Sample collection
Samples were collected from an evergreen broadleaf forest in central Taiwan (Lianhuachi Experimental Forest, Nantou County, 23°55′7″N 120°52′58″E) from January 2017 to March 2018. Permission to collect 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 present study complies with the International Union for Conservation of Nature 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 understory plants with a height of less than 3 m. Ant cadavers infected with O. unilateralis s. l. were removed carefully 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 collected.
Ophiocordyceps unilateralis sensu lato-infected (a) Polyrhachis moesta and (b) P. wolfi, with the stroma growing from the ant cadaver. The specimens were collected from the Lianhuachi Research Center, Taiwan and photographed in the laboratory by Wei-Jiun Lin.
Isolation and cultivation of bacteria
Ants on the leaves were first identified to species and then, using tweezers, each ant was placed carefully into a sterilized 1.5-mL microcentrifuge tube [see details in Lin et al. (2020)15. Samples were shaken one by one in 600 μL of sterilized water for a few seconds at 3000 revolutions/min (rpm) using a vortex mixer (AL-VTX3000L, CAE technology Co., Ltd., Québec, Canada), and were then soaked with 600 μL of 70% ethanol to sterilize the ant’s surface. The ethanol on the samples was washed twice with 600 μL of sterilized water, then vortexed in 400 μL of sterilized water. Next, 200 μL of the supernatant was spread homogeneously onto a Luria–Bertani (LB) agar plate (25 g Luria–Bertani broth and 15 g agar per liter) to confirm the absence of live bacteria.
Bacteria from inside the ant host were released by homogenizing the ant host in 200 μL of water and culturing on LB agar plates at 28 °C for 2 days. Bacteria from each of the ant individuals were cultured independently and approximately equal numbers of the isolates were picked randomly 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 for the purpose of comparing their resistance to naphthoquinones (see below) by using the aforementioned procedure but without initial cleaning and sterilizing of the sample surface.
Bacterial identification
Bacteria collected from the ant hosts were identified by gene marker sequencing. 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 Frost (2015)20. Conspecies/strains of the bacterial isolates from the same host were determined using the randomly amplified polymorphic DNA (RAPD) method with the primer 5′-GAGGGTGGCGGTTCT-3′. 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 bacterial isolates were characterized by fragment patterns. For each of the ant hosts, bacterial isolates with the same RAPD pattern were considered to be the same strain. In total, 106 and 178 strains were found from P. moesta and P. wolfi, respectively. One of the bacterial isolates was selected at random to represent the strain and coded with “JYCB” followed by a series of numbers (e.g., JYCB191). Taxonomic status of each strain was determined to species by 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. PCR products were first checked by running a gel, and were then sequenced at Genomics, Inc. (New Taipei City, Taiwan).
The sequences of the bacterial strains from each of the ant hosts were first analyzed by the unweighted pair group method with arithmetic mean (UPGMA) analysis and clustered into clades according to the sequence dissimilarity (< 0.01) by using MEGA X23. Species of each of the clades were judged by the basic local alignment search tool (BLAST) method against nucleotide sequences in the National Center for Biotechnology Information (NCBI) nucleotide database (https://ftp.ncbi.nlm.nih.gov/blast/db/), updated through 2021 May 17th. Because each of the clades contains one to several bacterial strains, each of the strains was first labeled by the species of the sequence with the highest BLAST identity, which was ranked by expected value, percentage of identical matches, and alignment length (https://www.ncbi.nlm.nih.gov/BLAST/tutorial/Altschul-1.html). If multiple sequences from the database were found to be same in the indexes of the identity, the bacterial strain was labeled by the species that appeared most frequently. Finally, the species for each of the clades were judged by the bacterial species found most frequently in the strains belonging to the clade.
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 PCR conditions suggested in Nakano et al. (2004)24. 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 using the abundance of bacterial isolates from each of the two ant host species25. The calculation was conducted using R26 with the “iNEXT” package27.
Biological properties of bacterial isolates from infected ants
Selected strains
For examining the biological properties of the most predominant species, B. cereus/thuringiensis (see results), 11 of 47 B. cereus/thuringiensis strains from P. moesta and 10 of 63 B. cereus/thuringiensis strains from P. wolfi 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, 6 of 15 strains of the second-most predominant Bacillus species (B. gibsonii) in P. wolfi were also selected randomly for examination, because B. gibsonii occupied approximately 20% of the individuals within the Bacillus isolates.
All the selected strains were used to examine biological properties including potential (1) capability of the isolate to lyse host tissue (hydrolytic enzymes); (2) defense against fungal competition for the ant cadaver, involving the presence of pathogenic and antibiotic genes; and (3) resistance to naphthoquinone derivatives. In addition to the repellence against entomopathogenic fungi, one strain of the B. cereus/thuringiensis and one strain from the secondarily predominant Bacillus clade from each of the hosts were selected at random for examining the potential impact on the invasion and consumption of ant cadavers by scavenger nematodes.
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 colonies28.
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, with 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 activity29,30; the skim milk agar medium was used to examine clearance zones for proteases activity31; and the lipase and esterase agar media were used to examine yellow zones, indicating lipase and esterase activity, respectively32.
Pathogenic and antibiotic genes
The total genomic DNA of Bacillus strains was extracted by using an AccuPrep genomic DNA extraction kit (Bioneer, Daejeon, Korea) 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 studies33,34,35,36. The primer sets used for the amplifications are listed in Table S2.
Lethal effects on Caenorhabditis elegans
Antagonistic effects of B. cereus/thuringiensis isolates on the model nematode, C. elegans, were examined by estimating the potential of hemolytic B. cereus/thuringiensis to prevent competition by scavengers for the resource-rich insect cadavers37. Daily mortality of C. elegans strain N2 was compared between randomly selected B. cereus/thuringiensis strains (B. cereus/thuringiensis JYCB227 in clade m4 from P. moesta and B. cereus/thuringiensis JYCB302 in clade w4 from P. wolfi) and Bacillus species of secondary predominance (Bacillus sp. JYCB252 in clade m8 from P. moesta and B. gibsonii JYCB395 in clade w30 from P. wolfi).
Synchronized fourth-stage larval (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 L) agar plates seeded with Escherichia coli OP50. The Bacillus isolates were prepared by inoculating in 3 mL LB liquid broth at 20 °C (the mean annual temperature in Lianhuachi Research Center, where the infected ants were collected) 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). We added 20 μL of bacterial culture 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 7 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 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 strains38. The MGI value from each of the bacteria co-cultured with each of the fungi was first tested by using Student’s t test and Holm–Bonferroni method to adjust P values. 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. cereus/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. Because fungal naphthoquinones are currently not purified and commercialized, the two naphthoquinones prepared for the experiment, plumbagin39 and lapachol40, were those found in plants. They were dissolved in a 30% dimethyl sulfoxide (DMSO) water solution39. 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.
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 h. The bacterial concentration was adjusted to ~ 1.5 × 108 cells/mL. Next, 10 μL of the bacterial suspension and 180 μL of the Mueller Hinton broth medium (Sigma-Aldrich, St. Louis, USA) were added to either 10 μL of the naphthoquinone solution or 10 μL of the 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, Waltham, USA) every hour for 12 h. Four bacterial isolates (one B. cereus/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 h). Thus, ten predominant Bacillus from the ant, eight Bacillus from the environment, and nine non-Bacillus bacteria from the environment were used to represent the naphthoquinone tolerances of each group. Each combination of bacterial isolate and naphthoquinone or control was replicated twice.
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. cereus/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 h) 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.
Source: Ecology - nature.com
