Onderzoek omtrent thiacloprid, essentiële oliën en dubbelstrengs RNA voor mogelijk gebruik in een veilig beheer van stuifmeelkever

A Thesis submitted to Estonian University of Life Sciences (EMÜ) and Ghent University (UGent) in partial fulfilment of the requirements for the joint degree of Doctor of Philosophy in Agriculture (EMÜ) and Bioscience Engineering (UGent).Throughout Europe, one of the most severely damaging threats to oilseed rape (Brassica napus) cultivation is the pollen beetle Brassicogethes aeneus; and routine use of pyrethroid insecticides has led to resistance development among B. aeneus populations. Moreover, exposure to pyrethroids can be detrimental to many nontarget insect taxa that inhabit agroecosystems. While studies have demonstrated some potential for relying on biological control (biocontrol) organisms, especially parasitoid wasps, for biosafe management of B. aeneus populations, the most effective B. aeneus management strategy will likely involve insecticide applications that show efficacy against B. aeneus while simultaneously minimising the impact on nontarget organisms. We examine the potential utility of three types of insecticide within a biosafe integrated B. aeneus management strategy. The first is the neonicotinoid thiacloprid, which has previously shown efficacy against B. aeneus, yet must be tested for biosafety in a model parasitoid wasp. Second, we examined potential for using seven different plant-based essential oils (EOs) in B. aeneus management. Last, we investigated the potential for applying B. aeneus-specific double-stranded ribonucleic acid (dsRNA), representing a potentially species-specific biopesticide option, within a B. aeneus management strategy. Exposure to residues of thiacloprid resulted in significant reductions in Aphelinus abdominalis (model biocontrol organism) survival and mobility. Moreover, simultaneous exposure to residues of thiacloprid and tebuconazole (a fungicide commonly tank-mixed with thiacloprid) resulted in synergistic reductions in A. abdominalis survival and mobility. Cinnamon (Cinnamomum verum) bark EO was the most effective EO tested against B. aeneus via topical application. However, subsequent assays using C. verum EO-sprayed leaf- and bud surfaces showed significant control efficacy only at concentrations too high for practical field application of an EO. Field relevant dietary routes of exposure to dsRNA targeting B. aeneus coatomer subunit alpha (αCOP) resulted in significant gene silencing and gene silencing-induced mortality. However, refinements to B. aeneus larval bioassay setups, as well as developing dsRNA formulations that enhance transport past the oilseed rape bud epithelium, and/or enhance dsRNA uptake in B. aeneus, represent critical steps to understanding the potential for RNAi-based management of B. aeneus. Most notably, we observed that RNAi efficacy is enhanced by chronic dsRNA feeding in B. aeneus. This result has implications for the economics and development of a potential dsRNA spray approach for managing B. aeneus; and it highlights the need for research into the development and potential future use of RNAi oilseed rape cultivars, given the enhanced RNAi efficacy resulting from chronic dsRNA feeding in B. aeneus.Rapsi (Brassica napus) kõige olulisemaks kahjuriks kogu Euroopas on naeri-hiilamardikas (Brassicogethes aeneus (syn. Meligethes aeneus, Coleoptera: Nitidulidae)), kelle tõrjeks kasutatakse peamiselt püretroididel baseeruvaid insektitsiide, mille tulemusel on neil väljakujunenud resistentsus selle tõrjevahendi suhtes. Lisaks sellele on püretroidid mürgised ka paljudele teistele mittesihtrühma putukatele, kelle elupaigaks on agroökosüsteemid. Kuigi paljud uurimused on tõestanud, et looduslikult esinevad röövtoidulised organismid (näiteks jooksiklased, lühitiiblased, parasitoidid) suudavad potentsiaalselt ohjata ka hiilamardikate arvukust rapsipõldudel hõlmab tõenäoliselt kõige efektiivsem hiilamardika tõrjestrateegia ka insektitsiidide kasutamist. Samas peaksid viimased olema võimalikult ohutud mitte-sihtrühma organismidele. Käesolevas töös uuriti kolme erinevat tüüpi insektitsiidi potentsiaalset rakendatavust hiilamardika tõrjestrateegias. Esiteks uuriti neonikotinoidi tiaklopriini, mis on efektiivne hiilamardika tõrjevahend, kuid bioloogilist ohutust parasitoidide, kui biotõrje agentide suhtes ei olnud uuritud. Teiseks uuriti seitsme taimse eeterliku õli potentsiaali hiilamardika tõrjevahendina. Ja viimaseks uuriti hiilamardika-spetsiifilise kaheahelalisel RNAl (dsRNA) põhineva biopestitsiidi potentstiaali võimalikku kasutamist hiilamardika tõrjestrateegias. Töödeldes parasitoidi mudelliiki Aphelinus abdominalis tiaklopriidi jääkidega vähendas oluliselt tema ellujäävust ja mobiilsust. Veelgi enam, tiaklopriini kasutamisel koos fungitsiidi tebukonasooliga (tavaline paagisegu rapsipõldude töötlemisel) ilmnes ainete sünergiline letaalne mõju parasitoidile. Taimsetest eeterlikest õlidest osutus kõige efektiivsemaks kaneelikooreõli (Cinnamomum verum), mis hiilamardikate välispidisel töötlusel vähendas oluliselt nende ellujäävust ja mobiilsust. Samas olid efektiivsed kontsentratsioonid liiga suured selleks, et neid soovitada põllutingimustes kasutamiseks. Katsed, kus kasutati põllukontsentratsioonidele relevantseid dsRNA koguseid, näitasid, et hiilamardikatele nii mikrosüstimisel kui suukaudsel dsRNA (mis oli suunatud hiilamardika coatomer alfa alam-üksuse (αCOP) proteiini vaigistamisele) manustamisel saavutasime olulise geenivaigistuse ja sellest põhjustatud suremuse. Vaatamata palju lubavatele esialgsetele tulemustele tuleb katseid jätkata, et leida võimalusi dsRNA stabiliseerimiseks, selle taimerakkudesse läbitavuse suurendamiseks, dsRNA mõju tõstmiseks B. aeneuse’l. Veelgi enam, katsete tulemusel täheldasime, et dsRNA kroonilisel manustamisel oli geenivaigistamise mõju suurem, millel on potentsiaalselt majanduslik mõju ja tõstatub suur vajadus edasiste uuringute järele RNAi sortide osas.This research was supported by European Social Fund’s Doctoral Studies and Internationalization Programme DoRa. The publication of this thesis was supported by the Estonian University of Life Sciences and the European Unions’s European Regional Development Fund (Estonian University of Life Sciences ASTRA project ‘Value-chain based bioeconomy’)

Case study on forage plants of the heath bumblebee (Bombus jonellus) in southwest Iceland

Bumblebees (Bombus spp.) are pollinators of especially high conservation interest. They have behaviours (pollen-gathering, buzz pollination), morphological structures (branched body hairs well-adapted for retaining pollen), and endothermic capabilities, that make them welladapted for transporting large amounts of pollen in subarctic regions (Heinrich & Vogt 1993, De Luca & Vallejo-Marín 2013). Recent findings indicate that 23.6% of bumblebee species in Europe are threatened with extinction, and that 45.6% of Europe’s bumblebee species are in decline (Nieto et al. 2014). These declines are likely due to multiple threats acting synergistically, but the primary threat is the loss and fragmentation of foraging and nesting resources (Kosior et al. 2007, Potts et al. 2010, Goulson et al. 2015). In Iceland, the aggressive spread of invasive non-native plant species such as Nootka lupine (Lupinus nootkatensis Donn) and cow parsley (Anthriscus sylvestris L.) (Magnússon 2011, Icelandic Institute of Natural History 2016) represents a serious threat to native forage-plant communities. Recent research suggests that Iceland’s only native bee, the heath bumblebee (B. jonellus Kirby), is at risk of serious declines in Iceland due to the spread of invasive plant populations (Willow 2016). To protect B. jonellus in Iceland, we need to not only manage invasive plant species, but also improve our knowledge of the native food plants that B. jonellus visits (Willow 2016). A range of flowering plant species used by B. jonellus in Iceland is given in Prŷs-Jones et al. (1981, 2016), with estimates of their significance as forage resources. However, further systematic observations of foraging preferences are required, as the plant-pollinator network throughout Iceland is undergoing changes, particularly due to the spread of invasive plant populations and the introduction of exotic bumblebee species (Magnússon 2011, Icelandic Institute of Natural History 2016, Prŷs-Jones et al. 2016). The primary aim of this study was to determine the significance of various plant species, across the forage season, as forage for B. jonellus in relatively natural environments in south-west Iceland. The importance of each forage plant species was estimated from the number of observed B. jonellus visits.Náttúruverndarsjóður Pálma JónssonarPeer Reviewe

Acute effect of low-dose thiacloprid exposure synergised by tebuconazole in a parasitoid wasp

Agricultural practices often involve tank-mixing and co-application of insecticides with fungicides to control crop pests. However, natural methods relying on biological control agents such as hymenopteran parasitoids have been shown to be highly effective in suppressing crop pest populations. The current body of insecticide risk assessment data accounting for fungicide co-application is very small, the present study being the first to examine this in a parasitoid wasp. Through low-dose exposure to dry residues of the neonicotinoid insecticide thiacloprid, we examined its mortal and knockdown effect on Aphelinus abdominalis when co-applied with increasing doses of the fungicide tebuconazole. Both of these acute effects of thiacloprid were synergised (toxicity increased to a greater-than-additive effect) by tebuconazole, resulting in significant mortality from low-dose co-applications of tebuconazole, and significant knockdown even without co-applied tebuconazole, the effect increasing as tebuconazole concentration increased. We show the highly toxic effect that a low dose of thiacloprid imposes on A. abdominalis populations, and a synergistic toxicity when co-applied with low doses of tebuconazole. Our work suggests a need for updating pesticide risk assessment methods, accounting for pesticide mixtures, in order to make these risk assessments more field relevant

Potential impact of Nootka lupine (Lupinus nootkatensis) invasion on pollinator communities in Iceland

Pollinator communities have an integral role in facilitating sexual reproduction within and between flowering plant populations. Declines in abundance and diversity of pollinating insects are widely documented throughout Europe, primarily the result of habitat loss and fragmentation (Fox 2013, Nieto et al. 2014, Goulson et al. 2015). In 1945, seeds of Nootka lupine (Lupinus nootkatensis Donn) were collected in Alaska, brought to Iceland, and introduced at reforestation sites. This plant’s invasiveness in Iceland was indicated by its tendency to replace native vegetation with homogeneous L. nootkatensis stands (Magnússon 2010). The lack of published research on Iceland’s broader pollinator community, combined with the dramatic changes in vegetation that have taken place over the last few decades, particularly with L. nootkatensis’s distribution, warrants an analysis of plant-pollinator relationships in Iceland, especially regarding L. nootkatensis. The present study aims to describe how pollinator communities differ between L. nootkatensis and the native flowering plants in heath habitat in south-west Iceland. The findings of this study will give an indication of whether L. nootkatensis can serve as an alternative food source for Iceland’s pollinator community, in the event that L. nootkatensis continues to replace native flowering plant communities throughout Iceland.Peer Reviewe

On the Non-Malleability of ECVRF in the Algebraic Group Model

ECVRF is a verifiable random function (VRF) scheme used in multiple cryptocurrency systems. It has recently been proven to satisfy the notion of non-malleability which is useful in applications to blockchains (Peikert and Xu, CT-RSA 2023); however, the existing proof uses the rewinding technique and has a quadratic security loss. In this work, we re-analyze the non-malleability of ECVRF in the algebraic group model (AGM) and give a tight proof. We also compare our proof with the unforgeability proof for the Schnorr signature scheme in the AGM (Fuchsbauer, Plouviez and Seurin, EUROCRYPT 2020)

First evidence of bud feeding-induced RNAi in a crop pest via exogenous application of dsRNA

An ecologically sustainable strategy for managing the pollen beetle Brassicogethes aeneus, a key pest of oilseed rape (Brassica napus) in Europe, is greatly needed. Gene silencing via RNA interference, through sprayed applications of target-specific double-stranded RNA, represents a potential alternative to conventional insecticides. We used dsRNA designed to target a vital gene in this pollen beetle species and allowed the beetles to feed on dsRNA-coated oilseed rape buds. We observed a significant silencing of the target gene; and this was followed by a significant, albeit delayed, reduction in pollen beetle survival rate. Further experiments are necessary in order to better understand the potential for developing a dsRNA-spray approach to pollen beetle management. Spray-induced gene silencing (SIGS) is a potential strategy for agricultural pest management, whereby nucleotide sequence-specific double-stranded RNA (dsRNA) can be sprayed onto a crop; the desired effect being a consumption of dsRNA by the target pest, and subsequent gene silencing-induced mortality. Nucleotide sequence-specificity is the basis for dsRNA's perceived biosafety. A biosafe approach to pollen beetle (Brassicogethes aeneus) management in oilseed rape (Brassica napus) agroecosystems is needed. We examined the potential for SIGS in B. aeneus, via bud feeding, a field-relevant dsRNA exposure route. Oilseed rape buds were uniformly treated with dsRNA designed to target alpha COP in B. aeneus. Our model control dsRNA (dsGFP) remained detectable on buds throughout the entire 3 d exposure period. When applied at 5 mu g/mu L, ds alpha COP induced significant alpha COP silencing 3 d after dietary exposure to buds treated with this ds alpha COP concentration. We also observed a trend of increased alpha COP silencing with increasing concentrations of ds alpha COP at both 3 and 6 d. Furthermore, we observed a marginally significant and significant reduction in B. aeneus survival at 10 and 15 d, respectively. Our results suggest potential for developing a SIGS approach to B. aeneus management-though further experiments are needed to more fully understand this potential

Towards dsRNA ‐integrated protection of medical Cannabis crops: considering human safety, recent‐ and developing RNAi methods, and research inroads

Owing to the expanding industry of medical Cannabis, we discuss recent milestones in RNA interference (RNAi)‐based crop protection research and development that are transferable to medical Cannabis cultivation. Recent and prospective increases in pest pressure in both indoor and outdoor Cannabis production systems, and the need for effective nonchemical pest control technologies (particularly crucial in the context of cultivating plants for medical purposes), are discussed. We support the idea that developing RNAi tactics towards protection of medical Cannabis could play a major role in maximizing success in this continuously expanding industry. However, there remain critical knowledge gaps, especially with regard to RNA pesticide biosafety from a human toxicological viewpoint, as a result of the medical context of Cannabis product use. Furthermore, efforts are needed to optimize transformation and micropropagation of Cannabis plants, examine cutting edge RNAi techniques for various Cannabis–pest scenarios, and investigate the combined application of RNAi‐ and biological control tactics in medical Cannabis cultivation. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry

Targeting a coatomer protein complex-I gene via RNA interference results in effective lethality in the pollen beetle Brassicogethes aeneus

he 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 (alpha COP), via both microinjection and dietary exposure to exogenous complementary dsRNA, on alpha COP-silencing and subsequent mortality in B. aeneus. Beetles injected with dsRNA targeting alpha COP (at 0.14 mu g/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 mu g dsRNA/mu L 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 alpha COP as a target of interest

Physiological stress and higher reproductive success in bumblebees are both associated with intensive agriculture

Free-living organisms face multiple stressors in their habitats, and habitat quality often affects development and life history traits. Increasing pressures of agricultural intensification have been shown to influence diversity and abundance of insect pollinators, and it may affect their elemental composition as well. We compared reproductive success, body concentration of carbon (C) and nitrogen (N), and C/N ratio, each considered as indicators of stress, in the buff-tailed bumblebee (Bombus terrestris). Bumblebee hives were placed in oilseed rape fields and semi-natural old apple orchards. Flowering season in oilseed rape fields was longer than that in apple orchards. Reproductive output was significantly higher in oilseed rape fields than in apple orchards, while the C/N ratio of queens and workers, an indicator of physiological stress, was lower in apple orchards, where bumblebees had significantly higher body N concentration. We concluded that a more productive habitat, oilseed rape fields, offers bumblebees more opportunities to increase their fitness than a more natural habitat, old apple orchards, which was achieved at the expense of physiological stress, evidenced as a significantly higher C/N ratio observed in bumblebees inhabiting oilseed rape fields

Mischaracterizing wildlife trade and its impacts may mislead policy processes

Overexploitation is a key driver of biodiversity loss but the relationship between the use and trade of species and conservation outcomes is not always straightforward. Accurately characterizing wildlife trade and understanding the impact it has on wildlife populations are therefore critical to evaluating the potential threat trade poses to species and informing local to international policy responses. However, a review of recent research that uses wildlife and trade-related databases to investigate these topics highlights three relatively widespread issues: (1) mischaracterization of the threat that trade poses to certain species or groups, (2) misinterpretation of wildlife trade data (and illegal trade data in particular), resulting in the mischaracterization of trade, and (3) misrepresentation of international policy processes and instruments. This is concerning because these studies may unwittingly misinform policymaking to the detriment of conservation, for example by undermining positive outcomes for species and people along wildlife supply chains. Moreover, these issues demonstrate flaws in the peer-review process. As wildlife trade articles published in peer-reviewed journals can be highly influential, we propose ways for authors, journal editors, database managers, and policymakers to identify, understand, and avoid these issues as we all work towards more sustainable futures