Bacterial community associated to the pine wilt disease insect vectors Monochamus galloprovincialis and Monochamus alternatus

Abstract

Monochamus beetles are the dispersing vectors of the nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD). PWD inflicts significant damages in Eurasian pine forests. Symbiotic microorganisms have a large influence in insect survival. The aim of this study was to characterize the bacterial community associated to PWD vectors in Europe and East Asia using a culture-independent approach. Twenty-three Monochamus galloprovincialis were collected in Portugal (two different locations); twelve Monochamus alternatus were collected in Japan. DNA was extracted from the insects' tracheas for 16S rDNA analysis through denaturing gradient gel electrophoresis and barcoded pyrosequencing. Enterobacteriales, Pseudomonadales, Vibrionales and Oceanospirilales were present in all samples. Enterobacteriaceae was represented by 52.2% of the total number of reads. Twenty-three OTUs were present in all locations. Significant differences existed between the microbiomes of the two insect species while for M. galloprovincialis there were no significant differences between samples from different Portuguese locations. This study presents a detailed description of the bacterial community colonizing the Monochamus insects' tracheas. Several of the identified bacterial groups were described previously in association with pine trees and B. xylophilus, and their previously described functions suggest that they may play a relevant role in PWD.

Figure 1. DGGE analysis of the V3 region from the 16S rRNA gene amplified from DNA of the trachea of Monochamus insects (MG – Monochamus galloprovincialis; MA – Monochamus alternatus) collected in Portugal (PC – Comporta or PM – Mortágua) and Japan (JH - Hikobe) respectively.

The dendrogram of DGGE patterns was created using UPGMA method (group average method) and is based on Pearson similarity correlation (0.0%–100% similarity). Blue squares represent samples from males and pink squares represent samples from females. Samples in red background were positive for Bursaphelenchus xylophilus. Cluster A has M. alternatus samples.

Figure 2. Relative abundance of the predominant bacterial orders for each insect sample (more than 0.1% of the total number of reads): (a) Monochamus galloprovincialis samples; (b) Monochamus alternatus samples.

Figure 3. Heatmap for the OTUs that represent more than 10% of the total number of reads.

The colour gradient goes from light yellow for the lower numbers of reads, to dark green for the higher numbers of reads. The numbers in the third top line represent the last two digits in the sample name (see Table 2).

Figure 4. Venn diagrams representing the number of (a) shared and unique OTUs and (b) shared and unique reads between Monochamus galloprovincialis (Mg) and Monochamus alternatus and the number of (c) shared and unique OTUs and (d) shared and unique reads between sampling locations (Comporta, Mortágua and Hikobe).

Figure 5. Bacterial community compositional structure in Monochamus galloprovincialis samples (blue) and Monochamus alternatus samples (red) indicated by non-metric multi-dimensional scaling (NMDS) with a stress value of 0.073, using Bray-Curtis distance.