# The conserved and high K-to-Na ratio in sunflower pollen: Possible implications for bee health and plant-bee interactions

Michal Filipiak, Morgan W Shields, Sarah M Cairns, Megan N C Grainger, Stephen D Wratten

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## To cite this version:

Michal Filipiak, Morgan W Shields, Sarah M Cairns, Megan N C Grainger, Stephen D Wratten. The conserved and high K-to-Na ratio in sunflower pollen: Possible implications for bee health and plant-bee interactions. Frontiers in Plant Science, 2022, 13, pp.1042348. 10.3389/fpls.2022.1042348. hal-03835937

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EDITED BY

Ji Zhang,
Yunnan Academy of Agricultural
Sciences, China

REVIEWED BY

Pierre Lau,
USDA, United States
Diana L. Six,
University of Montana, United States

*CORRESPONDENCE

Michał Filipiak
michal.filipiak@uj.edu.pl

†Deceased

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CITATION

Filipiak M, Shields MW, Cairns SM, Grainger MNC and Wratten SD (2022) The conserved and high *K*-to-*Na* ratio in sunflower pollen: Possible implications for bee health and plant-bee interactions.
*Front. Plant Sci.* 13:1042348.
doi: 10.3389/fpls.2022.1042348

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© 2022 Filipiak, Shields, Cairns, Grainger and Wratten. This is an open-access article distributed under the terms of the [Creative Commons Attribution License (CC BY)](https://creativecommons.org/licenses/by/4.0/). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

The conserved and high *K*-to-*Na*
ratio in sunflower pollen: Possible
implications for bee health and
plant-bee interactions

Michał Filipiak¹*, Morgan W. Shields², Sarah M. Cairns²,
Megan N. C. Grainger³ and Stephen D. Wratten²†

¹Institute of Environmental Sciences, Faculty of Biology, Jagiellonian University, Kraków, Poland,
²Bio-Protection Research Centre, Lincoln University, Lincoln, New Zealand, ³School of Science,
University of Waikato, Hamilton, New Zealand

Sodium (Na) concentrations are low in plant tissues, and its metabolic function in plants is minor; however, Na is a key nutrient for plant consumers. Previous studies have thus far focused on Na concentration. Nevertheless, a balanced potassium (K) to Na ratio (*K*:Na) is more important than Na concentration alone since food with high *K*:Na has detrimental effects on consumers irrespective of Na concentration. Therefore, plants may actively regulate *K*:Na in their tissues and products, shaping plant-insect interactions. Studies considering nutritional aspects of plant-insect interactions have focused on nonreproductive tissues and nectar. In this study, we consider pollen as serving a primary reproductive function for plants as well as a food of pollinivores. Plants might regulate *K*:Na in pollen to affect their interactions with pollinivorous pollinators. To investigate whether such a mechanism exists, we manipulated Na concentrations in soil and measured the proportion of K, Na, and 13 other nutrient elements in the pollen of two sunflower (Helianthus annuus) cultivars. This approach allowed us to account for the overall nutritional quality of pollen by investigating the proportions of many elements that could correlate with the concentrations of K and Na. Of the elements studied, only the concentrations of Na and K were highly correlated. Pollen *K*:Na was high in both cultivars irrespective of Na fertilization, and it remained high regardless of pollen Na concentration. Interestingly, pollen *K*:Na did not decrease as pollen increased the Na concentration. We hypothesize that high *K*:Na in pollen might benefit plant fertilization and embryonic development; therefore, a tradeoff might occur between producing low *K*:Na pollen as a reward for pollinators and high *K*:Na pollen to optimize the plant fertilization process. This is the first study to provide data on pollen *K*:Na regulation by plants. Our findings broaden the understanding of plant-bee interactions and provide a foundation for a better understanding of the role of the soil-plant-pollen-pollinator pathway in nutrient cycling in ecosystems. Specifically, unexplored costs and tradeoffs related to balancing the *K*:Na by plants and pollinivores might play a role in past and current shaping of pollination ecology.

KEYWORDS

ecological stoichiometry, ionomics, elementome, pollen, pollination, pollinivory, potassium, sodium
Introduction

Plant tissues and products differ in their chemical profiles but
have one thing in common: high concentrations of K and low
concentrations of Na (Kaspari, 2020). This unique plant feature
poses a challenge to consumers of plant matter, and an unbalanced
K:Na in food reduces the fitness of plant consumers (Kaspari, 2020;
Kaspari, 2021). The K:Na ratio in bee food resources may be more
important than the overall Na concentration since Na and K are
physiologically bound (Kaspari, 2020). Animals need a balanced K:
Na to maintain their phosphate homeostasis and sensing; they also
require Na-K ATPases, osmoregulatory ability, the ability to adapt
to cold and heat and to maintain their microbiomes, which also
need a balanced Na:K (Pedersen and Zachariassen, 2002; Dow,
2017; Kaspari, 2020). Overall, an unbalanced K:Na in food reduces
the fitness of plant consumers.

Regarding pollinivores, acute bee paralysis and even death may
be caused by an excessively high K:Na ratio in Western honey bee
food [(Horn, 1985); see also Kaspari (2020) who provided the
context and biological explanation for this finding]. Furthermore,
the ratio of K:Na in pollen consumed by wild bee larvae may affect
bee fitness, as feeding experiments have shown that concentrations
of both K and Na in wild bee larval food influence fitness-related
traits, such as mortality, body mass and cocoon development
(Filipiak and Filipiak, 2020; Filipiak et al., 2022). These effects
may potentially influence wild bee populations and communities
(Filipiak et al., 2022). Petanidou (2007) suggested that pollinivores
may be attracted to food sources having balanced K:Na. Indeed, in
contrast to earlier studies focusing on concentrations of individual
elements (Lau and Nieh, 2016; Bonoan et al., 2017; Bonoan et al.,
2018; de Sousa et al., 2022), our recent study (Cairns et al., 2021)
showed that the Western honey bee preference for “dirty water”, i.e.,
water enriched with nutrients, is driven by the K:Na ratio rather
than by the Na concentration alone.

Correspondingly, it was suggested that plants may regulate
concentrations of Na in their tissues and products in response to
their interactions with herbivores and pollinators (Kaspari, 2020;
Kaspari, 2021; Finkelstein et al., 2022). A literature review of the
elemental composition of pollen suggested that K concentrations
are among the most stable of the 11 elements for which data are
available, while Na concentrations are the most variable (Filipiak
et al., 2017). Additionally, plant tissues and products with higher
concentrations of Na, are preferred by herbivores (e.g. (Nicolson
and W.-Worswick, 1990; Borer et al., 2019); see (Finkelstein
et al., 2022) for experimental confirmation). We therefore
hypothesize that plants regulate K:Na in pollen by increasing
or decreasing the concentration of Na in pollen. Such regulation
might serve either (1) to encourage pollen consumption, making
pollen a nutritionally balanced reward for pollinivores and
improving animal pollination services, or (2) to discourage
pollinivores from pollen eating, making pollen nutritionally
inadequate for consumers and relying on wind pollination or

specialized pollination services by physiologically
adjusted consumers.

In this brief research report, we explored the idea that pollinivore-pollinated plant actively regulates K:Na in pollen while maintaining proportions of other elements for the overall nutritional balance of pollen for pollinators. To that end we increased an Na concentration in the soil assuming that such an increase would result in an increased Na concentration in pollen produced by a pollinivore-pollinated plant. We hypothesized that an increase in the Na concentration in pollen would result in a decreased K:Na ratio whilst maintaining the overall proportion of the remaining elements (i.e., elements other than K and Na), therefore improving the entire nutritional quality of pollen for pollinivores. To test this hypothesis, we investigated whether and how nutritional quality changes with an increased Na concentration in sunflower (Helianthus annuus L.) pollen with a focus on the K:Na ratio. Nutritional quality was reflected in the pollen stoichiometric phenotype, i.e., the proportion of vital nutrient elements [for detailed explanation see (Filipiak and Filipiak, 2022)]. Sunflower was chosen because it is a common monoculture and mass-flowering crop that provides large amounts of foraging resources for bees (Todd et al., 2016). We discuss the results within the context of a theory of sodium ecology, as introduced by Kaspari (2020), with a special focus on the nutritional needs of pollinivorous pollinators, i.e., bees.

Materials and methods

Experimental setup

To evaluate pollen nutritional quality, we measured the
concentrations and proportions of 15 vital elements in pollen:
Na, K, P, Mg, Ca, Fe, Mn, Cu, Zn, B, V, Cr, Co, Ni and Sr (note
that studied heavy metals are among vital elements (Fraústo da
Silva and Williams, 2001; Williams and Rickaby, 2012; compare
with Jager et al., 2013; Świątek et al., 2020; Filipiak and
Bednarska, 2021). This multi-element approach allowed us to
study the correlation of Na and K concentrations in pollen with
concentrations of other elements contributing to the overall
nutritional quality of pollen. To consider as much natural
variation in the concentrations of nutrients as possible, we
used two sunflower cultivars grown under different conditions.

Plants were grown in a closed greenhouse in May-October 2020
(winter-spring) in Lincoln, New Zealand, and involved two
sunflower (Helianthus annuus L.) cultivars, yellow pygmy
(hereafter YP) and Baltic, with three Na treatments comprising
NaCl (high: 10.728 g/pot, low: 7.152 g/pot, and control: no added
Na). There were six treatments in total, each with 10 replicates. The
NaCl concentrations were calculated based on findings from Bhatt
and Indirakutty (1973) on Na uptake by sunflowers. Therefore, the
concentrations of NaCl added were based on how much Na
sunflowers had been found to mobilize from soil. We used two staggered sowing dates (SDs, SD 1: 20/05/2020, and SD 2: 10/06/ 2020) to minimize the risks of environmental disturbance and inadequate pollen sample size. NaCl was administered as crystals sprinkled over the soil, followed by a 5-second, moderate-intensity flow of tap water to allow the NaCl to enter the soil profile. NaCl was applied at growth period stage 3 and reproductive stage 4 (R4) (Schneiter and Miller, 1981) at the beginning of pollen development when the developing pollen grains acquired nutrients during microsporogenesis. Growth period stage 3 (Robinson, 1971) was defined as the point at which the inflorescence head was visible, and yellow petals were visible underneath the unfurling bracts of the inflorescence. Reproductive stage R4 (Schneiter and Miller, 1981) was characterized as when the inflorescence began to open up and, when viewed from above, small ray florets were visible on the inflorescence head. Each replicate consisted of one 14 L pot with two plants per pot (in case one plant died or did not flower). Pots were randomized and watered 2-3 times per week.

Additionally, we grew plants under the same conditions and treatments as above but with 12 replicates and three sowing dates (SD 1: 20/05/2020, SD 2: 10/06/2020, and SD 3: 01/07/2020). Replicates at growth period stage 3 (Robinson, 1971) and reproductive stage R4 (Schneiter and Miller, 1981) were moved from the greenhouse to a fenced area with frost cloth to reduce wind and frost damage. Replicates were arranged in a randomized block design of 12 blocks, with one replicate of each treatment per block. Blocks were 1 m apart, and replicates within each block were 0.5 m apart.

## Pollen collection and elemental analysis

Inflorescences from the greenhouse were harvested in August at reproductive stage 5.5 (R5.5) when half the disc inflorescences were undergoing anthesis and pollen was available (Schneiter and Miller, 1981). Inflorescences from the field were harvested in September-October at R5.5 after one week of field exposure. Under laboratory conditions, inflorescences were tapped to allow pollen to drop onto greaseproof baking paper in a shallow tray. The pollen was then carefully transferred from the paper into sterile sieves to remove contaminants and stored in Falcon tubes. If less than 20 mg of pollen was collected from a single replicate, then it was pooled with additional pollen from replicates of the same treatment with the same sowing date to reach the minimum (20 mg) amount of pollen required for stoichiometric analyses.

Pollen samples were stored at -4°C. They were accurately weighed and digested with nitric acid (67%, 0.2 mL) at 80°C for 1 hour, followed by a further hour with the addition of hydrogen peroxide (30%, 0.1 mL). Distilled and deionized (Type 1) water (18 mΩ, 2 mL) was added after cooling so that the resulting solution contained 2% HNO₃ for instrument analysis. Samples were analyzed by an inductively coupled plasma mass spectrometer-QQQ (Agilent) controlled by MassHunter

Workstation (version 4.5) to measure chemical elemental concentrations (Grainger et al., 2020).

## Statistical analyses

To investigate whether and how the nutritional quality of pollen changes with increasing Na concentration in pollen, we first analyzed whether pollen samples obtained from different cultivars and various Na treatments differed in their stoichiometric phenotypes [proportions of elements; also called elementome (Peñuelas et al., 2019; Fernández-Martínez, 2022)] by performing permutational analysis of variance (PERMANOVA) in PAST 4.07b (Hammer et al., 2001). Furthermore, to explore how the Na concentration in pollen is related to the concentrations of the other measured elements, we performed redundancy analysis (RDA) in Canoco 5 (Smilauer and Lepš, 2014). In the case of RDA, we also performed independent analyses of variance (ANOVAs) for the 1st- and 2nd-axis scores of the RDA using Statistica 13 (TIBCO Software Inc.). The RDA showed that pollen Na concentration and pollen K concentration were highly correlated; thus, we further investigated the strength of this relationship by calculating the R² value of the correlation.

## Results

A higher concentration of Na in pollen did not translate into a higher nutritional value of pollen. Although the concentrations of the majority of studied elements were not associated with Na concentration (Table 1 and Figure 1), the concentration of K was highly correlated with that of Na (Figure 1), meaning that an increase in the Na concentration of pollen did not correspond to a considerable change in the K:Na of pollen.

Regarding multielemental composition, the two sunflower cultivars had significantly different (p<0.05) levels of nutritional quality, as reflected by the proportions of the studied nutritional elements (Table 1 and Figure 1), but within cultivars, the multielemental composition did not differ among the Na treatments (Figure 1). The majority of the observed variance (24.61%) was associated with the 2nd RDA axis and was driven by manganese (Mn, 2nd axis loading: 0.78), copper (Cu, 0.77),

<table><caption>TABLE 1 The results of two-way PERMANOVA.</caption><thead><tr><th></th><th>df</th><th>F</th><th>p</th></tr></thead><tbody><tr><td>Cultivar (A)</td><td>1</td><td>12.587</td><td>0.0001</td></tr><tr><td>Na-treatment (B)</td><td>2</td><td>1.445</td><td>0.2266</td></tr><tr><td>A x B interaction</td><td>2</td><td>0.244</td><td>0.8888</td></tr><tr><td>Residual</td><td>55</td><td></td><td></td></tr></tbody></table>

In sunflower pollen, the proportion of 15 elements differed between cultivars and did not differ between Na treatments. There was no cultivar x Na-treatment interaction.
<figure><img src="image_4.png" /><figcaption>FIGURE 1</figcaption></figure>

Multivariate analysis (RDA) of the relationships between the studied element concentrations and the proportion of Na in the studied pollen. N = 61, *p* = 0.002; (A) RDA plot, with the first two axes presented. The percentage of variation explained is given for both axes. The lengths of the vectors represent the contributions of the elements to the pattern shown (longer vector = greater contribution). The direction of the vector shows the axis to which it contributes. (B) ANOVA performed for both axis scores; different letters denote significant differences between the studied pollen groups. Different cultivars, but not Na treatments, were separated from each other. The addition of NaCl to soil increased the variation in concentrations of measured elements in pollen.

zinc (Zn, 0.74), and strontium (Sr, 0.73). The 1st axis, associated with Na concentration (independent variable in the RDA), explained 17.75% of the observed variance. Additionally, the K concentration also strongly contributed to this pattern (1st axis loading: 0.87). Regarding the Na and K concentrations, the YP cultivar was richer in these two elements than the Baltic cultivar, and the addition of Na (NaCl treatments: low and high) increased the variation in the concentrations of these two elements in the pollen of both cultivars (Figures 1, 2). Figure 2 shows the Na and K concentrations and descriptive statistics (median, interquartile range, min-max values) for these two elements in collected samples of pollen. Figure 3 shows the correlation between Na and K concentrations. The average K:Na ratio calculated for the concentrations was 269, and the molar average K:Na ratio was 158.

Discussion

Surprisingly, we did not find evidence supporting our
hypothesis that the K:Na ratio in sunflower pollen decreases

with increasing Na concentration in pollen. The sodium
concentration was correlated with the K concentration, and
the pollen K:Na ratio remained high regardless of the Na
concentration. The question arises ‘what K:Na ratio is optimal
for pollinivorous pollinators, and how much it differs from the
K:Na ratio measured in this study?’ Scarce literature data allow
for comparison of the sunflower pollen K:Na with nutritional
needs of two pollinivore species based on their stoichiometric
phenotypes (Filipiak et al., 2017; Filipiak, 2019). The optimal
food K:Na concentration ratio is approximately 15-20 for the
Western honey bee A. mellifera and approximately 50-90 for the
solitary wild bee Osmia bicornis L. (Filipiak et al., 2017; Filipiak,
2019). The K:Na concentration ratio in the pollen measured in
this study was 15 times (A. mellifera) and 4 times (O. bicornis)
higher than the optimal ratio. Therefore, we hypothesize that
bees, especially A. mellifera, must obtain Na from sources other
than pollen to decrease the overall K:Na ratio of their food and,
in the case of A. mellifera, to produce nutritionally balanced jelly
for their larvae. Jelly is rich in Na (Wang et al., 2016), and this
high concentration of Na in jelly cannot be obtained from pollen
alone (Filipiak et al., 2017).
<figure><img src="image_5.png" /><figcaption>FIGURE 2
Concentrations of Na and K in the studied pollen—variance and descriptive statistics. The addition of NaCl to the soil increased the variation in both Na and K in pollen. N = 61.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>FIGURE 3
Correlation between Na and K concentrations in the studied pollen. N=61.</figcaption></figure>

Our findings have implications for plant-pollinator interactions that are shaped by mismatches between the nutritional demand of pollinators and the nutritional supply offered by plants (Filipiak and Filipiak, 2022; Parreño et al., 2022), resulting from different or even conflicting interests of plants and pollinators (van der Kooi et al., 2021). The feeding strategy of pollinivores contrasts with that of other herbivores due to the exceptional nutritional richness of pollen. The nutritional quality of pollen is more similar to the quality of animal tissues than other plant products, making it an excellent food (Willmer, 2011). However, if a high and stable K:Na ratio is a general trait of pollen, regardless of its Na concentrations, pollen is nutritionally limiting for bees, which in turn might have negative consequences for bees (Kaspari, 2020). Plants selectively absorb certain nutrients in specific proportions according to the physiological requirements of the plant, and this process is part of

their environmental adaptation (Huang and Salt, 2016; Vaudo et al., 2022). Considering the minor role of Na in plant metabolism, one could expect that plants using pollen as a nutritional reward for pollinators would fill pollen with a large proportion of unnecessary Na to stimulate pollination services. This should result in decreased pollen K:Na. However, we did not observe the effect of low K:Na in sunflower pollen under various scenarios of Na fertilization of the two sunflower cultivars. One possible explanation is that high pollen K:Na is needed to optimize plant fertilization, embryo creation, and seed development, as K homeostasis plays an important role in pollen germination and embryo and seed development (White and Karley, 2010; Dresselhaus et al., 2016; Padmanaban et al., 2017). Indeed, the review of available data on K and Na concentrations suggests that pollen in general has a high K:Na ratio, similar to that of other plant tissues, even considering its high variability (Filipiak et al., 2017). It is
also possible that plant taxa differ in their pollen K:Na, depending on pollination syndromes [it was already suggested that concentrations of vital nutrients in zoophilous plants pollen may have been shaped by the nutritional needs of their pollinators (Ruedenauer et al., 2019)]; however, this possibility has yet to be investigated. Additionally, pollinivores may supplement Na from sources other than pollen to balance the overall K:Na in their progeny diets (Dorian and Bonoan, 2020; Khan et al., 2021), especially if direct larval food is produced by adults, as with Western honey bees (Filipiak et al., 2017). This can take place through, for example, cannibalistic behavior, drinking animal secreta, or puddling (Simpson et al., 2006; Bänziger and Bänziger, 2010; Dangles and Casas, 2012; de la Rosa, 2014; Dorian and Bonoan, 2020). It is possible that a tradeoff occurs between producing low K:Na pollen for pollinators and high K:Na pollen physiologically optimizing fertilization. The physiological mechanism of such a tradeoff and its ecological consequences constitute an interesting topic for future studies. Importantly, this study shows that, as suggested earlier (Filipiak and Filipiak, 2022; Lau et al., 2022; Parreño et al., 2022), a holistic approach to the chemical composition of pollen, complementary to the most common currently used examination of the concentration and proportion of proteins and lipids, is necessary to develop our understanding of the nutritional ecology of bees and consequently their health.

Although there are studies reporting both potassium and sodium concentrations in plant tissues [e.g. (Zhang et al., 2012; Zhao et al., 2014)], at present, the current study is the only one providing data on pollen K and Na regulation by plants. Therefore, although we do not exclude the possibility of flexible regulation of the K:Na ratio in pollen by plants, we are not able to perform substantive assessment of this possibility using sunflower. Interestingly, it has been shown recently on the example of Gentiana rigescens Franch. ex Hemsl. that concentration of K is higher in flowers than in other plant tissues [(Zhang et al., 2020); unfortunately Na was not considered in that study]. Future studies might elucidate the role of K:Na nutritional limitation in the ecology and evolution of bees. Currently, we do not know the role of bees and other pollinators and their interactions with plants in nutrient cycling in food webs and ecosystems (Ollerton, 2021a; Ollerton, 2021b). Given the high amount of biomass that bees move in ecosystems with the pollen they consume, we hypothesize that bees play a pivotal role in the mobilization and movement of nutrients from the soil through pollen and nectar, into food webs. Importantly, this crucial ecosystem service is shaped by nutritional interactions between bees and their host plants, including balancing K:Na in bee larval diet which is based on pollen.

## Conclusions

A balanced ratio of K:Na in food is a necessary condition for the health and fitness of herbivores. Previous studies suggested that plants manipulate their ionomes to shape their interactions with pollinators. Considering the minor role of Na in plant metabolism,

one could therefore expect that plants using pollen as a nutritional reward for pollinators would fill pollen with a large proportion of unnecessary Na to stimulate pollination services. This should result in decreased pollen K:Na. Contrary to expectations, we did not observe the effect of low K:Na in sunflower pollen under various scenarios of Na fertilization of the two sunflower cultivars. We show that the concentrations of K and Na in sunflower pollen are closely linked and an increased concentration of Na in pollen correlates with an increase in the concentration of K; therefore, the K:Na ratio in pollen is high irrespective of its Na concentration. High pollen K:Na is suboptimal for pollinivores and may be harmful for bees if additional Na is not supplemented from other food sources. It is possible that high pollen K:Na is needed to optimize plant fertilization, embryo creation, and seed development. Consequently, there may exist a tradeoff between producing pollen with a low K:Na optimal for pollinator health and development and pollen with a high K:Na optimal for plant fertilization and development. This observation suggests not-studied to date costs and tradeoffs related to pollinivory that may shape plant-pollinivore interactions and may influence pollination ecology.

## Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

## Author contributions

MF: Conceptualization, Methodology, Formal analysis, Validation, Writing—Original Draft, Writing—Review & Editing, Visualization, Funding acquisition. MS: Conceptualization, Methodology, Investigation, Validation, Writing—Review & Editing, Project administration. SC: Conceptualization, Methodology, Investigation, Validation, Writing—Review & Editing, Project administration. MG: Methodology, Investigation, Validation, Writing—Review & Editing. SW: Conceptualization, Methodology, Investigation, Funding acquisition, Project administration, Supervision. All authors made critical contributions to the drafts and gave permission for publication.

## Funding

This work was funded/supported by Bayer AG, Monheim, Germany; the Bio-Protection Research Centre (bioprotection.org.nz), Lincoln University, New Zealand; the Royal Society of New Zealand's James Cook Fellowship; Jagiellonian University, Faculty of Biology (N18/DBS/000003) and the National Science Centre, Poland (grant no. 2019/33/B/NZ8/01700).
## Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

## Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated

organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

## Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2022.1042348/full#supplementary-material

## References

Świątek, Z. M., Woźnicka, O., and Bednarska, A. J. (2020). Unravelling the ZnO-NPs mechanistic pathway: cellular changes and altered morphology in the gastrointestinal tract of the earthworm eisenia andrei. *Ecotoxicol. Environ. Saf.* 196, 110532. doi: 10.1016/j.ecoenv.2020.110532

Bänziger, H., and Bänziger, S. (2010). Mammals, birds and reptiles as hosts of foraminifera, the tear drinkers with the broadest host range (Hymenoptera, Apidae). *J. Swiss Entomol. Soc* 83, 271-282.

Bhatt, J. G., and Indirakutty, K. N. (1973). Salt uptake and salt tolerance by sunflower. *Plant Soil* 39, 457-460. doi: 10.1007/BF00014816

Bonoan, R. E., O'Connor, L. D., and Starks, P. T. (2018). Seasonality of honey bee (Apis mellifera) micronutrient supplementation and environmental limitation. *J. Insect Physiol* 107, 23-28. doi: 10.1016/j.jinsphys.2018.02.002

Bonoan, R. E., Tai, T. M., Tagle Rodriguez, M., Feller, L., Daddario, S. R., Czaja, R. A., et al. (2017). Seasonality of salt foraging in honey bees (Apis mellifera). *Ecol. Entomol.* 42, 195-201. doi: 10.1111/een.12375

Borer, E. T., Lind, E. M., Firn, J., Seabloom, E. W., Anderson, T. M., Bakker, E. S., et al. (2019). More salt, please: Global patterns, responses and impacts of foliar sodium in grasslands. *Ecol. Lett.* 22, 1136-1144. doi: 10.1111/ele.13270

Cairns, S. M., Wratten, S. D., Filipiak, M., Veronesi, E. R., Saville, D. J., and Shields, M. W. (2021). Ratios rather than concentrations of nutritionally important elements may shape honey bee preferences for 'dirty water'. *Ecol. Entomol.* 46, 1236-1240. doi: 10.1111/een.13067

Dangles, O., and Casas, J. (2012). The bee and the turtle: a fable from yasuni national park. *Front. Ecol. Environ.* 10, 446-447. doi: 10.1890/1540-9295-10.8.446

de la Rosa, C. L. (2014). Additional observations of lachryphagous butterflies and bees. *Front. Ecol. Environ.* 12, 210-210. doi: 10.1890/14.WB.006

de Sousa, R. T., Darnell, R., and Wright, G. A. (2022). Behavioural regulation of mineral salt intake in honeybees: a self-selection approach. *Philos. Trans. R. Soc B Biol. Sci.* 377(1853). doi: 10.1098/rstb.2021.0169

Dorian, N. N., and Bonoan, R. E. (2020). Stingless bees (Apidae: Meliponini) seek sodium at carrion baits in Costa Rica. *Ecol. Entomol.* 46(2):492-495. doi: 10.1111/een.12948

Dow, J. A. (2017). The essential roles of metal ions in insect homeostasis and physiology. *Curr. Opin. Insect Sci.* 23, 43-50. doi: 10.1016/j.cois.2017.07.001

Dresselhaus, T., Sprunck, S., and Wessel, G. M. (2016). Fertilization mechanisms in flowering plants. *Curr. Biol.* 26, R125-R139. doi: 10.1016/j.cub.2015.12.032

Fernández-Martínez, M. (2022). From atoms to ecosystems: Elementome diversity meets ecosystem functioning. *New Phytol.* 234, 35-42. doi: 10.1111/nph.17864

Filipiak, M. (2019). Key pollen host plants provide balanced diets for wild bee larvae: A lesson for planting flower strips and hedgerows. *J. Appl. Ecol.* 56, 1410-1418. doi: 10.1111/1365-2664.13383

Filipiak, Z. M., and Bednarska, A. J. (2021). Different effects of zn nanoparticles and ions on growth and cellular respiration in the earthworm eisenia andrei after long-term exposure. *Ecotoxicology* 30, 459-469. doi: 10.1007/s10646-021-02360-2

Filipiak, Z. M., Denisow, B., Stawiarz, E., and Filipiak, M. (2022). Unravelling the dependence of a wild bee on floral diversity and composition using a feeding experiment. *Sci. Total Environ.* 820, 153326. doi: 10.1016/j.scitotenv.2022.153326

Filipiak, Z. M., and Filipiak, M. (2020). The scarcity of specific nutrients in wild bee larval food negatively influences certain life history traits. *Biol. (Basel)*. 9, 462. doi: 10.3390/biology9120462

Filipiak, M., and Filipiak, Z. M. (2022). Application of ionomics and ecological stoichiometry in conservation biology: Nutrient demand and supply in a changing environment. *Biol. Conserv.* 272, 109622. doi: 10.1016/j.biocon.2022.109622

Filipiak, M., Kuszewska, K., Asselman, M., Denisow, B., Stawiarz, E., Woyciechowski, M., et al. (2017). Ecological stoichiometry of the honeybee: Pollen diversity and adequate species composition are needed to mitigate limitations imposed on the growth and development of bees by pollen quality. *PloS One* 12, e0183236. doi: 10.1371/journal.pone.0183236

Finkelstein, C. J., CaraDonna, P. J., Gruver, A., Welti, E. A. R., Kaspari, M., and Sanders, N. J. (2022). Sodium-enriched floral nectar increases pollinator visitation rate and diversity. *Biol. Lett.* 18(3). doi: 10.1098/rsbl.2022.0016

Frausto da Silva, J. J. R., and Williams, R. J. P. (2001). "The biological chemistry of the elements," in *The inorganic chemistry of life, 2nd ed* (Oxford University Press: New York, USA).

Grainger, M. N. C., Hewitt, N., and French, A. D. (2020). Optimised approach for small mass sample preparation and elemental analysis of bees and bee products by inductively coupled plasma spectrometry. *Talanta* 214, 120858. doi: 10.1016/j.talanta.2020.120858

Hammer, Ø., Harper, D. A. T., and Ryan, P. (2001). PAST: Paleontological statistics software package for education and data analysis. *Palaeontol. Electron.* 4, 9.

Horn, H. (1985). Die waldtrachtkrankheit der honigbiene. i. der einfluß des mineralstoffgehaltes in honigtauhonigen [The causes of paralysis in honeybees during a honeydew flow. 1. the effect of mineral content in honeydew honeys]. *Apidologie* 16, 139-156. doi: 10.1051/apido:19850204

Huang, X.-Y., and Salt, D. E. (2016). Plant ionomics: From elemental profiling to environmental adaptation. *Mol. Plant* 9, 787-797. doi: 10.1016/j.molp.2016.05.003

Jager, T., Barsi, A., and Ducrot, V. (2013). Hormesis on life-history traits: is there such thing as a free lunch? *Ecotoxicology* 22, 263-270. doi: 10.1007/s10646-012-1022-0

Kaspari, M. (2020). The seventh macronutrient: how sodium shortfall ramifies through populations, food webs and ecosystems. *Ecol. Lett.* 23, 1153-1168. doi: 10.1111/ele.13517

Kaspari, M. (2021). The invisible hand of the periodic table: How micronutrients shape ecology. *Annu. Rev. Ecol. Evol. Syst.* 52, 199-219. doi: 10.1146/annurev-ecolsys-012021-090118

Khan, K. A., Ghramh, H. A., Ahmad, Z., El-Niweiri, M. A. A., and Mohammed, M. E. A. (2021). Honey bee (Apis mellifera) preference towards micronutrients and their impact on bee colonies. *Saudi J. Biol. Sci* 28(6), 3362-3366. doi: 10.1016/j.sjbs.2021.02.084

Lau, P., Lesne, P., Grebenok, R. J., Rangel, J., and Behmer, S. T. (2022). Assessing pollen nutrient content: a unifying approach for the study of bee nutritional ecology. *Philos. Trans. R. Soc B Biol. Sci.* 377(1853), 20210510. doi: 10.1098/rstb.2021.0510

Lau, P. W., and Nieh, J. C. (2016). Salt preferences of honey bee water foragers. *J. Exp. Biol.* 219, 790-796. doi: 10.1242/ieb.132019

Nicolson, S. W., and W.-Worswick, P. V. (1990). Sodium and potassium concentrations in floral nectars in relation to foraging by honey bees. *South Afr. J. Zool.* 25, 93-96. doi: 10.1080/02541858.1990.11448196

Ollerton, J. (2021a). "Pollinators and pollination," in *Nature and society* (Exeter: Pelagic Publishing). Available at: https://pelagicpublishing.com/products/pollinators-and-pollination-ollerton-9781784272289.
Ollerton, J. (2021b). Protect the pollinators: pollinators have a critical, but largely unappreciated, role to play when it comes to climate change. *New Sci.* 3326(3), 23. doi: 10.1016/S0262-4079(21)00466-8

Padmanaban, S., Czerny, D. D., Levin, K. A., Leydon, A. R., Su, R. T., Maugel, T. K., et al. (2017). Transporters involved in pH and k+ homeostasis affect pollen wall formation, male fertility, and embryo development. *J. Exp. Bot.* 68, 3165–3178. doi: 10.1093/jxb/erw483

Parreño, M. A., Alaux, C., Brunet, J.-L., Buydens, L., Filipiak, M., Henry, M., et al. (2022). Critical links between biodiversity and health in wild bee conservation. *Trends Ecol. Evol.* 37, 309–321. doi: 10.1016/j.tree.2021.11.013

Pedersen, S. A., and Zachariassen, K. E. (2002). Sodium regulation during dehydration of a herbivorous and a carnivorous beetle from African dry savannah. *J. Insect Physiol.* 48, 925–932. doi: 10.1016/S0022-1910(02)00153-1

Peñuelas, J., Fernández-Martinez, M., Ciais, P., Jou, D., Piao, S., Obersteiner, M., et al. (2019). The bioelements, the elementome, and the biogeochemical niche. *Ecology* 0, e02652. doi: 10.1002/ecy.2652

Petanidou, T. (2007). "Ecological and evolutionary aspects of floral nectars in Mediterranean habitats," in *Nectaries and nectar* (Dordrecht: Springer Netherlands), 343–375. doi: 10.1007/978-1-4020-5937-7_8

Robinson, R. G. (1971). Sunflower phenology-year, variety, and date of planting effects on day and growing degree-day summations. *Crop Sci.* 11, 635–638. doi: 10.2135/cropsci1971.0011183X001100050009x

Ruedenauer, F. A., Spaethe, J., van der Kooi, C. J., and Leonhardt, S. D. (2019). Pollinator or pedigree: which factors determine the evolution of pollen nutrients? *Oecologia* 191, 349–358. doi: 10.1007/s00442-019-04494-x

Schneiter, A. A., and Miller, J. F. (1981). Description of sunflower growth stages. *Crop Sci.* 21, 901–903. doi: 10.2135/cropsci1981.0011183X002100060024x

Simpson, S. J., Sword, G. A., Lorch, P. D., and Couzin, I. D. (2006). Cannibal crickets on a forced march for protein and salt. *Proc. Natl. Acad. Sci. U. S. A.* 103, 4152–4156. doi: 10.1073/pnas.0508915103

Smilauer, P., and Lepš, J. (2014). *Multivariate analysis of ecological data using CANOCO* 5 (Cambridge: Cambridge University Press). doi: 10.1017/CBO9781139627061

Todd, K. J., Gardiner, M. M., and Lindquist, E. D. (2016). Mass flowering crops as a conservation resource for wild pollinators (Hymenoptera: Apoidea). *J. Kansas Entomol. Soc.* 89, 158–167. doi: 10.2317/0022-8567-89.2.158

van der Kooi, C. J., Vallejo-Marin, M., and Leonhardt, S. D. (2021). Mutualisms and (A)symmetry in plant–pollinator interactions. *Curr. Biol.* 31, R91–R99. doi: 10.1016/j.cub.2020.11.020

Vaudo, A. D., Erickson, E., Patch, H. M., Grozinger, C. M., and Mu, J. (2022). Impacts of soil nutrition on floral traits, pollinator attraction, and fitness in cucumbers (Cucumis sativus L.). *PREPRINT*. doi: 10.21203/rs.3.rs-2085798/v1

Wang, Y., Ma, L., Zhang, W., Cui, X., Wang, H., and Xu, B. (2016). Comparison of the nutrient composition of royal jelly and worker jelly of honey bees (Apis mellifera). *Apidologie* 47, 48–56. doi: 10.1007/s13592-015-0374-x

White, P. J., and Karley, A. J. (2010). "Potassium," in *Plant cell monographs*, 199-224. doi: 10.1007/978-3-642-10613-2_9. Heidelberg, New York, Dordrecht, London

Williams, R. J. P., and Rickaby, R. E. M. (2012). *Evolution's destiny: Co-evolving chemistry of the environment and life* (Cambridge: Royal Society of Chemistry). doi: 10.1039/9781849735599

Willmer, P. (2011). *Pollination and floral ecology* (Princeton and Oxford: Princeton university Press). Available at: https://press.princeton.edu/books/hardcover/9780691128610/pollination-and-floral-ecology.

Zhang, J., Wang, Y., and Cai, C. (2020). Multielemental stoichiometry in plant organs: A case study with the alpine herb gentiana rigescens across southwest China. *Front. Plant Sci.* 11. doi: 10.3389/fpls.2020.00441

Zhang, J., Yuan, T., Wang, Y., Zhao, Y., Zhang, J., and Jin, H. (2012). Determination of mineral elements in gentiana rigescens from different zones of yunnan, China. *Biol. Trace Elem. Res.* 147, 329–333. doi: 10.1007/s12011-011-9293-5

Zhao, Y.-L., Zhang, J., Yuan, T.-J., Zhang, J.-Y., Shen, T., Wang, Y.-Z., et al. (2014). Evaluation of mineral elements in gentiana rigescens in China. *Asian J. Chem.* 26, 4587–4590. doi: 10.14233/ajchem.2014.16123