Microbes were collected from a live cow calf (with consent and in the presence of the animal’s owner), and from the hide of a recently slaughtered adult cow. Samples were obtained from a front leg, back leg, and the back of both the live calf and the cow hide, because these areas are most frequented by stable flies. For microbe collections, cotton swabs (Puritan, Guilford, ME, USA) were dipped in sterile distilled water and then were firmly rubbed for 30 s against an approximately 6.5-cm² patch of cow skin/hide before being streaked for 30 s over the entire surface of Mueller Hinton, Yeast Extract, or Potato Dextrose agars in separate Petri dishes (d = 8.5 cm). These different agar types were meant to enable growth of as many bacterial species as possible. One sample was obtained from each body region per agar type, yielding nine samples each from the live calf and the cow hide. Morphologically distinct microbes growing on agar were isolated by continuous re-streaking. All microbial work was done in a biosafety cabinet (BSC; NUAIRE Biological Safety Cabinets, Class II type A2) using aseptic techniques. Microbe stock-samples were kept at −80°C in a solution of glycerol, distilled water, and liquid microbe culture (1:1:2).

Isolated microbes were identified using either Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) or genetic sequencing.

MALDI-TOF MS (Bruker Corp., Billerica, MA, USA) (Jimenez et al., 2017) was conducted using an extended Direct Transfer method. For each bacterial strain, two preparations were processed. Briefly, after growing unknown bacteria on agar overnight, single colonies were transferred to a well on a MALDI plate via a sterile toothpick, producing a heavy smear on the well. The same toothpick was then used to produce a lighter smear on the next well. Subsequently, 1 µL of 70% formic acid was applied to all microbe-treated wells and allowed to evaporate. Finally, each well received 1 µL of 2-cyano-3-(4-hydroxyphenyl) acrylic acid (HCCA matrix). After wells had dried, MALDI Biotyper measurements were taken. We used a Bruker bacterial test standard (Bruker Corp.) for calibration in accordance with manufacturer instructions. We analyzed all spectra using Biotyper software (Bruker Corp.). This Biotyper software calculates an arbitrary score for each sample between 0 and 3 by comparing sample mass spectra to reference mass spectra; we accepted species assignments at scores of >2.0 in accordance with the manufacturer’s recommended protocol.

In preparation for genetic sequencing of distinct bacterial colonies, Kodaq PCR Master Mix (Applied Biological Materials, Richmond, BC, CA) was used to amplify the V3-V4 loop of the 16S rRNA gene with the Universal Forward Primer (UniF) – 5’-CCTACGGGRBGCASCAG-3’ and the Universal Reverse Primer (UniR) – 5’-GGACTACNNGGGTATCTAAT-3’ (Takai and Horikoshi, 2000) by Polymerase Chain Reactions (PCR). Briefly, for a single colony of any unknown bacterium, a PCR mix (25 µL) was prepared using (i) 12.5 µL of Kodaq PCR Master Mix, (ii) 1 µL of UniF, (iii) 1 µL of UniR, and (iv) 10.5 µL of molecular grade water. The mix was then deposited in a PCR strip tube and a single colony of the unknown bacterium was added. A PTC-200 Peltier Thermal Cycler (MJ Research, Saint-Bruno-de-Montarville, QC, CA) was deployed to run PCR cycles on all samples. The program was set to 94°C for 2 min, followed by thirty 30-s cycles each at 94°C, 55°C, and 72°C, and a final extension step at 72°C for 5 min.

The presence of the expected band size at 466 base pairs (bps) and the success of the PCR amplification was checked on a 0.7% agarose gel with a 1 kb Plus Opti DNA Marker (ladder) (Applied Biological Materials). PCR amplicons were pooled and concentrated using the QIAquick Gel Extraction Kit and the QIA PCR & Gel Cleanup Kit (Qiagen, Venlo, NL). Amplicons were sequenced (Genewiz, South Plainfield, NJ, USA) and the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) was used to compare the sequenced region of individual isolates with known sequences. A species or genus was determined to be a match, if there was at least 95% coverage and 99% identity between a known sequence and the sequenced isolate.

Bioassays were run in still-air olfactometers, each with a central and two lateral chambers ( Figure 1A ), where treatment and control stimuli were placed. For each experimental replicate, 20 female flies that had been blood- and water-deprived for 24 h were released into the central chamber from which they could enter, but not exit, lateral chambers through mesh funnels. Experimental replicates were terminated and scored after 24 h by placing olfactometers in a freezer (−15°C) and counting the cold-euthanized flies in each chamber.

Of the 38 microbes collected from cattle skin and identified to genus and/or species (see Results; Table 1 ), those previously reported on vertebrate skin were tested for their attractiveness to stable flies. These microbes included four Staphylococcus congeners [S. sciuri, S. succinus, S. simulans, S. chromogenes (Group 1)] and three heterogeners [Glutamicibacter protophormiae, Corynebacterium stationis, Wautersiella sp. (Group 2)]. For Experiment 1 (n = 12) ( Table 2 ), each of the four Group-1 Staphylococcus species was grown overnight on agar in a petri dish (d = 8.5 cm), and then one quarter slice of the agar from each species was placed in a new sterile petri dish serving as the treatment stimulus, whereas four slices of sterile agar served as the control stimulus. Agar was sliced, and slices were transferred, using sterile (autoclaved) popsicle sticks. Test stimuli involving Risk Group 2 microbes (Government of Canada) were prepared either near a flame or in a biosafety cabinet. For Experiment 2 (n = 12), the same procedure was applied with the three Group-2 microbes except that only three quarter slices were used for the treatment stimulus and three corresponding sterile slices for the control stimulus.

Drawing on results that treatment stimuli in both experiments 1 and 2 attracted significantly more flies than corresponding control stimuli (see Results; 3.2.1), but that Group-1 microbes seemed more attractive (mean treatment to control response ratios: Group 1: 9.5 to 1; Group 2: 4.4 to 1), follow-up experiments were designed to determine the key microbe(s) in Group 1 that mediated attraction of flies. To this end, parallel experiments 3–7 (n = 10 each) tested all four Group-1 microbes (Exp. 3; positive control), and Group-1 microbes without S. chromogenes (Exp. 4), S. sciuri (Exp. 5), S. simulans (Exp. 6), or S. succinus (Exp. 7), all versus sterile agar controls. With evidence that the deletion of any one Staphylococcus species from Group 1 did not reduce the Group’s attractiveness (see Results; 3.2.1), experiments 8–12 (n = 10 each) then tested the four Group-1 Staphylococcus microbes in combination (Exp. 8; positive control) and singly (Exps. 9–12), again all versus sterile agar controls.

To determine whether Group-1 Staphylococcus microbes attract stable flies not only in small scale olfactometers but also over a long range, experiment 13 (n = 20) was set up in a laboratory room (230 × 230 × 225 cm high), with the treatment stimulus (one quarter agar slice of each of the four Staphylococcus microbes) and the control stimulus (sterile agar) prepared as described for experiment 1 (see 2.3.2). The room was kept at a temperature of approximately 26°C and lit by a combination of plant illumination lights (Standard Products Inc., Saint-Laurent, QC, CA) and day lights (Philips, Amsterdam, NL), set to maintain a photoperiod of 15 h L: 9 h D. The treatment and control plates were then placed on the room floor and surrounded by vertical black cardstock cylinders (9.5 × 28 cm high) ( Figure 1B ), which were coated on the inside with adhesive Tanglefoot^® (Tanglefoot, Marysville, USA). For each experimental replicate, 20 flies were released into the room, and fly captures in the cardboard cylinder traps were recorded 24 h later.

To test whether a Group-1 microbe affects the responses of flies not only in a room setting (see Results; 3.2.2) but also on an even larger scale, experiment 14 (n = 10) was set up in a greenhouse compartment (600 × 600 × 360 cm high) on the Burnaby campus of Simon Fraser University. Staphylococcus sciuri was selected for greenhouse bioassays, and subsequent field bioassays (see below), because it was as attractive as either one, and all four, of the Group-1 microbes (see Results; 3.2.1), and because it was the safest microbe (Risk Group 1 microbe; Government of Canada) for deployment in large-scale settings, being deemed unlikely to cause human or animal diseases.

In the greenhouse bioassay, the paired test stimuli consisted of barrels (38 × 64 cm high) covered in black cloth and placed on metal platforms 71 cm above ground, and 200 cm apart from each other ( Figure 1C ). In each replicate, three agar plates were secured with double-sided tape to each platform, with two plates at the front and one plate at the back of the barrels’ curved surface. Treatment and control plates were covered in S. sciuri (grown overnight) and kept sterile, respectively. To record alighting by flies on barrels and agar plates, video cameras (Akaso, Frederick, MD, USA) were mounted on stands 83 cm above ground and 100 cm away from both the front and the back of each barrel. To initiate a bioassay replicate, the cameras were turned on, and 100 blood- and water-deprived female flies were released into the compartment, 300 cm away from treatment and control stimuli. Ten minutes later, video recordings were stopped and flies were sweep-netted and released outside. Videos were subsequently examined to determine the number of times flies landed on each of the two barrels over the bioassay period.

With evidence that S. sciuri affected alighting responses by flies in the greenhouse compartment (see Results; 3.2.3), experiments 15–18 (n = 10 each) then tested the effect of S. sciuri on fly attraction at a livestock farm (Eagle Acres Dairy; Langley, BC, CA). A near-identical experimental design ( Figure 1D ) as in the greenhouse experiment was used, except that the platform of each paired barrel was fitted with 12 plates (Exp. 15), 4 plates (Exp. 16), 2 plates (Exp. 17), and 1 plate (Exp. 18). Barrels were placed approximately 300 cm away from the barn in all experiments. Experimental replicates were terminated after 5 min, and positions of treatment and control barrels were alternated between replicates.

Having shown that ammonia serves as an oviposition resource cue to stable flies (Nayani et al., unpubl.), here we tested whether ammonia also functions as a host-foraging cue for flies. To this end, we tested whether Staphylococcus microbes collected from cattle skin emit ammonia, and whether ammonia attracts host-foraging flies in bioassays. To test for ammonia emission, we grew microbes on agar overnight and measured ammonia emission, using a MultiRAE Wireless Portable Six-Gas Monitor (Honeywell, Charlotte, NC, USA). Three plates of each microbe were grown for measurement of gas emissions. Measurements were taken by placing the probe of the gas meter 0.5–1.0 cm above an agar plate, waiting 1 min for readings to stabilize, and then recording the ppm of ammonia. Between replicates, the gas meter was kept in regular laboratory air for 1 min, thus allowing readings to return to baseline ammonia levels in the atmosphere.

With evidence that microbes attractive to flies emit ammonia (see Results; Table 3 ), parallel still-air olfactometer experiments 19–21 (n = 20 each) then tested whether ammonia on its own is attractive to host-foraging flies. For both the treatment and the control stimulus in these experiments, sodium chloride solutions (50 g NaCl in 50 mL water) were prepared, with treatment solutions also containing ammonium bicarbonate (NH₄HCO₃) at 0.1 g (Exp. 19), 1 g (Exp. 20), or 10 g (Exp. 21). Aliquots (3 mL) of treatment solutions were pipetted into inverted bottle caps (2.5 × 1 cm; total volume: 4.9 mL), and ammonia ppm for each NH₄HCO₃ dose (0.1 g, 1.0 g or 10 g in 50 mL water) was measured 0.5 cm above the liquid surface. The mean ammonia ppm measured at 0.1 g, 1.0 g, and 10 g in seven replicates each was 0.42 ppm, 5.6 ppm, and >25 ppm (sensor overload), respectively, well within the ppm range of Staphylococcus microbes growing on agar ( Table 3 ). Carbon dioxide ppm was not measured.

Aliquots (2.5 mL) of treatment and control solutions were transferred to inverted bottle caps (see above), placed on jars (5.5 × 7 cm; total volume = 166 mL) filled two-thirds with water, and covered with a piece of wet black cloth ( Figure 1E ) (Nayani et al., unpubl.; Friesen et al., 2018). After treatment and control jars were randomly assigned to the lateral chambers of olfactometers, experimental replicates were initiated by releasing 20 blood- and water-deprived female flies into the central chamber of olfactometers, allowing them to enter, but not to exit, lateral chambers through mesh funnels. Replicates were terminated 24 h later by counting the number of flies in lateral treatment and control chambers.

Headspace volatiles were collected from the four strains of Staphylococcus bacteria that elicited significant behavioral responses from flies in olfactometer experiments. To this end, 10 agar plates were plated with a microbe of interest and incubated overnight. These plates, with open lids, were then placed into a glass chamber (diameter = 19 cm, height = 29.5 cm) connected to a vacuum pump (Neptune Dyna-pump). Charcoal-filtered air was drawn at a flow rate of 1 L · min⁻¹ for 24 h through the chamber and subsequently through a glass column (6 mm outer diameter × 150 mm) containing 200 mg of manufacturer-preconditioned Porapak-Q™ adsorbent (50–80 mesh; Waters Associates, Milford, MA, USA). Volatiles were desorbed from Porapak-Q with one rinse of pentane and ether (1:1; 2 mL), and volatile extracts were concentrated to 0.5 mL and kept at 4°C prior to analyses. All glassware was cleaned with Sparkleen (Thermo Fisher Scientific, MA, U.S.A), rinsed with distilled water, and oven-dried at 130°C prior to starting a new aeration.

Aliquots of Porapak-Q headspace volatile extracts were analyzed by gas chromatography-mass spectroscopy (GC-MS), using an Agilent 5977 Series 96MDS coupled to an Agilent 7890B GC (Agilent Technologies Inc., Santa Clara, CA, USA). The instrument was operated in full-scan electron ionization mode and fitted with a DB-5 GC-MS column (30 m × 0.25 mm ID, film thickness 0.25 µm; Agilent Technologies). The injector port, MS source, and MS quadrupole were set to 250, 230, and 150°C, respectively. Helium was used as a carrier gas (35 cm s⁻¹; 5:1 split ratio), with the following temperature program: 40°C (held 5 min), 10°C · min⁻¹ to 280°C (held 10 min). Compounds were identified by comparing their mass spectra and retention indices [relative to aliphatic alkanes (Van Den Dool and Kratz, 1963)] with those of authentic standards that were purchased or synthesized in our laboratory ( Table 3 ). Each compound was quantified by comparing its area count with that of an external standard run at 1, 10 and 100 ng/µL.

With evidence that microbe-derived ammonia attracts flies (see Results; 3.3.2), and that microbes also emit complex volatile blends (see Results, Table 3 ), experiments 22–25 were designed to test whether blends of synthetic microbial volatiles enhance the attractiveness of ammonia. Blend 1 consisted of all constituents common in the headspace of the four bioactive Staphylococcus species ( Table 3 , Table S1 ), whereas blend 2 consisted of all volatiles emitted by S. sciuri ( Table 3 , Table S1 ). Blends 1 and 2 were formulated separately in mineral oil to achieve sustained release of volatiles during the 24-h experimental period. Sustained release was tracked through capture and analyses of headspace volatiles from mineral oil formulations (as described in 2.4.4). Formulations were adjusted until their headspace blends matched those produced by bacteria. Both treatment and control stimuli consisted of ammonium bicarbonate solutions prepared and presented as in experiment 19 described above (see 2.4.2), whereas treatment stimuli also presented blend 1 or 2 in mineral oil ( Table 3 , Table S1 ), with plain mineral oil being the corresponding control stimulus.

To address potential effects of 3-dimensional scale on responses of flies (Nayani et al., unpubl.), experiments 22–24 (n = 12 each) were run in still-air olfactometers ( Figure 1E ), and experiment 25 (n = 10) was run in bioassay rooms (230 cm × 230 cm × 225 cm high) ( Figure 1F ), using experimental designs and protocols described in sections 2.3.1 and 2.3.3. As the bioactivity of synthetic volatile blends can be dose-dependent (Nyasembe et al., 2012; Wondwosen et al., 2021), we tested the synthetic “Staphylococcus blend” ( Table S1 ) formulated in mineral oil at three doses in parallel experiments 22–24: the dose described in Table S1 (Exp. 22), diluted 10× (Exp. 23), and diluted 100× (Exp. 24). By the time we tested the synthetic “S. sciuri blend” ( Table S1 ) in experiment 25, we did not know the results of experiments 22–24 (see Results 3.3.4), which would have prompted us to test the “S. sciuri blend” at a lower dose. However, re-running the S. sciuri blend at a lower dose was not possible due to logistic constraints.

Proportionally, more stable flies were attracted to Staphylococcus Group-1 bacteria and to heterogeneric Group-2 bacteria than to corresponding sterile agar controls ( Figure 2 , Exps. 1, 2; p < 0.05 each). As Group-1 microbes seemed more attractive to flies than Group-2 microbes (mean treatment to control response ratio of flies: Group 1: 9.5 to 1; Group 2: 4.4 to 1), all follow-up experiments were designed to determine the key microbe(s) in Group 1 that mediated fly attraction.

In parallel experiments 3–7 ( Figure 3 ), the four Group-1 microbes in combination (positive control) attracted, proportionally, more flies than sterile agar controls (Exp. 3; p < 0.05), as did Group-1 microbes without S. chromogenes (Exp. 4; p < 0.05), S. sciuri (Exp. 5; p < 0.05), S. simulans (Exp. 6; p < 0.05), or S. succinus (Exp. 7; p < 0.05). In parallel experiments 8–12 ( Figure 4 ), the four Group-1 microbes in combination attracted, proportionally, more flies than sterile agar controls (Exp. 8; p < 0.05), as did each of the four microbes singly (Exps. 9–12; p < 0.05 each).

All data combined indicate that single Staphylococcus species attract stable flies as effectively as all four Staphylococcus species in combination.

In a large laboratory bioassay room, adhesive-coated cardboard cylinder traps baited with the four Group-1 Staphylococcus microbes on agar captured, proportionally, more flies than cylinder traps baited with sterile agar controls ( Figure 5 , Exp. 13; p < 0.05), indicating that microbes affected the responses of flies also in a large bioassay room setting.

In a large greenhouse compartment, black barrels baited with S. sciuri on three agar plates prompted, proportionally, more alighting responses by flies than the black barrels baited with three sterile agar control plates ( Figure 6 , Exp. 14; p < 0.05), indicating that S. sciuri as a single microbe species modulated the responses of flies in a large-scale setting.

On a cattle farm, black barrels baited with S. sciuri on twelve agar plates (Exp. 15), four plates (Exp. 16), two plates (Exp. 17), and on one plate (Exp. 18), all did not prompt proportionally more alighting responses by flies than black barrels baited with the corresponding number of sterile agar control plates ( Figure 7 ; p > 0.05 for all experiments).

Each of the four Group-1 Staphylococcus species emitted ammonia, as follows: S. chromogenes: 8.0 ± 1.6 ppm; S. sciuri: 14.3 ± 2.6 ppm; S. simulans: 13.0 ± 0.0 ppm; S. succinus: 9.3 ± 0.9 ppm ( Table 3 ).

In still-air olfactometers ( Figure 1E ), all test stimuli containing ammonium bicarbonate, and thus emitting ammonia, attracted proportionately more flies than control stimuli lacking ammonium bicarbonate ( Figure 8 ; Exps. 19–21; p < 0.05 each). The amount of ammonium bicarbonate in test stimuli did not affect the proportional response level of flies (p > 0.05), suggesting that the emission of ammonia, rather than its concentration, affected behavioral responses of flies, at least in this experimental context.

Each of the four Group-1 Staphylococcus species emitted a complex volatile blend ( Table 3 ). Volatiles shared by all four species included two alcohols (isoamyl alcohol, phenylethyl alcohol), four pyrazines (2,5-dimethyl pyrazine, trimethyl pyrazine, 2-isopropyl-5-methyl-pyrazine, 2-ethyl-3,5(6)-dimethylpyrazine [composed of 50% 2-ethyl-3,5-dimethylpyrazine and 50% 2-ethyl-3,6-dimethylpyrazine]), and isoamyl acetate ( Table 3 ).

The “synthetic Staphylococcus blend” ( Table S1 ) – at a 100× dilution – enhanced attraction of flies to ammonia ( Figure 9 ; Exp. 24, p < 0.05), but the blend was not effective at a 10× dilution or without dilution (Exps. 22–23, p > 0.05 each), with either of these higher-dose blends being less attractive than the 100× diluted synthetic Staphylococcus blend ( Figure 9 ).

The “synthetic S. sciuri blend” ( Table S1 ) did not enhance attraction of flies to ammonia (mean ± SE number of flies responding to treatment and control stimuli: 1.6 ± 0.56 vs 1.0 ± 0.49; Exp. 25, p > 0.05), but in retrospect should also have been tested at lower doses (see above).