A scientific note on the strategy of wax collection as rare behavior of Apis mellifera

Wax collection behavior has not yet been described in the honeybee; it is typical of another Apis species. The wax collection was more difficult to observe due to the different biology of Apis mellifera and the fact that beekeepers do not display combs around the apiary to avoid the spread of bee diseases and robberies. The transport of wax in the pollen basket is typical of Apis florea; additionally, these bees have preferences for natal combs, which are significantly greater than for non-natal combs. Before they abandon their nests, these migratory bees following the nectar flow collect and transport some wax in their pollen baskets. This helps them to build a new nest quickly. The collection of available wax by both A. florea and A. mellifera is economically justified, as it reduces consumption of energy and honey supplies for the production of wax. The economic balance is believed to determine the collection of wax covers of Ceroplastes sp. soft scale insects by A. mellifera. This can be confirmed by the comparison of the energy value of wax (12.7 kcal/g) and honey (3.1 kcal/g), which indicates an over fourfold energy gain in favor of honey stored in the bee colony. The energetic trade-off between wax secretion and collection from an old nest may explain why A. florea is probably the only honeybee species known to recycle wax if the new breeding site is located at a distance lower than 100–200 m away from the nesting site. In such a case, it energetically pays off to recycle the wax. The same is probably true in A. mellifera, as we observed that wax was placed on the tops of the apiary hives at a distance lower than 100 m away from the colony. This is also reflected by the economic conversion rates, as a bee colony consumes from 4 to 8 kg of honey to produce 1 kg of wax. Our observations show that A. mellifera collect wax in pollen baskets. In addition, wax collection by honeybees is a static process occurring at the site where wax is present; bees do not have to flight, unlike in the case of pollen pellets, which are formed during flight. This paper addresses two completely unknown issues that make up two hypotheses: one is associated with collection of propolis into the pollen basket to stick the light wax fragments, and the other assumes saving energy required from the bee organism, which can be used to support colony functioning.

Thanks to the development of advanced technologies, humans are currently able to expand knowledge, also that related to the honeybee. Detailed data on the anatomy, morphology, or behaviors of bees contribute to the development of research techniques (Borsuk et al. 2017). Nevertheless, the bee colony has still many unexplored facets. There are still some rarely displayed behaviors of honeybees that have yet to be fully explained. One such unexpected behavior is the collection of wax into the pollen basket on the third pair of legs ( Figure 1), which was observed in the apiary of the University of Life Sciences in Lublin (51° 13′ N, 22° 38′ E). The collection of wax in this way has never been reported in the literature. It is documented in Figure 1a, b, c, and d. Similar behavior has been presented in a video film (Estrada Farms 2017), which shows a honeybee worker biting off pieces of wax from the wax foundation with mandibles and placing the wax fragments in the pollen baskets. Dimou and Thrasyvoulou (2007) reported that honeybees also collected wax covers of Ceroplastes sp. soft scale insects. This indicates that Apis mellifera bees are more heterospecific in terms of wax collection. Figure 1a, b, and c show workers collecting wax from a virgin/light honeycomb as well as dark wax scraped off by the beekeeper from the top bars of the nest frames ( Figure 1d) and forming a wax ball. The wax is darker than that from the light virgin honeycomb, as it may contain an admixture of propolis or wax collected by workers from older honeycombs, in which several generations of bees have been reared. Wax fragments removed from the bee nest during the inspection held at the end of July were left on the hive roof. They contained no honey or nectar. The wax pieces were left at different times so that the bees did not have to choose the type of wax to collect and that did not have mixed wax loads in their pollen baskets. The worker bees became interested in the wax after ca. 20 min. First, they bit off fragments of the wax with their mandibles and placed them in the pollen basket with the help of the combs of the first and second pairs of legs ( Figure 1b). The pollen basket is used by worker bees to collect and transport floral pollen. During the formation of the bee pollen pellet, bees rub the two combs on the basitarsus of the third pair of legs, lift the basitarsus, and press the pollen into the basket. They perform this activity during flight (as shown in video 1 in the Supplementary Material), as then they can move the third-pair legs freely (Thorp 2000;Goodman 2003;Stell 2012). The bees collecting the wax probably displayed two strategies: one may consist in collecting propolis into the pollen basket to stick the light wax fragments (Figure 1a). Bees may use the adherent properties of propolis (resins) to affix the wax faster to the pollen basket. The other strategy may involve direct attachment of the wax fragments to the basket (Figure 1c). It was observed that, during the collection of the wax pieces into the pollen baskets, the workers did not start the flight but lifted the front part of their body to detach both the first-and secondpair legs from the honeycomb. This facilitated transfer of the wax fragments onto the brushes on the second pair of legs, which are then used to attach the wax to the pollen basket ( Figure 1b).
Interestingly, honeybees collect wax and propolis in pollen baskets (Meyer 1956;Ghisalberti 1979;Kadhim et al. 2018) at the site where they find these materials, unlike in the case of pollen pellets, which are formed during flight (Thorp 2000;Goodman 2003;Stell 2012). During the production of scale wax, bees chew wax and add saliva, which contains enzymes exerting lipolytic activity, thereby reducing the pool of diesters in newly secreted wax and increasing the content of the monoester fraction in comb wax (Kurstjens et al. 1985). This is consistent with the previously described free fatty acid content in two kinds of waxes. The comb wax was shown to contain larger amounts of fatty acids released from esters (especially 16:0 and 24:0) compared to scale wax (Hepburn et al. 1991). In turn, fatty acids affect the mechanical properties of beeswax (e.g., stiffness) (Svečnjak et al. 2019). Probably, during wax collection, foraging bees add saliva to soften the wax fragments and to diminish its surface tension, which increases the adhesion strength of the wax to the pollen basket. This hypothesis corresponds to comparative analyses of the physicochemical properties of virgin wax scales and comb wax, which showed that the former had twofold higher strain and was characterized by significantly lower stiffness (Kurstjens et al. 1985).
Although the wax collection behavior has not yet been described in the honeybee, it is typical of another Apis species. The wax collection was more difficult to observe due to the different biology of Apis mellifera and the fact that beekeepers do not display combs around the apiary to avoid the spread of bee diseases and robberies (Sulborska et al. 2019). The transport of wax in the pollen basket is typical of Apis florea (Ruttner 1992(Ruttner , 1988; additionally, these bees have preferences for natal combs, which are significantly greater than for non-natal combs (Hepburn 2010). Before they abandon their nests, these migratory bees following the nectar flow collect and transport some wax in their pollen baskets. This helps them to build a new nest quickly. The collection of available wax by both A. florea and A. mellifera is economically justified, as it reduces consumption of energy and honey supplies for the production of wax. The economic balance is believed to determine the collection of wax covers of Ceroplastes sp. soft scale insects by A. mellifera (Dimou and Thrasyvoulou 2007). This can be confirmed by the comparison of the energy value of wax (12.7 kcal/g) and honey (3.1 kcal/g) (Colleen et al. 2002;Pirk et al. 2011), which indicates an over fourfold energy gain in favor of honey stored in the bee colony. The energetic trade-off between wax secretion and collection from an old nest may explain why A. florea is probably the only honeybee species known to recycle wax if the new breeding site is located at a distance lower than 100-200 m away from the nesting site. In such a case, it energetically pays off to recycle the wax (Pirk et al. 2011). The same is probably true in A. mellifera, as we observed that wax was placed on the tops of the apiary hives at a distance lower than 100 m away from the colony. This is also reflected by the economic conversion rates, as a bee colony consumes from 4 to 8 kg of honey to produce 1 kg of wax (Guderska 1983;Akratanakul et al. 1990;Prabucki 1998;Bradbear 2009).
Our observations show that A. mellifera collect wax in pollen baskets. In addition, wax collection by honeybees is a static process occurring at the site where wax is present; bees do not have to flight, unlike in the case of pollen pellets, which are formed during flight.
This paper addresses two completely unknown issues that make up two hypotheses: one is associated with collection of propolis into the pollen basket to stick the light wax fragments, and the other assumes saving energy required from the bee organism, which can be used to support colony functioning.

AUTHOR CONTRIBUTIONS
KO and GB conceived this research and designed experiments; KO, GB, PD, and MT participated in the design and interpretation of the data; GB and PD performed experiments and analysis; KO, GB, PD, and MT wrote the paper and participated in the revisions of it; all authors read and approved the final manuscript.

FUNDING
Financial support for this work was provided by the Ministry of Science and Higher Education ZIB/27/S/2021/ ZIR.

DATA AVAILABILITY
All data generated or analyzed during this study are included in this published article.

DECLARATIONS
Ethics approval No approval of the Research Ethics Committee was required to achieve the goals of this study, as the experimental work involved unregulated invertebrate species (Apis mellifera).

Competing interests The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.