Myrmica rubra colonies were collected at multiple sites in Acadia National Park Mount Desert Island, Maine (Table 3). Nests were located under natural (logs, rocks, etc.) and human (boards, plant pots, etc.) debris and exposed colonies were collected with an aspirator and transferred into plastic nest boxes (11 cm x 26 cm x 9 cm) or 19 L buckets with their nest soil. Colonies collected in buckets were stored at 4°C until used for experiments, when they would be transferred into nest boxes and held at 21°C. Each nest box contained a small portion of a cardboard egg carton covering a 3 x 3 x 3 cm3 piece of moistened sponge. The sponge was remoistened, and ants were fed ca. 2 g of sugar and tuna diet mix every 2 - 4 days.
Collection of nematodes: Ant colonies were inspected for dead individuals every 2-3 days. Dead worker ants were removed using sterile tweezers and surface sterilized by submerging in a 0.1% zephiran chloride solution for 30 sec , followed by two rinses in sterile dH2O, and drying on clean absorbent paper. After drying, cadavers from each nest were held in individual wells of 48-well microtitre plates, with cadavers from each colony maintained in their own separate plate. Plates were stored at ambient temperature (ca. 21°C) inside plastic bags containing damp paper towels to maintain high humidity . Cadavers were monitored for nematode emergence every other day. As nematodes emerged, they were pipetted from the wells and transferred to 50 mL centrifuge tubes with dH2O held at 10° C.
New M. rubra colonies were collected from field sites for each set of experiments to assure that the bacterial communities associated with the nematodes did not drift with repeated culturing. Nematode infections levels and successful recovery from different sites varied between collections. Nematodes recovered from colonies and locations that resulted in moderate to high ant mortality and high emergence of nematodes were chosen for each set of experiments.
G. mellonella infections and hemolymph collection: Last instar Galleria mellonella (Lepidoptera: Pyraidae) larvae were purchased from Petco (Bangor, ME) or Grubco Inc. (Fairfield, OH), and held at 5-10°C until used for nematode infections and/or bleeding experiments. For infection, larvae were placed in 100 mL cups or 100 mm diameter Petri dishes with 20 g sterilized sand moistened with sterile dH20. Nematodes in dH20 solution were pipetted directly onto the sand and/or the dorsal cuticle of the larvae, which were incubated without food at ambient temperature (approximately 21°C) and monitored daily for signs of infection and death. White traps [63,64] were used to harvest nematodes from dead larvae.
The hemolymph of larvae, infected and non-infected, was sampled for the presence of bacteria by bleeding caterpillars under aseptic conditions in a laminar flow hood. Caterpillars were removed from rearing/ infection cups, rinsed with dH20, and surface sterilized with 0.1% zephrin chloride as described previously  or with 70% ethanol and 1% sodium hypochlorite (NaClO) as described previously . After surface sterilization, a single proleg was cut with sterile, micro-dissecting scissors, and a microcapillary tube was used to draw the hemolymph that welled up from the wound. Slight pressure was applied to the body with soft forceps to exude as much sample as possible. Hemolymph samples were stored briefly at 4°C until bleeding of all specimens were completed, with plating or DNA extractions immediately following this process.
Isolation and identification of associated bacteria from nematodes emerged from M. rubra ant cadavers
Additional detail for experiment 1 can be found elsewhere . The conceptual schematic for this work is visualized in Figure S7.
Preparation of ant colonies
Multiple M. rubra colonies collected from different sites (Table 3) in September 2010, were maintained at 4°C for approximately 2 weeks prior to being transferred to nest boxes for use in bacterial culturing experiments. Colonies were maintained and monitored for dead ants and emergence of nematodes from ant cadavers. Colonies from two sites experiencing high levels of mortality and emergence of many nematodes were selected for use in this study, and nematodes were harvested from their corresponding cadaver plates.
Collection of externally located bacteria from nematodes
Approximately 50 μL of sterile distilled water was added to wells containing M. rubra cadavers and emerged nematodes. Nematodes from each well were pipetted into a 1.5 mL microcentrifuge tube filled with 1 mL of 1% Tween. Tubes were vortexed gently to mix and centrifuged for 10 sec at 13,000 RPM to concentrate the nematodes in the bottom of the tube. This stock solution was serial diluted four times, using a 1:10 ratio of rinse solution to distilled water. Three 300 μL aliquots of each 1×10-4 dilution were plated onto Trypticase Soy Agar (TSA). Plates were incubated at 29°C for 48 hr, after which, colonies were observed at 1-100X under a dissecting microscope and unique morphotypes were identified based on colony size, color, shape, and surface characteristics (Table S1; Figure S1a). Individual colony forming units of all unique morphotypes within a sample were transferred to fresh TSA plates. After two days of growth, monoculture plates were stored at 4°C.
Collection of digestive tract bacteria from nematodes (internal)
A total of 50 μL of sterile distilled water was added to selected wells in the 48-well plates housing the M. rubra cadavers and emerged nematodes. After gently mixing, three 5 μL aliquots were taken from each well and plated onto a contrasting black surface for counting. The number of nematodes in each aliquot was counted and averaged across each of the three aliquots for each site. The remaining 40 μL of each nematode solution was equilibrated to ½ the concentration of the least concentrated solution by adding sterile distilled water to each solution in the appropriate amount.
After standardization of nematode concentration, a 50 μL aliquot of nematode suspension was gently loaded onto a concavely folded piece of vacuum filter paper. The filter paper was loaded into an appropriately sized Buchner Funnel and attached to the laboratory vacuum system. Nematodes were continuously surfaced sterilized for 2-3 minutes by pipetting 1% bleach solution onto them, making sure not to spill the nematodes off of the filter paper. After surface sterilization, the nematodes were rinsed with sterile distilled water in the same manner for 2-3 minutes. The filter paper was then loaded into a small Petri dish and flooded with sterile distilled water to dislodge the nematodes from the filter paper. After nematode presence was confirmed using a dissecting microscope, 300 μL aliquots of the nematode suspension were plated onto TSA agar. To assure inoculation of a sufficient number of nematodes, individual nematodes were pipetted out of the remaining solution using a 200 μL pipette tip and added to the TSA plate with the nematode suspension. It was determined that holding nematodes at room temperature until pipetting was best, as nematodes tended to stick to the surface of the Petri dish if refrigerated for long periods. Nematodes feeding and tunneling on the agar gave rise to colonies of bacteria excreted from the nematode digestive tract (Figure S1b). Plates were incubated at 29°C for 48 hr, after which trails of feeding nematodes and bacterial colonies were observable. Morphotypes were identified and individual colony forming units of unique morphotypes were transferred, grown and stored as above.
Collection of bacteria in infected waxworm hemolymph
A total of 60 last instar G. mellonella larvae were inoculated with nematodes from the well plates of the two selected ant colonies/sites. Nematodes were harvested from six individual ants from each of the two colonies and transferred to individual infection dishes containing five larvae. Inoculated larvae were monitored daily and the dead were collected and surface sterilized in 0.1% zephiran chloride solution. Sterilized cadavers were placed in individually marked Petri dishes for each set of five larvae. All cadavers were stored for 24 - 72 hours at 4°C until caterpillars could be bled for hemolymph en masse. Hemolymph collected from each set of five larvae was pooled for one sample and placed into a 1.5 mL microcentrifuge tube filled with 1mL of sterile dH2O. Four-fold serial dilutions were made and quadrant streaks of each 1×10-4 dilution were plated onto two Trypticase Soy Agar (TSA) plates for each of the 12 samples. Plates were incubated at 29°C for 48 hr, after which morphotypes were identified and individual colony forming units of unique morphotypes were transferred, grown and stored as above.
DNA extraction and sequencing of bacterial isolates
Of the 45 bacterial isolates cultured (Table S1), 32 were selected for further evaluation via 16S rRNA sequencing. Due to the morphological similarities of isolates derived from samples from the same location and culture source (internal, external, hemolymph), samples originally derived from one well from each location were selected as representative samples of the external bacterial associates for sequencing. Fewer isolates were obtained from the internal and hemolymph samples and all were prepared for sequencing (Table S2).
All isolates were grown for 14-18 hr in 3 mL LB Broth, with a salt concentration of 5 g/L (Formedium), in 14 mL test tubes (VWR, USA) at 29°C and 90 rotations per minute (RPM). Broth cultures were centrifuged to pellet cells. Bacterial DNA was extracted using the Promega Wizard® Genomic DNA Purification Kit Cat no. A1120 (Promega, USA). DNA extract was stored at -20°C. Gel electrophoresis was used to assess presence of DNA extract using 0.8% Agarose gels made using 30 mL of TAE Buffer (tris base, acetic acid, and EDTA), 3 μL GelStar® GelStain (Lonza, USA) and 0.24 g Agarose (VSB Company, Cleveland, OH, USA). Samples were loaded using 5 μL of DNA and 1 μL of 6x loading dye (Gilbert). The Lambda HindIII ladder (Promega) was used as the standard and samples were run at 90V for approximately 1 hr. Gels were visualized on an ultraviolet (UV) transilluminator and recorded with a remote shooting camera.
Polymerase chain reaction (PCR) amplification of bacterial 16s rRNA was conducted using the primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-ACGGGCGGTGTGTAC-3’) (Lane 1991). Thermocycler conditions were as follows: 3 min at 95°C followed by 35 cycles of 15 s at 95°C, 30 s at 55°C, 1.5 min at 72°C, and a final step of 6.5 min at 72°C (Rae et al. 2008). Reactions were carried out at a volume of 20 μL. The PCR master mix recipe was derived from the original reaction conditions listed by Rae et al. (2008). After modifications, the final reaction mix included 9.6 μL H2O, 4 μL 5x PCR Buffer, 1.2 μL 25 mM MgCl2, 2 μL 2 mM dNTPs, 1 μL 10 μM 27f, 1 μL 10 μM 1492r, 0.2 μL GoTaq DNA Polymerase (Promega), and 1 μL of bacterial DNA.
PCR amplification was verified using 1.2% Agarose gels (30 μL of TAE Buffer, 3 μL GelStar® GelStain (Lonza, USA), and 0.36 g agarose). Invitrogen Low DNA Mass Ladder Cat. # 10068-013 (Invitrogen, USA) was used as a standard to detect the expected fragment length of 1465 bp; 5 μL of low mass ladder and 1 μL of 6x loading dye. Three microliters of PCR product and 2 μL of 2x loading dye was mixed and loaded for each sample. Gels were visualized on a UV transilluminator and recorded with a remote shooting camera. A fragment at the correct base pair size was taken to be a successful PCR run. PCR products were purified using a QiaQuick PCR Purification Kit (Qiagen, Venlo, Netherlands) following the Qiagen QiaQuick protocol, and stored at -20°C.
Purified PCR product quality was determined on a Nanodrop 1000 with version 3.3 software (ThermoScientific, USA). DNA concentrations were adjusted to 10 ng/μL using sterile water, as needed. Sanger sequencing was performed by the University of Maine Sequencing Facility (Orono, Maine, USA), using primers 27f and 1492r primers and ABI model 3730 DNA Sequencer with the XL Upgrade.
Quality-edited bacterial 16S rRNA gene sequences were obtained from the University of Maine Sequencing Facility and identified using the Basic Local Alignment Search Tool (BLAST) online database provided by the National Center for Biological Information (NCBI, accessed 2011). Proofed sequences can be accessed on NCBI (Bioproject PRJNA646935, GenBank accessions MT797825 - MT797845). A phylogenetic tree was generated by aligning isolates and their closest matches in MEGA ver X  using the MUSCLE algorithm, and calculating branch lengths with Maximum Likelihood algorithm. Tree was visualized in the Interactive Tree of Life .
Transfer potential of pathogenic bacteria from environment to nematodes to other insect hosts
Additional detail for all methods used in experiment 2 can be found elsewhere .
Harvest and storage of Pristionchus entomophagus nematodes
In September 2012, approximately 200 specimens were collected from each of 16 Myrmica rubra colonies located in eight sites in Acadia National Park and Orono, ME (Table 3) where Pristionchus entomophagus infection has previously been confirmed. Colonies were maintained and nematodes were collected from all colonies.
Virulence of P. entomophagus nematode populations
Galleria mellonella larvae were exposed to nematodes collected from ant cadavers from different colonies and monitored for mortality to assay nematode virulence. For each nematode population, larvae were exposed to low (20 - 25) and high (200 - 250) numbers of nematodes and compared to a control group exposed to no nematodes. Per site and dose assay, four replicate plates with 5 larvae per plate were exposed. The larvae were inoculated and maintained in 100 mm diameter Petri dishes, and monitored daily for mortality for 14 days. Survival analysis was conducted using a general parametric model based on the Weibull distribution to examine difference in time to death between sites and nematode treatments (JMP, SAS Institute Inc. 2012). Dead larvae were removed and placed into white traps , and nematodes were harvested once per week over 10 weeks by pipetting from the white traps into 50 mL centrifuge tubes. These nematode stocks were stored at 10° C. To replace old and dying nematode stocks 12 weeks after initial harvesting, nematode populations from the BNR site were exposed to G. mellonella as described above and collected via white traps (Figure S8).
Assessing uptake of RFP-labelled bacteria by P. entomophagus nematodes
To assess the ability of P. entomophagus to ingest and carry bacteria from their environment (Figure S2), nematodes from the BNR were used in the following experiments in which the nematodes were transferred to and held on culture media plates containing various bacteria treatments. For the first experiment, bacteria treatments included: 1) Escherichia coli HB101 (p6TT1) bacteria expressing a red fluorescent protein , and which have not been documented to be pathogenic towards nematodes; 2) bacterial-control plates containing non-labelled Paenibacillus sp. previously isolated from P. entomophagus; and 3) no-bacterial control plates without any additional bacteria added. For the second experiment bacteria treatments included: 1) the bacterial-control, non-labelled Paenibacillus plates; 2) no-bacterial control plates; and 3) plates containing a red fluorescent protein (p66TT1 plasmid d-tomato) labeled Pseudomonas aeruginosa (strain PA14) shown to be highly virulent in Caenorhabditis elegans worms and G. mellonella larvae with an LD50 of fewer than 10 bacteria , but not documented to be virulent in P. entomophagus nematodes.
To create treatment plates, 2.5 mL aliquots of nematode growth media (NGM; Carolina Biological, US) were poured into 45 mm diameter Petri plates under sterile conditions, streaked with bacteria, and incubated at 37° C for 48 - 72 hr to allow for bacterial growth. Four replicates were produced per treatment for a total of 12 plates. Approximately 200 - 400 nematodes in 140 - 150 μL of dH2O were pipetted onto each plate, and cultured at 20°C for 48 hr to allow nematode grazing, after which nematodes were sampled for mortality and the evidence of acquiring the fluorescent protein.
To determine mortality, total dead (indicated by straight, stiff or disintegrating nematodes) and living were counted in the entire plate (E. coli) or a subsample of ca. 50% of the plate sampling variable fields of view at 100X magnification under a dissecting microscope. To determine prevalence of uptake of environmental bacteria, 10 - 12 live juveniles and 10 - 12 live adults per plate with labeled E. coli or P. aeruginosa and unlabeled Paenibacillus sp. treatments were manually transferred to well slides and viewed on a Zeiss SteREO Discovery.V12 microscope with the Texas Red fluorescent filter (excitation 596/emission 615). The presence of external (cuticle) and internal fluorescence (digestive tract) was determined for adults and juveniles by visual observation (Figure S9). Repeated measures analysis of variance was conducted to examine the difference in the proportion survival and proportion with fluorescence between treatments over time in the E. coli trial, and a one way ANOVA was used to examine treatment differences in the P. aeruginosa trial (JMP, SAS Institute Inc. 2012). Proportion survival or fluorescence was transformed by arcsine square root for analysis (JMP, SAS Institute Inc. 2012).
Transference of RFP-labeled bacteria from P. entomophagus nematodes to G. mellonella larvae
To determine if nematodes are capable of transferring newly associated bacteria into their insect hosts, G. mellonella larvae were exposed to P. entomophagus nematodes carrying RFP-labeled bacteria. Three different treatments were established: 1) no nematode controls, 2) nematodes grown on plates with their naturally associated bacteria, and 3) nematodes grown on plates with RFP-labeled bacteria. There were 4 replicates per treatment for a total of 12 plates. Nine larvae were placed into each inoculation dish (100 mm2 diameter Petri dish with moistened autoclaved sand. Thirty-five live juvenile nematodes were transferred from their respective nematode growth agar to dish using a probe. Nematodes were added to a 50 μL sterile dH2O droplet within areas cleared of sand. Sand was gently pushed back into place to introduce nematodes into the moist sand. The petri dishes were stored in a humid chamber at ambient temperature (approximately 21° C) for 11 d, and larvae were monitored daily for mortality. Survival analysis was conducted using a general parametric model based on the Weibull distribution to examine difference in time to death between nematode treatments and control (JMP, SAS Institute Inc. 2012).
To assess bacterial transfer from nematodes, larvae were surface-sterilized with a 1% sodium hypochlorite solution, which had been confirmed as non-fluorescing via direct observation using the Zeiss SteREO Discovery V12 microscope. One to two microlitres of hemolymph were collected from three live larvae per plate 3 days after introduction of the nematodes, and then from each of the remaining larvae as they died over the 11-day duration of the experiment. Hemolymph was added to 20 μL of sterile dH2O on a standard microscope slide, covered with a 25×25 mm coverslip, and the edges were sealed with clear nail polish to prevent desiccation. The samples were viewed at 100X magnification on a Zeiss SteREO Discovery.V12 with the Texas Red fluorescent filter (excitation 543/emission 610) to determine presence/absence of fluorescence.
Microbial community profiling of bacteria associated with ants, nematodes, and infected G. mellonella larvae to determine in situ bacterial transfer between hosts
The design for this experiment is visualized in Figure S10. Three invasive ant colonies were collected from different sites on Mount Desert Island, Maine, in September 2015, held in rearing boxes in the laboratory. Ant colonies were maintained with regular watering and food, and dead ants were removed every 2-3 days and set up for emergence of nematodes as described above. Any adult nematodes that emerged were collected from wells and transferred to Petri dishes with Pasteur pipettes, reared in Baby Food (BF) agar according to procedures described previously , and identified via molecular and morphological assessment [63,69] .
Last instar G. mellonella larvae were inoculated with nematode cultures originating from each of the three ant colonies. Twenty-mL aliquots of nematode solution (7 nematodes/mL dH2O) were applied directly onto the dorsal surface of each of five larvae per inoculation dish with 5 replicate dishes per nematode culture and 5 dishes of untreated larvae. Larvae were monitored daily and two dishes per nematode culture plus two dishes with untreated larvae were selected for hemolymph sampling 3 days post exposure when treated larvae showed signs of septicemia, but had not yet died (bloated, discolored, with only minor movement when prodded). Larvae were surface-sterilized and hemolymph was collected and pooled from the five larvae per dish to yield 75-100 mL samples for DNA extraction.
Whole ants and nematodes were sampled from their original colonies and cultures for bacterial community analysis. Two samples of 5 ants were randomly collected from each nest box, transferred to sterile 1.5 mL snap tubes, and frozen for 15 min at -80°C prior to transfer to extraction tubes. Nematodes were collected by pipetting and manual transfer with a bent probe until two tubes with concentrations of 100 mixed aged nematodes in 500 mL dH2O were collected for each of the three nematode cultures. Neither ants nor nematode samples were surface sterilized.
DNA extraction and sequencing of bacterial communities
DNA was extracted from the whole ants, and whole nematodes samples, as well as from four individual whole G. mellonella larvae using the MoBio Soil Extraction kit (MoBio Laboratories, Inc., US) per the manufacturer’s protocol. DNA was extracted from hemolymph samples using QIAmp DNA Micro Kit for small sample volumes following the manufacturer’s protocols for Isolation of Genomic DNA from Small Volumes of Blood. A total of six nematode samples (2 per field site), six ant samples (2 per field site), and 13 G. mellonella larvae samples were selected for sequencing. Of the larvae samples, two were single whole larvae, two were single surface-sterilized larvae, and the remainder were hemolymph samples collected as described above from both nematode inoculated (infected, n = 6) and control (not inoculated, n = 2). Amplification of the 16S rRNA gene was done using eubacterial primers 27F (5’-AGRGTTTGATCMTGGCTCAG-3’)  and 519R (5′-GTNTTACNGCGGCKGCTG-3’) , and a 30-cycle PCR protocol per Molecular DNA Lab (MR DNA, Stillwater, TX). PCR products were pooled in equal concentrations and purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, US). Resulting library preparations were sequenced using the Illumina platform following the manufacturer’s protocols (MR DNA). Sequence data are publicly-available under BioProject Accession PRJNA646935, from the NCBI Sequence Read archive (SRA).
Sequence data processing
Raw data were denoised, barcodes were removed, and forward and reverse reads merged into contiguous sequences (i.e. ‘contigs’) by MR DNA. Resulting FASTQ files were processed in RStudio using R version 3.6.3 “Holding the Windsock” , following the DADA2 pipeline . Sequence quality was measured and plotted using the ShortRead package  and base R plotting. The first 15 bases and last 50 bases of reads were removed due to low sequence quality (sequence quality scores < 20). Error rates for each sequencing run were calculated, sequence variants identified, and chimeric reads were removed using DADA2. Taxonomy was assigned using the Silva ver. 138  taxonomic training database for DADA2, and any sequences which matched as eukaryotic mitochondria were removed using the dplyr package . Sequencing runs, along with taxonomy and metadata, were merged into one object using phyloseq the package . This phyloseq object was then subset into ant, nematode, and G. mellonella larvae groups, as needed for analytical comparisons. All subset groups were pruned to remove null samples or taxa (phyloseq) and rarefied to 5,000 SVs per sample for data analysis that included larvae only, or 10,000 SVs per sample for data analysis that included ants and nematodes.
Initial exploration of the data showed that two whole larvae samples that were prepared using surface sterilization prior to microbial DNA extraction had bacterial profiles that were distinct from the remainder of the larvae samples. These two samples were removed from the dataset along with a positive control sample that had been spiked with Bacillus bacteria.
Observed SV richness and evenness were calculated for all ant, nematode, and G. mellonella larvae samples in the package phyloseq. A Shapiro-Wilkes test was used to test if diversity data were normally distributed. Differences in observed richness and evenness between sample groups were tested using an ANOVA for normally distributed data, and a Wilcoxon test for those data that were not normally distributed. To determine differential abundance of bacteria SVs across sample groups, random forest classification algorithms were run using the rfpermute package . Principal Coordinate Analysis (PCoA) was performed using Jaccard’s distance to explore similarity of samples based on host species identity and host infection status. PCoA was visualized for all samples, then separately for G. mellonella larvae samples. The taxa belonging to the core microbial community between ant and nematode samples at an abundance of 1% and a prevalence of 70% -90%, noted in the respective results, were identified using the microbiome package .