**7. Sugar concentration in nectar**

Nectar is considered the main reward to pollinator (Delaplane & Mayer, 2000) and its sugar concentration is associated to different pollinators, wherever the frequency and duration of visits depend on rate of nectar production (Biernaskie et al., 2002; Shafir et al., 2003; Nicolson & Nepi, 2005).

Toledo et al. (2005) reported total sugar concentration presents variation during the day in *Macroptilium atropurpureum* Urb., and can be related to number of visitor insects, specially, bees that collect nectar and pollen. In *Citrus sinensis*, it was verified that the high sugar concentration is an attractive, in special for *Apis mellifera*, and the availability of concentrated nectar during the day keeps the attractivity to pollinators (Malerbo-Souza et al., 2003). Therefore, the high quantity of nectar can leads to greater pollination rate by increasing in number of visitors bees (Silva & Dean, 2000).

Sugars are principal components of the floral nectar (Baker & Baker, 1973; Baker, 1977). The three most common sugar in nectar are sucrose, glucose and fructose in varying proportions (Freeman et al., 1985; Endress, 1994; Proctor et al., 1996; Baker et al., 1998). The amount of sugar secreted by flowers and consumed by pollinators cause a variation in sugar concentration of the flowers, during their anthesis period. Floral nectar consists of sugar pure solutions, specially glucose, sucrose and fructose (Roberts, 1979), however it can be found traces of oligosacchrides (Harbone, 1998).

Wykes (1952a) reported two oligosaccharides in nectar composition, the trisaccharide raffinose, and disaccharide melibiose in some nectaries. In some varieties of clover the presence of disaccharide maltose also was identified, however, this maltose can be a contaminant from aphids (Furgala et al., 1958). Taufel & Reiss (1952) confirmed that sucrose, glucose, and fructose, sugars promptly accepted by honeybees are current compounds present in nectar but another sugars can be present.

Besides, fructose and glucose, the presence of the monosaccharide D-galactose, in very low quantities (traces) also already was related in honey samples (Goldschmidt & Burket, 1955). However, it is important to emphasize that this monosaccharide when in its free form is considered a toxic compound to the honeybees (Siddiqui, 1970). Moreira & De Maria (2001) reviewed about carbohydrates in honey, and reported several di-, tri-, and oligosaccharides presented in honey and they came from nectar.

High-fructose is commonly used as sugar substitutes in processed foods, especially in soft drinks, mainly for economical reasons (Long, 1991). These products high-fructose corn syrups (HFCS) are obtained by enzymatic isomerization of corn syrups by both acid and enzymatic hydrolysis of cornstarch. Three enzymes are needed to transform cornstarch into the simple sugars glucose and fructose, α-amylase, glucoamylase, and glucose-isomerase. Fructosyl-fructoses were mainly detected in honey from honeybees fed with high-fructose corn syrups but not from those honeys coming from free-flying foragers or workers fed with sugar syrup (Ruiz-Matute et al., 2010).

Percival (1961) examined 889 plant species and found three pattern of carbohydrates to the nectar: a) nectar with high sucrose, nectar with similar quantities of sucrose, glucose and fructose; and nectar with high glucose and fructose. The nectar with sucrose dominant was associated to flowers of long tubes in which the nectar was protected (clovers), wherever the

Nectar is considered the main reward to pollinator (Delaplane & Mayer, 2000) and its sugar concentration is associated to different pollinators, wherever the frequency and duration of visits depend on rate of nectar production (Biernaskie et al., 2002; Shafir et al., 2003;

Toledo et al. (2005) reported total sugar concentration presents variation during the day in *Macroptilium atropurpureum* Urb., and can be related to number of visitor insects, specially, bees that collect nectar and pollen. In *Citrus sinensis*, it was verified that the high sugar concentration is an attractive, in special for *Apis mellifera*, and the availability of concentrated nectar during the day keeps the attractivity to pollinators (Malerbo-Souza et al., 2003). Therefore, the high quantity of nectar can leads to greater pollination rate by increasing in

Sugars are principal components of the floral nectar (Baker & Baker, 1973; Baker, 1977). The three most common sugar in nectar are sucrose, glucose and fructose in varying proportions (Freeman et al., 1985; Endress, 1994; Proctor et al., 1996; Baker et al., 1998). The amount of sugar secreted by flowers and consumed by pollinators cause a variation in sugar concentration of the flowers, during their anthesis period. Floral nectar consists of sugar pure solutions, specially glucose, sucrose and fructose (Roberts, 1979), however it can be

Wykes (1952a) reported two oligosaccharides in nectar composition, the trisaccharide raffinose, and disaccharide melibiose in some nectaries. In some varieties of clover the presence of disaccharide maltose also was identified, however, this maltose can be a contaminant from aphids (Furgala et al., 1958). Taufel & Reiss (1952) confirmed that sucrose, glucose, and fructose, sugars promptly accepted by honeybees are current compounds

Besides, fructose and glucose, the presence of the monosaccharide D-galactose, in very low quantities (traces) also already was related in honey samples (Goldschmidt & Burket, 1955). However, it is important to emphasize that this monosaccharide when in its free form is considered a toxic compound to the honeybees (Siddiqui, 1970). Moreira & De Maria (2001) reviewed about carbohydrates in honey, and reported several di-, tri-, and oligosaccharides

High-fructose is commonly used as sugar substitutes in processed foods, especially in soft drinks, mainly for economical reasons (Long, 1991). These products high-fructose corn syrups (HFCS) are obtained by enzymatic isomerization of corn syrups by both acid and enzymatic hydrolysis of cornstarch. Three enzymes are needed to transform cornstarch into the simple sugars glucose and fructose, α-amylase, glucoamylase, and glucose-isomerase. Fructosyl-fructoses were mainly detected in honey from honeybees fed with high-fructose corn syrups but not from those honeys coming from free-flying foragers or workers fed with

Percival (1961) examined 889 plant species and found three pattern of carbohydrates to the nectar: a) nectar with high sucrose, nectar with similar quantities of sucrose, glucose and fructose; and nectar with high glucose and fructose. The nectar with sucrose dominant was associated to flowers of long tubes in which the nectar was protected (clovers), wherever the

**7. Sugar concentration in nectar** 

number of visitors bees (Silva & Dean, 2000).

found traces of oligosacchrides (Harbone, 1998).

present in nectar but another sugars can be present.

presented in honey and they came from nectar.

sugar syrup (Ruiz-Matute et al., 2010).

Nicolson & Nepi, 2005).

opened flowers had generally only glucose and fructose. These reports confirmed early researches (Wykes, 1953; Bailey et al., 1954) that suggested a relation between three monosaccharides and different species with flower. In another research, the nectar was divided in four different classes in function to sucrose/hexose rate - S/H: sucrose dominant - S/H >0.999, rich in sucrose - 0.5<0.999, rich in hexose - 0.1<0.499 and hexose dominant - S/H<0.1 (Baker & Baker, 1983).

In the research of Alves (2004) and Alves et al. (2010), the means of sucrose.hexose-1 (S/H) per flower for all treatments were: 0.91μg.μL-1, for covered area with Africanized honeybee colony – rich in sucrose; 0.74μg.μL-1, semicovered area with free insects visitation – rich in sucrose; 0.86μg.μL-1, uncovered area with free insect visitation – rich in sucrose; and 3.05μg .μL-1, for covered area without Africanized honeybee colony – sucrose dominant. However, Severson e Erickson (1984) reported in several cultivars of soybean values from 1.2:1.0:1.4 to 1.2:1.0:6.7, with sucrose predominance, which sucrose concentration in nectar ranged from 97 to 986μg.μL-1, these means are higher than those reported by Alves et al. (2010) who found 12.06μg.μL-1. This range suggests that sugar concentration in soybean nectar is influenced by other environment factors independently of pollinator action. Robacker et al. (1983) reported that edaphic and climatic factors affect the number of flowers and another floral characteristics during soybean growing. So, the environmental conditions that generate an increase in number and size of flowers, higher anthesis period, colourness more intense, and also greater nectar production are the factors responsible by became flowers more attractive to honeybees (Alves et al., 2010).

Cruden et al. (1983) suggested that the maximum nectar accumulation occurs before or at the beginning of pollination activity. Such fact can be verified in siratro, since the highest sugar concentration was found at 8:30 a.m. (Figure 5) time in which the bee visitation started (Toledo et al., 2005). Variations in the siratro nectar sugar content measured along the day were observed (Figure 6 – Toledo et al., 2005) and probably associated with the intensity of foraging by honeybees, which is directly related to the nectar quantity and quality (Heinrich, 1979; Hagler, 1990) or to its sugar composition (Waller, 1972; Abrol & Kapil, 1991).

In flowers exposed to pollinators, it is possible that nectar secretion ceases if there is not pollinator in the area or can be reabsorbed in old or pollinated flowers (Cruden et al., 1983). A nectar production without reabsorption may be have an impact on reproductive biology (Galleto & Bernardello, 1995). Therefore, plants reabsorb nectar from aging flowers and utilize its carbon in developing seeds and this is a reproductive advantage (Zimmerman, 1988).

Chiari et al. (2005) studying the pollination of Africanized honeybees on soybean flowers (*Glycine max* L. Merrill) var. BRS 133, measured through the manual refractometer the sugar concentration as total solids and concluded that data found presented a big uniformity, different of the results obtained by Sheppard et al. (1978) that observed big variations in these concentrations and attributed these differences to the variation in the soil composition and other environmental conditions, like precipitation. Despite this, the mean values found by Chiari et al. (2005) were 21.33 ± 0.22% in uncovered area and 22.33 ± 0.38% in covered area with honeybees and differed to each other (P=0.0001). Besides, the medium amounts of total sugar and glucose measured in the nectar of the flowers were 14.33 ± 0.96mg/flower and 3.61 ± 0.36mg/flower, respectively, in the same research.

Spectrophotometry as a Tool for Dosage Sugars in Nectar of Crops Pollinated by Honeybees 283

The study of 25 canola (*Brassica napus x Brassica campestris*) varieties carried out by Kevan et al. (1991) demonstrated that 23 of them had 0.95 or more glucose:fructose rates in their nectar. The same authors reported too that only three varieties had glucose in smaller quantities and none of the samples had detectable quantities of sucrose. Davis et al. (1998) reported higher glucose/fructose rate in lateral chambers than median in Brassicaceae.

Chromatography in paper showed that the nectar from siratro (*Macroptilium atropurpureum*  Urb.) is constituted exclusively of glucose, as may be seen in Figure 5 (Toledo et al., 2005). It can be explain the low percentage of honeybee visit on siratro flower - 4%, wherever the another bees were *Trigona spinipes -* 24%, *Bombus morio* - 8%, *Euglossa sp* – 20%, *Megachilidae* - 12%, *Pseudaugochloropsis graminea* – 8% and *Halictidae* - 8% (Vieira et al., 2002). From this, it can be concluded that the main visitor and pollinator in siratro was *Euglossini*, however, *Trigona spinipes* perfurated the external part of the flower avoiding the contact with the

Alves (2004) reported a variation of total sugar concentration in soybean nectar var. Codetec 207 from 21.33 to 27.47%, and the higher total sugar concentration was observed in covered area with Africanized honeybee colony. The sucrose concentration ranged from 9.63 to 13.61%, and the higher sucrose concentration was observed in covered area with Africanized honeybee colony. The glucose concentration in this variety was very low. The fructose concentration ranged from 7.93 to 13.75%, so the covered area with Africanized honeybee colony presented higher concentration too. Alves (2004) suggested that Africanized honeybees

Aproximately, 20% of all food-crop production and about 15% of seed crops require the help of pollinators for full pollination (Klein et al., 2007), but Kevan & Phillips (2001) reported that aproximately 73% of cultivated vegetals in the world would be pollinated by some bee species. Gallai et al. (2009) reported a bioeconomic approach, which integrated the production dependence ratio on pollinators, for the 100 crops used directly for human food worldwide as listed by FAO. The total economic value of pollination worldwide amounted to €153 billion, which represented 9.5% of the value of the world agricultural production used for human food in 2005. The honeybee is the most common insect used as agricultural

In United States, farmers rent more than 2 million honeybee colonies every year for pollination, but the honeybee is being threatened by several problems like Colony Colapse Desorder (James & Pitts-Singer, 2008). The cost of renting bees is up from US\$50 per hive in 2003 to US\$ 140 per hive in 2006 (Sumner & Boriss, 2006). Some crops require five to seven hives per hectare. In addition, no all crops are well pollinated by honeybees. For example, tomatoes require buzz pollination, which honeybees cannot achieve and alfalfa flowers are not properly worked by honeybees. Fortunately, honeybees are not the only bees that make

Therefore, Greenleaf & Kremen (2006) reported that behavioral interactions between wild and honeybees increase the pollination efficiency of honeybees on hybrid sunflower up to 5 fold, effectively doubling honeybee pollination services on the average field. These indirect contributions caused interspecific interactions between wild and honeybees were more than five times more important than the contributions wild bees make to sunflower pollination

estimulated the sugar secretion in soybean nectar var. Codetec 207 (Table 1).

pollinator in many parts of the world like Europe and United States.

good pollinators (James & Pitts-Singer, 2008).

directly.

pollen grains (Vieira et al., 2002).

Fig. 5. Total sugar and glucose contents in nectar of siratro flowers along two-day period reprinted from Toledo et al. (2005) with permission.

Fig. 6. Total sugar contents in nectar of siratro flowers along two-day period - reprinted from Toledo et al. (2005) with permission.

A- 20/05/2000 - Total Sugar B- 27/05/2000 - Glucose C- 20/05/2000 - Total Sugar D- 27/05/2000 - Glucose

8 10 12 14 16

Hour

6 8 10 12 14 16 18 Time (hour)

Fig. 6. Total sugar contents in nectar of siratro flowers along two-day period - reprinted

\* \*

\*

\*

\*

\*

Fig. 5. Total sugar and glucose contents in nectar of siratro flowers along two-day period -

0,0

0.0

from Toledo et al. (2005) with permission.

0.5

1.0

1.5

A

\*

B

\*

2.0

2.5

Total sugar (mg / flower)

3.0

3.5

4.0

4.5

0,5

1,0

1,5

2,0

Sugar Concentration

mg/flower

2,5

3,0

D B

C

reprinted from Toledo et al. (2005) with permission.

A

3,5

The study of 25 canola (*Brassica napus x Brassica campestris*) varieties carried out by Kevan et al. (1991) demonstrated that 23 of them had 0.95 or more glucose:fructose rates in their nectar. The same authors reported too that only three varieties had glucose in smaller

Chromatography in paper showed that the nectar from siratro (*Macroptilium atropurpureum*  Urb.) is constituted exclusively of glucose, as may be seen in Figure 5 (Toledo et al., 2005). It can be explain the low percentage of honeybee visit on siratro flower - 4%, wherever the another bees were *Trigona spinipes -* 24%, *Bombus morio* - 8%, *Euglossa sp* – 20%, *Megachilidae* - 12%, *Pseudaugochloropsis graminea* – 8% and *Halictidae* - 8% (Vieira et al., 2002). From this, it can be concluded that the main visitor and pollinator in siratro was *Euglossini*, however, *Trigona spinipes* perfurated the external part of the flower avoiding the contact with the pollen grains (Vieira et al., 2002).

quantities and none of the samples had detectable quantities of sucrose. Davis et al. (1998) reported higher glucose/fructose rate in lateral chambers than median in Brassicaceae.

Alves (2004) reported a variation of total sugar concentration in soybean nectar var. Codetec 207 from 21.33 to 27.47%, and the higher total sugar concentration was observed in covered area with Africanized honeybee colony. The sucrose concentration ranged from 9.63 to 13.61%, and the higher sucrose concentration was observed in covered area with Africanized honeybee colony. The glucose concentration in this variety was very low. The fructose concentration ranged from 7.93 to 13.75%, so the covered area with Africanized honeybee colony presented higher concentration too. Alves (2004) suggested that Africanized honeybees estimulated the sugar secretion in soybean nectar var. Codetec 207 (Table 1).

Aproximately, 20% of all food-crop production and about 15% of seed crops require the help of pollinators for full pollination (Klein et al., 2007), but Kevan & Phillips (2001) reported that aproximately 73% of cultivated vegetals in the world would be pollinated by some bee species. Gallai et al. (2009) reported a bioeconomic approach, which integrated the production dependence ratio on pollinators, for the 100 crops used directly for human food worldwide as listed by FAO. The total economic value of pollination worldwide amounted to €153 billion, which represented 9.5% of the value of the world agricultural production used for human food in 2005. The honeybee is the most common insect used as agricultural pollinator in many parts of the world like Europe and United States.

In United States, farmers rent more than 2 million honeybee colonies every year for pollination, but the honeybee is being threatened by several problems like Colony Colapse Desorder (James & Pitts-Singer, 2008). The cost of renting bees is up from US\$50 per hive in 2003 to US\$ 140 per hive in 2006 (Sumner & Boriss, 2006). Some crops require five to seven hives per hectare. In addition, no all crops are well pollinated by honeybees. For example, tomatoes require buzz pollination, which honeybees cannot achieve and alfalfa flowers are not properly worked by honeybees. Fortunately, honeybees are not the only bees that make good pollinators (James & Pitts-Singer, 2008).

Therefore, Greenleaf & Kremen (2006) reported that behavioral interactions between wild and honeybees increase the pollination efficiency of honeybees on hybrid sunflower up to 5 fold, effectively doubling honeybee pollination services on the average field. These indirect contributions caused interspecific interactions between wild and honeybees were more than five times more important than the contributions wild bees make to sunflower pollination directly.

Spectrophotometry as a Tool for Dosage Sugars in Nectar of Crops Pollinated by Honeybees 285

concentration in covered area with Africanized honeybee colony, reduction of sucrose concentration in uncovered area, and lower fructose concentration in covered area without honeybee colony and uncovered area. Besides, none difference among treatments in relation to glucose concentration. So, the high total sugar concentration observed in nectar of soybean var. Codetec 207, in covered area with Africanized honeybee colony suggests that the presence of *Apis mellifera* influenced in this composition, even though a variety with high grade of autopollination. However, the low fructose concentration in uncovered area can be related with low density of honeybees recorded or low presence of another preferential pollinator of fructose. For some researchers, the pollinators can affect the nectar composition (Canto et al., 2008; Herrera et al., 2009). In *Helleborus foetidus*, for example, some species of *Bombus* unchain modifications in sugar composition in nectar reducing the sucrose

Nectar is an important floral reward for the bee visitation in the flowers. That food is converted in honey into the hive and used as an energy source for the workers. The beeplant interaction is essential for the maintenance of genetic variability of plants as well as

Honeybees are very important for the pollination of both cultivated and native plants, and then understand the relationship between the collection of floral products (nectar, pollen, resins and oils), biology and behaviour of these insects help making better use for making bee products and agricultural products. Spectrophotometry is a tool to quantify and identify the components of floral products used by bees, especially nectar. These tests allow checking

The methods employed in this molecular tool began to be developed in the early 50s of last century. Currently, the main analyses performed with the nectar spectrophotometry are: total sugars, reducing sugars, fructose determination (furfural and resorcinol), glucose oxidase (glucose determination in foods), hexokinase (glucose and fructose) and sugar concentration in nectar. As well as, in honey are made several analyses too, like diastase

Among the tests that can be done is determining sugar concentration in the nectar. The quantification of these sugars allows developing a series of studies associated with the floral visitation by bees collecting nectar and pollination of several species of cultivated plants. These studies contribute to the use of *A. mellifera* to assist beekeepers in increasing the honey production and the farmer in agricultural production. The association between agriculture and beekeeping has been demonstrated by studies presented that showed a positive association between the type of nectar produced by plant and intensity collection by honeybees. Much is still to be undertaken because the number of plant species studied that

Especially in tropical regions, stingless native bees must be preserved and have great potential apicola and sustainable that is not operated, besides to the physicochemical analysis of the sugar composition of honey produced by these bees is not well known and do not have legislation around the world. Floral preferences of native stingless bees are not

are pollinated by bees is limited in relation to the number of known species.

the correlation between the type and intensity of nectar in flower visitation by bees.

percentage, and rising the fructose and glucose percentage (Canto et al., 2008).

increased production of grains and fruits on a commercial scale.

index, hydroxymethylfurfural and others.

**8. Conclusion** 


\*n- sample size; x-Averages; se-standard error; H - H test; DF-degrees of freedom; KW-Kruskal-Wallis Probability; Rx-Medium position; ns-non significant and probability of the interactions (T1, T2, T3 and T4 vs T2, T3 and T4)

Table 1. Means of total sugar, sucrose, glucose and fructose concentration (%), in soybean nectar (Glycine max L. Merrill) var. Codetec 207 for covered area with honeybee colony (T1); Semi-covered area (T2); uncovered area (T3) and covered area without honeybee colony (T4) – Reprinted from Alves (2004) and Alves et al. (2010) with permission

Analysis of sugar composition in nectar can be used for detecting variation between flowers or nectaries from different taxonomic varieties, and consequently generate differences in type and frequency of visitation of pollinators. Alves et al. (2010) studied the total sugar concentration in soybean nectar (*Glycine max* L. Merrill) var. Codetec 207 by spectrophotometry, using the general method for carbohydrates determination by phenolsulphuric technique (Dubois et al., 1956).

The research of Alves et al. (2010) was carried out in soybean plants in cages of 24m2 covered with an Africanized honeybee colony inside in which, semicovered area with free insect visits, uncovered area, covered area without Africanized honeybee colony. Each treatment was five repetitions. In this research, it was emphasized greater total sugar concentration in covered area with Africanized honeybee colony, reduction of sucrose concentration in uncovered area, and lower fructose concentration in covered area without honeybee colony and uncovered area. Besides, none difference among treatments in relation to glucose concentration. So, the high total sugar concentration observed in nectar of soybean var. Codetec 207, in covered area with Africanized honeybee colony suggests that the presence of *Apis mellifera* influenced in this composition, even though a variety with high grade of autopollination. However, the low fructose concentration in uncovered area can be related with low density of honeybees recorded or low presence of another preferential pollinator of fructose. For some researchers, the pollinators can affect the nectar composition (Canto et al., 2008; Herrera et al., 2009). In *Helleborus foetidus*, for example, some species of *Bombus* unchain modifications in sugar composition in nectar reducing the sucrose percentage, and rising the fructose and glucose percentage (Canto et al., 2008).
