Background

Recently, managed honeybee (Apis mellifera L.) colonies had been sharply declining all across the world. Declines of honeybee colonies are due to colony collapse disorder (CCD), the phenomenon that may occur because of many reasons; infections, pesticides, contaminated water, antibiotic use, inadequate diet and inappropriate breeding management have all been suggested as possible reasons of these large-scale losses (Borges and Goodwin 2021). Because honeybees are not only economically important for pollination, while they are also important producers of bee products. As pressures on honey bee colonies increase, probiotics bacteria-supplemented food can shield honey bees against American and European foulbrood and aid in pathogen defense, nutrition, and environmental protection (Mishukovskaya et al. 2020; Budge et al. 2016).

Probiotics are live microorganisms, when supplemented with food and ingested in adequate amounts, provide health benefits to the host by improving the intestinal microbial balance (Bonilla-Rosso and Engel 2018; Chernitskiy et al. 2019). Probiotics isolated from gut bacteria are frequently used in many dietary supplements and functional foods (Daisley et al. 2020; Kešnerová et al. 2020). Since antibiotics are forbidden in animal feed in many countries, natural substances are being investigated to improve animal health (Audisio 2017; Elzeini et al. 2021).

Probiotics are now widely used in a variety of animal husbandry fields. They are used to preventing and treat intestinal infections, as well as to restore the microflora of the digestive tract when antibiotics or antibacterial chemotherapeutic drugs have been used (Alberoni et al. 2016). Probiotics help to improve digestion and boost non-specific immunity (Elzeini et al. 2021; Safonov 2020; Schmidt and Engel 2016). Along with its direct pH-lowering impact, acidificants also have an antibacterial effect that stops the growth of potentially harmful bacteria and fosters the emergence of helpful species. Long-lasting benefits to honeybee longevity have been observed following relatively short probiotic supplementation periods and without the need for host colonization, demonstrating an intermittent dosing schedule that also reduced hive disturbance. The isolated lactic acid bacteria are among the probiotic microorganisms that are well known (LAB, such as Lactococcus, Lactobacillus, Streptococcus, and Enterococcus), and Bifidobacterium have a long history of safe use (Raymann and Moran 2018; Al-Ghamdi et al. 2018). Supplements with probiotics and prebiotics can raise colony productivity, and hence profits, when added to the spring stimulation feeding of bee colonies (Alberoni et al. 2016; Andreeva et al. 2018; Endo and Salminen 2013).

It is worth mentioning that research on the efficacy of probiotic bacteria extracted from the gastrointestinal tracts on honeybee colony activities is limited as a consequence. Thus, the purpose of this study was to evaluate the use of Hbs-LAB strains previously isolated from foragers honeybee workers’ gut as additives to the supplementary feed for honeybee activities, honey production, collecting pollen, covered combs with bees, sealed areas of worker as well as drone brood, and the effect of Hbs-LAB on protein level in the worker's hemolymph samples.

Methods

Preparation of Hbs-LAB strains

Tested strains were previously isolated from the honeybee workers gut and identified molecularly based on the 16S rRNA gene sequences as Lactobacillus brevisMH191230, Lactobacillus caseiKT273339, and Enterococcus Faecalis MG890204 (Elzeini et al. 2021; Gomaa and Rushdy 2014). Bacterial isolates were cultured in MRS medium at 37 ◦C in a 5% carbon dioxide prior to incubation for overnight (18 h). The inoculated count was adjusted at OD600 = 0.1 (107 CFU/mL) using spectrophotometer.

Experimental bee’s colonies

Twenty honeybee colonies of local first hybrid Carniolan bees (Apis mellifera carnica), divided into two experimental groups each of 10 colonies, first group were used as controls and only feed with sugar syrup, and the second group was feed with Hbs-LAB1 × 106 added to syrup. Each group approximately was at equal strength and headed by newly mated queens of the same age (Schmidt and Engel 2016).

The studies were carried out during the honeybees’ active season, at the Faculty of Agriculture's apiary yard in Giza, Egypt, from February to July of 2021. Control colonies were fed with sugar syrup only, two times every week on the other hand, the treated colonies were fed with 500 ml/colony sugar syrup supplemented with mixture of Hbs-LAB (Lactobacillus brevis-HBE2, Lactobacillus casei-HBE5, and Enterococcus Faecalis-HBE1), two times every week and the sugar syrup concentration (125 g/L) was chosen based on previous laboratory results (Pătruică et al. 2012).

Honeybee activity parameters

During the assays, the general state of the colonies was described using the activity parameters as a guiding principle. According to Fathy et al. (2017), the following variables were noted for each examined colony both treated and untreated with (Hbs-LAB):-

  • The average area of sealed worker’s brood (cm2) every 14 days intervals/colony.

  • The average area of sealed drone’s brood (cm2) every 14 days intervals/colony.

  • The average of stored honey (cm2) every 14 days intervals/colony.

  • The average of stored pollen and bee bread (cm2) every 14 days intervals/colony.

  • The average number of combs covered with bees every 14 days intervals/colony.

SDS polyacrylamide gel electrophoresis

The electrophoretic analysis was used to identify the changes that occurred in the protein structure in the hemolymph of honeybee workers which fed on Hbs-LAB. Hemolymph samples were collected from the treated and untreated honeybee workers by puncture of dorsal cuticle between the 2nd and 3rd tergites using fine capillary glass tubes with EDTA (ethylenediaminetetraacetic acid) according to Yowell and Flurkey (2006). Using the Laemmli method (Laemmli 1970), the total protein concentration (mg/dl) in honeybee workers hemolymph was determined. The computerized Gel was used to analyze the protein fraction, rate of flow, and molecular weight of electrophoretically separated serum protein, reporting using Gel- Pro- Analyzer V.3.0 (Mass. Comp., Cairo-Egypt).

Statistical analysis

Data are expressed as mean ± standard deviation (SD) values using the MSTAT-C program (MSTAT 1991) version 2.10; two-way analysis of variance (ANOVA) and Duncan's multiple range tests were used to examine changes in biological parameter activity.

Results

Determine the activity parameters of honeybee colonies

The average area of sealed worker’s brood (cm2/col.)

As shown in Fig. 1, there were clearly significant differences in the average area of sealed worker brood reared in the treated colonies fed with sugar syrup supplemented with Hbs-LAB than untreated colonies fed with sugar syrup only, and the control colonies were recorded 211.15 ± 5.12, 206.07 ± 6.10, 239.25 ± 3.33, 278.43 ± 3.45, 290.21 ± 2.44 and ,302.33 ± 4.10 cm2, respectively. Whereas the treated colonies reached higher worker brood areas in February, March, April, May, June, and July months, the mean values were scored 289.30 ± 6.22, 309.70 ± 5.36, 350.30 ± 6.21, 369.30 ± 5.34, 455.01 ± 4.34, and 520.70 ± 7.32 cm2, respectively.

Fig. 1
figure 1

The average (± SE) area of sealed workers brood/cm2 in control and treated colonies

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The average area of sealed drone’s brood (cm2/col.)

Data illustrated in Fig. 2 showed the average area of sealed drone brood reared in the treated colonies during the experimental period, the treated bee colonies obtained high value of drone brood area in April and May, 88.30 ± 3.45 and 82.30 ± 4.21 cm2, respectively. Whereas the control colonies reached high drone brood area in June and July months, 58.70 ± 3.10 cm2 and 62.30 ± 3.80 cm2, respectively.

Fig. 2
figure 2

The average (± SE) area of sealed drone’s brood/cm2 in control and treated colonies

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The average area of stored bee’s honey (cm2/col.)

The immature and capped honey kept in the apiary was extracted and collected at the end of the flowering season (February to July). The recorded honey yields were significantly higher in the colonies that fed on Hbs-LAB. In comparison with the control hives, the amounts of stored honey were reached in control colonies, 451.10 ± 4.21 and 457.40 ± 3.32 cm2 in June and July, respectively, whereas the stored honey area in the treated colonies was scored 533.70 ± 5.56 and 540.70 ± 6.33 cm2 in the same months (Fig. 3).

Fig. 3
figure 3

The average (± SE) of stored honey /cm2 in control and treated colonies

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The average area of stored pollen and bee bread (cm2/col.)

Results illustrated in Fig. 4 showed clearly significant differences in treated bee colonies with untreated colonies; untreated colonies scored lower values of pollen stored compared with treated colonies. The control colonies recorded 90.10 ± 3.21, 106.65 ± 4.33, 112.10 ± 3.56, 89.95 ± 4.22, 90.65 ± 4.01 and 78.00 ± 3.67 cm2 in February, March, April, May, June, and July, respectively, while treated bee colonies scored higher values, 98.53 ± 3.25, 114.75 ± 4.01, 135.22 ± 3.43, 160.10 ± 4.21, 132.10 ± 4.23, and 143.30 ± 5.33 cm2 in February, March, April, May, June, and July, respectively (Fig. 5).

Fig. 4
figure 4

The average (± SE) of stored pollen and bee bread/cm2 in control and treated colonies

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Fig. 5
figure 5

The average (± SE) no. of combs covered with bees in control and treated colonies

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The average no. of combs covered with bees (population density/colony)

In addition to the effect of Hbs-LAB in significantly improving the stored pollen and bee bread they also led to important modifications in the mean no. of combs covered with bees. In the treated hives, a greater mean no. of combs covered with bees was scored in May and June, 10.25 ± 0.09 and 10.33 ± 0.00, respectively. Whereas a greater mean no. of combs covered with bees in untreated bee colonies recorded 8.53 ± 0.14 and 7.30 ± 0.17 in the same months, respectively.

SDS polyacrylamide gel electrophoresis

As shown in Fig. 6, there were clear differences in the hemolymph protein pattern between untreated and treated colonies; higher numbers of the total protein bands were recorded in the hemolymph of treated worker bees. The number of total protein bands was recorded in the hemolymph of untreated workers 13 bands, while in treated bee workers no. of protein band reached 16 bands.

Fig. 6
figure 6

Electrophoretic of total proteins concentration in untreated and treated honey bee workers hemolymph samples. St: protein standard, 1: control colonies, 2: treated colonies

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The data in Table 1 showed that the untreated worker samples' hemolymph molecular low weight protein bands (KDa) were 70.990.3 and their high weight protein bands were 171.570.2 KDa. The hemolymph molecular low weight protein bands in treated worker samples were 99.240.3 KDa, whereas the high weight protein bands were 183.870.3 KDa.

Table 1 The hemolymph protein bands differentiation of control and treated bee workers in honeybee colonies
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Discussion

The use of probiotics has become need of the hour. Numerous studies insist the use for probiotics to improve workers as well as entire colony health because it is environment friendly methodology for rehabilitation of the host (Vergalito et al. 2020; Olofsson et al. 2014; Kwong and Moran 2016). The purpose of study was to evaluate the contribution of probiotics previously isolated from honeybee intestinal tract in the bee development under controlled experimental conditions. The degree of development of honeybees was judged by taking several measurements. To use a microorganism as a probiotic, it is important to determine with certainty its genus, species and subspecies, if applicable (Mustar and Ibrahim 2022; Zahoor et al. 2021). In the current study, the bacterial strains Lb.brevis, Lb. casei, and En. faecalis isolated from foragers bee worker intestinal tracts were used as natural supplements to beehives (Audisio 2017; Olofsson et al. 2016; Schwarz et al. 2016). Obtained data were similar with (Bonilla-Rosso and Engel 2018; Bemmo et al. 2021; Martinson et al. 2011) stated that when the bacterium was administered to the hives, the brood area was larger after 2nd month onward after application when compared with untreated hives. Honey includes a large amount of live and active LAB, which is applied to wounds. This is a good explanation on why honey is used to treat wounds (Heyndrickx et al. 1996; Adeniyi et al. 2015). Addition of probiotic products to feed improves colony health through intestinal colonization with beneficial bacterial (Lb. acidophilus, Bifidobacterium lactis, Lb. casei) compared with untreated colonies (BorgesD and Goodwin 2021). Numerous studies have shown that rising intestine secretory activity may account for the improved bee colony formation, as seen by the increased number of brood cells discovered. Furthermore, it has been demonstrated that decreasing intestinal pH results in a decline in the quantity of potentially harmful spores, colonization of the intestine with helpful bacteria, and an improvement in the efficiency of nutritional absorption from pollen and nectar. This clearly demonstrates a connection to the large rise in brood comb discovered following probiotic feeding. Given that intestinal villus development increases the surface area available for nutrient absorption and consequently improves digestive assimilation, the chance to promote its development in apicultural practice may be especially valuable. This may also improve the effectiveness of other pharmaceutical therapies added as feeding syrup additives (Borges and Goodwin 2021).The concentrations of proteins were increased in the EM 5% experimental group, 15 days after the experiment in control laboratory conditions started (Gajger et al. 2020). The results of this study are in line with past works on the effects of dietary sterols on several fitness indicators in insects (Chakrabarti et al. 2020). Treated of Hbs-LAB in bees feeding caused clear changes in the amino acids including their numbers and molecular weight, some amino acids were not found and others new were recorded in treated worker bee’s hemolymph samples (Glavinic et al. 2017). One means of improving colony health involves the addition of prebiotic and probiotic products to supplementary feeds (Martinson et al. 2011; Mudroňová et al. 2011).

Conclusions

The incorporation of probiotics Hbs-LAB in honeybee’s sugar syrup proves good for pollinator’s overall health. It is good and environment-friendly methodology to improve the efficiency of honeybee’s immunity and reproductivity. Our findings of the present study will be helpful to obtain higher storing honey and pollen yields. Future studies on the assessment of various other probiotic in colonies from other locations or other subspecies at pilot and commercial scales are needed for obtaining exact quantitative results at higher scales.