Nectar Abundance and Nectar Composition in Selected Rubus idaeus L. Varieties

: The increasing commercial production of R. idaeus offers insects nectar and pollen rewards, thus increasing the chance of cross-pollination, which enhances fruit yields. The knowledge of nectar characteristics may help farmers/beekeepers to improve the quality of their products. Therefore, we determined and compared nectar weight, sugar concentration and weight, and the qualitative and quantitative composition of sugars and amino acids in the nectar of six raspberry cultivars: three biennial and three repeated fruiting cultivars. The nectary abundance in these cultivars ranged between 20.2 ± 3.84 mg (‘Polka’) and 26.4 ± 7.3 mg (‘Glen Ample’) of nectar per ﬂower with a sugar concentration of 34.6 ± 5.61–47.3 ± 9.33%. The contents of glucose and fructose (g/100 g) were in the range from 42.96 ± 0.71 (‘Glen Ample’) to 46.94 ± 0.55 (‘Laszka’) and from 50.7 ± 1.43 (‘Polka’) to 54.2 ± 0.72 (‘Radziejowa’). Sucrose was detected only in ‘Glen Ample’ (5.6 ± 1.12 g/100 g) and ‘Polka’ (6.2 ± 0.95 g/100 g). Taking into account the increasing sugar weight in the nectar, the cultivars were ranked as follows: ‘Polka’ < ‘Polana’ < ‘Radziejowa’ < ‘Pokusa’ < ‘Laszka’ < ‘Glen Ample’. The nectar of the analyzed raspberry cultivars was classiﬁed as a hexose-dominant type. Aspartic acid, glutamic acid, and proline were the most dominant endogenous amino acids, whereas exogenous acids were dominated by lysine and leucine. The present results provide valuable information about the nutritious value of R. idaeus nectar for pollinators.


Introduction 1.Importance of Berry Fruits
Berries have been the staple food source for humans and animals since ancient times.Nowadays, the increasing importance of human health, nutrition, and protection against diseases has attracted global consumer demand for foods and raw materials with high nutritional value (especially in developed countries).Berries, especially the members of the families Rosaceae and Ericaceae, are regarded as excellent dietary sources of biochemical compounds.They have delicious taste and flavor and economic importance.Due to their antioxidant properties, they are of great interest to researchers, nutritionists, and food technology experts.In recent years, the consumer demand for healthy products has increased and many wild fruit species have begun to be cultivated in large areas [1].Berry fruits, e.g., black currants, black raspberries, blackberries, blueberries, cranberries, raspberries, and strawberries, contain phytotherapeutic nutrients with high biological activity, i.e., fiber, vitamins, minerals, phenolic compounds, including flavonoids (anthocyanins, flavonols, flavones, flavanols, flavanones, and isoflavonoids), tannins, and phenolic acids (polyphenols), which reduce the risk of various diseases [2][3][4].These compounds can be supplied to the organism with a diet based mainly on plants, e.g., vegetables, fruits, and berries, as well as whole grain products, fish, and a small amount of red meat with maintenance of the balance between the supply and consumption of energy [5].In the case of adults, consumption of at least 400-500 g of fruit and vegetables in five portions, including 250 g of fruits/berries, is recommended [5][6][7].
Rubus fruits (raspberries, blackberries, and cloudberries) are dominated by ellagitannins (mainly lambertianin C and sanguiin H-6), exhibiting health-enhancing effects [2,[8][9][10][11][12][13].These bioactive chemical compounds reduce oxidative stress and inflammation and are used in the prevention and phytotherapy of e.g., diabetes, neurological and cardiovascular diseases, and cancer [4,13,14].Hence, berry fruits have been recognized as "promising functional fruits" [2].A positive effect of modified atmosphere packaging and UV-C irradiation on the postharvest quality of red raspberries has been found.The methods contributed to a delay in the aging process, limitation of rotting, reduction of weight loss, maintenance of normal color and firmness, and an increase in the content of bioactive compounds (1.8 g kg −1  of gallic acid equivalents, 1.9 g kg −1 of catechin equivalents, and 0.3 g kg −1 of cyanidin 3-O-glucoside equivalents) [3].Currently, berries are gaining increasing consumer interest due to their therapeutic potential and the possibility to use them mainly in functional food and in the production of nutraceuticals [4,15].

Nectar Secretion and Its Impact on Pollination
The constantly increasing area of R. idaeus cultivation in Poland, Europe, and the world and the abundant and long flowering (from May to August) of the plants offers a continuous nectar and pollen supply [16][17][18].Data on the R. idaeus pollen reward were presented by Kostryco et al. [19] and Kostryco and Chwil [20].Nectar secretion increases the frequency of cross pollination in raspberry plantations, contributing to higher yields and quality of Rubus fruits [21][22][23][24].Cross-pollination was found to increase fruit weight from 90% to 252% and the number of seeds by 125-203% in R. idaeus 'Glen Ample', 'Laszka', 'Radziejowa', 'Polana', and 'Polka' cultivars and the size of fruit by 30% in 'Canby' and 'K81-6' [22,25].It also contributed to a threefold increase in the number of drupes in R. fruticosus [26].According to the forecasts and statistical estimates of the economic value of insect pollination (EVIP) coefficient, the effectiveness of pollination of R. idaeus flowers by insects has a significant influence on the fruit yield, as evidenced by the increase in the economic profit from EUR 48 million in 2011 to EUR 117.5 million in 2020 [27].Overall, pollination of crops by honeybees, wild bees, and flies generates revenues of around $577 billion annually and is an important factor in agricultural economy [28].The increasing commercial R. idaeus production ensures an adequate pollen and nectar supply to pollinators [20,29,30].Poland is one of the main raspberry fruit producers in the European Union in terms of acreage, and the share of global production in the world has been estimated at 13% with a sale value of approximately 930,100 EUR (euro) in 2019 [11,31].

Nectar Chemical Profile and Pollination Syndrome
Nectar contains many substances, and there is no doubt that its importance is associated with various ecological processes other than the food functions.This secretion plays an important role in the plant reproductive strategy [32].Nectar contains water (30-90%), sugars (from several to 70%), nitrogenous substances, organic acids, dyes, essential oils, vitamins, and mineral salts [33].In phylogenetically related species, the chemical profile of nectar can be adjusted to the requirements of specific pollinator guilds, revealing high phenotypic plasticity [34].

Qualitative Composition of Nectar Sugars
The chemical composition of nectar varies depending on the taxonomic affiliation at various systematic levels but there are also similarities in nectar features between taxonomically unrelated species in connection with the pollinator type [35].The chemical composition of this secretion, which is mainly a sugar solution, includes a representative fraction of two monosaccharides (glucose and fructose) and one disaccharide (sucrose) in various taxa and species.Less frequently, nectar contains small concentrations of mannose, arabinose, xylose, galactose, and sorbitol from the former group of sugars and maltose and melobiose from the latter group.It also contains oligosaccharides, i.e., raffinose, galactose, and melezitose [36][37][38].According to the proportion of glucose, fructose, and sucrose, the basic types of nectar have been distinguished in the scientific literature: sucrose-dominant (S/G + F > 1.0; % sucrose 51-100), sucrose-rich (S/G + F > 0.5-1.0;% sucrose 34-50), hexoserich (0.1-0.5; % sucrose 10-33), and hexose-dominant (S/G + F < 1.0; % sucrose 0-9) [38,39].
Sucrose contained in nectar is the main component of the phloem sap from which this secretion originates, while nectar glucose and fructose are produced in chemical reactions catalyzed by transglucosidases and transfructosidases localized in nectaries.A very wide range of total sugar content in the nectary, i.e., from 5% to 80%, has been reported [37].The variable qualitative and quantitative composition of sugars in nectar is closely correlated with the presence of pollinators specific for a given taxon [35][36][37].The floral nectar of Rubus species contains various amounts of sucrose (dominant or moderate concentrations) or exhibits a complete absence of this sugar [24,40,41].The content of sucrose in blackberry nectar was higher or close to the concentration of hexoses (glucose and fructose).In raspberries, the ratio of sucrose to the glucose and fructose sum was <0.1, which indicates the dominant content of hexoses, while this ratio in blackberries ranged between 0.37 (hexose-rich) and 0.78 (sucrose-rich) [42].
Honeybees choose flowers based on not only the amount of nectar available but also color and pattern of petals a well as the qualitative composition of sugars in the secretion [42][43][44][45][46][47][48].Floral color signaling is one of the main systems of communication between plants and pollinators [48].Honeybees prefer yellow, blue, and white flowers as well as combinations of these colors [45,[48][49][50].In the choice of blue or yellow flowers, the insects were guided only by the identification of the color of the petals, whereas in the case of blue and white flowers, the choice depended on the reward offered by the flower [45].Honeybees read differences in UV absorption in the UV range mainly of flavones and flavonols [48].These insects showed greater interest in flowers offering nectar with higher sugar concentrations [51].They also remembered color patterns as low-resolution images and landmarks with higher resolution for triangulation and localization of flowers [47,52].
Honeybees prefer nectar containing a combination of three sugars (sucrose, glucose, and fructose) than a sucrose-only solution [42,44].In terms of the content of single sugars in nectar, Apis mellifera exhibits preferences in the following order: sucrose > glucose > maltose > fructose [53].The nectar of many species from the Rosaceae family has been classified into different types, e.g., the secretion in Prunus laurocerasus, Crataegus, and Pyrus, has been classified as hexose-dominant nectar [54][55][56].In turn, the nectar of genus Malus species represents the sucrose-dominant and sucrose-rich type [43].The quality of nectar is determined not only by the sugar content but also by the presence of other components, especially proteins and amino acids [57,58].

Nectar Protein
Floral nectar contains only few specific proteins; these are mainly species-specific enzymes classified into glycosidases or so-called pathogenesis-related proteins, such as chitinase, glucanase, xylosidase, galactosidase, thaumatin-like proteins, and many others [57][58][59].Although the proteins identified in honey are mostly enzymes, their activity in this bee product is negligible.On the other hand, floral nectar does not exhibit the enzymatic activity found in honey [60].Furthermore, the content of two dominant proteins in Allium porrum L. floral nectar, i.e., mannose-binding lectin and allinase, which catalyzes the conversion of alliins to allicins, was found to be substantially reduced in honey [61].Therefore, nectar proteins are undoubtedly inactivated/degraded during nectar conversion into honey [59,61].Scientific reports on the natural variation of proteins identified in floral nectar indicate that nectar chemistry may have a complex function in plant-pollinatormicrobe interactions.Nectar proteins fulfill two basic functions: (1) defense against microorganisms [62-66] and (2) post-secretory hydrolysis of nectar sugars into fructose and glucose for protectors [67,68].
The protective role of nectar proteins against microorganisms is of great importance, as pollinators penetrating flowers in search of nectar and collecting pollen transmit pathogenic microorganisms that can substantially reduce plant fecundity.The nutrient-rich floral nectar secreted in the receptacle surrounding the ovary offers an excellent potential infection site [58].Non-specific lipid transfer protein BrLTP2.1 isolated from Brassica rapa was found to exhibit strong antimicrobial activity, especially against necrotrophic fungi [69].Floral nectar proteins in ornamental tobacco (Nicotiana langsdorffii x N. sanderae) exert antimicrobial effects through a biochemical pathway referred to as the "nectar redox cycle" [58,[70][71][72].The antimicrobial activity of GDSL lipase and nectarins (NECs) has been confirmed as well [65,68,73].GDSL lipase found in the floral nectary of Jacaranda minosifolia has lipolytic esterase activities and is involved in the process of release of free fatty acids, which plays a role in pollinator attraction.The antimicrobial activity of GDSL lyase is associated with disruption of spore membrane integrity [74].Nectarins (NEC) is the general name for proteins identified in the floral nectar of Nicotiana langsdorfii × Nicotaina sanderae classified as NEC1-NEC5 according to their increasing molecular weight [58,70,[74][75][76].The concentration of nectarins depends on the nectary development stage.These proteins were detected in nectary cells during nectar secretion and in ovary tissues but not in other flower elements and organs [68,74].
The antimicrobial activity of nectarins is a result of increased synthesis of reactive oxygen species (ROS), which directly inhibit microbial growth and indirectly activate signal transduction pathways inducing innate immunity in the plant.The NEC1 (nectarin 1) protein, previously classified as a germin-like protein, is a manganese-containing berberine superoxide dismutase involved in production of H 2 O 2 , i.e., a non-radical reactive oxygen form.In the case of the extracellular dermal glycoprotein (EDGP) known as Nectarin IV (NEC4), ROS are generated through the interaction of this protein with fungal protein endoglucanase XEG.The formation of the NEC4:XEG complex is a key step in induction of the catalytic activity of Nectarin V (NEC5), i.e., a flavin-containing berberine like protein, which converts D-glucose and molecular oxygen to D-gluconic acid and hydrogen peroxide [65].
The NEC3 protein with high sequence similarity to the antioxidant storage protein has both monodehydroascorbate reductase and carbonic anhydrase activities.This protein greatly contributes to the maintenance of pH and oxidative balance in nectar [74].In the floral nectar of the subtropical evergreen velvet bean (Mucuna sempervirens Hemsl), desiccation-related proteins called MS-desi have been identified.This major nectar protein (nectarin) in this bean plant composed of 306 amino acids with a molecular mass of 33.248 Da is exclusively expressed in the stylopodium, where the nectary is located.Purified MS-desi and raw floral nectar of the velvet bean showed dose-dependent citrate synthase inhibition activity and insensitivity to lactate dehydrogenase, suggesting that, unlike dehydrins, the protein does not act as a chaperone [77].Recently, a rubber elongation factor (REF) protein with a defensive role against microorganisms has been identified in Liriodendron tulipifera floral nectar.This protein with allergic characteristics showed high stage-specific expression in nectary tissues [68].
In addition to their protective function, floral nectar proteins are involved in the post-secretory hydrolysis of nectar sugars into fructose and glucose for protectors.Floral nectar has been shown to have high activity of invertase, which not only catalyzes the decomposition of sucrose into glucose and fructose, but also in some cases shows the activity of glucotransferase, catalyzing most often the synthesis of the 1-ketose trisaccharide.Under the influence of invertase, reactions with involvement of transglucosidases and transfructosidases take place in nectar.Their activity leads to changes in the qualitative composition and osmophilicity of nectar [78,79].

Floral Nectary Amino Acids
The quantities of amino acids in nectar are estimated at 0.02-4.8% of organic matter, but the evolutionary and ecological significance of their presence in this secretion is still being investigated and discussed [37,80,81].The qualitative and quantitative composition of amino acids varies between and within plant species and depends on the pollination modes as well as the systematic classification of plants [82][83][84].Nevertheless, it is believed that the amino acid composition is constant in plants of the same species, even if they grow in different environments.However, the latest research has provided evidence that the high variability within and between populations is related to nitrogen availability [37].A high concentration of amino acids has been noted in the nectar of various species producing flowers with well-developed morphological traits and complex pollination systems, in comparison with less specialized plants [82].
Amino acids are one of the major nutritional components for many consumers [64,85].Behavioral preferences for amino acids in nectar have been observed in bees, flies, and ants.These insects ingested nectar even with low amino acid content.As Gardener and Gillmann [86] claim, the concentration of nectar amino acids is closely related to the type of pollinating insects.A higher content of amino acids was found in flowers visited by butterflies, and lower concentrations of this nutrient were determined in nectar from flowers pollinated by bees, which obtain additional amounts of nitrogen by feeding on pollen [64,87].In turn, the proportion of amino acids in the nectar of plants pollinated by butterflies is higher than in the nectar of flowers pollinated by birds [86,87].Amino acids ingested by adults are incorporated into eggs.Male butterflies are believed to show no preference, but females prefer nectars spiced with amino acids to nectars with no content of these compounds [88].A high concentration of amino acids in the floral nectar of plants adapted to pollination by butterflies is the only source of nitrogen and energy for these insects.Appropriate qualitative and quantitative proportions of amino acids determine the condition, extend the lifespan, and increase the reproductive success of Lepidoptera [88][89][90][91].Amino acids, especially proline, which is preferentially used by nectar-feeding insects in the early stages of flight, play an important role in the maintenance of good muscle condition necessary for movement [37].This amino acid has another unique trait.It stimulates labellar salt receptor cells in some insect species, which most probably allows them to recognize tastes.Proline is the dominant amino acid in the hemolymph of insects, including honeybees.High concentrations of proline, even constituting 45-60% of all amino acids, have been detected in many types of nectar.In addition to proline, flower nectar contains large amounts of aromatic amino acids (phenylalanine, tyrosine), serine, and amides (asparagine, glutamine).Unlike pollinating birds, honeybees and butterflies prefer proline-enriched sugar solutions in the range of 2-6 mM but collect serine-rich nectar less eagerly [37,81].
The differences in the demand for amino acids of many pollinator groups are explained by the variable contents of these nutrients in nectar even in closely related taxa [91,92].These compounds have an impact on the preferences of pollinators [83, 84,86] and on the taste of nectar, which is more palpable in diluted than concentrated secretion, where it can be masked by sugars [83,86].Amino acids activate specific taste chemoreceptors and stimulate or inhibit the response of sensory cells in insects [93][94][95][96].Consequently, they attract pollinators and deter herbivores [63,86].Specific concentrations of amino acids inform insects about the intensity of nectar flavors [86].Hence, different pollinators often prefer nectar with a specific composition [97].There is a high degree of interspecies variability in the content of amino acids and sugars between nectar, honeydew, and sap leaking from damaged plant tissue, varying within the same plant [98].Insects and birds respond to the varying concentrations of amino acids [99,100].The composition of nectar amino acids may change in response to increased CO 2 concentrations, damage to the nectary, or nitrogen fertilization.Nitrogen has been shown to induce an increase in the total concentration of amino acids, especially proline and glutamic acid [83,101].
Although the nectar composition in various Rubus species has been described in the scientific literature, there are no data from the qualitative and quantitative composition of amino acids and sugars in common raspberry varieties cultivated in commercial production in Poland, i.e., 'Glen Ample', 'Laszka', 'Radziejowa', 'Pokusa', 'Polana', and 'Polka'.These cultivars are a valuable source of protein and energy necessary for the development of bee colonies and the production of honey.The amino acid and sugar profile serves as a carrier of the chemical information signal in the mutualistic insect-plant relationship, as it regulates the ecology of pollination, enhances the reproduction success, influences nectar consumption and pollinator behavior, and determines the amino acid composition and botanical origin of honey.
The aim of the study was to determine and compare (i) nectar abundance, (ii) total protein content, and the qualitative and quantitative composition of (iii) sugars and (iv) amino acids in the nectar of six R. idaeus cultivars, i.e., three biennial fruiting cultivars 'Glen Ample', 'Laszka', and 'Radziejowa' and three repeated fruiting cultivars 'Pokusa', 'Polana', and 'Polka'.

Plant Material
The experiment was carried out in 2016-2018 on Blinów II village plantation located in Lublin Province, Kraśnik County, Szastarka commune in south-eastern Poland (50 • 51 59 N 22 • 23 09 E).Six Rubus idaeus cultivars, i.e., three from the biennial fruiting group, 'Glen Ample', 'Laszka', and 'Radziejowa', and three from the repeated fruiting group, 'Pokusa', 'Polana', and 'Polka', were the biological objects of the study.Buds in the bud-burst stage and flowers on the first and last day of flowering were collected in the full flowering stage.

Scope of the Study
The lifespan of a single flower, i.e., the number of days from the anthesis onset to the appearance of the first withering symptoms, was determined.Nectar was collected throughout the life of the flowers of the six raspberry cultivars.Comparative analyses of nectar abundance were carried out by estimation of the nectar sugar concentration, nectar weight, and sugar weight throughout the flower lifespan.Additionally, the total protein content in the nectar was determined and the qualitative and quantitative compositions of sugars and amino acids in the nectar were compared.The type of nectar was identified as well.

Nectar Abundance
Before the beginning of flowering of the analyzed R. idaeus cultivars, branches were selected randomly and protected against insect visits with airy tulle insulators.For each cultivar, the lifespan of ten flowers was determined through observation thereof from the beginning of anthesis to the appearance of the first symptoms of wilting of corolla petals.Bursting buds intended for nectar collection after opening were marked with a marker.From each cultivar, nectar was collected from the entire life of flowers in 6 samples (6n) on three different dates in each year of the study (total n = 18).Nectar was collected between 9.30 a.m. and 10.30 a.m. with the pipette method as described by Jabło ński [102].This time period was dictated by the increased frequency of pollinator visits to the flowers [103,104].One sample of nectar was the amount of secretion accumulated throughout the flower lifespan and collected from 2-5 flowers depending on the volume of the solution and the capacity of the micropipette.The micropipette with the nectar was weighed on an analytical balance.The percentage of sugar was determined with an Abbe refractometer (RL-1 Polskie Zakłady Optyczne PZO, Warsaw, Poland) calibrated with distilled water.The weight of sugars (mg/flower) contained in the nectar of the cultivars was calculated based on the nectar weight (mg/flower) and the percentage of sugar (%).

Qualitative and Quantitative Composition of Nectar
The quantitative and qualitative analysis of sugars contained in the nectar collected throughout the lifespan of the flowers of the studied cultivars was performed using high-performance liquid chromatography (HPLC) with the method proposed by Bogdanov et al. [105] modified by Rybak-Chmielewska and Szczęsna [106] and Rybak-Chmielewska [107].Sugars were determined using an LC-10AD liquid chromatograph (Shimadzu Corporation, Kyoto, Japan) equipped with a LC-10ATVP pump, a DGU-14A degasser, a CTO-10AVP column thermostat, an RID-10A refractometric detector, a SUPELCOGEL™ chromatography column, and POL-LAB CHROMA 2001 software(creator POL-LAB Artur Dzieniszewski, Warsaw, Poland).The qualitative identification of nectar sugars was carried out by comparison of the retention times of each sugar in the standard solution.The quantitative determination was performed using the external standard method with comparison of peak areas of the sugars in the standard solution with peak areas of the same sugars in the nectar solution.The content of glucose, fructose, and sucrose was expressed as a percentage of the total sugar content of the nectar.The fructose-to-glucose ratio (F/G) and the ratio of sucrose to the sum of fructose and glucose (S/F + G) were calculated as well.The sugars were determined in three samples from each cultivar.

Determination of Total Protein and Amino Acid Content in Nectar
The total nitrogen content in the nectar of the tested R. idaeus cultivars (n = 3) in the first year of the study was determined with the Kjeldahl method.The total protein content was calculated by multiplying the nitrogen content by the protein factor 5.6 [108].
The amino acid composition in the nectar of the six analyzed R. idaeus cultivars was determined with the method proposed by Davies and Thomas [109].The nectar samples (n = 3) collected from each cultivar in each study year were placed in the INGOS hydrolyzer thimble (Prague, Czech Republic) and flooded with 6 M HCl.After closing with a valve, the solution and saturated with nitrogen and hydrolyzed at 110 • C for 20 h.After hydrolysis, the content of the thimble was cooled and filtered through a G-4 funnel.The hydrolyzate was evaporated on an RVO 400 SD vacuum evaporator at 50 • C, washed with 1 mL of distilled water, and evaporated again.The dry residue from the vacuum flask was dissolved in 5 mL of citrate buffer, pH 2.2.
The sample was dispensed onto a 35 cm long and 5-mm diam.column filled with ion exchange resin.Amino acids were separated using an AAA 400 amino acid analyzer at temperatures T1 = 60 • C and T2 = 63 • C. The amino acids were derivatized into colored amino acid-ninhydrin complexes.They were identified using a photometric detector at the wavelengths of 570 nm or 440 nm (for proline).The measurement was recorded as a chromatogram with the use of the Chromulan computer program.

Statistical Analysis
Statistical analyses were performed using a two-way analysis of variance (ANOVA), in which the study years and the cultivar were the dependent variables.The parameters studied: nectar sugar concentration, nectar weight, and nectar sugar weight as well as the glucose, fructose, and sucrose content and the concentration of individual amino acids were the independent variables.In turn, the effect of the cultivars on the protein content in the nectar was investigated using one-way analysis of variance.Tukey's posthoc comparison tests were performed.The statistical analyses were carried out using the integrated statistical software package SAS 9.2 and Statistica 6.0 at the significance level α = 0.05.

Nectar Abundance
Considering the mean value from the three years (2016-2018), it was found that the nectar of the six Rubus idaeus cultivars produced from 20.2 mg ('Polka') to 26.4 mg ('Glen Ample') of nectar per flower with a sugar concentration of 34.6-47.3%.These values calculated as the weight of sugars were 6.9 and 12.2 mg per flower, respectively.In terms of the significance of the differences, 'Glen Ample' was found to produce significantly greater amounts of nectar than the other cultivars.Concurrently, the concentration of sugars in the 'Pokusa' and 'Radziejowa' nectar was clearly higher than that in 'Polka' and 'Polana' but lower than in 'Laszka' and 'Glen Ample'.In turn, in comparison with the other cultivars, 'Glen Ample' was characterized by a significantly higher weight of nectar sugars per flower.The value of this parameter in 'Laszka' was comparable to that in 'Pokusa' and lower than in 'Radziejowa' 'Polka', and 'Polana'.Taking into account the significance of the differences in the nectar abundance parameters within the cultivars in the subsequent years, in 2017, the concentration of nectar sugar in 'Radziejowa' was higher but the sugar weight was lower than in 2016.The concentration of sugars in the 'Glen Ample' nectar in 2016 was significantly higher than in 2018 and lower than in 2017.Concurrently, the nectar weight and the sugar weight in this cultivar in 2018 were significantly higher than the values recorded in the other years.In the 'Polana' cultivar, the concentration of sugars in the nectar was significantly lower in 2018 and the nectar weight in 2016 was significantly higher than in the other years.In general, there were no statistically confirmed changes in the nectar abundance parameters of the 'Pokusa', 'Laszka', and 'Polka' cultivars in the subsequent years, with the exception of the significantly lower concentration of nectar sugars in 'Polka' in 2016 and in 'Laszka' in 2017 (Figure 1A-F).The statistically confirmed differences between the cultivars in the years of the study revealed different trends in the changes in the nectar abundance parameters of the analyzed raspberry cultivars.In terms of the percentage nectar sugar concentration, the taxa were ranked as follows: in 2016-'Glen Ample' = 'Laszka' > 'Pokusa' > 'Radziejowa' = 'Polana' > 'Polka', in 2017-'Glen Ample' > 'Radziejowa' = 'Laszka' > 'Pokusa' > 'Polana' = 'Polka', and in 2018-'Laszka' > 'Glen Ample' > 'Radziejowa' = 'Pokusa' > 'Polka' > 'Polana'.In 2016, the weight of nectar produced by the flowers of 'Laszka', 'Pokusa', and 'Polka' was significantly lower than in 'Glen Ample' and 'Pokusa' and lower than in The statistically confirmed differences between the cultivars in the years of the study revealed different trends in the changes in the nectar abundance parameters of the analyzed rasp-berry cultivars.In terms of the percentage nectar sugar concentration, the taxa were ranked as follows: in 2016-'Glen Ample' = 'Laszka' > 'Pokusa' > 'Radziejowa' = 'Polana' > 'Polka', in 2017-'Glen Ample' > 'Radziejowa' = 'Laszka' > 'Pokusa' > 'Polana' = 'Polka', and in 2018-'Laszka' > 'Glen Ample' > 'Radziejowa' = 'Pokusa' > 'Polka' > 'Polana'.In 2016, the weight of nectar produced by the flowers of 'Laszka', 'Pokusa', and 'Polka' was significantly lower than in 'Glen Ample' and 'Pokusa' and higher than in 'Radziejowa'.The weight of sugar in the nectar from 'Polana' and 'Pokusa' was significantly lower than in 'Glen Ample' and 'Laszka' but higher than in 'Radziejowa' and 'Polka'.In 2017, 'Polka' flowers produced substantially lower amounts of nectar than 'Radziejowa', 'Glen Ample', and 'Pokusa'.In turn, the latter cultivar produced higher amounts of nectar than 'Laszka' and 'Polana'.The 'Glen Ample' cultivar produced nectar with the highest weight of sugars, while the lowest values were calculated for 'Polka'.In turn, the value of this parameter in 'Polana' was significantly lower than in 'Laszka', 'Radziejowa', and 'Pokusa'.In the last study year (2018), 'Laszka' and 'Polka' produced substantially lower amounts of nectar than 'Glen Ample' and higher amounts than 'Polana', 'Pokusa', and 'Radziejowa'.Concurrently, the weight of sugar in the 'Polka' nectar was significantly lower than in 'Laszka' and comparable to that in the other repeated fruiting cultivars (Figure 1A-F).

Qualitative and Quantitative Composition of Nectar Sugars
The nectar of 'Glen Ample' and 'Polka' contained three main sugars: glucose (G), fructose (F), and small amounts of sucrose (S); sucrose was not detected in the other cultivars.The mean glucose content in the nectar in 2016-2017 ranged from 42.96 ('Glen Ample') to 46.94 ('Laszka') g/100 g.In general, there were no statistically confirmed differences in terms of glucose content within and between the cultivars in the study years.Compared to the other cultivars, it was clearly lower only in the 'Glen Ample' and 'Polka' nectar samples.The mean fructose content in the nectar ranged from 50.7 ('Polka') to 54.4 g/100 g ('Pokusa'), and the trend in the changes was similar to that in the glucose levels.The sucrose content in the nectar samples was in the range of 4.65-7.2g/100 g.The mean percentage content of sucrose in the 'Glen Ample' and 'Polka' nectar did not differ significantly, although the amount of this sugar in the former cultivar was clearly higher in 2016 than in the other years, whereas the latter cultivar was characterized by the highest value of this parameter in 2017.In comparison to the 'Polka' cultivar, the sucrose content in 'Glen Ample' was significantly lower in the first year, higher in the second year, and comparable in the third year (Figure 2A-C).
Taking into account the sugar content, the mean fructose-to-glucose (F/G) ratio, and the ratio of sucrose to glucose and fructose sum S/(G + F), the nectar of the analyzed raspberry cultivars was classified as a fructose-dominant hexose-rich type.The calculated F/G and S/(G + F) ratios had values above and below one, respectively.The mean value of the F/G ratio for 'Glen Ample' and 'Pokusa' was significantly higher than the value calculated for 'Laszka' and 'Polana'.Moreover, the F/G ratio for 'Radziejowa' and 'Polka' in 2017 and for 'Glen Ample' in 2016 was significantly lower than in the other years.Considering the differences between the cultivars in the subsequent years, it was shown that the F/G ratio in the 'Radziejowa', 'Pokusa', and 'Polka' nectar in the first study year (2016) was significantly higher than in 'Glen Ample' and 'Polka'.In the second year (2017), the ratio was higher in 'Glen Ample' and significantly lower in 'Polka' in comparison with the other cultivars.In the last year of the study (2018), 'Polana' exhibited a lower value of the ratio than the other cultivars.The mean sucrose-to-hexose ratio (S/G + F) was not significantly different in 'Glen Ample' and 'Polka', but there were clear differences within these cultivars in the individual years and between the cultivars in subsequent years.The trends in the changes were analogous to those in the sucrose content in the nectar (Figure 2D,E).Taking into account the sugar content, the mean fructose-to-glucose (F/G) ratio, and the ratio of sucrose to glucose and fructose sum S/(G + F), the nectar of the analyzed The total mean sugar concentration in the nectar samples within the biennial fruiting R. idaeus cultivars ranged from 36.14 to 50.71 g/100 g and decreased significantly as follows: 'Glen Ample' > 'Laszka' > 'Radziejowa'.The value of this parameter in the repeated fruiting cultivars ranged from 27.56 to 44.57g/100 g with the decreasing order: 'Polka' > 'Pokusa' > 'Polana'.In general, the nectaries of the biennial fruiting cultivars contained higher amounts of total sugars than the repeated fruiting cultivars.Moreover, within each cultivar, there were significant differences in the total sugar content in the study years.The highest values of this parameter were recorded for 'Glen Ample', 'Radziejowa', 'Pokusa', and 'Polana' in 2017 and in 'Laszka' and 'Polka' in 2016 (Figure 2F).

Nectar Protein Content
The total mean protein concentration (%) in the nectar samples in 2016-2018 ranged from 0.1233 ('Glen Ample') to 0.1633 ('Radziejowa') in the group of the biennial fruiting cultivars and from 0.2167 ('Pokusa') to 0.1433 ('Polana') in the repeated fruiting group.The nectar of 'Pokusa' and 'Polka' contained significantly higher amounts of proteins than 'Glen Ample', 'Laszka', and 'Polana'.According to the increasing content of the total protein in the nectar, the cultivars were ranked as follows: 'Glen Ample' < 'Polana' < 'Laszka' < 'Radziejowa' < 'Polka' < 'Pokusa' (Figure 3).ratio was higher in 'Glen Ample' and significantly lower in 'Polka' in comparison with the other cultivars.In the last year of the study (2018), 'Polana' exhibited a lower value of the ratio than the other cultivars.The mean sucrose-to-hexose ratio (S/G + F) was not significantly different in 'Glen Ample' and 'Polka', but there were clear differences within these cultivars in the individual years and between the cultivars in subsequent years.The trends in the changes were analogous to those in the sucrose content in the nectar (Figure 2D,E).
The total mean sugar concentration in the nectar samples within the biennial fruiting R. idaeus cultivars ranged from 36.14 to 50.71 g/100 g and decreased significantly as follows: 'Glen Ample' > 'Laszka' > 'Radziejowa'.The value of this parameter in the repeated fruiting cultivars ranged from 27.56 to 44.57g/100 g with the decreasing order: 'Polka' > 'Pokusa' > 'Polana'.In general, the nectaries of the biennial fruiting cultivars contained higher amounts of total sugars than the repeated fruiting cultivars.Moreover, within each cultivar, there were significant differences in the total sugar content in the study years.The highest values of this parameter were recorded for 'Glen Ample', 'Radziejowa', 'Pokusa', and 'Polana' in 2017 and in 'Laszka' and 'Polka' in 2016 (Figure 2F).

(A-F).
Total amino acid profiles in the six R. idaeus cultivars analyzed in the three study years Explanations: Ala-alanine, Asp-aspartic acid, Glu-glutamic acid, Gly-glycine, Pro-proline, Ser-serine, Tyr-tyrosine, Arg-arginine, his-histidine, Ile-isoleucine, Leu-leucine, Lys-lysine, Phe-phenylalanine, Thr-theronine, Val-valine, means followed by the same small letter are not significantly different within the cultivar in the years, and means followed by the same capital letter do not differ between the cultivars in each year of the study at a significance level α = 0.05 (two-way ANOVA, Tukey's test).
Taking into account the significance of the differences in the mean content of endogenous amino acids in the nectar between the cultivars during the three study years (2016-2018), it was shown that the concentration of glutamic acid was higher in 'Glen Ample' and lower in 'Polana' than in the other cultivars.The mean content of glycine in the nectar of 'Radziejowa' was significantly higher than in the nectar of 'Glen Ample', 'Pokusa', and 'Polka.The latter three cultivars were characterized by a significantly higher mean concentration of proline than its content in 'Laszka' and 'Radziejowa', and the serine content in 'Polana' was significantly higher than in 'Pokusa', 'Glen Ample', and 'Radziejowa'.Considering the significance of the changes in the mean concentration of exogenous amino acids in the nectar between the different cultivars, it was found that 'Polka' had higher content of leucine than 'Glen Ample', and 'Polana' had a lower concentration of lysine than the other cultivars.Concurrently, the histidine content in 'Laszka', 'Radziejowa', and 'Polana' was significantly higher than in the other cultivars.In turn, the content of exogenous phenylalanine was lower in the nectar of 'Polka' but higher in 'Glen Ample' in comparison with the other cultivars.There were no statistically confirmed differences in the mean concentration of endogenous alanine, aspartic acid, and tyrosine as well as exogenous threonine, valine, isoleucine, and arginine between the cultivars (Figure 4A-F).
Considering the significance of the differences in the amino acid concentration in the nectar from the analyzed cultivars in the consecutive years, noteworthy is the higher content of Ala, Gly, Arg, Leu, and Val in 'Glen Ample' in 2017 than in the other years as well as the lower content of Asp, Pro, and Phe and the significantly higher level of Ile and Lys in 2017 and 2018 than in 2016.In 2018, 'Laszka' exhibited a significantly lower amount of Ala, Leu, Lys, and Thr and a higher level of Asp than in the other years.Furthermore, its nectar was characterized by a clearly lower concentration of Pro in 2017 than in 2016 and 2018.In this cultivar, a statistically significant lower level of Arg and Ile and higher content of Phe were recorded in 2016 than in the subsequent years.The interesting findings in the case of the 'Radziejowa' cultivar include the significantly higher Glu level and the lower Pro and Thr content determined in 2017 than in the other years as well as the marked increase in the Gly and Phe levels and the reduced content of Leu recorded in 2018.The level of Arg and Thr in the nectar of the 'Pokusa' cultivar was significantly higher in 2016 than in the other two years, and the concentration of Ala and Val was significantly higher in 2017 than in 2016 and 2018.Additionally, in 2018, this cultivar had significantly higher contents of Asp, Glu, and Pro but lower amounts of His, Leu, and Lys than in the previous two years.An important finding of the analyses of 'Polana' is the significantly higher Pro level and the lower Ile content in 2016 than in the subsequent years.In comparison with the other two years, this cultivar was characterized by a significantly lower level of Phe in 2017 as well as a clearly lower Gly and Lys concentrations and higher Ser, Tyr, and His levels in 2018.In turn, statistically confirmed changes in the contents of amino acids in the 'Polka' nectar (a decrease in Gly, Pro, and Thr and an increase in Leu and Lys) were recorded only in 2018 (Figure 4A-F).
The weight of nectar sugars in the analyzed Rubus idaeus cultivars (7-12.17mg/flower) recorded in the present study was within the range found in 10 other cultivars of this species (8.4-14.5 mg/flower) [110] and higher than in R. idaeus (1.4-3.5 mg/flower) described by Szklanowska [111].As shown by literature data, the honey yield was estimated at 42.8-238.2kg/ha in R. idaeus [110,111] and 5.4-25.8kg/ha in R. fruticosus [111].These values indicate that, due to the production of nectar and pollen, R. idaeus plays not only an important role in the production of fruit but also a key role in the ecology of pollination.The increasing commercial production of these shrubs guarantees high supplies of this reward [20,117].In turn, the pollination process increases fruit yields [20,29,30,118].The involvement of insects in pollination of raspberry flowers was found to increase fruit yields by 30% [119,120].Concurrently, pollination with non-self pollen of self-pollinating raspberry varieties ensures even fruit yields [22].At full pollination, the economic value of fruit increases substantially [24].Raspberry flowers are mainly visited by bumblebees, honeybees, solitary bees, and flies.The proportion of honeybees pollinating raspberry flowers was estimated at 60% [112,[118][119][120][121][122].These insects utilize the pollen and nectar flow as a source of protein and energy for the development of bee colonies; their excess is used for production of honey [123][124][125][126].The antimicrobial properties of this product are associated with destabilization of the bacterial cell membrane structure [127].

Qualitative and Quantitative Composition of Nectar Sugars
The content of glucose and fructose in the nectar of the analyzed R. idaeus cultivars ranged from 42.96 g/100 g ('Glen Ample') to 46.94 g/100 g ('Laszka') and from 50.7 g/100 g ('Polka') to 54.2 g/100 g ('Radziejowa').Sucrose in this secretion was detected only in 'Glen Ample' (5.6 g/100 g) and 'Polka' (6.1 g/100 g).The qualitative analysis of sugars showed that the flowers of R. idaeus 'Glen Ample' and 'Polana' secreted hexose-dominated nectar.In other studies, this type of nectar was detected in the flowers of R. idaeus 'Autumn Bliss' and 'Malling Exploit' [24], R. caesius 'Fertődi Venus' [128], R. chamaemorus [40], Cearasus vulgaris [129], Prunus armeniaca, and P. persica [130][131][132][133], P. laurocerasus 'Schipkaensis' and 'Zabeliana' [56], and Fragaria sp.[43].Bordács et al. [134] demonstrated that self-sterile cultivars secreted hexose-rich nectar, whereas self-fertile cultivars produced nectar dominated by hexoses.As suggested by Percival [130], this type of nectar is associated with the open structure of the flower with an exposed nectary.In turn, other authors suggest that the sucrose concentration depends on the activity of invertase, i.e., low activity of this enzyme was accompanied by production of hexose-rich nectar [67,135].This relationship was confirmed by Lichtenberg-Kraag [136], who explained that it is also important in the honey ripening process.As reported by Braun and Hildebrand [137], low concentrations or an absence of sucrose represent a defense mechanism in R. idaeus against infection with a specific pathogen Erwinia amylovora, which is dangerous for this species.The pathogen requires the presence of sucrose to grow.
The values of the F/G ratio in the nectar of the analyzed R. idaeus cultivars ranged from 1.12 ('Laszka') to 1.20 ('Glen Ample').Similar values were reported for nectar produced by R. idaeus 'Autumn Bliss' and 'Malling Exploit', which either did not contain sucrose or had negligible amounts of this sugar [42,138].No sucrose was detected in nectar produced by R. fruticosus and R. longobaccus [139].Monosaccharides (fructose and glucose) and disaccharides (sucrose), which dominate in the nectar of many plant species, increase the attractiveness of flowers to pollinators [140].Bees most willingly collect nectar with a sucrose/fructose/glucose ratio of 1:1:1 [53].Apis mellifera, which is characterized by a high demand for energy during flight and high efficiency in collecting R. idaeus nectar and pollen, has been classified as the main pollinator in most ecosystems [141][142][143][144]. Nectar and pollen rewards meet this demand.Due to its high glycemic index (100), glucose is rapidly metabolized during flight, whereas fructose and sucrose with the lower glycemic index (14 and 65, respectively) are metabolized more slowly [145].As reported by Wykes [53], the order of Apis mellifera preferences for nectar sugars is as follows: sucrose > glucose > maltose > fructose.

Nectar Protein Content
Besides sugar, protein is the second important component in nectar, as it provides organic nitrogen to the consumers of this secretion [38,64,84,146,147].Pollinators compensate for the low availability of nitrogen in nectar through mechanisms increasing the consumption of this secretion [64,148].The mean percentage content of total protein in the nectar of the analyzed R. idaeus cultivars ranged from 0.12% in 'Glen Ample' to 0.21% in 'Pokusa'.In addition to the protein functions presented in the Section 1, i.e., (1) defense against microorganisms and (2) post-secretory hydrolysis of nectar sugars into fructose and glucose for protectors, literature reports show that PIN family proteins, especially PIN6 encoded by nectary-enriched gene PIN6, regulate the secretion of nectar and determine its proper function via auxins.The data also implicate the role of short stamens in the maturation of lateral nectaries [69,149].Mutants with reduced response to auxin produced 30-60% less nectar [149,150].Nepi [85] highlighted the ecological role of nectar proteins with special emphasis on selected amino acids.

Qualitative and Quantitative Composition of Nectar Amino Acids
The nectar of the analyzed Rubus idaeus cultivars was found to contain 15 protein amino acids.As reported in the literature, twenty, twelve, eight, and five amino acids were identified in Pyrus pyrifolia, Prunus spinosa, Crataegus monogyna, and R. fruticosus nectar, respectively [41,151].In turn, 17 amino acids were detected in Sanguisorba officinalis nectar [152], and 22 amino acids were identified in Crataegus pinnatifida [153].
As in other representatives of Rosaceae, endogenous aspartic acid, glutamic acid, and proline were the dominant amino acids in the nectar of the raspberries analyzed in the presented study [152][153][154][155].The amino acids in the floral nectar from Crataegus monogyna and Rubus fruticosus were dominated by aspartic acid and proline, whereas Prunus spinosa nectar had the highest concentrations of alanine, arginine, serine, and valine [41].Nectar from Crataegus exhibited the highest content of glutamic acid, which together with asparagine, glutamine, proline, and serine constituted over 65% of total amino acid content [153].Nectar from pear flowers was reported to contain the highest content of aspartic acid (3.9-7.8%)[156].
The percentage content of aspartic acid determined in the nectar of the six R. idaeus cultivars analyzed in the present study ranged between 14.2% ('Glen Ample') and 22.2% ('Laszka'), which was within the range or similar to the values determined in and Prunus persica (15.2-41.5%)[154], higher than in Crataegus pinnatifida (4.1-5.8%)[153], and lower than in Sanguisorba officinalis nectar (32%) [152].The content of glutamic acid in the nectar of the analyzed raspberry cultivars ranged from 10.8% in 'Polana' to 18.7% in 'Glen Ample'.These values were similar or within the range of this amino acid in the nectar of Crataegus pinnatifida (13.8-18.1%)[153] and Prunus persica (11.7-11.95%)[154], higher than the values reported for 11 species of the genus Pyrus (2.53-5.25%)[156].Glutamic acid also was identified in Prunus spinosa [40].Glutamic acid is an essential element in many metabolic pathways as a donor of amide groups for production of purine nucleotides and serotonin [157,158].Together with aspartic acid, cysteine, and lysine, it reduces oxidative stress in Apis mellifera larvae and adults [159,160].As reported by Lanza et al. [161], glutamic acid and hydroxyproline are the main determinants of the intraspecific variability in the qualitative composition of amino acids in nectar.Both these amino acids and proline are a source of nitrogen, carbon, and energy for insects [162][163][164].
Proline was the third most abundant amino acid in the nectar of the analyzed R. idaeus cultivars (from 6.8% in 'Laszka' to 14.8% in 'Pokusa').The levels of this amino acid were similar to those reported for Crataegus pinnatifida (11.6-23.2%)[153], higher than in Prunus persica (6.34-6.52%)[154] and Pyrus korshinskyi 'Lemon Bergomot' (2.12%), or partly similar to the range recorded for Pyrus longipes (8.06%) [156].Its content was lower than the concentrations determined in Crataegus pinnatifida nectar (from 15.5% in cultivar 'Geumsung' to 23.2% in cultivar 'Daemgeunsung') [153].As shown by literature data, proline is the dominant amino acid in the floral nectar of Crataegus monogyna, Prunus spinosa, Rubus fruticosus, and Sanguisorba officinalis [41,152].As reported by Kaczorowski et al. [165], the content of proline in nectar depends on the morphological structure of the flower.Higher proline levels have been detected in species with a shorter and wider corolla tube, which is most likely related to the fact that this structure increases the possibility of contact between nectar and pollen [82,166,167].In turn, Nicolson [64] suggests that the presence of pollen in nectar is not a source of amino acid variability.In plant tissues, proline is also produced under various abiotic stresses and is rapidly metabolized by pollinators [162,168].This amino acid stimulates receptor cells and, consequently, increases the nutritional requirements of insects [94,[169][170][171].Proline is preferred by honeybees, which eagerly collect nectar that is rich in this amino acid [172].It plays an important role in the reproduction and development of these insects and is a source of energy required for flight [71,172].Additionally, proline regulates the taste of nectar.It is also a link in metabolic pathways and is rapidly me-tabolized in the oxidation process, releasing large amounts of ATP [83,147,162,165,[169][170][171].According to one of the theories, an increase in the amount of proline in nectar is a coevolutionary strategy of plants aimed at attracting pollinators and, consequently, increasing yields [120].Proline and phenylalanine increase the concentration of sugars via the stimulation of bee chemosensors responsible for pollination [21].Moreover, proline is a component of antimicrobial peptides present in e.g., Apis mellifera hemolymph.These peptides have been shown to eliminate bacteria with no damage to the cell membrane [173].They are regarded as part of innate immunity and are present in all living organisms.By binding lipopolysaccharides (in vitro), they act selectively against gram-negative bacteria [174][175][176].
The concentration of alanine in the nectar of the analyzed R. idaeus cultivars ranged from 2.72% in 'Laszka' to 7.14% in 'Pokusa', with a mean value ranging from 5.2% in 'Glen Ample' to 6.4% in 'Polka'.The three-year mean contents of this amino acid presented in this study are lower or similar to those recorded in Crataegus pinnatifida (6.7-7.1%)[153].These values are partially consistent with the range reported for Prunus persica (5.85-15.21%)[154], Pyrus betulifolia (4.83%), Pyrus longipes (5.1%), Pyrus korshinskyi sp.(4.85-6.86%),Pyrus bretschneideri (4.13%), Pyrus boissieriana (5.13%), and Pyrus cossoni (4.98-5.1%),but they are lower than in Pyrus korshinskyi 'Lemon Bergamot' (7.88%) [156].Alanine contained in the nectar of Crataegus monogyna, Prunus species, Rubus fruticosus, and Sanguisorba officinalis has been identified as one of the dominant amino acids [41,152].Proline-, alanine-, and serine-enriched nutritional rations were preferred by Apis mellifera to a varying extent [172].Nectar with the addition of alanine was ingested by these insects on the first day of feeding, but later this amino acid had no significant effect on food preferences.Honeybees preferred nectar dominated by proline compared to that with dominance of alanine and serine, and nectar with higher alanine content was more willingly consumed by these insects than that enriched with serine [178,181].
In the group of endogenous amino acids, tyrosine was present in the lowest amounts in the nectar of the analyzed R. idaeus cultivars (from 1.13% in 'Glen Ample' to 3.47% in 'Polana').These levels were in a similar range to that reported by Caldwell and Gerhardt [154] in Prunus persica (0.97-3.26%) and higher than in all the species and varieties of Pyrus sp.investigated by Sharifani and Jackson [156] (<0.17%) and Chinese and Korean cultivars of Crataegus pinnatifida (0.4-0.6%) [153].Other authors reported low contents of tyrosine in the pool of all amino acids contained in nectar as well [41,153].An alternative to amino acids, which cannot be synthesized endogenously by bees, is the synthesis thereof from other amino acids, e.g., tyrosine from phenylalanine or cysteine from methionine.This so-called amino acid substitution has a favorable effect on the utilization of selected floral nectar compounds from this group (e.g., lysine and arginine or leucine and isoleucine) at the cellular level.The substitution of one amino acid with another one, where, e.g., tyrosine partially replaces phenylalanine and cysteine partially replaces methionine, and the reduction in the demand for these amino acids are characteristic traits of the honeybee metabolism [182].
In the nectar of the analyzed R. idaeus cultivars, leucine, lysine, and valine constituted a high percentage of exogenous amino acids versus the entire pool of these compounds, whereas threonine, phenylalanine, arginine, isoleucine, and histidine accounted for a smaller percentage share.This is in line with the results reported by Venjakob et al. [152] showing that the nectar of Sanguisorba officinalis was dominated by leucine and lysine, while valine was present in negligible amounts.As suggested by Dadd [95], both exogenous amino acids (leucine, isoleucine, lysine, valine, threonine, phenylalanine, methionine, arginine, and histidine) and endogenous amino acids (e.g., proline) are key compounds required for the development of many insects.
In the present study, the percentage content of valine in the R. idaeus nectar ranged from 4.4% in 'Glen Ample' to 5.3% in 'Polana'.These values were in the range reported for Prunus persica (3.9-5.43%)[154], and higher than in C. pinatinnatifida (2.5-4.0%)[153] and representatives of the genus Pyrus sp.(2.3-3.8%)[156].The considerable level of valine (18.5%), serine (16.7%), and phenylalanine in the apisimin protein contained in royal jelly contributes to activation of various cellular processes in bees [183].Some amino acids, e.g., valine and phenylalanine, regulate nectar preferences in insects by stimulation of sugar receptors, while others, e.g., proline, stimulate salt sensory cells; in turn, aspartic and glutamic acids and arginine delay their action [94,169,170].Pollinators sometimes react negatively to the content of glycine, leucine, valine, threonine, alanine, aspartic acid, methionine, and serine in nectar.Consequently, some amino acids exert an impact on the number of pollinator visits to flowers [84].
Among the exogenous amino acids analyzed in the present study, the concentration of threonine was ranked fourth, followed by phenylalanine and arginine.These three amino acids were reported to account for a small proportion of the total amino acid pool in Sanguisorba officinalis [152].In turn, Gyan and Woodell [41] demonstrated that threonine was present in small concentrations, but arginine dominated in Prunus spinosa and Crataegus monogyna nectar.
It is necessary to continue the search for key compounds in the chemical profile and analytical standard facilitating the development of a balanced diet that will be beneficial not only for bees, but also for humans, taking into account the positive effects on plantinsect interactions in the ecology of pollination.In future, given the perspective of a new field of research conducted with "omics" technologies, further studies should be focused on the development of genetic plant breeding to support the beneficial plant metabolome promoting interactions between flowering plants and pollinators.The present study results bring new data to the field of beekeeping.They provide information about the preferences and nutritional value of nectar determining the production of raspberry honey.Additionally, in the field of environmental protection, the results indicate that R. idaeus can be recommended for plantings in gardens and parks as a food base for insects in an urban environment.These data are also important for raspberry growers, as they indicate appropriate agrotechnical treatments that can ensure optimal flowering and provide food reward for honeybees and other pollinating insects, which will result in higher yields and a better quality of fruit and, consequently, higher economic profits.

Conclusions
In terms of the increasing sugar weight in the nectar, the studied cultivars were ranked as follows: 'Polka' < 'Polana' < 'Radziejowa' < 'Pokusa' < 'Laszka' < 'Glen Ample'.Their nectar was classified as hexose-dominant.In terms of the increasing content of total protein in the nectar, the following ranking of the cultivars was determined: 'Glen Ample' < 'Polana' < 'Laszka' < 'Radziejowa' < 'Polka' < 'Pokusa'.The nectar of the R. idaeus cultivars contained the highest amounts of aspartic acid, glutamic acid, and proline (endogenous amino acids) as well as lysine and leucine (exogenous amino acids).The information presented in the paper is valuable and particularly important to beekeepers and raspberry growers.Further investigations should focus on the biochemistry of nectar in association with food preferences of honeybees and other pollinators.Additionally, key compounds in the chemical profile and analytical standard should be determined in order to develop a beneficial balanced diet not only for bees, but also for humans.Given the new dynamically developing "omics" technologies, further interest should be focused on the development of genetic breeding to support the beneficial plant metabolome promoting interactions between flowering plants and their pollinators.

Figure 1 .
Figure 1.(A-F).Nectar abundance: nectar weight, nectar sugar percentage concentration, and nectar sugar weight of the six R. idaeus cultivars analyzed in the three study years.Explanations: means followed by the same small letter are not significantly different within the cultivar in the years, and means followed by the same capital letter do not differ between the cultivars in each year of the study at a significance level α = 0.05 (two-way ANOVA, Tukey's test).

Figure 1 .
Figure 1.(A-F).Nectar abundance: nectar weight, nectar sugar percentage concentration, and nectar sugar weight of the six R. idaeus cultivars analyzed in the three study years.Explanations: means followed by the same small letter are not significantly different within the cultivar in the years, and means followed by the same capital letter do not differ between the cultivars in each year of the study at a significance level α = 0.05 (two-way ANOVA, Tukey's test).

Figure 2 .
Figure 2. (A-F).Qualitative and quantitative composition of the nectar of the six R. idaeus cultivars analyzed in the three study years.Explanations: means followed by the same small letter are not significantly different within the cultivar in the years, and means followed by the same capital letter do not differ between the cultivars in each year of the study at a significance level α = 0.05 (two-way ANOVA, Tukey's test).

Figure 2 .
Figure 2. (A-F).Qualitative and quantitative composition of the nectar of the six R. idaeus cultivars analyzed in the three study years.Explanations: means followed by the same small letter are not significantly different within the cultivar in the years, and means followed by the same capital letter do not differ between the cultivars in each year of the study at a significance level α = 0.05 (two-way ANOVA, Tukey's test).

Figure 3 .
Figure 3.Total protein concentration in the nectar of the six R. idaeus cultivars analyzed in the three study years.Explanations: means followed by the same letter are not significantly between the cultivars at a significance level α = 0.05 (one-way ANOVA, Tukey's test).

Figure 3 .
Figure 3.Total protein concentration in the nectar of the six R. idaeus cultivars analyzed in the three study years.Explanations: means followed by the same letter are not significantly between the cultivars at a significance level α = 0.05 (one-way ANOVA, Tukey's test).