Research of Sugar from Vegetables to Supply Carbohydrate Sources to Honeybee, Apis mellifera

Samples

Sugar samples extracted from agricultural by-products such as watermelon, and cabbage were the ones extracted, refined, and manufactured from the research team from Chonnam National University (Table 1). Watermelon extract, watermelon extract + fructose (10%), watermelon extract + fructose (5%) + pollen (5%), cabbage extract, cabbage extract + fructose (10%), cabbage extract + fructose (5%) + pollen (5%) were used (Fig. 1). In order to apply the honeybees in such samples, brix was corrected to 50 consistently, and fifty young worker bees were kept into the paper cage to supply water and feed, respectively (Fig. 1).

Fig. 1.

Test of sugar source from plants. Plants extract sugar solutions was supply to honey bee in paper cages.

Northern blotting

Northern blotting was separated with total RNA by using Trizol proceeding electrophoresis with formamid gel, transferring them to nitrocellulose membrane for 18 hours, and cross-linked with UV. Membrane was proceeded with pre-hybridization with hybridization solution and hybridized for 18 hours afterwards. Probe was exposed to X-ray film for 24 hours by labeling the radiation isotope p³² on PCR outcome of immue-related genes (abaecin, dafencin, apidaecin, hymanopteacin) and printed by using development solution and fixer.

cDNA synthesis and Quantitative PCR (qPCR)

In order to confirm the gene expression related to immune of honeybees samples supplied with cabbage extracts, honeybee samples were separated with total RNA by using Trizol. After freezing and pulverizing the samples with liquid nitrogen, 200μl chloroform and Trizol 1ml were mixed with 100mg sample and vortexed for fifteen seconds and reacted in the room temperature for two to three minutes. After the reaction, sample was centrifuged in conditions of 12,000xg and in 4°C for fifteen minutes separating the supernatant and mixing it with 500μl isopropanol to react in the room temperature for ten minutes. Then, they were centrifuged in conditions of 12,000xg and in 4°C for fifteen minutes, and pellet was cleaned with 80% EtOH. Final pellet was used for cDNA synthesis by melting it in the DEPC water. cDNA synthesis was reacted in 65°C for five minutes with PrimeScript II 1^st strand cDNA Synthesis kit by using oligo dT primer with extracted total RNA as a mold. Then, they were cooled down in ice, and cDNA was synthesized in 42°C for sixty minutes.

Synthesized cDNA was used as a mold to amplify four types of honeybee immune-related genes, defencin, hymenopteacin, apidaecin, and abaecin. As for abeacin cene, the forward primer, 5’-CGCACTACTCGCCATGCGCAGC-3’, Reverse primer 5’-GGACGATTAATAGCCTTGAGGCC-3’, was used. As for hymenoptaecin gene, forward primer, 5’-GTGCCGTTGC ATACGTTTCTGCTC-3’, and the reverse primer, 5’-GTCCACCATAGGCGTCTCCTGTC-3’, were used. As for apidaecin gene, forward primer, 5’-GAACCGCTGCTACCACTACG-3’, and reverse primer, 5’-GTCGAGTAGGCGGATCTAG-3’ were used. As for defencin gene, forward primer, 5’-GAACCGCTGCTACCACTACG-3’, and reverse primer, 5’-CCATTTCTGCAACTACCGC-3’, were used. As for control gene, β-actin gene was used. qPCR was set to have the final volume as 25μl that 10 pmol primer, 1×SYBR Green (Applied Biosystems), and 1^st strand cDNA 1μl were mixed to react. With analysis of Ct value of final result of reaction, the amount of expressed gene was compared (Applied Biosystems, user bulletin #2).

Fig. 2.

Impact of honey bee on agricultural byproducts. lane 1: watermelon, lane 2: watermelon + fructose (10%), lane 3: watermelon + fructose (5%) + pollen (5%), lane 4: cabbage, lane 5: cabbage + fructose (10%), lane 6: cabbage + fructose (5%) + pollen (5%), lane 7: sugar syrup.

Fig. 3.

Preference for sugar components extracted from agricultural byproducts. lane 1: watermelon, lane 2: watermelon + fructose (10%), lane 3: watermelon + fructose (5%) + pollen (5%), lane 4: cabbage, lane 5: cabbage + fructose (10%), lane 6: cabbage + fructose (5%) + pollen (5%), lane 7: sugar syrup.

Preference of honeybees and death rate of sugar extracted from agricultural byproducts

In order to analyze influence of dietary treatments on the honeybees, we recorded the mortality (number of dead bees) and preference (feed consumption rate) of honeybees samples. According to the result of supplying samples to the honeybees for three days, the death rate of bees was 88% for watermelon extract, 59.3% for watermelon extract + fructose, 71.3% for watermelon extract + fructose (5%) + pollen (5%), 80.7% for cabbage extract, 52.7% for cabbage extract + fructose (10%), 50.0% for cabbage extract + fructose (5%) + pollen (5%), and 1.3% for sugar liquid as a control solution.

In addition, according to the result of feed preference, the amount of food ingested by honeybees was 1.3g for watermelon extract, 1.2g for watermelon extract + fructose (105), 1.1g for watermelon extract + fructose (5%) + pollen (5%), 5.2g for cabbage extract, 5.2g for cabbage extract + fructose (10%), 6.9g for cabbage extract + fructose (5%) + pollen(5%), and 2.2g for sugar liquid as a control solution. Therefore, cabbage extract has lower death rate than watermelon but with higher feed preference. Especially, cabbage extract turned out to have higher feed preference than the sugar liquid, the control solution, which could be chosen as the alternative feed.

As watermelon and cabbage extracts have the black color, it seems that their samples contain various foreign substances. It also seems that there are cases where such foreign substances lower the preference in honeybees and serve as toxicity. Therefore, it is important to refine toxic substances on honeybees through many refining procedures and improving preference. At the same time, it seems to be inevitable to develop the process in consideration of economic feasibility to guarantee the possibility of it as an alternative of sugar. In recent years, with the steady rise in sugar prices in Korea, it is very necessary to develop honeybee feed that can substitute sugar. However, until now, it is very difficult to develop a sugar of cheaper price than the sugar price. However, if we can produce substitution sugar using agricultural byproducts, we think that it will be able to have price competitiveness. If we simplify the production process and reduce the cost in glycation process, it will be competitive with sugar. I Korea, sugar is not registered as a feed for bees. Because sugar bees are registered as feed for bees, some beekeepers may produce sugar honey. So, expensive sugar is used as feed for bees. Therefore, the use of alternative sugar as a bee feed is expected to reduce the administrative costs of beekeepers because the government subsidizes the cost of the feed. So, even if the substitute feed is higher than the sugar price, beekeepers will be able to feed the feed at a cheaper price than sugar.

Since the mixture of cabbage extract and fructose and also the one of fructose and pollen among cabbage extracted tested in this study turned out to provide the best result, gene expression test has been conducted on immunization of honeybees by collecting honeybees supplied with their samples. As a result, expression of four types of antibacterial proteins in regard of immunization (Apidaecin, Defencin, Hymenopteacin, Abaecin) turned out to all increase (Fig. 4). Apidaecin, Defencin, Hymenopteacin, and Abaecin are major antibacterial protein in honeybee. As for causes of an increase of expressed anti-bacterial protein, if samples being taken serve as a toxicity on honeybees causing the stress on honeybees, the amount of expression increases. Therefore, if deriving the expression of immune proteins without causing toxicity in honeybees, it seems that they will have the value as a functional feed.

Fig. 4.

Transcriptional expression of the antibacterial genes in cabbage sugar treated colony (upper). Northern blot analysis of the defencin (A), Hymenopteacin (B), Apideacin (C), Abaecin (D) in A. mellifera. Total RNAs were isolated from the worker (lane 1), cabbage sugar eat worker (lane 2), cabbage sugar and fructose eat worker (lane 3). The RNAs were separated by 1.0% formaldehyde agarose gel electrophoresis, transferred onto a nylon membrane and hybridized with radiolabled 1,695-bp Juvenile Hormone Esterase like (Acjhelike) gene. (Lower) Transcript levels of antimicrobial genes in different treats by qPCR (t-test, P<0.001).

When extracting sugar from agricultural byproducts, the extracted sugar is rich in color when it does not go through many processes. It is presumed that the cause is due to a lot of impurities such as various minerals in the plant. These impurities may be toxic to bees in some cases, but may also act as functional substances with an effect of improving the physiology of bees. Therefore, as the result of this study, it has been confirmed that the expression of immune-related substances of bees is increased, and thus it is expected to act as a material having a specific function to bees. However, it is a problem that food preference is very low due to unique flavor and taste. So, if bee prey preference for alternative feeds improves, it will be available in apiaries.