Spinosad-associated modulation of select cytochrome P450s and glutathione S-transferases in the Colorado potato beetle, Leptinotarsa decemlineata.

The Colorado potato beetle (Leptinotarsa decemlineata (Say)) is an insect pest that threatens potato crops. Multiple options exist to limit the impact of this pest even though insecticides remain a primary option for its control. Insecticide resistance has been reported in Colorado potato beetles and a better understanding of the molecular players underlying such process is of utmost importance to optimize the tools used to mitigate the impact of this insect. Resistance against the insecticide spinosad has been reported in this insect and this work thus aims at exploring the expression of targets previously associated with insecticide response in Colorado potato beetles exposed to this compound. Amplification and quantification of transcripts coding for cytochrome P450s and glutathione S-transferases were conducted via qRT-PCR in insects treated with varying doses of spinosad and for different time duration. This approach notably revealed differential expression of CYP6a23 and CYP12a5 in insects exposed to low doses of spinosad for 4 h as well as modulation of CYP6a13, CYP6d4, GST, GST1, and GST1-Like in insects treated with high doses of spinosad for the same duration. RNAi-based targeting of CYP4g15 and CYP6a23 was associated with marked reduction of transcript expression 7 days following dsRNA injection and reduction of the former had a marked impact on insect viability. In general, results presented here provide novel information regarding the expression of transcripts relevant to spinosad response in Colorado potato beetles and reveal a novel target to consider in the development of RNAi-based strategies aimed at this potato pest.

Characterization of Insecticide Response-Associated Transcripts in the Colorado Potato Beetle: Relevance of Selected Cytochrome P450s and Clothianidin.

The Colorado potato beetle (Leptinotarsa decemlineata (Say)) is known for its capacity to cause significant damages to potato crops worldwide. Multiple approaches have been considered to limit its spread including the use of a diverse arsenal of insecticides. Unfortunately, this insect frequently develops resistance towards these compounds. Investigating the molecular bases underlying the response of L. decemlineata against insecticides is of strong interest to ultimately devise novel and targeted approaches aimed at this pest. This work aimed to characterize, via qRT-PCR, the expression status of targets with relevance to insecticide response, including ones coding for cytochrome P450s, glutathione s-transferases, and cuticular proteins, in L. decemlineata exposed to four insecticides; chlorantraniliprole, clothianidin, imidacloprid, and spinosad. Modulation of levels associated with transcripts coding for selected cytochrome P450s was reported in insects treated with three of the four insecticides studied. Clothianidin treatment yielded the most variations in transcript levels, leading to significant changes in transcripts coding for CYP4c1, CYP4g15, CYP6a13, CYP9e2, GST, and GST-1-Like. Injection of dsRNA targeting CYP4c1 and CYP9e2 was associated with a substantial decrease in expression levels and was, in the case of the latter target, linked to a greater susceptibility of L. decemlineata towards this neonicotinoid, supporting a potential role for this target in clothianidin response. Overall, this data further highlights the differential expression of transcripts with potential relevance in insecticide response, as well as generating specific targets that warrant investigation as novel dsRNA-based approaches are developed against this insect pest.

Adventive Larval Parasitoids Reconstruct Their Close Association with Spotted-Wing Drosophila in the Invaded North American Range.

Two species of larval parasitoids of the globally invasive fruit pest, Drosophila suzukii (Diptera: Drosophilidae), Leptopilina japonica, and Ganaspis brasiliensis (both Hymenoptera: Figitidae), were detected in British Columbia, Canada in 2016 and 2019, respectively. Both are presumed to have been unintentionally introduced from Asia; however, the extent of their establishment across different habitats with diverse host plants used by D. suzukii was unclear. In addition, there was no knowledge of the temporal dynamics of parasitism of D. suzukii by these two parasitoids. To address these gaps, we repeatedly sampled the fruits of known host plants of D. suzukii over the entire 2020 growing season in British Columbia. We documented the presence of L. japonica and G. brasiliensis and estimated the apparent percentage of D. suzukii parasitized among host plant species. Across a large region of southwestern British Columbia, both L. japonica and G. brasiliensis were found to be very common across a variety of mostly unmanaged habitats over the entire course of the season (May-October) in the fruits of most host plants known to host D. suzukii larvae. Parasitism of D. suzukii was variable (0-66% percent parasitism) and appeared to be time-structured. Our study demonstrates that the close association between the two larval parasitoids and D. suzukii that exists in Asia has evidently been reconstructed in North America, resulting in the highest parasitism levels of D. suzukii yet recorded outside of its area of origin.

Exposure to Temperature and Insecticides Modulates the Expression of Small Noncoding RNA-Associated Transcripts in the Colorado Potato Beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The Colorado potato beetle (Leptinotarsa decemlineata (Say)) is an insect that can adapt to various challenges, including temperature fluctuations or select insecticide treatments. This pest is also an ongoing threat to the potato industry. Small noncoding RNAs such as miRNAs, which can control posttranscriptionally the expression of various genes, and piRNAs, which can notably impact mRNA turnover, are modulated in insects under different conditions. Unfortunately, information regarding the expression status of key players involved in their synthesis and function is for the most part lacking. The current study thus aims at assessing the levels of such targets in L. decemlineata exposed to hot and cold temperatures as well as treated to the insecticides chlorantraniliprole, clothianidin, imidacloprid, and spinosad. Transcript expression levels of Ago1, Ago2, Ago3, Dcr2a, Dcr2b, Expo-5, Siwi-1, and Siwi-2, components of pathways associated with small noncoding RNA production or function, were measured by qRT-PCR and revealed modulation of select transcripts in response to temperature challenges and to select insecticides. RNAi-mediated reduction of Ago2 transcript levels in L. decemlineata injected with Ago2-targeting dsRNA and exposed to cold and warm temperatures was also conducted. Changes in survival rates were observed for the latter condition in dsRNA- versus saline-injected insects. These results showcase the differential expression of select targets involved in small noncoding RNA homeostasis and provide leads for the subsequent assessment of their involvement during stress response in L. decemlineata using RNAi-based approaches.

A Coordinated Sampling and Identification Methodology for Larval Parasitoids of Spotted-Wing Drosophila.

We provide recommendations for sampling and identification of introduced larval parasitoids of spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae). These parasitoids are either under consideration for importation (aka classical) biological control introductions, or their adventive (presumed to have been accidentally introduced) populations have recently been discovered in North America and Europe. Within the context of the ecology of D. suzukii and its parasitoids, we discuss advantages and disadvantages of estimating larval parasitism levels using different methods, including naturally collected fruit samples and sentinel baits. For most situations, we recommend repeated sampling of naturally occurring fruit rather than using sentinel baits to monitor seasonal dynamics of host plant-Drosophila-parasitoid associations. We describe how to separate Drosophilidae puparia from host fruit material in order to accurately estimate parasitism levels and establish host-parasitoid associations. We provide instructions for identification of emerging parasitoids and include a key to the common families of parasitoids of D. suzukii. We anticipate that the guidelines for methodology and interpretation of results that we provide here will form the basis for a large, multi-research team sampling effort in the coming years to characterize the biological control and nontarget impacts of accidentally and intentionally introduced larval parasitoids of D. suzukii in several regions of the world.

Field Assessment of the Host Range of Aculus mosoniensis (Acari: Eriophyidae), a Biological Control Agent of the Tree of Heaven (Ailanthus altissima).

Tree of heaven (Ailanthus altissima) is a fast-growing deciduous tree native to China, considered a serious invasive species worldwide, with several socio-economic and ecological impacts attributed to it. Chemical and mechanical methods have limited efficacy in its management, and biological controls may offer a suitable and sustainable option. Aculus mosoniensis (Ripka) is an eriophyid mite that has been recorded to attack tree of heaven in 13 European countries. This study aims to explore the host range of this mite by exposing 13 plant species, selected either for their phylogenetic and ecological similarity to the target weed or their economic importance. Shortly after inoculation with the mite, we recorded a quick decrease in mite number on all nontarget species and no sign of mite reproduction. Whereas, after just one month, the population of mites on tree of heaven numbered in the thousands, irrespective of the starting population, and included both adults and juveniles. Significantly, we observed evidence of damage due to the mite only on target plants. Due to the specificity, strong impact on the target, and the ability to increase its population to high levels in a relatively short amount of time, we find A. mosoniensis to be a very promising candidate for the biological control of tree of heaven.

Transcriptomics-Based Approach Identifies Spinosad-Associated Targets in the Colorado Potato Beetle, Leptinotarsa decemlineata.

The Colorado potato beetle Leptinotarsa decemlineata is an insect pest that threatens potato crops globally. The primary method to control its damage on potato plants is the use of insecticides, including imidacloprid, chlorantraniliprole and spinosad. However, insecticide resistance has been frequently observed in Colorado potato beetles. The molecular targets and the basis of resistance to imidacloprid and chlorantraniliprole have both been previously quantified. This work was undertaken with the overarching goal of better characterizing the molecular changes associated with spinosad exposure in this insect pest. Next-generation sequencing was conducted to identify transcripts that were differentially expressed between Colorado potato beetles exposed to spinosad versus control insects. Results showed several transcripts that exhibit different expression levels between the two conditions, including ones coding for venom carboxylesterase-6, chitinase 10, juvenile hormone esterase and multidrug resistance-associated protein 4. In addition, several microRNAs, such as miR-12-3p and miR-750-3p, were also modulated in the investigated conditions. Overall, this work reveals a molecular footprint underlying spinosad response in Colorado potato beetles and provides novel leads that could be targeted as part of RNAi-based approaches to control this insect pest.

Differential expression of transcripts with potential relevance to chlorantraniliprole response in the Colorado potato beetle, Leptinotarsa decemlineata.

The Colorado potato beetle (Leptinotarsa decemlineata [Say]) is an insect pest that can significantly harm potato plants worldwide. Control of this insect relies heavily on chemical insecticides such as chlorantraniliprole. Nevertheless, the complete molecular signature associated with response to this compound is lacking in L. decemlineata. In this study, amplification and quantification by qRT-PCR (quantitative reverse transcription-polymerase chain reaction) of targets relevant to chlorantraniliprole were undertaken in insects exposed to this chemical. This approach showed modulation of numerous cytochrome P450s, such as CYP350D1 and CYP4Q3, as well as upregulation of microRNAs (miRNAs), including miR-1-3p and miR-305-5p, in chlorantraniliprole-exposed insects. Functional assessment of transcript targets predicted to be regulated by these miRNAs was performed and revealed their likely impact on transcriptional regulation. RNAi-based targeting of CYP350D1 notably provided preliminary evidence of its underlying implication for chlorantraniliprole response in L. decemlineata. Overall, this study strengthens the current knowledge of the molecular changes linked to chlorantraniliprole response in L. decemlineata and provides novel targets with potential relevance to chlorantraniliprole susceptibility in this insect pest of global relevance.

Expression status of heat shock proteins in response to cold, heat, or insecticide exposure in the Colorado potato beetle Leptinotarsa decemlineata.

The Colorado potato beetle (Leptinotarsa decemlineata (Say)) is an agricultural pest that threatens the potato industry worldwide. This insect is widely regarded as one of the most difficult-to-control pests, as it can thrive in a wide range of temperature conditions and routinely develops resistance towards various insecticides. The molecular changes associated with response to these challenges have not been fully investigated in L. decemlineata. While differential expression and characterization of heat shock proteins (HSPs) in response to stress have been conducted in several insects, data regarding HSPs in L. decemlineata are limited. The overarching objective of this study consisted of evaluating the expression of various HSPs in L. decemlineata exposed to different temperatures or treated with the insecticides imidacloprid and chlorantraniliprole. Expression levels of HSP60, HSP70, HSP90, and HSP Beta-1 were evaluated by qRT-PCR and insect mortality was assessed using dsRNAs aimed at select HSP targets. Elevated HSP70 and HSP90 transcript levels were observed in heat-exposed L. decemlineata while downregulation of HSP70 transcript levels was measured in insects submitted to cold conditions. Chlorantraniliprole exposure was associated with reduced HSP Beta-1 transcript levels while no change in expression was monitored in insects exposed to imidacloprid. RNAi-based knockdown of HSP60 levels correlated with significant insect mortality 14 days after dsRNA injection. These results highlight the modulation of HSPs that occur in L. decemlineata exposed to fluctuating temperatures and position HSPs as interesting candidates in the identification of novel molecular leads that could be targeted to control this insect.

Rethinking biological control programs as planned invasions.

Biological control of pests with their natural enemies essentially consists of planned invasions, with the opportunity to select both the invader and the invaded environment. Recent advances in invasion science link 'intrinsic invasion factors' (life history and behavioral traits) with invader success; connect 'extrinsic invasion factors' (abiotic and biotic aspects of the invaded environment) with environmental invasibility; and demonstrate that their interaction leads not only to ecologically driven variability but also to rapid evolutionary change in biocontrol systems. However, current theory and empirical evidence from invasion science have not yet been extensively adopted into biological control research and practice.

Morphology delimits more species than molecular genetic clusters of invasive Pilosella.

UNLABELLED: • PREMISE OF THE STUDY: Accurate assessments of biodiversity are paramount for understanding ecosystem processes and adaptation to change. Invasive species often contribute substantially to local biodiversity; correctly identifying and distinguishing invaders is thus necessary to assess their potential impacts. We compared the reliability of morphology and molecular sequences to discriminate six putative species of invasive Pilosella hawkweeds (syn. Hieracium, Asteraceae), known for unreliable identifications and historical introgression. We asked (1) which morphological traits dependably discriminate putative species, (2) if genetic clusters supported morphological species, and (3) if novel hybridizations occur in the invaded range.• METHODS: We assessed 33 morphometric characters for their discriminatory power using the randomForest classifier and, using AFLPs, evaluated genetic clustering with the program structure and subsequently with an AMOVA. The strength of the association between morphological and genotypic dissimilarity was assessed with a Mantel test.• KEY RESULTS: Morphometric analyses delimited six species while genetic analyses defined only four clusters. Specifically, we found (1) eight morphological traits could reliably distinguish species, (2) structure suggested strong genetic differentiation but for only four putative species clusters, and (3) genetic data suggest both novel hybridizations and multiple introductions have occurred.• CONCLUSIONS: (1) Traditional floristic techniques may resolve more species than molecular analyses in taxonomic groups subject to introgression. (2) Even within complexes of closely related species, relatively few but highly discerning morphological characters can reliably discriminate species. (3) By clarifying patterns of morphological and genotypic variation of invasive Pilosella, we lay foundations for further ecological study and mitigation.