DOI: 10.5061/dryad.qnk98sfmd
title: Fertilizer and herbicide alter nectar and pollen quality with consequences for pollinator floral choices

description: Flower-visiting insects in agroecosystems forage on weeds exposed to
 agrochemicals that may compromise the quality of their floral resources.
 We conducted complementary field and greenhouse experiments to evaluate:
 1) the effect of low concentrations of agrochemical exposure on nectar and
 pollen quality and 2) the relationship between floral resource quality and
 insect visitation. We found pollen amino acid concentrations were lower in
 plants exposed to low concentrations of herbicide, and pollen fatty acid
 concentrations were lower in plants exposed to low concentrations of
 fertilizer, while nectar amino acids were higher in plants exposed to low
 concentrations of either fertilizer or herbicide. Exposure to low
 fertilizer concentrations also increased the quantity of pollen and nectar
 produced per flower. The responses of plants exposed to the experimental
 treatments in the greenhouse helped explain insect visitation in the field
 study. The insect visitation rate correlated with nectar amino acids,
 pollen amino acids, and pollen fatty acids. An interaction between pollen
 protein and floral display suggested pollen amino acid concentrations
 drove insect preference among plant species when floral display sizes were
 large. We show that floral resource quality is sensitive to agrochemical
 exposure and that flower-visiting insects are sensitive to variation in
 floral resource quality.
description: Greenhouse Study Design
 We
 selected seven plant species for our experiment (<em>Cirsium
 vulgare</em>, (Savi) Ten. Asteraceae<em>, Epilobium
 hirsutum</em>, L. Onagraceae<em>, Filipendula
 ulmaria</em>, (L.) Maxim.<em> </em>Rosaceae<em>,
 Hypochaeris radicata</em>, L.<em>
 </em>Asteraceae<em>, Origanum vulgare,</em> L.<em>
 </em>Lamiaceae<em>, Phacelia tanacetifolia,</em>
 Benth.<em> </em>Boraginaceae, and <em>Plantago
 lanceolata,</em> L. Plantaginaceae). These comprised six native
 perennial and one non-native annual herbaceous species (<em>P.
 tanacetifolia</em>), selected as pollinator-attractive
 (Clifford, 1962; Russo et al.,
 2022), and likely to be
 found on agricultural field edges in Europe. They represent a diverse
 group of plant families with regard to floral resource quality (Ruedenauer
 et al., 2019b; Vaudo et al., 2020; Zu et al., 2021). To
 collect sufficient quantities of pollen and nectar for nutritional
 analyses from each species in each treatment, and to avoid interrupting
 natural patterns of insect visitation in the field, we conducted a
 concurrent greenhouse study. We collected 20 wild individuals of each
 perennial species in the spring of 2017 and planted them in pots with
 field soil in the greenhouse. The annual species (<em>P.
 tanacetifolia</em>) was planted in potting media in the greenhouse
 with 20 seeds (purchased from a regional seed supplier: QuickCrop Ireland
 ©) to each pot. After
 the plants were established, we randomly assigned five individuals of each
 species to each treatment (see below). Treatments were applied with a
 watering can holding 10 litres of water, applied across the five
 individuals of each of the seven species once a week. The plants were also
 treated with an insecticidal/fungicidal product (SB Plant Invigorator ©)
 once a week to control pest outbreaks. The insecticidal treatment was
 applied evenly across all plant species and
 treatments. The four
 experimental treatments were designed to simulate non-target agrochemical
 exposure on field edges: (1) control (20 L water), (2) run-off
 concentration of NPK fertilizer (in 10 L water plus 10 L untreated water),
 (3) low concentrations of herbicide (glyphosate in 10 L water plus 10 L
 untreated water), or (4) a combination treatment (same low concentrations
 of NPK in 10 L water and glyphosate in 10 L water). The treatments were
 mixed with 10 litres of water used for a foliar application once a week
 for three months. The first four weeks of application were the highest
 concentration, the second four weeks lower, and the last four weeks the
 lowest (Table 1). These applications were based on estimates of field-edge
 exposure; there is commonly a high concentration spring application of
 chemical fertilizer and herbicide, followed by decreasing exposure later
 in the growing season. Concentrations were selected using published
 studies of fertilizer run-off (Korsaeth & Eltun, 2000; Bertol et al., 2007; Craig & Mannix, 2009; Russo et al., 2020). Because glyphosate is not mobile in the groundwater, we based our highest glyphosate application on the US EPA’s Maximum Contaminant Level (MCL) for safe drinking water (United States Environmental Protection Agency, 2003). The highest concentration we applied was less than half the maximum level detected in (Silva et al., 2019), or roughly 7.6 % of a standard annual field application (1440 g/ha) (Dupont, Strandberg & Damgaard, 2018). Outside of this treatment regime, the plants in our experiments received only water. In the greenhouse, we collected pollen and nectar daily between the hours 0600–1000. We collected sufficient quantities of pollen for nutritional analyses (8 to 16 mg per species in each treatment) from six of the seven species (all except <em>O. vulgare</em>, which produced very little pollen in the greenhouse), and sufficient quantities of nectar for nutritional analyses from three of the seven species. <em>Cirsium vulgare, H. radicata, F. ulmaria</em>, and <em>P. lanceolata</em> either did not produce nectar or had small inflorescences from which we were not able to obtain sufficient quantities of nectar. We collected nectar and pollen from greenhouse plants to avoid interrupting normal insect foraging behaviour in the field. We counted the inflorescences from which we collected pollen and nectar on each collection day. Pollen was collected from dehisced anthers directly into Eppendorf tubes and transferred immediately to a -20°C freezer. For <em>F. ulmaria, E. hirsutum</em>, and <em>P. tanacetifolia</em> we collected whole anthers; while for <em>H. radicata, C. vulgare</em>, and <em>P. lanceolata</em>, we collected fresh pollen. We collected nectar with microcapillary tubes and measured the filled volume before transferring them to a -20°C freezer. We calculated the average amount of nectar per inflorescence. Because pollen was collected fresh and later dried for analysis, we measured the total dry weight of pollen divided by the total flowers sampled for each species and treatment at the end of the season (Table S1). Field Study Design We conducted a field experiment to measure the effects of non-target agrochemical exposure on plant growth and pollinator visitation from 2017–2018 in Dublin, Ireland (Russo et al., 2020). The study consisted of four experimental treatment plots (2 x 2m) replicated across eight sites over two years (four sites in 2017 and four different sites in 2018). The sites were located in urban Dublin and selected based on space availability in collaboration with businesses and research entities, as well as the absence of outside exposure to herbicide or fertilizer. Each plot contained the same plant community with equal densities of individuals of the same seven plant species as the greenhouse experiment (above). The same experimental treatments as described above for the greenhouse experiment, with the same concentrations of fertilizer and herbicide, were used in the field experiment in both years of the study (Table 1). Treatments were randomly assigned to plots within a site at the beginning of the season. For the purposes of this study, we were primarily interested in the pollinator visitation from the field experiment. Once the plants in the field began to flower, we sampled insects that came in contact with the reproductive parts of the inflorescences for at least 1 s. On each sample day at each site, we collected flower-visiting insects on each flowering plant species at each plot for five minutes using an insect vacuum (total of 96 sample days, 623 date-plot-samples, or 2036 five-minute samples (approximately 170 h)). Each site was visited between 12–14 times for collections in both 2017 and 2018; the number of site collections varied due to variation in the timing of flowering between different sites. We sampled between the hours of 0700 and 1800 (84 % of the samples were collected between from 1000 to 1600). The order in which we visited sites, plots within sites, and species within plots was randomized during each sampling event. We also recorded the number of inflorescences of each species during each sampling event. Insect species that could be identified in the field (specifically <em>Apis mellifera</em>, Linneaus Apidae<em>, Episyrphus balteatus</em>, De Geer Syrphidae<em>, Bombus pascuorum</em>, Scopoli Apidae<em>, B. lapidarius</em>, Linnaeus, and <em>B. pratorum, </em>Linnaeus) were released alive at the end of the sampling event. Collected specimens were transferred to a freezer and identified at the end of the field season [53,54]. Bee identifications were verified by Dr. Úna Fitzpatrick of the National Biodiversity Data Centre (Waterford, Ireland), while hoverfly specimens were identified by Dr. Martin Speight (Trinity College Dublin, Ireland). Chemical Analyses We quantified amino acids in 3-6mg of pollen of each of six plant species and four treatments and analysed three subsamples of each pollen sample for amino acids (72 samples). We used high-performance liquid chromatography (HPLC) and a spectrum analyser to identify the peaks of the individual amino acids, and the area under the curve of the spectra corresponded to the quantity of individual amino acids (full description in Supplemental Materials).  We quantified fatty acids in 5–10mg of pollen of each of six plant species and four treatments and analysed two subsamples of each pollen sample (48 samples) (Trinkl et al., 2020). The fatty acids were analysed via gas chromatography/mass spectrometry (GCMS, Supplemental Materials). We quantified the amino acids and sugars of the nectar from three plant species and four treatments (12 samples) as described in Venjakob et al. (2020) at the University of Freiburg in Freiburg, Germany. The analysis of the nectar amino acids and sugars was carried out chromatographically with an HPLC system (Agilent Technologies 1260 Series; Agilent, Böblingen, Germany, Supplemental Materials). Data Analysis Our ultimate goal was to determine whether the treatments in the greenhouse resulted in changes in pollen and nectar quality and whether these changes corresponded to the changes in pollinator visitation we observed in the field. As such, we aggregated the field visitation data over time to each plant species in each treatment. First, we tested for differences between treatments among plant species in terms of the concentrations of (a) pollen total amino acids (summed concentrations of all amino acids), (b) pollen total fatty acids (summed concentrations of all fatty acids), (c) number of different pollen fatty acids, (d) pollen production per flower, (e) nectar total amino acids, (f) nectar total sugars, and (g) nectar production per flower. We used generalized linear mixed effect models (GLMMs, R package “lme4”) with treatment as a fixed effect and subsample nested within plant species as the random effect (Bates et al., 2014). Note these are not true replicates because we pooled pollen and nectar across individuals of a species within treatments from the greenhouse to have a sufficient quantity to analyse. Instead, these numbers represent variation within and between samples relative to variation within our subsamples. We provide results from among species comparisons in the supplement (Table S2). Next, we tested for a correlation between the pollen or nectar attributes, or between the pollen and nectar attributes and flower visitation in the subgroups laid out above for data aggregated at the species and treatment level. We used visitation rate (abundance of visitors in a given sample divided by the size of the floral display (inflorescence size*number)) as a normalized measure for comparing visitation among plant species with variable floral displays. When visitation rate increases, it indicates a per floral unit preference (Russo et al., 2019b, 2020). We also tested the relationship between visitation rate and the ratio of proteins:lipids in the pollen (here pollen amino acids vs pollen fatty acids). We separately evaluated the following groups of flower-visiting insects: (1) pollen-collecting bees (females of non-parasitic species, 1320 observations), (2) all bees (1755 observations), (3) bumblebees (1178 observations), (4) honeybees (386 observations), (5) hoverflies (Syrphidae, 677 observations), and (6) all flower-visiting insects (2567 observations). We hypothesized pollen-collecting bees would be most sensitive to pollen quality because they are provisioning offspring, and that bumblebees would be sensitive to protein:lipid ratios in the pollen as found in previous studies (e.g. (Vaudo et al., 2016a; Russo et al., 2019b)). Next, we tested whether any of the attributes of pollen or nectar significantly improved the fit of the visitation data in the field, compared to published models of pollinator visitation (Russo et al., 2020). These tested whether pollinator visitation was influenced by pollen/nectar quality beyond previously established variables. We used a model selection process, choosing the model with the lowest AICc (function <em>dredge</em> in the package “MuMin” (Barton, 2009)). The site and plant species were treated as random effects, while the floral display and experimental treatment were fixed effects (Table 2 for full model structures). We then tested for interactions between the fixed effects in the model with the lowest AICc, and removed fixed effects that were not significant. We reported the marginal and conditional R<sup>2</sup> for all models. Because the field visitation data were zero-inflated, we ran two sets of models. First, we ran a model with a binary presence/absence response variable. Next, we ran a model using only samples where flower-visiting insects were recorded, with insect abundance as the response variable.