Insect maintenance and antibiotic treatment
In September 2019 and 2020, the third instar larvae of M. alternatus were collected from the beetle-infested pine trees (Pinus massoniana) in the Jiuhua Mountain (30°5′N, 117°8′E), Chizhou city, Anhui province, China. Larvae were placed in 6-cm disposable petri dishes with artificial medium and fed with an artificial diet (made of 15 g agar mixed with 210 g powder of pine, then added to 1 L boiled distilled water) , at 25 ± 1 °C. The artificial diet was refreshed each day.
For antibiotic treatment, M. alternatus larvae were washed with 75% alcohol for 30 s and rinsed with sterile water, then transferred on artificial diet containing streptomycin sulfate, ampicillin sodium salt, tetracycline HCl and nystatin each at 1 mg/mL, respectively. Germ-free (axenic) larvae were obtained after rearing for 4 days.
To determine whether gut and cuticle microbes were eliminated completely, third instar larvae were dissected under sterile conditions and gut and cuticle samples were collected in 1.5 mL DNase/RNase-free centrifuge tube with 200 μL 10% phosphatic buffer solution. Three gut samples from M. alternatus with antibiotic treatment were assigned as axenic, and three from untreated larvae were non-axenic. The suspension was serially diluted 1000 times with sterile water, plated onto Luria–Bertani media (LB) agar plates and tryptic soy agar (TSA). In addition, we also measured gut bacteria by 16s rRNA qPCR with 16sF (5′-TCCTACGGGAGGCAGCAGT-3′) and 16sR (5′-GGACTACCAGGGTATCTAATCCTGTT-3′) primers (qPCR method is specifically described below). Results showed we successfully obtained axenic insects (Additional file 1: Fig. S1).
To obtain B. bassiana (Bb3275) blastospores, Sabouraud’s Dextrose Agar (1% polypeptone, 4% glucose, 1.5% agar) supplemented with 1% yeast extract (SDAY) was inoculated with the fungus and incubated at 26 °C for 14 days. Then, conidial suspensions were dispersed in a sterile solution of 0.05% (v/v) Tween 80 and increasing concentrations of 1 × 106–1 × 109 conidia mL−1 were determined by direct counting using a hemocytometer.
To infect M. alternatus, 25 axenic and 25 nonaxenic M. alternatus larvae were dipped for 10 s into B. bassiana conidial suspension respectively, and the larvae dipped with sterile Tween 80 solution were set as controls, resulting in four separate treatment groups (Axenic; Nonaxenic; Bb + axenic; Bb + Nonaxenic). The larvae fed with the artificial medium/diet were kept at 26 °C and 90 ± 5% relative humidity. The number of surviving larvae was recorded daily.
Microbiome analysis by 16S rRNA gene amplicon sequencing
Five days after infection, 10 gut and cuticle samples from each treatment of M. alternatus larvae challenged by Tween 80 and three levels of concentration (1 × 107; 1 × 108; 1 × 109 conidia mL−1) Bb3275 were dissected (only live larvae were collected). All gut samples were processed with a DNA extraction kit (Fast DNA SPIN Kit for Soil) to extract the total community DNA. The quality and concentration of DNA were determined by 1.0% agarose gel electrophoresis and a NanoDrop® ND-2000 spectrophotometer (Thermo Scientific Inc., USA) and kept at − 80 °C prior to further use.
The DNA samples were sent to a sequencing company (Majorbio Bio-Pharm Technology Co. Ltd., Shanghai, China). 16s rRNA of distinct regions V3–V4 were amplified using universal primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR reaction mixture including 4 μL 5 × Fast Pfu buffer, 2 μL 2.5 mM dNTPs, 0.8 μL each primer (5 μM), 0.4 μL Fast Pfu polymerase, 10 ng of template DNA, and ddH2O to a final volume of 20 µL. PCR amplification cycling conditions were as follows: 95 °C, 3 min; 27 cycles of 95 °C, 30 s; 55 °C, 30 s; 72 °C, 45 s; 72℃, 10 min (ABI GeneAmp 9700). PCR products were detected by 2% agarose gel electrophoresis.
Analysis of bacterial microbiota
The raw 16S rRNA gene sequencing reads were demultiplexed and quality-filtered with Trimmomatic. Then, the paired-end (PE) reads were merged into a sequence using FLASH (V1.2.7) with a minimum overlap of 10 bp. Meanwhile, the quality of reads and the effect of merging were filtered, and the maximum error ratio allowed for overlapping area was 0.2. Samples were distinguished according to the barcode and primers, and the sequence direction was adjusted. The maximum number of barcodes was 0 and primer mismatches was 2, and the reads containing ambiguous characters were removed.
The optimized sequences were clustered into Operational Taxonomic Units (OTUs) with a similarity cutoff of 97% using Uparse (version 7.0.1090 http://drive5.com/uparse/). The OTUs were further subjected to a taxonomy-based analysis using the Ribosomal Database Project (RDP) algorithm (version 2.11 http://sourceforge.net/projects/rdp-classifier/) and confidence threshold set to 0.7. Silva (Release138 http://www.arb-silva.de) was used as an annotated database.
Alpha diversity using ACE, Chao1, Shannon and Simpson’s index as indicator of species richness and diversity, and beta diversity (weighted UniFrac, PCoA and NMDS) were analyzed by QIIME, then figures were made using R language (Version 3.3.1). Compositional differences in NMDS were tested using ANOSIM with 1000 permutations. A Permutational Multivariate Analysis of Variance based on the weighted UniFrac distance (PERMANOVA) was used to test for differences in PCoA between treatments.
Quantification of total or specific bacteria
To confirm the variation of total bacteria and amounts of Pseudomonas and Serratia in the gut and on the cuticle of M. alternatus larvae after different treatments, a quantitative PCR approach was used. DNAs from gut and cuticle of the beetle larvae were extracted using TIANamp Genomic DNA kit (TIANGEN, Beijing, China). Then all samples were adjusted to the same concentration according to quantifications with a Nanodrop 2000. Quantitative Real-Time PCR was performed by a CFX96™ Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA) in a volume of 20 μL using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and the protocol was as follows: 95 °C for 30 s, then 40 cycles of 95 °C for 10 s, and 60 °C for 15 s. Data was analyzed using the 2−ΔΔt method. The primer pair used for all bacteria was 16s-F (5′-TCCTACGGGAGGCAGCAGT-3′) and 16s-R (5′-GGACTACCAGGGTATCTAATCCTGTT-3′). The primer pair used for Pseudomonas quantification was Pse434F (5′-ACTTTAAGTTGGGAGGAAGGG-3′) and Pse686R (5′-ACACAGGAAATTCCACCACCC-3′), Sm-F2 (ACGTTCATCAATTGACGTTACTCGCA) and Sm-R2 (AACCGCCTGCGTGCGCTTTA) for Serratia. In addition, the larvae’s ActinF (5′-TGGGTATGGAATCTTGCGGT-3′) and ActinR (5′-GGCGGTGATTTCCTTTTGCA-3′) were used as reference genes.
Identification of bacteria
Monochamus alternatus larvae associated bacterial isolates were identified by PCR amplification and sequencing of conserved 16S ribosomal RNAs. DNA was isolated using TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China) as specified by the manufacturer for use as template in PCR. Nearly full length 16S rRNA was amplified for each isolate using primers universal to prokaryote 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). Thirty-five PCR cycles were conducted. The PCR final products were tested by electrophoresis at a 1% concentration of agarose gel, and band watched under UV reader.
DNA samples were sent to Sangon Biotech (Shanghai) Co., Ltd for sequencing. Then sequences of DNAs were edited and assembled (SeqMan component) and compared with those of known bacteria on NCBI-Genbank database. Finally, the phylogenetic tree was analyzed in Mega11 edition software (Additional file 1: Fig. S2).
Antifungal activity assay
To determine the inhibitory effect of bacteria on fungal spore germination, Pseudomonas protegens (B1-S1-1L) and Serratia marcescens (C1-1-2L) were cultivated on LB (1% tryptone, 0.5% yeast extract, 0.5% NaCl) at 28 °C with shaking. The bacterium Enterobacter soli (C4-2-2L), isolated from M. alternatus, was incubated with the same method and set as a negative control. Adjusted to an optical density (OD) of 0.3 (OD600 = 0.3), we then inoculated the 10 μL 1:1 mixture of bacterial fluid and fungal spore suspension (105 conidia mL−1) on PDA medium . The plates were incubated for 12 h in the dark at 28 ± 1 °C, and germination was observed at 400× magnification . A total of 100 conidia were scored for each treatment each time. Three independent repetitions of the experiment were performed.
To test antifungal activity, 20 μL bacterial fluid of each strain (OD600 = 0.3) was inoculated in 5 mm inoculation hole on 60 mm PDA medium which was initially coated with B. bassiana (107 conidia mL−1) and the experiment was repeated three times for each treatment [18, 26]. The inhibition was measured after 3 days of incubation.
qPCR analysis of immune-related genes and phenoloxidase activity assay
Since a similar antagonistic effect of M. alternatus-associated bacteria against B. bassiana was found among three dilutions of fungal treatments, we chose the concentration of B. bassiana spores at 1 × 107 conidia mL−1 for further analysis. Five days after post-infection (dpi), the larval fat body samples from the four groups (Axenic; Nonaxenic; Bb + axenic; Bb + Nonaxenic) were taken; these were collected using a dissecting shear under aseptic conditions from five larvae and pooled as one sample (n = 3). All samples were stored at − 80 °C. Total RNA was extracted using RNA isolater Total RNA Extraction Reagent (Nanjing Vazyme Biotech Co, Ltd) following the manufacturer’s instructions. One μg RNA was reverse transcribed using HiScript® III RT SuperMix for qPCR (Vazyme, Nanjing, China). The q-PCR method was the same as described above. Each experiment was repeated three times independently. The primers are described in Additional file 1.
The hemocytes of axenic and nonaxenic M. alternatus larvae (14 larvae per group) were collected under aseptic conditions after 5 dpi. Then, using an insect peroxidase (PO) ELISA kit (JingMei Biotechnology, Jiangsu, China), we assayed the enzymatic reaction in different samples following the manufacturer’s instructions. Each sample and standard substance were placed in 96-well plates, at 37 °C for 60 min. Then, plates were washed five times by eluent for 30 s each, and ELISA HRP A, B was added and maintained at 37 °C for 15 min. The optical density value at 450 nm was measured after a stop reaction.
Statistical analysis of data
Bacterial community composition was analyzed at phylum and genus levels, and species with an abundance ratio of less than 0.01 in all samples were classified as “other”. Kruskal–Wallis H-test was used to test for significant differences between groups among the top 10 genera according to their relative abundance.
Survival curves were analyzed using the Kaplan–Meier method, and the log-rank test was used to evaluate significant differences between two groups. As for other data, prior to analysis, we tested the residuals normality with the Shapiro–Wilk test for each level of variable. Data consisting of two treatments were analyzed using Student’s t-test and data consisting of more than two treatments were analyzed with one-way ANOVA coupled with a post hoc Waller–Duncan (equal variances) or Dunnett’s T3 (unequal variances) test. A value of P < 0.05 was considered significantly different. Data were analyzed using SPSS 19.0 and figures were made using GraphPad Prism 8.