RNA interference (RNAi) is a powerful technology that offers new opportunities for pest control through silencing of genes that are essential for the survival of arthropod pests. The approach relies ...on sequence-specificity of applied double-stranded (ds) RNA that can be designed to have a very narrow spectrum of both the target gene product (RNA) as well as the target organism, and thus allowing highly targeted pest control. Successful RNAi has been reported from a number of arthropod species belonging to various orders. Pest control may be achieved by applying dsRNA as foliar sprays. One of the main concerns related to the use of dsRNA is adverse environmental effects particularly on valued non-target species. Arthropods form an important part of the biodiversity in agricultural landscapes and contribute important ecosystem services. Consequently, environmental risk assessment (ERA) for potential impacts that plant protection products may have on valued non-target arthropods is legally required prior to their placement on the market. We describe how problem formulation can be used to set the context and to develop plausible pathways on how the application of dsRNA-based products could harm valued non-target arthropod species, such as those contributing to biological pest control. The current knowledge regarding the exposure to and the hazard posed by dsRNA in spray products for non-target arthropods is reviewed and suggestions are provided on how to select the most suitable test species and to conduct laboratory-based toxicity studies that provide robust, reliable and interpretable results to support the ERA.
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•Floral food sources can be utilized by the invasive Harmonia axyridis.•Low quality prey can support full development when combined with floral food.•Differences in food quality are ...consistently visible in lab and semi-field studies.•Chemical analysis of the insects’ energetic budget corroborates biological measures.
The ability to utilize plant-derived food sources and suboptimal prey when the main prey is scarce may enhance competitiveness and invasiveness of entomophagous species such as Harmonia axyridis. Alternative food sources are particularly abundant in flower strips and other agri-environment schemes to promote biodiversity and may thus also benefit the invasive species. We investigated the effects of alternative food sources on the development and reproduction of H. axyridis. Laboratory experiments demonstrated that larvae of H. axyridis developed into adults and produced offspring when reared solely on aphids, lepidopteran eggs or maize pollen but not when they were fed only lepidopteran caterpillars or buckwheat flowers. When fed a combination of the latter two suboptimal food sources, however, some H. axyridis larvae developed into fertile adults. Flowering plant species differed in their food quality to sustain ladybird survival and development when fed alone or in combination with suboptimal prey. Differences in food quality of flower species were confirmed in field-cage studies where newly emerged adults were exposed for six days to different plant species and their energetic compartments were analyzed subsequently. Overall Fagopyrum esculentum and Centaurea cyanus provided a higher food quality than Calendula arvensis in those experiments and mixing flower species did not provide an additional benefit. The results show that the harlequin ladybird can sustain itself not only on optimal prey, but also utilize alternative, animal- and plant-derived diets. This could provide H. axyridis a competitive advantage over those native ladybird species that depend on aphids for their reproduction.
A rape seed pollen-based diet was developed and found to be suitable for use in a dietary exposure assay for Propylea japonica. Using the diet, we established and validated a dietary exposure assay ...by using the protease inhibitor E-64 as positive control. Dose-dependent responses were documented for all observed life-table parameters of P. japonica including survival, pupation and eclosion rates, development time and adult weight. Results suggested that the dietary assay can detect the effects of insecticidal compounds on the survival and development of P. japonica. Using the established dietary assay, we subsequently tested the toxicity of Cry1Ab, Cry1Ac and Cry1F proteins that are expressed by transgenic maize, cotton or rice plants to P. japonica larvae. The diet containing E-64 was included as a positive control. Survival and development of P. japonica larvae were not adversely affected when the diet contained purified Cry1Ab, Cry1Ac, or Cry1F at 500 µg/g diet representing a worst-case exposure scenario. In contrast, P. japonica larvae were adversely affected when the diet contained E-64. The bioactivity and stability of the Cry proteins in the diet and Cry protein uptake by the ladybird larvae were confirmed by bioassay with a Cry-sensitive insect species and by ELISA. The current study describes a suitable experimental system for assessing the potential effects of gut-active insecticidal compounds on ladybird beetle larvae. The experiments with the Cry proteins demonstrate that P. japonica larvae are not sensitive to Cry1Ab, Cry1Ac and Cry1F.
Genetically engineered (GE) crops with stacked insecticidal traits expose arthropods to multiple Cry proteins from Bacillus thuringiensis (Bt). One concern is that the different Cry proteins may ...interact and lead to unexpected adverse effects on non-target species. Bi- and tri-trophic experiments with SmartStax maize, herbivorous spider mites (Tetranychus urticae), aphids (Rhopalosiphum padi), predatory spiders (Phylloneta impressa), ladybeetles (Harmonia axyridis) and lacewings (Chrysoperla carnea) were conducted. Cry1A.105, Cry1F, Cry3Bb1 and Cry34Ab1 moved in a similar pattern through the arthropod food chain. By contrast, Cry2Ab2 had highest concentrations in maize leaves, but lowest in pollen, and lowest acquisition rates by herbivores and predators. While spider mites contained Cry protein concentrations exceeding the values in leaves (except Cry2Ab2), aphids contained only traces of some Cry protein. Predators contained lower concentrations than their food. Among the different predators, ladybeetle larvae showed higher concentrations than lacewing larvae and juvenile spiders. Acute effects of SmartStax maize on predator survival, development and weight were not observed. The study thus provides evidence that the different Cry proteins do not interact in a way that poses a risk to the investigated non-target species under controlled laboratory conditions.
The invasive vinegar fly
Drosophila suzukii
not only infests and damages numerous fruit crops but also develops in many wild fruit species in semi-natural habitats. Biological control in these ...refuges could reduce
D.
suzukii
populations and minimize their dispersal into fruit crops. We investigated parasitization of
D. suzukii
by the pupal parasitoid
Trichopria drosophilae
in seven common wild fruit species. Development and nutrient content of
D.
suzukii
differed among the fruit species. Nevertheless,
T. drosophilae
significantly reduced
D.
suzukii
numbers in all fruits. The development of
T.
drosophilae
was affected by both the fruit species and the quality of the pupal host (size, nutrient content). In olfactometer assays, parasitoid females preferred infested fruits to an empty control and to most non-infested fruits, but spent an equal amount of time on the walking arena above the infested and non-infested fruits of
Rhamnus cathartica
and
Viscum album
. Our results show that
T.
drosophilae
can utilize
D.
suzukii
hosts from a variety of wild fruit species and therefore has the potential to be used as a control agent of
D.
suzukii
in semi-natural habitats.
Genetic modification of apple cultivars through cisgenesis can introduce traits, such as disease resistance from wild relatives, quickly and without crossing. This approach was used to generate the ...cisgenic apple line C44.4.146, a 'Gala Galaxy' carrying the fire blight resistance gene FB_MR5. In contrast to traditionally bred apple cultivars, genetically modified (GM) plants need to undergo a regulatory risk assessment considering unintended effects before approval for commercial release. To determine potential unintended effects of C44.4.146, we assessed major leaf components and effects on the fitness of the decomposers Drosophila melanogaster (fruit fly) and Folsomia candida (collembolan), which were fed a diet amended with powdered apple leaf material. Leaf material of 'Gala Galaxy', several natural 'Gala' mutants, and the unrelated apple cultivar 'Ladina' were used for comparison. The genetic modification did not alter major leaf components and did not adversely affect survival, growth, or fecundity of the two decomposers. Consistent with previous studies with other GM crops, the differences between conventionally bred cultivars were greater than between the GM line and its non-GM wild type. These data provide a baseline for future risk assessments.
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•Leaf composition of cisgenic fire blight-resistant apple similar to parental ‘Gala’.•Apple leaf powder was mixed into the diet of springtails and fruit flies.•Effects of the cisgenic line were comparable to those of six non-GM ‘Gala’ lines.•Differences among non-GM lines exceeded those between cisgenic and parental line.•GM plant risk assessments should consider natural variation among non-GM cultivars.
This paper provides recommendations on experimental design for early-tier laboratory studies used in risk assessments to evaluate potential adverse impacts of arthropod-resistant genetically ...engineered (GE) plants on non-target arthropods (NTAs). While we rely heavily on the currently used proteins from Bacillus thuringiensis (Bt) in this discussion, the concepts apply to other arthropod-active proteins. A risk may exist if the newly acquired trait of the GE plant has adverse effects on NTAs when they are exposed to the arthropod-active protein. Typically, the risk assessment follows a tiered approach that starts with laboratory studies under worst-case exposure conditions; such studies have a high ability to detect adverse effects on non-target species. Clear guidance on how such data are produced in laboratory studies assists the product developers and risk assessors. The studies should be reproducible and test clearly defined risk hypotheses. These properties contribute to the robustness of, and confidence in, environmental risk assessments for GE plants. Data from NTA studies, collected during the analysis phase of an environmental risk assessment, are critical to the outcome of the assessment and ultimately the decision taken by regulatory authorities on the release of a GE plant. Confidence in the results of early-tier laboratory studies is a precondition for the acceptance of data across regulatory jurisdictions and should encourage agencies to share useful information and thus avoid redundant testing.
The ladybird beetle, Coleomegilla maculata (DeGeer), is a common and abundant predator in many cropping systems. Its larvae and adults are predaceous, feeding on aphids, thrips, lepidopteran larvae ...and plant tissues, such as pollen. Therefore, this species is exposed to insecticidal proteins expressed in insect-resistant, genetically engineered cotton expressing Cry proteins derived from Bacillus thuringiensis (Bt). A tritrophic bioassay was conduced to evaluate the potential impact of Cry2Ab- and Cry1Ac-expressing cotton on fitness parameters of C. maculata using Bt-susceptible and -resistant larvae of Trichoplusia ni as prey. Coleomegilla maculata survival, development time, adult weight and fecundity were not different when they were fed with resistant T. ni larvae reared on either Bt or control cotton. To ensure that C. maculata were not sensitive to the tested Cry toxins independent from the plant background and to add certainty to the hazard assessment, C. maculata larvae were fed artificial diet incorporated with Cry2Ab, Cry1Ac or both at >10 times higher concentrations than in cotton tissue. Artificial diet containing E-64 was included as a positive control. No differences were detected in any life-table parameters between Cry protein-containing diet treatments and the control diet. In contrast, larvae of C. maculata fed the E-64 could not develop to the pupal stage and the 7-d larval weight was significantly negatively affected. In both feeding assays, the stability and bioactivity of Cry proteins in the food sources were confirmed by ELISA and sensitive-insect bioassays. Our results show that C. maculata is not affected by Bt cotton and is not sensitive to Cry2Ab and Cry1Ac at concentrations exceeding the levels in Bt cotton, thus demonstrating that Bt cotton will pose a negligible risk to C. maculata. More importantly, this study demonstrates a comprehensive system for assessing the risk of genetically modified plants on non-target organisms.
Among herbivorous insects with a complete metamorphosis the larval and adult stages usually differ considerably in their nutritional requirements and food ecology. Often, feeding on plant structural ...tissue is restricted to the larval stage, whereas the adult stage feeds primarily or exclusively on plant-provided food supplements such as nectar and pollen. Research on herbivore nutritional ecology has largely been divided along these lines. Most studies focus on actual herbivory by larval stages, while nectar and pollen feeding by adult herbivores has been addressed mainly in the light of plant-pollinator interactions. Only recently have we started to realize that the two phenomena are closely interlinked and that nectar and pollen feeding by adult herbivores can have a strong impact on plant-herbivore interactions. Here we address this largely ignored aspect of multitrophic level interactions and discuss its wide-ranging implications.
Background Hundreds of studies on environmental effects of genetically modified (GM) crops became available over the past 25 years. For maize producing insecticidal proteins from Bacillus ...thuringiensis (Bt), potential adverse effects on non-target organisms are a major area of concern and addressed in risk assessments. Reviews and meta-analyses have helped various stakeholders to address uncertainties regarding environmental impacts of the technology. Many field studies from Europe and other parts of the world have been published in the last decade, and those data are often not covered by previous meta-analyses. Therefore, we conducted a systematic review to answer the question: "Does the growing of Bt maize change abundance or ecological function of non-target animals compared to the growing of non-GM maize?" Methods Literature published until August 2019 was searched systematically in 12 bibliographic databases, 17 specialized webpages, and reference sections of 78 review articles. Defined eligibility criteria were applied to screen titles, abstracts, and full texts of the retrieved references. A custom-made database was developed with quantitative data on invertebrate abundance, activity density, or predation/parasitism rates. Eligible data that did not fit the quantitative database were captured in detailed tables and summarized narratively. For the first time, a critical appraisal scheme for field studies on non-targets in GM crops was developed to estimate the risk of bias (internal validity) and the suitability to answer the review question (external validity) of all primary data. Meta-analyses on different taxonomic levels, functional groups, and types of Bt maize were conducted. Untreated Bt maize was either compared with untreated non-Bt maize, or with insecticide-treated non-Bt maize. The influence of contributions by private sector product developers on reported effects was investigated. Review findings The database on non-target effects of Bt maize field trials contains more than 7200 records from 233 experiments and 120 articles. Meta-analyses on different taxonomic levels revealed only few and often non-robust significant effect sizes when both Bt maize and non-Bt maize were untreated. Bt maize harboured fewer parasitoids (Braconidae, Tachinidae) of the European corn borer, the main target pest of Lepidoptera-active Bt maize, compared with non-Bt maize. Similarly, sap beetles (Nitidulidae), that are associated with Lepidoptera damage, were recorded less in Bt maize. In some analyses, a negative effect of Bt maize was observed for rove beetles (Staphylinidae) and hoverflies (Syrphidae) and a positive effect for ladybeetles (Coccinellidae), flower bugs (Anthocoridae), and lacewings (Neuroptera). However, those effects were not consistent for different analyses and often related to individual articles. When untreated Bt maize was compared with pyrethroid-treated non-Bt maize, more effect sizes were significant. In particular, populations of predators were reduced after pyrethroid treatment, while few data were available for other insecticides. Funnel plots showed no evidence for publication bias and the analyses of private sector contribution revealed no evidence for influence of vested interests. Conclusions about potential effects of Bt maize on vertebrates or on animals inhabiting off-crop habitats were not possible, because only few such studies fitting the format of direct Bt/non-Bt comparisons on plot or field level were identified. Conclusions The current work largely confirmed previously published results. The effects of Bt maize on the community of non-target invertebrates inhabiting maize fields were small and mostly neutral, especially when compared with the effects of broad-spectrum pyrethroid insecticide treatments. Keywords: Bacillus thuringiensis, Corn, Critical appraisal, Cry protein, Environmental risk assessment, Genetic engineering, Meta-analysis, Non-target organisms, Sensitivity analysis, Systematic literature search
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