Impact of different pollen protein diets on the physiology of Apis mellifera L. (Hymenoptera: Apidae) workers from essential plant sources

2.1 Pollen loads trapping

Pollen trapping is a simple method to get pollen samples gathered by honey bee foragers (Dimou and Thrasyvoulou, 2006). Pollen traps have been used worldwide as a method to assess the pollen flora of any area (Andrada and Tellería, 2007). Twelve typical Afghani hives with rather uniform colonies of Apis mellifera L. were chosen for the current investigation, and they were placed in four different sites to catch pollen loads. Standard pollen traps obtained from the National Agriculture Research Center in Islamabad, with an extraction effectiveness of 50 % were used in the study. Every-two weeks, pollen-loads from coming foragers were captured for two days, providing the colonies with 13 days of inbound pollen. The pollen loads were collected from mid-January to the end of December. The weighted and colour sorted pollen loads were divided into groups, placed in polyethylene bags, and stored at −20 °C in a freezer.

2.2 Harvesting of bee bread

The bee bread was harvested in the active season by cutting the pieces of comb full of bee bread. These pieces were than stored in plastic boxes at 20  degreesC. For feeding bees the pieces were taken and placed for four hours in room temperature before feeding.

2.3 Determination of total protein and moisture content

For the purpose of analyzing pollen for protein content, the Kjeldahl technique was used (Table 1). Approximately-four hours were spent heating one gram of pollen and 20 ml of sulfuric acid (95–97 %) in the presence of a catalyst. A 90-ml solution of 30 % NaOH was used for distillation. For 2 min, the H3BO3 solution (4 %) volume was kept at 30 ml. The nitrogen content was then calculated after measuring the amount of HCl solution (0,1 M) consumed throughout the titration. The 5.60 factor was used for estimation of crude protein content and expressed as % dry matter (Rabie et al., 1983) was used for estimation of crude protein content and expressed as % dry matter. In addition to this, it is mandatory to know how much dry matter is still left and be able to quantify the protein value as a % dry matter. The level of moisture in the samples was used as a proxy for the dry matter content. The value of this parameter was determined by placing one gram of pollen in an evaporating dish made of porcelain and heating it in an oven set at 103 °C for two hours. It was determined that the moisture content of the sample was equal to the difference in weight between the samples after they had been dehydrated and before they were placed in the drying oven.

2.4 Formulation of pollen diets

The impacts of the primary flora of Dera Ismail Khan, which is located in Khyber Pakhtunkhwa, Pakistan, were investigated and measured for their effects on lifespan. Pollen sources, i.e., Trifolium alaxandrinum L. (Berseem), Zea mays L. (maize), Acacia modesta L. (Pulai), and Brassica napus L. (Canola), were employed. Each diet was designed using a single sample drawn from a larger pool (Table 1). An initial mix of 6 gm divided into two portions measuring 3gm/ portion was provided to the caged bees. The diet container was placed on the floor of the cage. To create a wet, kneadable consistency, pollen, sugar, and lukewarm water (2:1:1, weight) were combined and soaked for 12 h. The creation of corbicular loads typically involves 30 % nectar mixing by the bees. We used the same methodology and added 30 % sugar by weight before combining with water to get a sucrose level that was almost identical to that which was typically provided by bees for the manufacture of pollen pellets.

2.5 Food consumption of pollen diets

Bees were selected from strong colonies at the apiary at Gomal University DIK's Faculty of Agriculture and hatched in a warm environment, gently brushed into a cage for about a day before being used in the experiment. After a day, the newly hatched bees were randomly placed in cages (with 60 bees per cage) at a temperature of 35 °C and 50 % relative humidity. Wood was used to construct the confinement cages (70 mm 90 mm 100 mm), and the Plexiglas door was designed to glide open and down to reveal feeding chambers where 5 ml plastic medical syringes could be hung for feeding. A pollen diet's intake was evaluated by weighing the residual food and subtracting it from the amount supplied, then adding the water evaporated from a comparable diet in a bee-free cage in the environmental chamber. Both the controls and the experimental groups were given sugar solution, thus that was the only noticeable difference. Each experiment was repeated three times with bees from three different colonies. In this case, pollen feeding continued for another 21 days until it stopped completely.

2.6 Pollen diets’ impact on longevity of Apis mellifera L.

The sucrose solution and water was diluted to a 1:1 (w/v) ratio and placed into the vials. A little piece of wax comb was hung from the ceiling of the hives' confinement chambers to familiarize the bees with their surroundings and encourage them to remain still (Williams et al., 2013). Caged honey bees live longer if they have access to a comb (Rinderer and Elliott, 1977), which also reduces stress and restless appetitive behavior (Craig, 1917). Each day, fresh water and a 50 % sucrose solution were provided to the bees.

By removing and counting the dead bees from each cage, the longevity of the workers from the trial's beginning was estimated for each treatment group. Three-day intervals were used for this examination until half of the test insects' original population had died (LT50).

2.7 Acini size measurement for determination of hypopharyngeal glands development

Ten honey bee workers were chosen at random between the ages of 3 and its multiples up to 18 days old to study the variance in morphometry of the feeding glands. Upon capturing bees in Epondwarf tubes, we chilled them for 10 min in the fridge. In the Plant Breeding & Genetics Lab at the DIK Faculty of Agriculture, the heads of carefully chosen worker bees were slashed off with a razor blade. Fine forceps were used to split the HPGs into their appropriate age groups. Overnight at 4 °C, the glands were preserved in 4 % formaldehyde in phosphate-buffered saline (PBS, pH 7.5). HPGs were washed three times in PBS to remove any debris. These were cleaned, then left in an incubator with 30 % glycerol in PBS at 40 °C for a full 24 h. The glands were re-incubated for 2 h at 40 °C with 50 % glycerol in PBS before to mounting. Slides were prepared from samples using a mounting medium of 50 % glycerol in PBS. Translucent nail polish was used to seal the transparency's edges so they wouldn't dry off. Under a binocular light microscope equipped with a 10x objective, we captured these pictures of acini from mounted gland specimens (M165C, Leica, Germany). Images captured with the high-resolution Vivo-S1 camera were analyzed using the image J software. Ten acini were randomly picked from glands in different places, and their pictures were analyzed using the Image J program. Following the method shown in, morphological measurements were extracted from these photographs. We measured the bees at 3, 6, 9, 12, 15, and 18 days of age. The selected acini/slide/bee had its length (L) and width (W) measured (W). Each group had 270 acini measured. The following formula was examined in order to calculate the acinar surface area (Maurizio and Hodges, 1951). $$\mathit{Acinalsurfacearea}=(\pi \ast ((a\ast b)/2))$$

Where a is the full length and b is the full width of the acini and π has a constant value of 3.14.

2.8 Measurement of rectal weight contents (RWCs) of the workers

The same bees that were employed to gauge Hypopharyngeal gland maturation also weighed in on the rectal contents. Each bee had its whole digestive system removed with delicate forceps before its head was dissected. The rectum was carefully removed with a blade from the stomach and placed on a weighted coverslip. Once again, the coverslip was weighed, and the rectum's mass was determined by the following formula. $$\mathit{Wr}=WC2-WC1$$

In this scenario, WC1 represents the beginning weight of coverslips without a rectum and WC2 represents the ultimate weight of coverslips with a rectum. In order to get the means, analysis of variance was utilized. The DMR test was used to compare means (Duncan, 1955).

2.9 Statistical analysis

Diet and age of bees were included as independent factors into a general linear model with food intake, longevity, HPG sizes, and RWCs as the dependent variables. The post hoc method for mean comparisons was Tukey’s HSD. Two-way analysis of variance was used to determine statistically significant mean differences.

3.1 Nutritional analyses for crude protein and moisture content

The nutritional assessments of the four species under investigation's mono-floral pollens are shown in Table 2 along with their moisture and crude protein contents. Maize had the lowest protein levels and canola had the highest protein levels. All of polen kinds that were investigated had protein values > 4 %, ranging from 4.06 % to 25.1 %. Similar results were reported for moisture contents, for maize showing the highest values (72.23 %) and canola showing the lowest (8.28 %). Berseem and phulai had protein contents of 23.45 % and 4.14 %, respectively. With readings of 19.89 % and 19.94 %, phulai and berseem practically had the same moisture content.

3.2 Food consumption

Table 3 and Fig. 1 show that there are statistically significant differences between the treatments at the P < 0.05 level. Sixty bees were monitored every-three days, and their food intake was recorded in milligrams per day. Experiments were conducted using freshly emerging bees given a variety of mono-floral pollen regimes as a comparison to two controls: Sucrose syrup, which is typically supplied during the dearth period, and bee bread, which was collected from hives during the active season. According to the average pollen intake, confined bees fed bee bread consumed 1.46 gm of pollen in the first three days after emergence, and this number grew to 1.81–2.01 gm/50 bees in the next three-day interval. After that, between 16 and 18 days, the quantity dropped to a minimum of 0.50gm/60 bees. The amount of each experimental diet was consumed in the same way by the nursing bees. Over the course of the 9 days, the nursing bees gradually ate less and less of their food. It continued till the experiment was completed. For all experimental diets, the shortest period of time between the onset of starvation and the first occurrence of food consumption was 16–18 days.

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Fig. 1

Effects of different pollen diets on FC & RWCs of Apis mellifera L.

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With a mean of 25.279 mg, this study found that worker bees consumed 3.03 mg of pollen per bee per day from canola which is comparable to the 3.37 mg of pollen per bee per day found in Bee bread. Berseem's pollen diet came in second with a consumption of 2.76 mg per bee per day, followed by phulai with a consumption of 2.40 mg per bee per day. The confined bees were found to ingest the least amount of maize pollen diet. In contrast to pollen diets, control 1′s sugar syrup content (1.65 mg/bee/day) was not as high. The current study calculated the average longevity of confined workers that consumed various mono-floral pollen diets. Four different pollen loads were fed to honey bee workers as protein-rich meals when they were a day old (Table 1). Every subject was housed in a little cage, and freshly emerging workers were given experimental meals with sucrose solution (1:1). Both the overall death rate and the bees' life cycle in each experiment were recorded. LT50 is the expected number of days needed for 50 % of the bees to perish. Through the use of Probit analysis, the lethal time for 50 % mortality was determined (Table 4, Fig. 2). In contrast to the negative control sugar syrup, which required 10 days, diet 4 took the longest time for 50 % mortality, followed by diets 1, 3, and 2. The positive control, which consisted of a bees' normal diet, required the longest time to reach 50 % mortality (34 days).

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Fig. 2

Graph showing lethal time for 50% mortality of worker bees fed on different pollen diets.

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3.3 Hypo-Pharyngeal glands (HPGs) sizes and rectal weight contents (RWCs)

Fig. 3 illustrate the influence of various pollen diets at various time intervals (3,6,9,12,15, and 18 days), in regard to the sizes of HPG. The three treatments produced statistically significant changes (P 0.05) when comparing the HPG diameters of worker bees. HPGs had sizes ranging from 107.65 to 332.77 µm. Natural diet bee bread had the largest size (332.77 µm), followed by diets including canola, berseem, phulai, maize, and sugar syrup, which had respective sizes of 278.47, 261.73, 237.49, 124.38, and 107.65 µm.

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Fig. 3

Hypopharyngeal glands development on different days.

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On HPGs of worker bees, the interactional effect revealed substantial changes in size based on both the days and the treatment. The sizes varied from 68.62 to 448.47 µm. The largest size was measured on day 6 of treatment 6 (Bee bread), with the same treatment producing the largest size on day 9 (437.04 m), and treatment 4 producing the smallest size on day 6 (429.48 m). On day 18, the treatment 5 reading of 68.62 m was recorded as having the lowest value.

On various days, there was a very significant fluctuation in the HPG sizes of the worker bees. According to the research, HPGs may have any size between 101.33 and 326.59 µm. In 6 day old bees, maximum sizes (326.59 µm) were noted. After six days, HPG sizes began to decline, reaching 289.21 µm in nine days, 246.28 µm in twelve days, 139.24 µm in fifteen days, and 101.33 µm in eighteen days old workers, at which point the bees began to act as foragers. The acinal size is age-related, according to the data above. Up to the age of six days, glands grow larger; after that, they begin to contract. At six days, HPGs have a diameter that is double that of freshly emerging bees.

3.4 RWCs as indicators of protein utilization

In Table 5 and Fig. 1, the findings of the current study that demonstrate the impact of various pollen diets at various time intervals (3, 6, 9, 12, 15, and 18 days) in relation to RWCs are shown. For worker bees' rectal weight contents, various treatments showed significant changes (P < 0.05). The information in Table 5 compares the RWCs (mg/bee/3 days) of freshly emerging bees fed various mono-floral pollen diets to a natural diet of bee bread, which served as the control. The data were taken from the combs of honeybee colonies during the active season. The mean RWC value demonstrates the digestibility of the pollen diet and is inversely related to the weight of the rectal contents. Starting on day one, the weight would grow until finally, on day fifteen, feces flights would be conducted and employees would be required to empty their rectums. Honeybee workers fed bee bread had the lowest average rectal contents (3.95 mg), indicating the highest digestibility, while in pollen diets, canola diet was consumed and digested more efficiently than berseem (4.81), maize (7.01), and phulai (11.01 mg), indicating the highest level of digestibility (5.25). The mean number of days showed a peak at about day 3, with a gradual decline afterwards with age.

The contents of the rectal cavity varied in weight from 10.60 mg to 1.24 mg. Overall, maize pollen was digested the least (7.01 mg), followed by phulai (5.25 mg), berseem (4.82 mg), canola (4.43 mg), and finally, natural diet bee bread (3.95 mg), which showed the most digestion of pollen. Rectal weight contents of worker bees differed significantly (P0.05) among days. In the research it was found that the rectums' weight might be anything from 1.33 mg to 8.18 mg. Bees between the ages of 13 and 15 days were measured to be the largest (8.18). The rectums' weights began falling after day 15, and by day 16–18, they had dropped by 5.53 mg.