Operational taxonomic unit (OTU) sequencing results
A total of 9174 OTUs were obtained from the 60 samples of M. alternatus and its habitat niche. According to the rarefaction curves, the number of sequences obtained was able to reflect the main bacterial information in each sample (Additional file 1: Fig. S1). There were 1573 OTUs shared among all samples. 1778 and 1922 unique OTUs were detected in samples from healthy and infected pines, respectively. Only 195 unique OTUs were found in samples from M. alternatus (Fig. 1A). Instar II larvae feed on phloem, and the number of OTUs shared by the instar II larvae midgut and the phloem of infected pine (346) was close to the number shared by instar II larvae midgut and the phloem of healthy pine (325) (Fig. 1B). Instar III larvae feed on xylem, and the number of OTUs shared by instar III larvae midgut and the xylem of infected pine (233) was approximately twice that shared by instar III larvae midgut and the xylem of healthy pine (114). There were 1328 unique OTUs in the xylem of infected pines, which was far more than the 237 unique OTUs in the xylem of healthy pines (Fig. 1C). There were 84 shared OTUs in the samples from midgut of adult M. alternatus, healthy pine bark, and infected pine bark. The number of unique OTUs (not found in the adult M. alternatus) in infected pine bark was about 2.5 times that in healthy pine bark (Fig. 1D).
Linear discriminant analysis effect size (LEfSe) analysis
Species distribution analysis at the phylum level indicated that the main bacteria in the M. alternatus midgut belonged to Proteobacteria and Firmicutes. Infected pines mainly harbored Bacteroidetes, Armatimonadetes, Actinobacteria, Acidobacteria, and Proteobacteria (Additional file 1: Fig. S2). The Acidobacteria in infected pines was highly similar to that in healthy pines, while the Proteobacteria in infected pines was highly similar to that in the midgut of M. alternatus (Fig. 2).
Bacterial community compositions inM. alternatus and its habitat niche
There were significant differences in species composition between infected and healthy pines. The Streptophyta of Cyanobacteria/Chloroplast was the dominant in healthy pines, due to the V3-V4 region cannot distinguish 16s rDNA from bacteria and Cyanobacteria/Chloroplast. Regarding the infected pines, the most abundant genera were Sphingomonas (7.66%), followed by Burkholderia (6.51%) and Acidobacteria subgroup 1 (Gp1) (6.51%). In the midgut and frass of M. alternatus, the most abundant genera were Serratia (25.25%), Enterobacter (12.42%), Halotalea (8.81%), and Stenotrophomonas (6.68%). The relative abundance of Acidobacteria subgroup 1 (Gp1), subgroup 2 (Gp2), and subgroup 3 (Gp3) in surface soil and rhizosphere soil exceeded 50%, with no differences between infected and healthy pines (Fig. 3) (Additional file 1: Fig. S3, S4).
Regarding the frass of different stages of M. alternatus after feeding, Granulicella was the most abundant genus (12.15%) in the frass of instar II larvae, followed by genus Sphingomonas (10.11%). Saccharibacteria was the most abundant genus in the frass of instar III larvae (12.57%), followed by genus Burkholderia (11.68%). The relative abundance of genus Pseudoxanthomonas (5.31%) in the frass of instar III larvae was higher than in the frass of instar II larvae and the midgut of various instars (total: 0.03%) (Fig. 3 and Additional file 1: Fig. S5, S6). After feeding by M. alternatus adults, the most abundant genera in the bark from infected pines were Sphingomonas and Granulicella (Additional file 1: Fig. S7). The bark, phloem, and xylem of infected pines contained more putative pathogenic bacteria (mainly Saccharibacteria, Burkholderia, and Granulicella) than the corresponding tissues in healthy pines (Fig. 3). These results indicate that the dominant bacteria were similar between the frass of larvae and infected pines.
Specific bacterial genera in the habitat niche of M. alternatus
The heatmap shows that genera Escherichia/Shigella, Pseudomonas, and Spartobacteria were mainly distributed in pines, and their overall level was constant in healthy and infected pines (Fig. 4, labeled green). Several bacterial genera were mainly found in the infected pines and soil of healthy pines, including Dyella, Burkholderia, Bradyrhizobium, Mycobacterium, and Mucilaginibacter (Fig. 4, labeled pink). The genera Rhizobium, Terriglobus, Nocardioides, and Saccharibacteria were mainly found in infected pines and the phloem of healthy pines (Fig. 4, labeled light blue). In addition, the genus Pseudoxanthomonas was mostly distributed in the phloem and root of healthy pines (14% in both tissues) and infected pines (39% and 2.56%, respectively) (Fig. 4, labeled light blue). Granulicella and Sphingomonas genera were mainly distributed in the bark of healthy pines compared to the other health pine tissues, and their relative abundances were increased in all infected pines tissues (Fig. 4, labeled yellow). The genus Gryllotalpicola was only found in the phloem (0.1%) of healthy pines (rather than any other of the healthy pine tissues), but it was increased in the bark (4.1%), phloem (3.1%), xylem (1.6%) and root (0.6%) in infected pines, and was also found with low relative abundance in the midgut and frass of M. alternatus (Fig. 4, labeled orange). Interestingly, the genus Cellulomonas was not found in the midgut of M. alternatus, and the highest relative abundance occurred in the phloem of infected pines (2.9%), followed by the phloem of healthy M. alternatus (0.8%). Its relative abundance was also low (<0.01%) in the needle, root, and surface soil of healthy pines, as well as in the needle, bark, xylem, root, surface soil, and rhizosphere soil of infected pines (Fig. 4, labeled blue).
Specific bacterial genera in the midgut of M. alternatus
The bacterial genera Serratia, Enterobacter, Achromobacter, and Stenotrophomonas were dominant in the midgut of M. alternatus (Fig. 4, labeled red). Serratia was the most abundant bacterial genus in the midgut of instar II larvae. Enterobacter was the most abundant genus in the midgut of instar III larvae (65%), and it was also highly abundant in the midgut of adult insects (10.30%). Halotalea was the most abundant bacterial genus in the pupae midgut (47.69%) (Fig. 5A).
Interestingly, the relative abundance of genus Serratia was different in various instars of M. alternatus. In the habitat niche, Serratia was detected in all samples, but with low relative abundance (<0.5%). However, Serratia was enriched in the midgut of M. alternatus larvae; it peaked at 72.11% in the instar II larvae, decreased in the instar III larvae (23.46%), increased again in the pupae (32.85%), and was lowest in adults (22.71%). Additionally, Serratia was found in the frass of the instar II and III larvae (<0.6%). These results indicate a close relationship between genus Serratia and M. alternatus (Fig. 5B).
The colony-forming unit assays showed that Serratia sp. was present in midgut of instars I–V regarding both M. alternatus reared on an artificial diet and wild-caught M. alternatus. Serratia sp. peaked in instar II (about 81% in both), was at a minimum in instar III (9% in the larvae reared on the artificial diet and 11% in the wild-caught larvae), and was relatively stable for instars I and IV between the reared on artificial diet and wild-caught groups. However, in instar V (diapause), Serratia sp. in larvae reared on the artificial diet was higher than in wild-caught larvae (Fig. 5C and D). The results suggest that food has little effect on the relative abundance of Serratia sp. in the midgut of M. alternatus larvae, but further research is needed on its abundance pattern and whether it is related to the larval metabolic mechanisms.
The heatmap of Spearman’s rank correlation coefficients at the genus level shows that the relative abundance of Serratia was positively correlated with Stenotrophomonas, Gryllotalpicola, and Pseudoxanthomonas, and negatively correlated with Gp1 Gp2 Gp3, Escherichia/Shigella, Burkholderia. Bradyrhizobium, Sphingomonas, Granulicella, and Mucilaginibacter (Additional file 1: Fig. S8).