1 Scientific RepoRts | 6:23908 | DOI: 10.1038/srep23908 www.nature.com/scientificreports Bacterial community associated to the pine wilt disease insect vectors Monochamus galloprovincialis and Monochamus alternatus Marta Alves 1,2 , Anabela pereira 1 , patrícia Matos 1 , Joana Henriques 3 , Cláudia Vicente 4,6 , takuya Aikawa 5 , Koichi Hasegawa 6 , Francisco Nascimento 4 , Manuel Mota 4,7 , António Correia 1 & Isabel Henriques 1,2 Monochamus beetles are the dispersing vectors of the nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD). PWD inficts signifcant damages in Eurasian pine forests. Symbiotic microorganisms have a large infuence 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 diferent 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. Signifcant diferences existed between the microbiomes of the two insect species while for M. galloprovincialis there were no signifcant diferences between samples from diferent Portuguese locations. This study presents a detailed description of the bacterial community colonizing the Monochamus insects’ tracheas. Several of the identifed 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. Monochamus (Cerambycidae: Coleoptera) is a genus of sapro-xylophagous sawyer beetles 1,2 . In nature, they have a role in the processes of wood decomposition and nutrient cycling. Sawyer beetles can cause a signifcant loss in pine value due to the tunnels their larvae bore 3 . Yet, the major concern about Monochamus insects is their role as vectors of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae), the Pine Wilt Disease (PWD) causing agent 4,5 . Tis disease has been devastating pine trees since the beginning of the twentieth century with tremen- dous economic and environmental impacts 6–9 . Te tree-to-tree natural transmission of B. xylophilus is only possible through Monochamus beetles. Te pine- wood nematode (PWN) enters in its dispersal form into Monochamus when the beetle larvae molts to the late pupa 10,11 . Bursaphelenchus xylophilus concentrates mainly in the vectors’ tracheal system 12 , where it stays in a phoretic commensalism through insects’ development and emergence 9 . When the adult insect feeds, the PWN enter in a new host tree through the feeding wounds 13 . Once inside the host tree, B. xylophilus migrates through the vascular system and obstructs water conductance resulting in wilting of the pine host 8 . 1 Departamento de Biologia e centro de estudos do Ambiente e do Mar (ceSAM), Universidade de Aveiro, Aveiro, 3810-193, Portugal. 2 Departamento de Biologia e instituto de Biomedicina (iBiMeD), Universidade de Aveiro, Aveiro, 3810-193, Portugal. 3 iniAV/Unidade estratégica de investigação e Serviços de Sistemas Agrários e Florestais e Sanidade Vegetal, Av. da República, Quinta do Marquês, Oeiras, 2780-157, Portugal. 4 NemaLab-ICAAM, Universidade de Évora, Núcleo da Mitra, Ap. 94, Évora, 7002-554, Portugal. 5 FFPRI - Forestry and Forest Products Research Institute, Tohuku, Japan. 6 Environmental Biology Department, Chubu University, Kasugai, Japan. 7 Dep. Ciências da Vida, EPCV, Universidade Lusófona de Humanidades e Tecnologias (ULHT), Av. Campo Grande 376, 1749-024 Lisboa, Portugal. Correspondence and requests for materials should be addressed to A.P. (email: anabela. pereira@ua.pt) Received: 07 October 2015 Accepted: 10 March 2016 Published: 05 April 2016 OPEN