DOI: 10.6086/d1fx0d
title: Data from: Pollinators and plant nurseries: how irrigation and pesticide treatment of native ornamental plants impact solitary bees

description: A key conservation goal in agroecosystems is to understand how management
 practices may affect beneficial species, such as pollinators. Currently,
 broad gaps exist in our knowledge as to how horticultural management
 practices, such as irrigation level, might influence bee reproduction,
 particularly for solitary bees. Despite the extensive use of ornamental
 plants by bees, especially little is known about how irrigation level may
 interact with insecticides, like water-soluble neonicotinoids, to
 influence floral rewards and bee reproduction. We designed a two-factor
 field cage experiment in which we reared Megachile rotundata (Fabricius)
 (Hymenoptera: Megachilidae) on containerized ornamental plants grown under
 two different irrigation levels and imidacloprid treatments (30% label
 rate dosage of a nursery formulation or an untreated control). Lower
 irrigation was associated with modest decreases in nectar volume and
 floral abundance in untreated plants, whereas irrigation did not affect
 plants treated with imidacloprid. Furthermore, higher irrigation decreased
 the amount of imidacloprid entering nectar. Imidacloprid application
 strongly reduced bee foraging activity and reproduction, and higher
 irrigation did not offset any negative effects on bees. Our study
 emphasizes the impact of a nursery neonicotinoid formulation on solitary
 bee foraging and reproduction, while highlighting interactions between
 irrigation level and neonicotinoid application in containerized plants
 themselves.
description: We examined the effects of
 imidacloprid application and irrigation level on floral nectar and ALCB
 reproduction using a fully crossed randomized block design. From March
 2018 to July 2020, we maintained 20 field cages (each 5.8
 m<sup>3</sup>)<sup> </sup>in a 0.30-ha plot at
 University of California Riverside Agricultural Operations (33.965 °N,
 117.341 °W). We used four cages for the<i> </i>nectar
 experiment and 16 for the ALCB reproduction experiment. Each cage served
 as a replicate mesocosm simulating conditions at a containerized
 nursery.  
 <i>Nectar experiment</i>
       We began growing lacy phacelia (Boraginaceae:
 <i>Phacelia tanacetifolia</i>) from seed in UC Soil Mix III
 (agops.ucr.edu/soil-mixing) in 2-L pots in a greenhouse on 29 October
 2019. We focused on containerized plants, as soil dynamics for plants in
 containers likely differ from those experienced by plants growing in the
 ground. We selected phacelia due to its attractiveness to bees, including
 ALCBs [32], and its abundant floret production [33]. We assigned 180
 plants to one of six treatments resulting from the crossing of irrigation
 level and imidacloprid treatment (table 1), organized into two
 experimental blocks of two cages each (figure S1<i>a</i>),
 resulting in 45 plants per cage. Plants were moved into cages on 14
 January 2020, and we inserted an individual high- or low-flow irrigation
 spike (Primerus Products, Encinitas, CA) into each pot. These spikes are
 widely used in nurseries for container irrigation, and we selected spikes
 representing the lowest and highest flow rates for 2-L pots. High-flow
 spikes emitted 2.6 times more water as low-flow spikes (figure S2),
 resulting in 23% higher average midday volumetric water content (VWC;
 figure S3<i>a</i>). All pots were automatically irrigated
 simultaneously over the soil surface once per day at 0600 h for 60 s,
 increased to 120 s whenever any plants visibly wilted due to heat. The
 ground inside each cage was lined with fabric barrier to prevent plants
 from rooting outside pots. For imidacloprid treatments, we
 applied Marathon<sup>®</sup> 1% Granular (OHP, Bluffton, SC),
 a commercial nursery formulation, to pots on 28 January 2020. Formulated
 for use in greenhouses and nurseries, Marathon<sup>®</sup>
 consists of 1% imidacloprid and 99% inert ingredients by mass. Granular
 formulations allow the active ingredient to leach more slowly from potting
 soil than liquid drenches [34]. The label rate dosage for a 2-L pot
 equates to 1.4–2.0 g formulation. We used ~30% the label rate as our
 “high” dosage to be conservative, as near total ALCB mortality occurred
 when separate plants were treated at label rate (unpublished data). In
 this experiment, we also included a “low” dosage treatment of ~3% the
 label rate (table 1). Phacelia<i> </i>bloom
 began in early May 2020 and lasted six weeks. On each of six separate days
 during the bloom period, we measured floral nectar volume between 1030 and
 1330 h (daily high temperatures between 22.2 to 36.1 °C) in randomly
 selected flowers in each treatment using microcapillary tubes (Drummond
 Scientific, Broomall, PA) and handheld calipers (<i>N</i> = 13
 flowers x 4 cages x 6 days = 312 flowers). We also quantified total sugar
 concentration of samples on three of these days using a refractometer
 (Eclipse, Bellingham + Stanley) (<i>N</i> = 7 to 8 flowers x 4
 cages x 3 days = 94 flowers). On each of eight separate days during bloom,
 we collected nectar during the same time of day, pooled samples within
 treatments within cages (<i>N</i> = 3 pooled samples x 8 days
 = 24 samples (4 per treatment), and quantified imidacloprid residues in
 nectar via an ELISA. We used a QuantiPlate<sup>™</sup> kit
 (EnviroLogix, Portland, ME) and microplate spectrophotometer (Thermo
 Fisher, Waltham, MA), which provide similar quality data to HPLC/MS-MS at
 less cost [16]. Imidacloprid metabolites, also toxic to bees [29],
 cross-react in the assay. Thus, assay results reflect the total
 concentration of the parent compound and its metabolites. Samples were
 diluted 10- to 100-fold before analysis as needed to complement the kit’s
 quantification range.  
 <i>ALCB reproduction experiment</i> In the other 16 field cages, we
 placed the California-native ornamental plants <i>Erigeron
 glaucus</i>, <i>Sphaeralcea ambigua</i>, and
 <i>Baileya multiradiata</i> in 2-L pots (not previously
 treated with insecticides), purchased from a local native plant nursery
 (Valley Center, CA) in 2019 and 2020. We selected these species based on
 their popularity at nurseries, drought tolerance to ensure bloom in low
 irrigation conditions, and from surveys of wild bee visitation at
 nurseries (Cecala and Wilson Rankin, in review). We also included pots of
 lacy<i> </i>phacelia<i> </i>and alfalfa
 (<i>Medicago sativa</i>). The ratio of plant species in each
 cage varied slightly between years due to availability (table S1). We
 divided field cages into four experimental blocks, with one cage assigned
 to each of four treatments (table 1, figure S1<i>b</i>). The
 low dosage (~3% label rate) imidacloprid treatment was not included in the
 bee reproduction experiment. We manipulated plant irrigation
 levels as in the phacelia nectar experiment. Although soil VWC varied
 across plant species, VWC in high irrigation pots was 44% higher than that
 of soil in low irrigation pots (figure S3<i>b</i>). In
 imidacloprid treatment cages, we applied Marathon<sup>®</sup>
 four weeks before introducing bees to allow for translocation [as in 14].
 We applied Marathon<sup>®</sup> to each plant species
 <i>except</i> alfalfa, which we anticipated would serve as the
 principal leaf clipping source for nesting female ALCBs, to ensure bees
 were primarily exposed to imidacloprid through consumption of pollen and
 nectar, and not via leaf tissue clipping. In each cage, we provided one nest
 block constructed according to USDA-ARS specifications [35] facing
 southeast. Each block contained 60 drilled tunnels into which we inserted
 paper straws (diameter 5 mm, length 12.7 cm). We purchased commercially
 reared ALCB pre-pupae from Canada (JWM Leafcutters, Parkside, SK, Canada)
 and allowed them to develop into adults in an incubator at 30.3 ± 0.1 °C,
 57.2 ± 0.4% relative humidity (mean ± s.e.) and a 12-hour light-dark
 cycle. Emergence occurred after 21 days. In mid-June 2019 and 2020, we
 introduced 30 male and 20 female ALCBs inside each cage to approximate sex
 ratios in commercial populations [36]. Over the following six weeks, two
 to three times per week, we recorded floral abundance (for each plant
 species and the entire cage) and ALCB foraging activity in each cage using
 ordinal indices. For floral abundance, we assigned: ‘0’ if no flowers were
 present, ‘1’ if a few flowers were present, ‘2’ if flowers covered 10–50%
 the cage area, and ‘3’ if flowers covered &gt; 50% the cage area. For
 bee foraging activity, we visually monitored the inside of each cage for
 10 seconds [similar to 32] and assigned: ‘0’ if no foraging bees were
 visible, ‘1’ if 1–3 bees were visible, ‘2’ if 4–10 bees were visible, and
 ‘3’ if &gt; 10 bees were visible. We also recorded ambient temperature
 during observations. After six weeks, we collected all
 straws and individually labeled and weighed each. After three weeks of
 storage at 22 °C, straws were kept at 5 °C over the winter. After at least
 four months, straws were incubated again. However, no bees emerged in
 either year, potentially due to the lack of a fluctuating temperature
 regime during cold storage [37], insufficient quantity or quality of
 pollen provisions, or adults not entering diapause (though we did not
 observe any non-diapausing adults emerging) [38]. To assess reproduction,
 we dissected straws and inspected the contents, quantifying incomplete
 cells (leaf pieces not fully formed into a cell), empty cells (fully
 constructed, but with no contents), cells containing pollen provisions but
 no brood, and cells with brood (and the developmental stage).
  
 <i>Statistical analysis</i> We conducted all analyses in R
 (version 3.3.3) [39]. All means are reported ± s.e. In all models, we
 checked for collinearity using function ‘vif’ (<i>car</i>)
 [40]. To assess how treatments impacted volume, sugar concentration, and
 imidacloprid concentration of phacelia nectar, we constructed linear mixed
 models (LMMs) using function ‘lmer’ (<i>lme4</i>) [41]. We
 included as fixed effects irrigation level and imidacloprid treatment (and
 their interaction) and number of days elapsed since imidacloprid
 application. In the volume and sugar concentration models only, we
 included daily high temperature, known to influence nectar secretion in
 phacelia [33]. Cage nested within block served as random effects. Volume
 and imidacloprid concentration were
 log<sub>10</sub>(<i>x</i>+1)-transformed. In all
 models, we used function ‘emmeans’ (<i>emmeans</i>) [42] for
 post-hoc comparisons (Tukey’s HSD tests) as appropriate. To assess treatment effects on
 indices of cage-level floral abundance and ALCB foraging activity, we
 constructed additional LMMs. We included as fixed effects irrigation
 level, imidacloprid treatment, and year (and all interactions), and number
 of days elapsed since bees were added to cages. For the bee foraging
 activity model, we also included ambient temperature during the
 observation. We again included cage nested within block as random effects.
 To assess how treatments influenced nest initiation by ALCBs, we
 constructed a generalized linear mixed model (GLMM) using function ‘glmer’
 (<i>lme4</i>) and a logit link. In this model, we treated each
 straw (empty or not) as a replicate, noting whether there was
 <i>any</i> evidence of nest construction or not. We included
 the same fixed and random effects as the bee foraging activity model.
 Furthermore, to determine if treatments influenced (per cage) the number
 of cells (incomplete or complete) constructed, number of cells
 <i>containing brood</i>, or <i>proportion</i> of
 cells containing brood, we constructed LMMs with the aforementioned
 predictor variables. We square-root transformed the number of cells per
 cage and number of cells containing brood per cage. Finally, to determine
 if female ALCBs clipped plants <i>other</i> than alfalfa
 (which would further expose them to imidacloprid), we used Fisher’s exact
 tests. We tested if the number of cells (incomplete or complete)
 constructed with versus without alfalfa (never treated with imidacloprid)
 differed between imidacloprid or irrigation treatments.
description: "phacelia_nectar_data.xlsx" contains data from the
 experiment examining the effects of irrigation level and imidacloprid
 dosage on <em>Phacelia tanacetifolia</em> nectar
 characteristics.
 "alcb_reproduction_data.xlsx" contains data from the
 experiment examining the effects of irrigation level and imidacloprid
 dosage on foraging and reproduction of alfalfa leafcutting bees.
 Descriptions of tabs and variables are provided in the first tab
 (metadata) of each Excel file.