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.

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.

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.

Transcriptome and proteome analyses to investigate the molecular underpinnings of cold response in the Colorado potato beetle, Leptinotarsa decemlineata.

The Colorado potato beetle (Leptinotarsa decemlineata (Say)) is an insect that can cope with prolonged periods of low temperatures exposure. The molecular changes required to adapt to such conditions have not been thoroughly investigated in this insect. The current work aims at characterizing deregulated transcripts and proteins in adult L. decemlineata exposed to 15 °C and -5 °C using RNA-sequencing-based transcriptomics and liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomics approaches, respectively. RNA-sequencing highlighted the differential expression of several transcripts, including ubiquilin-1 and ubiquitin carboxyl-terminal hydrolase 5, in insects submitted to low temperatures when compared with control insects. In addition, proteomics approach detected 2840 proteins in cold-exposed beetles including elevated levels for 409 proteins and reduced levels for 200 proteins. Cuticular proteins CP1, CP4, CP5 and CP7 as well as eukaryotic translation initiation factor 4B were notable proteins with elevated levels in cold insects. Functional analysis of targets modulated at low temperatures using DAVID indicated processes likely affected under cold conditions including select metabolic cascades and RNA-associated processes. Overall, this work presents molecular candidates impacted by low temperatures exposure in L. decemlineata and builds on the current knowledge associated with response to these conditions in this insect.

Effects of fasting and refeeding on protein and glucose metabolism in Arctic charr.

Refeeding, following a period of food deprivation will often lead to compensatory growth. Although many studies have focused on molecular mechanisms behind this accelerated growth response in fish, little is known on the roles of protein and metabolism. We also assessed, for the first time, the potential roles of miRNAs in regulating compensatory growth. Artcic charr, Salvelinus alpinus, a northern freshwater species, was deprived of food for 101 days and then fed to satiety for 126 days. The refeeding period resulted in compensatory growth, with a partial compensation of body mass. The feed deprivation period lead to a decrease in hepatosomatic index (HSI) and intestinal somatic index (ISI). HSI and ISI were then gradually replenished during early refeeding, following a lag phase prior to the compensatory growth response. mRNA transcripts regulating protein degradation via the autophagy pathway (Cathepsin D and Cathepsin L) in muscle were upregulated during feed restriction and downregulated after refeeding, which could allow for greater protein accretion in muscle, facilitating compensatory growth. Transcript levels from the ubiquitin proteasome pathway (Mafbx and Murf1) and the calpain system (Calpain 7 and Calpastatin) suggested that these pathways were not involved in regulating compensatory growth. Furthermore, we've shown that miRNAs (miR-29a and miR-223) could be involved in fish glycogen homeostasis during the early stages of refeeding. These findings provide a deeper understanding of the molecular mechanisms regulating growth in fish.

Investigating Nrf2-associated non-coding RNAs in the hibernating ground squirrel, Ictidomys tridecemlineatus.

Small mammals hibernate to deal with environmental conditions associated with the winter season. Numerous physiological changes occur during a typical torpor-arousal cycle including variations in heart rate and blood flow. Such cycle possesses characteristics of ischemia-reperfusion cycles that can lead to oxidative stress in non-hibernating models. Interestingly, hibernators can cope with these conditions and the complete molecular picture underlying this adaptation is not fully understood. Non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), can impact expression and activity of various targets and have been associated with oxidative stress response. This work was aimed at assessing expression of oxidative stress-associated non-coding RNAs and their targets during hibernation. Measurement of miRNAs miR-93, miR-141, miR-144 and miR-200a, lncRNAs Mhrt and ODRUL, as well as of several targets associated with the Nrf2 signaling cascade including Keap1 was conducted using qRT-PCR in hibernating hearts of the thirteen-lined ground squirrel, Ictidomys tridecemlineatus. Elevated Nrf2 levels and reduced miR-200a levels were notably observed in hibernating versus euthermic samples. Functional analysis of targets predicted to be regulated by the investigated miRNAs was performed and revealed transcriptional regulation and phosphorylation as relevant processes. These results highlight a potential interplay between non-coding RNAs and targets associated with oxidative stress response during hibernation and further strengthen the underlying importance of non-coding RNAs in cold torpor.

Identification of Differentially Expressed miRNAs in Colorado Potato Beetles (Leptinotarsa decemlineata (Say)) Exposed to Imidacloprid.

The Colorado potato beetle (Leptinotarsa decemlineata (Say)) is a significant pest of potato plants that has been controlled for more than two decades by neonicotinoid imidacloprid. L. decemlineata can develop resistance to this agent even though the molecular mechanisms underlying this resistance are not well characterized. MicroRNAs (miRNAs) are short ribonucleic acids that have been linked to response to various insecticides in several insect models. Unfortunately, the information is lacking regarding differentially expressed miRNAs following imidacloprid treatment in L. decemlineata. In this study, next-generation sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were used to identify modulated miRNAs in imidacloprid-treated versus untreated L. decemlineata. This approach identified 33 differentially expressed miRNAs between the two experimental conditions. Of interest, miR-282 and miR-989, miRNAs previously shown to be modulated by imidacloprid in other insects, and miR-100, a miRNA associated with regulation of cytochrome P450 expression, were significantly modulated in imidacloprid-treated beetles. Overall, this work presents the first report of a miRNA signature associated with imidacloprid exposure in L. decemlineata using a high-throughput approach. It also reveals interesting miRNA candidates that potentially underly imidacloprid response in this insect pest.

Substituted Caffeic and Ferulic Acid Phenethyl Esters: Synthesis, Leukotrienes Biosynthesis Inhibition, and Cytotoxic Activity.

Glioblastoma multiforme (GBM) is an aggressive brain tumor that correlates with short patient survival and for which therapeutic options are limited. Polyphenolic compounds, including caffeic acid phenethyl ester (CAPE, 1a), have been investigated for their anticancer properties in several types of cancer. To further explore these properties in brain cancer cells, a series of caffeic and ferulic acid esters bearing additional oxygens moieties (OH or OCH3) were designed and synthesized. (CAPE, 1a), but not ferulic acid phenethyl ester (FAPE, 1b), displayed substantial cytotoxicity against two glioma cell lines. Some but not all selected compounds derived from both (CAPE, 1a) and (FAPE, 1b) also displayed cytotoxicity. All CAPE-derived compounds were able to significantly inhibit 5-lipoxygenase (5-LO), however FAPE-derived compounds were largely ineffective 5-LO inhibitors. Molecular docking revealed new hydrogen bonds and π-π interactions between the enzyme and some of the investigated compounds. Overall, this work highlights the relevance of exploring polyphenolic compounds in cancer models and provides additional leads in the development of novel therapeutic strategies in gliomas.

Identification and profiling of miRNAs in the freeze-avoiding gall moth Epiblema scudderiana via next-generation sequencing.

The rapid development of high-throughput next-generation sequencing approaches in recent years has facilitated large-scale discovery and expression analysis of non-coding RNAs, including miRNAs, in traditional and non-traditional animal models. Such an approach has been leveraged to amplify, identify, and quantify miRNAs in several models of cold adaptation. The present study is the first to investigate the status of these small RNAs in an insect species that uses the freeze avoidance strategy of cold hardiness, the gall moth Epiblema scudderiana. To characterize the overall miRNA expression profile and to identify cold-modulated miRNAs in control (5 °C) and cold-exposed (-15 °C) E. scudderiana larvae, a next-generation sequencing-based approach was undertaken. A total of 44 differentially expressed miRNAs were identified between the two conditions; 21 up-regulated miRNAs and 23 down-regulated miRNAs in -15 °C-exposed larvae as compared with controls. Among the most significant changes observed in miRNAs with potential relevance to cold adaptation were elevated miR-1-3p, miR-92b-3p, and miR-133-3p levels as well as reduced miR-13a-3p and miR-13b-3p levels in E. scudderiana larvae exposed to cold temperatures. Expression values obtained from next-generation sequencing were also validated by a quantitative PCR approach for five miRNAs; miR-34-5p, miR-274-5p, miR-275-3p, miR-307a-3p, and miR-316-5p. Overall, this work provides the first description of a miRNA signature for subzero survival by a freeze-avoiding insect using a high-throughput approach and positions a new group of miRNAs at the forefront of the molecular changes underlying cold adaptation.

Investigating a signature of temozolomide resistance in GBM cell lines using metabolomics.

Glioblastoma multiforme (GBM) is the most common form of malignant glioma. Current therapeutic approach to treat this malignancy involves a combination of surgery, radiotherapy and chemotherapy with temozolomide. Numerous mechanisms contributing to inherent and acquired resistance to this chemotherapeutic agent have been identified and can lead to treatment failure. This study undertook a metabolomics-based approach to characterize the metabolic profiles observed in temozolomide-sensitive and temozolomide-resistant GBM cell lines as well as in a small sub-set of primary GBM tumors. This approach was also utilized to explore the metabolic changes modulated upon cell treatment with temozolomide and lomeguatrib, an MGMT inhibitor with temozolomide-sensitizing potential. Metabolites previously explored for their potential role in chemoresistance including glucose, citrate and isocitrate demonstrated elevated levels in temozolomide-resistant GBM cells. In addition, a signature of metabolites comprising alanine, choline, creatine and phosphorylcholine was identified as up-regulated in sensitive GBM cell line across different treatments. These results present the metabolic profiles associated with temozolomide response in selected GBM models and propose interesting leads that could be leveraged for the development of therapeutic or diagnostic tools to impact temozolomide response in GBMs.

Expression of miRNAs in response to freezing and anoxia stresses in the freeze tolerant fly Eurosta solidaginis.

Insect cold hardiness is associated with substantial metabolic rate suppression, often including developmental diapause as well as metabolic suppression imposed by freezing and freeze-associated oxygen limitation. MicroRNAs, small non-coding transcripts that bind to mRNA, are known modulators of hypometabolism in freeze tolerant insects. To further contribute to the growing signature of stress-responsive miRNAs, this study amplified and quantified changes in the expression levels of four microRNA species, miR-8, miR-9, miR-92b and miR-277, in response to freezing or anoxia exposures of freeze tolerant gall fly larvae, Eurosta solidaginis. MiR-92b levels were significantly elevated by 1.57-fold in frozen E. solidaginis at -15°C as compared with 5°C controls, whereas miR-92b levels were significantly reduced in anoxic E. solidaginis to levels that were 0.77-fold as compared with larvae held under normoxic conditions. The other miRNAs investigated showed no significant changes in stressed larvae. These data demonstrate differential miR-92b expression in frozen/anoxic versus control insect larvae and position this miRNA as a stress responsive marker in this model insect.

Differential expression of miRNAs with metabolic implications in hibernating thirteen-lined ground squirrels, Ictidomys tridecemlineatus.

Mammalian hibernators undergo significant physiological and biochemical changes when confronted with cold temperatures. Metabolic depression and translational repression are two examples of the various processes impacted during a torpor bout. MicroRNAs (miRNAs), non-coding transcripts that bind to mRNAs, are known regulators of mRNA translation and a growing number of these molecules have been found to be differentially expressed during hibernation. We hypothesized that a group of six miRNAs, with targets involved in various metabolic cascades, is modulated in selected tissues of the hibernating thirteen-lined ground squirrel Ictidomys tridecemlineatus. Expression levels of these miRNAs were assessed in the liver, heart, and skeletal muscle ground squirrel tissues using qRT-PCR. miR-29a, miR-152, miR-195, miR-223, and miR-486 were shown to be up-regulated in the hibernating liver, while miR-378 was shown to be down-regulated in hibernating skeletal muscle tissue samples. Interestingly, fatty acid synthase (FAS), an enzyme involved in fatty acid biosynthesis and a miR-195 target, was shown to be down-regulated in hibernating squirrel liver. This data add to the growing signature of differentially expressed miRNAs during hibernation and puts the light on the potential regulation of fatty acid homeostasis by a miRNA in torpid animals.

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.

NMR metabolomics analysis of the effects of 5-lipoxygenase inhibitors on metabolism in glioblastomas.

Changes across metabolic networks are emerging as an integral part of cancer development and progression. Increasing comprehension of the importance of metabolic processes as well as metabolites in cancer is stimulating exploration of novel, targeted treatment options. Arachidonic acid (AA) is a major component of phospholipids. Through the cascade catalyzed by cyclooxygenases and lipoxygenases, AA is also a precursor to cellular signaling molecules as well as molecules associated with a variety of diseases including cancer. 5-Lipoxygenase catalyzes the transformation of AA into leukotrienes (LT), important mediators of inflammation. High-throughput analysis of metabolic profiles was used to investigate the response of glioblastoma cell lines to treatment with 5-lipoxygenase inhibitors. Metabolic profiling of cells following drug treatment provides valuable information about the response and metabolic alterations induced by the drug action and give an indication of both on-target and off-target effects of drugs. Four different 5-lipoxygenase inhibitors and antioxidants were tested including zileuton, caffeic acid, and its analogues caffeic acid phenethyl ester and caffeic acid cyclohexethyl ester. A NMR approach identified metabolic signatures resulting from application of these compounds to glioblastoma cell lines, and metabolic data were used to develop a better understanding of the mode of action of these inhibitors.

1H NMR metabolomics analysis of glioblastoma subtypes: correlation between metabolomics and gene expression characteristics.

Glioblastoma multiforme (GBM) is the most common form of malignant glioma, characterized by unpredictable clinical behaviors that suggest distinct molecular subtypes. With the tumor metabolic phenotype being one of the hallmarks of cancer, we have set upon to investigate whether GBMs show differences in their metabolic profiles. (1)H NMR analysis was performed on metabolite extracts from a selection of nine glioblastoma cell lines. Analysis was performed directly on spectral data and on relative concentrations of metabolites obtained from spectra using a multivariate regression method developed in this work. Both qualitative and quantitative sample clustering have shown that cell lines can be divided into four groups for which the most significantly different metabolites have been determined. Analysis shows that some of the major cancer metabolic markers (such as choline, lactate, and glutamine) have significantly dissimilar concentrations in different GBM groups. The obtained lists of metabolic markers for subgroups were correlated with gene expression data for the same cell lines. Metabolic analysis generally agrees with gene expression measurements, and in several cases, we have shown in detail how the metabolic results can be correlated with the analysis of gene expression. Combined gene expression and metabolomics analysis have shown differential expression of transporters of metabolic markers in these cells as well as some of the major metabolic pathways leading to accumulation of metabolites. Obtained lists of marker metabolites can be leveraged for subtype determination in glioblastomas.

PF-8380 and closely related analogs: synthesis and structure-activity relationship towards autotaxin inhibition and glioma cell viability.

A series of PF-8380 analogs, a recently developed autotaxin inhibitor, was explored. Inhibition of autotaxin by these analogs, as well as by all PF-8380 synthetic intermediates, shows the importance of meta-dichlorobenzyl and benzo[d]oxazol-2(3H)-one fragments. However, analogs 8 and 9, bearing only the benzo[d]oxazol-2(3H)-one moiety, are more cytotoxic on the LN229 glioblastoma cell line than PF-8380 and temozolomide (TMZ).

Leveraging RNA Interference to Impact Insecticide Resistance in the Colorado Potato Beetle, Leptinotarsa decemlineata.

The Colorado potato beetle, Leptinotarsa decemlineata Say, is a potato pest that can cause important economic losses to the potato industry worldwide. Diverse strategies have been deployed to target this insect such as biological control, crop rotation, and a variety of insecticides. Regarding the latter, this pest has demonstrated impressive abilities to develop resistance against the compounds used to regulate its spread. Substantial work has been conducted to better characterize the molecular signatures underlying this resistance, with the overarching objective of leveraging this information for the development of novel approaches, including RNAi-based techniques, to limit the damage associated with this insect. This review first describes the various strategies utilized to control L. decemlineata and highlights different examples of reported cases of resistances against insecticides for this insect. The molecular leads identified as potential players modulating insecticide resistance as well as the growing interest towards the use of RNAi aimed at these leads as part of novel means to control the impact of L. decemlineata are described subsequently. Finally, select advantages and limitations of RNAi are addressed to better assess the potential of this technology in the broader context of insecticide resistance for pest management.

Application of annotation-agnostic RNA sequencing data analysis tools for biomarker discovery in liquid biopsy.

RNA sequencing analysis is an important field in the study of extracellular vesicles (EVs), as these particles contain a variety of RNA species that may have diagnostic, prognostic and predictive value. Many of the bioinformatics tools currently used to analyze EV cargo rely on third-party annotations. Recently, analysis of unannotated expressed RNAs has become of interest, since these may provide complementary information to traditional annotated biomarkers or may help refine biological signatures used in machine learning by including unknown regions. Here we perform a comparative analysis of annotation-free and classical read-summarization tools for the analysis of RNA sequencing data generated for EVs isolated from persons with amyotrophic lateral sclerosis (ALS) and healthy donors. Differential expression analysis and digital-droplet PCR validation of unannotated RNAs also confirmed their existence and demonstrates the usefulness of including such potential biomarkers in transcriptome analysis. We show that find-then-annotate methods perform similarly to standard tools for the analysis of known features, and can also identify unannotated expressed RNAs, two of which were validated as overexpressed in ALS samples. We demonstrate that these tools can therefore be used for a stand-alone analysis or easily integrated into current workflows and may be useful for re-analysis as annotations can be integrated post hoc.

Differential expression of microRNA species in a freeze tolerant insect, Eurosta solidaginis.

Freeze tolerance in insects is associated with a variety of adaptations including production of cryoprotectants, specialized proteins that regulate ice formation, and energy-saving mechanisms that strongly suppress the rates of metabolic processes in the oxygen-limited frozen state. We hypothesized that microRNAs (miRNAs), small non-coding transcripts that bind to mRNA, could play a role in the global regulation of energy-expensive mRNA translation in frozen insects and would be modulated at subzero temperatures. Expression levels of miRNA species were evaluated in control (5 °C) and frozen (-15 °C) goldenrod gall fly larvae, Eurosta solidaginis, using a miRNA microarray. Levels of miR-11, miR-276, miR-71, miR-3742, miR-277-3p, miR-2543b and miR-34 were significantly reduced in frozen larvae whereas miR-284, miR-3791-5p and miR-92c-3p rose significantly in frozen larvae. Target prediction for two miRNAs, miR-277-3p and miR-284, revealed potential regulation of transcripts involved in translation and the Krebs cycle. These data constitute the first report that differential expression of miRNAs occurs in a freeze tolerant insect and suggest a mechanism for reversible gene regulation during prolonged periods of freezing over the winter months, a mechanism that can be rapidly reversed to allow renewed translation of mRNA when temperatures rise and insects thaw.

Silencing of Molecular Targets with Relevance to Insecticide Resistance in Colorado Potato Beetle Using dsRNA.

Various approaches based on RNA interference (RNAi) have garnered significant attention in the field of insect pest management in recent years. For example, the use of double-stranded RNA (dsRNA) has notably been investigated to target transcripts of interest with relevance to insecticide resistance in multiple pests and has emerged as a potential tool to be deployed in agricultural fields in the near future. A careful characterization of a given dsRNA in a laboratory setting, including the assessment of dsRNA-mediated molecular and phenotypical changes observed in the targeted pest upon dsRNA exposure, is nevertheless essential prior to its use in field-based study. The current chapter thus describes the process via which a dsRNA, aimed at a molecular target underlying insecticide response in the Colorado potato beetle Leptinotarsa decemlineata, is conceived, synthesized and injected. Assessment of knockdown efficiency in injected insects is further presented.