The antioxidant system was unexpectedly strongly suppressed in apis mellifera worker bees emerged from larvae reared on combs adulterated with paraffin or stearin

Abstract

The bee-wax combs are "the biggest organ of the bee colony body" as, in addition to their structural functions, they transfer information - pheromones and sounds. The lack of quality control procedures for bee-wax foundation, leads to a deterioration of this raw material, among others with the addition of paraffin and/or stearin. The adulteration of beeswax, from which wax foundation is produced, affects the mechanical strength of the combs and the development of the brood. Little is known about the effects of these adulterants on bees' biochemistry and physiology. Therefore, the activity of the antioxidant system (SOD, CAT, GPx, GST and Vg) was determined in the hemolymph of bees reared on pure wax and wax adulterated with paraffin (10%, 30% or 50%) or stearin (10%, 30% or 50%). Additionally, the level of global DNA methylation in the brain of these bees was identified. We showed for the first time that paraffin or stearin strongly suppressed the activity of the antioxidant system, including Vg, in honeybee workers emerged from larvae reared on combs adulterated with these compounds. Stearin was much more harmful and may cause serious metabolic disturbances, including an increase in the global DNA methylation. This is important new information that serves as a warning to wax foundation producers and beekeepers. Therefore, there is an urgent need to introduce proper procedures and regulations for the routine quality evaluation of wax intended for the production of the bee comb building foundation.

Keywords: Antioxidative enzymes; DNA methylation; Honeybee; Vitellogenin; Wax adulteration.

Fig. 1 The effect of wax foundation adulteration with paraffin or stearin on the activities of the antioxidant enzymes (mean ± SD; n = 30) within the three age classes of the worker bees. Explanations: SOD – superoxide dismutase; CAT – catalase; GST – glutathione S- transferase; GPx – glutathione peroxidase. The wax foundations were adulterated with 10%, 30%, and 50% of paraffin (groups PA10%, PA30%, and PA50%), respectively. The wax foundations were adulterated with 10%, 30%, and 50% of stearin (groups ST10%, ST30%, and ST50%), respectively. Pure wax foundations with no additives were used (the control group C). (*) – the difference between the control group and a given experimental group was statistically significant (p ≤ 0.01) when evaluated within each age class. The horizontal green lines indicate that the specific group means differ significantly (p ≤ 0.01) for a given adulteration level when the bees reared on the stearin-adulterated combs are compared with those reared on the combs with paraffin, separately within each age class. a, b, c – the differences between the age groups for the control group and the particular levels of paraffin and stearin adulteration are statistically significant at p ≤ 0.01. The vertical bars indicate standard deviation.

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Fig. 2 The effects (mean ± SD; n=30) of wax foundation adulteration with paraffin or stearin on the total antioxidant capacity (TAC) within the three age classes of the worker bees. Explanations: The wax foundations were adulterated with 10%, 30%, and 50% of paraffin (groups PA10%, PA30%, and PA50%), respectively. The wax foundations were adulterated with 10%, 30%, and 50% of stearin (groups ST10%, ST30%, and ST50%), respectively. Pure wax foundations with no additives were used (the control group C). (*) – the difference between the control group and a given experimental group was statistically significant (p ≤ 0.01) when evaluated within each age class. The horizontal green lines indicate that the specific group means differ significantly (p ≤ 0.01) at a given adulteration level when the bees reared on the stearin-adulterated combs are compared with those raised on the combs with paraffin, separately within each age class. a, b, c – the differences between the age groups for the control group and the particular levels of paraffin and stearin adulteration are statistically significant at p ≤ 0.01. The vertical bars indicate standard deviation.

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Fig. 3 The effects of wax foundation adulteration with paraffin or stearin on the vitellogenin (Vg) titers (mean ± SD; n = 30) within the three age classes of worker bees. Explanations: The wax foundations were adulterated with 10%, 30%, and 50% of paraffin (groups PA10%, PA30%, and PA50%), respectively. The wax foundations were adulterated with 10%, 30%, and 50% of stearin (groups ST10%, ST30%, and ST50%), respectively. Pure wax foundations with no additives were used (the control group C). (*) – the difference between the control group and a given experimental group was statistically significant (p ≤ 0.01) when evaluated within each age class. The horizontal green lines indicate that the specific group means differ significantly (p ≤ 0.01) for a given adulteration level when the bees reared on the stearin-adulterated combs are compared with those reared on the combs with paraffin, separately within each age class. a, b, c – the differences between the age groups for the control group and the particular levels of paraffin and stearin adulteration are statistically significant at p ≤ 0.01. The vertical bars indicate standard deviation.

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Fig. 4 The effects of wax foundation adulteration with paraffin or stearin on the global DNA methylation level (mean ± SD; n = 30) within the three age classes of worker bees Explanations: The wax foundations were adulterated with 10%, 30%, and 50% of paraffin (groups PA10%, PA30%, and PA50%), respectively. The wax foundations were adulterated with 10%, 30%, and 50% of stearin (groups ST10%, ST30%, and ST50%), respectively. Pure wax foundations with no additives were used (the control group C). (*) – the difference between the control group and a given experimental group was statistically significant (p ≤ 0.01) when evaluated within each age class. The horizontal green lines indicate that the specific group means differ significantly (p ≤ 0.01) for a given adulteration level when the bees reared on the stearin-adulterated combs are compared with those raised on the combs with paraffin, separately within each age class. a, b, c – the differences between the age groups for the control group and the particular levels of adulteration with paraffin and stearin are statistically significant at p ≤ 0.01. The vertical bars indicate standard deviation.

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