using Bayesian inference, a non-mechanistic modeling approach based on state space reconstruction, and a novel hybrid methodology composed of the two for an age-structured population data set. The data come from cannibalistic flour beetles, in which it is observed that the adults preying on the eggs and pupae result in non-equilibrium population dynamics. Uncertainty quantification methods for the hybrid models are outlined and illustrated for these data. We perform an analysis of the results from Bayesian inference for the mechanistic model and hybrid models to suggest reasons why hybrid modeling methodology may enable more accurate forecasts of multivariate systems than traditional approaches.

The hispid leaf beetle, Octodonta nipae (Maulik), (Coleoptera: Chrysomelidae), is a devastating pest of palm cultivation worldwide. Endosymbiotic bacteria in the genus Wolbachia are arguably one of the most abundant bacterial group associated with arthropods. Owing to its critical effects on host reproduction, Wolbachia has garnered much attention as a prospective future tool for insect pest management. However, their association, infection dynamics, and functionality remain unknown in this insect pest. Here, we diagnosis for the first time, the infection prevalence, and occurrence of Wolbachia in O. nipae. Experimental evidence by the exploration of wsp gene vindicate that O. nipae is naturally infected with bacterial symbiont of genus Wolbachia, showing a complete maternal inheritance with shared a common Wolbachia strain (wNip). Moreover, MLST (gatB, fbpA, coxA, ftsZ, and hcpA) analysis enabled the detections of new sequence type (ST-484), suggesting a particular genotypic association of O. nipae and Wolbachia. Subsequently, quantitative real-time PCR (qPCR) assay demonstrated variable infection density across different life stages (eggs, larvae, pupae and adult male and female), body parts (head, thorax, abdomen), and tissues (ovaries, testes, and guts). Infection density was higher in egg and female adult stage, as well as abdomen and reproductive tissues as compared to other samples. Interestingly, Wolbachia harbored dominantly in a female than the male adult, while, no significant differences were observed between male and female body parts and tissues. Phylogeny of Wolbachia infection associated with O. nipae rectified from all tested life stages were unique and fall within the same monophyletic supergroup-A of Wolbachia clades. The infection density of symbiont is among the valuable tool to understand their biological influence on hosts, and this latest discovery would facilitate the future investigations to understand the host-symbiont complications and its prospective role as a microbiological agent to reduce pest populations., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Previously, transgenic trichome-bearing (hairy leaf) Brassica napus lines expressing either the Arabidopsis thaliana GL3 gene (line AtGL3+) [1] or the AtGL3 gene in combination with an RNAi construct to down-regulate TTG1 (line K-5-8) [2] were developed. The leaves of these lines exhibited altered insect feeding (flea beetle) and oviposition (diamondback moth) behaviour compared to the non-transgenic semi-glabrous leaves of B. napus cv. Westar. Interestingly, the cotyledons of these lines remained glabrous, but also showed reduced feeding by flea beetles. Here we examine the composition and global transcriptome of the glabrous cotyledons from these transgenic lines to ascertain the mechanism(s) underlying this unexpected phenomenon., Results: Approximately, 7500 genes were up-regulated in cotyledons of each hairy line, compared with < 30 that were down-regulated. The up-regulated genes included those involved in cell wall synthesis, secondary metabolite production, redox, stress and hormone-related responses that have the potential to impact host plant cues required to elicit defense responses toward insect pests. In particular, the expression of glucosinolate biosynthetic and degradation genes were substantially altered in the glabrous cotyledons of the two hairy leaf lines. The transcriptomic data was supported by glucosinolate and cell wall composition profiles of the cotyledons. Changes in gene expression were much more extreme in the AtGL3+ line compared with the K-5-8 line in terms of diversity and intensity., Conclusions: The study provides a roadmap for the isolation and identification of insect resistance compounds and proteins in the glabrous cotyledons of these hairy leaf lines. It also confirms the impact of mis-expression of GL3 and TTG1 on types of metabolism other than those associated with trichomes. Finally, the large number of up-regulated genes encoding heat shock proteins, PR proteins, protease inhibitors, glucosinolate synthesis/breakdown factors, abiotic stress factors, redox proteins, transcription factors, and proteins required for auxin metabolism also suggest that these cotyledons are now primed for resistance to other forms of biotic and abiotic stress.

Olfaction is a pivotal sense for insects and granivorous pests may exploit grain volatiles for food selection. Tribolium confusum, is a secondary pest of stored cereals that benefits from primary pests' infestation, as other secondary feeders, triggering competition. This study aimed to evaluate the preferences of T. confusum females toward different-infested paddy rice, highlighting the impact of intra- and interspecific competition. Tribolium confusum showed positive chemotaxis toward rice infested by larvae of a primary pest (Sitophilus zeamais), but not for grain attacked by adults alone. Furthermore, kernels concurrently infested by a primary (S. zeamais) and a secondary pest (T. confusum or Cryptolestes ferrugineus) were evaluated in Y-tube bioassays, highlighting that both food-sources were innately attractive for T. confusum females. Moreover, females positively oriented toward rice infested by conspecifics, while they avoided grain infested by C. ferrugineus, averting an extremely competitive habitat. Behavioural responses of T. confusum females and volatile emissions of different-infested rice highlighted the occurrence of plant-mediated interactions among insects from the same trophic guild. Seventy volatiles were identified and significant differences among the tested food-sources were recorded, emphasizing the presence of 6 putative attractants and 6 repellents, which may be useful biocontrol tools.

Costalimaita ferruginea (Coleoptera: Chrysomelidae) attacks Eucalyptus plants causing severe damage through netting of the leaves. Recently, this Coleoptera has been reported attacking Myrtaceae in Mato Grosso State and, studies about the occurrence of this beetle in commercial plantations of eucalypts has been the subject of researchers through monitoring programmes in the forest protection area. With the beginning of the rainy season, adults were observed causing damage in eucalypt plantations in four cities that are part of the transition region of Amazon and Cerrado Biomes. The spots where these insects were observed are located in Feliz Natal, Lucas do Rio Verde, Sorriso and Vera. The purpose of this study was to report the new occurrences and to characterize the attack period of the beetle C. ferruginea in Eucalyptus plantations in Middle-North region of Mato Grosso State, region of Biomes Transition.

Chemical induction can drive tree susceptibility to and host range expansions of attacking insects and fungi. Recently, mountain pine beetle (Dendroctonus ponderosae Hopkins; MPB) has expanded its host range from its historic host lodgepole pine (Pinus contorta var. latifolia Douglas ex Loudon) to jack pine (Pinus banksiana Lamb) in western Canada. Beetle success in jack pine forests likely depends upon the suitability of tree chemistry to MPB and its symbiotic phytopathogenic fungi. In particular, how rapid induced defenses of jack pine affect MPB colonization and the beetle's symbionts is unknown. In the field, we characterized and compared differences in rapid induced phloem monoterpenes between lodgepole and jack pines in response to various densities of Grosmannia clavigera (Robinson-Jeffery and Davidson)-a MPB symbiotic fungus used to simulate beetle attack-inoculations. Overall, lodgepole pine had higher limonene and myrcene, but lower α-pinene, concentrations than jack pine. However, myrcene concentrations in jack pine increased with inoculation density, while that in lodgepole pine did not respond to density treatments. We compared the growth and reproduction of MPB's symbiotic fungi, G. clavigera, Ophiostoma montium (Rumford) von Arx and Leptographium longiclavatum Lee, Kim and Breuil, grown on media amended with myrcene, α-pinene and limonene at concentrations reflecting two induction levels from each pine species. Myrcene and α-pinene amendments inhibited the growth but stimulated the reproduction of G. clavigera, whereas limonene stimulated its growth while inhibiting its reproduction. However, the growth and reproduction of the other fungi were generally stimulated by monoterpene amendments. Overall, our results suggest that jack pine rapid induction could promote MPB aggregation due to high levels of α-pinene (pheromone precursor), a positive feedback of myrcene (pheromone synergist) and low levels of limonene (resistance). Jack pine is likely as susceptible to MPB-vectored fungi as lodgepole pine, indicating that jack pine induction will likely not adversely affect symbiont activities enough to inhibit the invasion of MPB into jack pine forests., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Both mountain pine beetle (MPB) Dendroctonus ponderosae Hopkins and fire leave scars with similar appearance on lodgepole pine Pinus contorta Dougl. ex Loud. var. latifolia Engelm. that have never been compared microscopically, despite the pressing need to determine the respective effects of MPB and fire injury on tree physiology. We analysed changes in wood formation in naturally caused scars on lodgepole pine, and tested the hypotheses that (i) MPB and fire injury elicit distinct anomalies in lodgepole pine wood and (ii) anomalies differ in magnitude and/or duration between MPB and fire. Mountain pine beetle and fire injury reduced radial growth in the first year post-injury. Otherwise, radial growth and wood density increased over more than 10 years in both MPB and fire scars. We found that the general increase in radial growth was of greater magnitude (up to 27%) and of longer duration (up to 5 years) in fire scars compared with MPB scars, as shown in earlywood width. We also observed that the increase in latewood density was of greater magnitude (by 12%) in MPB scars, but of longer duration (by 4 years) in fire scars. Crystallinity decreased following MPB and fire injury, while microfibril angle increased. These changes in fibre traits were of longer duration (up to 4 years) in MPB scars compared with fire scars, as shown in microfibril angle. We found no significant changes in carbon and nitrogen concentrations. In conclusion, we stress that reduced competition and resistance to cavitation play an important role alongside cambial injury in influencing the type and severity of changes. In addition, more research is needed to validate the thresholds introduced in this study. Our findings serve as a foundation for new protocols to distinguish between bark beetle and fire disturbance, which is essential for improving our knowledge of historical bark beetle and fire regimes, and their interactions., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

RNA interference (RNAi) in transgenic maize has recently emerged as an alternative mode of action for western corn rootworm (Diabrotica virgifera virgifera) control which can be combined with protein-based rootworm control options for improved root protection and resistance management. Currently, transgenic RNAi-based control has focused on suppression of genes that when silenced lead to larval mortality. We investigated control of western corn rootworm reproduction through RNAi by targeting two reproductive genes, dvvgr and dvbol, with the goal of reducing insect fecundity as a new tool for pest management. The results demonstrated that exposure of adult beetles, as well as larvae to dvvgr or dvbol dsRNA in artificial diet, caused reduction of fecundity. Furthermore, western corn rootworm beetles that emerged from larval feeding on transgenic maize roots expressing dvbol dsRNA also showed significant fecundity reduction. This is the first report of reduction of insect reproductive fitness through plant-mediated RNAi, demonstrating the feasibility of reproductive RNAi as a management tool for western corn rootworm.

Insect-plant interactions are governed by a complex equilibrium between the mechanisms through which plant recognize insect attack and orchestrate downstream signaling events that trigger plant defense responses, and the mechanisms by which insects overcome plant defenses. Due to this tight and dynamic interplay, insight into the nature of the plant defense response can be gained by analyzing changes in the insect herbivores digestive system upon plant feeding. In this work we have identified a Solanum melongena miraculin-like protease inhibitor in the midgut juice of Colorado potato larvae feeding on eggplant plants treated with the natural inducer of plant defenses hexanoic acid. We analyzed the corresponding gene expression by qRT-PCR and our results showed that this eggplant miraculin-like gene enhanced induction contributes to the hexanoic acid priming effect in this Solanaceae species. Moreover, our data evidencing that OPDA might be involved in this gene regulation highlights its potential as biomarker in eggplant plant responses to stress mediated this oxylipin signaling pathway., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Lobiopa insularis is a newly reported pest of strawberry in Argentina. We investigated characteristics of its biology in the laboratory, including survivorship and reproduction. We also estimated population growth for L. insularis fed ripe strawberry fruits. Lobiopa insularis was not observed ovipositing on strawberry fruits. A higher proportion of egg masses were recorded from a depth of 1 cm within the soil than on either the soil surface or deeper than 1cm (i.e. between 1and 2 cm) within the soil. The duration of preimaginal developmental stages represented ~18.5% of the total life cycle, while the adult stage represented 81.5%. Survival from egg to adult was 64.20% and mean longevity of females and males adults was 121.84, (SE = 8.86) and 118.58 (SE = 5.90) days, respectively. Females laid eggs only when they were with a male, so reproductive period was dependent on male presence. The number of eggs/female/day was 18.01 (SE = 1.71); and total fecundity was 1655 (ES = 249.53) eggs/female. The long life span of adults and high reproductive output, i.e high fecundity and long reproductive period, indicate that availability and concentration of suitable developmental resources are important factors in the population dynamics of Lobiopa insularis associated with strawberry crops.

Crops expressing Bacillus thuringiensis (Bt)-derived insecticidal protein genes have been commercially available for over 15 years and are providing significant value to growers. However, there remains the need for alternative insecticidal actives due to emerging insect resistance to certain Bt proteins. A screen of bacterial strains led to the discovery of a two-component insecticidal protein named AfIP-1A/1B from an Alcaligenes faecalis strain. This protein shows selectivity against coleopteran insects including western corn rootworm (WCR). Transgenic maize plants expressing AfIP-1A/1B demonstrate strong protection from rootworm injury. Surprisingly, although little sequence similarity exists to known insecticidal proteins, efficacy tests using WCR populations resistant to two different Cry proteins show that AfIP-1A/1B and mCry3A differ in their mode of action while AfIP-1A/1B and the binary Cry34Ab1/Cry35Ab1 protein share a similar mode. These findings are supported by results of competitive binding assays and the similarity of the x-ray structure of AfIP-1A to Cry34Ab1. Our work indicates that insecticidal proteins obtained from a non-Bt bacterial source can be useful for developing genetically modified crops and can function similarly to familiar proteins from Bt.

American Elms were devastated by an outbreak of Dutch Elm Disease is caused by the fungus Ophiostoma novo-ulmi Brasier that originated in Asia and arrived in the early 1900s. In spite of decades of study, the specific mechanisms and disease resistance in some trees is not well understood. the fungus is spread by several species of bark beetles in the genus Scolytus, during their dispersal and feeding. Our objective was to understand elm responses to beetle feeding in the absence of the fungus to identify potential resistance mechanisms. A colony of Scolytus multistriatus was established from wild-caught beetles and beetles were co-incubated with susceptible or resistant American elm varieties in a controlled environment chamber. Beetles burrowed into the auxillary meristems of the young elm shoots. The trees responded to the beetle damage by a series of spikes in the concentration of plant growth regulating compounds, melatonin, serotonin, and jasmonic acid. Spikes in melatonin and serotonin represented a 7,000-fold increase over resting levels. Spikes in jasmonic acid were about 10-fold higher than resting levels with one very large spike observed. Differences were noted between susceptible and resistant elms that provide new understanding of plant defenses.

Sugarcane, a major crop grown in the tropical and subtropical areas of the world, is produced mainly for sucrose, which is used as a sweetener or for the production of bioethanol. Among the numerous pests that significantly affect the yield of sugarcane, the sugarcane rhizome borer (Migdolus fryanus, a cerambycidae beetle) is known to cause severe damage to the crops in Brazil. The absence of molecular information about this insect reinforces the need for studies and an effective method to control this pest. In this study, RNA-Seq technology was employed to study different parts of M. fryanus larvae. The generated data will help in further investigations about the taxonomy, development, and adaptation of this insect. RNA was extracted from six different parts (head, fat body, integument, hindgut, midgut, and foregut) using Trizol methodology. Using Illumina paired-end sequencing technology and the Trinity platform, trimming and de novo assembly was performed, resulting in 44,567 contigs longer than 200 nt for a reunion of data from all transcriptomes, with a mean length of 1,095.27 nt. Transcripts were annotated using BLAST against different protein databanks (Uniprot/Swissprot, PFAM, KEEG, SignalP 4.1, Gene Ontology, and CAZY) and were compared for similarity using a Venn diagram. Differential expression patterns were studied for select genes through qPCR and FPKM comprising important protein families (digestive peptidases, glucosyl hydrolases, serine protease inhibitors and otopetrin), which allowed a better understanding of the insect's digestion, immunity and gravity sensorial mechanisms.

Background and Methodology: There is a continuing need to express new insect control compounds in transgenic maize against western corn rootworm, Diabrotica virgifera virgifera (LeConte) (WCR). In this study three experiments were conducted to determine cross-resistance between the new insecticidal DvSnf7 dsRNA, and Bacillus thuringiensis (Bt) Cry3Bb1; used to control WCR since 2003, with field-evolved resistance being reported. Laboratory susceptible and Cry3Bb1-resistant WCR were evaluated against DvSnf7 dsRNA in larval diet-incorporation bioassays. Additionally, the susceptibility of seven field and one field-derived WCR populations to DvSnf7 (and Cry3Bb1) was assessed in larval diet-overlay bioassays. Finally, beetle emergence of laboratory susceptible and Cry3Bb1-resistant WCR was evaluated with maize plants in the greenhouse expressing Cry3Bb1, Cry34Ab1/Cry35Ab1, or DvSnf7 dsRNA singly, or in combination., Principal Findings and Conclusions: The Cry3Bb1-resistant colony had slight but significantly (2.7-fold; P<0.05) decreased susceptibility to DvSnf7 compared to the susceptible colony, but when repeated using a field-derived WCR population selected for reduced Cry3Bb1 susceptibility, there was no significant difference (P<0.05) in DvSnf7 susceptibility compared to that same susceptible population. Additionally, this 2.7-fold difference in susceptibility falls within the range of DvSnf7 susceptibility among the seven field populations tested. Additionally, there was no correlation between susceptibility to DvSnf7 and Cry3Bb1 for all populations evaluated. In greenhouse studies, there were no significant differences (P<0.05) between beetle emergence of susceptible and Cry3Bb1-resistant colonies on DvSnf7 and Cry34Ab1/Cry35Ab1, and between DvSnf7 and MON 87411 (DvSnf7 + Cry3Bb1) for the Cry3Bb1-resistant colony. These results demonstrate no cross-resistance between DvSnf7 and Cry3Bb1 against WCR. Therefore, pyramiding DvSnf7 with Bt proteins such as Cry3Bb1 and Cry34Ab1/Cry35Ab1 will provide a valuable IRM tool against WCR that will increase the durability of these Bt proteins. These results also illustrate the importance of using appropriate bioassay methods when characterizing field-evolved resistant WCR populations., Competing Interests: Competing Interests: All authors are employed by Monsanto Co. and the goal of this manuscript was to support the insect resistant management (IRM) components of the next generation of corn rootworm control product; SmartStax® PRO. This does not alter our adherence to PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Background: Relatively little is known about the genomic basis and evolution of wood-feeding in beetles. We undertook genome sequencing and annotation, gene expression assays, studies of plant cell wall degrading enzymes, and other functional and comparative studies of the Asian longhorned beetle, Anoplophora glabripennis, a globally significant invasive species capable of inflicting severe feeding damage on many important tree species. Complementary studies of genes encoding enzymes involved in digestion of woody plant tissues or detoxification of plant allelochemicals were undertaken with the genomes of 14 additional insects, including the newly sequenced emerald ash borer and bull-headed dung beetle., Results: The Asian longhorned beetle genome encodes a uniquely diverse arsenal of enzymes that can degrade the main polysaccharide networks in plant cell walls, detoxify plant allelochemicals, and otherwise facilitate feeding on woody plants. It has the metabolic plasticity needed to feed on diverse plant species, contributing to its highly invasive nature. Large expansions of chemosensory genes involved in the reception of pheromones and plant kairomones are consistent with the complexity of chemical cues it uses to find host plants and mates., Conclusions: Amplification and functional divergence of genes associated with specialized feeding on plants, including genes originally obtained via horizontal gene transfer from fungi and bacteria, contributed to the addition, expansion, and enhancement of the metabolic repertoire of the Asian longhorned beetle, certain other phytophagous beetles, and to a lesser degree, other phytophagous insects. Our results thus begin to establish a genomic basis for the evolutionary success of beetles on plants.

Genetically modified (GM) crops have been developed and commercialized that utilize double stranded RNAs (dsRNA) to suppress a target gene(s), producing virus resistance, nutritional and quality traits. MON 87411 is a GM maize variety that leverages dsRNAs to selectively control corn rootworm through production of a 240 base pair (bp) dsRNA fragment targeting for suppression the western corn rootworm (Diabrotica virgifera virgifera) Snf7 gene (DvSnf7). A bioinformatics assessment found that endogenous corn small RNAs matched ∼450 to 2300 unique RNA transcripts that likely code for proteins in rat, mouse, and human, demonstrating safe dsRNA consumption by mammals. Mice were administered DvSnf7 RNA (968 nucleotides, including the 240 bp DvSnf7 dsRNA) at 1, 10, or 100 mg/kg by oral gavage in a 28-day repeat dose toxicity study. No treatment-related effects were observed in body weights, food consumption, clinical observations, clinical chemistry, hematology, gross pathology, or histopathology endpoints. Therefore, the No Observed Adverse Effect Level (NOAEL) for DvSnf7 RNA was 100 mg/kg, the highest dose tested. These results demonstrate that dsRNA for insect control does not produce adverse health effects in mammals at oral doses millions to billions of times higher than anticipated human exposures and therefore poses negligible risk to mammals., (Copyright © 2016 Monsanto Company. Published by Elsevier Inc. All rights reserved.)

MON 87411 maize, which expresses DvSnf7 RNA, was developed to provide an additional mode of action to confer protection against corn rootworm (Diabrotica spp.). A critical step in the registration of a genetically engineered crop with an insecticidal trait is performing an ecological risk assessment to evaluate the potential for adverse ecological effects. For MON 87411, an assessment plan was developed that met specific protection goals by characterizing the routes and levels of exposure, and testing representative functional taxa that would be directly or indirectly exposed in the environment. The potential for toxicity of DvSnf7 RNA was evaluated with a harmonized battery of non-target organisms (NTOs) that included invertebrate predators, parasitoids, pollinators, soil biota as well as aquatic and terrestrial vertebrate species. Laboratory tests evaluated ecologically relevant endpoints such as survival, growth, development, and reproduction and were of sufficient duration to assess the potential for adverse effects. No adverse effects were observed with any species tested at, or above, the maximum expected environmental concentration (MEEC). All margins of exposure for NTOs were >10-fold the MEEC. Therefore, it is reasonable to conclude that exposure to DvSnf7 RNA, both directly and indirectly, is safe for NTOs at the expected field exposure levels., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

T1C-19 is newly developed transgenic rice active against lepidopteran pests, and expresses a synthesized cry1C gene driven by the maize ubiquitin promoter. The brown planthopper, Nilaparvata lugens, is a major non-target pest of rice, and the rove beetle (Paederus fuscipes) is a generalist predator of N. lugens nymphs. As P. fuscipes may be exposed to the Cry1C protein through preying on N. lugens, it is essential to assess the potential effects of transgenic cry1C rice on this predator. In this study, two experiments (a direct feeding experiment and a tritrophic experiment) were conducted to evaluate the ecological risk of cry1C rice to P. fuscipes. No significant negative effects were observed in the development, survival, female ratio and body weight of P. fuscipes in both treatments of direct exposure to elevated doses of Cry1C protein and prey-mediated exposure to realistic doses of the protein. This indicated that cry1C rice had no detrimental effects on P. fuscipes. This work represents the first study of an assessment continuum for the effects of transgenic cry1C rice on P. fuscipes. Use of the rove beetle as an indicator species to assess potential effects of genetically modified crops on non-target arthropods is feasible.

Plants have developed biochemical responses to adapt to biotic stress. To characterize the resistance mechanisms in poplar tree against Apripona germari, comprehensive metabolomic changes of poplar bark and xylem in response to A. germari infection were examined by gas chromatography time-of-flight mass spectrometry (GC-TOF/MS). It was found that, four days after feeding (stage I), A. germari infection brought about changes in various metabolites, such as phenolics, amino acids and sugars in both bark and xylem. Quinic acid, epicatechin, epigallocatechin and salicin might play a role in resistance response in bark, while coniferyl alcohol, ferulic acid and salicin contribute resistance in xylem. At feeding stages II when the larvae fed for more than one month, fewer defensive metabolites were induced, but levels of many intermediates of glycolysis and the tricarboxylic acid (TCA) cycle were reduced, especially in xylem. These results suggested that the defense strategies against A. germari might depend mainly on the early defense responses in poplar. In addition, it was found that bark and xylem in infected trees accumulated higher levels of salicylic acid and 4-aminobutyric acid, respectively, these tissues displaying a direct and systemic reaction against A. germari. However, the actual role of the two metabolites in A. germari-induced defense in poplar requires further investigation.

Several insect species pose a serious threat to different plant species, sometimes becoming a pest that produces significant damage to the landscape, biodiversity, and/or the economy. This is the case of Rhynchophorus ferrugineus Olivier (Coleoptera: Dryophthoridae), Semanotus laurasii Lucas (Coleoptera: Cerambycidae), and Monochamus galloprovincialis Olivier (Coleoptera: Cerambycidae), which have become serious threats to ornamental and productive trees all over the world such as palm trees, cypresses, and pines. Knowledge about their flight potential is very important for designing and applying measures targeted to reduce the negative effects from these pests. Studying the flight capability and behaviour of some insects is difficult due to their small size and the large area wherein they can fly, so we wondered how we could obtain information about their flight capabilities in a controlled environment. The answer came with the design of flight mills. Relevant data about the flight potential of these insects may be recorded and analysed by means of a flight mill. Once an insect is attached to the flight mill, it is able to fly in a circular direction without hitting walls or objects. By adding sensors to the flight mill, it is possible to record the number of revolutions and flight time. This paper presents a full description of a computer monitored flight mill. The description covers both the mechanical and the electronic parts in detail. The mill was designed to easily adapt to the anatomy of different insects and was successfully tested with individuals from three species R. ferrugineus, S. laurasii, and M. galloprovincialis.

Micraspis discolor (Fabricius) (Coleoptera: Coccinellidae) is a widely distributed coleoptera predator in southern Asia in rice ecosystem, and adult M. discolor feed on both rice pollen and soft-bodied arthropods. Bitrophic bioassay and tritrophic bioassay were conducted to evaluate the potential impact of Cry1Ac/Cry1Ab-expressing rice Huahui 1 and its non-transgenic counterpart Minghui 63 on fitness parameters of adult M. discolor. The results showed that the survival, and fecundity of this beetle' adults were not different when they fed on Bt rice or non-Bt rice pollen or Nilaparvata lugens (Stål) reared on Bt rice or non-Bt rice. Toxicity assessment to ensure M. discolor adults were not sensitive to Cry1Ab or Cry1Ac protein independent from the pollen background, M. discolor adults were fed with an artificial diet containing Cry1Ac, Cry1Ab or both protein approximately 10 times higher concentration than in Huahui 1 rice pollen. No difference was detected for any of the life-table parameters tested between Cry protein-containing and pure diet. Artificial diet containing E-64 (N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide) was included as a positive control. In contrast, the pre-oviposition and fecundity of M. discolor were significantly adversely affected by feeding on E-64-containing diet. In both bioassays, the uptakes of Cry protein by adult M. discolor were tested by ELISA measurements. These results indicated that adults of M. discolor are not affected by Cry1Ab- or Cry1Ac-expressing rice pollen and are not sensitive to Cry protein at concentrations exceeding the levels in rice pollen in Huahui1. This suggests that M. discolor adults would not be harmed by Cry1Ac/Cry1Ab rice if Bt rice Huahui 1 were commercialized.