Dendroctonus valens is the main pest of the genus Pinus. To facilitate gene expression analyses, suitable reference genes for adults and mature larvae of D. valens under different temperature conditions were determined. In particular, we obtained the sequences of candidate reference genes, ACT, TUB, SHDA, PRS18, 18S rRNA, and CYP4G55, from transcriptome data. Real-time quantitative PCR was used to analyze gene expression, and geNorm, NormFinder, and BestKeeper were used to evaluate expression stability. Under different temperature conditions, the expression levels of 18S rRNA, PRS18, and TUB were stable in adults, in which 18S rRNA > PRS18 > TUB. In mature larvae, the expression levels of TUB, 18S rRNA, and SDHA were stable, in which TUB > 18S rRNA > SDHA. The combination of 18S rRNA and PRS18 is recommended for studies of gene expression in adults and the combination of 18S rRNA and TUB is effective for studies of gene expression in mature larvae of D. valens under different temperature conditions.

Dendroctonus valens LeConte, an invasive bark beetle, has caused severe damage in pine forests and has the potential to disperse into new geographic ranges in China. Although the gut microbiota of D. valens and its fundamental role in host fitness have been investigated widely, little is known about the relationship between the seasonal shifts of both cold tolerance and the gut microbiome of D. valens during overwintering, which occurs at the larval stage. In this study, to examine seasonal variations in the composition of the microbiome, we collected D. valens larvae in September (autumn), January (winter), and May (spring), and then analyzed the bacterial and fungal communities of the gut via sequencing of partial 16S rRNA and ITS genes. In addition, changes in the supercooling capacity and antioxidant enzyme activities of D. valens larvae collected in the different seasons were evaluated. Overwintering resulted in changes to microbial communities. In particular, the abundances of Enterobacter, Serratia, Erwinia, and Klebsiella decreased during overwintering. Concurrent with these changes, the cold tolerance of D. valens larvae was enhanced during overwintering, and the activities of the antioxidant enzymes catalase and peroxidase were reduced. We hypothesize that seasonal shifts in the gut microbiome may be connected to changes in cold tolerance and antioxidant enzyme activity in D. valens. It will be worthwhile to confirm whether seasonal changes in the microbiome contribute to the success of host overwintering.