DOI: 10.15482/usda.adc/29345147.v1
title: Data from: Predation and immunocompetence: Grasshoppers increase Mormon cricket resistance to a fungal pathogen

description: Data DescriptionsfungalExpt.csv<i>Beauveria bassiana</i> GHA is registered for application to grasshoppers and Mormon crickets on rangeland and improved pasture. Conidia were obtained as a dry technical grade powder (Laverlam International, Butte, Montana). A concentrated <i>B. bassiana</i> stock suspension in paraffin crop oil (Orchex 796, Calumet Specialty Product Partners) was prepared from the dry conidia and the concentration determined by hemocytometer counts of kerosene-diluted samples (Srygley and Jaronski 2011, 2018). A suspension of 3 x10<sup>9</sup> viable spores per ml oil was adjusted for conidial viability.On July 24, 2019, we collected 98 female and 90 male Mormon crickets from the cages (detailed below) with each cricket placed in an Eppendorf tube marked with sex and cage of origin. Mormon crickets were weighed, and fungal-treated insects were inoculated with 1 µl of the fungal suspension applied with an electric micro-applicator at the bases of each of two forelegs (6x10<sup>6</sup> spores per treated insect) in a camper on the site. Note that in laboratory conditions, that dose was sufficient to exceed 50% mortality in four days and resulted in 100% mortality in six days of application. The same amount of paraffin crop oil without spores was applied to the 51 control insects. Controls and <i>Beauveria</i>-treated Mormon crickets (listed as ‘c’ and ‘f’, respectively under the header ‘Treatment’) were held in 50 ml Eppendorf tubes with a hole in the top for air exchange and distributed to the 0.025 m<sup>2</sup> cages, but not released until the following day to confirm that none molted and may have shed the spore inoculation. We added five grasshoppers to each of 25 controls and 67 inoculated insect cages, and attached the cage tops.Cages with fungus-treated and sham-treated controls were checked on July 29, five days after fungal treatments were applied. Six controls and 22 fungus-infected Mormon crickets had escaped or were otherwise missing. Cages were checked again the following day, and each day thereafter until August 15. Cadavers were recorded and placed individually in uniquely marked petri dishes. Petri dishes for controls and fungus-treated insects were separated into 100% humidity chambers, and transported to the laboratory in Sidney to visually check for <i>B. bassiana</i> sporulation on the surface of the cuticle. On August 4, 6, and 8, grasshoppers were added to grasshopper-provisioned cages to restore the number to five per cage.Because mortality in the field was lower than we expected, we brought the 42 fungus-treated insects that were alive on August 15 to the laboratory in Sidney, Montana to confirm that they were indeed infected with <i>B. bassiana</i>. Each was housed in a cup with a screen top, provided with dry food and water ad lib, and placed in<b> </b>an environmental chamber at 28 C and 15:9 h day:night lights.<b> </b>Daily, they were checked for mortality when given fresh Romaine lettuce.<br>Immunity.csvOn July 2, 22 days prior to the fungal treatments, we erected 28 cages at a site on Forest Service Road (FSR) 14 (44.8264 N, 107.8280 W, 2773 m<b> </b>a.s.l.). Lucite cages (1 m<sup>2</sup> x 0.7 m in height) were erected, fastened to the ground, and cleared of all grasshoppers, katydids, and spiders. On July 2-3, we collected Northern grasshoppers <i>Melanoplus borealis</i> nymphs and Mormon cricket nymphs from a meadow on Paint Rock Road, Bighorn County, Wyoming (WY, 44.4644 N, 107.4592 W, 2653 m<b> </b>a.s.l.). Nearly all of the 390 Mormon crickets were wild-caught 4<sup>th</sup> instar nymphs (only 12 were 5<sup>th</sup> instars). Grasshoppers were sorted to exclude anything less than a 3<sup>rd</sup> instar. On July 3, cages were stocked with two Mormon cricket density treatments: 10 and 20 Mormon crickets; and two grasshopper density treatments: 0 and 45 grasshoppers (listed as ‘MC trt’ and ‘GH trt’ in the data set). Hence, some of the Mormon crickets were provisioned with grasshoppers and some were not, and these treatments were continued when the insects were separated into individual cages later (detailed above). Cages were open at the bottom which gave the Mormon crickets and grasshoppers a diversity of grasses, sedges, and broad-leaved plants for forage.Beginning six days after the fungal treatment, hemolymph was collected from the Mormon crickets in sequential order by cage number so that sampling was randomized. We punctured the arthrodial membrane at the base of each insect’s hindleg with a 26 gauge hypodermic needle. Hemolymph was collected from the wound with a 20 µl capillary and 8 µl was diluted 1:50 with phosphate buffered sailine (PBS) solution for assays of spontaneous phenoloxidase (PO) and total phenoloxidase (PO and prophenoloxidase combined) enzymatic activities, and total hemolymph protein. To complete hemolymph sampling for the insects in all 188 cages required four days. Of the 188 cages, 14 cages had missing insects, and the Mormon cricket had died in 3 cages.For spontaneously-active phenoloxidase (listed as mean PO in the data sheet), we followed the protocol in Srygley et al. (2009). Briefly, each sample of hemolymph diluted in PBS was centrifuged and activated with 10 mM dopamine solution. We loaded the plate into a temperature-controlled microplate reader (Biotek) set to 25 C, and absorbance at 492 nm was read between 5-15 min, the time period during which the sample absorbance was linearly related with time. We calculated mean V as the change in absorbance per min. One unit of PO activity per ml hemolymph is defined as the amount of enzyme resulting in a 0.001 increase in V. In order to measure total PO activity (listed as mean proPO in the data sheet), we dissolved 1 mg alpha-chymotrypsin from bovine pancreas (Sigma) in 1 ml PBS, and combined it with an equal volume of centrifuged hemolymph diluted in PBS, and incubated the solution for 30 min. In the plate wells, 5 µl of the incubated solution was added to 195 µl 10 mM dopamine. Mean V was calculated from the plate readings between 5-15 min to measure total PO activity in units per ml hemolymph. Total hemolymph protein in mg protein per ml hemolymph was measured with a Total Protein Kit, Micro (Sigma) compared to a serial dilution of the human albumin standard.ReferencesSrygley, R. B. & Jaronski, S. T. (2011) Immune response of Mormon crickets that survived infection by <i>Beauveria bassiana</i>. <i>Psyche</i>, <i>849038</i>, 1-5<i>.</i> DOI: 10.1155/2011/849038.Srygley, R. B. & Jaronski, S. T. (2018) Protein deficiency lowers resistance of Mormon crickets to the pathogenic fungus <i>Beauveria bassiana</i>. <i>Journal of Insect Physiology</i> 105: 40-45 DOI: 10.1016/j.jinsphys.2018.01.005Srygley, R. B., Lorch, P. D., Simpson, S. J., & Sword, G. A. (2009) Immediate protein dietary effects on movement and the generalized immunocompetence of migrating Mormon crickets. <i>Ecological Entomology,</i> <i>34</i>, 663-668.