Simulation of seasonal temperatures
A thermal gradient plate (TGP) (Mclaughlin et al. 1985; Ritchie et al. 2021; Stumborg et al. 2021) was used to simulate seasonal temperatures and contain experimental units (Petri plates containing beetles). The particular TGP TCMCI-176 model comprised 176 - 115mm diameter x 45mm deep cells, each of whose temperature can be controlled separately over a range from -5C to +40C. For this experiment, 72 TGP cells were programmed to oscillate between simulated mean daily minimum and mean daily maximum air temperatures based on 30-year climate normals (1985-2015; weather.gc.ca) at Agassiz, British Columbia (N 49.2400o, W 121.7567o) for 15-30 April (‘April’), May, and June i.e. periods during spring and early summer when click beetles are active and vulnerable to surface-applied control efforts (Kabaluk 2016). The TGP was programmed so that the minimum temperature occurred at 4AM and the maximum temperature at 4PM. The temperature changed every 10min as it oscillated in a sine wave pattern between these two set temperatures. Twenty-four cells were programmed for each of the three seasonal temperatures, with these 72 temperatures randomly assigned to the cells. Because there are no apparent physical gradients when using the TGP, the assignment of temperatures among the 72 cells was completely random. Thermocouples emanating from a Campbell Scientific 21X data logger (Edmonton, AB) logged the actual temperatures every 15min inside the experimental units in two randomly selected TGP cells for each of the three seasonal temperatures, as temperatures were expected to deviate from set temperatures. The actual temperatures (y) of daily maximum and minimum values were plotted against maximum and minimum set temperatures (x) to validate the ability of the TGP to reproduce set temperatures in the experimental units. The mean logged temperatures (y) were plotted against day (x) and used in all analyses as they were temperatures to which the beetles were actually exposed.
Beetle collection and housing
Male Agriotes obscurus and A. lineatus click beetles were collected during April and May 2015 from an experimental farm field located near Agassiz, BC using pitfall traps baited with pheromone bubble caps (Scotts Canada Ltd. Mississauga, ON). Beetles were retrieved daily from the traps and transferred to ventilated plexiglass boxes containing long grass leaves for cover, a wet paper towel for moisture, and apple pieces for food. The boxes were kept in a growth chamber at 8C (van Herk et al. 2008) until active beetles were selected for use in the experiments.
Strain origins and culture
Metarhizium brunneum LRC112 (‘MbLRC112’) originally isolated from an A. obscurus larval cadaver (Kabaluk et al., 2007) was mass produced on rice using a two stage fermentation process (Jackson and Jaronski 2012), yielding conidiated rice granules. M. brunneum F52 (‘MbF52’) is a widely recognized commercial biocontrol agent originating from a codling moth Cydia pomonella cadaver (European Food Safety Authority 2020). For the experiment, it was isolated from the granular product Met52 provided by Novozymes (Franklinton, NC, USA), and mass produced as for MbLRC112. Conidiated rice granules were dried to ca. 8% MC, vacuum packed in plastic bags and stored at ca. 8C. Prior to use in the experiments, conidia viability was assessed by suspending conidiated granules in sterile water containing 0.01% Tween 20 to release and disperse conidia. A 100ul conidia suspension sample was spread on a Petri plate containing potato dextrose agar and incubated at 27C for 20h, after which the percent viability was determined.
M. brunneum inoculation, apportionment of beetles, and dose assessment
To inoculate beetles with M. brunneum, 5g of conidiated rice granules were placed on filter paper contained in a 9cm Petri plate. After the plate was shaken to release spores from the granules, the granules were removed, and eleven beetles were placed onto the filter paper. After the lid was placed to prevent escape, the beetles crawled on the paper for 3min to acquire conidia. Ten of the inoculated beetles were then transferred to a clean modified Petri plate (see Experimental layout and unit apparatus, below) containing a cigarette filter moistened with distilled water and a 5mm x 10mm cylinder of apple. The eleventh beetle was placed in a microcentrifuge tube containing 250ul of 0.05% Tween 20 water solution to assess conidia dose. This procedure was repeated 48 times – once for each M. brunneum strain (2) x click beetle species (2) x seasonal temperature (3) x Petri plate replicate (4) combination. The eight control groups were subjected to the same procedure, but without conidia. These comprised four groups of ten beetles for each click beetle species. Each Petri plate of 10 beetles constituted an experimental unit. In terms of timeline, day 0 was the day of conidia inoculation.
Ten of the 12 beetles representing each click beetle species x M. brunneum strain combination in the microcentifuge tubes were vortexed for 30s to release conidia from their bodies, after which a suspension subsample was placed on a haemocytometer to assess the dose of conidia that each beetle acquired. The total conidia dose was adjusted to the viable conidia dose based on the percent germination determined for each M. brunneum strain. Data were log transformed and subjected to Shapiro-Wilk’s test for normality and Levene’s test for equal variances. The log transformed conidia dose data were subject to analysis of variance using R ver. 4.0.4 (R Core Team 2021) using the model:
log[conidia dose (conidia/beetle)] = click beetle species + M. brunneum strain + Cbs x Mbs
Means of interaction terms were separated using Tukey’s HSD test (α = 0.05).
Experimental layout and unit apparatus
Twenty-four experimental units (Petri plates), each comprising 10 beetles for each click beetle species (2) x M. brunneum strain (2; plus control = 3) x replicate (4) combination were individually and randomly assigned to TGP cells of a common seasonal temperature. With this assignment, and that of seasonal temperatures to TGP cells, the experiment was a completely random design. The Petri plates containing the beetles were modified by opening a 1cm square ventilation hole covered in nylon mesh to permit air exchange. This was referred to as the ‘lower plate’. An additional empty Petri plate was placed on top of the lower plate, referred to as the ‘upper plate’. On the bottom of the upper plate three small rubber ‘feet’ (kitchen cupboard door bumper pads) elevated them from the top of the lower plate so that air could circulate through the ventilation hole in the lower plate. With the addition of moistened filter paper, the upper plate was used for incubating beetles found dead in the lower plate.
Mortality and disease development assessment and experiment maintenance
Beetles in the lower plate were scored as alive or dead when assessed every Monday, Wednesday, and Friday. Dead beetles were immediately placed in the upper Petri plate and scored as dead-asymptomatic, mycelial outgrowth, or sporulating during subsequent evaluations. On each evaluation day, the cigarette filter in the lower plate and filter paper in the upper plate were moistened as required, and apple replaced if it was moldy or deteriorating. On day 31, beetles maintained at April temperatures were removed and placed in a 25C incubator to verify the contribution of degree days to mortality and disease development. All beetles were assessed until day 35. Mean beetle mortality outcomes (y) with standard deviation were plotted against evaluation day (x) for each seasonal temperature x beetle species x M. brunneum strain. Zero-intercept transition function (sigmoid) models were fit to the data using TableCurve 2D (Systat Software, San Jose, CA, USA). The percent of subdivided dead categories were plotted against evaluation day.
Degree day calculations and model plotting
Cumulative degree days for each seasonal temperature were calculated over the duration of the experiment using the mean logged temperature from each pair of randomly selected TGP cells. The base temperature (T0) of 5C was previously derived from i) thermal profiles of MbLRC112 and MbF52 colony growth (Kabaluk et al. 2007b); and ii) empirical data assessing the mortality of A. obscurus and A. lineatus caused by MbLRC112 and MbF52 over a range of low temperatures (T. Kabaluk, unpublished). The cumulative degree day calculation was:
∑ [(recorded time point temperature – 5)/96*]
*temperatures were logged every 15min, or 1/96 of a 24h day
To plot the data, cumulative degree days was used as the x coordinate, and the associated mean beetle mortality from four replicate Petri plates for each M. brunneum strain x beetle species as the y coordinate. In doing so, data from the three seasonal temperature treatments were pooled. Zero-intercept transition function (sigmoid) models were fit to the data using TableCurve 2D. The 95% confidence interval shading was generated in R using the TableCurve 2D model fit results. Degree-day regressions were analyzed with a generalized linear model followed by pairwise comparisons using the package emmeans in R with a Tukey’s adjustment.
Hypothetical field application of M. brunneum
The number of degree days to kill 50% of the beetle population (LDD50) were interpolated from data predicted by transition function models of the beetle mortality vs. degree days plots. Three hypothetical, but realistic M. brunneum field application dates were then chosen and the number of accumulated degree days (using actual outdoor temperatures in Agassiz, BC in 2019) from each application date plotted (the degree days for the start of each application date was set to zero). LDD50 for each species*strain combination were used to interpolate the time (days) to kill 50% of the beetle population (LT50), and matched to an associated date.
Figures were created using Microsoft Powerpoint and R.