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Insecticidal RNAi is a targeted pest insect population control measure. The specificity of insecticidal RNAi can theoretically be enhanced by using symbiotic bacteria with a narrow host range to deliver RNAi, an approach termed symbiont-mediated RNAi (SMR), a technology we have previously demonstrated in the globally-invasive pest species Western Flower Thrips (WFT). Here we determine a very limited distribution of the two predominant bacterial symbionts of WFT, BFo1 and BFo2, among genome-sequenced insects. Moreover, we have challenged two non-target insect species with both bacterial species, namely the pollinating European bumblebee, Bombus terrestris, and an insect predator of WFT, the pirate bug Orius laevigatus. Our data indicate that whereas BFo1 could establish itself in both these non-natural hosts, albeit with no significant effects on insect fitness, BFo2 was unable to persist in either species. In terms of biosafety, these data, together with the more specific growth requirements for BFo2, vindicate the choice of this species for precision pest management of WFT using SMR.

In addition to its role in the digestive system, the peritrophic membrane (PM) provides a physical barrier protecting the intestine from abrasion and against pathogens. Because of its sensitivity to RNA interference (RNAi), the notorious pest insect, the Colorado potato beetle (CPB, Leptinotarsa decemlineata), has become a model insect for functional studies. Previously, RNAi-mediated silencing of Mannosidase-Ia (ManIa), a key enzyme in the transition from high-mannose glycan moieties to paucimannose N-glycans, was shown to disrupt the transition from larva to pupa and the metamorphosis into adult beetles. While these effects at the organismal level were interesting in a pest control context, the effects at the organ or tissue level and also immune effects have not been investigated yet. To fill this knowledge gap, we performed an analysis of the midgut and PM in ManIa-silenced insects.As marked phenotype, the ManIaThe observed decrease in PM pore size could be a response to prevent potential pathogens to access the midgut epithelium. This hypothesis is supported by the strong increase in transcription levels of the anti-fungal peptide drosomycin-like in ManIa

A first step in any pest management initiative is recognizing the existing problem - identifying the pest species and its abundance and dispersal capacities. This is not simple and even more challenging when insidious (invasive) species are involved constituting a pest complex. Understanding a species' population diversity and structure can provide a better understanding of its adaptation and relative pest potential. Such is the need for the native rice stink bug Oebalus poecilus and the invasive O. ypsilongriseus in low and high flatlands of South America.The genetic structure differed between both rice stink bug species (FDespite the pointed peculiarities, the obtained results indicate overlap in both species' occurrence and similar genetic structure allowing for a compound problem to be dealt with as the complex requires managing without, as yet, a prevailing species or a niche specialization. © 2022 Society of Chemical Industry.

Aphids feed on plant phloem sap that contains massive amounts of sucrose; this not only provides vital nutrition for the aphids but also produces high osmotic pressure. To utilize this carbon source and overcome the osmotic pressure, sucrose is hydrolyzed into the monosaccharides, glucose and fructose. In the green peach aphid (Myzus persicae), we show that this process is facilitated by a key α-glucosidase (MpAgC2-2), which is abundant in the aphid salivary gland and is secreted into leaves during feeding. MpAgC2-2 has a pH optimum of 8.0 in vitro, suggesting it has adapted to the environment of plant cells. Silencing MpAgC2-2 (but not the gut-specific MpAgC3-4) significantly increased the amount of sucrose ingested and hindered aphid feeding on the phloem of tobacco seedlings, resulting in a smaller body size, as well as lower α-glucosidase activity and glucose levels. These effects could be rescued by feeding aphids on tobacco plants transiently expressing MpAgC2-2. The transient expression of MpAgC2-2 also led to the hydrolysis of sucrose in tobacco leaves. Taken together, these results demonstrate that MpAgC2-2 is a salivary protein that facilitates extra-intestinal feeding via sucrose hydrolysis. Our findings provide insight into the ability of aphids to digest the high concentration of sucrose in phloem, and the underlying mechanism of extra-intestinal digestion.

The soybean stem fly, Melanagromyza sojae, an Asian native insect, has successfully established in Brazil, Argentina, Paraguay and Bolivia. These countries are among the lead global soybean producing nations, being collectively known as the soybean belt of South America. Infestation levels of M. sojae grow by the year, facilitated by the lack of efficient management strategies. Previous studies have revealed a high number of maternal lineages in M. sojae populations from Southern Brazil and Paraguay, but a comprehensive survey on genetic diversity combining samples from all countries within the South American soybean belt remains absent. We used the mitochondrial DNA cytochrome oxidase I partial gene (mtCOI) to characterize specimens of M. sojae collected in fourteen Brazilian sites and one Argentine site, and then combined our mtCOI data with previously published data from Australia, Bolivia, Paraguay, and other Brazilian sites, to investigate genetic diversity in this invasive agricultural pest species. Based on the molecular characterisation of the mtCOI gene, haplotypes Msoj-COI-01 and Msoj-COI-02 have the highest frequencies in the continent. The high genetic diversity found is evidence of introductions involving multiple female founders into the continent, and the high proportion of unique mtDNA haplotypes identified from Brazil, Paraguay and Bolivia (~ 50%) suggests potential novel introductions have taken place. The findings from our study will contribute to a better understanding of M. sojae genetic diversity in South America, supporting the development of management strategies for this highly invasive pest and assisting with biosecurity preparedness of other emerging Agromyzidae flies of economic importance.

The invasion of non-native bees to new ecological territories could spread novel pathogens causing emerging infectious diseases (EIDs) in native species. We provide novel information on the prevalence, load, and co-infection network of honey bee viruses, trypanosoma, microsporidia and neogregarinorida pathogens in native Bombus dahlbomii and non-native Bombus terrestris and Bombus ruderatus. Apicystis bombi and Crithidia bombi were highly prevalent (> 78%) in three bumble bee species, with high loads of these pathogens. Nosema bombi was detected only in B. terrestris (37%) and B. ruderatus (15%). Lotmaria passim was detected in low prevalence (

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.

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a persistent agricultural pest in many areas of the world, and it successfully invaded China in 2019. Like many lepidopteran insects, S. frugiperda pupates live in soil for protection under harsh environmental conditions. We conducted a serial experiment to understand the pupation behavior of S. frugiperda, as well as the effect of soil moisture on pupation. A digital video camera system was used to monitor larval pupation behavior of S. frugiperda. Four different soil moisture choices (5, 25, 50, and 80%) were used to determine the pupation location and behavior of S. frugiperda under laboratory conditions. The four-choice tests showed that most of the larvae prefer to pupate at 25–50% in saturated soil, and other few larvae burrow and pupate under dry (5%) or wet (80%) soils. In the no-choice tests, S. frugiperda larvae did not prefer the wet soil (80%) to pupate. However, soil moisture did not significantly affect the emergence rates. This study helps to increase the knowledge on pupation ecology of S. frugiperda and implies management strategies for this destructive pest.

The pollen beetle Brassicogethes aeneus is a serious pest of oilseed rape (Brassica napus) in Europe. Management of this pest has grown difficult due to B. aeneus’s development of resistance to pyrethroid insecticides, as well as the pressure to establish control strategies that minimise the impact on nontarget organisms. RNA interference represents a nucleotide sequence-based, and thus potentially species-specific, approach to agricultural pest control. The present study examined the efficacy of targeting the coatomer gene coatomer subunit alpha (αCOP), via both microinjection and dietary exposure to exogenous complementary dsRNA, on αCOP-silencing and subsequent mortality in B. aeneus. Beetles injected with dsRNA targeting αCOP (at 0.14 µg/mg) showed 88% and 100% mortality at 6 and 10 days post-injection, respectively; where by the same time after dietary exposure, 43%–89% mortality was observed in the 3 µg dsRNA/µL treatment, though the effect was concentration-dependent. Thus, the effect was significant for both delivery routes. In working towards RNA-based management of B. aeneus, future studies should include αCOP as a target of interest.

Double-stranded RNA (dsRNA) designed to target genes in a sequence-specific manner can be valuable in pest control. However, in scenarios where a nontarget could be susceptible and is predicted to be exposed to insecticidal dsRNA in the environment, it is useful to understand possible gene silencing effects in the nontarget. In this study, a sequence complementarity-based approach was used to evaluate potential gene silencing effects in the bumblebee, Bombus terrestris, following oral exposure to a fusion dsRNA product (Ba-dsCOP-vg) specifically designed to target both alpha-coatomer (alphaCOP) and vitellogenin (vg) in the pest pollen beetle, Brassicogethes aeneus. The susceptibility of bumblebees to ingested dsRNA was first evaluated by feeding them with bumblebee-specific dsRNA (Bt-dsCOP-vg) (1 µg/µl) targeting its alphaCOP and vg. A respective 50% and 47% reduction in the transcripts of alphaCOP and vg at 96 h post exposure confirmed its susceptibility to the ingested dsRNA. Using computational tools, a pool of all possible continuous 22-nucleotide stretches from Ba-dsCOP-vg was generated and mapped to all potential off-target genes in the B. terrestris genome, with at least a 13-nt match within the 22-nt stretch. A set of criteria based on sequence complementarity was then used to select 24 potential off-targets for transcript analysis in bumblebees. Following ingestion of Ba-dsCOP-vg (1 µg/µl), no reduction was found in the transcript level for all off-targets, including an off-target with 20-continuous-nt matches. Our findings provide guidance to future risk analysis of dsRNA products and for risk assessments frameworks that incorporate sequence complementarity-based analysis for off-target predictions.

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.

Crangon crangon is economically a very important species. Recently, promising culture attempts have been made, but a major problem is the uncontrollable mortality during the grow-out phase. As of yet, the life cycle of C. crangon is not closed in captivity so wild-caught individuals are used for further rearing. Therefore, it is important to investigate the virome of C. crangon both in wild-caught animals as in cultured animals. In recent years, next-generation-sequencing (NGS) technologies have been very important in the unravelling of the virome of a wide range of environments and matrices, such as soil, sea, potable water, but also of a wide range of animal species. This will be the first report of a virome study in C. crangon using NGS in combination with the NetoVIR protocol. The near complete genomes of 16 novel viruses were described, most of which were rather distantly related to unclassified viruses or viruses belonging to the Picornavirales, Bunyavirales Nudiviridae, Parvoviridae, Flaviviridae, Hepeviridae, Tombusviridae, Narnaviridae, Nodaviridae, Sobemovirus. A difference in virome composition was observed between muscle and hepatopancreatic tissue, suggesting a distinct tissue tropism of several of these viruses. Some differences in the viral composition were noted between the cultured and wild shrimp, which could indicate that in sub-optimal aquaculture conditions some viruses become more abundant. This research showed that a plethora of unknown viruses is present in C. crangon and that more research is needed to determine which virus is potentially dangerous for the culture of C. crangon.

Insecticides can affect development and survival of non-target and beneficial arthropods like honey bees (Apis mellifera L.). Thiamethoxam and spinosad are widely used as pesticides in agriculture but they have become an important concern for beekeepers and researchers focusing on bee health; multiple reports stressed adverse effects on bees, notably on honey bees. The present study aims to evaluate the impact of these two insecticides on the development of the HPGs and on the survival of Apis mellifera intermissa a native African subspecies of honey bee present in Algeria. Newly emerged workers were acutely and chronically exposed to thiamethoxam and spinosad through sugar syrup and pollen pastry. The effects of these insecticides were assessed by measuring the size of HPGs acini and the total head protein content. The survival of the workers was also evaluated over 60 days when they were chronically exposed to the insecticides at concentrations corresponding to LC25 and LC10. We found that the insecticide-treated workers, after both acute and chronic exposure, exhibited smaller and irregularly shaped HPG acini. The total head protein content also decreased in treated individuals with the two concentrations of insecticides at day 6 and 9 compared to the respective controls. While the control group exhibited an LC25 (i.e. the time needed to kill 50% of the tested workers) of 22 days, the LC25 was only 3 days for the workers exposed to the LC25 of spinosad and all workers were dead at day 17. In contrast, thiamethoxam exposure at LC25 had no significant detrimental effect on honey bee survival. This study demonstrated the toxicity of thiamethoxam and spinosad to workers of A. mellifera intermissa and highlighted potential detrimental effects of the bioinsecticide spinosad on HPGs and survival of the bee workers.

Pollination service in agricultural crops increases significantly with pollinator diversity and wild pollinator abundance. Differences in the foraging behaviour of pollinating insects are one of the reasons why pollinator diversity and abundance enhances crop pollination. Here, we focused on the foraging behaviour of honey bees and bumble bees in sweet cherry orchards. In addition, we studied the influence of bee diversity and abundance on the foraging behaviour of honey bees and bumble bees. Honey bees were found to visit fewer flowers than bumble bees. Bumble bees also showed a higher probability of changing trees between rows than honey bees. Both visitation rate and probability of row changes of honey bees increased with bumble bee diversity and with bumble bee abundance. We also found that the probability of row changes of honey bees increased with increasing bumble bee abundance. These effects of bumble bee richness and abundance on the pollination behaviour of honey bees can improve the pollination performance of honey bees in crops that depend on cross pollination. Our results highlight the higher pollination performance of bumble bees and the facilitative effect of wild pollinators to crop pollination.

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.

As one of the most abundant and destructive pests in agriculture, aphids cause significant damage to crops due to their sap-taking and as virus vectors. Chemical insecticides are the most effective method to control aphids, but they bring insecticide resistance problems and harm nontarget organisms, especially bees, therefore the search for novel eco-friendly aphid control agents with low bee toxicity is urgent. Insect kinins are a class of small neuropeptides that control important functions in insects. In our previous study, we found insect kinin analog IV-3 has good aphicidal activity and the location of the aromatic ring on the side chain of PheIn this project, 44 insect kinin analogs with the PheThe residue Phe

Commercial apple production relies on managed honey bees for pollination, and on intensive management for pest control. Previous studies revealed the detrimental effects of these factors on wild bee diversity in agroecosystems, and the pollination services they provide. However, the extent to which honey bee dominance and management interact under field-realistic conditions to shape wild communities has never been investigated. We measured the species richness and functional and phylogenetic diversity of wild bees associated with apple blossoms, in 46 organic and non-organic orchards, and along a climatic gradient across Western Europe and Morocco. Our results consistently show a strong and negative association between honey bee dominance and all diversity indices, regardless of local practices and surrounding landscape cover. Collectively, we show that wild bee diversity decreases with increasing honey bee density, with alternative management practices (i.e., organic) having no significant influence on wild bee communities in commercial apple orchards.

The quality of acclimatized in vitro cultivated plants is essential to ensure good survival and growth after planting in field conditions. After two months of acclimatization, this study revealed a significant variation in survival rate, shoot characteristics, root traits, and biomass features between 13 clones of Melia volkensii Gürke. A number of quality indices such as the Dickson Quality Index (DQI), shoot dry weight: root dry weight ratio (S:R), and sturdiness quotient (SQ) also showed a large variation. The survival rate was genotype-dependent, with an average of 85%. Extreme genotypes were tall, had long internodes, a lot of leaves, and a large leaf area. At the other side of the spectrum, there were small clones with short internodes and a reduced number of leaves and leaf areas. The high S:R values (>2) of the evaluated clones indicate an imbalance between the shoot and root system, negatively influencing the quality of the in vitro produced M. volkensii plants. The SQ for all clones was below the threshold value of 6, implying a good and expected survival rate. This study illustrates that some clones might systematically score better for SQ, S:R, plant volume, and DQI after acclimatization and that these factors could form the basis for selection.

Commercial apple production relies on managed honey bees (Apis mellifera) for pollination, and on intensive management for pest control. Previous studies have highlighted the potentially detrimental effects of intensive crop management on wild bee diversity in agroecosystems, potentially jeopardizing the pollination services they provide. However, the extent to which honey bee dominance and crop management interact under field-realistic conditions and drive the structure of wild bee assemblages has not been investigated so far. In this study, we measured species richness, as well as the functional and phylogenetic diversity of wild bee assemblages in 36 paired organic and non-organic apple orchards during their flowering season and along a geographic gradient across western Europe. Our results show a strong significant and negative association between honey bee dominance and all wild bee diversity metrics, regardless of local management. Semi-natural habitats had a significant and positive effect on functional diversity, while urbanization and crop cover around the orchards showed no effect on all measured diversity metrics. A greater number of species exhibited less common, or frequent, combinations of functional traits at sites with high honey bee dominance, especially larger bee species with longer tongues. Collectively, we show that wild bee diversity decreases with increasing honey bee dominance, and that this negative association is not buffered by alternative (i.e., organic) management practices in commercial apple orchards. Although organic farming can bring about biodiversity benefits, our study demonstrates that, in the context of commercial apple production, other measures are needed to enhance and harness biodiversity for sustainable and profitable crop production. In particular, a lowered reliance on honey bees and a redesign of orchards through configurational crop heterogeneity and/or the restoration of in-field semi‐natural elements are required beyond agricultural input substitution.

Bees, both wild and domesticated ones, are hosts to a plethora of viruses, with most of them infecting a wide range of bee species and genera. Although viral discovery and research on bee viruses date back over 50 years, the last decade is marked by a surge of new studies, new virus discoveries, and reports on viral transmission in and between bee species. This steep increase in research on bee viruses was mainly initiated by the global reports on honeybee colony losses and the worldwide wild bee decline, where viruses are regarded as one of the main drivers. While the knowledge gained on bee viruses has significantly progressed in a short amount of time, we believe that integration of host defense strategies and their effect on viral dynamics in the multi-host viral landscape are important aspects that are currently still missing. With the large epidemiological dataset generated over the last two years on the SARS-CoV-2 pandemic, the role of these defense mechanisms in shaping viral dynamics has become eminent. Integration of these dynamics in a multi-host system would not only greatly aid the understanding of viral dynamics as a driver of wild bee decline, but we believe bee pollinators and their viruses provide an ideal system to study the multi-host viruses and their epidemiology.

Editorial on the Research Topic: Advances and Challenges of RNAi Based Technologies for Plants—Volume 2

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.

N-glycosylation is one of the most common and important post-translational modifications in the eukaryotic cell. The study of protein N-glycosylation in several model insects confirmed the importance of this process in insect development, immunity, survival and fertility. The Colorado potato beetle (Leptinotarsa decemlineata) (CPB) is a common pest of Solanaceae crops. With the infamous title of champion of insecticide resistance, novel pest control strategies for this insect are needed. Luckily this pest insect is reported as very sensitive for the post-genomic technology of RNA interference (RNAi).In this project, we investigated the importance of N-glycosylation in the survival and development of CPB using RNAi-mediated gene silencing of N-glycosylation-related genes (NGRGs) during the different transition steps from the larva, through the pupa to the adult stage. High mortality was observed in the larval stage with the silencing of early NGRGs, as STT3a, DAD1 and GCS1. With dsRNA against middle NGRGs, abnormal phenotypes at the ecdysis process and adult formation were observed, while the silencing of late NGRGs did not cause mortality.The lethal phenotypes observed on silencing of the genes involved in the early processing steps of the N-glycosylation pathway suggest these genes are good candidates for RNAi-mediated control of CPB. Next to the gene-specific mechanism of RNAi for biosafety and possible implementation in integrated pest management, we believe these early NGRGs provide a possible alternative to the well-known target genes Snf7 and vacuolar ATPases that are now used in the first commercial RNAi-based products and thus they may be useful in the context of proactive resistance management. © 2021 Society of Chemical Industry.

Simple Summary Over the past decades, Integrated Pest Management (IPM) strategies have been widely adopted in commercial fruit production in Europe, supporting natural pest control as an ecosystem service. At the same time, there has been a growing awareness of the importance of pollinating insects, leading to the concept of Integrated Pest and Pollinator Management (IPPM). Here we present the outcomes of a 4-year case study as a valuable illustration of an IPPM strategy in a commercial intensive pear orchard. We show how the added-value of local biodiversity measures can be visualized in front of growers, linking ecological measures to economic benefits. This scientifically-based as well as practice-oriented demonstrative case study supports the acceptance and adoption of IPPM principles in commercial intensive pear production cultivation. Abstract Recently, the concept of Integrated Pest Management (IPM) was further extended into Integrated Pest and Pollinator Management (IPPM). Implementation of IPPM strategies entails the combination of actions for pest and pollinator management providing complementary or synergistic benefits for yield and/or quality of the harvest. The aim of this study was to examine IPPM elements (i.e., mixed hedgerow, nesting boxes for mason bees, Osmia spp.) and demonstrate their impact in the practical context of modern commercial fruit cultivation in a 4-year case study in an intensive ‘Conference’ pear orchard. The outcomes of visual observations during transect walks and molecular analysis of pollen collected by mason bees, showed the importance of additional floral resources for the presence of mason bees and other pollinating insects in the orchard environment. Pear quality assessments indicated that insect-mediated pollination had a significant positive impact, with a tendency for higher quality pears in the close vicinity of Osmia nesting boxes. However, despite the fact that pear pollen was also detected in Osmia spp. nest cells, the amount and frequency of pear pollen collection for their nest built-up turned out to be rather low. In the same intensive pear orchard studied for pollination effects, we simultaneously demonstrate the impact of a mixed hedgerow to enhance integrated pest control.

The African sweet potato weevil, Cylas puncticollis, and fall armyworm, Spodoptera frugiperda, are insect pests of economic importance that have a negative impact on sweet potato and maize production, respectively. In this study, we aimed to evaluate the potential of Melia volkensii extracts to protect sweet potato and maize plants against damage by both insect pests. We evaluated extracts from the bark, leaves, pulp and nuts of Melia volkensii for antifeedant activity against C. puncticollis and S. exigua (used as a substitute for S. frugiperda), under laboratory conditions. Interestingly, extracts of all plant parts showed antifeedant activity. These results led us to investigate the effectiveness of nut and pulp extracts to protect sweet potato and maize crops in greenhouse conditions. Against C. puncticollis, the sweet potato plants treated with nut extracts showed the lowest tuber damage (18%) when compared to pulp extracts (30%), positive control (33%) and negative control (76%). Nut extracts, pulp extracts and positive control reduced maize leaf and whorl damage by S. frugiperda compared to the negative control. Altogether, this study highlights the potential of M. volkensii extracts and their application in integrated insect pest management

Although dibenzoylhydrazine-type non-steroidal ecdysone agonists, such as fufenozide, have an excellent performance record, the emergence of resistance could severely compromise the efficacy of these compounds in integrated pest management programs. To investigate possible mechanisms of resistance, we investigated the regulation of the expression of the PxEcR-B gene encoding the ecdysone receptor isoform B (PxEcR-B), which is the specific target of fufenozide in P. xylostella. Bioinformatics analysis revealed a putative miR-189942 binding site in the 3’-UTR of PxEcR-B mRNA. In a PxEcR-B 3’-UTR luciferase reporter system, miR-189942 downregulated the luciferase activity, and these effects were abolished by a deletion mutation in the putative miR-189942 binding site. Moreover, at 96 h after treatment with an agomir (mimic) or antagomir (inhibitor) of miR-189942, PxEcR-B expression was decreased by 71 ± 4% and increased by 4.19- fold respectively. Furthermore, overexpression or knockdown of miR-189942 changed the sensitivity of P. xylostella to fufenozide in vivo but had no influence on the sensitivity to chlorantraniliprole, which does not target PxEcR-B. These data indicate that miR-189942 suppressed PxEcR-B expression via binding at the 3’-UTR of PxEcR-B, thus increasing the tolerance of P. xylostella to fufenozide. These findings provide empirical evidence of the involvement of miRNAs in the regulation of insecticide resistance.