The Effects of Landscape Structure and Stand-Scale Factors on Dendroctonus Valens Damage Under Disturbance Conditions in North China

Background In recent years, the red turpentine beetle (Dendroctonus valens, RTB), an invasive pest species has spread northward along the distribution of pine forests, forming a potential threat to healthy pine forests in North China. Previous studies have shown that natural (e.g., fire) and human (e.g., felling) disturbances can significantly promote bark beetle damage. However, few studies have considered the effect of multi-scale factors on bark beetle damage under disturbance conditions. Here, we investigated RTB damage (entrance holes) in 98 forest stands with and without disturbance (fire or stolen felling) in the Heilihe National Nature Reserve, Inner Mongolia, which is considered to be in the early stage of RTB outbreak. We assessed the effects of forest landscape structure (forest proportion and host connectivity) and stand-scale characteristics on RTB damage under different disturbance conditions (presence or absence). In addition, we also explored the effects of fire and stolen felling disturbance on RTB damage and the significant differences between them. Result Disturbance (i.e., fire and stolen felling) could significantly promote the occurrence of RTB and there was no significant difference between the two types of disturbance. In the absence of disturbance, small stand-scale factors (i.e., aspect and canopy cover) played important roles in the prediction of RTB damage. In the presence of disturbance, forest proportion within a radius of 250 m was the main factor affecting RTB invasion. Higher forest coverage could reduce the migration of RTB from the surrounding environment to the disturbance area, thus reducing RTB damage. In addition, we observed a positive relationship between elevation and RTB invasion. Conclusion Landscape structure and stand-scale factors had different effects on RTB invasion under different disturbance conditions. This study not only provides new insights into understanding the roles played by multi-scale factors in RTB damage but also assists in the implementation of pest management programs.

Seasonal Shifts in Cold Tolerance and the Composition of the Gut Microbiome of Dendroctonus valens LeConte Occur Concurrently

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.

Effects of Forest Structure On Dendroctonus Valens Damage Under Natural and Human Disturbances

Background In recent years, the red turpentine beetle (Dendroctonus valens, RTB), an invasive pest species has spread northward along the distribution of pine forests, forming a potential threat to healthy pine forests in North China. Previous studies have shown that natural (e.g., fire) and human (e.g., felling) disturbances can significantly promote bark beetle damage. However, few studies have considered the effect of forest landscape structure on bark beetle damage under disturbance conditions. Here we used generalized linear models and generalized linear mixed models to explore the effects of multi-scale factors on RTB damage under different disturbance conditions (presence or absence) in 109 forest stands in the Heilihe National Nature Reserve, Inner Mongolia. Result Disturbance (i.e., fire and stolen felling) could significantly promote the occurrence of RTB. In the absence of disturbance, small-scale stand factors (i.e., aspect and canopy density) played important roles in the prediction of RTB damage. In the presence of disturbance, forest landscape structure (i.e., forest isolation) was the main factor affecting RTB invasion. Conclusion In the presence of disturbance, the forest structure played an important role in the process of the diffusion of RTB from the surrounding habitat to the disturbance. This study, which emphasizes the potential importance of forest landscape structure on RTB spread, not only provides new insights into understanding the roles of large-scale factors but also assists in the implementation of pest management programs.

Expression analysis of genes related to cold tolerance in Dendroctonus valens

Pine beetles are well known in North America for their widespread devastation of pine forests. However, Dendroctonus valens LeConte is an important invasive forest pest in China also. Adults and larvae of this bark beetle mainly winter at the trunks and roots of Pinus tabuliformis and Pinus sylvestris; larvae, in particular, result in pine weakness or even death. Since the species was introduced from the United States to Shanxi in 1998, its distribution has spread northward. In 2017, it invaded a large area at the junction of Liaoning, Inner Mongolia and Hebei provinces, showing strong cold tolerance. To identify genes relevant to cold tolerance and the process of overwintering, we sequenced the transcriptomes of wintering and non-wintering adult and larval D. valens using the Illumina HiSeq platform. Differential expression analysis methods for other non-model organisms were used to compare transcript abundances in adults and larvae at two time periods, followed by the identification of functions and metabolic pathways related to genes associated with cold tolerance. We detected 4,387 and 6,091 differentially expressed genes (DEGs) between sampling dates in larvae and adults, respectively, and 1,140 common DEGs, including genes encoding protein phosphatase, very long-chain fatty acids protein, cytochrome P450, and putative leucine-rich repeat-containing proteins. In a Gene Ontology (GO) enrichment analysis, 1,140 genes were assigned to 44 terms, with significant enrichment for cellulase activity, hydrolase activity, and carbohydrate metabolism. Kyoto Encyclopedia of Genes and Genomes (KEGG) classification and enrichment analyses showed that the lysosomal and purine metabolism pathways involved the most DEGs, the highly enriched terms included autophagy—animal, pentose and glucuronate interconversions and lysosomal processes. We identified 140 candidate genes associated with cold tolerance, including genes with established roles in this trait (e.g., genes encoding trehalose transporter, fructose-1,6-bisphosphatase, and trehalase). Our comparative transcriptome analysis of adult and larval D. valens in different conditions provides basic data for the discovery of key genes and molecular mechanisms underlying cold tolerance.

Fungal associates of an invasive pine-infesting bark beetle, Dendroctonus valens, including seven new Ophiostomatalean fungi

The red turpentine beetle (RTB; Dendroctonus valens ) is a bark beetle that is native to Central and NorthAmerica. This insect is well-known to live in association with a large number of Ophiostomatalean fungi. The beetle is considered a minor pest in its native range, but has killed millions of indigenous pine trees in China after its appearance in that country in the late1990s. Inorder to increase the base of knowledge regarding the RTB and its symbionts, surveys of the beetle's fungal associates were initially undertaken in China, and in a subsequent study in its native range in North America. A total of 30 Ophiostomatalean species that included several undescribed taxa, were identified in these surveys. In the present study, seven of the undescribed taxa collected during the surveys were further characterised based on their morphological characteristics and multi-genephylogenies. We proceeded to describe five of these as novel Leptographium spp. and two as new species of Ophiostoma. Four of the Leptographium spp. resided in the G. galeiformis-species complex, while one formed part of the L. olivaceum species complex. One Ophiostoma sp. was a member of the O. ips-species complex, while the only new species from China was closely related to O. floccosum. Two of the previously undescribed taxa from North America were shown to be congeneric with L. terebrantis, implying that this species was most often isolated in association with the RTB in North America. The undescribed taxon from North America was identified as O. ips, and like L. terebrantis, this species was also not recognized during the initial North American survey. Resolving the identities of these taxa provides essential baseline information to better understand the movement of fungal pathogens with this beetle. This then enhances our ability to accurately assess and predict the risks of invasions by these and related fungi.

Reference Gene Selection for Expression Analyses by qRT-PCR in Dendroctonus valens

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.

Gut Bacterial Communities of Dendroctonus valens and D. mexicanus (Curculionidae: Scolytinae): A Metagenomic Analysis across Different Geographical Locations in Mexico

Dendroctonus bark beetles are a worldwide significant pest of conifers. This genus comprises 20 species found in North and Central America, and Eurasia. Several studies have documented the microbiota associated with these bark beetles, but little is known regarding how the gut bacterial communities change across host range distribution. We use pyrosequencing to characterize the gut bacterial communities associated with six populations of Dendroctonus valens and D. mexicanus each across Mexico, determine the core bacteriome of both insects and infer the metabolic pathways of these communities with Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) to evaluate whether these routes are conserved across geographical locations. Our results show that the β-diversity with UniFrac unweighted varies among locations of both bark beetles mainly due to absence/presence of some rare taxa. No association is found between the pairwise phylogenetic distance of bacterial communities and geographic distance. A strict intraspecific core bacteriome is determined for each bark beetle species, but these cores are different in composition and abundance. However, both bark beetles share the interspecific core bacteriome recorded previously for the Dendroctonus genus consisting of Enterobacter, Pantoea, Providencia, Pseudomonas, Rahnella, and Serratia. The predictions of metabolic pathways are the same in the different localities, suggesting that they are conserved through the geographical locations.