Abstract
The six-spined ips, Ips calligraphus, is a North American bark beetle that can exploit most eastern North American Pinus species and can cause mortality. Biotic and abiotic disturbances weaken trees, creating breeding substrate that promotes rapid population growth. Management historically relied on silvicultural practices, but as forests become increasingly stressed, innovative management is needed. Manipulation of the cellular RNA interference (RNAi) pathway to induce gene silencing is an emerging means of insect suppression, and is effective for some bark beetles. Quantitative PCR (qPCR) is a powerful tool for analysis of gene expression, and is essential for examining RNAi. To compare gene expression among individuals, stably expressed reference genes must be validated for qPCR. We evaluated six candidate reference genes (18s, 16s, 28s, ef1a, cad, coi) for stability under biotic (beetle sex, developmental stage, and host plant), and abiotic (temperature, photoperiod, and dsRNA exposure) conditions. We used the comprehensive RefFinder tool to compare stability rankings across four algorithms. These algorithms identified 18s, 16s, and 28s as the most stably expressed. Overall, 16s and 28s were selected as reference genes due to their stability and moderate expression levels, and can be used for I. calligraphus gene expression studies using qPCR, including those evaluating RNAi.
Highlights
The six-spined ips, Ips calligraphus, is a North American bark beetle that can exploit most eastern North American Pinus species and can cause mortality
The double-stranded RNA (dsRNA) is cleaved by the enzyme dicer into 21–23 base pair strands known as small-interfering RNA, which are incorporated into the RNAiinduced silencing complex (RISC)[25]
Discussion Quantitative PCR (qPCR) is an indispensable tool for evaluating gene expression, an important aspect of many genetic studies, and essential for demonstrating RNA interference (RNAi)-induced gene silencing, as its sensitivity permits detection of even minor differences between samples[27,29,30]
Summary
The six-spined ips, Ips calligraphus, is a North American bark beetle that can exploit most eastern North American Pinus species and can cause mortality. I. calligraphus has historically been invasive in the Philippines and Jamaica[5,13], and has more recently established populations in south mainland China[14], well outside of its native range, and is classified as a Scolytinae at high risk of becoming invasive in the southern hemisphere[15] This invasiveness, coupled with human-mediated disturbances and rapidly changing climatic conditions, has increased the risk that I. calligraphus poses to forest ecosystems, necessitating development of innovative, sustainable, and affordable management tools. An established method of demonstrating gene silencing through the introduction of exogenous dsRNA involves evaluation of relative gene expression via quantitative PCR (qPCR) This requires selection of stably expressed endogenous controls, or reference genes, that can be used to interpret gene expression levels across samples that may have different mRNA levels due to methodological variation, rather than biological c auses[26,27]. The stability of a candidate reference gene can be quantitatively evaluated via algorithms such as GeNorm, NormFinder, delta-Ct, and BestKeeper, allowing for selection of the most reliable endogenous controls
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