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.

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.

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.

Differential expression and emerging functions of non-coding RNAs in cold adaptation.

Several species undergo substantial physiological and biochemical changes to confront the harsh conditions associated with winter. Small mammalian hibernators and cold-hardy insects are examples of natural models of cold adaptation that have been amply explored. While the molecular picture associated with cold adaptation has started to become clearer in recent years, notably through the use of high-throughput experimental approaches, the underlying cold-associated functions attributed to several non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), remain to be better characterized. Nevertheless, key pioneering work has provided clues on the likely relevance of these molecules in cold adaptation. With an emphasis on mammalian hibernation and insect cold hardiness, this work first reviews various molecular changes documented so far in these processes. The cascades leading to miRNA and lncRNA production as well as the mechanisms of action of these non-coding RNAs are subsequently described. Finally, we present examples of differentially expressed non-coding RNAs in models of cold adaptation and elaborate on the potential significance of this modulation with respect to low-temperature adaptation.