Effect of Rearing in Small-Cell Combs on Activities of Catalase and Superoxide Dismutase and Total Antioxidant Capacity in the Hemolymph of Apis mellifera Workers

Honeybee nests constructed without man-made wax foundation have significantly more variability of cell widths/sizes than those in commercially-kept colonies. The effects of this natural variability in comb cell widths on individual and colony traits have not been explained to date. The investigation of this problem can lead to new findings about the biology, physiology, and possibly, the evolution of the honeybee. The aim of the study was to compare the catalase and superoxide dismutase activities and the total antioxidant capacity levels in the hemolymph of honeybee workers reared in small-cell combs and standard-cell combs in colonies kept simultaneously on standard- and small-cell combs. The ratio of the small-cell combs to the standard-cell combs in the nest was 1:1. The workers reared in small-cell combs were characterized by higher antioxidant activities in the hemolymph than those reared in standard-cell combs. Consequently, their hemolymph had a greater antioxidant capacity, which indicates that they may be better predisposed to be foragers than workers reared in standard-cell combs. To describe the physiological differences between worker bees reared in small- and standard-cell combs in the same colony, the role of the considerable variation in the cell width in natural combs built without the use of artificially produced wax foundation is worth elucidating. The comparison of the apiary and cage experiments indicated that changes in antioxidant activities predominantly result from worker activities, especially those requiring the intensification of metabolism, rather than the age of the worker bees. To reduce the impact on the results of random environmental factors potentially present in one-season studies of honeybee research, investigations should preferably be carried out over a few consecutive years.


Introduction
An adequate nutrient supply and resistance to the pressure posed by parasites, predators, and other adverse environmental factors are the main determinants of survival for all animals, including the superorganism constituted by a colony of social insects [1]. The honeybee is a social insect, and the main problems in contemporary apiculture are caused by external environmental stressors and internal nest factors that affect both individual bees and the colony as a whole [2]. The greatest risk of exposure to stressors in the external environment is faced by the forager caste during flights for food and water. Contaminants encountered by these bees in the environment around the bee colony are transported on their bodies and enter the colony along with food and water, thus affecting the other bees and the brood in the nest [3,4].
The maintenance of homeostasis in individual honeybees and, consequently, in entire colonies, is ensured by, e.g., the mitigation of the impact of negative factors by the immune SMC-workers reared in small-cell combs; STC-workers reared in standard-cell combs.

Determination of Antioxidant Activities
We decided to analyze hemolymph in the present study, as its parameters are a good source of information about the physiological status of the organism [36]. The antioxidant activities were assessed with the spectrophotometric method using commercial kits. Catalase enzyme (CAT) activity in the worker hemolymph was determined using the Sigma-Aldrich kit (CAT-100). One unit of CAT is the amount of the enzyme that decomposes 1 µmol of H 2 O 2 per minute at 25 • C. The H 2 O 2 decomposition was measured at a wavelength of 520 nm and the results were expressed as U/mL of the catalase enzyme in the sample volume. The activity of superoxide dismutase (SOD) was determined using the Sigma-Aldrich 19160 SOD determination kit, with one unit inhibiting the rate of reduction of cytochrome c by 50% in a coupled system using xanthine and xanthine oxidase in the sample volume. The total antioxidant capacity (TAC) level in the hemolymph of the worker bees was assessed using a Cell Biolabs STA-360 kit. The antioxidant capacity of unknown samples was expressed as µM of copper reducing equivalents and was proportional to the sample's total antioxidant capacity in the sample volume. The samples and sample volumes were prepared according to the aforementioned enzyme analysis protocols.

Infection of Worker Bees by Nosema spp.
Neither of the bee samples for which hemolymph was collected in the apiary and laboratory experiments contained Nosema spp. spores. This allows the analysis to eliminate from the findings the effects of infestation with this parasite.

SOD and CAT Activities and TAC Levels
In both the SMC and STC workers, the year (i.e., 2020, 2021, and 2022) had a significant effect on the SOD and CAT activities and TAC levels in the hemolymph ( Table 2). In all years, the age (1 d, 7 d, 14 d, and 21 d) exerted a significant effect on the SOD and CAT activities and the TAC levels in the hemolymph of both the SMC and STC workers ( Table 2). The SOD activity in the STC workers in 2020 was an exception. In each year of the apiary experiment, the SOD and CAT activities and TAC levels were higher in the 7-, 14-, and 21-day-old SMC workers than in the STC workers (Figures 1-3, Table 3). The highest number of statistically significant differences was noted in the case of the SOD and CAT activities. Opposite results were obtained in the analysis of the CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significantly lower values of these parameters were recorded in the group of the SMC workers vs. the STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old workers were higher in the STC group, but the differences were statistically significant only in 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in the groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiary experiment (Figures 4-6, Table 3). oxidant capacity; -green arrow indicates that the value of the trait is statistically significantly higher in the SMC workers than the STC workers; -red arrow indicates that the value of the trait is statistically significantly lower in the SMC workers than the STC workers; n.s.-statistically insignificant difference between the SMC and STC workers; -black arrow indicates that the value of the trait is higher in the SMC workers than the STC workers but without statistical significance; =-equality sign indicates that the value of the trait in the SMC workers is close or equal to the trait value in the STC workers.
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activity; TAC-Total antioxidant capacity; -green arrow indicates that the value of the trait is statistically significantly higher in the SMC workers than the STC workers; -red arrow indicates that the value of the trait is statistically significantly lower in the SMC workers than the STC workers; n.s.-statistically insignificant difference between the SMC and STC workers; -black arrow indicates that the value of the trait is higher in the SMC workers than the STC workers but without statistical significance; =-equality sign indicates that the value of the trait in the SMC workers is close or equal to the trait value in the STC workers. CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significantly lower values of these parameters were recorded in the group of the SMC workers vs. the STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old workers were higher in the STC group, but the differences were statistically significant only in 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in the groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiary experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activity; TAC-Total antioxidant capacity; -green arrow indicates that the value of the trait is statistically significantly higher in the SMC workers than the STC workers; -red arrow indicates that the value of the trait is statistically significantly lower in the SMC workers than the STC workers; n.s.-statistically insignificant difference between the SMC and STC workers; -black arrow indicates that the value of the trait is higher in the SMC workers than the STC workers but without statistical significance; =-equality sign indicates that the value of the trait in the SMC workers is close or equal to the trait value in the STC workers.

Figure 1.
Apiary experiment-Superoxide dismutase (SOD) activities in the hemolymph of workers in three consecutive years. SMC-workers reared in small-cell combs; STC-workers reared in standard-cell combs; *-differences between the SMC and STC workers within the age group are significant at p ≤ 0.05; vertical bars indicate standard deviation.
CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significant lower values of these parameters were recorded in the group of the SMC workers vs. th STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old worke were higher in the STC group, but the differences were statistically significant only 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in th groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiar experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activity; TAC-Total an oxidant capacity; -green arrow indicates that the value of the trait is statistically significant higher in the SMC workers than the STC workers; -red arrow indicates that the value of the tra is statistically significantly lower in the SMC workers than the STC workers; n.s.-statistically i significant difference between the SMC and STC workers; -black arrow indicates that the valu of the trait is higher in the SMC workers than the STC workers but without statistical significanc =-equality sign indicates that the value of the trait in the SMC workers is close or equal to the tra value in the STC workers.

Figure 1.
Apiary experiment-Superoxide dismutase (SOD) activities in the hemolymph of wor ers in three consecutive years. SMC-workers reared in small-cell combs; STC-workers reared standard-cell combs; *-differences between the SMC and STC workers within the age group a significant at p ≤ 0.05; vertical bars indicate standard deviation.
CAT activities and TAC levels in the 1-day-old workers, i.e., statistically si lower values of these parameters were recorded in the group of the SMC work STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-o were higher in the STC group, but the differences were statistically significa 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends obser groups of the 7-, 14-, and 21-day-old workers were very similar to those in experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activity; TACoxidant capacity; -green arrow indicates that the value of the trait is statistically higher in the SMC workers than the STC workers; -red arrow indicates that the valu is statistically significantly lower in the SMC workers than the STC workers; n.s.-sta significant difference between the SMC and STC workers; -black arrow indicates th of the trait is higher in the SMC workers than the STC workers but without statistical =-equality sign indicates that the value of the trait in the SMC workers is close or equa value in the STC workers. CAT activities and TAC levels in the 1-day-old workers, i.e., statis lower values of these parameters were recorded in the group of the S STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the were higher in the STC group, but the differences were statistically 2021 (Figures 1-3, Table 3). In the laboratory experiment, the tren groups of the 7-, 14-, and 21-day-old workers were very similar to experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activ oxidant capacity; -green arrow indicates that the value of the trait is sta higher in the SMC workers than the STC workers; -red arrow indicates th is statistically significantly lower in the SMC workers than the STC workers significant difference between the SMC and STC workers; -black arrow in of the trait is higher in the SMC workers than the STC workers but without =-equality sign indicates that the value of the trait in the SMC workers is clo value in the STC workers. CAT activities and TAC levels in the 1-day-old w lower values of these parameters were recorded in STC workers (Figures 1-3, Table 3). In turn, the SO were higher in the STC group, but the differences 2021 (Figures 1-3, Table 3). In the laboratory exp groups of the 7-, 14-, and 21-day-old workers we experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dism oxidant capacity; -green arrow indicates that the va higher in the SMC workers than the STC workers; -re is statistically significantly lower in the SMC workers th significant difference between the SMC and STC worker of the trait is higher in the SMC workers than the STC w =-equality sign indicates that the value of the trait in the value in the STC workers. CAT activities and TAC levels in the 1-day-old w lower values of these parameters were recorded in STC workers (Figures 1-3, Table 3). In turn, the SO were higher in the STC group, but the difference 2021 (Figures 1-3, Table 3). In the laboratory exp groups of the 7-, 14-, and 21-day-old workers we experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dism oxidant capacity; -green arrow indicates that the va higher in the SMC workers than the STC workers; -re is statistically significantly lower in the SMC workers th significant difference between the SMC and STC worker of the trait is higher in the SMC workers than the STC w =-equality sign indicates that the value of the trait in th value in the STC workers.
CAT-Catalase enzyme activity; SO oxidant capacity; -green arrow in higher in the SMC workers than the S is statistically significantly lower in t significant difference between the SM of the trait is higher in the SMC work =-equality sign indicates that the va value in the STC workers.
CAT-Catalase enzyme act oxidant capacity; -green higher in the SMC workers is statistically significantly significant difference betwe of the trait is higher in the S =-equality sign indicates th value in the STC workers. CAT activities and TAC lower values of these para STC workers (Figures 1-3 were higher in the STC g 2021 (Figures 1-3, CAT-Catalase enzyme acti oxidant capacity; -green higher in the SMC workers t is statistically significantly l significant difference betwee of the trait is higher in the S =-equality sign indicates th value in the STC workers.      CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significantly lower values of these parameters were recorded in the group of the SMC workers vs. the STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old workers were higher in the STC group, but the differences were statistically significant only in 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in the groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiary experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activity; TAC-Total antioxidant capacity; -green arrow indicates that the value of the trait is statistically significantly higher in the SMC workers than the STC workers; -red arrow indicates that the value of the trait is statistically significantly lower in the SMC workers than the STC workers; n.s.-statistically insignificant difference between the SMC and STC workers; -black arrow indicates that the value of the trait is higher in the SMC workers than the STC workers but without statistical significance; =-equality sign indicates that the value of the trait in the SMC workers is close or equal to the trait value in the STC workers. CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significantly lower values of these parameters were recorded in the group of the SMC workers vs. the STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old workers were higher in the STC group, but the differences were statistically significant only in 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in the groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiary experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activity; TAC-Total antioxidant capacity; -green arrow indicates that the value of the trait is statistically significantly higher in the SMC workers than the STC workers; -red arrow indicates that the value of the trait is statistically significantly lower in the SMC workers than the STC workers; n.s.-statistically insignificant difference between the SMC and STC workers; -black arrow indicates that the value of the trait is higher in the SMC workers than the STC workers but without statistical significance; =-equality sign indicates that the value of the trait in the SMC workers is close or equal to the trait value in the STC workers. CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significant lower values of these parameters were recorded in the group of the SMC workers vs. th STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old worke were higher in the STC group, but the differences were statistically significant only 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in th groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiar experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activity; TAC-Total an oxidant capacity; -green arrow indicates that the value of the trait is statistically significant higher in the SMC workers than the STC workers; -red arrow indicates that the value of the tra is statistically significantly lower in the SMC workers than the STC workers; n.s.-statistically i significant difference between the SMC and STC workers; -black arrow indicates that the valu of the trait is higher in the SMC workers than the STC workers but without statistical significanc =-equality sign indicates that the value of the trait in the SMC workers is close or equal to the tra value in the STC workers. CAT activities and TAC levels in the 1-day-old workers, i.e., statistically si lower values of these parameters were recorded in the group of the SMC work STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-o were higher in the STC group, but the differences were statistically significa 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends obser groups of the 7-, 14-, and 21-day-old workers were very similar to those in experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activity; TACoxidant capacity; -green arrow indicates that the value of the trait is statistically higher in the SMC workers than the STC workers; -red arrow indicates that the valu is statistically significantly lower in the SMC workers than the STC workers; n.s.-sta significant difference between the SMC and STC workers; -black arrow indicates th of the trait is higher in the SMC workers than the STC workers but without statistical =-equality sign indicates that the value of the trait in the SMC workers is close or equa value in the STC workers. CAT activities and TAC levels in the 1-day-old workers, lower values of these parameters were recorded in the group STC workers (Figures 1-3, Table 3). In turn, the SOD activit were higher in the STC group, but the differences were st 2021 (Figures 1-3, Table 3). In the laboratory experiment, groups of the 7-, 14-, and 21-day-old workers were very si experiment (Figures 4-6, Table 3).   CAT activities and TAC levels in the 1-day-old workers, lower values of these parameters were recorded in the grou STC workers (Figures 1-3, Table 3). In turn, the SOD activi were higher in the STC group, but the differences were st 2021 (Figures 1-3, Table 3). In the laboratory experiment groups of the 7-, 14-, and 21-day-old workers were very s experiment (Figures 4-6, Table 3).   CAT activities and TAC levels in the 1-day-old w lower values of these parameters were recorded in STC workers (Figures 1-3, Table 3). In turn, the SO were higher in the STC group, but the difference 2021 ( Figures 1-3, Table 3). In the laboratory exp groups of the 7-, 14-, and 21-day-old workers we experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dism oxidant capacity; -green arrow indicates that the va higher in the SMC workers than the STC workers; -re is statistically significantly lower in the SMC workers th significant difference between the SMC and STC worker of the trait is higher in the SMC workers than the STC w =-equality sign indicates that the value of the trait in th value in the STC workers.            CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significantly lower values of these parameters were recorded in the group of the SMC workers vs. the STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old workers were higher in the STC group, but the differences were statistically significant only in 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in the groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiary experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activity; TAC-Total antioxidant capacity; -green arrow indicates that the value of the trait is statistically significantly higher in the SMC workers than the STC workers; -red arrow indicates that the value of the trait is statistically significantly lower in the SMC workers than the STC workers; n.s.-statistically insignificant difference between the SMC and STC workers; -black arrow indicates that the value of the trait is higher in the SMC workers than the STC workers but without statistical significance; =-equality sign indicates that the value of the trait in the SMC workers is close or equal to the trait value in the STC workers. CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significantl lower values of these parameters were recorded in the group of the SMC workers vs. th STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old worker were higher in the STC group, but the differences were statistically significant only i 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in th groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiar experiment (Figures 4-6, Table 3). Table 3. Changes in antioxidant activities in workers reared in small-cell combs (SMC) in compa ison with workers reared in standard-cell combs (STC) at the ages of 1, 7, 14, and 21 days in th apiary and laboratory experiments.
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activity; TAC-Total ant oxidant capacity; -green arrow indicates that the value of the trait is statistically significant higher in the SMC workers than the STC workers; -red arrow indicates that the value of the tra is statistically significantly lower in the SMC workers than the STC workers; n.s.-statistically in significant difference between the SMC and STC workers; -black arrow indicates that the valu of the trait is higher in the SMC workers than the STC workers but without statistical significanc =-equality sign indicates that the value of the trait in the SMC workers is close or equal to the tra value in the STC workers. Figure 1. Apiary experiment-Superoxide dismutase (SOD) activities in the hemolymph of work ers in three consecutive years. SMC-workers reared in small-cell combs; STC-workers reared standard-cell combs; *-differences between the SMC and STC workers within the age group ar significant at p ≤ 0.05; vertical bars indicate standard deviation. n.s.
Antioxidants 2023, 12, x FOR PEER REVIEW CAT activities and TAC levels in the 1-day-old workers, i.e., statistically si lower values of these parameters were recorded in the group of the SMC work STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-ol were higher in the STC group, but the differences were statistically significa 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends obser groups of the 7-, 14-, and 21-day-old workers were very similar to those in experiment (Figures 4-6, Table 3).   CAT activities and TAC levels in the 1-day-old workers, i.e., statistically si lower values of these parameters were recorded in the group of the SMC work STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-o were higher in the STC group, but the differences were statistically significa 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends obser groups of the 7-, 14-, and 21-day-old workers were very similar to those in experiment (Figures 4-6, Table 3).   CAT activities and TAC levels in the 1-day-old workers, lower values of these parameters were recorded in the group STC workers (Figures 1-3, Table 3). In turn, the SOD activit were higher in the STC group, but the differences were st 2021 (Figures 1-3, Table 3). In the laboratory experiment, groups of the 7-, 14-, and 21-day-old workers were very si experiment (Figures 4-6, Table 3).   CAT activities and TAC levels in the 1-day-old w lower values of these parameters were recorded in STC workers (Figures 1-3, Table 3). In turn, the SO were higher in the STC group, but the differences 2021 (Figures 1-3, Table 3). In the laboratory exp groups of the 7-, 14-, and 21-day-old workers we experiment (Figures 4-6, Table 3).   CAT activities and TAC levels in the 1-day-old w lower values of these parameters were recorded in STC workers (Figures 1-3, Table 3). In turn, the SO were higher in the STC group, but the difference 2021 (Figures 1-3, Table 3). In the laboratory exp groups of the 7-, 14-, and 21-day-old workers we experiment (Figures 4-6, Table 3).     1-3, Table 3). In t groups of the 7-, 14-, and 21-day experiment (Figures 4-6, Table 3)                CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significantly lower values of these parameters were recorded in the group of the SMC workers vs. the STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old workers were higher in the STC group, but the differences were statistically significant only in 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in the groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiary experiment (Figures 4-6, Table 3).   CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significantly lower values of these parameters were recorded in the group of the SMC workers vs. the STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old workers were higher in the STC group, but the differences were statistically significant only in 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in the groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiary experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activity; TAC-Total antioxidant capacity; -green arrow indicates that the value of the trait is statistically significantly higher in the SMC workers than the STC workers; -red arrow indicates that the value of the trait is statistically significantly lower in the SMC workers than the STC workers; n.s.-statistically insignificant difference between the SMC and STC workers; -black arrow indicates that the value of the trait is higher in the SMC workers than the STC workers but without statistical significance; =-equality sign indicates that the value of the trait in the SMC workers is close or equal to the trait value in the STC workers.  CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significant lower values of these parameters were recorded in the group of the SMC workers vs. th STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old worke were higher in the STC group, but the differences were statistically significant only 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in th groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiar experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activity; TAC-Total an oxidant capacity; -green arrow indicates that the value of the trait is statistically significant higher in the SMC workers than the STC workers; -red arrow indicates that the value of the tra is statistically significantly lower in the SMC workers than the STC workers; n.s.-statistically i significant difference between the SMC and STC workers; -black arrow indicates that the valu of the trait is higher in the SMC workers than the STC workers but without statistical significanc =-equality sign indicates that the value of the trait in the SMC workers is close or equal to the tra value in the STC workers. CAT activities and TAC levels in the 1-day-old workers, i.e., statis lower values of these parameters were recorded in the group of the S STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the were higher in the STC group, but the differences were statistically 2021 (Figures 1-3, Table 3). In the laboratory experiment, the tren groups of the 7-, 14-, and 21-day-old workers were very similar to experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activ oxidant capacity; -green arrow indicates that the value of the trait is sta higher in the SMC workers than the STC workers; -red arrow indicates th is statistically significantly lower in the SMC workers than the STC workers significant difference between the SMC and STC workers; -black arrow in of the trait is higher in the SMC workers than the STC workers but without =-equality sign indicates that the value of the trait in the SMC workers is clo value in the STC workers.  CAT activities and TAC levels in the 1-day-old w lower values of these parameters were recorded in STC workers (Figures 1-3, Table 3). In turn, the SO were higher in the STC group, but the differences 2021 (Figures 1-3, Table 3). In the laboratory exp groups of the 7-, 14-, and 21-day-old workers we experiment (Figures 4-6, Table 3).    CAT activities and TAC levels in the 1-d lower values of these parameters were rec STC workers (Figures 1-3, Table 3). In tur were higher in the STC group, but the d 2021 (Figures 1-3, Table 3). In the labor groups of the 7-, 14-, and 21-day-old wo experiment (Figures 4-6, Table 3).              CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significantly lower values of these parameters were recorded in the group of the SMC workers vs. the STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old workers were higher in the STC group, but the differences were statistically significant only in 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in the groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiary experiment (Figures 4-6, Table 3).    CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significantly lower values of these parameters were recorded in the group of the SMC workers vs. the STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old workers were higher in the STC group, but the differences were statistically significant only in 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in the groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiary experiment (Figures 4-6, Table 3).   CAT activities and TAC levels in the 1-d lower values of these parameters were rec STC workers (Figures 1-3, Table 3). In tur were higher in the STC group, but the d 2021 (Figures 1-3, Table 3). In the labor groups of the 7-, 14-, and 21-day-old wo experiment (Figures 4-6, Table 3).       CAT activities and TAC lower values of these para STC workers (Figures 1-3 were higher in the STC g 2021 (Figures 1-3, Table  groups of the 7-, 14-, and experiment (Figures 4-6,        CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significantly lower values of these parameters were recorded in the group of the SMC workers vs. the STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old workers were higher in the STC group, but the differences were statistically significant only in 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in the groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiary experiment (Figures 4-6, Table 3).    -red arrow indicates that the value of the trait is statistically significantly lower in the SMC workers than the STC workers; n.s.-statistically insignificant difference between the SMC and STC workers; -black arrow indicates that the value of the trait is higher in the SMC workers than the STC workers but without statistical significance; =-equality sign indicates that the value of the trait in the SMC workers is close or equal to the trait value in the STC workers.  CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significant lower values of these parameters were recorded in the group of the SMC workers vs. th STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old worke were higher in the STC group, but the differences were statistically significant only 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in th groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiar experiment (Figures 4-6, Table 3).
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activity; TAC-Total an oxidant capacity; -green arrow indicates that the value of the trait is statistically significant higher in the SMC workers than the STC workers; -red arrow indicates that the value of the tra is statistically significantly lower in the SMC workers than the STC workers; n.s.-statistically i significant difference between the SMC and STC workers; -black arrow indicates that the valu of the trait is higher in the SMC workers than the STC workers but without statistical significanc =-equality sign indicates that the value of the trait in the SMC workers is close or equal to the tra value in the STC workers. CAT activities and TAC levels in the 1-day-old workers, lower values of these parameters were recorded in the group STC workers (Figures 1-3, Table 3). In turn, the SOD activit were higher in the STC group, but the differences were st 2021 (Figures 1-3, Table 3). In the laboratory experiment, groups of the 7-, 14-, and 21-day-old workers were very si experiment (Figures 4-6, Table 3).     CAT activities and TAC levels in the 1-d lower values of these parameters were rec STC workers (Figures 1-3, Table 3). In tur were higher in the STC group, but the d 2021 (Figures 1-3, Table 3). In the labor groups of the 7-, 14-, and 21-day-old wo experiment (Figures 4-6, Table 3).   CAT activities and TAC levels in the 1-d lower values of these parameters were rec STC workers (Figures 1-3, Table 3). In tur were higher in the STC group, but the d 2021 (Figures 1-3, Table 3). In the labor groups of the 7-, 14-, and 21-day-old wo experiment (Figures 4-6, Table 3).      CAT activities and TAC lower values of these para STC workers (Figures 1-3 were higher in the STC g 2021 (Figures 1-3,         CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significantly lower values of these parameters were recorded in the group of the SMC workers vs. the STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old workers were higher in the STC group, but the differences were statistically significant only in 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in the groups of the 7-, 14-, and 21-day-old workers were very similar to those in the apiary experiment (Figures 4-6, Table 3).    -green arrow indicates that the value of the trait is statistically significantly higher in the SMC workers than the STC workers; Antioxidants 2023, 12, x FOR PEER REVIEW CAT activities and TAC levels in the 1-day-old workers, i.e., statis lower values of these parameters were recorded in the group of the SM STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the were higher in the STC group, but the differences were statistically 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trend groups of the 7-, 14-, and 21-day-old workers were very similar to experiment (Figures 4-6, Table 3).

  
CAT-Catalase enzyme activity; SOD-Superoxide dismutase enzyme activ oxidant capacity; -green arrow indicates that the value of the trait is sta higher in the SMC workers than the STC workers; -red arrow indicates tha is statistically significantly lower in the SMC workers than the STC workers; significant difference between the SMC and STC workers; -black arrow in of the trait is higher in the SMC workers than the STC workers but without s =-equality sign indicates that the value of the trait in the SMC workers is clos value in the STC workers. -red arrow indicates that the value of the trait is statistically significantly lower in the SMC workers than the STC workers; n.s.-statistically insignificant difference between the SMC and STC workers; Antioxidants 2023, 12, x FOR PEER REVIEW 7 CAT activities and TAC levels in the 1-day-old workers, i.e., statistically significa lower values of these parameters were recorded in the group of the SMC workers vs STC workers (Figures 1-3, Table 3). In turn, the SOD activities in the 1-day-old wor were higher in the STC group, but the differences were statistically significant on 2021 (Figures 1-3, Table 3). In the laboratory experiment, the trends observed in groups of the 7-, 14-, and 21-day-old workers were very similar to those in the ap experiment (Figures 4-6, Table 3).       Different trends were evident in the SOD activities in the apiary and laboratory experiments each year (Figures 1 and 4). In the SMC workers in the apiary experiment conducted in 2020, the 1-day-old workers exhibited the lowest activities of the enzymes, but they increased with age ( Figure 1). Similar values of the SOD activities were recorded for the STC workers. In the group of the SMC workers in 2021, the highest SOD activities were determined for the hemolymph of the 1-and 7-day-old workers, but the activities declined in the older workers. In turn, the activity persisted at a similar level in the group of the STC workers. In 2022, the highest SOD activities were noted in the 1-day-old SMC and STC workers, but their values decreased with age. In the laboratory experiment conducted in 2020 and 2022, the lowest SOD activities were recorded in the samples collected from the 1-day-old SMC and STC workers. The SOD activities in the hemolymph of the older workers were higher and persisted at a similar level (Figure 4). In 2021, the lowest SOD activities were noted in the groups of the 1-day-old SMC and STC workers. The highest activity was recorded for the 7-day-old workers, but the activities in the hemolymph of the older workers decreased.  Different trends were evident in the SOD activities in the apiary and laboratory experiments each year (Figures 1 and 4). In the SMC workers in the apiary experiment conducted in 2020, the 1-day-old workers exhibited the lowest activities of the enzymes, but they increased with age ( Figure 1). Similar values of the SOD activities were recorded for the STC workers. In the group of the SMC workers in 2021, the highest SOD activities were determined for the hemolymph of the 1-and 7-day-old workers, but the activities declined in the older workers. In turn, the activity persisted at a similar level in the group of the STC workers. In 2022, the highest SOD activities were noted in the 1-day-old SMC and STC workers, but their values decreased with age. In the laboratory experiment conducted in 2020 and 2022, the lowest SOD activities were recorded in the samples collected from the 1-day-old SMC and STC workers. The SOD activities in the hemolymph of the older workers were higher and persisted at a similar level (Figure 4). In 2021, the lowest SOD activities were noted in the groups of the 1-day-old SMC and STC workers. The highest activity was recorded for the 7-day-old workers, but the activities in the hemolymph of the older workers decreased.
With the exception of the apiary experiment conducted in 2020, in all of the apiary and laboratory experiments, the CAT activities were higher in the 1-day-old workers than in the older workers (Figures 2 and 5). In turn, in the apiary experiment, the values of the  Different trends were evident in the SOD activities in the apiary and laboratory experiments each year (Figures 1 and 4). In the SMC workers in the apiary experiment conducted in 2020, the 1-day-old workers exhibited the lowest activities of the enzymes, but they increased with age ( Figure 1). Similar values of the SOD activities were recorded for the STC workers. In the group of the SMC workers in 2021, the highest SOD activities were determined for the hemolymph of the 1-and 7-day-old workers, but the activities declined in the older workers. In turn, the activity persisted at a similar level in the group of the STC workers. In 2022, the highest SOD activities were noted in the 1-day-old SMC and STC workers, but their values decreased with age. In the laboratory experiment conducted in 2020 and 2022, the lowest SOD activities were recorded in the samples collected from the 1-day-old SMC and STC workers. The SOD activities in the hemolymph of the older workers were higher and persisted at a similar level (Figure 4). In 2021, the lowest SOD activities were noted in the groups of the 1-day-old SMC and STC workers. The highest activity was recorded for the 7-day-old workers, but the activities in the hemolymph of the older workers decreased.
With the exception of the apiary experiment conducted in 2020, in all of the apiary and laboratory experiments, the CAT activities were higher in the 1-day-old workers than With the exception of the apiary experiment conducted in 2020, in all of the apiary and laboratory experiments, the CAT activities were higher in the 1-day-old workers than in the older workers (Figures 2 and 5). In turn, in the apiary experiment, the values of the enzyme declined in the older SMC and STC workers after the 14th day of life in 2021 and 2022 and increased in 2020 (Figure 2). In the laboratory experiment, the activities of the enzyme persisted at the same level in both the SMC and STC workers ( Figure 5).
In the laboratory experiment, the trends in the TAC parameter were very similar in the SMC and STC workers in each year. The highest TAC levels were recorded in the 1-day-old workers; next, they decreased significantly and remained at similar levels in the older workers ( Figure 6). In the apiary experiments conducted in 2020 and 2022, the SMC and STC workers exhibited similar trends in TAC levels ( Figure 3). The highest values were recorded in the 1-day-old workers; they declined or remained at similar levels in the older workers. The 21-day-old workers in 2020 were an exception, as the TAC levels increased in comparison with the 1-and 7-day-old workers. In the STC group analyzed in 2021, the highest TAC values were found in the 1-day-old workers. Their levels decreased significantly in the samples collected from the 7-day-old workers, increased significantly in the hemolymph of the 14-day-old insects, and declined again in the group of the 21-day-old insects. The TAC levels in the 7-day-old STC workers also decreased significantly in comparison with the 1-day-old workers; on day 14, the levels increased and exceeded the values recorded for the 1-day-old group. Next, the TAC levels decreased in the 21-day-old group, as they did in the SMC workers.

SOD and CAT Activities and TAC Levels in the SMC and STC Workers
The present study showed that the CAT and SOD activities and TAC levels on days 7, 14, and 21 were usually higher in the hemolymph of the SMC workers in comparison with the STC group. The same trends were observed both in the apiary experiment and in the laboratory cage tests. With the exception of the SOD activities, opposite results were obtained in the group of 1-day-old insects in comparison with the 7-, 14-, and 21-day-old worker bees. Thus, we demonstrated a significant effect of the width of worker comb cells on the activities of antioxidants in worker bee hemolymph. We described very similar trends in our previous study, showing a significant effect of different widths of worker comb cells (small vs. standard) on the total protein content and the activities of proteolytic enzymes and their inhibitors in the hemolymph [31]. As in the case of the activities of antioxidants on days 7, 14, and 21, the activities of proteolytic enzymes and their inhibitors in the hemolymph were higher in the worker bees reared in small-cell combs than in standard-cell combs. In contrast, the total protein content in the hemolymph was lower. Opposite results were obtained in the group of 1-day-old workers in comparison with the 7-, 14-, and 21-day-old bees.
Higher CAT and SOD activities and TAC levels determined in the 7-, 14-, and 21-day-old SMC workers in the apiary and laboratory conditions contributed to higher ROS scavenging capacity in comparison with the STC workers. Interestingly, with the exception of SOD activity, opposite results were obtained in the group of the 1-day-old workers, as in the case of the previously reported activities of proteases and their inhibitors [31]. Elevated activities of antioxidant enzymes in worker bees and thus the ability to neutralize free radicals through an increased expression of antioxidant genes may result from the quality of nutrition, including the protein content in the larval diet [37,38]. The samples collected from the 1-day-old SMC workers exhibited higher protein content than those from the STC workers. The protein level declined with age, as it may have been utilized for, e.g., the synthesis of proteolytic enzymes and their inhibitors [31].

Age-Related Changes in SOD and CAT Activities and TAC Levels in the Workers
In many organisms [39][40][41], including bees [13,42], the ability to stimulate the activities of heat shock proteins and antioxidants decreases with age. Different results were obtained by Paleolog et al. [21] and Skowronek et al. [34] in apiary experiments and by Skowronek et al. [25] in laboratory analyses; they demonstrated an age-related increase in the activity of the antioxidant system in worker bees. The findings obtained in the apiary experiments may reflect the adaptation of the worker bees to function as foragers outside the nest in an environment exposed to pollution [43][44][45]. Furthermore, the increased activities of detoxification enzymes in older worker bees seem to indicate their biochemical adaptation to the forager work, compensating for the age-related decline in protein content [43]. However, in the case of pesticide poisoning, e.g., with imidacloprid, the activity of the antioxidant system in workers exposed to the pesticide was found to decline with age (20 days old) in comparison with imidacloprid-untreated 1-day-old workers [21]. In contrast, the activity of the antioxidant system in laboratory experiments was reported to increase with age [25].
However, a direct comparison of the present results with the findings reported by Paleolog et al. [21] and Skowronek et al. [25] is difficult, as the authors calculated the an-tioxidant activity per protein mass unit without specifying the protein content per solution volume unit in the worker age groups. This method of data presentation does not provide explicit information about whether the significant increase in the antioxidant activity is related to an actual increase in the activities of the antioxidants or to the significant decrease in the protein content without a significant change in the antioxidant activities. Such a relationship was indicated in other studies that compared changes in protein concentrations in the hemolymph solution with changes in the activities of proteases [23,24,31]. In the present study, to exclude the impact of fluctuations in protein concentrations on antioxidant activities, we calculated the SOD, CAT and TAC values per solution volume unit, without converting the result into a protein mass unit.
In the apiary experiment conducted in the present study, the antioxidant activities tended to decrease with age in both the SMC and STC worker groups. The greatest decrease was recorded between days 1 and 7. In the cage experiment, with the exception of SOD, the activity of antioxidants decreased significantly between days 1 and 7 (as in the apiary experiment), but then persisted at a similar level. The decrease in the antioxidant activities between days 1 and 7 may be associated with the change in the habitat from the conditions prevailing in the capped comb cell with a constant high temperature (34.44-35.39 • C) [46] to the less precisely regulated conditions of the nest and external environment. We collected hemolymph from 1-day-old worker bees immediately after their emergence from the comb cells; therefore, their metabolism may not yet have reacted to the change in the conditions. In turn, the significant decrease in the CAT activities and TAC levels between days 1 and 7 may have been a result of the decrease in the ambient temperature. In contrast, the gradual decrease in SOD and CAT activity in the older workers (between days 7 and 21) was probably caused by the age-related decline in the ability to stimulate antioxidant activity induced by prolonged exercise [13]. Similarly, Williams et al. [47] found that the ability of the antioxidant system to deactivate ROS decreased with age in nurses and foragers up to the loss of the ability in aged honeybees (30-32 days). This resulted in reduced productivity and a shorter lifespan [47]. Our assumption was confirmed by the laboratory experiment results, which showed that the CAT activities and TAC levels declined between days 1 and 7 in the SMC and STC workers and then persisted at a similar level between days 7 and 21, probably because these bees did not take up any activities in accordance with their role in the colony. These observations indicate that changes in the antioxidant activity are largely a consequence of the activities performed by worker bees, especially those requiring an intensification of metabolism, rather than their age.
The trends in SOD activity were opposite to those in CAT activities in both the apiary and laboratory experiments. No decline in SOD activity was observed between days 1 and 7. In the laboratory experiment, the SOD activities increased and persisted at a similar level between days 7 and 21. No significant changes in this parameter in caged workers between days 9 and 26 were found by [13]. In turn, the activities of SOD in our apiary experiment were dependent on the year. In 2020, they increased with age in the SMC worker group and persisted at a similar level in the samples collected from the STC workers. In 2021 and 2022, the activities decreased with age in both the SMC and STC workers, and the decline noted in 2021 was substantially lower than in 2022. Different trends were observed in the age-related CAT and TAC changes in the bees in the apiary experiment in the particular years of the study. In the laboratory experiment, the trends in the levels of all the antioxidants were similar. This indicates a significant impact of environmental factors on the results of our apiary experiments; therefore, to reduce the risk of an effect of random environmental factors on the results of one-season investigations of honeybees, it is advisable to continue the research over a few consecutive years.

Conclusions
Workers reared in small-cell combs exhibit higher hemolymph antioxidant activities than those from standard-cell combs. Consequently, their hemolymph has a greater antioxidant capacity.
In terms of physiological differences between worker bees reared in small-and standard-cell combs in the same bee colony, it is necessary to elucidate the role of the considerable variation in the cell width in natural combs built without the artificial wax foundation.
To reduce the effect of random environmental factors that may be encountered in one-season studies on the results of research on honeybees, it is advisable to continue such investigations over several consecutive years.
Author Contributions: Conceptualization, research, discussion, statistical analysis, and writing, P.D. and K.O.; Laboratory analysis, P.D.; research, writing, A.S. and G.B. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.