No abstract

Ambient temperature is an important factor limiting the abundance and distribution of insects, and heat shock protein (Hsp) gene expression is sensitive to extremes of cold and heat. In order to explore the role of Hsps during thermal stress and development in Monochamus alternatus Hope (Coleoptera: Cerambycidae), we cloned and characterized full-length Hsp genes, including MaHsp60, MaHsp70, and MaHsp90. M. alternatus were exposed to different temperatures (−15, −5, 5, 15, 25, 35, and 40℃) for 1 h and was allowed to recover at 25℃ for 1 h. Following the treatments, we investigated the expression of the Hsps by quantitative real-time polymerase chain reaction. In third instar larvae, MaHsp60, MaHsp70, and MaHsp90 expression was upregulated in response to cold and heat, but the three Hsps were especially sensitive to heat, specifically at 35℃ and 40℃. After heating M. alternatus to 35℃, the expression of MaHsp60, MaHsp70, and MaHsp90 was higher than at 5℃ and 25℃ in nearly all developmental stages. MaHsp60, MaHsp70, and MaHsp90 expression was highest in later pupal, early adult, and early adult stages, respectively. These results suggest that compared with normal ambient temperatures, thermal stress could induce high expression of the three Hsps.

The Japanese pine sawyer, Monochamus alternatus Hope (Coleoptera: Cerambycidae), is a major pest of coniferous forests. Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (Bt) is an ecosystem-friendly agent for the control of pest insects. To identify the genes that are potentially associated with Bt toxicology, differentially expressed gene (DGE) libraries of M. alternatus after 20 h of exposure to a sublethal concentration of Bt were prepared from dissected midgut tissue. The transcripts were sequenced using the Illumina sequencing platform. After quality checks, the total numbers of clean reads were 29.20 and 41.53 million in the control and Bt-exposed libraries, respectively. We found that 947 and 8,895 unique genes were significantly up-and down-regulated, respectively. The annotation of the non-redundant sequences using the Gene Ontology database identified 3,837 sequences involved in cellular components, 3,190 sequences involved in molecular functions, and 6,930 sequences involved in biological processes, and showed that the genes were distributed across 50 categories. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the top 20 enriched pathways included metabolism of xenobiotics by the cytochrome P450 pathway and steroid hormone biosynthesis. Seventy-four DEGs were enriched in the metabolism of xenobiotics by the cytochrome P450 pathway, including the DEGs encoding Cyp1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), Cyp1A2 (cytochrome P450, family 1, subfamily A, polypeptide 2), Cyp3A (cytochrome P450, family 3, subfamily A), aldehyde dehydrogenase (NAD[P] +) and ADH1_7 (alcohol dehydrogenase 1/7), all of which were up-regulated. However, the DEGs encoding cathepsin L and CBR1 (carbonyl reductase 1) were down-regulated. Therefore, the effects of Bt on M. alternatus are complex and time dependent. This study is a first step toward achieving an understanding of the profile of the secondary targets of Bt in M. alternatus, and the results may provide insights for further exploration of the functions of the target genes in detoxification and resistance to Bt.

Among the three existing targeted gene editing technologies, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9), the latter is widely used owing to its simplicity, efficiency, and low cost. Here, we routinely infected A172 and U251 cells with lentiviral vectors, in which aquaporin-8 (AQP8) was knocked out using CRISPR/Cas9. Our results indicated that cryopreservation did not significantly alter the viral infection efficiency, but influenced AQP8 expression in the infected cells at both protein and mRNA levels compared with the non-cryopreserved samples. Further, AQP8 expression at protein and mRNA levels in recovered cryopreserved infected cells did not significantly differ from those in the blank and negative controls, indicating that the lentivirus was still infectious at low temperatures. However, it failed to release the AQP8-targeting guide RNA in the infected cells, or the guide RNA was released, but underwent changes that caused it to malfunction in the cells with CRISPR/Cas9-mediated AQP8 knock-out. Our findings possibly provide some insights into the reliability of lentiviruses as CRISPR/Cas9 vectors.