DOI: 10.5061/dryad.4mw6m90j1
title: Tarnished plant bug and eastern bumble bee in strawberry

description: Land use change affects both pollinator and herbivore populations with
 consequences for crop production. Recent evidence also shows that land use
 change affects insect traits, with intraspecific body size of pollinators
 changing across landscape gradients. However, the consequences on crop
 production of trait changes in different plant interactors have not been
 well-studied. We hypothesized that changes in the body size of key species
 can be enough to affect crop productivity, and therefore looked at how the
 field-realistic variation in body size of both an important
 pollinator, Bombus impatiens (Cresson), and a key pest
 herbivore, Lygus lineolaris (Palisot), can affect fruit size and
 damage in strawberry. First, we determined if pests vary in body size
 along land use gradients as prior studies have documented for pollinators;
 and second, we tested under controlled conditions how the individual and
 combined changes in the size of an important pollinator and a key
 herbivore pest affect strawberry fruit production. The key herbivore pest
 was smaller in landscapes with more natural and semi-natural habitats,
 confirming that herbivore functional traits can vary along a land-use
 gradient. Additionally, herbivore size, and not pollinator size,
 marginally affected fruit production—with plants exposed to larger pests
 producing smaller fruits. Our findings suggest that land use changes at
 the landscape level affect crop production not just through changes in the
 species diversity of insect communities that interact with the plant, but
 also through changes in body size traits.
description: Tarnished plant bug size across the landscape
 gradient In the summer of 2019, 10 small farms growing
 strawberries as one of their crops in the Finger Lakes region of New York
 State (within a 50-mile radius of 42.697047, -76.712271) were selected
 across a landscape gradient. The landscape surrounding these sites
 consisted of 15% to 42% natural area at a 750m radius. Each site was
 visited once between August 12–30, and three of those sites were also
 visited once on July 2–3, though as tarnished plant bug populations were
 not large enough to enable enough specimens to be caught at that time,
 most individuals were caught in mid-late August. At each site, tarnished
 plant bugs were sweep-net collected both from within strawberry fields and
 in the weedy field margins around the strawberry crop. Landscape
 composition within a 500, 750, and 1000-meter radius around each
 collection site was determined via the 2019 USDA NASS CropScape Cropland
 Data Layer (nassgeodata.gmu.edu/CropScape). Multiple scales were chosen in order to determine which was most predictive of tarnished plant bug size, and these particular scales were chosen based on the most indicative scales found in a prior landscape study of this species [source]. The Cropland Data Layer provides crop-level land cover, which we then aggregated into broader land cover categories. Those land cover categories were agricultural, agricultural with pasture, natural forested, naturally open, pasture, and urban (urban being defined as buildings and other developed areas like roads) cover. Strawberry field cover was included in the “agricultural” category but was not analyzed separately because it is not an industry large enough in this area for it to be possible to analyze the proportion of strawberries in the landscape. A Mantel test revealed no spatial autocorrelation of sampling sites (Mantel <em>R</em> = -0.1275, <em>P</em> = 0.689, n = 10) Each tarnished plant bug’s body size was measured, using pronotum width at its widest point as a proxy for whole body size [source]. Size was measured using an Olympus SZX10 stereo microscope and DP22 microscope digital camera, with Olympus CELLSENS Standard v.1.16 software used to measure bugs to the nearest 10 μm. Effect of bee size and tarnished plant bug size on strawberry fruit size A greenhouse bioassay was conducted in the spring of 2021 to examine the effects of bee and tarnished plant bug size on strawberry pollination and damage, respectively. A total of 300 strawberry plants (<em>Fragaria x ananassa</em>, variety: Seascape, bare-root plants sourced from Nourse Farms) were grown in the greenhouse (14 hours daylight, 70–74℉ day/70-66℉ night) until they flowered. Three managed <em>Bombus impatiens</em> bumble bee colonies were sourced from Biobest (Biobest Standard hives). From these three hives, we constructed ten microcolonies of 8 female workers each, all from the same source colony. Five microcolonies contained individuals with the smallest body sizes, selected by eye, and five contained individuals with the largest body sizes. Microcolonies were allowed to acclimate for one week with supplemental pollen and a 30% sugar solution. Post-experiment, bee body size was measured to confirm that large and small colonies were significantly different in size by measuring intertegular distance (ITD), a standard measure of body size in bees [source], Welch two-sample t-test comparing mean sizes of large versus small colonies, <em>t</em> = 6.4746, <em>df</em> = 4.7168, <em>P</em> = 0.001628). To ensure these colonies represented a standard range of bee sizes for the region, we compared them with other commercial hives used in other regional studies. In total, our average bee ITD was 3.47 ± 0.36mm (mean ± 1SD). Miller et al. [source] used commercial hives in the same region of New York State and found an average bee ITD of 3.34 ± 0.45mm (mean ± 1SD) and in nearby Southern Quebec, Gervais et al. [source] found an average ITD of 3.943 ± 0.318mm (mean ± 1SD). Both studies measured body size later in the season, to ensure that body size (a plastic trait affected by available resources) was a reflection of the landscape’s resources and not the method by which they were commercially raised. Thus, these were used as a suitable proxy for regional bee size for this species and confirmed that our colonies fell within an average range. All microcolonies were ‘trained’ on strawberry plants for a minimum of three days in order to ensure each individual had experience visiting strawberry flowers and had a built-up pollen load. Training consisted of colonies being kept open in a 24x36” mesh cage, with three or four blooming strawberry plants. These plants were individually switched out if no blooms were present at any point, and none of the training plants were used in the following trials. All colonies were kept in a growth chamber set at 14 hours of daylight and 70–74°F day temperature, and 66–70°F night temperature. Pollination and herbivory trials were conducted within mesh cages placed outdoors and in the greenhouse, respectively. Pollination cages (24x36” white mesh pop-ups) were set up outdoors in the morning starting at around 9 am each day–these trials were run outside in order to provide the most natural environmental conditions possible for the bees (wind, direct sun, etc.). Only plants with at least two open secondary flowers were used. Experimental plants were brought from the greenhouse and one or two were carefully inserted into each cage containing a microcolony. These plants were then observed until the first visit by a bee to one of the exposed secondary flowers. The plant was then removed from the cage, the pollinated flower was marked with yarn, and the other unpollinated secondary flower (at approximately the same stage of development) was marked as unpollinated. Pollination trials were run until around 12 pm each day, after which time visitation activity was negligible. Pollination trials were run for four days until at least 60 pollinated plants per pollinator size group were acquired. Once plants were pollinated, they were transported back to the greenhouse and allocated to their plant bug trial group. To determine the effect of <em>L</em>. <em>lineolaris</em> size on fruit damage and size, we conducted an experiment exposing developing fruits to individual bugs across a range of body sizes. These trials were conducted in the greenhouse. Approximately half of the plants from each pollination size group, 30 plants pollinated by large bees and 34 plants pollinated by small bees, were placed in empty cages (no damage treatment). Each of the remaining pollinated plants, 30 pollinated by large bees and 30 pollinated by small bees, were placed in individual 12x12 cages along with a single wild-caught tarnished plant bug individual. The bugs for this experiment were collected from a subset of four sites used for the landscape trials, and a simple linear model was run to show that size did not differ between these sites (<em>F</em><sub>3,50</sub> = 1.841, <em>P</em> = 0.1518), eliminating the possibility of confounding effects from populations. These individuals varied in size from 1.930 to 2.328mm (pronotal width) and were randomly selected. This range is similar to the range seen across local landscapes—in 2019, field collections of tarnished plant bugs made across 10 sites of varying landscape complexity showed a range in size from 1.67 to 2.27mm (pronotal width). Due to plant bug mortality during trials, the final count of usable plants was 22 plants pollinated by large bees and 29 plants pollinated by small bees. As we were unable to determine exactly when the bugs in the cages had died, we were thus unable to say with certainty that the plant had been exposed for the full 24 hours–hence why these plants were removed from the experiment. The bugs were left to feed on the plants for 24 hours, at which point they were removed, frozen, and their pronotal width measured to the nearest 10 μm using an Olympus SZX10 stereo microscope and DP22 microscope digital camera, with Olympus CELLSENS Standard v.1.16 software. Plants were then removed from small cages and placed together in large cages. This was done to enable greenhouse staff to water the plants, which were inaccessible in the small cages, and to eliminate the risk of stray pollinators in the greenhouse accessing them. Plants were watered regularly and monitored until fruits were fully developed. Once that stage was reached, each fruit was removed and weighed.
description: # Tarnished plant bug and eastern bumble bee in strawberry
 [https://doi.org/10.5061/dryad.4mw6m90j1](https://doi.org/10.5061/dryad.4mw6m90j1) This repository contains the R file containing analysis and 4 .csv files that contain the data needed to run those analyses. The .csv files are contained within the Archive.zip file. ## Description of the data and file structure The R file has the necessary code to import and clean the data files provided. The user will have to set their working directory to the folder into which they've downloaded the data files, at which point the code should be fully functional. #### Individual file descriptions ###### **Bee colony sizes.csv** This file** **contains body size data for each bumble bee microcolony used in the study. Columns: “Colony #” is the randomly assigned  ID number for the microcolony, “Mean size” is the mean intertegular distance (in mm) of bees in that microcolony, and “colony_s_or_l” is whether the colony was assigned the category “large” or “small” based on the mean size of the bees within it. **bug-size-site-for-bioassay** This file contains the data for the tarnished plant bugs (TPBs) used in the bioassay portion of the study. Columns: “cup #” is the randomly assigned ID number for each TPB, “date caught” is the day the bug was collected, “location” was the site at which the bug was collected (from within or directly adjacent to a strawberry field), and “TPB width” was the width in millimeters of each bug measured as the widest part of the pronotum. **Lygus body size by landscape data** This file contains the data for the first analysis of the paper, looking at tarnished plant bug size across a landscape gradient.  Each row is for one individual tarnished plant bug collected. Columns: “Date” is the day the TPB was collected, “Cup#” is the randomly assigned  ID number for the bug, “Location” is the site where the bug was collected, “Sex” is the sex of the bug. “Pronotal width” and “Pronotal height” are the length in millimeters of the widest and longest parts of the bug’s pronotum, respectively. Likewise, “scutellum height” and “scutellum width” are the length in millimeters of the longest and widest parts of the scutellum, respectively. “Tibia L length (rear)” and “Tibia R length (rear)” are the lengths of the back left and right legs of the bug. The final columns are all proportion date, detailing the proportions of different land cover types surrounding each site where the bug was collected. For each column, the land cover type is the first part of the name, and the second part of the name is the distance in meters of the circular buffer around each site that was measured. This data was acquired from the USDA-NASS CropScape Data Layer. For each landcover type: “AG” = agricultural cover, “AGwPAST” = agricultural and pasture cover combined, “NAT_FOR” = natural forested cover, “NAT_OPEN” = natural open cover, “PAST” = pasture cover, and “URB” = urban cover (buildings, paved surfaces). Data is included at buffer scales of 500, 750, and 1000 meters. Description of NA values: For both tibia length columns (left and right legs), NA values are present when a bug was missing one or both of its' hind legs.  **TPBee.csv** This file contains the data collected during the bioassay, when strawberry plants were exposed to both pollination/no pollination and tarnished plant bug damage/no TPB damage. The columns are as follows: “Plant_num” is the randomly assigned  ID number for each strawberry plant, “TPB_num” is the randomly assigned  ID number for each tarnished plant bug. “TPB_present_absent” is whether the plant was exposed to a TPB individual or not. “TPB_width” and “TPB_height” were the width and height, respectively, of the pronotum of the TPB (if one was present). “TPB_deadORnot” was whether the TPB died during the bioassay or not. “microcolony_num” was the microcolony used to pollinate the plant (if a plant was in the not pollinated treatment, this column contains an NA). “bee_size” was the size of the microcolony used (see more in the “bee colony sizes.csv” description). “Date_pollinated” was the day the plant was pollinated - or if it was in the "not pollinated" category, the day was when it was placed in a separate cage to develop fruit. “pollination_trt” was whether the plant was pollinated or not. “size_and_poll” combines the date_pollinated and pollination_trt columns, and consists of three levels: unvisited, pollinated by a large microcolony (“large_visited”) or pollinated by a small microcolony (“small_visited”). “date_harvested” was the day that the fruit was picked, “fruit_weight_g” was the weight of each fruit in grams, “percent_pollinated” was the percent of seeds on each fruit that were determined to be viable, and “percent_tpb_damaged” was the percent of each fruit with visible TPB damage. Description of NA values: For TPB number, width, height, "percent_tpb_damage," and "TPB_deadORnot," NAs were present when the treatment called for no TPB to be present (when the column "TPB_present_absent" was "absent). "Date_pollinated" contains NA values when the date the plant was separated was not recorded (since all plants were grown at the same time and we did not look at time to fruit development, the date they were separated was not important). ## Code/Software Analyses were conducted in RStudio 2021.09.0, using packages nlme, and stats. Graphs were constructed in RStudio 2021.09.0, using packages ggplot2 and cowplot