{"id":3399,"date":"2019-06-21T20:03:01","date_gmt":"2019-06-21T20:03:01","guid":{"rendered":"http:\/\/beefree.es\/?p=3399"},"modified":"2019-07-17T18:09:43","modified_gmt":"2019-07-17T18:09:43","slug":"comb-construction-in-mixed-species-colonies-of-honeybees-apis-cerana-and-apis-mellifera","status":"publish","type":"post","link":"https:\/\/resistantbees.es\/?p=3399","title":{"rendered":"Comb construction in mixed-species colonies of honeybees, Apis cerana and Apis mellifera"},"content":{"rendered":"

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Ming-Xian Yang<\/span>,\u00a0Ken Tan<\/span>,\u00a0Sarah E. Radloff<\/span>,\u00a0Mananya Phiancharoen<\/span>,\u00a0H. Randall Hepburn<\/span><\/span><\/div>\n
Journal of Experimental Biology\u00a0<\/span>2010\u00a0<\/span>213:\u00a0<\/span>1659-1664;\u00a0<\/span>doi:\u00a010.1242\/jeb.035626<\/span><\/div>\n<\/div>\n
https:\/\/jeb.biologists.org\/content\/213\/10\/1659<\/a><\/div>\n<\/div>\n<\/div>\n<\/div>\n
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SUMMARY<\/h2>\n

Comb building in mixed-species colonies of\u00a0Apis cerana<\/em>\u00a0and\u00a0Apis mellifera<\/em>\u00a0was studied. Two types of cell-size foundation were made from the waxes of these species and inserted into mixed colonies headed either by an\u00a0A. cerana<\/em>\u00a0or an\u00a0A. mellifera<\/em>\u00a0queen. The colonies did not discriminate between the waxes but the\u00a0A. cerana<\/em>\u00a0cell-size foundation was modified during comb building by the workers of both species. In pure\u00a0A. cerana<\/em>\u00a0colonies workers did not accept any foundation but secreted wax and built on foundation in mixed colonies. Comb building is performed by small groups of workers through a mechanism of self-organisation. The two species cooperate in comb building and construct nearly normal combs but they contain many irregular cells. In pure\u00a0A. mellifera<\/em>\u00a0colonies, the\u00a0A. cerana<\/em>\u00a0cell size was modified and the queens were reluctant to lay eggs on such combs. In pure\u00a0A. cerana<\/em>\u00a0colonies, the\u00a0A. mellifera<\/em>\u00a0cell size was built without any modification but these cells were used either for drone brood rearing or for food storing. The principal elements of comb-building behaviour are common to both species, which indicates that they evolved prior to and were conserved after speciation.<\/p>\n

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INTRODUCTION<\/h2>\n

Nest-construction behaviour in social insects is a very complex and highly cooperative phenomenon (Wilson 1971<\/a>;\u00a0Belic et al., 1986<\/a>). In honeybee colonies, the nests result from numerous kinds of operations performed by many individual bees (Hepburn, 1986<\/a>). Although the task of comb construction, like many tasks performed in a honeybee colony, requires concerted actions by many nestmates, individuals are in fact very poorly informed and lack a central controller. Coordination relies on subtle mechanisms combining individual decision rules with specialised signals and informative local cues (Pratt, 2004<\/a>). It has been suggested that the comb building of honeybees can be interpreted as a model of self-organisation (Belic et al., 1986<\/a>;\u00a0Bonabeau et al., 1997<\/a>;\u00a0Hepburn, 1998<\/a>).<\/p>\n

Indeed, regulation of behaviour through self-organisation (Bonabeau et al., 1997<\/a>;\u00a0Boomsma and Franks, 2006<\/a>;\u00a0Detrain and Deneubourg, 2006<\/a>), specifically in honeybee societies, can be used to interpret behaviours including comb construction (Belic et al., 1986<\/a>;\u00a0Hepburn, 1998<\/a>), as well as the arrangement of food storing and brood rearing on the combs (Camazine et al., 1990<\/a>;\u00a0Camazine, 1991<\/a>) and the regulation of food collection behaviour (Jenkins et al., 1992<\/a>).<\/p>\n

Several studies on comb building in\u00a0Apis mellifera<\/em>\u00a0have shown that some very simple building rules (Darchen, 1954<\/a>;\u00a0Hepburn and Whiffler, 1991<\/a>), coupled to the physico-chemical properties of beeswax as a building material (Pirk et al., 2004<\/a>;\u00a0Buchwald et al., 2006<\/a>), can parsimoniously explain several aspects of comb-building behaviour. However, the two sister-species,\u00a0Apis cerana<\/em>\u00a0and\u00a0A. mellifera<\/em>, not only differ in the chemical components of their waxes (Aichholz and Lorbeer, 1999<\/a>) but also have different worker cell sizes [5.2\u20135.4 mm in diameter for\u00a0A. mellifera<\/em>\u00a0(Winston, 1987<\/a>); 4.2\u20134.8 mm in diameter for\u00a0A. cerana<\/em>\u00a0(Ruttner, 1988<\/a>)]. In order to induce the colonies to build combs more quickly and with more regularity, artificial beeswax foundation embossed with the average cell sizes are commonly used for a particular species.<\/p>\n

In other studies of mixed species, it has been shown that\u00a0A. cerana<\/em>\u00a0and\u00a0A. mellifera<\/em>\u00a0can decipher their differing dance languages (Su et al., 2008<\/a>;\u00a0Tan et al., 2008<\/a>) but differ in their thermoregulatory fanning behaviour (Yang et al., 2010b<\/a>). So, mixed colonies offer us a valuable opportunity for the integrative investigation of the relationships of the two species and provide us with a new perspective to study the theories of self-organisation in honeybees as well as in investigations of the evolution of behaviour. Division of labour in mixed-species colonies remains an intriguing issue not previously considered. In this paper we report studies on the comb-construction behaviour of mixed colonies of\u00a0A. cerana<\/em>\u00a0and\u00a0A. mellifera<\/em>\u00a0to answer several questions: (1) will the mixed colonies accept the waxes of both species? (2) Will pure colonies of\u00a0A. cerana<\/em>\u00a0accept\u00a0A. mellifera<\/em>\u00a0wax and\u00a0vice versa<\/em>? (3) Given that the bees are presented with beeswax foundation of different cell base sizes, are these accepted as such or are they modified? (4) Do\u00a0A. cerana<\/em>\u00a0workers and\u00a0A. mellifera<\/em>\u00a0workers cooperate heterospecifically in comb building or do they form separate, conspecific festoons? (5) Under the various conditions above, what cell sizes would result in the newly constructed combs? (6) Once constructed, how are these cells used in the economy of the nest?<\/p>\n

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MATERIALS AND METHODS<\/h2>\n
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Organisation of the mixed colonies<\/h3>\n

Mixed colonies of both\u00a0Apis cerana<\/em>\u00a0L. and\u00a0Apis mellifera<\/em>\u00a0L. workers were established. Three colonies were headed by\u00a0A. cerana<\/em>\u00a0queens; and, reciprocally, three colonies were headed by\u00a0A. mellifera<\/em>\u00a0queens. Sealed brood about to emerge as young adults of each species was placed in the colonies of the other species (Tan et al., 2006<\/a>). Wax-building behaviour was investigated when the newly emerged workers of the two species were about 10\u201318 days old, the peak age of wax secretion (R\u00f6sch, 1927<\/a>;\u00a0Hepburn et al., 1984<\/a>;\u00a0Seeley, 1995<\/a>). Each of three pure\u00a0A. cerana<\/em>\u00a0and\u00a0A. mellifera<\/em>\u00a0colonies with the same age cohort of workers were selected as control groups. The colonies were equalised for size, numbers of combs and adult bees, nectar and pollen stores and brood. This was achieved simply by adding or removing combs.<\/p>\n

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Wax foundation<\/h3>\n

The experiments on the utilisation of the newly built combs in the pure\u00a0A. cerana<\/em>\u00a0and\u00a0A. mellifera<\/em>colonies were done at an apiary of Yunnan Agricultural University, Kunming, China, to refine the final experimental protocol. In these experiments, beeswax was extracted from the combs of both\u00a0A. cerana<\/em>and\u00a0A. mellifera<\/em>\u00a0and then used to make small sheets of beeswax foundation of two worker cell sizes:\u00a0A. cerana<\/em>, about 4.75 mm diameter and\u00a0A. mellifera<\/em>, 5.35 mm diameter using a silicon rubber mould (Hepburn et al., 2009<\/a>). The foundations were about 25 mm long \u00d7 80 mm wide \u00d7 2 mm thick for the\u00a0A. mellifera<\/em>\u00a0queen-headed mixed colonies, and about 12.5 mm long \u00d7 80 mm wide \u00d7 2 mm thick for the pure-species colonies and the\u00a0A. cerana<\/em>\u00a0queen-headed mixed colonies, because they were more concentrated in the middle areas, the foundation widths were cut by half (honeycomb foundation used in commercial beekeeping is made from beeswax by pressing a blank sheet of wax between metal moulds on which hexagons are embossed so as to create a facsimile of natural comb).<\/p>\n

We inserted both\u00a0A. cerana<\/em>\u00a0cell-size (4.75 mm diameter) foundation and\u00a0A. mellifera<\/em>\u00a0cell-size (5.35 mm diameter) foundation into pure\u00a0A. cerana<\/em>\u00a0colonies and pure\u00a0A. mellifera<\/em>\u00a0colonies. The experiments on cell size and wax discrimination, and comb-building cooperation were conducted with colonies of\u00a0A. cerana cerana<\/em>\u00a0and\u00a0A. mellifera ligustica<\/em>\u00a0at an apiary at the Ratchaburi Campus of King Mongkut’s University of Technology Thonburi, Thailand. The same four types of beeswax foundation sheets (two cell sizes and two wax types) were fixed on the top bar of a frame, their relative positions determined by random number assignment. They were then inserted into the centre of the hives.<\/p>\n

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Observations<\/h3>\n

We used a video camera (Sony handycam DCR-DVD803E PAL) and DVD recorder (Sony DVD+RW, DPW60DSS2) to record the comb-building behaviour of the test and control colonies for 10 s intervals three times a day, the videos were taken every day for all of the replicates (Table 1<\/a>). On replay of the video clips, we were able to obtain detailed information on: (1) how many workers of each species were engaged in which type of comb building; (2) how many starting sites were used to extend the building of new combs; (3) whether they formed a mixed-species building chain and cooperated with each other in comb building; (4) how many workers of each species were in each festoon; and (5) when building was complete. When the foundation sheets had been extended beyond their original lengths by the addition of several centimetres of new wax, the combs were removed from the hive and represented one sample for that colony. These combs were replaced by a new top bar with the same four kinds of foundation. The cell sizes and cell patterns were measured.<\/p>\n

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Statistical analyses<\/h3>\n

Chi-square tests were used to test for differences in the numbers of modified cells and patterns of cell orientation between the four types of colonies of\u00a0A. mellifera<\/em>\u00a0and\u00a0A. cerana<\/em>\u00a0queen-headed mixed colonies, and pure\u00a0A. cerana<\/em>\u00a0and\u00a0A. mellifera<\/em>\u00a0colonies. To test for differences in the mean numbers of workers engaged in comb building and the mean cell size of the newly built combs between the four types of colonies, we used analysis of variance (ANOVA) and Tukey\u00a0post-hoc<\/em>\u00a0tests. Homogeneity of the variances between types of colonies was checked using Levene’s test. Paired samples\u00a0t<\/em>-tests were used to compare the mean number and proportions of\u00a0A. cerana<\/em>\u00a0workers with\u00a0A. mellifera<\/em>\u00a0workers in the\u00a0A. mellifera<\/em>\u00a0and\u00a0A. cerana<\/em>\u00a0queen-headed mixed colonies (Johnson and Wichern, 2002<\/a>). The means and standard deviations of each variable were calculated. All tests were performed using Statistica (version 8,\u00a0www.statsoft.com<\/a>; StatSoft).<\/p>\n

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RESULTS<\/h2>\n
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Cell-size and wax discrimination<\/h3>\n

Pure\u00a0A. cerana<\/em>\u00a0colonies ignored all sheets of beeswax foundation and began building new combs either from the top bar or from the lower edges of the foundation sheets (Fig. 1A<\/a>). By contrast, the pure\u00a0A. mellifera<\/em>\u00a0colonies accepted all sheets of both\u00a0A. cerana<\/em>\u00a0and\u00a0A. mellifera<\/em>\u00a0foundation and built cells on both cell sizes (Fig. 1B<\/a>). In the two types of mixed colonies, all the four types of foundations were accepted (Fig. 1C,D<\/a>); workers of both species were seen building cells on the foundations (Fig. 2<\/a>,\u00a0Table 1<\/a>). None of these colonies showed any preference for a particular type of foundation with respect to wax type or cell size (repeated-measures ANOVA:\u00a0P<\/em>>0.05,\u00a0Table 1<\/a>).<\/p>\n

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Table 1.<\/span><\/p>\n

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Mean numbers (\u00b1s.d.) of worker bees engaged in comb building on the four types of foundation<\/p>\n

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Fig. 1.<\/span><\/p>\n

Combs built in the four types of colonies. (A) Pure\u00a0Apis cerana<\/em>; (B) pure\u00a0Apis mellifera<\/em>; (C)\u00a0Apis cerana<\/em>\u00a0queen-headed; and (D)\u00a0Apis mellifera<\/em>\u00a0queen-headed colony (photographed in a horizontal perspective due to different widths). Abbreviations on the top bars are: CC=A. cerana<\/em>\u00a0cell-size foundation made from\u00a0A. cerana<\/em>\u00a0wax; CM=A. cerana<\/em>\u00a0cell-size foundation made from\u00a0A. mellifera<\/em>\u00a0wax; MM=A. mellifera<\/em>\u00a0cell-size foundation made from\u00a0A. mellifera<\/em>\u00a0wax; MC=A. mellifera<\/em>\u00a0cell-size foundation made from\u00a0A. cerana<\/em>\u00a0wax; cell direction patterns of newly built combs, V=vertical, H=horizontal, T=tilted. AC3=an example comb built by a pure\u00a0A. cerana<\/em>\u00a0colony (colony 3). AM1=an example comb built by a pure\u00a0A. mellifera<\/em>\u00a0colony (colony 1). CMX3=an example comb built by a mixed colony\u00a0A. cerana<\/em>\u00a0queen headed (colony 3). MMX2=an example comb built by a mixed colony\u00a0A. mellifera<\/em>\u00a0queen headed (colony 2).<\/p>\n

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Cell-size modification of foundation sheets<\/h3>\n

All of the\u00a0A. mellifera<\/em>\u00a0cell-size sheets of foundation were built to their original size without any modification (Table 2<\/a>) but the\u00a0A. cerana<\/em>\u00a0cell-size foundation sheets were modified in all colonies except the pure\u00a0A. cerana<\/em>\u00a0colonies. Some of these cells were squeezed to make space for enlarging neighbouring cells. The percentages of combs that had modified cells in the test and control groups are shown in\u00a0Table 2<\/a>. In\u00a0A. mellifera<\/em>\u00a0queen-headed mixed colonies, all of the\u00a0A. cerana<\/em>\u00a0foundation sheets were modified and nearly all in the pure\u00a0A. mellifera<\/em>\u00a0colonies, which is significantly different from the\u00a0A. cerana<\/em>\u00a0queen-headed mixed colonies and pure\u00a0A. cerana<\/em>\u00a0colonies (Chi-square: \u03c72<\/sup>3<\/sub>=71.7,\u00a0P<\/em><0.001).<\/p>\n

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Freely built combs<\/h3>\n

On completion of the trials of comb building on the artificial foundation sheets, the workers from the four types of colonies were observed to start building new combs at several sites (Table 3<\/a>). Pure\u00a0A. mellifera<\/em>\u00a0colonies and\u00a0A. mellifera<\/em>\u00a0queen-headed mixed colonies had significantly more festoons at new comb-building sites than\u00a0A. cerana<\/em>\u00a0and pure\u00a0A. cerana<\/em>\u00a0queen-headed colonies (ANOVA:\u00a0F<\/em>3,44<\/sub>=15.9,\u00a0P<\/em><0.001;\u00a0Table 3<\/a>). In\u00a0A. cerana<\/em>\u00a0queen-headed mixed colonies, workers of both species were seen working together in festoons, although significantly more\u00a0A. mellifera<\/em>\u00a0workers were involved (42.1\u00b16.2%\u00a0A. cerana<\/em>\u00a0workers, 57.9\u00b16.2%\u00a0A. mellifera<\/em>\u00a0workers; paired\u00a0t<\/em>-test:\u00a0t<\/em>13<\/sub>=4.9,\u00a0P<\/em><0.001). Similarly, in\u00a0A. mellifera<\/em>\u00a0queen-headed mixed colonies, significantly more\u00a0A. mellifera<\/em>\u00a0workers than\u00a0A. cerana<\/em>\u00a0workers were engaged in comb building in the festoons (32.5\u00b14.8%\u00a0A. cerana<\/em>\u00a0workers, 67.5\u00b14.8%,\u00a0A. mellifera<\/em>\u00a0workers; paired\u00a0t<\/em>-test:\u00a0t<\/em>9<\/sub>=9.8,\u00a0P<\/em><0.001;\u00a0Table 3<\/a>). In total, significantly more workers were engaged in comb building in the mixed colonies than in the pure\u00a0A. cerana<\/em>\u00a0and pure\u00a0A. mellifera<\/em>\u00a0colonies (ANOVA:\u00a0F<\/em>3,44<\/sub>=11.3,\u00a0P<\/em><0.001;\u00a0Table 3<\/a>).<\/p>\n

As for irregular cells on the new combs, pure\u00a0A. cerana<\/em>\u00a0and pure\u00a0A. mellifera<\/em>\u00a0colonies built significantly fewer irregular cells (0.8% and 2.7%, respectively), than did the mixed colonies (9.1% and 10.8%, respectively), most of which were located at the seams of combs which had been started at different sites (ANOVA:\u00a0F<\/em>3,44<\/sub>=30.0,\u00a0P<\/em>=0.003;\u00a0Table 3<\/a>). The\u00a0A. cerana<\/em>\u00a0queen-headed mixed colonies showed significantly greater variation in the patterns of cell orientation on the newly built combs than\u00a0A. mellifera<\/em>\u00a0queen-headed colonies, pure\u00a0A. cerana<\/em>\u00a0and\u00a0A. mellifera<\/em>\u00a0colonies; different festoons on one comb may build patterns different from others (Chi-squared: \u03c72<\/sup>6<\/sub>=27.9,\u00a0P<\/em><0.001;\u00a0Fig. 1C<\/a>;\u00a0Table 3<\/a>).\u00a0Apis mellifera<\/em>\u00a0queen-headed colonies built new combs mainly in vertical and horizontal patterns (Fig. 1D<\/a>); in pure\u00a0A. cerana<\/em>\u00a0and\u00a0A. mellifera<\/em>\u00a0colonies, the patterns of cell orientation were more homogeneous and mainly vertical (Figs 1A,B<\/a>;\u00a0Table 3<\/a>).<\/p>\n

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