Sirex noctilio (Hymenoptera: Siricidae) is an invasive pest that has spread and established in many regions worldwide. However, its cold tolerance strategy is still unclear. We measured the supercooling point (SCP) and the lower lethal temperature (LLT) of overwintering S. noctilio larvae during three overwintering periods in four geographically separated populations in China. In addition, using the statistical analysis of the local historical temperature data, we also conducted comprehensive studies of S. noctilio cold tolerance variations and strategies. We measured the SCP of all samples as S. noctilio could survive at its SCP during a short period of exposure (<48 h) and its cold tolerance strategy was freeze-avoidance. The average SCPs of the groups in different spatiotemporal scales were significantly related to average temperature variation with most individuals exhibiting stronger cold hardiness at low ambient temperatures. S. noctilio exhibited a strong cold tolerance and it has the ability to withstand lower temperatures in cold environments. The geographic population showed a positive tendency as the ambient temperature decreased, which would increase its chance of developing in cold regions.

Hylurgus ligniperda (Fabricius) (Curculionidae: Scolytinae) is a new invasive pest beetle in China, which colonized the Shandong province, causing devastating damage. Originating in Europe, it has spread to Oceania, Asia, North and South America. Bacterial associates have been frequently reported to play a vital role in strengthening the ecological adaptations of bark and ambrosia beetles. The environmental adaptability of H. ligniperda may be supported by their associated bacteria. Bacterial communities colonizing different body parts of insects may have different functions. However, little is known about the bacteria associated with H. ligniperda and their potential involvement in facilitating the adaptation and invasion of the beetles into new environments. In this study, we employed high-throughput sequencing technology to analyze the bacterial communities associated with male and female adults of H. ligniperda by comparing those colonizing the elytra, prothorax, and gut. Results showed that the bacterial communities of male and female adults were similar, and the elytra samples had the highest bacterial diversity and richness, followed by the gut, while the prothorax had the lowest. The dominant phyla were Proteobacteria, Firmicutes, and Actinobacteriota, while the dominant genera were Serratia, Lactococcus, Rhodococcus, unclassified Enterobacteriaceae, and Gordonia. Among these, Rhodococcus and Gordonia were the specific genera of endobacteria and ectobacteria, respectively. Differences in the distribution of associated bacteria may suggest that they have different ecological functions for H. ligniperda. The results of functional prediction showed that bacteria were enriched in terpenoid backbone biosynthesis, degradation of aromatic compounds, limonene and pinene degradation, neomycin, kanamycin and gentamicin biosynthesis, indicating that they may assist their beetles in synthesizing pheromones, degrading toxic secondary metabolites of host trees, and antagonizing pathogenic fungi. These results help us understand the interaction between H. ligniperda and bacteria and highlight possible contributions to the invasion process.

The mutualistic symbiosis relationship between the gut microbiome and their insect hosts has attracted much scientific attention. The native woodwasp, Sirex nitobei, and the invasive European woodwasp, Sirex noctilio, are two pests that infest pines in northeastern China. Following its encounter with the native species, however, there is a lack of research on whether the gut microbiome of S. noctilio changed, what causes contributed to these alterations, and whether these changes were more conducive to invasive colonization. We used high-throughput and metatranscriptomic sequencing to investigate S. noctilio larval gut and frass from four sites where only S. noctilio and both two Sirex species and investigated the effects of environmental factors, biological interactions, and ecological processes on S. noctilio gut microbial community assembly. Amplicon sequencing of two Sirex species revealed differential patterns of bacterial and fungal composition and functional prediction. S. noctilio larval gut bacterial and fungal diversity was essentially higher in coexistence sites than in separate existence sites, and most of the larval gut bacterial and fungal community functional predictions were significantly different as well. Moreover, temperature and precipitation positively correlate with most of the highly abundant bacterial and fungal genera. Source-tracking analysis showed that S. noctilio larvae at coexistence sites remain dependent on adult gut transmission (vertical transmission) or recruitment to frass (horizontal transmission). Meanwhile, stochastic processes of drift and dispersal limitation also have important impacts on the assembly of S. noctilio larval gut microbiome, especially at coexistence sites. In summary, our results reveal the potential role of changes in S. noctilio larval gut microbiome in the successful colonization and better adaptation of the environment.

Dastarcus helophoroides Fairmaire, a parasitic natural enemy widely used in the biological control of Monochamus alternatus Hope, possesses strong abilities to search for hosts and prey. To enhance the biological control efficacy of D. helophoroides against M. alternatus, this study systematically evaluated the effectiveness of enhancing control through methods such as drilling holes in lure logs and introducing D. helophoroides carrying Pyemotes zhonghuajia or Beauveria bassiana under controlled indoor conditions. The results showed that: (1) Releasing D. helophoroides eggs (after 30 days) and releasing adults (after 120 days) were both effective in controlling M. alternatus, with the best control effects achieved when 50 eggs/m and 3 adults/m were released. The release of adults took longer to show effects but had better results than the release of eggs in the later stages. (2) The location and number of holes drilled in lure logs significantly influenced the efficiency of D. helophoroides in searching for and parasitizing M. alternatus, while the diameter of the holes was not significant. Considering the highest control efficacy and minimizing damage to the tree, the optimal conditions were releasing D. helophoroides adults 2 cm above M. alternatus fecal holes, with 0.4 cm in diameter and 2 holes/m release density, which significantly promoted the invasion of D. helophoroides adults into the borer tunnels and increased the M. alternatus mortality rate. (3) Releasing D. helophoroides adults carrying P. zhonghuajia or B. bassiana significantly improves the control efficacy against M. alternatus. The higher the number of D. helophoroides adults released per log segment and the greater the quantity of P. zhonghuajia or B. bassiana that they carry, the higher the mortality rate of M. alternatus. After 60 days of release, the highest mortality rate of M. alternatus was observed in treatments where D. helophoroides (released at densities of 3 adults per meter of log segment) carried P. zhonghuajia (0.6 million/m) or were sprayed with B. bassiana (3 g/m). The synergistic effect of carrying P. zhonghuajia was evident, but the overall control efficacy of spraying B. bassiana was limited. In summary, optimizing drilling techniques and combining biological release strategies under laboratory conditions can significantly enhance the control efficacy against M. alternatus, providing data support for D. helophoroides future application in forest pest control.

Pinus massoniana Lamb. is an economically important conifer native to China. However, it is highly susceptible to the pine wood nematode (Bursaphelenchus xylophilus, PWN), the causal agent of pine wilt disease (PWD), resulting in substantial ecological and economic losses. To elucidate potential molecular defense mechanisms, 50 NAC (NAM, ATAF1/2, and CUC2) transcription factors (PmNACs) were identified in the P. massoniana genome. Phylogenetic analysis divided these PmNACs into seven subfamilies, and motif analysis identified ten conserved motifs associated with stress responses. Twenty-three genes were selected for expression analysis in various tissues and under exogenous salicylic acid (SA), methyl jasmonate (MeJA), and PWN infection. Six genes (PmNAC1, PmNAC8, PmNAC9, PmNAC17, PmNAC18, and PmNAC20) were significantly up-regulated by both hormonal treatment and PWN infection, implying their involvement in JA/SA-mediated immune pathways. Functional characterization showed PmNAC8 is a nuclear-localized transcription factor with autoactivation activity. Furthermore, transient overexpression of PmNAC8 in Nicotiana benthamiana induced reactive oxygen species (ROS) accumulation and necrotic lesions. Collectively, these results elucidate NAC-mediated defense responses to PWN infection in P. massoniana and identify candidate genes for developing PWD-resistant pine varieties.