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Insect neuropeptides control essential physiological metabolic activities. In our previous studies, Capability/CAP2b (PK/CAPA) analog 1895 applied alone or as a combination of CAPA analogs (1895 + 2315) was reported to decrease aphid fitness. While this was obtained with the combination of two peptide analogs of the same neuropeptide class, the effect of combining peptide analogs of different neuropeptide classes has not been explored so far.In this study, we assessed the effect of combinations of the PK/CAPA analog 1895 with neuropeptide analogs of four different classes [adipokinetic hormone (AKH) analog: 2271; myosuppressin analog: 2434; kinin analog: 2460; tachykinin-related peptide analog: 2463] on the fitness of aphids. We found that the combination of 1895 and AKH analog 2271 was the most effective one to control Myzus persicae. The triple combination 1895 + 2271 + 2315 provided a synergistic effect by further increasing aphid mortality and reducing reproduction relative to 1895 + 2315. Additionally, a biosafety assessment of the combination 1895 + 2271 + 2315 showed no significant lethal nor sub-lethal effects on survival rates and food intake for the pollinator (Bombus terrestris) and the two representative natural enemies (Harmonia axyridis and Nasonia vitripennis).These results could facilitate establishment of the triple combination 1895 + 2271 + 2315, and/or inclusion of second generation analogs, as alternatives to broad spectrum and less friendly insecticides. © 2022 Society of Chemical Industry.

We provide a culturomics analysis of the cultivable bacterial communities of the crop, midgut and hindgut compartments, as well as the ovaries, of the invasive insect Vespa velutina, along with a cultivation-independent analysis of samples of the same nest through 16S rRNA amplicon sequencing. The Vespa velutina bacterial symbiont community was dominated by the genera Convivina, Fructobacillus, Lactiplantibacillus, Lactococcus, Sphingomonas and Spiroplasma. Lactococcus lactis and Lactiplantibacillus plantarum represented generalist core lactic acid bacteria (LAB) symbionts, while Convivina species and Fructobacillus fructosus represented highly specialised core LAB symbionts with strongly reduced genome sizes. Sphingomonas and Spiroplasma were the only non-LAB core symbionts but were not isolated. Convivina bacteria were particularly enriched in the hornet crop and included Convivina intestini, a species adapted towards amino acid metabolism, and Convivina praedatoris sp. nov. which was adapted towards carbohydrate metabolism.

International audience; The present study aimed to characterise the bacterial, fungal and parasite gut community of the invasive bee Megachile sculpturalis sampled from native (Japan) and invaded (USA and France) regions via 16S rRNA and ITS2 amplicon sequencing and PCR detection of bee microparasites. The bacterial and fungal gut microbiota communities in bees from invaded regions were highly similar and differed strongly from those obtained in Japan. Core amplicon sequence variants (ASVs) within each population represented environmental micro-organisms commonly present in bee-associated niches that likely provide beneficial functions to their host. Although the overall bacterial and fungal communities of the invasive M. sculpturalis in France and the co-foraging native bees Anthidium florentinum and Halictus scabiosae, were significantly different, five out of eight core ASVs were shared suggesting common environmental sources and potential transmission. None of the 46 M. sculpturalis bees analysed harboured known bee pathogens, while microparasite infections were common in A. florentinum, and rare in H. scabiosae. A common shift in the gut microbiota of M. sculpturalis in invaded regions as a response to changed environmental conditions, or a founder effect coupled to population re-establishment in the invaded regions may explain the observed microbial community profiles and the absence of parasites. While the role of pathogen pressure in shaping biological invasions is still debated, the absence of natural enemies may contribute to the invasion success of M. sculpturalis.

Aphids are important agricultural pests, vectors of many plant viruses and have sophisticated relationships with symbiotic microorganisms. Abundant asymptomatic RNA viruses have been reported in aphids due to the application of RNA-seq, but aphid-virus interactions remain unclear. Bunyavirales is the most abundant RNA virus order, which can infect mammals, arthropods, and plants. However, many bunyaviruses have specific hosts, such as insects. Here, we discovered 18 viruses from 10 aphid species by RNA-seq. Importantly, a widespread presence bunyavirus, Aphid bunyavirus 1 (ABV-1), was determined to have a wide host range, infecting and replicating in all 10 tested aphid species. ABV-1 may be transmitted horizontally during feeding on plant leaves and vertically through reproduction. In a comparison of the physiological parameters of ABV-1

Carotenoids are involved in many essential physiological functions and are produced from geranylgeranyl pyrophosphate through synthase, desaturase, and cyclase activities. In the pea aphid (Acyrthosiphon pisum), the duplication of carotenoid biosynthetic genes, including carotenoid synthases/cyclases (ApCscA-C) and desaturases (ApCdeA-D), through horizontal gene transfer from fungi has been detected, and ApCdeB has known dehydrogenation functions. However, whether other genes contribute to aphid carotenoid biosynthesis, and its specific regulatory pathway, remains unclear. In the current study, functional analyses of seven genes were performed using heterologous complementation and RNA interference assays. The bifunctional enzymes ApCscA-C were responsible for the synthase of phytoene, and ApCscC may also have a cyclase activity. ApCdeA, ApCdeC, and ApCdeD had diverse dehydrogenation functions. ApCdeA catalyzed the enzymatic conversion of phytoene to neurosporene (three-step product), ApCdeC catalyzed the enzymatic conversion of phytoene to ζ-carotene (two-step product), and ApCdeD catalyzed the enzymatic conversion of phytoene to lycopene (four-step product). Silencing of ApCscs reduced the expression levels of ApCdes, and silencing these carotenoid biosynthetic genes reduced the α-, β-, and γ-carotene levels, as well as the total carotenoid level. The results suggest that these genes were activated and led to carotenoid biosynthesis in the pea aphid.

Insect CAPA neuropeptides are considered to affect water and ion balance by mediating the physiological metabolism activities of the Malpighian tubules. In previous studies, the CAPA-PK analogue 1895 (2Abf-Suc-FGPRLamide) was reported to decrease aphid fitness when administered through micro-injection or via topical application. However, a further statistically significant decrease in the fitness of aphids and an increased mortality could not be established with pairwise combinations of 1895 with other CAPA analogues. In this study, we assessed the topical application of new combinations of 1895 with five CAPA-PVK analogues on the fitness of aphids. We found that 1895 and CAPA-PVK analogue 2315 (ASG-[β3 L]-VAFPRVamide) was statistically the most effective combination to control the peach potato aphid Myzus persicae nymphs via topical application, leading to 72% mortality. Additionally, the combination (1895+2315) was evaluated against a selection of beneficial insects, i.e. a pollinator (Bombus terrestris) and three natural enemies (Chrysoperla carnea, Nasonia vitripennis and Adalia bipunctata). We found no significant influence on food intake, weight increase and survival for the pollinator and the three representative natural enemies. These results could facilitate to further establish, and generate CAPA analogues as alternatives to broad spectrum and less friendly insecticides. This article is protected by copyright. All rights reserved.

Long non-coding RNAs (lncRNAs) generally display tissue-specific distributions, and testis-specific lncRNAs form the highest proportion of lncRNAs in many species. Here, we presented a detailed analysis of testis-specific lncRNAs in the melon fly, Zeugodacus cucurbitae, a highly destructive insect pest of cucurbitaceous and other related crops. Most testis-specific lncRNAs were found to be long intergenic non-coding RNAs (lincRNA). The size distribution of these lncRNAs ranged between 600 and 1000 nucleotides. Testis-specific lncRNAs that harboured one isoform number and two exons were the most abundant. Compared to other male tissues, the testis had more highly expressed lncRNAs. The quantitative real-time polymerase chain reaction results of 10 randomly selected testis-specific lncRNAs showed expression patterns consistent with RNA-seq data. Further analysis of the most highly expressed testis-specific lncRNA, lnc94638, was undertaken. Fluorescent in situ hybridization assays localized lnc94638 to the apical region of the testis that contains mature spermatozoa. RNA interference-mediated knockdown of lnc94638 expression reduced spermatozoa numbers and impaired the fertility of Z. cucurbitae male. This study provides a catalogue of testis-specific lncRNAs, shows that the testis-specific lnc94638 is involved in spermatogenesis and has the potential to be used for treating male sterility.

Recently N-glycosylation was found to be required for the postembryonic development and metamorphosis of the holometabolous beetle Tribolium castaneum. However, the role of N-glycosylation in the development of hemimetabolous insects is unknown. To further elucidate the role of N-glycosylation in the development of insects, a functional characterization of the N-glycosylation-related genes (NGRGs) was performed in a model insect for hemimetabolous development, namely the brown planthopper Nilaparvata lugens. In this project, we report the effects of RNAi-mediated silencing of 15 NGRGs on the postembryonic development of N. lugens. Two major observations were made. First, interruption of the early steps of N-glycan processing led to a lethal phenotype during the transition from nymph to adult as was observed in T. castaneum. Second, we report here on an essential function for the α-1,6-fucosyl transferase in the ecdysis event of N. lugens between nymphal instars, since gene-silencing by RNAi led to failure of ecdysis and subsequent mortality of the treated insect.

The Plusiinae subfamily has many polyphagous species, many of which occur in South America. Chrysodeixis includens and Rachiplusia nu are two representatives that mainly occurs in soybeans, cotton, common beans, sunflower and alfalfa. A population genetic study of C. includens and R. nu collected in the Southern Cone of America was performed using a partial COI gene sequencing data and compared with specimens from other American countries. Six haplotypes were identified in C. includens populations of Brazil, Argentina, Paraguay and Uruguay, organized within a star-like haplotype network, with the most common haplotype identified as Chin_MC. R. nu populations are more diverse and stable in comparison to C. includens. Populations from Argentina and Uruguay had the highest haplotype diversity, sharing five haplotypes and putatively indicating haplotype exchange. Demographic change analysis suggested a recent population expansion of C. includens over the American continent. Some C. includens haplotypes were country-specific, suggesting population expansion in the countries where specimens were collected.

BACKGROUND: The southern green stinkbug, Nezara viridula (Hemiptera: Pentatomidae), is an important emerging polyphagous pest infesting soybean in the United States, Brazil and Argentina. The indiscriminate use of synthetic insecticides to control stinkbugs has limited the effectiveness of current management strategies. Alternatively, RNA interference (RNAi) has emerged as a novel mode of action to control pests in an eco‐friendly manner. RESULTS: Here, we assessed the potential of RNAi technology by oral delivery of double‐stranded RNA (dsRNA) for the control of N. viridula. Initially, ten candidate genes were tested by microinjection assay to select the best target genes for oral delivery. Seven genes resulted in more than 90% mortality after microinjection. To evaluate RNAi efficacy by oral delivery of dsRNA, five genes were tested by feeding the insects on gene‐specific dsRNA mixed with an artificial diet. Significant mortality of 43% and 45% was observed after 14 days of treatment with dsαCop and dsvATPase A, respectively. To elucidate the lower RNAi efficacy via oral delivery of dsRNA, ex vivo dsRNA degradation in the saliva and the midgut juice was performed, which indicated that the reduced RNAi efficacy is accompanied by a rapid degradation of dsRNA by digestive secretions. CONCLUSION: This study proves that RNAi can be triggered by orally delivered dsRNA in N. viridula and can be exploited to control this economically important pest. The reduced stability of dsRNA in saliva and midgut that was observed indicates a need to further improve RNAi efficacy, for example by use of specific formulations.

Background Insect Capability neuropeptides (CAP2b/CAPA-PKs) play a critical role in modulating different physiologies and behavior in insects. In a previous proof-of-concept study, the CAP2b analogues 1895 (2Abf-Suc-FGPRLamide) and 2129 (2Abf-Suc-ATPRIamide) were reported to reduce aphid fitness when administered by injection. In the current study, the insecticidal efficacy of 1895 and 2129 on the peach potato aphid Myzus persicae was analyzed by topical application, simulating a spray application scenario in the field. Additionally, the selectivity of the tested analogues was evaluated against a selection of beneficial insects, namely three natural enemies (Adalia bipunctata, Chrysoperla carnea and Nasonia vitripennis) and a pollinator (Bombus terrestris). Results Within 3-5 days post topical exposure of aphids to 1895, higher mortality (33%) was observed, as was the case for the treatment with 2129 (17%) and the mixture of 1895 + 2129 (47%) compared to the control (3%). 1895 and the mix 1895 + 2129 showed the strongest and comparable insecticidal effects. Additionally, surviving aphids treated with 1895 showed a reduction in total lifetime reproduction (GRR) of 30%, 19% with 2129 and 39% with the mix 1895 + 2129. Of interest from a biosafety perspective is that by using the same delivery method and dose, no significant effects on survival, weight increase and food intake was observed for the representative natural enemies and the pollinator. Conclusion This study highlights the potential of exploiting CAP2b analogues such as 1895 (core structure FGPRL) as aphicides. Additionally, the CAP2b analogues used in this study were selective as they showed no effects when applied on four representative beneficial insects.

Biocontrol agents (BCAs) are commonly sprayed on flowering pipfruit trees to prevent them from getting infected by various pathogens. By entomovectoring, BCAs can be directly delivered onto the flowers. However, we currently lack knowledge on the distribution dynamics of BCAs by pollinators. Here, managed bees, both bumble bees (Bombus terrestris) and mason bees (Osmia bicornisandOsmia cornuta), were placed in the vicinity of flowering pipfruit trees (pear -'Conference', and apple-'Svatava' and 'Jonagold'), and this allowed us to investigate the distribution of a model BCA, namely, Serenade (R) MAX, from spray-inoculated flowers of a centralized tree to non-inoculated flowers of surrounding receiver trees by bees in an experimental setup in outdoor conditions. One hour after inoculation, we detected an enrichment of BCA in the flowers of the receiver trees and this for each tested pipfruit. The distribution of BCA from treated to untreated flowers was homogenous between the receiver trees for 'Svatava', while significantly different loads were detected for both 'Conference' and 'Jonagold', which might be due to differences in environmental factors, and/or bee characteristics. More research is needed to understand the distribution dynamics of BCAs by pollinators in field conditions, such as in commercial orchards or crop fields, and how this could result in an efficient control.

Wild bees are key providers of pollination services in agroecosystems. The abundance of these pollinators and the ecosystem services they provide rely on supporting resources in the landscape. Spatially explicit models that quantify wild bee abundance and pollination services in food crops are built on the foundations of foraging and nesting resources. This dependence limits model implementation as land-cover maps and pollination experts capable of evaluating habitat resource quality are scarce. This study presents a novel approach to assessing crop pollination services using remote sensing data (RSD) as an alternative to the more conventional use of land-cover data and local expertise on spatially explicit models. We used landscape characteristics derived from remote sensors to qualify nesting resources in the landscape and to evaluate the delivery of pollination services by mining bees (Andrena spp.) in 30 fruit orchards located in the Flemish region of Belgium. For this study, we selected mining bees for their importance as local pollinators and underground nesting behavior. We compared the estimated pollination services derived from RSD with those derived from the conventional qualification of nesting resources. We did not observe significant differences (p = 0.68) in the variation in mining bee activity predicted by the two spatial models. Estimated pollination services derived from RSD and conventional characterizations explained 69% and 72% of the total variation, respectively. These results confirmed that RSD can deliver nesting suitability characterizations sufficient for estimating pollination services. This research also illustrates the importance of nesting resources and landscape characteristics when estimating pollination services delivered by insects like mining bees. Our results support the development of holistic agroenvironmental policies that rely on modern tools like remote sensors and promote pollinators by considering nesting resources.

Eukaryotic cells can decorate their proteins with carbohydrate structures or glycans, significantly affecting the properties and activities of these proteins. Despite the importance of protein glycosylation in numerous biological processes, our knowledge of this modification in insects is far from complete. While N-glycosylation is the most studied, the study of O-glycans in insects is still very fragmentary and these studies are limited to a specific developmental stage or a specific tissue. In this article, matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) technology was used to analyze the O-glycan profile for the different developmental stages of egg, larva, pupa, and adult of the red flour beetle Tribolium castaneum, an important insect model and pest worldwide. The results on the O-glycan profile showed that the mucin-type glycans dominate the O-glycome of the red flour beetle. Interestingly, some of the more complex mucin-type O-glycans, such as a tetra- (O-GalNAcGalGlcAGalNAc) and pentasaccharide O-glycan (O-GalNAc(GalGlcA)GalNAcGlcA), were highly abundant during the pupa stage, the intermediate stage between larval and adult stage in holometabolous insects, demonstrating that insect metamorphosis is accompanied with a change in the insect O-glycan profile. Together with the N-glycan profile, the current data are a foundation to better understand the role of protein glycosylation in the development of insects.

Crop pollination generally increases with pollinator diversity and wild pollinator visitation. To optimize crop pollination, it is necessary to investigate the pollination contribution of different pollinator species. In the present study, we examined this contribution of honey bees and non-Apis bees (bumble bees, mason bees and other solitary bees) in sweet cherry. We assessed the pollination efficiency (fruit set of flowers receiving only one visit) and foraging behaviour (flower visitation rate, probability of tree change, probability of row change and contact with the stigma) of honey bees and different types of non-Apis bees. Single visit pollination efficiency on sweet cherry was higher for both mason bees and solitary bees compared with bumble bees and honey bees. The different measures of foraging behaviour were variable among non-Apis bees and honey bees. Adding to their high single visit efficiency, mason bees also visited significantly more flower per minute, and they had a high probability of tree change and a high probability to contact the stigma. The results of the present study highlight the higher pollination performance of solitary bees and especially mason bees compared with bumble bees and honey bees. Management to support species with high pollination efficiency and effective foraging behaviour will promote crop pollination.

BACKGROUND With the growing number of available aphid genomes and transcriptomes, an efficient and easy-to-adapt tool for gene function study is urgently required. RNA interference (RNAi), as a post-transcriptional gene silencing mechanism, is important as a research tool for determining gene functions and has potential as a novel insect control strategy. However, these applications have been hampered by the lack of effective dsRNA delivery approaches in aphids. RESULTS Here, we developed a convenient and efficient dsRNA delivery method, topical RNAi, in aphids. An investigation of its dose and time-dependent RNAi efficiencies revealed that with as little as 60 ng dsRNA per adult pea aphid (Acyrthosiphon pisum), the indicator gene, Aphunchback, could be significantly silenced within 2 h of exposure. The method was further validated by successfully silencing other different genes, and it was also efficient toward two other aphid species, Aphis citricidus and Myzus persicae. Furthermore, a noticeable mortality was also observed in pea aphids using topical RNAi-mediated gene silencing, within 4 days post-dsRNA application for four out of seven tested genes. CONCLUSION Compared with the currently used dsRNA delivery methods in aphids, microinjection and ingestion, topical RNAi is time- and cost-effective, which could greatly influence RNAi-based gene functional studies and potential candidate gene selection for developing RNAi-based aphid control strategies in the future. © 2019 Society of Chemical Industry.

Background Cytochrome P450-mediated detoxification plays an important role in the development of insecticide resistance. Previous studies have demonstrated that overexpression of CYP6BG1 was responsible for permethrin resistance in Plutella xylostella, and our experiments also showed that upregulation of this gene is associated with chlorantraniliprole resistance in P. xylostella. However, the transcriptional regulation involved in the expression of CYP6BG1 remains unknown. To further investigate the regulation of CYP6BG1 expression, the promoters of this gene were cloned and analyzed from one susceptible and four different resistant populations of P. xylostella. Results First, the promoter region of P. xylostella CYP6BG1 was compared in five populations, and three types of 5'-flanking region were found. Second, the region between -562 and +49 of CYP6BG1 in a field population (TH) of P. xylostella showed the highest promoter activity and could be induced by chlorantraniliprole. Third, the transcriptional factor FTZ-F1, which is an orphan nuclear receptor and binds to the fushi tarazu (ftz) gene, was predicted by the online software Alggen and Jaspar. It was proved to regulate the expression of CYP6BG1 by RNAi. The expression levels of FTZ-F1 and CYP6BG1 could be induced by chlorantraniliprole and were significantly higher in the resistant populations. Conclusions These data give a better understanding of the transcriptional regulation of an important insecticide detoxification enzyme gene, and therefore will help in understanding the molecular mechanisms of insecticide resistance in P. xylostella. © 2018 Society of Chemical Industry.

Peel and seeds of red-skinned passion fruit, mango, longan, rambutan, white-flesh and red-flesh dragon fruit were screened for their in vitro antioxidant activity, and determination of a detailed profile of phenolic compounds. Rambutan peel and mango seed extracts exhibited the highest total phenolic content and antioxidant activities (DPPH, ABTS and FRAP values). By using UPLC-QTOF-MS/MS analysis, the profiles of soluble and bound phenolics in the fruit by-products were obtained. Ellagic acid, geraniin, quercetin hexoside, gallic and galloyshikimic acid were predominant in rambutan peel, whereas, mangiferin, ellagic acid and galloy(di)glucoside were the major phenolic compounds in mango seed. Main phenolic compounds in longan peel were ellagic acid, galloyldiglucoside, and gallic acid, while in dragon fruit peel this was isorhamnetin glycoside, isorhamnetin glucorhamoside. Meanwhile, rutin and quercetin hexoside were predominant in passion fruit peel. These findings contribute significantly to the database of phenolic profiles of by-products of tropical fruits.

One of the main functions of the piwi-interacting RNA pathway is the post-transcriptional silencing of transposable elements in the germline of many species. In insects, proteins belonging to the Tudor superfamily proteins belonging to the Tudor superfamily play an important role in to play an important role in this mechanism. In this study, we identified the tudor gene in the oriental fruit fly, Bactrocera dorsalis, investigated the spatiotemporal expressional profile of the gene, and performed a functional analysis using RNA interference. We identified one transcript for a tudor homologue in the B. dorsalis transcriptome, which encodes a protein containing the typical 10 Tudor domains and an Adenosine triphosphate (ATP) synthase delta subunit signature. Phylogenetic analysis confirmed the identity of this transcript as a tudor homologue in this species. The expression profile indicated a much higher expression in the adult and pupal stages compared to the larval stages (up to a 60-fold increase), and that the gene was mostly expressed in the ovaries, Malpighian tubules and fat body. Finally, gene knockdown of tudor in B. dorsalis led to clearly underdeveloped ovaries in the female adult and reductions in copulation rate and amount of oviposition, indicating its important role in reproduction. The results of this study shed more light on the role of tudor in ovary development and reproduction.

Background The parasitic wasps Eretmocerus mundus, Eretmocerus eremicus and Encarsia formosa are important natural enemies of whiteflies. A broad understanding of their biology, ecology and behavior has been achieved, but the composition and role of their microbiota is not fully determined. The knowledge of the bacteria present in insects might be useful to manage species of human concern such as natural enemies or pests. Here, we performed a residual contact test to study a possible change in the susceptibility of E. mundus adults to abamectin insecticide after antibiotic treatment. Moreover, we assessed the microbiota present in adults of E. eremicus, E. formosa and two strains of E. mundus by MiSeq 16S rRNA amplicon sequencing. Finally, enzymatic tests were done to determine the influence of Arthrobacter species in the susceptibility of E. mundus to pesticides. Results The assays showed that when E. mundus adults were pretreated with antibiotic, the toxicity of abamectin was significantly higher. Among the different bacteria associated with parasitic wasps, Arthrobacter has been shown to be involved in the degradation of several kinds of pesticides. Four Arthrobacter species were detected in all the studied insects and the presence of esterases in this bacterial species was confirmed. Conclusions The results suggest that the microbiota can modify the susceptibility of E. mundus to pesticides, which in turn supports the importance of the microbial community in natural enemies that it should be considered as a factor in risk assessment tests of pesticides. © 2018 Society of Chemical Industry.

Background The Neotropical stink bug Euschistus heros is a major pest in soybean fields. Development of highly species-specific pesticides based on RNA interference (RNAi) could provide a new sustainable and environmentally friendly control strategy. Results Here, the potential of RNAi as a pest control tool against E. heros was assessed. First, target gene selection using a microinjection approach was performed. Seven of the 15 candidate genes tested exhibited > 95% mortality after hemolymph injection of 27.5 ng dsRNA. Subsequently, dsRNA was administered orally using different formulations: naked dsRNA, liposome-encapsulated-dsRNA and dsRNA formulated with EDTA. Liposome-encapsulated dsRNA targeting vATPase A and muscle actin led to significant mortality after 14 days (45% and 42%, respectively), whereas EDTA-formulated dsRNA did so for only one of the target genes. Ex vivo analysis of the dsRNA stability in collected saliva indicated a strong dsRNA-degrading capacity by E. heros saliva, which could explain the need for dsRNA formulations. Conclusion The results demonstrate that continuous ingestion of dsRNA with EDTA or liposome-encapsulated dsRNA can prevent dsRNA from being degraded enzymatically and suggest great potential for using these formulations in dsRNA delivery to use RNAi as a functional genomics tool or for pest management of stink bugs. © 2018 Society of Chemical Industry.

The interaction between the insect host and its microbiota plays a central role in insect health and is mostly studied in relation to the digestive system. Nonetheless, there are numerous microorganisms occupying multiple habitats in and on insects. We studied microbial communities in the gut and fat body of bumblebees (Bombus terrestris) using the V4 region of the 16S rRNA gene on the Illumina MiSeq platform. In one of the two study locations, the fat body microbial composition was marked by the dominant presence of Arsenophonus sp. and Phyllobacterium sp. Bumblebees infected with Apicystis bombi, a eukaryotic parasite multiplying in the fat body, had a significant higher relative abundance of Arsenophonus sp. compared with the non-infected individuals. In general, the infection of A. bombi correlated with a more interlinked microbial association network, as we observed an increase of significant associations between the relative abundance of bacteria present in the gut and fat body of infected bumblebees. The causality within this potential prokaryotic-eukaryotic relation is important when assessing the health impact on bees.

Mites comprise a group of key agricultural pests on a wide range of crops. They cause harm through feeding on the plant and transferring dangerous pathogens, and the rapid evolution of pesticide resistance in mites highlights the need for novel control methods. Currently, RNA interference (RNAi) shows great potential for insect pest control. Here, we review the literature regarding RNAi in mite pests. We discuss different target genes and RNAi efficiency in various mite species, a promising Varroa control program using RNAi, the synergy of RNAi with plant defense mechanisms and microorganisms, and current understanding of systemic movement of double-stranded RNA (dsRNA). On the basis of this evidence, we can conclude that there is clear potential for application of RNAi-based mite control, but further research on several aspects of RNAi in mites is needed, including: (i) the factors influencing RNAi efficiency, (ii) the mechanism of environmental RNAi and cross-kingdom dsRNA trafficking, (iii) the mechanism of possible systemic and parental RNAi, and (iv) non-target effects, specifically in predatory mites, which should be considered during RNAi target selection. © 2018 Society of Chemical Industry.

In recent years, research in life sciences has been remarkably revolutionized owing to the establishment, development and application of genome editing technologies. Genome editing has not only accelerated fundamental research but has also shown promising applications in agricultural breeding and therapy. In particular, the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology has become an indispensable tool in molecular biology owing to its high efficacy and simplicity. Genome editing tools have also been established in silkworm (Bombyx mori), a model organism of Lepidoptera insects with high economic importance. This has remarkably improved the level and scope of silkworm research and could reveal new mechanisms or targets in basic entomology and pest management studies. In this review, we summarize the progress and potential of genome editing in silkworm and its applications in functional genomic studies for generating novel genetic materials.

Scientists have made significant progress in understanding and unraveling several aspects of double-stranded RNA (dsRNA)-mediated gene silencing during the last two decades. Now that the RNA interference (RNAi) mechanism is well understood, it is time to consider how to apply the acquired knowledge to agriculture and crop protection. Some RNAi-based products are already available for farmers and more are expected to reach the market soon. Tailor-made dsRNA as an active ingredient for biopesticide formulations is considered a raw material that can be used for diverse purposes, from pest control and bee protection against viruses to pesticide resistance management. The RNAi mechanism works at the messenger RNA (mRNA) level, exploiting a sequence-dependent mode of action, which makes it unique in potency and selectivity compared with conventional agrochemicals. Furthermore, the use of RNAi in crop protection can be achieved by employing plant-incorporated protectants through plant transformation, but also by non-transformative strategies such as the use of formulations of sprayable RNAs as direct control agents, resistance factor repressors or developmental disruptors. In this review, RNAi is presented in an agricultural context (discussing products that have been launched on the market or will soon be available), and we go beyond the classical presentation of successful examples of RNAi in pest-insect control and comprehensively explore its potential for the control of plant pathogens, nematodes and mites, and to fight against diseases and parasites in beneficial insects. Moreover, we also discuss its use as a repressor for the management of pesticide-resistant weeds and insects. Finally, this review reports on the advances in non-transformative dsRNA delivery and the production costs of dsRNA, and discusses environmental considerations. © 2017 Society of Chemical Industry.

Vitellogenin (Vg) and its receptor (VgR) play a key role in the reproductive process and development of insects. Aphids are a group of high-fecundity insect species with pseudoplacental viviparity, but the roles of their Vg and VgR genes have not been investigated yet. The brown citrus aphid, Aphis (Toxoptera) citricidus, is a major insect pest of citrus and the main vector of Citrus tristeza closterovirus. In this study, we identified and characterized these two genes, designated as AcVg and AcVgR, from the brown citrus aphid. We found that AcVg has lost the DUF1943 domain that is present in other insect Vgs. Silencing of AcVg and AcVgR led to a delay in the nymph-adult transition, a prolonged prereproductive period, and a shortened reproductive period, which in turn resulted in slower embryonic development and fewer new-born nymphs. Interestingly, silencing of AcVg decreased the transcript level of AcVgR, but silencing of AcVgR resulted in increased transcript levels of AcVg. In addition, silencing of Vg/VgR had similar phenotypes between alate and apterous morphs, suggesting that the functions of these two genes are the same in the two wing morphs of the aphid. Our results demonstrate that Vg and VgR are involved in various aspects of aphid development and reproduction. Further studies on the synthesis of Vg could help to elucidate the reproductive mechanism and provide information that will be useful for developing new pest control strategies.

As the RNA interference mechanism was discovered, researchers have made huge strides in unravelling biological mechanisms at the gene level in a wide range of organisms. RNAi is a simple and rapid method that allows silencing gene expression at the post-transcriptional level and is triggered by the introduction of double-stranded RNA into the cell, leading to the degradation of the corresponding mRNA. In this review, we will provide an overview of successful RNAi experiments to date in Crustacea, and shrimp in particular, with special focus on various potential applications of RNA interference in research and aquaculture. An important aspect of RNAi in any species is the delivery of the double-stranded RNA and the barriers that have to be overcome to get the dsRNA into the target cells. Different options, experimental procedures, success rates and challenges in using RNAi are discussed in this review.

Although the gut microbial communities in adult bumblebees and their associated functionalities are widely studied, descriptive data on the larval gut microbiota are still limited. The gut microbiota of a fully sampled wild Bombus pascuorum nest has been characterized, using the multiplexed Illumina MiSeq 16S ribosomal RNA amplicon sequencing technique. The nesters and foragers inhabiting the same nest showed the typical core bacterial sequences and only marginal differences in their characterized gut microbiota. The gut microbial communities within the adult and larval specimens differed strongly, as the typical core gut bacteria in the adult bumblebees are absent in the larval bumblebees. The bacterial communities within the larval gut are dominated by bacterial phylotypes of Enterobacteriaceae and Lactobacillaceae, supplemented with genera belonging to Corynebacteriales and Bacillales. The function of this larval gut microbiota, being different from the adult, remains to be determined.

BACKGROUND RNA interference (RNAi) technology can potentially serve as a suitable strategy to control the African sweet potato weevil Cylas puncticollis (SPW), which is a critical pest in sub-Saharan Africa. Important prerequisites are required to use RNAi in pest control, such as the presence of an efficient RNAi response and the identification of suitable target genes. RESULTS Here we evaluated the toxicity of dsRNAs targeting essential genes by injection and oral feeding in SPW. In injection assays, 12 of 24 dsRNAs were as toxic as the one targeting Snf7, a gene used commercially against Diabrotica virgifera virgifera. Three dsRNAs with high insecticidal activity were then chosen for oral feeding experiments. The data confirmed that oral delivery can elicit a significant toxicity, albeit lower compared with injection. Subsequently, ex vivo assays revealed that dsRNA is affected by degradation in the SPW digestive system, possibly explaining the lower RNAi effect by oral ingestion. CONCLUSION We conclude that the full potential of RNAi in SPW is affected by the presence of nucleases. Therefore, for future application in crop protection, it is necessary constantly to provide new dsRNA and/or protect it against possible degradation in order to obtain a higher RNAi efficacy. © 2016 Society of Chemical Industry

Summary The pathogen Fusarium graminearum, producer of the mycotoxin deoxynivalenol, and Sitobion avenae aphids both reside on wheat ears. We explored the influence of an earlier aphid infestation on the expression profile of specific molecular markers associated with F. graminearum infection. Using reverse transcription-quantitative polymerase chain reaction analysis, we followed the expression of wheat defence genes on S. avenae infestation and explored the effect on a subsequent F. graminearum infection. This was performed by the assessment of disease symptoms, fungal biomass, mycotoxin production and number of aphids at several time points during disease progress. Wheat ears infected with F. graminearum showed more disease symptoms and higher deoxynivalenol levels when ears were pre-exposed to aphids relative to a sole inoculation with F. graminearum. Aphids induced defence genes that are typically induced on F. graminearum infection. Other defence genes showed earlier and/or enhanced transcription after exposure to both aphids and F. graminearum. In the discussion, we link the symptomatic and epidemiological parameters with the transcriptional induction pattern in the plant. Our study suggests that pre-exposure of wheat ears to aphids affects the plant response, which plays a role in the subsequent attack of F. graminearum, enabling the fungus to colonize wheat ears more rapidly.

The current bumblebee decline leads to inbreeding in populations that fosters a loss of allelic diversity and diploid male production. As diploid males are viable and their offspring are sterile, bumblebee populations can quickly fall in a vortex of extinction. In this article, we investigate for the first time a potential premating mechanism through a major chemical reproductive trait (male cephalic labial gland secretions) that could prevent monandrous virgin queens from mating with diploid males. We focus our study on the cephalic labial gland secretions of diploid and haploid males of Bombus terrestris (L.). Contrary to initial expectations, our results do not show any significant differentiation of cephalic labial gland secretions between diploid and haploid specimens. Queens seem therefore to be unable to avoid mating with diploid males based on their compositions of cephalic labial gland secretions. This suggests that the vortex of extinction of diploid males could not be stopped through premating avoidance based on the cephalic labial gland secretions but other mechanisms could avoid mating between diploid males and queens.

RNA interference (RNAi)-based technology has emerged as a potential tool for controlling insect pests, however, previous studies found that the efficiency of RNAi in Bactrocera dorsalis was variable. In nature, insects often meet various challenges, such as pathogen infections, extreme temperatures, lack of nutrition and heavy metals. To better understand the association of the stressors with efficiency of RNAi, in the current study we tested the expression of three core genes, dicer2 (Bddcr2), r2d2 (Bdr2d2) and argonaute2 (Bdago2), of the small interfering RNA (siRNA) pathway of B. dorsalis upon various stressors. Our results showed that all three genes were upregulated by the infection of invertebrate iridescent virus 6, which suggested a function of the siRNA pathway against viral infection. The loading of FeCl3 could also increase the expression of Bddcr2. The treatments of Escherichia coli, extremely high (40°C) and low (0°C) temperatures, as well as starvation, could negatively influence the expression of Bddcr2 and/or Bdago2. In total, our results showed that various stressors could influence the expression of core components of B. dorsalis siRNA pathway. This highlights further speculation on the RNAi efficiency upon these stressors. Considering the complexity and variation of RNAi efficiency in different conditions, these results provide initial aspects in possible environmental stressors to influence the activity of the siRNA pathway, but the real impact of RNAi efficiency posed by these stressors requires further studies.

In this study, the cDNAs of five cytochromes P450 genes (named CYP345P1, CYP358B1, CYP4FD2, CYP4CD2, and CYP6JN1) contained open reading frames from 1,500 to 1,554 nucleotides that encoded 499 to 517 amino acids were cloned from the psocid Liposcelis entomophila. They are characterized by predicted molecular weights from 57.67 to 59.64 kDa and theoretical isoelectric points of 5.57-9.07. Quantitative real-time PCR analysis showed these five genes were expressed at all tested developmental stages and higher expressions were observed in adults. CYP358B1 was expressed at higher levels in egg and adult compared to the larval stages. mRNA abundances of five genes were detected in both sexes and were relatively more abundant in adult females than in adult males. Synergism bioassay showed that the synergic ratio was 2.20 and 2.45 when insects were treated with the mixture of deltamethrin or malathion with the synergist piperonyl butoxide (PBO). Because PBO induces cytochrome P450s in some insects, this suggested to us that cytochromes P450 might participate in detoxification of these insecticides. The transcripts of the five cytochromes P450 genes in adult psocids could be induced to the highest level at 12 h after the exposure to malathion. After exposure to deltamethrin, CYP358B1 reached maximum expression at 24 h. The maximum expression of the other four genes occurred at 36 h. Treatments with the carbamate propoxur did not influence transcription of the cytochromes P450 gene. The induction profiles suggested that these five cytochrome P450 genes may be associated with deltamethrin and malathion metabolism in psocids.

RNA interference (RNAi) is a powerful tool in entomology and shows promise as a crop protection strategy, but variability in its efficiency across different insect species limits its applicability. For oral uptake of the double-stranded RNA (dsRNA), the RNAi trigger, two different mechanisms are known: systemic RNA interference deficient-1 (Sid-1) transmembrane channel-mediated uptake and clathrin-mediated endocytosis. So far, a wide range of experiments has been conducted, confirming the involvement of one of the pathways in dsRNA uptake, but never both pathways in the same species. We investigated the role of both pathways in dsRNA uptake in the Colorado potato beetle, Leptinotarsa decemlineata, known to have an efficient RNAi response. Through RNAi-of-RNAi experiments, we demonstrated the contribution of two different sid-1-like (sil) genes, silA and silC, and clathrin heavy chain and the 16kDa subunit of the vacuolar H(+) ATPase (vha16), elements of the endocytic pathway, to the RNAi response. Furthermore, the sid-1-like genes were examined through phylogenetic and hydrophobicity analysis. This article reports for the first time on the involvement of two pathways in dsRNA uptake in an insect species and stresses the importance of evaluating both pathways through a well-devised reporter system in any future experiments on cellular dsRNA uptake.

Scope: Intestinal cells are constantly exposed to luminal toxins. In this study, we investigated the effect of cellular stress caused by valinomycin, which is structurally and functionally similar to the bacterial toxin cereulide, on quercetin metabolism and cellular localization in undifferentiated cells. Methods and results: Coadministration of quercetin and valinomycin (50 mu M quercetin/0.05 mu M valinomycin) reduced intracellular reactive oxygen species content and increased cell viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) of Caco-2 cells compared to valinomycin-only (0.05 mu M) treatment. Quercertin was effectively metabolized into methyl, glucuronide, and sulfate conjugates, which were mostly secreted into to the culture medium. Three different O-methylated quercetin isomers were detected. Two were exported from the cells and one remained intracellularly. Further, valinomycin caused an increase in the intracellular accumulation of O-methylated quercetin metabolites compared to cells treated only with quercetin. In valinomycin-untreated cells, quercetin and O-methylated quercetin metabolite were localized in the cell membrane, whereas valinomycin treatment resulted in their uptake by the cells. Conclusion: This is the first report on the change in metabolism, localization, and accumulation of O-methylated quercetin metabolites in undifferentiated Caco-2 cells as a response during stress caused by valinomycin. These results indicate a potential cellular stress response mechanism in undifferentiated Caco-2 cells, which adds novel insights into the mechanisms of flavonoid cellular bioactivity.

Social bees, like honeybees and bumblebees, have a close contact with nest mates of different developmental stages and generations. This could enhance bacterial transfer between nest mates and offers opportunities for direct transfer of symbionts from one generation to the next, resulting in a stable host specific gut microbiota. Gut symbionts of honeybees and bumblebees have been suggested to contribute in digestion and protection against parasites and pathogens. Here we studied the impact of contact with the bumblebee colony on the colonization potential of the bacterial families (i.e. Neisseriaceae, Orbaceae, Lactobacillaceae and Bifidobacteriaceae) occurring in the gut of adult bumblebees (Bombus terrestris). Bacterial profiles of the gut microbiota of B. terrestris were determined based on the hypervariable V4 region of the 16S rRNA using paired-end Illumina sequencing. In our experiments, we created different groups in which we gradually reduced the contact with nest mates and hive material. We made three observations: (i) reducing the contact between the colony and the bumblebee during adult life resulted in a significant drop in the relative abundance of Lactobacillus bombicola and Lactobacillus bombi. (ii) Bifidobacteriaceae required contact with nest mates to colonize the gut of B. terrestris and a significant lower bacterial diversity was observed in bumblebees that were completely excluded from colony contact during the adult life. (iii) Snodgrassella and Gilliamella were able to colonize the gut of the adult bumblebee without any direct contact with nest mates in the adult life stage. These results indicate the impact of the colony life on the diversity of the characteristic bumblebee gut bacteria.

The aim of this review is to provide a comprehensive discussion on the various human/animal cell-based models used to study the absorption, transport, and metabolism of flavonoids. Flavonoids are plant-based bioactive compounds that have been extensively investigated for their active role in health alleviation and disease prevention. For this purpose, cell lines isolated from various human and animal tissues have been routinely used as an in vitro model to assess the bioavailability and bioactivity of these compounds. This paper reviews for the first time various transporters (SLCT, SGLT, bilitranslocase, and ABC transporters), metabolic routes (deglycosylation, glucuronidation, sulfation, and deconjugation), and accumulation of flavonoids in different cell lines commonly used in flavonoid research. Also, the use of co-culture systems to study flavonoid bioactivity will be discussed. To date, no definite mono-culture or co-culture formulation has been generally accepted to be the most accurate representation of the in vivo situation. Therefore, further investigation and improvement of cell-based in vitro models for flavonoid research merit further investigation.

Summary Many weeds that are closely associated with horticul-tural activities are known as natural reservoirs of plantviruses. However, whether these weeds can also serveas hosts of pospiviroids is not well known. Pospivi-roids are naked, non-coding RNA pathogens thatcause severe economic damage in many solanaceouscrops. In this study, we examined the overall risk ofpospiviroid spreading from weeds to economicallyimportant crops, by combining the results from previ-ous inoculation studies with new results coming froma survey, a contact experiment and an inoculationexperiment. A survey of commercial ornamental glass-houses revealed that ornamental plants mainly belong-ing to the Solanaceae harbour pospiviroids, in contrastto weed species sampled in the same places. No newweed hosts could be identified after testing weeds thatgrew in contact with Tomato apical stunt viroid(TASVd)-infected plants of tomato (Solanum lycoper-sicum) and jasmine nightshade (Solanum jasminoides)in an experimental glasshouse. Finally, in mechanicalinoculation experiments with TASVd, none of the sixtested weed species were determined to be a host at6 weeks after inoculation. Commonly occurring weedspecies therefore do not appear to play a significantrole as reservoir hosts for pospiviroids. This does notrule out other potential weed hosts that have not yetbeen tested. Inoculation studies should include rigor-ous experimental protocols with a sufficient number ofreplicated as well as adequate positive controls. Theinformation gained through this study may prove use-ful in future risk assessments for the pospiviroidgroup.Keywords: Pospiviroidae, ornamental plants, mechani-cal inoculation, biolistic inoculation, natural reservoirs,risk assessment, Tomato apical stunt viroid.V

Because of their natural origin, biopesticides are assumed to be less harmful to beneficial insects, including bees, and therefore their use has been widely encouraged for crop protection. There is little evidence, however, to support this ingrained notion of biopesticide safety to pollinators. Because larval exposure is still largely unexplored in ecotoxicology and risk assessment on bees, an investigation was performed on the lethal and sublethal effects of a diet treated with 2 bioinsecticides, azadirachtin and spinosad, on the stingless bee, Melipona quadrifasciata, which is one of the most important pollinators in the Neotropics. Survival of stingless bee larvae was significantly compromised at doses above 210 ng a.i./bee for azadirachtin and 114 ng a.i./bee for spinosad. No sublethal effect was observed on larvae developmental time, but doses of both compounds negatively affected pupal body mass. Azadirachtin produced deformed pupae and adults as a result of its insect growth regulator properties, but spinosad was more harmful and produced greater numbers of deformed individuals. Only spinosad compromised walking activity of the adult workers at doses as low as 2.29 ng a.i./bee, which is 1/5000 of the maximum field recommended rate. In conclusion, the results demonstrated that bioinsecticides can pose significant risks to native pollinators with lethal and sublethal effects; future investigations are needed on the likelihood of such effects under field conditions. Environ Toxicol Chem 2015;34:2149–2158. © 2015 SETAC

Although invertebrates generally have a low public profile, the honey bee, Apis mellifera L., is a flagship species whose popularity likely derives from the products it provides and its perceived ecological services. Therefore, the raging debate regarding honey bee decline has surpassed the realm of beekeepers, academia, industry and regulatory agencies and now also encompasses non-governmental agencies, media, fiction writers and the general public. The early interest and concern about honey bee colony collapse disorder (CCD) soon shifted to the bigger issue of pollinator decline, with a focus on the potential involvement of pesticides in such a phenomenon. Pesticides were previously recognised as the potential culprits of the reported declines, particularly the neonicotinoid insecticides owing to their widespread and peculiar use in agriculture. However, the evidence for the potential pivotal role of these neonicotinoids in honey bee decline remains a matter of debate, with an increased recognition of the multifactorial nature of the problem and the lack of a direct association between the noted decline and neonicotinoid use. The focus on the decline of honey bee populations subsequently spread to other species, and bumblebees became another matter of concern, particularly in Europe and the United States. Other bee species, ones that are particularly important in other regions of the world, remain the object of little concern (unjustifiably so). Furthermore, the continuous focus on neonicotinoids is also in need of revision, as the current evidence suggests that a broad spectrum of compounds deserve attention. Here we address both shortcomings. © 2015 Society of Chemical Industry

Esterase has been reported to be involved in malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel). However, the underlying molecular mechanism of the esterase-mediated resistance remains largely unknown in this species. Here, with the use of a strain selected for malathion resistance in the laboratory (MR), we found that two overexpressed α-esterase genes, namely BdCarE4 and BdCarE6, predominant in the adult midgut and fat body, function in conferring malathion resistance in B. dorsalis. Notably, these two genes were found to be mostly close to the esterase E3, which are usually implicated in detoxifying organophosphate insecticides. The transcript levels of BdCarE4 and BdCarE6 were investigated and compared between the MR and a susceptible (MS) strain of B. dorsalis. Both genes were significantly up-regulated in the MR strain, which was consistent with the enhanced esterase activity in the MR strain. However, no changes in either the coding sequence or gene copy number were observed between the two strains. Subsequently, heterologous expression combined with cytotoxicity assay in Sf9 cells demonstrated that BdCarE4 and BdCarE6 can probably detoxify malathion. Furthermore, RNA interference-mediated knockdown of each of these two genes significantly increased malathion susceptibility in the MR strain adults. In conclusion, these results expand our molecular understanding of the important role of α-esterases during the development of resistance to organophosphorous insecticides in B. dorsalis.