Microhabitat Governs the Microbiota of the Pinewood Nematode and Its Vector Beetle: Implication for the Prevalence of Pine Wilt Disease

Abstract

Our understanding of environmental acquisition of microbes and migration-related alteration of microbiota across habitats has rapidly increased. However, in complex life cycles, such as for many parasites, exactly how these microbes are transmitted across multiple environments, such as hosts and habitats, is unknown. Pinewood nematode, the causal agent of the globally devastating pine wilt disease, provides an ideal model to study the role of microbiota in multispecies interactions because its successful host invasion depends on the interactions among its vector insects, pine hosts, and associated microbes. Here, we studied the role of bacterial and fungal communities involved in the nematode's life cycle across different micro- (pupal chamber, vector beetle, and dispersal nematodes) and macrohabitats (geographical locations). We identified the potential sources, selection processes, and keystone taxa involved in the host pine-nematode-vector beetle microbiota interactions. Nearly 50% of the microbiota in vector beetle tracheae and ~60% that of third-stage dispersal juveniles were derived from the host pine (pupal chambers), whereas 90% of bacteria of fourth-stage dispersal juveniles originated from vector beetle tracheae. Our results also suggest that vector beetles' tracheae selectively acquire some key taxa from the microbial community of the pupal chambers. These taxa will be then enriched in the dispersal nematodes traveling in the tracheae and hence likely transported to new host trees. Taken together, our findings contribute to the critical information toward a better understanding of the role of microbiota in pine wilt disease, therefore aiding the knowledge for the development of future biological control agents. IMPORTANCE Our understanding of animal microbiota acquisition and dispersal-mediated variation has rapidly increased. In this study, using the model of host pine-pinewood nematode-vector beetle (Monochamus sp.) complex, we disentangled the routes of microbial community assembly and transmission mechanisms among these different participants responsible for highly destructive pine wilt disease. We provide evidence that the microhabitat is the driving force shaping the microbial community of these participants. The microbiota of third-stage dispersal juveniles (L_III) of the nematodes collected around pupal chambers and of vector beetles were mainly derived from the host pine (pupal chambers), whereas the vector-entering fourth-stage dispersal juveniles (L_IV) of the nematodes had the simplest microbiota community, not influencing vector's microbiota. These findings enhanced our understanding of the variation in the microbiota of plants and animals and shed light on microbiota acquisition in complex life cycles.

Keywords: bacterial and fungal community; community assembly; microbial transmission; microhabitat; pinewood nematode; plant-nematode-vector beetle.

FIG 1

Diversity and composition of bacterial and fungal communities in four pinewood nematode-associated microhabitats. (A) Schematic presentation of the complex life cycle of pinewood nematode. The gray circle represents different larval stages and colored arrows represent the different habitats of nematodes. (B) Microbial alpha diversity across all five sites in pupal chamber, tracheae, L_III, and L_IV samples based on the observed ASVs. (C) Composition of bacterial classes and fungal phylum at different microhabitats. Kruskal-Wallis with FDR adjusted, *, P < 0.05; **, P < 0.01; ***, P < 0.001. PC, pupal chamber; TR, trachea; L_III, third-stage dispersal juveniles; L_IV, fourth-stage dispersal juveniles.

FIG 2

Taxonomic classification of different bacteria and fungi between pupal chambers and tracheae. The Manhattan plot shows ASVs enriched in tracheae samples for bacteria (A) and fungi (B). Each dot or triangle represents a single ASV. Venn diagrams above show the shared and unique ASVs between pupal chambers and tracheae. ASVs enriched or depleted in tracheae are represented by filled or empty triangles, respectively (FDR adjusted P < 0.05). ASVs are grouped by taxonomic order and colored according to the phylum (class for Ascomycota and Proteobacteria). “(2, 15)” means the number of enriched (2) and depleted (15) ASVs. PC, pupal chamber; TR, trachea; CPM, counts per million.

FIG 3

Microbial community assembly processes of the pinewood nematode life cycle. Shown is the Sloan neutral model for bacterial (A) and fungal (B) communities for tracheae samples across three southern sites. Gray dashed lines represent 95% confidence intervals around the model prediction and the ASVs fitting the model are colored gray. The ASVs occurring more frequently than predicted by the model are colored blue, and ASVs that occur less frequently than predicted are colored red. Letters in blue show the MST value based on Bray-Curtis distances. (C) The cumulative relative abundance distribution of three types of ASVs of the neutral model in northern (TRLS) and southern (TRAZJ) sites. (D) Source route of host microbiota showing the potential source of bacterial and fungal communities for different host types involved in the pinewood nematode life cycle. U, unknown source; TRLS, trachea samples from Liaoning and Shaanxi; TRAZJ, trachea samples from Anhui, Zhejiang, and Jiangsu.

FIG 4

Keystone taxa across the pinewood nematode life cycle. (A and B) Taxonomic composition of the core microbiota taxa at class level across the five sampling sites for L_III and L_IV. (C and D) Heatmap for keystone taxa enriched in L_III and L_IV. The ASVs above or below prediction in the neutral model are marked with triangles. PCLS, pupal chamber samples from Liaoning and Shaanxi; PCAZJ, pupal chamber samples from Anhui, Zhejiang, and Jiangsu.