Rearing EPNs
S. carpocapsae (All strain) and S. feltiae (SN strain) IJs (ARBICO Organics, Tucson, AZ), S. scapterisci, S. riobrave (355), S. glaseri (11a&7b strain), S. diaprepesi, H. bacteriophora (HP88 strain), H. indica (HOM1 strain) and H. floridensis (K22 strain) have been kept in culture using Galleria mellonella in the laboratory. To maintain cultures, commercially obtained G. mellonella larvae (Wax worms, Grubco, Hamilton, OH or Vanderhorst Wholesale Inc. St. Marys, OH) were exposed to 100 IJs per larva. Infected G. mellonella larvae were incubated for 4 days at RT (20 + 1 °C) and insect cadavers were transferred to White traps for IJ collection1,52,53.
Removing pheromones and storing IJs in the absence of dispersal pheromones from consumed host cadavers
To detect a pheromone response, nematodes need to be sensitized to pheromones by removing them22,23,30,48. Pheromones and other metabolites were removed from IJs according to the method established by Kaplan et al.22,37,48,54. Briefly, S. carpocapsae or S. feltiae IJs were removed from the White trap after 4 days from the beginning of emergence and were rinsed 3 times in deionized water or ELGA Purelab Ultra (High Wycombe, UK) to remove residual cadaver-derived pheromones. Rinsed IJs were stored at 20 ± 1 °C until used in all experiments with a density of 24,000 IJs/ml unless otherwise stated. The two exceptions are followings; IJs in in Fig. 6 were stored at 14± 1 °C for 4 days and IJs in Fig. 5 were stored at 23± 1 °C for over 60 days.
Mechanical disturbance, like shaking, increases S. feltiae dispersal temporarily for 24 hours. On the other hand, S. carpocapsae IJ quiescent period were not affected by mechanical disturbances. To eliminate the effect of mechanical disturbance on dispersal, the IJs were placed in a 6 cm petri dish in 5 ml of water to provide a shallow water for aeration without shaking or mechanically disturbing the dish.
Quiescent period quantification
The IJs (~200) in 10 µL of water were placed in the center of an agar plate and excess water was absorbed by the media. The quiescent period was considered to begin when the water was absorbed by the agar medium and IJs became motionless. Quiescence ended when the first IJs resumed motion and started moving away from the 1.3 cm diameter IJ placement site (Fig. 3A).
Dispersal assays and quantification
Dispersal assays were conducted as described by Kaplan et al.22 with two modifications to the assay run time. Briefly, residual dispersal pheromones were removed from the IJs by rinsing them in deionized water 3 times, and the IJs were then stored in deionized water at 20 °C in all experiments unless stated otherwise. On day 0 (the day pheromone was removed) and at 2 day intervals thereafter, ~200–300 IJs of either species in 10 µL of water were placed in the center of an agar plate (Fig. 3A) with 6 cm diameter Petri dishes, waited until the water was absorbed by the media and IJs were free to move from the deployment site. The assays were conducted at the same time of the day in the mornings. The experiment was stopped by collecting IJs after 30 minutes; IJs remaining inside the 1.3 cm ring were considered non-dispersed (Fig. 3A), and those that left the placement ring were considered to have dispersed. If there was a quiescent period, IJs were allowed to disperse for 30 min after the quiescent period ended. Agar was at 0.9% with a gel strength >/= 900 g/cm2 (Caisson Agar, Type I, Smithfield, UT).
Preparation of pheromone extract
Pheromones were extracted from S. carpocapsae, S. feltiae, S. scapterisci, S. riobrave, S. glaseri, S. diaprepesi, H. bacteriophora, H. indica and H. floridensis infected and consumed G. mellonella grubs in 70% methanol as by Kaplan et al.22. Infected cadavers were harvested within 10 days of IJ emergence. Then the cadavers were mixed with 70% methanol (one cadaver in 1 mL of 70% methanol) in an incubator shaker (New Brunswick Scientific,) with a speed of 150 rpm shaker at RT for 10 minutes. The supernatant was collected by centrifugation at 5000 g for 15 min and dried in a rotary evaporator. The extract was then resuspended in 10X concentration using purified water (ELGA Purelab Ultra, High Wycombe, UK) and centrifuged at 6,000 g for 15 min. The supernatant was lyophilized in a Labconco Freeze Dryer (Labconco floor model Casscade FreeZone 12 L, Kansas City, MO) and stored at −80 °C.
Pheromone treatment
A physiologically relevant concentration of pheromone extract was used for the pheromone treatment as described in Kaplan et al.22,23. Briefly, each G. mellonella were considered as 200 µL and extracts from consumed waxworms were resuspended in water to prepare 10X stock. When 10 µL water containing suspended S. carpocapsae or S. feltiae IJs was placed on the agar, a 1 µL aliquot of 10X pheromone extracts was added to the suspension. It took 10–15 min for the media to absorb the liquid and for the IJs to thus be able to move. IJs were allowed to disperse for 30 min for quantification.
Experiments at temperatures from 15 °C to 30 °C for dispersal and quiescent period
S. feltiae 12-day time course experiment
S. feltiae IJ dispersal was observed every other day during a 12-day period starting on day 0 and ending on day 12 or 14 at room temperature (RT), 20 ± 1 °C, in the absence of dispersal pheromones from host cadavers (Fig. 3B). Assays were conducted twice with separate culture batches of nematodes (presented on Fig. 3B and Supplemental Table S2), and started on different days with 3 or 4 replicate plates per treatment per time point per run; a total of 53 plates were assessed. Statistical differences were analyzed using linear regression model (R Studio with R version 6.3.1). R2 is 0.93 for S. feltiae dispersal.
S. carpocapsae temperature experiments
S. carpocapsae IJ quiescent period and dispersal were measured (Fig. 4A,C, Supplemental Fig. S1, Table S5) twice, once for a period of 60 days (0, 4, 21, 32, 46, 60 days), and once for 30 days (0, 1, 4, 10, 18, 30 days) at temperatures from 30 °C to 15 °C in 5 °C increments. S. carpocapsae IJ quiescent period was determined a third time for 12 days (0, 2, 4, 6, 8, 10, 12) at temperatures from 30 °C to 15 °C in 5 °C increments using a different methodology (Supplemental Table S4). The IJs storage temperature was at 20 ± 1 °C for all the experiments unless otherwise stated. Agar plates were conditioned to test temperatures prior to assay as 15, 20, 25, 30 °C. IJs were placed in 10 µl of water onto temperature conditioned agar plates and then into incubators with respective temperatures. Quantification of the quiescent period was followed by 30 min dispersal. Only the 15 °C and 20 °C temperature treatment were set up with a paired pheromone treatment (Fig. 4B,D and Supplemental Table S5) because we were testing stimulation of dispersal during the quiescent period and IJs at 25 and 30 °C did not show a quiescent period to test.
For temperatures 25 and 30 °C, since there was no quiescence for high temperatures, their dispersal was quantified and presented in Supplemental Fig. S1A and Table S5. For temperatures 15 and 20 °C in Fig. 4A,C and Supplemental Tables S4 and S5, 248 agar plates were analyzed in the presence and absence of pheromone extracts. Because populations of IJs with no pheromone extracts (96 plates) showed quiescence and in a side by side experiment, populations of IJs treated with pheromone extracts (96 plates) showed no quiescent period and dispersed, the IJ dispersal reported in Fig. 4B and 4D was analyzed with linear regression; R2 of 0.95 and 0.64, respectively. The assays for 60-day, 30-day and 12-day temperature experiments were conducted using different batches of nematodes at different times. Assays were conducted for 60-days (presented in Fig. 4A–D and Supplemental Fig. S1), with 4 replicate plates per treatment per run; a total of 144 plates were assessed. Assays were conducted for 30-days (Supplemental Table S5), with 2 (only for day 30 of 25 °C and 30 °C treatments) or 4 replicate plates per treatment per run (total of 116 plates) and for 12-days (Supplemental Table S4) with 4 replicate plates per treatment per run (total of 112). The sum of 3-time course experiments at 4 different temperatures with and without pheromones resulted in the analysis of 372 plates.
S. feltiae experiment in the absence and presence of pheromones
S. feltiae IJ dispersal (Fig. 5) was determined at 20 ± 1 °C for a period of 60 days; 11, 17, 24, 45 and 60 days after removal of residual pheromones from host cadavers. The IJs were reared and stored at 23 ± 1 °C. The assays were scored qualitatively as dispersed and non-dispersed after 30 min. The day 60 data collection method was different where we used a pre-post research design. The control was water treatment since the solvent for the cadaver extracts is water. Replications (n) are presented in Fig. 5 and a total of 35 plates were analyzed.
Crawling assays with agar with sand for testing pheromone extracts’ effect on S. carpocapsae ambushing behaviors
Assays of crawling prevalence were conducted on 90 mm Petri plates containing 30 mL of 2% water agar. Trials were conducted either on Petri plates without sand (agar only) or on plates that had been surface sprinkled with 0.5 g of air-dry sand (sand + agar)25,42. The sand used was commercial play sand passed through a 35-mesh sieve (500 micron opening) in the laboratory.
Deconditioned IJs (see the pheromone removal section above) that had been stored in tissue culture flasks at 14 °C for 4 days were suspended in 500 µL of distilled water and then exposed either to pheromone (50 µL of 10X S. carpocapsae dispersal pheromone extracts) or to 50 µL of DI water and then shaken regularly for a minimum of 20 minutes. Individual 550 µL IJ suspensions were then filtered through a 25 mm diameter P8 filter paper disc (Fisher Scientific, Pittsburgh, PA) by vacuum. IJs were transferred from the filter paper to the Petri plates using a laboratory probe.
Behavioral assessments began 10 min after IJs were transferred to the plate. The assay temperature was 22 °C. IJs were recorded as crawling or not crawling (which included jumping [attached to the lid within the field of view], waving, or tail standing). Once 100 worms had been counted, another field of view within the same plate was selected and assessed in a similar fashion. Assessments were made of three separate groups of 100 IJs per plate (total of 300 worms assessed per plate). Assays were conducted twice, with either 2 (in one case) or 3 replicate plates per treatment (factorial combination of +/− pheromone, +/− sand) per run; a total of 23 plates were assessed.
Dispersal plate assays with agar and sand + agar for testing pheromone extracts’ effect on S. carpocapsae
Dispersal assays were conducted on 90 mm Petri plate arenas with 30 mL of 2% water agar. Trials were conducted either in arenas without sand (agar only) or on plates that had been surface sprinkled with 0.5 g of air-dry sieved commercial play sand (sand + agar), as described above. Deconditioned IJs (IJs kept in the absence of pheromone in water at 14 °C for 4 days) were treated as described above. Individual 550 µL IJ suspensions were then filtered through a 25 mm diameter P8 filter paper disc (Fisher Scientific, Pittsburgh, PA) by vacuum. IJs were applied to the plate by gently pressing the filter paper disc on the center of the agar surface. The disc was then removed, leaving the IJs behind; if a dish was to receive sand, the sand was then sprinkled over the agar surface. The assay temperature was 22 °C.
S. carpocapsae IJs were allowed to disperse across the surface of the 90 mm arena for 30 minutes; at the conclusion of the test, 30 mm and 59 mm diameter stainless steel rings were used to separate the inner, middle, and outer “rings” of the arena. IJs were washed from each of the separate rings, as well as from the lid of the arena, and counted under a stereomicroscope. A dispersal index was determined for each arena, using the number of IJs found in the outermost ring (“Outer”), the number found in the middle ring (“Middle”) and the total number of IJs recovered from the surface of the arena (“Total” – note that while this number includes Inner, Middle, and Outer IJs, it excludes IJs found on the lid, see statistical analysis section below). The formula used to calculate the Dispersal Index was ((59*Outer) + (30*Middle))/Total. Assays were conducted twice, with 3 replicate plates per treatment (factorial combination of +/− pheromone, +/− sand) per run; a total of 24 plates were assessed.
Cross species test to determine whether S. carpocapsae IJs respond to pheromones from infected host cadavers of other EPN species
S. carpocapsae IJs were rinsed 3 times and tested for quiescence using the dispersal assay described in “dispersal assays and quantification” described above when exposed to physiologically relevant concentration of dispersal pheromone extracts from consumed host cadavers of S. carpocapsae (positive control), S. feltiae, S. scapterisci, S. riobrave, S. glaseri, S. diaprepesi, H. bacteriophora, H. indica, H. floridensis, and water (negative control) on day 0. Assays were conducted twice, with either 4 or 5 replicate plates per treatment per run; a total of 90 plates were assessed in Fig. 7A. Assays conducted at 21.5 ± 1 °C. Since pheromone treated IJs did not show a quiescent period, they were allowed to disperse 30 min. Five replicates for each treatment were quantified for dispersal (a total of 50 plates) in Fig. 7B. ANOVA and Tukeys HSD test was done for pairwise comparisons (Supplemental Table 8A–C).
Statistical analysis
Linear regression, ANOVA, Tukey’s Honestly Significant Difference (HSD), or Students’ t-tests were used to determine statistical differences using R version 3.6.1 (or 3.5.1) run in R Studio. Details of statistical analysis are in the Supplemental Methods.
Source: Ecology - nature.com
