The effects of dietary protein different levels and Silymarin on population growth and hypopharyngeal gland of Iranian honey bee workers (Apis mellifera meda)
Pollen substitute diets are a valuable resource for maintaining strong and healthy honey bee colonies. Current study was performed to investigate the effects of dietary protein different levels and Silymarin (SM) on honey bee colonies, specifically the population growth and hypopharyngeal gland of honey bee workers in Khuzestan Ramin Agricultural and Natural Resources University, Ahwaz, Iran from November to February 2015. Diets containing four different levels of crude protein (0, 20, 30 and 40 %), two levels of SM (0 and 0.2 Mm) and pure pollen (control) were evaluated. The artificial diets were included soybean meal, corn gluten, wheat gluten and yeast bread. Twenty colonies of honey bees with sister queens were investigated. Effects of the different bee diets were compared by measuring population growth, emergent worker weight, and hypopharyngeal gland development. Based on the present results the SM supplementation improved honeybee survival at a dose of 0.2 mM. Moreover, significantly greater emergent worker weight and hypopharyngeal gland acini were observed in the colonies fed the colonies fed the control diet and diet containing 30% crude protein (CP) compared to the other crude protein levels. The study concluded a dietary crude protein content of 30% is recommended to maximize the reproduction rate of honey bee colonies.
Keywords: dietary protein, population growth, hypopharyngeal gland, honey bee workers.
According to the study of vanEngelsdorp et al. (2009) Honey bees as major pollinators of many food and wild plants, their population has declined. A poor diet, due to land use changes reducing the availability and diversity of floral resources, might help drive these declines (Vanbergen and Initiative, 2013). In keeping with this idea, nutrition is a key determinant of honeybee survival (Altaye et al., 2010; Archer et al., 2014). Understanding the association between diet and honeybee survival is essential to protect declining and threatened honeybee populations (Pirk et al., 2014).
Oxidative stress suppresses animal health, performance, and production, subsequently impacting economic feasibility; hence, maintaining and improving oxidative status especially through natural nutrition strategy are essential for normal physiological process in animals (Li et al., 2012). Antioxidants, which neutralize Reactive Oxygen Species (ROS), help maintain this balance and a poor diet could, in theory, disrupt it. Diets high in protein or low protein could push cells into oxidative stress by increasing ROS production from the mitochondria, impairing antioxidant defenses against ROS or reducing the repair of oxidised molecules (López-Torres and Barja, 2008). Silymarin (SM) as a natural antioxidant could be considered as one of the most promising materials used in animal diets in various forms. SM is the bioactive extract from Silybum marianum L. seeds (Asteraceae) and contains 65-85% flavonolignans like silychristin, isosilychristin, silydianin, silybin A and B, isosilybin A and B), and also 20- 35% fatty acids, flavonoids, and other polyphenolics. The major source of SM is fruits and seeds from this plant, but traces of these compounds can occur in all plant parts (Ramasamy and Agarwal, 2008). Worker bees start to consume pollen just a few hours after emerging (Hagedorn and Moeller, 1967; Dietz, 1969) and an enough supply of proteins particularly during the first two weeks after emergence is required to sustain their normal growth and development, and for them to be able to rear larvae (Haydak, 1963). Pollen as a natural and protein-rich food source for honey bees (Schäfer et al., 2006) is essential for the production of royal jelly; a high-protein food used to feed bee larvae and adult queens (Crailsheim, 1992). Royal jelly is secreted by two hypopharyngeal glands (HPGs), situated in the head, reaching maximum development in nurse bees, around 6–12 days after emergence, and afterwards degenerate in forager bees (Deseyn and Billen, 2005). A decline in accessibility and protein content of bee-collected pollen might result in the development of lower HPGs (DeGrandi-Hoffman et al., 2010; Di Pasquale et al., 2013), less brood reared (Herbert et al., 1977; DeGrandi-Hoffman et al., 2008), shorter longevity (Schmidt et al., 1987; Di Pasquale et al., 2013) and recruitment of bees at a young age (Sagili and Pankiw, 2007), eventually entailing a decreased lifespan (Khoury et al., 2011). During times of pollen scarcity, the pollen reserves in the combs and protein reserves in bees are quickly expended. Consequently, supplementary pollen or pollen substitutes are needed to preserve the colony’s strength for pollination services or honey production (Herbert et al., 1977). The availability of pollen depends on the plants’ growing seasons during the year. Brood production could decrease or even stop completely in the colonies if there is no pollen or not available at desirable pollen substitute.
The pollen might contain spores of pathogens causing diseases such as chalkbrood (Flores et al., 2005) or American foulbrood in bees and/or larvae. Provision of artificial diets is a safe way to feed bees protein. Beekeepers often provide pollen substitute diets to colonies, although these are often formulated without considering the costs of the diet components versus the benefits of providing such diets (Herbert et al., 1977; Li et al., 2012; Morais et al., 2013).
This study aims to investigate the effects of dietary protein levels on honey bee colonies, especially the population growth and hypopharyngeal gland of honey bee workers during fall.
Material and methods
Effects of Silymarin dose on the survival of worker bees fed sucrose solution
To identify an appropriate dose of SM for supplementation, brood frames were collected from three different colonies at the Department of honey bee, Animal Sciences Research Institute of Iran and incubated at 34°C in constant darkness. On the day of their emergence from the brood comb, freshly emerged (<24 h) workers were caged in groups of 100 individuals. Each group received a diet consisting of 0.68 M sucrose solution and one of six SM doses (0, 0.2, 0.4, 0.6, 0.8 and 2.4 mM). Diets were made every two weeks to ensure that the Silymarins did not deteriorate and were frozen in aliquots at -20 °C and defrosted on the day of use. Each colony received all six diets; therefore, a total of 18 groups were fed in standard laboratory hoarding cages (Köhler et al., 2013) following standard procedures (Köhler et al., 2012). The liquid diet and water were provided fresh daily, when survival was also measured and dead bees removed from cages. To calculate the consumption of diets, the mass changes between the experimental were measured and then were retrieved twenty-four hours later diets.
Current study was performed in Khuzestan Ramin Agricultural and Natural Resources University, Ahwaz, Iran from November to February 2015. The study was terminated when flowering plants became available for the bees and supplemental feeding was no longer necessary. A set of equalized honey bee (Apis mellifera Medda) colonies were selected during the fall. The colonies were randomly assigned to different protein-level treatments. Prior to the experiment, each colony consisted of a 4-mo-old queen that had been reared and mated naturally and the same quantity of bees. Three empty comb frames were applied for each brood chamber, and two comb frames were full of honey. According to Burgett (1985) the population of winter bees was evaluated. To prevent pollen from entering the vent, all of the hives were installed with a pollen trap.
The effects of four diets containing different levels of crude protein (CP) were tested. The dietary formulas and the approximate compositions of four isocaloric test diets (diet 1-diet 4); two levels of SM (0 or 0.2 Mm) and pollen (control diet) are presented in Table 1. The CP of the diets was measured using the Kjeldahl nitrogen procedure. The experiment was consisted of testing 36 colonies (9 diets by 4 colonies). The defatted soybean meal, corn gluten, wheat gluten and yeast bread components of the diet were sieved through a 185 μm mesh. The diets were supplemented by providing the colonies with prepared feed patties weighing 400 g each consisting of the dry feeds. The patties were wrapped in ordinary waxed paper and placed on top of the frames over the brood clusters. Also, the honey and patties were checked every 3-4 d, and new honey and patties were supplied when the levels were inadequate.
Coding and Sampling the Bees
Emerging brood combs were put in single-combs isolator from the colonies, to obtain bees of defined ages. Fifty newly emerged adult worker bees were collected per colony within 6 h, and their thoraces were marked with shellac paint (von Frisch, 1965). To minimally disturb the colonies, the sampling was carried out without the use of smoke, inducing workers to feed on honey (Free, 1968; Hrassnigg and Crailsheim, 1998b) and affects the trophallactic behavior of the workers (Farina and Núñez, 1991).
Monitoring Brood Rearing Activity in the Colonies
Capped broods number was evaluated to determine the workers number reared in each colony. Assessments were made at 12-d intervals, once before and four times after treatment. The pupating workers remained in the sealed cells for 12 d (Winston 1987), and the sealed broods mortality was so little (generally, the workers mortality in sealed broods was lower than 3%; according to Fukuda and Sakagami (1968)). Thus, a new set of pupating workers was counted in each assessment. The capped broods number was monitored via a modified square grid system (Mattila and Otis, 2007). The grids with characteristic of 189 squares and each with an area of 4 cm2were placed over the brood frames, and the comb area occupied by the capped brood was measured. The total number of bees reared in each colony was evaluated using a factor of 4.29 worker cells per square centimeter (Seeley and Visscher, 1985).
Newly Emerged Workers
The wet weight of 20 bees per colony was obtained within 2 h after emergence. Nutrition quality was measured via CP level and this index is correlated with the physiological conditions of worker bees (Standifer et al., 1960; Pernal and Currie, 2000).
Hypopharyngeal Gland Measurements
Five honey bee workers from each colony were selected at 9 d of age to assess HPG development. The HPGs were removed and placed in a Petri dish with wax depressions each containing a droplet of ice-cold sodium chloride solution (0.85%, isotonic to the hemolymph). Micrographs of the HPGs were taken using a microscope equipped with a camera. For calibrating, an image of a 1-mm scale bar was obtained at the same magnification. The analysis of HPG acini was carried out by measuring the areas of five acini cells selected randomly for each bee using the Photoshop (Adobe) pixel counting routine. Hereupon, 25 acini cells were surveyed for each colony for a total of 125 acini cells per treatment.
The data were analyzed as a completely randomized design by one-way ANOVA using Proc MIXED. All statistical analyses were performed with SAS, version 9.4 (SAS Institute, 2014). Protein level and SM was the main effect and colonies in each group were the random factor. The significance of differences among treatments (P < 0.05) was tested using Duncan’s new multiple range test of SAS.
Effects of silymarin dose on survival of worker bees fed a single, pure carbohydrate diet
The dose of SM that bees were fed affected their survival (Fig. 1). SM improved survival with dosage of 0.2 mM, compared to other treatment. The other SM doses (0.4, 0.6, 0.8 and 2.4 mM) had no significant effect on lifespan.
The Brood rearing area in sealed broods during the experiment is shown in Fig. 1. Furnishing diets with different CP levels to the experimental colonies had a significant effect on the timing of the increase in brood rearing activity as the season progressed (P < 0.05). The average number of workers brood cell by the colonies quickly increased. At the beginning of the experiment, all of the brood colonies were similar. Treatments supplements with pollen and 30% CP colonies tended to have the greatest number of workers in sealed broods at each colony census, and brood rearing activity was not significantly different from that observed with other treatments (P > 0.05). Treatment of 20% CP and 40% CP colonies represents more workers than sugar treatment, but were statistically insignificant (P > 0.05).
Newly Emerged Workers-Emergent Worker Weight
The average emergent worker weight was significantly affected by dietary protein levels (P < 0.05; Fig. 2). The greatest emergent worker weights were obtained with honey bees fed the 30% CP diet, but there was no significant difference between the weights of honey bees fed the 30% CP and 30% CP with SM diet and pollen diet, and the lowest emergent worker weights was observed in the treatment of sugar, although it was statistically insignificant (P > 0.05).
The highest protein concentrations in body were obtained using the 30% CP with SM diet (Fig. 3), but there was no significant difference between protein in body of the bees fed the 30% CP diet, 20% CP diet and the control diet . There was significant difference between the protein in the body, the 40% CP diet and 0% diet against other treatments.
Development of hypopharyngeal glands
The largest HPG acini in the nurse bees were obtained at 30.0% CP with SM (P < 0.05). There were no significant differences in HPG development in bees fed 30.0% CP versus one receiving mix pollen (P > 0.05; Fig. 3). However, the average area of the HPG acini was significantly greater in bees fed the 30.0% CP diet and the bees fed the mix pollen diet than other treatments.
Caged honey bees survived favourably when fed sucrose and 0.2 mM SM solutions. Antioxidant supplementation at low doses improved honey bee survival. According to (Surai, 2015), there are many possible mechanisms by which SM can improve the antioxidant defence mechanisms in the body
In the current study, we provided experimental evidence for a link between dietary protein and important parameters in honey bees. Somerville (2005) showed that what is probably more important for the growth rate and development of bees is the total protein intake of a colony but not simply food consumption. The high-protein diets also increased colony growth parameters during periods of scarcity of pollen resources, in the field experiments, as also reported by Mattila and Otis (2007). Bee bread and the artificial protein diets were well accepted by the bees. Cremonez et al. (1998) also found the highest protein values in bees fed bee bread.
Body weight is not an appropriate index of measuring the protein status of bees fed with high crude protein and low crude protein. Proteins are responsible for 66-74% of the dry matter of adult workers (Hrassnigg and Crailsheim, 1998a). This protein content increases during the first days due to protein anabolism and decreases as the workers age(Crailsheim, 1992). Measurement protein content (especially hemolymph protein concentration) in honey bees is an effective method to evaluate the protein diet quality (De Jong et al., 2009). It is therefore reasonable to conclude that there is a strong association between dietary protein content and worker protein content. Current results showed that increase in dietary protein from 20% to 30% of colonies led to enhancement of number of workers in sealed broods at each colony census, and brood rearing activity was noticeable however, increase in dietary protein from 30% to 40% was associated with decrease number of workers in sealed broods at each colony census, and brood rearing activity was observed. Garcia et al. (1986) supplied various protein foods with 20, 30 and 40% crude protein; they reported that CP negatively affects food collection. To evaluate the diet efficiency precisely, total protein content in newly emerged workers was tested. Increase in dietary protein from 20% to 30% of colonies in present study had significantly positive effect in the protein concentrations of body weight, but increase in dietary protein from 30% to 40% substantially decreased the protein concentrations of body weight. significant difference was not observed between protein supplement of 30% treatment and pollen treatment. Similar to current results bees fed protein supplement 15% diet had lower body protein concentration than bees fed protein supplement 30.5% and protein supplement 35% diet, indicating that protein level in protein supplement 15% diet was not supportive for the optimum growth and development of bees (Li et al., 2012)
Herbert et al. (1977) reported that pollen substitutes containing 50% protein depressed brood rearing. Although the 40% CP diet is rich in protein, the extra protein might inhibit the absorption of other nutrients or otherwise result in fitness costs. Current study demonstrated that 30% protein level is optimal for meeting the nutritional requirements for brood rearing during fall. The bee development enhancement with increase in protein intake is probably due to honey bees requiring protein for producing cuticle (Campbell, 1929), muscle, and other tissues (Somerville 2000). The second possible explanation is that a lack of dietary protein could reduce the immunity in honey bees (Alaux et al., 2010).
Results indicated that increase in dietary protein from 20% to 30% of colonies had significant effect on HPG acini, whereas increase in dietary protein from 30% to 40% significantly decreased the HPG asinal surface. Moreover, the nurse bees fed with 30% CP had larger HPG acini, compared to those fed with pollen but was not significant. The effect of SM was not significant, but the HPG asinal surface to improve. Our study indicates that protein supplements can closely resemble pollen in nutritional value and because of their effects on protein concentrations, population growth and HPG development can play a significant part in reducing colony losses. Similar results were obtained when comparing honey bees fed pollen patties versus Mega Bee patties (DeGrandi-Hoffman et al., 2010).
Contrary to current results, (Zheng et al., 2014) reported that nurse bees fed pollen supplements had significantly larger HPG acini than the control group, which was fed pure pollen and confirmed that the superficial area of HPG acini increased with the bees age regardless of whether the colonies were fed pollen or pollen supplements. It is seemed that the pollen supplements activated HPG development similarly to that activated by bee bread. Our observations indicated that bees fed the 30 % CP diet had the largest HPG acini and brood rearing activities.
In general, current results suggest that an adequate provision of protein is required to sustain normal development of bees. The results of the current study demonstrate that dietary protein levels strongly affect the population growth, performance, and physiological status of worker bees.
In present study, protein supplements can closely resemble pollen in nutritional value and because of their effects on protein concentrations and HPG development can play a significant part in colony. Diet containing 30% protein were recognized as an excellent one to promote honey bee colonies development. The optimum protein content in field applications might differ according to dietary ingredient compositions and feeding methodology. These findings are particularly important for the successful bee keeping (colonies management) using pollen supplements when natural pollen is unavailable. In conclusion, population growth, body protein content and surface Hypopharyngeal gland of emerging workers and worker quality were significantly affected by dietary composition and could be manipulated as metabolic tools to assess the optimal concentration of dietary protein in the honey bees feeding.
Ingredients and chemical compositions of the experimental diets (on dry matter basis)
|Dietary protein levels|
|Ingredients (%)||0 (diet 1)||20 % (diet 2)||30 % (diet 3)||40 % (diet 4)||Control diet (control)|
|Crude protein (%)||0||20.09||29.96||39.84||22.52|
|Gross energy (MJ/kg)||4030||4220.9||4303.9||4388.45||4660|
Gross energy (kJ/g diet) = (%Crude protein × 23.6) + (%Crude lipids × 39.5) + (%Carbohydrates × 17.3).
honey bee workers survival fed 0.68 M sucrose without SM (control) and with five SM concentrations until bee population decline by half. diet 1: suger, diet 2: suger with 0.2 Mm SM, diet 3: suger with 0. 4 Mm SM, diet 4: suger with 0. 6 Mm SM, diet 5: suger with 0.8 Mm SM and diet 6: suger with 2.4 Mm SM. Different letters signify significant differences at P<0.05.
Mean number of Sealed pupa by colonies fed mix pollen and diets with different protein levels (0, 20, 30 and 40 %). (N = 5 colonies per treatment). Different letters signify signiﬁcant differences at P < 0.05.
Mean emergent worker weight (A ±SE) bees fed pollen and diets containing different protein levels (0, 20.09, 29.96, and 39.84%), two levels of silymarin (0 or 0.2 Mm) N=4 colonies per treatment). Different letters signify significant differences at P<0.05.
1= 20.09% CP, 2= 29.96% CP, 3= 39.84% CP, 4= 20.09% CP and 0.2 Mm SM, 5= 29.96% CP and 0.2 Mm SM, 6= 39.84% CP and 0.2 Mm SM, 7= Mixed pollen, 8= Flour sugar and 9= Flour sugar and 0.2 Mm SM.
The mean Protein Concentration (±SE) of workers fed pollen and the various dietary protein levels (0, 20.09, 29.96, and 39.84%), two levels of silymarin (0 or 0.2 Mm) N=4 colonies per treatment). Different letters signify significant differences at P<0.05.
1= 20.09% CP, 2= 29.96% CP, 3= 39.84% CP, 4= 20.09% CP and 0.2 Mm SM, 5= 29.96% CP and 0.2 Mm SM, 6= 39.84% CP and 0.2 Mm SM, 7= Mixed pollen, 8= Flour sugar and 9= Flour sugar and 0.2 Mm SM.
Development of hypopharyngeal glands during the nursing period of honey bee workers (at 9 d; N=4 colonies per treatment). The control diet is mixed pollen, and the dietary protein levels in the diets are as following: diet 1, 20.09% CP; diet 2, 29.96% CP; diet 3, 39.84% CP; diet 4, 20.09% CP with SM; diet 5, 29.96% CP with SM; diet 6, 39.84% CP with SM diet 7, mixed pollen, diet 8, suger and diet 9, suger with SM. Different letters signify significant differences at P<0.05.