Supplementary file 1
Supplementary Tables S1-6.
Table S1: Geographic location and other abiotic data of sampled beetle galleries. Table S2: Code and description of each microbe isolated from bessbug galleries. Antagonism assay results are represented in size of the zone of inhibition in millimeters. Table S3: Compounds detected in the frass of each gallery with their respective observed m/z of different adducts, retention time (Rt), peak height, and presence of MS2 spectrum. Compounds 1-13, 24 and 26 were annotated at level 1 identification if a MS2 spectrum of at least one adduct was detected, otherwise, they were annotated at level 2 identification. Compounds 16 and 17 were annotated at level 2 identification. Compounds 14 and 15 were annotated at level 3 identification. Table S4: Peak heights of compounds detected in each technical replicate of the LC-MS analyses of the environmental frass. Table S5: Details on the annotation of each compound. Table S6: GenBank accession number of each sequenced gene for microbial strains that were included in the phylogenetic study.
Supplementary file 2
Extracted ion chromatogram (EIC, showing retention time) and MS2 spectra of each compound annotated at identification level 1 (actinomycin D, actinomycin X2, STA-21, rubiginone B2, cycloheximide, nonactin, monactin, dinactin, trinactin, tetranactin, filipin I, filipin II, filipin III, antimycin A, nocardamine, bafilomycin A1, bafilomycin B1, novobiocin, piericidin A, nigericin, beauvericin), comparing a commercial standard (top) to the culture extract of an exemplary microbe or environmental frass extract (bottom).
Supplementary file 3
MS2 spectra of each compound annotated at identification level 2 (alteramide A, alteramide B, surugamide A), comparing a spectrum detected in the culture extract of an exemplary microbe (top) to a publicly available spectrum on the MassIVE repository (bottom).
Publicly available spectra can be found at: alteramide A, alteramide B: f.MSV000079516/ccms_peak/Labelled/R5_lab_J1074_pre.mzXML; surugamide A: f.MSV000079519/ ccms_peak/Unlabelled/A1_unlab_J1074_pre.mzXML (accessed on June 2020).
Supplementary file 4
MS2 spectrum of each compound putatively annotated at identification level 3 (filipin IV, fungichromin), detected in the culture extract of an exemplary microbe.
Annotations were made based on fragmentation similarities with other analogs of the same family annotated at identification level 1 (filipins I–III).
Supplementary file 5
Three exemplary total ion chromatograms (TICs) of the LC-MS/MS analysis performed on environmental samples.
(A) Pupal chamber material; (B, C) old frass, plus the extracted ion chromatogram (EIC) of an exemplary compound detected in each sample. The MS1 spectrum refers to the main peak detected on each EIC, highlighting two adducts of each compound.
Supplementary file 6
Strains of Streptomyces padanus were isolated from 19/22 of the sampled O. disjunctus galleries.
(A) S. padanus P333 growing on an ISP2-agar plate after 7 days of incubation at 30°C. (B) Galleries that S. padanus was isolated from.
Supplementary file 7
Average coverage distribution of Streptomyces-derived genes identified in the metagenome of the different gut compartments of O. disjunctus.
The figure shows the prevalence of the Streptomyces-derived genes in all four gut compartments with significantly higher normalized-coverage in the posterior hindgut (PHG), the region where frass is compacted prior to its excretion. In each boxplot, a point represents a single gene per category and its detected coverage, and the diamond symbols represent the mean. The box boundaries represent the first and third quartiles of the distribution, and the median is represented as the horizontal line inside each box. Boxplots whiskers span 1.5 times the interquartile range of the distribution. FG: foregut; MG: midgut; AHG: anterior hindgut. Statistical differences were evaluated with Kruskal–Wallis test, and pairwise comparisons were done using a two-sided Wilcoxon test with p-values adjusted using the Benjamini–Hochberg method. Contigs and RPKM-normalized coverage data reported in Ceja-Navarro et al., 2019 were used to generate this figure. Contigs were aligned against the NCBI non-redundant database using the DIAMOND software (Buchfink et al., 2015) and the ‘long reads’ option. The obtained alignment was imported into MEGAN (Huson et al., 2018) and the taxonomy assigned using MEGAN’s LCA algorithm for long reads. Coverage data across the four regions of O. disjunctus’ gut was retrieved for contigs identified as taxonomically derived from Streptomyces sp.
Supplementary file 8
Metarhizium anisopliae strains P016 and P287 phenotypes after 10 days growing on PDA plates incubated at 25°C under constant light.