The spatial dynamics of nutrient limitation in communities of generalist predators of crop pests

Field surveys Field surveys were carried out at Cockle Park, Morpeth, UK (55.214° N, 1.685° W), a farm site operated by Newcastle University. Sampling took place across two rounds in July and August 2026 (14-15th July 2026 and 10-11th August 2026) in three fields. Two fields contained oats, which were desiccated during round one and harvested prior to round two, and one field contained peas, which were ‘fresh’ during round one and desiccated during round two. Pairs of dry 500 mL pitfall traps were set in the north margin at the approximate centre of each field, and then 5 m, 20 m, 50 m and 100 m into the crop. The selected margins were all relatively simple, short grass margins (~ 5 m wide) adjacent to either hedgerow or, in one case, a small patch of woodland. Pitfall traps contained clay balls to provide refuge to trapped invertebrates and prevent within-trap predation. Traps were collected after 24 h and the contents of each transferred to a 125 mL collection pot for transport to the lab. The beetles from one trap were frozen and stored at -20 °C for macronutrient analysis. The beetles from the other trap were taken forward for nutritional feeding assays. Nutritional feeding assays Nutritional feeding assays followed the ‘double-test’ procedure of pre-acclimation (i.e., following collection) and post-acclimation (i.e., following a week of acclimation under controlled conditions) feeding trials (e.g., see Christensen et al., 2020; Nielsen et al., 2022; Toft et al., 2019). The protocol used is broadly presented by Croft et al. (2026). Feeding arenas were prepared by pouring a ~5 mm thick plaster-charcoal base (5 g activated charcoal powder, 40 g plaster of Paris and 45 mL water per 90 mL) into 9 cm Petri dishes, which were allowed to set at room temperature for 24 h. Base moisture was maintained throughout by applying ~2.5 mL of water to each arena when beginning to dry (determined by light grey colour of dried charcoal-plaster mix). Following field collection, beetles were separated into feeding arenas and left for 24 h before feeding assays were initiated. All beetles were kept in incubators at 25 °C with 12:12 light:dark cycles during initial incubation, feeding assays and satiation periods. For the feeding trials, three artificial diets were prepared: high-carbohydrate, high-lipid and high-protein. Each of these diets consisted of 50% dried, powdered mealworm, prepared by grinding commercially sold dried mealworms with a pestle and mortar, and sieving the fine powder. The remaining 50% consisted of an equal mixture of sucrose and dextrose powder, lard oil and casein for the high-carbohydrate, high-lipid and high-protein diets, respectively. These were stored at 4 °C for immediate use or -20 °C for longer-term storage. All artificial diets were precautionarily dried in an oven at 50 °C for 3 h prior to use, although none should have contained water. Into each feeding trial arena, four inverted screw-cap 2 mL microcentrifuge cap lids were placed as feeding dishes. The inner recess of each lid was filled with one of the three foods, and the fourth was filled with water. Lids were labelled on their underside with the food mixture they contained, and each was weighed immediately before the assay. The lids were placed at cardinal points in the Petri dish, each ~10 mm from the Petri dish edge to allow beetles to freely follow the edge of the dish. Beetles were weighed immediately before being placed in the centre of the arena. Beetles were allowed to feed for 24 h (pre-acclimation feeding trial), at which point the foods were collected, dried for 3 h in an oven at 50 °C (or up to 6 h if mass was still decreasing) and weighed. Beetles were allowed to feed ad libitum on dried mealworms and water for seven days (acclimation). The feeding trial was then repeated (post-acclimation feeding trial) as above. Following the conclusion of this second feeding trial, beetles were weighed and killed by freezing. Beetles were identified to species level using a morphological key (Luff, 2007) and were digitally imaged on an Epson Perfection V850 Pro charged coupled device scanner using the settings described by Ong et al. (2025). The mass change of each artificial diet was calculated by subtracting the post-feeding trial mass from the pre-feeding trial mass for both pre- and post-acclimation feeding trials. In very few instances where artificial diet mass was apparently gained during a feeding trial, results were discounted unless the gain was less than 2 mg (to account for potential instrument error), at which point it was recorded as no mass change. Some data were lost due to beetles dying during one of the assays or between the two. Macronutrient analysis Macronutrient analysis followed the Macronutrient Extraction and Determination from Invertebrates (MEDI) protocol (Cuff et al., 2021; Cuff & Wilder, 2023) which streamlines sulfo-phospho-vanillin, anthrone and bicinchoninic acid (BCA) colorimetric assays for lipid, carbohydrate and protein estimation, respectively. As per Christopher et al. (2026), assay volumes were adjusted for 384-well plates to increase throughput. Individual ground beetles were placed in 2 mL tubes of a 96-well rack and dried at 60 °C for 3 h. To each sample, 1 mL of 1:9 chloroform:methanol solution was added and incubated for 24 h at room temperature. After 24 h, 500 μL of the chloroform:methanol solution was removed and retained for lipid analysis. A further 500 μL of chloroform:methanol solution was added to the sample and incubated for 24 h at room temperature to reduce the prevalence of potential residual lipids. Following incubation, all chloroform:methanol solution was removed by pipetting, and any remaining solution evaporated by incubation at 60 °C for ~ 2 h. To each sample, 1 mL 0.1 M NaOH was added and the samples incubated at 80 °C for 60 min, followed by room temperature incubation overnight (~ 16 h). From each sample, the supernatant was transferred directly into assay plates for protein and carbohydrate determination immediately following the overnight incubation. All assays were carried out in 384-well plate format, with all samples run in triplicate. For each assay, a stock standard dilution series of a known concentration was made with lard oil, glucose and bovine serum albumin (BSA) for lipid, carbohydrate and protein quantification, respectively. Stock solutions of 2 mg mL-1 glucose and BSA were made using distilled water and subsequently diluted in 0.1 M NaOH, whereas 2 mg mL-1 lard oil was made and diluted with 1:9 chloroform:methanol. Dilution series comprised 0-2 mg mL-1 in eight increments (0, 25, 100, 300, 800, 1000, 1500, 2000 μg ml-1). A quarter of each 384-well assay plate (96 wells) was reserved for repeats of these standards, except in one plate where 72 wells were used for standards to include eight additional samples in triplicate. Lipid quantification The sulfo-phospho-vanillin method was used to determine lipid content. The vanillin reagent was prepared by mixing 1.08 mg of vanillin with 180 μL of hot water and 720 μL 85 % phosphoric acid for every 1 mL of reagent. From each sample, three repeats of 20 μL were added to a 384-well plate alongside standards, as above, and heated to 90 °C for 10 min to evaporate the chloroform:methanol carrier. To each well, 10 μL of concentrated sulfuric acid was added, mixed briefly by vortexing and incubated at 90 °C for 10 min. Following this, 75 μL of vanillin reagent was added to each well and incubated at room temperature for 5 min. Absorbance at 490 nm was determined for each well in a spectrophotometer. Carbohydrate quantification The anthrone method was used to determine carbohydrate content. The anthrone reagent was prepared by mixing 1 mg of anthrone with 1 mL of concentrated sulfuric acid for every 1 mL of reagent. From each sample, three repeats of 20 μL were added to a 384-well plate alongside standards, mixed with 80 μL of the anthrone reagent and incubated at 90 °C for 6 min. The plate was incubated at room temperature for 5 min and absorbance at 620 nm was determined for each well in a spectrophotometer. Protein quantification The BCA method was used to determine protein content. From each sample, three repeats of 20 μL were diluted with 180 μL NaOH and 20 μL of the diluted sample were added to a 384-well plate alongside standards. A BCA working reagent was prepared according to the manufacturer (Pierce™ BCA Protein Assay Kit, ThermoFisher). To each well, 80 μl of BCA reagent was added and mixed briefly on a plate shaker. The plate was incubated at 37 °C for 10 min and absorbance at 562 nm was determined for each well in a spectrophotometer. Nutrient content data processing Absorbance values generated by the spectrophotometer were blank corrected by subtracting the mean absorbance for blank controls (i.e., controls with a concentration of 0 mg mL-1) within each plate. A linear relationship between absorbance and concentration was assumed and a line of best fit generated for the standards included in each plate. If the R2 for that line was below 0.95, outliers were identified and removed (e.g., blank readings for a high concentration standard). Where most of the points for a given standard concentration were associated with a raw absorbance of 4.000 (the maximum reading), that standard concentration was discarded (which was only true for high concentrations of carbohydrate standards). The equation for the line of best fit was used to calculate concentration values for each sample based on their absorbance values. Negative concentration values were excluded, and mean concentrations calculated for each nutrient of each sample. Macronutrient data were generated for 389 beetles, of which data for five beetles were removed due to a lack of detections of at least one nutrient, and data for two beetles were removed due to detected nutrient contents exceeding 10 % above the highest standard concentration for at least one nutrient. Data analysis All data analyses were conducted in R version 4.5.2 (R Core Team, 2025). Data were processed and formatted using ‘tidyverse’ for reproducibility (Wickham et al., 2019). Beetle abundances and community structure were compared against distance into the field, crop stage, crop type and interactions between crop type and distance into the crop, and crop stage and distance into the crop. These relationships were tested in a multivariate generalised linear model (MGLM) using the ‘manyglm’ function in ‘mvabund’ with a Poisson distribution function (Wang et al., 2012). The model was analysed with Monte Carlo resampling and adjustment of p-values for univariate results to account for multiple testing using a step-down resampling procedure. Community differences were visualised using non-metric multidimensional scaling (NMDS) using the ‘metaMDS’ function in the ‘vegan’ package (Oksanen et al., 2022) with 999 ‘tries’ and a Bray-Curtis dissimilarity matrix. Nutrient intake data from the feeding trials were compared against distance into the field, crop stage, beetle species, crop type and interactions between beetle species and distance into the crop, beetle species and crop stage, crop type and distance into the crop, and crop stage and distance into the crop. Nutrient intake was represented as proportions of the total mass of feed consumed to account for differences in beetle size. These relationships were tested in two multivariate linear models using the ‘manylm’ function in ‘mvabund’. One model included data from the pre-acclimation feeding trials, whereas the other included data from the post-acclimation feeding trials. The models were analysed in 1000 interactions with p-values for univariate results adjusted for multiple testing using a step-down resampling procedure. The difference in nutrient intake mass between pre-acclimation and post-acclimation (i.e., pre-acclimation intake subtracted from post-acclimation intake) for each of the three foods was compared against distance into the field, crop stage, beetle species, crop type and interactions between beetle species and distance into the crop, beetle species and crop stage, crop type and distance into the crop, and crop stage and distance into the crop. Intake mass differences were scaled using the ‘scale’ command and all values made positive by subtracting the minimum scaled value to adjust negative values. The relationships were tested in a multivariate linear model using the ‘manylm’ function in ‘mvabund’, with models analysed as above. The total food intake mass consumed during pre-acclimation and post-acclimation feeding trials were separately compared against distance into the field, crop stage, beetle species, crop type and interactions between beetle species and distance into the crop, beetle species and crop stage, crop type and distance into the crop, and crop stage and distance into the crop. These relationships were tested using generalised linear models, with total intake mass during each feeding trial as the dependent variable. Model assumptions were checked using the ‘testResiduals’ function of the ‘DHARMa’ package (Hartig, 2022). Beetle nutrient content data were compared against distance into the field, crop stage, beetle species, crop type and interactions between beetle species and distance into the crop, beetle species and crop stage, crop type and distance into the crop, and crop stage and distance into the crop. Beetle nutrient content data were represented as μg per beetle since proportional data violated the model assumptions, particularly normality. These relationships were tested in a multivariate linear model using the ‘manylm’ function in ‘mvabund’, with models analysed as above. To meet the model assumptions, particularly normality, nutritional data were log-transformed. Nutritional data were visualised using ternary plots with the ‘ggtern’ package (Hamilton & Ferry, 2018) and scatterplots using ‘ggplot2’ (Wickham, 2016). README