Small-Cell Combs Offer as Favorable Conditions of Rearing Worker Bees as Standard-Cell Combs in the Temperate Climate in Spring

: During the spring development of bee colonies, small-cell combs were found to create equally favorable conditions for worker bee rearing as standard-cell combs, since the workers reared in the small-cell combs did not differ significantly in the majority of morphometric traits, including the length of wings and the sum of the widths of the third and fourth tergites, from those reared in standard-cell combs. Moreover, they had a significantly longer and wider thorax. It can be assumed that the workers reared in small-cell combs collect nectar as effectively as those reared in standard-cell combs, as both groups did not differ in the proboscis length. It was confirmed that the body size of workers is relatively constant and is less influenced by the width of comb cells than was assumed previously, as the values of their morphometric parameters did not increase proportionally with the increasing cell width. The colony kept on small-cell combs provided worse rearing conditions for workers reared in standard-cell combs than the colony kept on standard-cell combs, which may have been related to the less abundant feeding of larvae by workers reared in small-cell combs.


Introduction
Early spring in central Europe coincides with unfavorable conditions for the spring development of honeybee colonies.Usually, there are alternating cool and warm periods, with cool days predominating.Spring cold snaps have been correlated with interruptions in flight activity, reduced pollen intake, and increased brood cannibalism, resulting in the reduction in nurse populations that then have to carry heavier nursing loads [1].Under such conditions, spring development depends on environmental factors outside the colony, such as temperature and the availability of fresh pollen [2,3].However, the factors of a bee colony nest environment, such as the strength of the colony or the quality of nursing bees, are equally important [4].Another crucial and still poorly elucidated factor is the bee comb as an element of the environment of the bee's nest [5].
The identification of honeybee subspecies relies on the exhibition of distinctive morphometric traits which are highly heritable and are therefore a good identification tool [6].However, the value of these traits undergoes seasonal fluctuations associated with larval nutrition, as demonstrated by [6,7].This opinion has been confirmed by Brodschneider et al. [8], who found that worker bees that had emerged from more poorly nourished larvae had a lower body weight and a smaller surface area of the first pair of wings than those emerging from optimally nourished larvae.In turn, Michailoff [9] found that workers that had developed from malnourished larvae had a shorter proboscis and shorter first-pair wings.Due to seasonal changes in the body size, worker bees should be sampled for morphometric analysis in summer months when they are larger [6,7].They should not be Appl.Sci.2024, 14, 4566 2 of 8 reared in very dark combs, as the size of the worker bee also depends on the size of the cell [6].
A study conducted in South America by Maggi et al. [10] on colonies with nest combs built without the use of a wax foundation showed a considerable variation in the width of comb cells, ranging from 4.17 to 8.07 mm even in the same colony.At present, the width of standard cells is usually in the range of 5.40-5.50mm.The width of worker comb cells in natural nests of the European dark bee (Apis mellifera mellifera) was reported to range between 4.90 and 5.10 mm [11].Currently, 4.90 mm wide comb cells are referred to as "small cells" [11][12][13].
It was expected that an increase in the width of comb cells via the use of an artificially produced wax foundation would result in a proportional increase in the worker body size [11] and, consequently, in an increase in the honey yield of colonies.The belief that the worker body size changes proportionally to changes in the cell width still prevails [6,7,11].However, it has been shown that keeping bee colonies on small-cell combs exerts a significant effect on the morphological traits and the biology of worker bees.McMullan and Brown [11] found that a 7-8% reduction in the comb cell width resulted in a mere 1% decrease in the width of the worker head and thorax.Similar results were reported by Seeley and Griffin [14].Our previous work also supports these findings, indicating that the rearing of workers in small-cell combs in the colony kept on standard-cell combs resulted in an increase in the value of the fill factor (thorax width to cell width ratio) [15].Additionally, it contributes to a higher effectiveness of hygienic behavior [16,17] and a higher rate of springtime colony development [18] as well as an extension of the lifespan of workers [19] and has an impact on the activities of the proteolytic and antioxidant system in the hemolymph of workers [20,21].
Therefore, the aim of this study was to find out whether small-cell combs provide as favorable conditions for rearing worker bees as standard-cell combs during the spring development, expressed as a lack of statistically significant changes in morphological traits.

Worker Rearing
The experiment was conducted in the apiary of the University of Life Sciences in Lublin (Poland; 51.224039 N-22.634649E).Six drawn honeycombs in which no brood was reared were selected.Two of the combs were drawn out on a small-cell foundation (smComb) (cell width 4.90 mm) and four were drawn on a standard-cell foundation (STComb) (cell width 5.50 mm).
The length of the horizontal segments (one in each quarter of the comb) was measured on each side of each comb.In the central part of the comb, the widths of 10 adjacent cells in contact with the vertical side walls were measured.Each of the 10 cells was measured separately [11].The measurements were made on photographs with the use of the Multi Scan digital image analysis system v. 14.02 (segment measurement option) supplied by Computer Scanning System II, Warsaw [15].
In spring, more specifically in the last ten days of April, four honeybee colonies with similar strength and structure were selected.Two colonies were kept on small-cell combs (smColony) and the other two were kept on standard-cell combs (STColony).The colonies were kept in Ostrowska-type vertical hives and were headed by naturally mated Buckfast sister-queens.Buckfast bee colonies were selected for the study, as they had adapted successfully to living on small-cell combs in the apiary of the University of Life Sciences in Lublin [15].In each colony, the workers populated one brood chamber and one honey super, each with 10 frames (360 × 230 mm).Two brood combs from the center of the brood chamber of each smColony (colony kept on small-cell combs) were transferred to the honey super.A queen-excluder frame cage divided into two parts containing one comb each was placed in this space.The smComb (small-cell comb) was placed in one part and the STComb (standard-cell comb) was placed in the other.Next, the queen was kept on each comb for 24 h.After this time, the frame cage was removed from each colony, and the removed brood combs were placed in the center of the brood chamber.
One brood comb was transferred from the center of the brood chamber to the honey super from the nest of each STColony (colony kept on standard-cell combs).The queenexcluder frame cage containing one comb was placed in this space.A STComb on which the queen was kept for 24 h was placed in the frame cage.After this time, the frame cage was removed from each colony and the removed brood comb was placed in the center of the brood chamber.After 19 days, each brood comb was placed in a separate mesh frame cage and transferred into an incubator (34.5 • C and 60% RH), where it was kept until the emergence of workers.Approximately 100 workers from each comb were transferred into a separate cage and left in the incubator (26 • C and 60% RH) for 7 days to allow their chitin cuticle to harden.Bees in the cage were fed sugar syrup with sugar to water in a 1:1 ratio.Three groups of workers corresponding to the combinations of the comb type and the foster colony type were obtained: bees reared in small-cell combs in colonies kept on small-cell combs (smComb + smColony), bees reared in standard-cell combs in colonies kept on small-cell combs (STComb + smColony), and bees reared in standard-cell combs in colonies kept on standard-cell combs (STComb + STColony).
The scheme for obtaining worker bees is shown in Figure 1.
of the brood chamber of each smColony (colony kept on small-cell combs) were transferred to the honey super.A queen-excluder frame cage divided into two parts containing one comb each was placed in this space.The smComb (small-cell comb) was placed in one part and the STComb (standard-cell comb) was placed in the other.Next, the queen was kept on each comb for 24 h.After this time, the frame cage was removed from each colony, and the removed brood combs were placed in the center of the brood chamber.One brood comb was transferred from the center of the brood chamber to the honey super from the nest of each STColony (colony kept on standard-cell combs).The queen-excluder frame cage containing one comb was placed in this space.A STComb on which the queen was kept for 24 h was placed in the frame cage.After this time, the frame cage was removed from each colony and the removed brood comb was placed in the center of the brood chamber.After 19 days, each brood comb was placed in a separate mesh frame cage and transferred into an incubator (34.5 °C and 60% RH), where it was kept until the emergence of workers.Approximately 100 workers from each comb were transferred into a separate cage and left in the incubator (26 °C and 60% RH) for 7 days to allow their chitin cuticle to harden.Bees in the cage were fed sugar syrup with sugar to water in a 1:1 ratio.Three groups of workers corresponding to the combinations of the comb type and the foster colony type were obtained: bees reared in small-cell combs in colonies kept on small-cell combs (smComb + smColony), bees reared in standard-cell combs in colonies kept on small-cell combs (STComb + smColony), and bees reared in standard-cell combs in colonies kept on standard-cell combs (STComb + STColony).
The scheme for obtaining worker bees is shown in Figure 1.

Morphometric Assessment of Worker Bees
Fifteen workers from each comb of each group were examined (smComb + SMColony n = 30, STComb + SMColony n = 30, STComb + STColony n = 30) [6,7].The following analysis procedure was employed to measure each worker: (1) Anesthesia with ethyl acetate.(2) Measurement of the thorax width and length on the dorsal side under an Olympus SZX16 stereoscopic microscope.(3) Transfer of the bee head onto a glass slide to measure its width and height under an Olympus SZX16 stereoscopic microscope.(4) Dissection of the proboscis, the right fore wing, and the 3rd and 4th abdominal tergites [6] and transfer thereof onto a glass slide.(5) Measurement of the body parts specified in point 4 under an Olympus SZX16 stereoscopic microscope.The following linear measurements were obtained: the proboscis length, the length and width of the fore wing, and the distances a and b of the cubital vein [6,7] .The cubital index was calculated as the cubital vein distance a/b ratio as proposed by Goetze [6] .The percentage of the thorax/cell width ratio, i.e., the fill factor, was calculated [11,15] .
(2) Measurement of the thorax width and length on the dorsal side under an Olympus SZX16 stereoscopic microscope.(3) Transfer of the bee head onto a glass slide to measure its width and height under an Olympus SZX16 stereoscopic microscope.(4) Dissection of the proboscis, the right fore wing, and the 3rd and 4th abdominal tergites [6] and transfer thereof onto a glass slide.( 5) Measurement of the body parts specified in point 4 under an Olympus SZX16 stereoscopic microscope.The following linear measurements were obtained: the proboscis length, the length and width of the fore wing, and the distances a and b of the cubital vein [6,7].The cubital index was calculated as the cubital vein distance a/b ratio as proposed by Goetze [6].The percentage of the thorax/cell width ratio, i.e., the fill factor, was calculated [11,15].
We checked whether the differences between the values of the morphometric traits of the smComb + smColony and STComb + smColony groups, expressed as a percentage and calculated vs. the STComb + STColony group, corresponded to the percent difference in the cell width between the small-cell and standard-cell combs.

Statistical Analysis
The results were analyzed statistically using Statistica software formulas, version 13.3 (2017) for Windows, StatSoft Inc., Tulsa, OK, USA.The distribution of the data was analyzed with the Shapiro-Wilk test.The widths of the cells in the small-cell combs (each group n = 12) and in the standard-cell combs (each group n = 12) were compared with the Kruskal-Wallis test for independent samples.The significance of differences between the groups for the values of individual morphometric traits (smComb + smColony n = 30, STComb + smColony n = 30, STComb + STColony n = 30) for data with normal distribution was compared with the Tukey HDS test.Data with no normal distribution were compared with the Wilcoxon's signed-rank test (dependent samples) and the Mann-Whitney U test (independent samples).

Results
The width of cells in the small-cell combs was significantly smaller (p ≤ 0.01) than that of the standard-cell combs (Table 1).Workers reared in the smComb + smColony variant had a significantly longer and wider thorax and a significantly wider third tergite than those reared in the STComb + smColony and STComb + STColony variants.The larger thorax width with the smaller width of the comb cells resulted in a significantly higher value of the fill factor (thorax width to cell width ratio).The workers reared in the smComb + smColony combination had a significantly longer fore wing and a higher value of the head height than the STComb + smColony workers but did not differ from those reared in the STComb + STColony.In turn, their head width did not differ significantly from that of workers reared in the STComb + smColony variant but was significantly smaller than in the case of the STComb + STColony workers.In comparison with the STComb + STColony group, the STComb + smColony workers exhibited lower values of the following parameters: head width and height, thorax width, fore wing width, fore wing length, and fill factor.There were no significant differences between the groups in the proboscis length, distances a and b of the cubital vein, cubital index, fourth tergite width, and the sum of the widths of the third and fourth tergites.smComb + smColony-workers reared in small-cell combs in colonies kept on small-cell combs; STComb + SMColony-workers reared in standard-cell combs in colonies kept on small-cell combs; STComb + STColonyworkers reared in standard-cell combs in colonies kept on standard-cell combs; a, b differences in the rows are significant at p ≤ 0.05; A, B, C, D differences in the rows are significant at p ≤ 0.01.
The values of the morphometric parameters of the STComb + smColony and STComb + STColony workers, compared with their values in the workers reared in the smComb + smColony, did not increase proportionally to the increase in the width of the comb cells (13%) in which they were reared (Table 2).The range of the changes in the values of most traits did not exceed 3%.A surprising finding was the significant decrease in the thorax width, thorax height, and the widths of the third tergite.The fill factor declined significantly as well, which was related to the reduced thorax width and the greater width of the comb cells.STComb + smColony-workers reared in standard-cell combs in colonies kept on small-cell combs; STComb + STColony-workers reared in standard-cell combs in colonies kept on standard-cell combs.

Discussion
As proposed by Ruttner [7], the fore wing length and the sum of the widths of the third and fourth tergites are measures of the worker's body size.Based on this suggestion, we found that the small-cell combs offered equally favorable conditions expressed as a lack of statistically significant changes in morphological traits describing bee body dimensions used for species identification.This is supported by the findings that the workers from the small-cell combs did not differ significantly in the fore wing length and the sum of the widths of the third and fourth tergites from worker bees reared in the standard-cell combs.It is surprising that they had a significantly longer and wider thorax.A contrasting result was reported by Seeley and Griffin [14] in workers collected from colonies in September: a significantly higher value of the thorax width was recorded in workers reared in standardcell combs.These findings may indicate that small-cell combs provide comparable rearing conditions only in spring.Moreover, the workers reared in the small-cell combs in the present study did not differ significantly in the proboscis length from those reared in the standard-cell combs.This suggests that they can collect nectar with equal efficiency from flowers with a long and thin floral tube and deep-seated nectaries.
The values of the morphometric parameters of the STComb + STColony workers compared to those reared in the smComb + smColony variant did not increase proportionally to the increase in the width of their comb cells (13%).The range of changes in the values of most traits did not exceed 3% and was equal to the range of seasonal changes [6,7].Similar results were obtained by McMullan and Brown [11] and Seeley and Griffin [14].The values of traits of the STComb + STColony workers compared to those reared in the smComb + smColony regarded by Ruttner [6,7] as a measure of the body size of workers, i.e., the fore wing length and the sum of the widths of the third and fourth tergites, increased by 1.8% (fore wing length) and declined by 1.4% (sum of tergites).We observed a similar relationship in our pervious study [15] where the fore wing length of the STComb + STColony workers compared to those reared in the smComb + smColony increased by 3.4%, which slightly exceeded the range of seasonal changes [6,7].On the other hand, rearing bees in the STComb in STColony in summer months resulted in the increased value of the sum of the widths of the third and fourth tergites by 3.3% compared to those reared in the smComb + smColony [15].Such a difference supports our hypothesis that the small-cell combs offered equally favorable conditions in spring for colony development by affecting the bee body dimensions as providing better brood-rearing conditions.The lack of significant differences in the sum of the widths of the third and fourth tergites in spring and the decline in the sum of the tergites in summer could be indicated by the higher density of cells in the area of the comb, which ensures better temperature conditions for rearing a bigger amount of brood in the same area of the comb in smComb compared to STComb.The present study, likewise the experiments conducted by McMullan and Brown [11] as well as Seeley and Griffin [14] and our previous findings [15], indicated the relatively constant body size of workers and a lesser effect of the width of comb cells than was previously assumed.However, the results are not in agreement with findings reported by Ruttner [6,7], who observed an increase in the worker body size corresponding to changes in the width of the rearing cells.
The small-cell combs provided comparable rearing conditions to the standard-cell combs; however, the colony kept on the small-cell combs ensured worse conditions for the brood reared in the standard-cell combs.Bees reared in the STComb + smColony variant exhibited a lower value of the head width, head height, thorax width, and fore wing width and length than those reared in the STComb + STColony combination.In turn, they did not differ in the sum of the widths of the third and fourth tergites, i.e., one of the measures of the worker's body size proposed by Ruttner [6,7].The values of most morphometric parameters of the STComb + smColony workers were lower than those in the smComb + smColony and STComb + STColony groups.This indicates that the width of the comb cells does not determine the size of the worker's body.Other elements of rearing are regarded as equally important, e.g., the quality of feeding larvae, which was confirmed by Brodschneider et al. [8].Willem et al. [22] reported that workers reared in standard-cell combs feed larvae more abundantly than workers reared in small-cell combs.
We find it particularly advantageous to place small-cell combs in a colony maintained on standard-cell combs.In our other studies, it resulted in a significantly higher value of the functional traits of such colonies compared to colonies kept only on combs of one type, with only small-or only standard-cells [23].We assume that such a combination may modify the division of labor in the worker caste (Dziechciarz,[15,24]. The absence of significant differences in the values of traits most often used in the identification of worker subspecies, i.e., the proboscis length, cubital index, and the sum of the widths of the third and fourth tergites, between the assessed worker groups (smComb + smColony, STComb + smColony, and STComb + STColony) indicates the low dependence of these parameters on changes in rearing conditions.This confirms the high suitability of these parameters for the identification of honeybee subspecies.
The results of the morphometric measurements of the F1 generation of Buckfast worker bees analyzed in the present study indicate that they represent large-body size bees.In the STComb + STColony variant, the values of the most frequently assessed morphometric parameters, e.g., the proboscis length (6.71 mm) or the length and width of the fore wing (9.56 mm and 3.22 mm, respectively) were within the upper limits of the range for honeybees.In turn, the sum of the widths of the third and fourth tergites (4.84 mm) exceeded the range for honeybees (value range: proboscis length 5.02-7.26mm, fore wing length 7.46-9.75mm, fore wing width 2.64-3.31mm, sum of the widths of the third and fourth tergites 3.63-4.76mm) [6].Such high values may be associated with the fact that we assessed F1-generation workers derived from naturally mated queens, which may have caused heterosis.In a study of the morphometric traits of pure Buckfast bees, Borsuk and Olszewski [25] reported slightly lower values of the sum of the widths of the third and fourth tergites (4.79 mm) and proboscis length (6.36 mm).

Conclusions
During the spring development of colonies in temperate climates, small-cell combs ensure equivalent fostering terms as standard-cell combs.
The colony kept on the small-cell combs provided worse rearing conditions for the brood reared in the standard-cell combs than the colony kept on the standard-cell combs, which may have been associated with the less abundant feeding of larvae by workers reared in the small-cell combs.
It can be assumed that the spring-reared worker bees from the small-cell combs were equally efficient in the collection of nectar from flowers with a long and thin floral tube and deep-seated nectaries as those reared in standard-cell combs, as the groups did not differ in the proboscis length.
We confirmed that the body size of workers is relatively constant and is less influenced by the width of the comb cells than was assumed previously, as the values of the morphometric parameters of the workers did not increase proportionally with the increasing cell width.
The values of the traits that are most often used in the identification of worker subspecies, i.e., the proboscis length, cubital index, and the sum of the widths of the third and fourth tergites, indicate the low dependence of these parameters on changes in rearing conditions related to changes in the width of comb cells.This confirms their high suitability for the identification of honeybee subspecies.

Table 1 .
Values of the width of comb cells [mm], morphometric parameters of workers [mm], and fill factor [%].

Table 2 .
Percentage changes in comb cell width, values of morphometric parameters, and fill factor calculated in relation to workers reared in small-cell combs in the colony kept on the small-cell combs (smComb + smColony).