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bicolor schencki (Apidae; Meliponini) castes in brood
combs in southern Brazil
Ney Jr, Betina Blochtein, José Serrão
To cite this version:
Seasonal production and spatial distribution
of
Melipona bicolor schencki (Apidae; Meliponini) castes
in brood combs in southern Brazil
Ney Telles FERREIRA JR1,Betina BLOCHTEIN1,José Eduardo SERRÃO2
1Laboratório de Entomologia, Faculdade de Biociências, Pontifícia Universidade Católica do Rio Grande do Sul,
90619-900 Porto Alegre, RS, Brazil
2
Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brazil
Received 23 May 2012– Revised 3 September 2012 – Accepted 23 September 2012
Abstract– Melipona bicolor schencki is currently considered a threatened stingless bee in southern Brazil, and studies about its ecology may provide new insights to enable its conservation. This study evaluated the seasonal variability in the sex/caste proportion, numbers of combs and brood cells, and spatial distribution of the individuals present in emerging combs in five polygynous colonies of M. bicolor schencki for 1 year. Throughout the year, the colonies showed similar variations. Individual combs exhibited a higher number of brood cells in spring and summer, although there was no variation in the number of combs over the four seasons. Workers (79.7 %), queens (15.6 %), and drones (4.7 %) were produced in all months sampled. Analysis of spatial distribution indicated no clear predominance of a specific sex or caste in a specific comb area, contrary to other species of Melipona.
Melipona / larvae / stingless bee / queen / male
1. INTRODUCTION
Stingless bees (Meliponini) are widespread in tropical regions, predominantly in the subtrop-ics in the Southern Hemisphere (Michener
2000). Species of Melipona Illiger, 1806, as well as other genera of stingless bees, build nests with horizontal brood combs (Michener
1974; Cepeda2006; Roubik2006), which have offspring in different developmental stages (Kerr et al. 2001). The production of brood combs by stingless bees may be affected by the available space inside the nests (Sakagami
1982), environmental conditions (Ribeiro et al.
2003; Borges and Blochtein 2006), and food availability (Winston 2003).
The diameter of brood combs from Meliponini can be used to calculate the number of brood cells. Thus, intraspecific differences in the production of brood cells can be determined, which allows for the classification of colonies from the same environment. Colonies can be classified as weak, median, or strong for those with a few, intermediate, or high number of brood cells, respectively (Kleinert-Giovannini and Imperatriz-Fonseca 1986; Hilário et al.
2000; Kleinert 2005; Fidalgo and Kleinert
2007). However, the seasonal variations in the number of brood cells in Meliponini nests are poorly studied (Pick and Blochtein 2002; Borges and Blochtein 2006).
In Melipona bicolor bicolor Lepeletier, 1836, the production of brood cells was shown to be
Corresponding author: B. Blochtein,
betinabl@pucrs.br; J.E. Serrão, jeserrao@ufv.br Manuscript editor: James Nieh
higher at 30–32 °C than at lower temperatures (Velthuis et al. 1999). In southern Brazil, colonies of Melipona marginata obscurior Moure, 1971, showed the highest number of combs and brood cells during the spring and summer (Borges and Blochtein 2006). In M. bicolor, which has permanent polygynous col-onies (Kerr 1949; Silva et al. 1972; Nogueira-Neto1997; Velthuis et al.2001; Cepeda2006), the production of brood cells is also influenced by the number of physogastric queens (Velthuis et al.1999).
In contrast to other stingless bees, in the nests of Melipona species, queens, workers, and males develop until the adult stage in brood cells of the same size (Kerr 1950; Kerr et al.
1966; Engels and Imperatriz-Fonseca 1990; Nogueira-Neto 1997; Roubik 2006; Menezes et al. 2007). M. bicolor produces a similar number of queens and males, whereas Plebeia remota (Holmberg 1903) and Schwarziana quadripunctata (Lepeletier 1836), which are monogynic species with brood cells for queen production that are larger than those for pro-duction of males and workers, have a lower proportion of queens compared with males (Santos-Filho et al.2006).
Seasonal changes may affect the number of queens, workers, and males in the colonies. Melipona beecheii Bennett, 1831, produces a higher number of males in the spring, followed by a gradual decrease until summer. In Melipona favosa (Fabricius 1798) colonies, the production of males increases in autumn (Van Veen et al.1997) or summer (Sommeijer et al.
2003). The population of queens in the combs of M. favosa in field conditions was similar throughout the year (Sommeijer et al.2003).
In species of Melipona, there may be variation in the ratios of queens, males, and workers in the combs. In brood combs of Melipona subnitida Ducke 1910, the workers represent 91.8 % of the offspring, while queens and males make up 6.96 % and 1.24 %, respectively (Koedam et al. 1999). In Melipona trinitalis Cockerell 1919, the propor-tion of workers was shown to be 87.6 %, queens 4.8 %, and males 7.7 % (Sommeijer et al.
2003). In M. favosa, brood combs were found to have 78.4 % workers, 5.1 % queens, and 17.3 % males (Koedam1999; Sommeijer et al.2003).
Unlike other species of stingless bees that build brood cells for the production of queens in the periphery of the brood combs (Nogueira-Neto
1997), in species of Melipona, the distribution of brood cells for queens, workers, and males in the combs cannot be estimated because there is no apparent differentiation of size of the brood cells. However, in M. favosa, brood cells containing queens are more frequently placed in the peripheral region of the comb, although they can also be found in the center, while males are predominantly displayed in the center of the comb (Koedam1999). In M. subnitida, males are grouped in the central area of the combs (Koedam et al. 1999). Similarly, in combs of M. beecheii, males are more often found in the central and medium areas, whereas queens are randomly distributed, and the workers are more frequently at the edges of the comb (Moo-Valle et al. 2004).
Melipona bicolor schencki Gribodo, 1893, is a stingless bee threatened in southern Brazil (Blochtein and Harter-Marques 2003; IBAMA
2003). This study evaluated the seasonal brood production, contributing to the management, and conservation of this bee.
2. MATERIALS AND METHODS 2.1. Area
The study was carried out at the Research and Nature Conservation Center Pró-Mata of PUCRS ("Pro-Forest") (29°27’S/29°S, 50°08’W/50°15’W, 900 m above sea level) in São Francisco de Paula, state of Rio Grande do Sul, Brazil, with predominant vegetation of Araucaria Forest. The climate is humid to super-humid, with 1,750–2,500 mm annual rainfall (Bertoletti and Teixeira1995).
2.2. Bee colonies
wooden boxes, with external sizes of 26×28×37 cm, covered by transparent glass lids. Colonies were maintained in a room at 25±2 °C. Each colony had access to the external environment by means of a plastic tube (15 mm in diameter) that crossed the room wall. When necessary, colonies were fed on 50 % sucrose solution (Nogueira-Neto1997).
2.3. Sampling of combs and seasonality
The wax covering the combs (involucrum) of each colony was removed to access the brood combs. The number of combs was counted monthly, and one mature brood comb was removed for analyses of offspring. This procedure was performed monthly from November, 2006 to October, 2007, and the results were analyzed according to the four seasons.
2.4. Determination of the number of workers, queens, and males in the brood combs
The quantities and spatial distribution of brood cells containing workers, queens, and males were analyzed one comb removed from colony, according to the procedures described by Koedam (2003). Briefly, the operculum of each brood cell was opened with the aid of a cutting blade, and it was possible to determine workers, queens, and males according to the head morphology of the pupae. The combs were then replaced into their respective nests, and the brood cells were sealed by workers. The total number of individual representatives of each sex and caste was estimated on basis of the number of each individual in the examined brood comb multiplied by total number of brood combs in each colony.
Taking into account the 12 months sampled in all colonies, the mean number of combs in each colony as well as the number of mature brood cells in the combs of each colony, the results showed normal distribution and homogeneity of variances, which were submitted to Tukey’s test at 5 % significance level.
Estimates of the total number of brood cells per colony per month, calculated from the number of brood cells and combs, were submitted to one-way ANOVA, with month being the explanatory variable and the number of brood cell per colony the response variable.
The statistical similarity found allowed the clus-tering of the of the monthly data obtained from the five colonies as well as a clustering according the seasonal production (spring–summer and autumn– winter) of the number of combs and brood cells. The number of combs and brood cells in spring–summer and autumn–winter periods were compared by the Student’s t test at 5 % significance level.
2.5. Seasonal quantification of workers, queens, and males in mature brood combs
The presence of workers, queens, and males in each comb was estimated from the percentage of individuals and the total number of brood cells per comb, for a total of 11,317 brood cells. The data set was submitted to the Kolmogorov–Smirnov test. To evaluate the homogeneity and normal distribution and because both conditions were not obtained, sex per caste per month dataset were showed as median, first and third quartiles, and standard error of the median. Otherwise the percentage of workers, queens, and males per colony, along the entire period, had normal distribution and were submitted to the Tukey`s test at 5 % significance level to test whether the percentage of sex and castes change according the year period.
2.6. Proportion of workers, queens, and males in the brood combs
homogeneous and normal distribution. Worker and male data showed non-normal distribution and were submitted to Kruskal–Wallis test, and queen data, with normal distribution, submitted to Tukey's test, both at 5 % significance level.
2.7. Spatial distribution of workers, queens, and males in the combs
The spatial distribution of brood cells containing workers, queens, and males was analyzed in each colony. The combs were divided into three concentric areas (central, medium, and edge portions) with the same number of cells in each area. While there were clear differences in the pupal development in the central portion, this portion was defined by the older pupae. The three portions of combs from each colony were compared with regard to the percentage of workers, queens, and males. Since samples were from three paired groups with normal distribution (Kolmogorov–Smirnov test), data were submitted to the Friedman test at 5 % significance level to test whether queens, males, and workers are preferentially produced in the different areas of the brood combs. Data with were analyzed descriptively by median, minimum, and maximum values for the representa-tive offspring in the different comb areas.
3. RESULTS
3.1. Number of combs and brood cells The number of brood combs (Tukey's test, P00.23) and brood cells (Tukey's test, P00.38) were similar in the five colonies throughout the year (Figure 1a, b). The estimated number of brood cells per colony, obtained from the number of brood cells and combs sampled monthly, showed no significant difference in the total production of brood cells among the colonies (Tukey's test, P00.7).
The number of brood cells in the combs of colonies was higher in spring and summer, decreased during autumn, and reached a mini-mum in winter (Figure2). Despite the seasonal differences found in the number of brood cells, the number of combs sampled was similar during the spring–summer and autumn–winter
periods (Student's t test P00.09) (Figure 3). However, comparatively, the number of brood cells in mature combs during spring and summer periods was higher than in autumn and winter (Student's t test P<0.001) (Figure4).
3.2. Seasonal quantification of workers, queens, and males in mature combs Throughout the period studied, we found variations in the production of workers, queens, and males in the five colonies. Although the number of workers (Tukey's test, P00.65) and queens (Tukey's test, P00.83) was similar
between the five colonies, the number of males was more numerous in colony 5 (Tukey's test, P00.001) than in the other colonies (Figure5a–c). During the 12 months studied, the produc-tion of workers in all colonies gradually increased from November to April, with a peak of 260 individuals, followed by a decrease in subsequent months with lowest number of workers in August (Figure 6a).
Queens were found in the combs in all months, with 64 individuals in March and 70 in April (Figure 6b). The months of reduced production of queens were November, August,
September, and October with five, seven, four, and five queens, respectively.
The evaluation of the five colonies together showed that males, as well as queens, were present in samples from all months but in smaller quantities than workers (Figure 6c).
3.3. Proportion of workers, queens, and males in mature combs
In the four seasons throughout the year, the estimated proportions of workers were highest, followed by the queens, while the proportions of males were lowest (TableI).
Figure 4. Number (mean and standard error) of brood cells per brood comb sampled in five colonies of M. bicolor schencki in two different periods of year. São Francisco de Paula, RS, Brazil.
Figure 3. Number (mean and standard error) of brood combs and in five colonies of M. bicolor schencki in two different periods of year. São Francisco de Paula, RS, Brazil.
Variations in the percentage of workers, queens, and males in brood combs of the five colonies occurred over the 12 months (Figure7a–e). The
workers had the highest percentage of individuals in all months, always above 50 %. The queens were present in all months analyzed, except in
colony 3 in September. In May, the queens represented 25 % of all offspring sampled in the colony 4. In colonies 1–4, male individuals were only present in a few months. In contrast, in colony 5, males were present in all months evaluated in the highest proportion com-pared with the other colonies, comprising 35.7 % of the estimated brood of this colony in August. The comparison of sexes and castes ratios among colonies in the seasons showed similar proportions for workers (Kruskal–Wallis' test, P 00.32), queens (Tukey's test, P00.13), and males (Kruskal–Wallis' test, P00.43).
The analysis of the percentages of indi-viduals showed that colony 5 differed from the other colonies (Tukey's test, P00.001) in that it exhibited a lower percentage of workers, while the percentage of males was higher (Kruskal–Wallis' test, P<0.001) in relation to the other colonies. The percentage of queens was similar between all five colonies.
3.4. Spatial distribution of workers, queens, and males in mature combs In the five colonies, the workers were the predominant individuals in all three areas examined in the combs. In colonies 1–4, the median number of queens was higher than males in all three areas. Only colony 5 differed from this pattern, with a greater number of males than queens (Table II).
In colonies 1, 2, 3, and 5, the three areas of the comb in each individual colony had similar proportions of workers, queens, and males (Friedman's test, P00.32). Only colony 4 showed differences in the distribution of individ-uals. Queens predominated in the central portion
Figure 6. Boxplot of estimated number of workers (a), queens (b), and males (c) of M. bicolor schencki on basis of the percentage of each caste per sex and the total number of brood cells in a brood comb per colony per month in five colonies from November, 2006 to October, 2007. Horizontal lines 0 median; bars 0 standard error; boxes 0 first and third quartiles; asterisks 0 highest values sampled. São Francisco de Paula, RS, Brazil.
of the combs (Friedman's test, P00.01), while workers and males were equally distributed in the three areas.
4. DISCUSSION
The colonies were similar in the number of brood combs sampled during the 12 months as well as between the seasons spring–summer and autumn–winter. The variations in the number of brood cells during spring–summer and autumn– winter periods were similar in all five colonies. These data differ from those found for other species of Melipona (Lacerda et al. 1991; Kleinert-Giovannini and Imperatriz-Fonseca
1986; Hilário et al. 2000; Kleinert 2005). Although the number of brood cells per comb was 47 % in the autumn–winter in comparison to the spring–summer, the number of combs was similar in the colonies. This finding corroborates the hypothesis that even with a small number of brood cells the horizontal arrays of brood combs play a role in the thermoregulation process with adult workers accommodating the spaces between combs (Roubik2006).
Despite the adverse environmental conditions in autumn–winter, which are characteristic of Southern Brazil, and the reduction in the number of brood cells, M. bicolor schencki has no reproductive diapauses. This corroborates data from Freitas et al. (2006) in which the colonies of this species reduce their forager activities but
maintain reproduction, contrary to findings for M. marginata obscurior, which in the same geographic region, has facultative diapauses yet under controlled laboratory conditions (Borges and Blochtein 2006). Colonies of M. bicolor schencki were kept in a climatized room, which may have interfered with offspring productivity taking into account that the thermoregulation effort was reduced. However, external conditions during the autumn–winter period, especially the decrease in food resources, daily photophase, and temperature, are unfavorable for bee foraging, resulting in reduction in the number of brood cells in the combs. The number of brood cells produced is affected by the temperature inside the colony, with low number of offspring in low temperatures (Velthuis et al.1999). In this sense, the energetic cost for thermoregulation and decreased metabolic rates of insects at low temperatures (Kitazawa et al.1963; Danielviskii
1965; Tauber et al.1986; Xu et al.1995) results in a reduction of investment in brood production, because the laying rate depends on both the queen and workers tasks, such as brood cell building and provisioning.
The number of pupae workers in the combs increased from November to April, when there was a peak of these individuals. This period corresponds to late spring until early autumn and is closely related to the period when food resources are abundant and bees are stimulated to produce more workers for foraging of food and nest materials. From May to August, the number of workers produced was gradually reduced. This period corresponds to mid-autumn to mid-winter, in which the environ-mental conditions including food resources are adverse, suggesting that bees use their reserves in order to produce the minimal number of workers just to maintain the colony. From September, the number of workers in the combs began to increase, and in October, there was an equivalent quantity of workers in the combs compared with the first sampling month (November 2006). This suggests that the pro-cess of production of workers is cyclical, with peaks of high and low production associated with seasons.
Table I. Estimated seasonal proportion (%) of sexes and castes in a brood comb per month in five colonies of M. bicolor schencki from November, 2006 to October, 2007, São Francisco de Paula, RS, Brazil.
Season Worker Queen Male
Spring 83.1 13 3.9
Summer 79.7 15.1 5.2
Autaunm 75.3 20.9 3.8
Winter 80.6 13.6 5.8
The number of queens varied differently than the number of workers, in that queens were
produced in all months of the year, and in March and April, queens were present in high
numbers in the combs. For males, March was the month of highest occurrence, which corresponds to the late summer and early autumn. These findings are similar to that found for M. favosa (Van Veen et al.1997), with higher occurrence of males in autumn. Since in Melipona species males leave their nests when they are 18.6 days old (Van Veen et al.1997), males of M. bicolor schencki that emerged in March would already be reproductively mature when queens emerged in March and April. Because both queens and males are found throughout the 12 months in combs of M. bicolor schencki, we suggest that mating may occur in any season. However, the high rate of males and queens in autumn may be a response of M. bicolor schencki to adverse environmental conditions for this season in southern Brazil, suggesting a mitigation mecha-nism for the mating risk by increasing production of reproductive individuals.
Considering all the colonies evaluated in the 12 months of research, M. bicolor schencki workers, queens and males represent 79.7 %, 15.6 %, and 4.7 % of the offspring, respectively, similar to other Melipona species with a higher rate of workers in the combs (Koedam 1999; Koedam et al. 1999; Sommeijer et al. 2003). However, the number of queens was higher than males in M. bicolor schencki, corroborating the findings for M. subnitida but contrary to the findings for M. trinitalis and M. favosa with a higher proportion of males than queens (Sommeijer et al.2003). In M. bicolor schencki, there is no seasonal effect in the pattern of sex and caste ratio, with workers more frequent and higher numbers of queens than males in all seasons (see Table I), as well as there no variations according the colony evaluated. These findings suggests that the similarity in the proportionality patterns between queens and
Table II. Percentage of brood cells with workers (W), queens (Q), and males (M) in the center, middle, and border regions from brood combs in five colonies of M. bicolor schencki from November, 2006 to October, 2007, São Francisco de Paula, RS, Brazil.
Colony (combs) (pupae)
Center region Middle region Border region
males is an intrinsic feature of the species not affected by environmental conditions, as is found in colonies of M. favosa in The Netherlands (Koedam 1999) and Trinidad and Tobago (Sommeijer et al.2003). All together, these data suggest that Melipona species maintain their proportions of sex and caste independent of environmental conditions. Alternatively, our food supplementation to enhance colony survival during cold periods may have affected brood production and contributed to our finding no seasonal effects.
In M. bicolor schencki colonies, the spatial distribution of castes and sex is similar between the three comb areas analyzed, such as found in M. beecheii (Moo-Valley et al. 2004). In Melipona species, haploid eggs for males are usually laid in the central portion of the combs (Kerr 1950). Melipona quadrifasciata and M. favosa exhibit male concentration at the center of combs and queens at the edges (Koedam1999). The intriguing finding in colony 4 of a higher concentration of queens in the center of combs may be due to sample size in this nest, since the quantity of queens in the combs of this colony were low in comparison to other colonies. The result is that each individual has great percentage representation during the analysis.
M. bicolor schencki exhibits facultative po-lygyny, unique among species of Melipona (Kerr 1949; Silva et al. 1972; Nogueira-Neto
1997; Velthuis et al. 2001; Cepeda 2006). In some observations, we found three physogastric queens in the same comb, but it remains unknown whether the simultaneous occurrence of physogastric queens may play a role in the patterns of spatial distribution of sex and castes. In conclusion, M. bicolor schencki produces a higher number of brood cells in the spring and summer and constant number of combs and production of workers, queens and males throughout the year.
ACKNOWLEDGMENTS
This research was supported by Brazilian Research Agencies National Council of Research (CNPq).
Production saisonnière et distribution spatiale des castes dans les rayons de couvain deMelipona bicolor schencki (Apidae; Meliponini), dans le sud du Brésil Melipona / larve / abeille sans aiguillon / reine / mâle Jahreszeitabhängige Produktion und räumliche Ver-teilung der Kasten auf Brutwaben der Stachellosen BieneMelipona bicolor schencki (Apidae; Meliponini) in Südbrasilien
Melipona / Larven / Stachellosen Biene / Königin / Männchen
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