Response of Superoxide Dismutase (SOD) to Homogeneous and Heterogeneous Food Sources in Bumblebees ( Bombus terrestris ) and Honeybees ( Apis mellifera )

: The conception of “ﬂoral strips” is a strategy to provide more and different food sources for pollinators. The impact of “homogeneous” Phacelia tanacetifolia (“Phacelia”) and “heterogeneous” (ﬂower mix) food sources on the enzyme activity of bumblebees ( Bombus terrestris ) and honeybees ( Apis mellifera ) under urban conditions has not been reported. Organisms responding to challenging environmental conditions are known to exhibit increases in oxidative stress parameters which in turn affect both physiological and metabolic parameters. A ﬁeld study was conducted in Berlin-Dahlem, Germany, using the response of the “marker” enzyme superoxide dismutase (SOD) on food sources for assessment. SOD data is also shown from the wild bee Megachile rotundata (Fabricius 1787), obtained from three different locations in the federal state Brandenburg, Germany. The results demonstrate that the enzyme activity of SOD signiﬁcantly increased in bumblebees visiting the ﬂower mix compared to the Phacelia. The experimental approach had individual effects at the level of the species, bumblebees and honeybees, respectively. The activity of the biomarker SOD could be successfully used to assess the effects of the compositions of homogeneous and heterogeneous ﬂower ﬁelds.


Introduction
Bees (Hymenoptera: Apoidea) are the main group of pollinators, with approximately 20,000 species worldwide [1]. In Germany, more than 566 bee species with differences in their habitats, feeding habits, and nesting sources are known [2]. The decline of insects, including bees, is widely documented [3,4] and has been estimated at 9% decrease per decade [5]. Several factors could act as drivers of pollinator decline, e.g., land use changes, weather conditions, environmental pollution, but also diseases and pathogens [3,6]. These drivers affect the availability of pollen, nectar, and nesting sites and also pollinator health [3,[7][8][9]. Several studies provide evidence that pollinator health, physiology, microbiota, reproductive capacity, and longevity are linked to the availability and diversity of floral sources [3,7,10,11]. Insects are exposed to diverse environmental stressors (agrochemicals, ultraviolet radiation, bacteria, and viruses), which are responded to by the production of "Reactive Oxygen Species" (ROS) [12]. ROS are free radicals, such as superoxide anion, hydroxyl, nitric oxide radical, and hydrogen peroxide. These metabolites are produced up to a certain level during physiological and metabolic processes, such as digestion, energy production, and Physiologia 2023, 3 273 respiration, as well as at a higher level for detoxification and immune responses. Reactive species can be scavenged by several enzymatic and nonenzymatic detoxification systems [13]. In invertebrates' systems, ROS are produced by the response of plasma membrane nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, intracellular cytosolic xanthine oxidase, peroxisomal oxidases, endoplasmic reticular oxidases, and mitochondrial electron transport components [14], and references therein. Apart from this, the auto-oxidation of catecholamines, ubihydroquinone, hemoproteins, and flavin enzymes also generates ROS. However, in cases of hyperproduction of ROS or decreased antioxidative defense, oxidative stress develops [15]. In response to nutrient effects and/or stress, cells enter autophagy, which can lead to adaptation. Additionally, a number of small molecules also play a role in scavenging ROS, and some of these small molecules are plant-derived, whereas others such as carotenoids, α-tocopherol, ascorbic acid, and glutathione (GSH) can be synthesized by the insect itself.
Nutritional effects, such as the "availability" of pollen [16], result in a measurable increase in the antioxidative defense system. However, a distinction should be made here between the diversity of pollen and the amount of pollen. Thus, insects are continually subjected in different ways and intensities to oxidative stress from ROS. Superoxide dismutase (SOD, EC 1.15.1.1) is a key enzyme that plays a primary role in removing ROS, which has been identified in lepidopteran model insects (https://metazoa.ensembl.org/ (accessed on 1 January 2023)) [12]. Three types of SOD protein have been reported in insects. SOD1 is a major cytoplasmic enzyme, SOD2 is a mitochondrial enzyme, and an extracellular, SOD3, has been identified in the hemolymph and molting fluid of insects. However, through a series of experiments, [12] four more unique and previously not-reported SODs were identified with different responses to physiology and different types of oxidative stress in Bombyx mori, the silk spinner, a lepidopteran insect.
The available information indicates that the use of "flower strips" has mitigated the negative impacts on bees and other pollinators caused by the scarcity and diversity of floral sources in agricultural landscapes, including urban areas [17]. Flower strips are areas that should provide food sources with mixtures of wild and cultivated plants exclusively for insects [18]. These flower strips are not subject to commercial use. Despite the success of flower strips to enhance the abundance and diversity of pollinators [11,19], the knowledge of how bees respond in physiological terms to a diverse availability of food sources on flower strips is limited. Agri-Environment Schemes (AES) [20] are being implemented in agricultural systems to stop the decline of pollinators. Scientific assessments of these schemes are still lacking, and only a few studies have examined the extent to which insect pollinators use the floral enhancements that are a part of AES and on which floral components they feed (i.e., pollen and/or nectar). To our knowledge, no field study in an urban area has been reported to date on the impact of "homogeneous" food supply (Phacelia) compared to "heterogeneous" food supply (flower mix) on the volume activity ("activity") of the "marker" enzyme SOD in bumblebees and honeybees. It is generally assumed that the quality of the environment in rural areas is higher than in urban ones; this fact was the motivation for setting up trap nests under "natural conditions" in the federal state Brandenburg. SOD data from the wild bee Megachile rotundata (Fabricius 1787), obtained from three different locations in the federal state Brandenburg, Germany, are shown in this study.

Materials and Methods
2.1. Construction of Trap Nests ("Insect Nesting Aids"/"Insect Hotels") and Places of Installation in Federal State Brandenburg, Germany For identification, wild bees are caught with so-called "trap nests". The trap nests were constructed according to the methodology of [21]. They were inexpensive, made from natural materials, and can be easily placed in different habitats. A 30 cm long, 10 cm diameter PVC pipe forms the outer casing. Nest tubes were made of reeds (Phragmites) with a diameter of 4-10 mm and a length of 15 cm. The trap nests were protected from Physiologia 2023, 3 274 predators by protective grilles over the entrance and were placed on wooden posts 1.5 m above the ground (Figure 1). from natural materials, and can be easily placed in different habitats. A 30 cm lon diameter PVC pipe forms the outer casing. Nest tubes were made of reeds (Phr with a diameter of 4-10 mm and a length of 15 cm. The trap nests were protect predators by protective grilles over the entrance and were placed on wooden po above the ground (Figure 1).

Treatment before and after Overwintering of Trap Nests from Different Sites in the F State of Brandenburg, Germany
After deinstallation, the complete trap nests from the three locations in Bran (Table 1) were stored in a cold chamber at 6 °C for overwintering until March April 2021, the nest tubes were stored at constant temperatures of 24 °C and 60% humidity in an incubator (Memmert GmbH & Co. KG, Germany) until the w hatched (emerged between 14 and 28 days). Until hatching, the nest tubes were co daily and the adult wild bees, corresponding to M. rotundata (Fabricius 1787), we mL microcentrifuge tubes (Eppendorf ® , Hamburg, Germany) snap frozen i nitrogen, and stored at −80 °C until processing and analysis (3.5). All handlings accordance with the "Code of Honor for Entomological Fieldwork" of the Committee of Experts (BFA) Entomology at NABU-Naturschutzbund Deutschlan

Experimental Design for Different Food Sources for Bees
Two food source plots for bees (each ~600 m 2 ) were established in 202 experimental fields of the Free University Berlin (FU) and the Albrecht Danie Institute (ADTI at the Humboldt University of Berlin, HU) (52.47° N, 13.30° E, h a.s.l.) under Berlin s urban landscape, close to forest remnants and green areas. (23 April 2021), the plot with Phacelia tanacetifolia (Phacelia), cultivar "Be ("homogeneous food resource") with a seed density of 12 kg/ha was planted on of FU (Figure 2), and the peak of flowering was observed during June. In May

Treatment before and after Overwintering of Trap Nests from Different Sites in the Federal State of Brandenburg, Germany
After deinstallation, the complete trap nests from the three locations in Brandenburg (Table 1) were stored in a cold chamber at 6 • C for overwintering until March 2021. In April 2021, the nest tubes were stored at constant temperatures of 24 • C and 60% relative humidity in an incubator (Memmert GmbH & Co. KG, Germany) until the wild bees hatched (emerged between 14 and 28 days). Until hatching, the nest tubes were controlled daily and the adult wild bees, corresponding to M. rotundata (Fabricius 1787), were in 1.5 mL microcentrifuge tubes (Eppendorf ® , Hamburg, Germany) snap frozen in liquid nitrogen, and stored at −80 • C until processing and analysis (3.5). All handlings were in accordance with the "Code of Honor for Entomological Fieldwork" of the Federal Committee of Experts (BFA) Entomology at NABU-Naturschutzbund Deutschland e.V.

Experimental Design for Different Food Sources for Bees
Two food source plots for bees (each~600 m 2 ) were established in 2021 at the experimental fields of the Free University Berlin (FU) and the Albrecht Daniel Thaer-Institute (ADTI at the Humboldt-University of Berlin, HU) (52.47 • N, 13.30 • E, h = 51 m a.s.l.) under Berlin's urban landscape, close to forest remnants and green areas. In April (23 April 2021), the plot with Phacelia tanacetifolia (Phacelia), cultivar "Beehappy" ("homogeneous food resource") with a seed density of 12 kg/ha was planted on the area of FU (Figure 2), and the peak of flowering was observed during June. In May (11 May 2021), the plot with the flower mix "Veitshöchheimer Bienenweide" ("heterogeneous food resource"), obtained from Feldsaaten Freudenberger GmbH & Co. KG, 47800 Krefeld, with a seed density of 12 kg/ha was planted at the area of ADTI (Figure 2), and the peak of flowering was observed during July. Both months correspond to the "food gap" for bees. The Veitshöchheimer Bienenweide is a bee pasture, consisting of flowering annual and perennial wild and cultivated species (69% herbs; 22% legumes; and 9% of a mixture of buckwheat (Fagopyrum), mallow (Malva), and sunflower (Helianthus)). Annual species, such as the marigold (Targetes) and the sunflower flower in the year of sowing. The mullein (Verbascum thapsus) blooms in the second year, as well as perennial species such as meadow sage (Salvia pratensis) and clover (Trifolium L.). The more detailed composition, especially for the herbs, is shown at www.freudenberger.net. Surrounding both plots, crops of rapeseed, wheat, potatoes, and aromatic herbs were found. The two plots were about 1 km apart (Figure 2). Physiologia 2023, 3, FOR PEER REVIEW with a seed density of 12 kg/ha was planted at the area of ADTI (Figure 2), and the p of flowering was observed during July. Both months correspond to the "food gap" bees. The Veitshöchheimer Bienenweide is a bee pasture, consisting of flowering an and perennial wild and cultivated species (69% herbs; 22% legumes; and 9% of a mix of buckwheat (Fagopyrum), mallow (Malva), and sunflower (Helianthus)). An species, such as the marigold (Targetes) and the sunflower flower in the year of sow The mullein (Verbascum thapsus) blooms in the second year, as well as perennial spe such as meadow sage (Salvia pratensis) and clover (Trifolium L.). The more deta composition, especially for the herbs, is shown at www.freudenberger.net. Surround both plots, crops of rapeseed, wheat, potatoes, and aromatic herbs were found. The plots were about 1 km apart (Figure 2).

Bee Collection of Bombus terrestris and Apis mellifera from Phacelia and Flower Mix Plo
The sampling of bees was conducted following the "Code of Honor Entomological Fieldwork" of the Federal Committee of Experts (BFA) Entomolog NABU-Naturschutzbund Deutschland e.V. Individuals were collected at midmorn during sunny days using sweep nets, on two sampling days separated by one wee June (5 individuals from B. terrestris and 5 individuals from A. mellifera collected at e sampling day) from Phacelia, and the same was done in July from the flower Taxonomic identification was done a priori on site, considering that the most abund bumblebee at the study site was B. terrestris. Both species were confirmed using the h and thorax of each individual using taxonomic keys. Subsequently, individuals w immediately introduced into 1.5 mL microcentrifuge tubes (Eppendorf ® ) and shock fro in liquid nitrogen during transportation to the laboratory where they were finally sto at −80 °C until further processing ( Figure 3) and analysis (2.5).

Bee Collection of Bombus terrestris and Apis mellifera from Phacelia and Flower Mix Plots
The sampling of bees was conducted following the "Code of Honor for Entomological Fieldwork" of the Federal Committee of Experts (BFA) Entomology at NABU-Naturschutzbund Deutschland e.V. Individuals were collected at midmorning during sunny days using sweep nets, on two sampling days separated by one week in June (5 individuals from B. terrestris and 5 individuals from A. mellifera collected at each sampling day) from Phacelia, and the same was done in July from the flower mix. Taxonomic identification was done a priori on site, considering that the most abundant bumblebee at the study site was B. terrestris. Both species were confirmed using the head and thorax of each individual using taxonomic keys. Subsequently, individuals were immediately introduced into 1.5 mL microcentrifuge tubes (Eppendorf ® ) and shock frozen in liquid nitrogen during transportation to the laboratory where they were finally stored at −80 • C until further processing ( Figure 3) and analysis (2.5).

Measuring Superoxide Dismutase (SOD) Activity
SOD activity was measured by LABOKLIN, 97688 Bad Kissingen (https://laboklin.de (accessed on 1 January 2022)), using a Superoxide Dismutase Assay Kit (Cayman Chemical, Ann Arbor, MI, USA, product no. 706002 [16]. The assay measures the three types of SOD (Cu/Zn, Mn, and FeSOD). Ten µL of the supernatant was diluted 1:500 with the supplied sample buffer, and the remainder of the assay was conducted following the manufacturer's instructions. SOD in the bee samples was compared to a standard curve of SOD (provided by the manufacturer) and absorbance was read at 450 nm. SOD activity was expressed as units per mL.

Measuring Superoxide Dismutase (SOD) Activity
SOD activity was measured by LABOKLIN, 97688 Bad Kissingen (https://lab (accessed on 1 January 2022)), using a Superoxide Dismutase Assay Kit (Cayman cal, Ann Arbor, MI, USA, product no. 706002 [16]. The assay measures the three SOD (Cu/Zn, Mn, and FeSOD). Ten µL of the supernatant was diluted 1:500 with plied sample buffer, and the remainder of the assay was conducted following th facturer's instructions. SOD in the bee samples was compared to a standard curve (provided by the manufacturer) and absorbance was read at 450 nm. SOD acti expressed as units per mL.

Statistical Analysis
Standard statistical analyses (mean, standard error (SE), multiple compa means) was performed using IBM SPSS V29, using the one-way ANOVA with th HSD-test (Table 2), and the two-sided test, testing whether the means are differe 0.05 (Table 3).

Statistical Analysis
Standard statistical analyses (mean, standard error (SE), multiple comparison of means) was performed using IBM SPSS V29, using the one-way ANOVA with the Tukey HSD-test (Table 2), and the two-sided test, testing whether the means are different at p = 0.05 (Table 3).

SOD Activity in Hutched Wild Bee Megachile Rotundata from Different Sites in the Federal State Brandenburg
It is well known that cities have various problems related to emissions they put into the environment and the so-called "carbon footprint". This can affect the quality of the environment to varying degrees. It can be generally assumed that the quality of the environment in rural areas is higher than in urban ones; this fact was the motivation for setting up trap nests under "natural conditions" in the federal state Brandenburg. From trap nests in Brandenburg, the species M. rotundata was dominant (~90%). This "alfalfa-leafcutting bee" is distributed in Southern and Central Europe. The preferred habitat includes inland dunes, sand and clay pits, flood dams in contact with alluvial forests, south-facing and structurally rich forest edges, and also parks and gardens in urban areas [22]. Flowers and leaves, e.g., of Pelargonium, Hortensia, Rosa, Euphorbia, Reseda, Syringa, and Vitis are used as food sources and nesting materials. In the United States and Canada, M. rotundata is the main pollinator of alfalfa (Medicago sativa L.) for seed production [23].
The SOD activity in hutched bees of M. rotundata (Table 2)-without any contact to flowers-was on average (± SE) 24.87 (±1.81), 29.36 (±3.42), and 28.62 (±4.09) U/mL for the three sites, Alt Madlitz, Groß Schönebeck, and Schenkendöbern. For this species, there are no statistical differences in the SOD activity between the three rural sites. Therefore, the mean value of the pooled SOD activity (n = 20) for the three locations in Brandenburg was 28.05 (±2.13) U/mL (Table 2). However, it should be considered that M. rotundata is a different species compared with B. terrestris and A. mellifera, because it is a solitary bee species, which does not build colonies or store honey.
It should be considered [20,24] that both B. terrestris' and A. mellifera's most flower visits were for collecting nectar. Only 22% of honeybee visits and 3% of bumblebee worker visits were for pollen collection. Nectar properties vary with environmental factors, pollinator visits, and microbial contamination [25]. Nectar is a viscous liquid, rich in glucose, fructose, and sucrose, that contains minerals and fragrances, free amino acids, proteins, inorganic ions, and secondary plant compounds (plant defense chemicals). The naturally occurring ratios of each sugar vary between plant species; bee-pollinated plants tend to be sucrosedominant and glucose-poor, whereby sucrose-rich nectars are more common in protected, tubular flowers [26], and references therein, [25]. This behavior may lead to lower SOD activity values for the homogeneous stand of Phacelia in A. mellifera, compared to the flower mix.
A further explanation lies in the purple flower color of Phacelia, typical to the occurrence of anthocyanins. These are water-soluble plant pigments in the cell sap of almost all higher plants and they give flowers, fruits, and vegetables an intense red, violet, or blue color. Anthocyanins are among the most effective antioxidants, which primarily act as free-radical scavengers, but also absorb short-wave UV light and pass on radiant energy as heat.
Results on honeybees [15] kept in traditional, "natural accommodation" hives ("trmka hives") without any human influence, compared to commercially managed standard hives, showed that both hive types resulted in marked differences of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and malondialdehyde (MDA). Only the SOD activity in the traditional hives was significantly higher compared to bees from commercial hives, where the CAT, GST, and MDA concentration was significantly increased in comparison to the traditional hives. It is known that SOD catalyzes the dismutation of the superoxide radical (O 2 − ) into hydrogen peroxide (H 2 O 2 ), the first-line defense against ROS; it was assumed, that significantly higher values of oxidative stress parameters (CAT, GST, and MDA) in commercial hives resulted from higher burdens of parasites.
Flower contact is inevitably linked to pollen contact. The crude protein (CP) content of pollen is reported in the wide range from 2.5% to 61%. The protein content of the pollen of blooming plants may influence the floral choice of pollinators [27]. In contrast to many wild bee species, which depend on certain flowers, the honeybee flies to everything that is in bloom, making it very flexible. For A. mellifera this may be the reason for the wider adaptation range of SOD activity when visiting homogeneous or heterogeneous flower plots, 35.19 (±2.34) U/mL and 51.92 (±14.15) U/mL, respectively (not significant, Table 3).
Summarized, the reasons for the marked upregulation of the SOD activity for B. terrestris for the flower mix plot can be explained as follows: (a) Superoxide inactivates in the citric acid cycle the enzyme aconitase (EC 4.2.1.3) (catalyze isomerization of citrate to isocitrate via cis-aconitate); (b) For the inactivation of other ROS and RNS (Reactive Nitrogen Species); (c) And/or protection against diseases and so to maintaining health and fitness. The halflife of superoxide is less than one second in the mmol range and~14 h in the nmol range. Superoxide dismutase is one of the few known catalytically perfect enzymes (~7 × 10 9 M −1 s −1 ), because a catalytic reaction occurs at almost every encounter with the substrate and the reaction is only limited by the rate of diffusion [30]. The activity of the SOD, thus, represents the current metabolic status of B. terrestris and A. mellifera.

Conclusions
Our experimental approach-monofloral vs. polyfloral field plots combined with measurement of SOD activity-had individual effects at the level of the species, bumblebees and honeybees, respectively. Therefore, the activity of the biomarker SOD could also be used to assess the effects of different compositions of mixed flower fields on other wild bees.
To improve the assessment of the benefit from different wild and cultivated plants for wild bees, the content and profile of anthocyanins, the total phenol content, and the antioxidant capacity of nectar should be analyzed. With such an approach, the spectrum of flower colors from white to yellow/orange and red to dark purple could be selected first, with the flower morphology being considered secondarily. This could improve the decision for plant selection for the composition of flower strips, based on the "quality" of nectar.  Institutional Review Board Statement: Sampling of bees was conducted following the "Code of Honor for Entomological Fieldwork" of the Federal Committee of Experts (BFA) Entomology at NABU-Naturschutzbund Deutschland e.V.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data of this study available in the text.