Cyantraniliprole and Thiamethoxam Exposure Changes Expression of Transcripts Associated with Small Non-Coding RNA Processing in the Colorado Potato Beetle.

The Colorado potato beetle (Leptinotarsa decemlineata (Say)) can cause extensive damage to agricultural crops worldwide and is a significant insect pest. This insect is notorious for its ability to evade various strategies deployed to control its spread and is known for its relative ease in developing resistance against different insecticides. Various molecular levers are leveraged by L. decemlineata for this resistance to occur, and a complete picture of the genes involved in this process is lacking. While small non-coding RNAs, including miRNAs, are differentially expressed in insects exposed to insecticides, levels of transcript coding for proteins underlying their synthesis remain to be characterized fully. The overarching objective of this work aims to fill that gap by assessing the expression of such targets in L. decemlineata exposed to cyantraniliprole and thiamethoxam. The expression status of Ago1, Ago2, Ago3, Dcr2a, Dcr2b, Expo-5, Siwi-1 and Siwi-2 transcripts were quantified via qRT-PCR in adult L. decemlineata treated with low and high doses of these compounds for different lengths of time. Variation in Ago1 and Dcr2b expression was notably observed in L. decemlineata exposed to cyantraniliprole, while thiamethoxam exposure was associated with the modulation of Dcr2a and Siwi-1 transcript levels. The down-regulation of Ago1 expression in L. decemlineata using dsRNA, followed by cyantraniliprole treatment, was associated with a reduction in the survival of insects with reduced Ago1 transcript expression. Overall, this work presents the insecticide-mediated modulation of transcripts associated with small non-coding RNA processing and showcases Ago1 as a target to further investigate its relevance in cyantraniliprole response.

Regional differences in susceptibility to spinosyn insecticides registered for Colorado potato beetle management in Canada.

The Colorado potato beetle (CPB) is the most economically important pest of Canadian potato, and if left uncontrolled, it can completely consume the crop. In the past decade, the control of CPB has relied heavily on systemic insecticides, principally the neonicotinoids thiamethoxam and clothianidin. Resistance to neonicotinoids in CPB has been well documented in the past 2 decades and mechanisms underlying the resistance better understood. In contrast, resistance to other insecticide classes, including spinosyns (spinosad and spinetoram) and anthranillic diamides (chlorantraniliprole and cyantraniliprole), have not been studied to the same degree in CPB. Spinosyns are the only insecticide certified for organic potato growers in Canada and are frequently applied as a mid-season foliar spray by conventional growers when seed treatments with neoniconitoid or diamide experience control breaks. Improved knowledge on resistance to spinosyns in CPB would allow for the development of regional management strategies. A survey of insecticide susceptibility in CPB populations from 6 potato growing regions between 2018 and 2022 observed: 1) spatial and temporal resistance trends; 2) cross-resistance; and 3) evidence of regional differences in susceptibility to spinosyns. The proportion of populations within each province considered resistant to spinosyns was, in descending order: Québec (16%) > Ontario (14%) > Manitoba (13%) > New Brunswick (9%) > Prince Edward Island (2%) > Alberta (0%). There was a significant change in CPB mortality at the diagnostic concentration (DC = LC90) for spinosad and spinetoram in the 6 provinces but only for year 5 relative to the previous 4 years. Moderate cross-resistance was determined between spinosad and spinetoram with the DC mortality for all populations based on a positive and significant correlation (adjusted R2 = 0.3758; P = 1.263e-13). There was also a positive relationship observed between the number of spinosyn applications (years applied at the sampling location) and declining susceptibility to spinosad (R2 = 0.0927; P < 0.002). Cross-resistance was observed between spinosyns and insecticides in the other two classes, the more significant correlation was between spinosad and tetraniliprole (R2 = 0.3025; P < 0.0002). In Québec, the greater spinosad use in organic potato farms led to resistance in those CPB populations, but spinosyn resistance at conventional farms was not related to greater application of neonicotinoids and diamides. Spinosyns remain relatively effective, nevertheless growers should be concerned over the increasing cases of reduced susceptibility in conventional potato farms and resistance where organic production occurs. Resistance management should continue to encourage rotation with products from the other classes in season and between years in order to extend spinosyn use for CPB control.

Can species naming drive scientific attention? A perspective from plant-feeding arthropods.

How do researchers choose their study species? Some choices are based on ecological or economic importance, some on ease of study, some on tradition-but could the name of a species influence researcher decisions? We asked whether phytophagous arthropod species named after their host plants were more likely to be assayed for host-associated genetic differentiation (or 'HAD'; the evolution of cryptic, genetically isolated host specialists within an apparently more generalist lineage). We chose 30 arthropod species (from a Google Scholar search) for which a HAD hypothesis has been tested. We traced the etymologies of species names in the 30 corresponding genera, and asked whether HAD tests were more frequent among species whose etymologies were based on host-plant names (e.g. Eurosta solidaginis, which attacks Solidago) versus those with other etymologies (e.g. Eurosta fenestrata, from Latin fenestra, 'window'). Species with host-derived etymologies were more likely to feature in studies of HAD than those with other etymologies. We speculate that the etymology of a scientific name can draw a researcher's attention to aspects of life-history and thus influence the direction of our scientific gaze.

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.

Evidence for Cardiac Glycosides in Foliage of Colorado Potato Beetle-Resistant Solanum okadae.

Leptinotarsa decemlineata, the Colorado potato beetle (CPB), is a herbivore that primarily feeds on Solanum foliage and is a global pest of the potato agricultural industry. Potato breeding through cross-hybridization with CPB-resistant wild relatives is used for genetic improvement. The wild species Solanum okadae was demonstrated to deter CPB feeding in choice and no choice feeding assays. Liquid chromatography-mass spectrometry (LC-MS) was used for comparative metabolite profiling between S. okadae and CPB-susceptible domesticated potato variety, Solanum tuberosum cv. Shepody. Major foliar metabolites detected were steroidal glycoalkaloids (SGAs) with tomatine and dehydrotomatine produced in S. okadae and solanine and chaconine in S. tuberosum cv. Shepody. Cardiac glycosides were also detected in the foliar metabolite profile of S. okadae but not S. tuberosum cv. Shepody. This class of plant compounds have known insecticidal activity through inhibition of animal Na+/K+ ATPase. Thin-layer chromatography (TLC) separation of foliar extracts also provided evidence for cardiac glycosides in S. okadae. Cardiac glycosides are known inhibitors of Na+/K+ ATPase, and foliar extracts from S. okadae (OKA15), but not S. tuberosum cv. Shepody, were able to inhibit the Na+/K+ ATPase of CPB. These findings suggest a novel mechanism of plant resistance against CPB involving production of cardiac glycosides in S. okadae.

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.

Flexible Thermal Sensitivity of Mitochondrial Oxygen Consumption and Substrate Oxidation in Flying Insect Species.

Mitochondria have been suggested to be paramount for temperature adaptation in insects. Considering the large range of environments colonized by this taxon, we hypothesized that species surviving large temperature changes would be those with the most flexible mitochondria. We thus investigated the responses of mitochondrial oxidative phosphorylation (OXPHOS) to temperature in three flying insects: the honeybee (Apis mellifera carnica), the fruit fly (Drosophila melanogaster) and the Colorado potato beetle (Leptinotarsa decemlineata). Specifically, we measured oxygen consumption in permeabilized flight muscles of these species at 6, 12, 18, 24, 30, 36, 42 and 45°C, sequentially using complex I substrates, proline, succinate, and glycerol-3-phosphate (G3P). Complex I respiration rates (CI-OXPHOS) were very sensitive to temperature in honeybees and fruit flies with high oxygen consumption at mid-range temperatures but a sharp decline at high temperatures. Proline oxidation triggers a major increase in respiration only in potato beetles, following the same pattern as CI-OXPHOS for honeybees and fruit flies. Moreover, both succinate and G3P oxidation allowed an important increase in respiration at high temperatures in honeybees and fruit flies (and to a lesser extent in potato beetles). However, when reaching 45°C, this G3P-induced respiration rate dropped dramatically in fruit flies. These results demonstrate that mitochondrial functions are more resilient to high temperatures in honeybees compared to fruit flies. They also indicate an important but species-specific mitochondrial flexibility for substrate oxidation to sustain high oxygen consumption levels at high temperatures and suggest previously unknown adaptive mechanisms of flying insects' mitochondria to temperature.