DOI: 10.13102/sociobiology.v61i1.60-67Sociobiology 61(1): 60-67 (March, 2014) Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 Colony performance of Melipona quadrifasciata (Hymenoptera, Meliponina) in a Green- house of Lycopersicon esculentum (Solanaceae) BF Bartelli, AOR Santos, FH Nogueira-Ferreira Introduction Worldwide, there are approximately 20,000 described bee species (Michener, 2007). Among them, the stingless bees are highly social organisms that constitute an important group due to the ecological and economic roles they play. These bees belong to the subtribe Meliponina, which consists of approximately 400 species grouped in about 50 genera, dis- tributed across tropical and subtropical regions, in America, Southeast Asia, Africa, Madagascar and Australia (Silveira et al., 2002). Stingless bees are responsible for the pollination of many native (Michener, 2007) and cultivated (Heard, 1999) plant species. As a result, since the 1990s, the number of studies involving the introduction of these bees in greenhouses to evaluate their pollina- tion efficiency on different crops has grown steadily. Particularly noteworthy among these are: eggplant pollination, using Melipona quadrifasciata Lepeletier, 1836 (Bispo dos Santos, 2008); basil pollination, tested with Nannotrigona testaceicornis Lepeletier, Abstract The use of stingless bees in greenhouses has provided tremendous benefits to diverse crops in terms of productivity and fruit quality. However, knowledge about management techniques in these environments is still scarce. The present study aimed to evaluate colony performance of Melipona quadrifasciata Lepeletier, 1836 in a greenhouse of Lycopersicon esculentum Mill. and its potential use in pollinating this crop. Six nests of M. quadrifasciata were introduced in a green- house in Araguari, Minas Gerais state, Brazil. The development of colonies inside the greenhouse was investigated and the foraging behavior of the workers was assessed before introduction, into the greenhouse and after the nests had been removed from the greenhouse. The vital activi- ties of colony maintenance were performed unevenly throughout the day inside and outside the greenhouse, but with confinement the daily period of foraging decreased and bees started col- lecting pollen from the flowers after approximately six months. The difficulty in orienting to and identifying flowers by the workers was attributed to sunlight diffusion and blockage of ultraviolet radiation caused by the cover on the greenhouse. Structural changes in the greenhouses, as well as improvements in management techniques, are required for better utilization of stingless bees for pollination of plant species grown in greenhouses. Sociobiology An international journal on social insects Universidade Federal de Uberlândia, Instituto de Biologia, Uberlândia, Minas Gerais, Brazil Article History Edited by: Celso F Martins, UFPB, Brazil Received 01 July 2013 Initial Acceptance 27 August 2013 Final Acceptance 29 October 2013 Keywords stingless bees, management of bees, external activity, resource collection Corresponding Author Fernanda Helena Nogueira-Ferreira Universidade Federal de Uberlândia Instituto de Biologia Ceará St., Umuarama Uberlândia-MG, Brazil, 38400-902 Phone: (34) 3218-2243 Fax: (34) 3218-2243 E-Mail: ferferre@inbio.ufu.br 1836 (Bispo dos Santos, 2008); strawberry pollination, tested with Tetragonisca angustula Latreille, 1811 (Malagodi-Braga, 2002), Scaptotrigona aff. depilis Moure, 1942 (Roselino et al., 2009) and N. testaceicornis (Roselino et al., 2009); sweet pepper pollination, using M. subnitida Ducke, 1910 (Cruz et al., 2005), M. quadrifasciata anthidioides Lepeletier, 1836 (Roselino et al., 2010) and M. scutellaris Latreille, 1811 (Roselino et al., 2010); and tomato pollination, tested with N. perilampoides Cresson, 1878 (Cauich et al., 2004; Palma et al., 2008b) and M. quadrifasciata (Del Sarto et al., 2005; Bispo dos Santos et al., 2009). Tomato, Lycopersicon esculentum Mill. (Solanaceae), is one of the most widespread vegetable crops in the world, being cultivated in almost all parts of the world under dif- ferent cropping systems and various levels of management. It is a self-fertilizing plant and its flowers are bisexual, do not produce nectar and present poricidal anthers. Therefore, in order to release the pollen, vibration of the anthers with consequent opening thereof is necessary (Buchmann, 1983). Pollination can be performed by the shaking of the anthers RESEARCH ARTICLE - BEES Sociobiology 61(1): 60-67 (March, 2014) 61 by the wind, but cross-pollination is ensured through visits of bees that exhibit vibratory behavior or buzz-pollination (Buchmann & Hurley, 1978; Heard, 1999; Nunes-Silva et al., 2010). Tomato can be grown in open areas or in greenhouses. When grown in open areas, the released pollen is carried by the wind (McGregor, 1976; Free, 1993) and/or natural pol- linators, especially bees, which have free access to the flowers. As for cultivation in greenhouses, pollination is normally per- formed by the mechanical method of vibrating the flowers, to compensate for the absence of wind and natural pollinators. However, studies involving the management of pollinators in greenhouses have indicated improvements in productivity and fruit quality (Cruz & Campos, 2009). Among the pollina- tors of tomato in greenhouses, M. quadrifasciata proved to be efficient in pollinating the long-lived variety (Del Sarto et al., 2005), but studies testing colony performance and pollination efficiency of this bee on the grape variety, grown mostly in greenhouses, are yet to be performed. Stingless bees are considered particularly promising for use as commercial pollinators (Cruz & Campos, 2009), given that they do not present a functional sting, are easy to handle (usually low aggressive-nonstinging), have populous and pere- nnial nests, present a marked worker recruitment behavior and stock a large amount of food (Heard, 1999; Malagodi-Braga et al., 2004). Nevertheless, the lack of techniques for management and multiplication of nests has hindered the availability and use of these bees in agriculture on a large-scale (Imperatriz- Fonseca et al., 2006). The present study sought to advance knowledge about management of stingless bees in greenhouses for use as com- mercial pollinators. We have specifically evaluated colony performance of M. quadrifasciata in a greenhouse of grape tomato, L. esculentum. Material and Methods Study area The study was performed at the “Meliponário da Universidade Federal de Uberlândia (UFU)”, located at the “Fazenda Experimental do Glória” (FEG) (18°56’57”S - 48°12’14”'W), Uberlândia, Minas Gerais state, Brazil, and in the “Chácara Paraíso” (CP) (18°39’3.55”S - 48°11’7.51”W), located in the municipality of Araguari, Minas Gerais state, Brazil. At FEG, agricultural and cattle raising activities are developed, but preserved fragments of cerrado and semide- ciduous forest can be found. CP consists of areas of cultiva- tion and pasture, fragments of cerrado and 12 greenhouses where grape tomatoes are grown. One of these was used for the experiments. The climate of the regions of Uberlândia and Araguari is marked by two distinct seasons, the rainy season that ex- tends from October to March and a dry season from April to September. Annual rainfall varies between 1,160 and 1,460 mm/year and the average annual temperature is between 23 and 25°C, being uniform throughout the year (Alves & Rosa, 2008). The greenhouse used in the experiment comprised ap- proximately 1,344 m2 (48 m x 28 m), being covered at the top with an Extra Long Life (ELL) Diffuser Antivirus plas- tic diffuser and fully enclosed on the sides with anti-aphid screens (Fig 1A). The greenhouse presented 24 planting rows and each of these had an average of 112 tomato plants, adding up 2,688 plants. Introduction of nests of M. quadrifasciata Six nests of M. quadrifasciata with similar population sizes were introduced at the onset of flowering, in March 2012. The nests were kept in wooden boxes with an approximate size of 40x25x25cm (Fig 1B). Before introduction, due to the difficulty in bee orientation inside the greenhouse (Bartelli, personal communication; Del Sarto, 2005), the population of old foragers was removed to avoid their loss in the enclosed space, following the methodology used by Cauich et al. (2004). Moreover, for the same reason, the nests were placed in the greenhouse after dark (Cuypers, 1968). In order to allow a homogeneous distribution of the bees on the flowers, nests were arranged in the central region of the greenhouse (Free, 1993) and supported by plastic boxes installed in eucalyptus logs located within the planting rows. To increase the number of reference points for the bees, since the uniformity of greenhouses may hinder orientation (Dyer, 1994), the entrance to each nest was painted with different color patterns. Containers with water, mud and cerumen (alternative source for plant resin) of T. angustula were placed on the plas- tic boxes (Fig 1C). Nests were sporadically fed with A. mel- lifera pollen macerated with sugar and water until the onset of flower visitation, and fed weekly with syrup (a mixture of honey of A. mellifera, sugar and water in the ratio 1:1:1) over the entire period of confinement. For pesticide application, a common management practice, the entrances of all nests were sealed with paper, the nests themselves were protected with plastic bags and the containers covered with cardboard boxes. These protections were removed only after the pesticide had dried; then, the entrances of the nests were unobstructed after dark. Furthermore, to increase luminosity inside the green- house, another common management practice, the ceiling and sides of the greenhouse were washed with soap and water in late August. In order to facilitate bee orientation, one mercury va- pour lamp and two mixed lamps (alternative sources of light and ultraviolet radiation) were installed inside the greenhouse in one of the corridors where some of the nests were placed (Fig 1D). The lamps were connected to a digital timer, which was programmed to switch them on at 6 am and switch them BF Bartelli et al. - Colony performance of M. quadrifasciata in Greenhouse of L. esculentum62 off at 6 pm. They remained in the greenhouse from early June to mid-July. The lamps’ efficiency, as well as the influence of the diffuser film on solar radiation focused on the greenhouse, was evaluated by analysing the pattern of reflectance of the flowers. 10 flowers were photographed between 10 and 11 am in a white background with a Sony Cyber-shot DSC-H20 dig- ital camera. Each flower had its picture taken in three different circumstances: inside the greenhouse and next to the lamps; inside the greenhouse and 12m far from the lamps; and out- side the greenhouse. Photoshop software CS3 10.0 was used to quantify patterns of chromatic (RGB - red, green and blue in the light spectrum) and achromatic (luminosity) saturation of flower corollas. Colony performance of M. quadrifasciata To assess colony performance, the internal conditions of nests, behavior of workers in flight and on flowers, foraging ac- tivities and temperature inside and outside the greenhouse were analyzed. Figure 2 outlines the arrangement of nests inside the greenhouse. Nests D and F remained in the greenhouse for three months and then they were relocated to the outside area. Internal conditions Biweekly, from March to October 2012, we assessed qualitative features of nests, such as the presence of cells in construction (yes / no), number of workers (very low / low / medium / high) and presence of guards (yes / no). Behavior of workers Biweekly, from May to October 2012, the flight behavior of bees and the behavior of workers visiting tomato flowers were observed inside the greenhouse. These behaviors were assessed every 15 minutes during each hour (from 6 am to 6 pm). Foraging activities Foraging activities of bees were assessed through direct observations of the flow of workers from nests. The quantity and quality of resources (pollen, nectar/water or water, resin, mud and garbage) that entered and/or left the nests were re- corded every 10 minutes during each hour (from 6 am to 6 pm). In order to compare the daily pattern of resource collection of workers inside and outside the greenhouse, the procedures described above were performed: before introduction in three nests (B, D and F, Fig 2), over three non-consecutive days in late February 2012 at FEG; inside the greenhouse in two nests (C and E, Fig 2), biweekly from May to October 2012; and outside the greenhouse in one (nest F, Fig 2) of the nests removed from the greenhouse at CP, biweekly from June to October 2012. Temperature Through the use of a digital thermohygrometer, the temperature inside and outside the greenhouse was measured every day at 8 am, 11 am and 3 pm from April to October 2012. Statistical analyses To verify whether the patterns of reflectance of tomato flowers depended on the presence of the lamps and diffuser film or not, we conducted an analysis of variance (ANOVA) followed by Tukey's test, since there was no difference be- tween blocks (flowers) for RGB (F = 1.118; df = 9; P = 0.399) and luminosity (F = 1.037; df = 9; P = 0.449) (Zar, 2010). In order to evaluate the daily pattern of resource collection of bees inside and outside the greenhouse, circular analyses were performed using Oriana 4.1 software (Kovach, 2011), using the Rayleigh test for the calculation of probabilities of distri- bution of bees throughout the day. To investigate whether the temperature differed inside and outside the greenhouse during the months the nests were confined, we conducted a paired t test (Zar, 2010). Fig 1. A) Greenhouse used in the experiment at Chácara Paraíso in Araguari; B) Nest of Melipona quadrifasciata installed inside the greenhouse; C) Containers with water, mud and cerumen available inside the greenhouse; D) Lamps installed inside the greenhouse. Fig 2. Diagram representing the arrangement of nests inside and out- side the greenhouse. Sociobiology 61(1): 60-67 (March, 2014) 63 Results Internal conditions Two nests (A and B, Fig 2) did not survive the con- ditions of confinement and died after about two months. To avoid their loss, after three months of confinement, two other nests (D and F, Fig 2) were removed from the greenhouse and placed outside due to the absence of cells in construction, the low number of workers and the absence of guard workers. One of these nests (F, Fig 2) survived and increased the number of workers to “high” after two months, but the other died (D, Fig 2), attacked by phorids (Diptera). Nests C and E (Fig 2) survived the conditions of confinement and remained in the greenhouse during the entire experiment, from March to Octo- ber 2012, with cells in construction, guard workers and a high number of workers. Behavior of workers Inside the greenhouse, flight activities of bees began af- ter 22 days of confinement. However, workers were limited only to the removal of garbage from the colonies and many of them clashed against the ceiling or sides of the greenhouse, where they remained and ended up dying. This flight behav- ior toward the ceiling and sides of the greenhouse decreased over time during confinement but did not cease, even after the beginning of collection activities in the flowers. The beginning of pollen collection from the tomato flowers occurred in late July, but there was only one record during that period and none in the following month. Intensive visits of workers to flowers (six records of pollen collection per nest) occurred in early September, after nearly six months of confinement, and the ceiling and sides of the greenhouse had been washed with soap and water. Bees landed on the anthers of a flower and bowed around or at the apex of the anthers cone to grab it (Fig 3A). Thus, they transmitted vibra- tions to the anthers through their thorax and legs to release the pollen. For the transfer of pollen from the body to the corbicula, some bees remained stuck to the base of the anthers cone by the legs and/or jaws, while others performed the transfer during flight or on other parts of the plant, such as the leaves and fruits (Fig 3B). Pattern of reflectance of the flowers The lamps installed inside the greenhouse did not alter the flight behavior of the bees and did not influence the RGB and luminosity of tomato flower corollas. However, the pat- terns of chromatic (F = 10.51; df = 2; P< 0.001) and achromatic (F = 8.44; df = 2; P = 0.001) saturation of the flowers were sig- nificantly different inside and outside the greenhouse (Fig 4). Foraging activities Before introduction, at Fazenda Experimental do Glória (FEG), the bees began to forage at around 5:40 am. The number of forager workers coming in and out of nests was not uniform throughout the day and the period of highest activity occurred at 6-8 am. The relative frequency of collection per resource varied considerably between the nests. Of the workers observed, 7.7-18.2% transported pollen, 17.0-81.4% nectar/ water, 3.6-55.7% resin and 0.2-11.1% mud. The resources were not obtained uniformly throughout the day. Pollen col- lection occurred only in the morning, with a peak at 6-8 am. The highest mean frequency observed for nectar/water oc- curred between 6 and 7 am and the collection of this resource gradually decreased throughout the day. The peaks of collec- tion of resin and mud were, respectively, at 8-10 am and at 8-11 am (Fig 5, Table 1). Fig 3. A) Melipona quadrifasciata worker collecting pollen in tomato flower; B) M. quadrifasciata worker transferring pollen from the body to corbicula on a fruit. Fig 4. Mean values (± standard error) of patterns of chromatic (or RGB - A) and achromatic (or luminosity - B) saturation of tomato flowers in the different treatments (Near: inside the greenhouse and next to the lamps; Far: inside the greenhouse and far from the lamps; and Outside: outside the greenhouse) at Chácara Paraíso in Araguari. Distinct letters indicate significant differences between treatments. BF Bartelli et al. - Colony performance of M. quadrifasciata in Greenhouse of L. esculentum64 Inside the greenhouse, the activity of bees began ap- proximately at 7 am and was not uniform throughout the day, with the greatest movement of workers occurring between 5 and 6 pm for nest C, and between 12 and 1 pm for nest E. The relative frequency of collection per resource varied between the nests. From the workers observed over the entire period of confinement, 14.8-21.4% transported pollen, 37.5-60.7% water and 7.8-23.3% resin (cerumen). There was no collec- tion of mud. Workers obtained pollen and water in a hetero- geneous way throughout the day and there was no defined peak of resin collection. Pollen collection was limited to the morning, with a peak between 8 and 9 am. The highest mean frequency observed for water occurred between 5 and 6 pm (Fig 5, Table 1). After the nests were removed from the greenhouse and placed in the outer area, it was possible to observe work- ers carrying pollen in their corbiculas on the following day. The movement of bees was not uniform throughout the day and peak activity occurred between 6 and 7 am. Of workers observed over the months, 31.4% carried pollen, 57.5% nec- tar/water and 7.0% resin. The resources were obtained in a heterogeneous way throughout the day, except resin. Pollen collection, limited again to the morning, and nectar/water col- lection had peaks between 7 and 8 am, and between 6 and 7 am, respectively. There was no collection of mud (Fig 5, Table 1). Temperature The temperature inside the greenhouse was significantly higher over the entire period of confinement of the nests of M. quadrifasciata (t = 6.99; df = 6; P< 0.001; Fig 6). Discussion The results of our experiments showed that acclimation of Melipona quadrifasciata to conditions inside greenhouses are colony-dependent and, besides foraging activities varied a little inside and outside the greenhouse, the daily period of foraging into the greenhouse decreased and bees took a long time to visit flowers consistently for pollen collection. The foraging behavior of stingless bees is related both to factors intrinsic to the nest, including the ability to com- municate and population size, and extrinsic factors, such as the abundance and distribution of resources in the environment and susceptibility to abiotic factors (Fidalgo & Kleinert, 2007). However, despite the methodological differences used in the introduction of the nests and the different conditions of con- finement (size and structure of the greenhouses, crop type, etc), our results about the foraging behavior of M. quadrifasciata were similar to results found in other studies that evaluated the intro- duction of stingless bees in greenhouses (Del Sarto et al., 2005; Nunes-Silva et al., 2013). The workers concentrated pollen collection in the morning. Inside the greenhouse, the foraging behavior of M. quadrifasciata showed little variation compared to the out- side. However, stronger patterns (represented by smaller probability values, Table 1) in the external activities of bees were observed at FEG, before confinement. Additionally, the daily period of foraging in the greenhouse decreased, as well as for N. perilampoides (Cauich et al., 2004; Palma et al., 2008a), and bees visited flowers consistently for pollen col- lection only after approximately six months of confinement. The time required for acclimation to protected environments Table 1. Mean time vector (with the number of observations (n) and probability values (p), according to Rayleigh test) for resources collec- tion and total external activity throughout the day by Melipona quadrifasciata at Fazenda Experimental do Glória (FEG), in Uberlândia, and inside and outside the greenhouse at Chácara Paraíso, in Araguari. Resource FEG Inside Outside Nest B Nest D Nest F Nest C Nest E Nest F Pollen 7:56 am (n = 31) (P < 0.001) 6:51 am (n = 60) (P < 0.001) 6:44 am (n = 67) (P < 0.001) 7:40 am (n = 3) (P = 0.036) 8:10 am (n = 6) (P < 0.001) 7:29 am (n = 10) (P < 0.001) Nectar/water or Water 7:18 am (n = 329) (P < 0.001) 7:44 am (n = 79) (P < 0.001) 7:52 am (n = 117) (P < 0.001) 4:00 pm (n = 3) (P = 0.042) 2:45 pm (n = 6) (P = 0.011) 7:33 am (n = 19) (P < 0.001) Resin 10:41 am (n = 15) (P < 0.001) 9:50 am (n = 259) (P < 0.001) 9:39 am (n = 136) (P < 0.001) ----- 11:30 am (n = 2) (P = 0.144) 6:30 am (n = 2) (P = 0.144) Mud 9:00 am (n = 1) (P = 0.512) 9:42 am (n = 50) (P < 0.001) 10:38 am (n = 23) (P < 0.001) ----- ----- ----- Total external activity 7:32 am (n = 801) (P < 0.001) 9:00 am (n = 910) (P < 0.001) 8:36 am (n = 719) (P < 0.001) 11:43 am (n = 17) (P < 0.001) 11:28 am (n = 39) (P < 0.001) 7:45 am (n = 62) (P < 0.001) (-----) Insufficient data for analysis or resource not collected Sociobiology 61(1): 60-67 (March, 2014) 65 varies both between species and between colonies of the same species (Malagodi-Braga, 2002). However, this timing was probably determined by the presence of the diffuser film on the coverage of the greenhouse. As evidence of this, pollen collection observed in nests placed in the outer area started immediately after their removal from the greenhouse. Due to the dispersant effect of plastic films used in greenhouses coverage, solar radiation is one of many environ- mental factors that can be changed by using protected crops (Schwengber et al., 1996). This could be evidenced via the different reflectance patterns found on tomato flowers inside and outside the studied greenhouse. This dispersant effect is favorable to plants, since the fraction of diffuse solar radia- tion is more effective for photosynthesis (Farias et al., 1993), but may have hindered bee orientation and identification of flowers by worker bees, affecting foraging activities inside the greenhouse and thereby delaying the beginning of pollen collection. In relation to the external environment, global solar ra- diation (measured by luminous flux density) and diffuse solar radiation (multidirectional) are respectively lower and higher inside the greenhouse as a result of reflection and absorption by the material of the plastic coverage. In turn, this reflection and absorption are determined, for example, by the conditions of coverage at the time of use and dust deposition (Farias et al., 1993). This explains why visits to flowers by M. quadri- fasciata workers intensified when the ceiling and sides of the greenhouse were washed with soap and water as part of the mana-gement of the protected tomato crop. For orientation, bees use elements like the sun, polarized light, visual cues present in the environment and ultraviolet ra- diation (Kerr, 1973; Dyer, 1994; Briscoe & Chittka, 2001). The presence of clouds and fog cause dispersion of light rays and reduces the polarization signal (Shashar & Cronin, 1998). Thus, greenhouse cover can produce a similar effect to that promoted by clouds and restrict bee activity to a period in which light rays are less dispersed by the cover (Malagodi- Braga, 2002). Besides the dispersant effect, the film ELL Diffuser Antivirus eliminates the entry of ultraviolet (UV) to hinder the vision of tomato pest insects (Electro Plastic, 2013). This could also have been an aggravating factor hindering the orientation of M. quadrifasciata forager workers inside the greenhouse. Although this species present a wide distribu- tion throughout Brazil (Camargo & Pedro, 2012), foraging in places relatively poor in UV, such as under the canopies of dense tropical forests (Briscoe & Chittka, 2001), the nests were in an open environment at FEG and were accustomed to high UV exposure before introduction to the greenhouse. In temperate areas, where bumble bees are largely used for greenhouse tomato pollination, showing high efficiency (Banda & Paxton, 1991; Morandin et al., 2001), the struc- ture of the greenhouses are similar to those used in this study. However, in the case of bumblebees, it seems that the reduc- tion in UV light can be compensated and as a result their visit to flowers is not affected by the type of greenhouse coverage (Dyer & Chittka, 2004). In the present study, as in Del Sarto et al. (2005), the performance of M. quadrifasciata to conditions of confine- Fig 5. Mean number of workers for each resource collected through- out the day by Melipona quadrifasciata before introduction (A), at Fazenda Experimental do Glória, in Uberlândia, and inside (B) and outside (C) the greenhouse at Chácara Paraíso, in Araguari. Fig 6. Mean values (± standard error) of temperature (°C) inside and outside (environment) the greenhouse during the period of con- finement of nests of Melipona quadrifasciata at Chácara Paraíso in Araguari. Distinct letters indicate significant differences for each month. BF Bartelli et al. - Colony performance of M. quadrifasciata in Greenhouse of L. esculentum66 ment proved to be colony-dependent. Studies have shown that stingless are tolerant to high temperatures and capable of cooling the inside of their colony by ventilation generated by beating their wings in the nest entrance. However, we believe that the high temperatures inside the greenhouse may have been an aggravating factor for colony development, since the intense heat decreases the density of larval food, causing the eggs to sink and death of larvae by drowning (Amano et al., 2000). From results obtained in this study and considering the benefits that the use of stingless bees has provided to diverse crops grown in greenhouses (Cruz & Campos, 2009), the con- trol of temperature and humidity inside greenhouses would be an important step. Another idea would be to exchange the plastic greenhouse coverings for materials that interfere less significantly in solar radiation, or, alternatively, a less drastic alternative would be to intercalate the plastic cover with such materials, allowing at least partial diffusion of solar radiation to occur in the greenhouse. For the farmer, taking into account the duration of the cycle of grape tomato in greenhouses, which is eight months, using stingless bees is not practical if these bees require a great deal of time to acclimate and initiate foraging activities. However, studies asses-sing the cost-benefit relationship of such structural changes in greenhouses must still be made and more information that will permit improve- ments in management techniques for stingless bees in green- houses is needed. Acknowledgements The authors are grateful to Conselho Nacional de Desen- volvimento Científico e Tecnológico (CNPq), to Fundação de Amparo à Pesquisa do estado de Minas Gerais (FAPEMIG) and to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support, to the farmers Edson, Clóvis and João, who allowed and gave full support to this study, to Dr. Paulo Eugênio Alves Macedo de Oliveira for sugges- tions given to this study and to the biologists Isabel Farias Aidar and Jaqueline Eterna Batista for contributions in the field. References Alves, K.A. & Rosa, R. (2008). Espacialização de dados climáticos do Cerrado mineiro. Horizonte Científico, 8: 1-28. Amano, K., Nemoto, T. & Heard, T. (2000). What are sting- less bees and why and how to use them as crop pollinators? A review. Japan Agricultural Research Quarterly, 34: 183-190. Banda, H.J. & Paxton, R.J.(1991). Pollination of greenhouse tomatoes by bees. Acta Horticulturae, 288: 194-198. Bispo dos Santos, S.A. (2008). Polinização em culturas de manjericão, Ocimum basilicum L. (Lamiaceae), berinjela, Solanum melongena L. (Solanaceae) e tomate Lycopersicon esculentum (Solanaceae) por espécies de abelhas sem ferrão (Hymenoptera, Apidae, Meliponini). Dissertation (MSc in Entomology), Universidade de São Paulo, Ribeirão Preto. Bispo dos Santos, S.A., Roselino, A.C., Hrncir, M. & Bego, L.R. (2009). Pollination of tomatoes by the stingless bee Melipona quadrifasciata and the honey bee Apis mellifera (Hymenoptera, Apidae). Genetics and Molecular Research, 8: 751-757. Briscoe, A.D. & Chittka, L. (2001).The evolution of color vi- sion in insects. Annual Review of Entomology, 46: 471-510. Buchmann, S.L. (1983). Buzz pollination in angiosperms. In: Jones, C.E. & Little, R.J. (Eds.), Handbook of Experimental Pollination Biology (pp. 73-113). New York: Scientific and Academic Editions. Buchmann, S.L. & Hurley, J.P. (1978). A biophysical model for buzz pollination in angiosperms. Journal of Theoretical Biology, 72: 639-657. doi: 10.1016/0022-5193(78)90277-1. Camargo, J. M. F. & Pedro, S. R. M. (2012). Meliponini Lepe- letier, 1836. In: Moure, J. S., Urban, D. & Melo, G. A. R. (Eds.), Catalogue of bees (Hymenoptera, Apoideae) in the neotropical region – online version (Available in: http://www.moure.cria. org.br/catalogue). Cauich, O., Quezada-Euán, J. J. G., Macias-Macias, J. O., Reyes-Oregel, V., Medina-Peralta, S. & Parra-Tabla, V. (2004). Behavior and pollination efficiency of Nannotrigona perilampoides (Hymenoptera: Meliponini) on greenhouse tomatoes (Lycopersicon esculentum) in subtropical México. Journal of Economic Entomology, 97: 475-481. Cruz, D.O. & Campos, L.A.O. (2009). Polinização por abelhas em cultivos protegidos. Revista Brasileira de Agrociência, 15: 5-10. Cruz, D.O., Freitas, B.M., Silva, L.A., Silva, E.M.S. & Bom- fim, I.G.A. (2005). Pollination efficiency of the stingless bee Melipona subnitida on greenhouse sweet pepper. Pesquisa Agropecuária Brasileira, 40: 1197-1201. Cuypers, J. (1968). Using honeybees for pollinating crops under glass. Bee World, 49: 72-76. Del Sarto, M.C.L. (2005). Avaliação de Melipona quadrifasciata Lepeletier (Hymenoptera: Apidae) como polinizador da cul- tura do tomateiro em cultivo protegido. Dissertation (MSc in Entomology), Universidade Federal de Viçosa, Viçosa. Del Sarto, M.C.L., Peruquetti, R.C. & Campos, L.A. O. (2005). Evaluation of the neotropical stingless bee Melipona quadrifasciata (Hymenoptera: Apidae) as pollinator of green- house tomatoes. Journal of Economic Entomology, 98: 260- 266. doi: 10.1603/0022-0493-98.2.260. Dyer, F.C. (1994). Spatial cognition and navigation in insects. In: Real, L.A. (Ed.), Behavioral mechanisms in evolutionary ecology (pp. 66-98). Chicago: University of Chicago Press. Sociobiology 61(1): 60-67 (March, 2014) 67 Dyer, A.G. & Chittka, L.(2004). Bumblebee search timewith- out ultraviolet light. Journal of Experimental Biology, 207: 1683-1688. doi:10.1242/jeb.00941. Electro Plastic. (2013). E.L.V – Difusor Antivírus. Available at: Farias, J.R.B., Bergamaschi, H., Martins, S.R. & Berlato, M.A. (1993). Efeito da cobertura plástica de estufa sobre a radiação solar. Revista Brasileira de Agrometeorologia, 1: 31- 36. Fidalgo, A.O. & Kleinert, A.M.P. (2007). Foraging behavior of Melipona rufiventris Lepeletier (Apinae, Meliponini) in Ubatuba/SP, Brazil. Brazilian Journal of Biology, 67: 137- 144. Free, J.B. (1993). Insect pollination of crops. San Diego: Aca- demic. Heard, T.A. (1999). The role of stingless bees in crop pol- lination. Annual Review of Entomology, 44: 183-206. doi: 10.1146/annurev.ento.44.1.183. Imperatriz-Fonseca, V.L., Saraiva, A.M., De Jong, D. (2006). In- formation technology and pollinators iniciatives. In: Imperatriz- Fonseca, V.L., Saraiva, A.M. & De Jong, D. (Eds.), Bees as pollinators in Brazil: assessing the status and suggesting best practices (pp. 20-20). Ribeirão Preto: Holos Editora. Kerr, W.E. (1973). Sun compass orientation in the stingless bees Trigona (Trigona) spinipes (Fabricius, 1793) (Apidae). Anais da Academia Brasileira de Ciências, 45: 301-308. Kovach, W.L. (2011). Oriana – Circular Statistics for Windows, ver. 4. Kovach Computing Services, Pentraeth, Wales, U.K. Malagodi-Braga, K.S. (2002). Estudo de agentes poliniza- dores em cultura de morango (Fragaria x ananassa Duchesne – Rosaceae). Thesis (Doctorate in Sciences - Ecology Area), Universidade de São Paulo, São Paulo. Malagodi-Braga, K.S., Kleinert, A.M.P. & Imperatriz-Fonseca, V.L. (2004). Abelhas sem ferrão e polinização. Revista Tecnologia e Ambiente, 10: 59-70. McGregor, S.E. (1976). Insect pollination of cultivated crop plants. Washington: United States Department of Agriculture. Michener, C.D. (2007). The bees of the world. Baltimore: The Johns Hopkins University Press. Morandin, L.A., Laverty, T.M. & Kevan, P.G. (2001). Bumble bee (Hymenoptera-Apidae) activity and pollination levels in commer- cial tomato greenhouses. Journal of Economic Entomology, 94: 462-467. Nunes-Silva, P., Hrncir, M. & Imperatriz-Fonseca, V.L. (2010). A polinização por vibração. Oecologia Australis, 14: 140-151. doi: 10.4257/oeco.2010.1401.07. Nunes-Silva, P., Hrncir, M., Silva, C.I., Roldão, Y.S. & Imperatriz- Fonseca, V.L. (2013). Stingless bees, Melipona fasciculata, as efficient pollinators of egg plant (Solanum melongena) in greenhouses. Apidologie, 44: 537-546. doi: 10.1007/s13592- 013-0204-y. Palma, G., Quezada-Euán, J.J.G., Meléndez-Ramirez, V., Irig- oyen, J., Valdovinos-Nuñez, G.R. & Rejón. M. (2008a). Com- parative Efficiency of Nannotrigona perilampoides, Bombus impatiens (Hymenoptera: Apoidea), and Mechanical Vibration on Fruit Production of Enclosed Habanero Pepper. Journal of Economic Entomology, 101: 132-138. doi: 10.1603/0022- 0493(2008)101[132:CEONPB]2.0.CO;2. Palma, G., Quezada-Euán, J.J.G., Reyes-Oregel, V., Meléndez, V. & Moo-Valle, H. (2008b). Production of greenhouse to- matoes (Lycopersicon esculentum) using Nannotrigona perilampoides, Bombus impatiens and mechanical vibration (Hym.:Apoidea). Journal of Applied Entomology, 132: 79-85. Roselino, A.C., Santos, S.B., Hrncir, M. & Bego, L.R. (2009). Differences between the quality of strawberries (Fragaria x ananassa) pollinated by the stingless bees Scaptotrigona aff. depilis and Nannotrigona testaceicornis. Genetics and Molecular Research, 8: 539-545. Roselino, A.C., Bispo dos Santos, S.A. & Bego, L.R. (2010). Qualidade dos frutos de pimentão (Capsicum annuum L.) a partir de flores polinizadas por abelhas sem ferrão (Melipona quadrifasciata anthidioides Lepeletier 1836 e Melipona scutellaris Latreille 1811) sob cultivo protegido. Revista Bra- sileira de Biociências, 8: 154-158. Schwengber, F.E., Peil, R.M.N., Martins, S.R. & Assis, F.N. (1996). Comportamento de duas cultivares de morangueiro em estufa plástica em Pelotas – RS. Horticultura Brasileira, 14: 143-147. Shashar, N. & Cronin, T.W. (1998).The polarization of light in a Tropical Rain Forest. Biotropica, 30: 275-285. Silveira, F.A., Melo, G.A.R. & Almeida, E.A.B. (2002). Abelhas brasileiras: sistemática e identificação. Belo Horizonte: Fundação Araucária. Zar, J.H. (2010). Biostatistical Analysis - Fifth Edition. New Jersey: Prentice-Hall/Pearson.