Sample collection
Samples were collected from an evergreen broadleaf forest in central Taiwan (Lienhuachi Experimental Forest, Nantou County, 23°55'7''N 120°52'58''E) from January 2017 to March 2018; The permission to collect the plants for the study was obtained from the Lianhuachi Research Center, Taiwan Forestry Research Institute, Council of Agriculture, Executive Yuan, Taiwan (Permission no.: 1062272538). The authors confirm that the present study complies with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. Ant cadavers with fungal growth were collected from the canopy of understory plants with a height of < 3 meters. Ant cadavers infected by O. unilateralis s. l. were carefully removed by cutting the leaf and placing it into a 50 mL conical centrifuge tube, which was then transported to the laboratory. Only cadavers in which the fungal growth stage preceded the development of perithecia, which theoretically has the highest biological activity, were collected (Fig. 1). In total, 24 infected P. moesta and 20 infected P. wolfi samples were pooled, respectively, and examined in this study.
Isolation and cultivation of bacteria
Ants on the leaves were first identified to species, and then each ant was carefully placed using tweezers into a sterilized 1.5 mL microcentrifuge tube (see details in Lin et al.15). Samples were vortexed in 600 μL sterilized water for a few seconds at 3,000 revolutions/min (rpm) using a vortex mixer (AL-VTX3000L, CAE technology Co., Ltd.), and were then soaked with 600 μL 70% ethanol to sterilize the ant’s surface. The ethanol on the samples was washed out twice with 600 μL of sterilized water, then finally vortexed in 400 μL of sterilized water. Next, 200 μL of the supernatant was spread homogeneously onto an LB agar plate (25 g Luria-Bertani broth and 15 g agar per liter) to confirm the absence of live bacteria.
Bacteria from the inside of the ant host were released by homogenizing the ant host in 200 μL water and culturing on LB agar plates at 28°C for two days. Approximately 45 (43-46) isolates were randomly picked from each plate with sterile toothpicks, and were suspended in the LB medium supplemented with 15% v/v glycerol and maintained at -80°C until the time of examination. In total, 247 bacterial isolates from P. moesta and 241 bacterial isolates from P. wolfi were collected.
In addition to the bacterial isolates from the ant bodies, 60 bacterial isolates from soil, leaves, and air in the same forest were collected with the same aforementioned procedure, for the purpose of comparing their resistance to naphthoquinones (see below).
Bacterial identification
Bacteria collected from the ant hosts were identified by gene marker sequencing. The bacterial isolates were cultured in LB medium at 28°C overnight to reach the log-phase, and genomic DNA was extracted following the methods described in Vingataramin and Frost20. Conspecies/strain of the bacterial isolates from the same host was determined using the randomly amplified polymorphic DNA (RAPD) method with the primer 5'-GAGGGTGGCGGTTCT-3'. The PCR amplification was performed as follows: initial denaturation at 95°C for 5 min, 40 cycles of amplification including denaturation at 95°C for 1 min, annealing at 42°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. PCR products were run in 2% agarose gel and the bacterial isolates were characterized by the fragment patterns. For each of the ant hosts, bacterial isolates with the same RAPD pattern were considered as the same strain. In total, 106 and 178 strains were found from each of the ant hosts, respectively. One of the bacterial isolates was selected at random to represent the strain and coded with JYC followed by a series of numbers. Taxonomic status of each strain was identified to species by the selected isolates and identifying it using the V3/V4 region of the 16S rDNA gene. PCR amplification with the primer set (8F: 5’-AGAGTTTGATCCTGGCTCAG-3’ and 1541R: 5’-AAGGAGGTGATCCAGCCGCA-3’)21-22 was performed under the following conditions: initial denaturation at 95°C for 5 min, 40 cycles of amplification including denaturation at 95°C for 1 min, annealing at 55°C for 30 s, and extension at 72°C for 1 min 45 s, followed by a final extension at 72°C for 10 min. The PCR products were first checked by running a gel, and were then sequenced at Genomics, Inc. (New Taipei City, Taiwan). Taxa for the sequences were judged by the BLAST method against nucleotide sequences in the NCBI database (https://www.ncbi.nlm.nih.gov/). Genbank accession numbers of the sequences uploaded to the NCBI database were provided in Supplementary file 1.
The 60 bacterial isolates collected from the environment were examined using the RAPD method and a Bacillus-specific primer set (5’-CTTGCTCCTCTGAAGT TAGCGGCG-3’ and 5’-TGTTCTTCCCTAATAACAGAGTTTTACGACCCG-3’), with the PCR conditions suggested in Nakano et al.23. Twenty of the bacterial isolates (10 Bacillus and 10 non-Bacillus) with different RAPD patterns were collected for further experiments.
Bacterial diversity of the two ant host species
Three biodiversity indexes (Chao1 richness, exponential of Shannon entropy, and inverse Simpson concentration) of bacterial species were estimated by the sample size-based rarefaction/extrapolation sampling curve based on the abundance of bacterial isolates from each of the two ant host species24. The calculation was conducted using R25 with the “iNEXT” package26.
Biological properties of bacterial isolates from infected ants
For examining the biological properties of the most predominant species, B. thuringiensis (see results), eleven from 47 and ten from 63 of the B. thuringiensis strains from P. moesta and P. wolfi, respectively, were selected. The strains were selected according to the UPGMA analysis of the sequence. One to three strains grouped in the same cluster were selected (Fig. S1). In addition, six from 15 strains of the second predominant Bacillus species (B. gibsonii) in P. wolfi, as it occupied approximately 20% of the individuals among Bacillus, were also randomly selected for the examinations.
All the selected strains were used to examined the biological properties including potential 1) capability of the isolate to lyse host tissue (hydrolytic enzymes); 2) defense against fungal competition for the ant cadaver, presence of pathogenic and antibiotic genes; and 3) resistance to naphthoquinone derivatives. In addition to the repellence against entomopathogenic fungi, one of the B. thuringiensis strains from each of the hosts was selected at random for examining the potential impact on the nematode invasion.
Hemolysis reaction
Hemolysis reaction tests were conducted on tryptic soy agar (TSA) plates (15 g pancreatic digest of casein, 5 g soybean meal, 5 g NaCl, and 15 g agar, with final pH of 7.3) mixed with 5% defibrinated sheep blood, which was added to the TSA after it had cooled down to approximately 50°C. One 3 µL drop of the log-phase bacterial suspension was placed onto each TSA plate and incubated at 28°C for 1-2 days.
The hemolysis reaction was determined by the formation of clean (β-hemolysis) or greenish (α-hemolysis) hemolytic zones, or no such zone (γ-hemolysis, non-hemolytic) around the bacterial colonies27.
Production of hydrolytic enzymes
The production of hydrolytic enzymes was examined by culturing a 3 µL drop of the exponential-phase bacterial suspension on four different types of plated media: chitinase detection medium (solid medium with 0.3 g MgSO4.7H2O, 3 g (NH4)2SO4, 2 g KH2PO4, 1 g citric acid monohydrate, 0.15 g bromocresol purple, 200 μL Tween 80, 4.5 g colloidal chitin, and 1 L deionized water with 1.5% [w/v] agar and final pH of 4.7); skim milk agar (solid medium with 2% [w/v] agar, 28 g skim milk powder, 5 g casein enzymic hydrolysate (Tryptone), 2.5 g yeast extract, 1 g dextrose, and 1 L deionized water); lipase agar (solid medium with 2% [w/v] agar, 0.1 g phenol red, 1 g CaCl2, 10 mL olive oil, and 1 L deionized water, with final pH of 7.4); and esterase agar (solid medium with 2% [w/v] agar, 0.1 g phenol red, 1 g CaCl2, 10 mL tributyrin, and 1 L deionized water, with final pH of 7.4). The chitinase detection medium was used to examine purple zones, indicating chitinase activity28-29; the skim milk agar medium was used to examine clearance zones for proteases activity30; and the lipase and esterase agar media were used to examine yellow zones, indicating lipase and esterase activity, respectively31.
Pathogenic and antibiotic genes
The total genomic DNA of Bacillus strains was extracted by using an AccuPrep genomic DNA extraction kit (Bioneer) for PCR amplification. The specific screening primers for amplifying the genes, including cry, cyt, Iturin, Chitinase, Bacillomycin, Fengycin, Surfactin, vip, and Zwittermicin A, were used under PCR conditions suggested in previous studies32-35. The primer sets used for the amplifications are listed in Table S2-1.
Lethal effects on Caenorhabditis elegans
Antagonistic effects of B. thuringiensis isolates on the model nematode, Caenorhabditis elegans, were examined by estimating the potential of hemolytic B. thuringiensis to prevent competition by scavengers for the resource-rich insect cadavers36. Daily mortality of C. elegans strain N2 was examined in response to one random selected B. thuringiensis strain (B. thuringiensis JYCB227 from P. moesta and B. thuringiensis JYCB302 from P. wolfi) in comparing with one strain of the second predominated Bacillus species (B. drentensis JYCB252 from P. moesta and B. gibsonii JYCB395 from P. wolfi).
Synchronized L4 nematodes were grown on nematode growth medium (NGM; 3 g NaCl, 2.5 g peptone, 17 g agar, 5 mg cholesterol, 1 mL 1 M CaCl2, 1 mL 1 M MgSO4, 25 mL 1 M KH2PO4, and H2O to 1 liter) agar plates seeded with Escherichia coli OP50. The Bacillus isolates were prepared by inoculating in 3 mL LB liquid broth at 20°C overnight, and then adjusting to an absorbance of optical density (O.D.) 0.2 at a wavelength of 600 nm.
To test the survival rate of C. elegans in the presence of various bacteria, L4 nematodes were co-cultured with 1) a hemolytic bacterial strain, 2) a non-hemolytic bacterial strain, 3) a hemolytic strain + E. coli OP50, 4) a non-hemolytic strain + E. coli OP50, and 5) E. coli OP50 only (control). Twenty μL of bacterial culture was added to a 35 mm NGM agar plate and spread evenly with a glass rod. For each treatment, 30 L4 larvae were cultured on the NGM agar plate and their survival was monitored daily for seven days. Each treatment was replicated three times.
Survival curves were compared using a survival analysis with treatment as the fixed effect. The significance of fixed effect was assessed by model reduction and the likelihood ratio test. Post hoc multiple comparisons were conducted with Tukey’s all-pair comparisons. The model building and hypothesis tests were conducted by using the“survival” and “multcomp” packages in R.
Antagonism to entomopathogenic fungi
We examined the response of three entomopathogenic fungi, including Aspergillus nomius (isolated from the ant Dolichoderus thoracicus), Trichoderma asperellum (isolated from the litchi stink bug, Tessaratoma papillosa), and Purpureocillium lilacinum (isolated from T. papillosa), to co-cultured Bacillus strains. The entomopathogenic fungi were prepared by culturing on potato dextrose agar (PDA) plates for 4 (A. nomius, T. asperellum) or 10 (P. lilacinum) days at 28°C, until the mycelia covered approximately 80% of the plate.
A piece of mycelium (approximately 5 × 5 mm2) was seeded in the center of a TSA plate and surrounded by three equidistant 3 μL drops of exponential-phase bacterial suspension. Plates were incubated at 20°C for 7-10 days. After incubation, areas of the mycelium occupying the plate surface were photographed and measured using Image J. Each pair of bacteria and entomopathogenic fungi, plus the control (a piece of mycelium not surrounded by the bacterial suspension) was replicated 3-4 times.
Antagonism was estimated based on the percentage of mycelial growth inhibition (MGI), which was calculated using the formula ([Rc – Rexp]/Rc) × 100%, where Rc is the mean area of the control fungus and Rexp is the mean area of the examined entomopathogenic fungus co-cultured with each of the Bacillus strains37. The MGI values among all entomopathogenic fungi were compared using a beta regression model with the Bacillus species as the fixed effect. The significance of the Bacillus species effect was tested by comparing the full model with a model that removed the fixed effect term by using a likelihood ratio test. Post hoc tests were conducted using a Tukey-adjusted pairwise comparison. The statistical analysis was conducted using the R packages “betareg,” “emmeans,” “lmtest,” and “multcomp.”
Resistance of bacterial isolates to naphthoquinones
To examine the resistance of bacterial isolates to naphthoquinones, the growth of 11 predominant B. thuringiensis strains isolated from the principal ant host was compared with the growth of 20 environmental bacterial isolates (10 Bacillus and 10 non-Bacillus) using two naphthoquinones, respectively. The two naphthoquinones prepared for the experiment, plumbagin38 and lapachol39, were dissolved in 30% dimethyl sulfoxide (DMSO) water solution38. Naphthoquinone concentrations were determined from the serial dilutions in which three randomly selected bacteria from the ant host and three from the environment had the most distinctive growth rate. Based on these results, concentrations of 45 µg/mL (plumbagin) and 64.5 µg/mL (lapachol) were used (Fig. S2).
In this experiment, the bacterial isolates were first inoculated in LB medium at 20°C overnight and were then refreshed to the exponential phase with LB medium for 3 hours. The bacterial concentration was adjusted to ~1.5 × 108 cells/mL. Next, 10 μL of the bacterial suspension and 180 μL Mueller Hinton broth medium (Sigma-Aldrich) were added to either 10 μL naphthoquinone solution or 10 μL 30% DMSO water solution for the control. The growth of bacterial isolates at 20°C was monitored by measuring the O.D. value at 600 nm with a Multiskan GO microplate spectrophotometer (Thermo Scientific) every hour for 12 hrs. Each combination of bacterial isolate and naphthoquinone or control was replicated twice. Four bacterial isolates (one B. thuringiensis from the ant host, plus two Bacillus and one non-Bacillus from the environment) were omitted from the analysis due to low growth rate in the media with DMSO (O.D. value lower than 0.05 at the end of 12 hr).
The resistance index of each bacterial isolate was calculated by the normalized difference of the O.D. value in the naphthoquinone-treated medium versus the control medium ([naphthoquinone – DMSO]/[naphthoquinone + DMSO]). Values closer to 1 represent higher resistance to the presence of naphthoquinone. Resistance index was compared among the bacterial isolates from different resources (B. thuringiensis from the ant host, Bacillus from the environment, and non-Bacillus from the environment) using a linear mixed model with resource as the fixed effect, bacterial isolate as a random effect, and growth time (5-12 hrs) as a nest effect. The significance of resource as a fixed effect was assessed by model reduction and the likelihood ratio test. Post hoc multiple comparisons were made using Tukey’s all-pair comparisons. The model building and hypothesis tests were conducted using the “lme4” and “multcomp” packages in R.