Pollen Substitute Diet for Apis Mellifera: Consumption and Effects on Colony Parameters in Sub-Tropical Himalaya

Beekeeping plays an important role in Indian economy, as it directly impacts agricultural sector through pollination. As per report published for the year 2016-17 by the Ministry of Agriculture and Farmer’s Welfare, Govt. of India, the total geographical area of India is 328.7 million hectares, out of which 200.9 million hectares is the gross cropped area and 141.4 million hectares is the net sown area (source: http://agricoop.nic.in/sites/default/files/Annual rpt 2016-17 E.pdf). As per an estimate, it requires approximately 50 million honey bee colonies to pollinate this crop area, but we have only 30 lac bee colonies in our country (source: http://pib.nic.in/newsite/printrelease.aspx?relid). There are several factors responsible for less bee colonies and decline in honey bee health/population in India, main of which is the dietary problem. Like all other animals, honey bees do have specific nutritional requirements. Pollen collected by bees from flowers are the major sources of protein, carbohydrates, minerals and vitamins whereas, nectar is the source of energy.
       
During prolonged dearth periods, conditions of starvation, quick dwindling and perishing of honey bee colonies have been observed due to non-availability of flowers and rapid consumption of stored honey and pollen (Mishra, 1995; Kumar et al., 2013). In India, natural bee flora starts disappearing in the month of May causing dearth of food (pollen and nectar) for bees. The periodical dearth periods of pollen result into low nutritional reserves which reduces the longevity, poor growth of hypopharyngeal gland, less royal jelly production, inferior growth of worker larvae and young bees (Zaytoon et al., 1988; Saffari et al., 2010a). Also due to poor  strength, colonies  may be attacked by various bee enemies like black ants, bee eating birds,Varroa mite. All these factors lead to quick dwindling and sometimes even perishing of bee colonies (Kumar and Agrawal, 2013).
       
To avoid this situation, colonies are either physically moved to a bee flora rich region or provision of feeding pollen are made. There are some disadvantages to both migration and pollen feeding. The shifting of bee colonies is a tedious work, which causes huge loss to honey bees in term of mortality during transportation (Mishra, 1995). Also a lot of time and labor is required for packaging and moving the colonies to distant places. Secondly, beekeepers usually neither collect and feed natural pollen to bee colonies, nor pollen is available in the market commercially. Further, commercial pollen may be contaminated with various pathogens (Herbert and Shimanuki, 1978; Kumar and Agrawal, 2014).
       
Therefore, efforts should be made to formulate a highly palatable and nutritionally balanced pollen substitute to help the colonies over dearth period so as to further strengthen commercial beekeeping in India. The necessity of artificial diets to honey bees has been the long-standing interest of the beekeeping industry and most enduring apicultural research problem (De-Grandi et al., 2008; Saffari et al., 2010a,b; Sihag and Gupta, 2011; Kumar et al., 2013; Morias et al., 2013; Gemeda, 2014; Kumar and Agrawal, 2014; Pande et al., 2015; Shehata, 2016; Abd el-Wahab et al., 2016). Haydak (1967) observed a variety of pollen substitutes and reported a mixture containing soy flour, brewer’s yeast and skimmed milk powder as best formulation. This combination has been suggested by National Bee Board, India in the advisory issued to beekeepers for beekeeping management practices (https://nbb.gov.in/pdf/Advisory.pdf).
       
Soybeen flour and its prodcuts have been widely used by number of researchers all over the globe and reported to show great benefits to honey bees (Rana et al., 1996; Schmidt and Hanna, 2006; Dastouri et al., 2007; Saffari et al., 2010a; Kumar and Agrawal, 2014). Though, some pollen substitutes are available commercially in international market (De Grandi et al., 2008; De Jongn et al., 2009, Saffari et al., 2010a), but they are formulated and tested in different eco-climatic conditions and may not prove much effective under Indian conditions. The effect and efficacy of many of such substitutes have been reported poor, as measured by brood rearing, bee population and honey production etc., in comparision to natural pollen (Saffari et al., 2010a). The pollen substitutes must be palatable and digestible to bees and should satisfy the nutritional requirements of honey bees. It should have proper taste and texture so that it is consumed by bees in optimal amount (Wilson et al., 2005; Saffari et al., 2010). Once consumed, diet must provide complete nutrition similar to that of pollen, be free from toxic materials and have long shelf life (Herbert, 2000; Saffari et al., 2010a; Kumar and Agrawal, 2014; Kumar and Agrawal, 2014). Further, no such formulation is available in India that can be fed to honey bees during pollen scarce periods. Therefore, there is an urgent demand for development of satisfactory and cost-effective formulation for honey bees to strengthen beekeeping industry in India.
       
The objective of current research work was to formulate and compare the efficacies of two diets (bee sup and Haydak’s diet) when fed to Apis mellifera colonies on top bars of hives. The following assessments were made: (i) The consumption of diets in patty form (ii) The relative efficacy of two feeds on brood rearing, bee covered franes, bee population and honey stores in comparison with sugar fed and un-fed control colonies.

Feeding experiments were conducted during summer 2017 and 2018 in the experimental colonies. Bees were kept in Langstroth wooden hives and equalized in term of brood and honey stores. The details of method adopted are mentioned below.
 
Colony equalization
 
For feeding experiment, Apis mellifera colonies (single story hives) were procured from registered beekeeper and maintained in Arni University campus, Himachal Pradesh, India, located at latitude 32° 8’4.6752’’ N and longitude 75° 41’21.0156’’ E. Twenty four colonies of almost similar strength were selected, numbered and labelled. Fresh queens were maintained in all colonies and were given equal brood and honey stores measured by gridding method (Seelley and Mikheyev, 2003; Amir and Peveling, 2004). Similar equlaization of colonies was made during both the years before the start of experiment.
 
Diet preparation
 
The protein and carbohydrate ingredients were selected keeping in mind the nutritional requirements of honey bees. These ingredients were mixed in specific ratio to prepare a complete formulation (Table 1).


 
This formulation was named bee sup during the current study. The diet was prepared by adding suitable quantity of water and left overnight so that all components may get properly mixed. Next day, patties (dough form) of diet were prepared and wrapped in butter paper to prevent moisture loss. Another diet composed of soy flour, brewer’s yeast and skimmed milk powder (Haydak, 1967), was also prepared so as to compare the efficacy of bee sup diet.
 
Top bar feeding and consumption
 
Weighed amount (100 g) of diet patties was placed on top bars, inside the hives for feeding, with few small holes on both sides (Fig 1). The patties were replaced with fresh patties after every seven days interval and the left out diet patties were weighed using electronic weighing balance.


                                         
Measuring colony parameters
 
Egg laying, area of brood coverage and honey stores in the colonies were measured after every 21 days interval, with the help of wire grid frame consisting of squares of one inch² (Seeley and Mikheyev, 2003; Amir and Peveling, 2004) (Fig 2).


       
The values were then converted into cm² by multiplying with a factor of 6.45. Bee strength in terms of total number of bee covered frames was also checked and recorded. Bee population was recorded by photographic print method (Jefree, 1951).
 
Statistical analysis
 
The experimental design used in the study was Completely Randomised Design (CRD). Data on feed consumption, sealed and unsealed brood area, bee population, bee covered frames, honey area was tabulated, transformed and statistically analyzed by two way ANOVA. Means were compared using critical differences (C.D.) at 0.05 per cent level of significance. 

The consumption and efficacy of formulated diet was assessed by patty feeding method conducted with experimental colonies. The colonies were fed with Haydak’s diet (T1) and bee-sup (T2). The results obtained during both the years i.e. 2017 and 2018, revealed the superiority of bee-sup over Haydak’s diet.
 
Consumption of diet during 2017
 
The average consumption of bee-sup (T2) was 80.60 g/colony/week, which was significantly greater (p<0.05) than Haydak’s diet (T1) whose consumption was 50.22 g/colony/week (Fig 3). It was observed that bees consumed bee-sup with great interest, complete quantity of diet was consumed by bees right from beginning of experiment to first week of July, values being 100, 92.60, 96.49, 100, 93.54 and 88 g on 14 May, 21 May, 28 May, 04 June, 11 June, 18 June, 25 June and 02 July, respectively.


       
The consumption of diet decreased after onset of monsoon season due to re-appearing of bee flora. Almost similar results for consumption of diet (s) were obtained in summer 2018. The total mean consumption of T1 was recorded to be 88.45 g/colony/week which was significantly different (p<0.05) from T2 (58.71 g/colony/week). Maximum consumption of bee-sup (97.33 g/colony) was recorded on 11 June and least consumption (59.38 g/colony) was recorded at the end of experiment. Results obtained for the net consumption of diet formulations are in accordance with that of Saffari et al., (2004);  Mattila and Otis (2006); De Grandi et al., (2008) and  Kumar and Agrawal, (2014) who reported that in some instances, protein supplements were consumed at higher rates as compared to that of pollen / pollen supplement. Feeding of pollen substitute to honey bee colonies improved their overall performance. However, the potential was more in colony maintenance and build up during pollen dearth periods.
 
Sealed brood area
 
The sealed brood area recorded during summer 2017 is presented in Fig 4. Mean sealed brood area (1938.3 cm² per colony) of colonies treated with T2 sup was significantly higher (p<0.05) than the colonies treated with T1 (1000.9 cm² per colony), sugar fed (T3) and control colonies (T4). In summer 2018, mean sealed brood (3934.60 cm² per colony) was again recorded higher in T2 fed colonies as compared to the colonies fed with T1 (2873.33 cm² per colony).


 
Unsealed Brood Area
 
The data recorded on the effect of feeding diet formulations on unsealed brood area is shown in Fig 4. Maximum unsealed brood area (1121.03 cm² per colony) was recorded in T2 fed colonies, followed by 740.13 cm² per colony in colonies fed T1. Minimum unsealed brood was recorded in control colonies. During summer 2018, unsealed brood area was again recorded in all experimental as well as control colonies and presented in Fig 5.


       
Maximum mean unsealed brood area (2108.07 cm² per colony) was recorded in the honey bee colonies fed T2, which was significantly different (p<0.05) from all other treatments. Such an enhancement in brood rearing clearly indicated the practical utility of protein rich diet formulations to the bee colonies.
       
Sealed and unsealed brood area in the colonies fed artificial diet increased significantly, Partially similar results were obtained by Chhuneja et al., (1993); Saffari et al., (2010a,b) and Sihag and Gupta, (2011) who reported an increase in brood area in the colonies provided various pollen substitutes and supplements.
 
Number of frames covered by bees
 
The data obtained for the number of frames covered by bees from May to July 2017 is presented in Fig 6. The results revealed that maximum number of frames (8.29) covered by bees was recorded in the colonies given T2, which was statistically higher (p<0.05), as compared to Haydak’s diet, sugar syrup as well as control colonies. Colonies given T1 had 7.36 frames covered by bees per colony followed by 7.11 in the colonies given T3 and 6.61 in T4. Almost similar results were obtained during summer 2018. Significant increase in average number of frames covered by bees was recorded in the colonies given T2 (7.87), in comparison to T1, T3 and T4.


       
The results obtained in case of total number of frames covered by bees are in accordance with Guler, (1999); Kumova, (2000) and Kumar and Agrawal, (2014), who reported positive effect of different artificial diets on total number of frames covered by bees.
 
 Bee population
 
The data recorded on the effect of feeding diet formulations on the population of bee colonies is presented in Fig 7. During summer 2017, significantly maximum bee population was recorded in the bee-sup (T2) fed colonies (17801.13 bees per colony) followed by T1, T3 and T4, values being 15583.99, 13795.86 and 12688.33 bees per colony, respectively. Almost similar results were obtained during summer 2018, where maximum average bee population (27977 bees per colony) was recorded in the colonies given T2 followed by T1 (21318.67 bees per colony) and T3 (18205 bees per colony).


       
The differences among these values were statistically different. However, significantly minimum bee population i.e. 15859 bees per colony was recorded in the control colonies (T4). Feeding of honey bees with nectar and pollen substitutes and supplements during dearth period in general has been found to increase the population of honey bee colonies. Stranger and Grip, (1972); Peng et al., (1984); Nabors, (2000); Sharma, (2002); De Grandi et al., (2008); and Saffari et al., (2010b) reported that colonies fed on protein patties had significantly higher adult bee population than the unfed control colonies.
 
Honey stores
 
Honey stores were also measured in all the colonies during summer 2017 as well as 2018 (Fig 8). The results revealed that honey stores were statistically maximum (p<0.05) in the colonies receiving T2 during dearth periods (1963.46 cm² per colony). Honey stores in these colonies differed significantly from the colonies given T1 (1235.33 cm² per colony), T3 (1007.99 cm² per colony) and T4 (845.33 cm² per colony). During 2018, honey stores were again recorded statistically different (p<0.05) in the colonies given T2 (1131.67 cm² per colony) followed by T1 (893.67 cm² per colony), T3 (1004.33 cm² per colony) and T4 (585 cm² per colony).


       
In case of honey store area, observations are comparable with those of Wahl, (1963); Forster, (1966); Stranger and Grip, (1972); Erickson and Herbert, (1980); Silva and Silva, (1985); De Grandi et al., (2008); and Kumar et al., (2014) who reported that colonies fed artificial diet produced significantly more honey as compared to unfed control colonies.

From patty feeding experiments carried out on A. mellifera colonies during summer dearth periods in Himachal Pradesh (India), it is concluded that bee-sup, a diet formulated in the laboratory was consumed in higher quantity as compared to standard formulation suggested by Haydak, (1967) and National Bee Board, New Delhi. The difference in acceptibility of bee-sup and Haydak’s diet probably reflects palatability of diets only and not the nutritional value (Saffari et al., 2010b). The effectiveness of formulated diet as nutrional food for honey bees is that it should result in positive/ similar influence on various colony attributes during dearth period., as that of natural pollen in blooming season. Therefore, this diet may effectively help to save colonies during dearth period so that full advatange of next honey flow season and beekeeping venture can be availed.

Authors are highly grateful to Himachal Pradesh Council for Science Technology and Environment, Shimla (India), for providing financial support to carry out this research work.