DOI: 10.13102/sociobiology.v60i4.436-440Sociobiology 60(4): 436-440 (2013) Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 Pollen Content in Honey of Apis mellifera Linnaeus (Hymenoptera, Apidae) in an Atlantic Forest Fragment in the Municipality of Piracicaba, São Paulo State, Brazil DFD Araujo1, ACCC Moreti2, TA Silveira1, LC Marchini1, IP Otsuk2 Introduction The population growth of a bee colony depends entirely on the quality and quantity of nectar and pollen sources col- lected by workers, since honey and pollen, a carbohydrate and a protein source, respectively, are essential for the nutrition of larvae and adults of Apis mellifera (L.) (Zerbo et al., 2001). The composition and quality of food sources as well as other factors that affect the colony growth may vary according to the region and year season (Funari et al., 2003; Marchini et al., 2006). To obtain an adequate food storage and high popula- tion growth rate, colonies need to build an optimal food in- ventory. The climatic conditions and food availability in the region directly affects the productive and reproductive char- acteristics of bee colonies. Therefore, food storage (honey and pollen), oviposition and occupation of combs are subject to seasonal variations (Modro et al., 2011). Abstract The productive and reproductive characteristics of Apis mellifera L. bees are directly affected by climatic conditions and food availability in the region where the bees are reared or kept. Therefore, food storage (honey and pollen), oviposition and population growth of these bees are subject to seasonal variations. These variations lead the bees to constantly search for food, making exploratory trips, called “foraging”. This study investigated the botanical origin of nectar sources collected by A. mellifera bees for six consecutive months, from October/2011 to March/2012 in six bee colonies. The study was carried out in the experimental apiary of the Entomology and Acarology Department of the College of Agriculture “Luiz de Queiroz”, from the University of São Paulo, in the municipality of Piracicaba, São Paulo State. The study site has a pre- dominant vegetation of Semideciduous Forest (Atlantic Forest). In each sampling month, we analyzed the pollen types in the honey samples. We used the acetolysis method to prepare the samples for melissopalynology. We carried out the quantitative analysis by successive count of 900 sample grains. The samples were grouped in terms of botanical species, families and/or pollen types. The results show that bees used several plants from the region as a nectar source. However, the Arecaceae, Fabaceae/Mimosoideae and Myrtaceae families were predominant throughout the sampling period. The occur- rence of these plant species was significant and essential for the maintenance of the bee colonies. Sociobiology An international journal on social insects 1 - Universidade de São Paulo, ESALQ /USP, Piracicaba-São Paulo, Brazil. 2 - Secretaria de Agricultura e Abastecimento, Nova Odessa, São Paulo, Brazil. rESEArCH ArTICLE - BEES Article History Edited by Celso F. Martins, UFPB, Brazil received 13 may 2013 Initial acceptance 26 June 2013 Final acceptance 18 October 2013 Keywords Melissopalynology; Nectar; Foraging; Apiculture; Food source. Corresponding author: Diogo Feliciano Dias Araujo Departo. de Entomologia e Acarologia Universidade de São Paulo Campus “Luiz de Queiroz” Avenida Pádua Dias, 11, São Dimas, 13418-900, Piracicaba, São Paulo - SP, Brazil E-Mail: diogofdaraujo@gmail.com The biological characterization of the honey plays an important role in ecological and commercial aspects, contribut- ing to the process for quality control of the honey and even to the standardization to honey products used in food and pharma- ceutical industries (Lins et al., 2005; Neves et al., 2009). Melissopalynology is the science that deals with the morphological characteristics of pollen grains and spores, as well as their dispersion and use (Barth, 1989). It is a very important tool to identify plant species visited by bees. This science branch contributes to research by providing informa- tion on the food source location and allows to identify the range that bees travel to collect food in the year seasons. This study investigated the pollen in honey produced by A. mel- lifera bees during six consecutive months, between October 2011 and March 2012. Sociobiology 60(4): 436-440 (2013) 437 Material and Methods Study site We carried out the experiment at the experimental api- ary of the Laboratory of Beneficial Insects from the Entomol- ogy and Acarology Department of the College of Agriculture “Luiz de Queiroz”, at the University of São Paulo. The re- search facility is located in the municipality of Piracicaba, São Paulo State at 22º 42’ 02” S / 47º 37’ 35.18” O, at 539 meters above sea level. The site has predominant vegetation of Semideciduous Forest (Atlantic Forest). Honey bee colonies We installed six colonies of Africanized bees (A. mel- lifera) in Langstroth hives. Initially, we set up a nest for ovipo- sition and pollen and nectar storage for colony maintenance. During the sampling period, we added supers to collect honey. Ten days before each collection, the supers were added to the colonies for a complete clean and reconstruction of alveolus and later honey storage. Sampling The samples were collected on a monthly basis from October 2011 to March 2012. Afterwards, we added the supers to the nests, each with ten plots, and carried out inspections every15 days. In case there was honey deposited into the su- pers, we removed it, bagged it individually in sterilized plas- tic bags, centrifuged it, stored about 200g in sterile plastic flasks and placed in acclimatized chambers of BOD (Biologic Oxygen Demand) at constant temperature of 20 °C, with the respective identification of each colony. Melissopalynological analyses The melissopalynological analyses of the samples were carried out according to the standard methodology using ace- tolysis (Erdtman, 1952). The identification of the pollen types was based mainly on the reference collection of microscopy blades from the Pollen Database of the Laboratory of Ben- eficial Insects of the Entomology and Acarology Department of College of Agriculture “Luiz de Queiroz”. We also used specialized catalogs of pollen morphology of several floral species (Barth, 1989; Roubik & Moreno, 1991; Carreira et al., 1996; Colinvaux et al., 1999; Moreti et al., 2002; Carreira & Barth, 2003; SANTOS, 2006). The term used for identifica- tion of pollen types is not related to the International Code for Botanical Nomenclature; however, it gives an approxi- mate identification of the samples with an existing taxonomic group (Joosten & Klerk, 2002). The scientific nomenclature used follows the norms proposed by APG II (Angiosperm Phylogeny Group II), according to Souza and Lorenzi (2008), and the nomenclature is in accordance to Tropicos.Org (2011). We identified and counted about 900 pollen grains per sample and each pollen grain was photomicrographed using a Zeiss photomicroscope. After counting the pollen grains, we grouped them ac- cording to the following international criteria: Predominant Pollen (PP) – more than 45% of total pollen grains counted; Accessory Pollen (AP) – from 16 to 45%; Isolate Pollen (IP) – up to 15%, subdivided into: Important Isolate Pollen (IIP): 3 to 15% and Occasional Isolate Pollen (OIP): less than 3% (Louveaux et al., 1978). Data analysis The experimental design was completely randomized with six replications (represented by the colonies) and data were repeated to study the families and pollen types of the plants visited by the bees during the six months of study. After classification, the data were analyzed through the MIXED procedure of the SAS (Statistical Analysis System), SAS Institute (2001) to determine structure of matrix for vari- ance and covariance at 5% probability. The data were con- verted to square root of (X+1) to obtain homocedasticidity in the analysis of variance. Results and Discussion We identified 26 pollen types in the honey samples (Table 1) belonging to 15 botanical families and the families Arecaceae, Fabaceae and Myrtaceae (Figure 1) were the main food sources identified. Comparing our findings to results ob- tained by Modro (2011) and Silveira et al. (2012), carried out in the same region to identify pollen types collected by A. mellifera bees for one year, in our study, we observed a smaller number of pollen types collected by this bee species. The authors identified between 60 and 80 pollen types; however, despite the smaller number of pollen identified, the melisso- palynological analysis in study was carried out in two sea- sons, showing an even greater diversity of botanical species visited by the bees during the experiment. The variation in foraging of the botanical sources used by the bees, regardless the source collected (honey or pollen), can vary according to several factors such as flowering, climate and competition with other bee species (Gonzalez et al., 1995; Villanueva & Roubik 2004; Webby, 2004; Keller et al., 2005). The pollen type from the Arecaceae family was found in the months of November and December of 2011 and January of 2012. However, its occurrence was AP only in November and December of 2011, remained as AP and IP in the other months. This is surprising because species of this family normally have greater occurrence during palynological studies on pollen types collected by the bees. The Arecaceae family shows a high pol- len production rate due to the pollination syndrome occurring in most species (Barfod et al., 2011). However, bees collect DFD Araujo et al - Melissopalynology of A. mellifera in an Atlantic Forest438 nectar from Arecaceae because, in addition to great pollen production, its flowers are used in nectar production (Küch- meister et al., 1997; Mantovani & Morellato, 2000; Venturi- eri, 2008; Kidyoo & McKey, 2012). The Arecaceae species are also important pollen sources for bees. This can contaminate honey and mask the significance of their relative high occurrence in honey sam- ples. Similarly, many Mimosa species provide pollen grains that are identified in the melissopalynological analysis. De- spite its importance for honey characterization, Mimosa is not the main nectar source collected for honey production. This fact may explain the occurrence of pollen types of the Arecaceae family during the palynological studies in honey samples. The Arecaceae family is an important food source for bees in the study area. The pollen types of the Fabaceae family, such as that of Anadenanthera sp., showed greater occurrence in October/2011, and did not show significance in the other months. The Mimosa scabrella type was significant only in October/2011 and December/2011. In the same family, Mimo- sa caesalpinifolia was PP and AP in the months of February and March/2012, respectively, presenting itself as an impor- tant food source for all colonies, given that in February, this species was PP in 83.33% of the colonies, according to the classification of Louveaux et al. (1978). The same condition was found in March. In the Myrtaceae family, we observed two pollen types, Eucalyptus sp. and Myrcia. The Myrcia type presented itself as an important food source for the bees, because this family showed significant occurrence (PP and PA) from October/2011 to January/2012, and can be considered one of the main species used for colony maintenance. Eucalyptus sp. showed occurrence after January/2012 and also considered an important food source for bees during the period. Other studies in the region showed that the Fabaceae/ Mimosoideae and Myrtaceae families are the main food sources for A. mellifera bees, showing the importance of this species for the colonies, both as nectar and pollen sources during certain periods of the year (Modro et al., 2011). We identified species that were relevant as food sources based on the pollen types found, such as plants with potential use in management programs of apiculture in pasture. Despite the occurrence of other pollen types, bees showed intensive collecting activities of these plant species. In a study on geographic location of food sources used by bees, the pollen considered as isolated occurrence can also serve as an important indicative to monitor the site, even showing low relevance in terms of quantity of nectar collected. Conclusion Although poorly used as a nectar source, species from the Arecaceae family are important food sources for bees during some seasons in the year. Species from the families Arecaceae, Fabaceae/Mimo- soideae and Myrtaceae are the most indicated for the imple- mentation or improvement of apiculture pastures in the re- gion. The family Myrtaceae, represented by the pollen type Myrcia, which encompasses the genera Psidium, Eugenia, Myrcia and Myrciaria), show great importance as food source during the productive period of bees in the region. Acknowledgments The authors wish to thank CNPq for granting a schol- arship to the first author. References Barfod, A.S., Hagen, M.,Borchsenius, F. (2011). Twenty-five years of progress in understanding pollination mechanisms in palms (Arecaceae). Ann. Bot., 108(8): 1503-1516. DOI: 10.1093/aob/mcr192. Barth, O.M. (1989). O pólen no mel brasileiro. Rio de Ja- neiro: Gráfica Luxor. Carreira, L.M.M.& Barth, O.M. (2003). Atlas de pólen da vegetação de Canga da Serra dos Carajás, Pará. Belém: Mu- seu Paraense Emilio Goeldi. Carreira, L.M.M., Silva, M.F. da, Lopes, J.R.C., Nasci- mento, L.A.S. (1996). Catalogo de pólen das leguminosas da Amazônia Brasileira. Belém: Museu Paraense Emilio Goeldi. Figure 1 – Representation of the main pollen types found in the hon- ey samples, collected from October/2011 to March/2012 from the Atlantic Forest fragment in the municipality of Piracicaba, São Paulo State, Brazil. (1) Mimosa scabrella Type; (2) Mimosa caesalpinifo- lia; (3) Myrcia Type; (4) Eucalyptus sp.; (5); Anadenanthera sp.; (6) Arecaceae Type. Sociobiology 60(4): 436-440 (2013) 439 Colinvaux, P., Oliveira, P.E. de, Patiño, J.E.M. (1999). Amazon pollen manual and atlas. Amsterdam: Harwood Academic. Erdtman, G. (1952) Pollen morphology and plant taxonomy: Angiosperms. Stockholm: Almqvist & Wiksell. Funari, S.R.C., Rocha, H.C., Sforcin, J.M., Curi, P.R., Funari, A.R.M., Orsi, R. O. (2003) Efeitos da coleta de pólen no de- senvolvimento de colônias e na composição bromatológica de pupas de Apis mellifera L. Arch. Latinoam. Prod. Anim., 11 (2): 80-86. Gonzalez, A., Rowe, C.L., Weeks, P.J., Whittle, D., Gilbert, F.S. Barnard, C.J. (1995). Flower choice by honey bees (Apis mellifera L.): sexphase of flowers and preferences among nectar and pollen foragers. Oecologia, 101: 258-264. Joosten, H. & Klerk, P. (2002) What´s in name?some thoughts on pollen classification, identification, and nomenclature in quaternary palynology. Rev. Palaeobot. Palynol., 122: 29-45. Keller, I., Peter, F.,Imdorf, A. (2005) Pollen nutrition and colony development in honey bees: part I. Bee World, 86 (1): 3-10. Kidyoo, A.M. & McKey, D. (2012) Flowering phenology mimicry on the rattan Calamuscastaneus (Arecaceae) in southern Tailand. Botany, 90: 856-865. DOI:10.1139/B2012- 058 Küchmeister, H.,Silberbauer-Gottsberger, I.,Gottsberger, G. (1997) Flowering, pollination, nectar standing crop, and nec- taries of Euterpe precatoria(Arecaceae), an Amazonian rain forest palm. Pl. Syst. Evol., 206: 71-97. Lins, A.C.S., Silva, T.M.S., Camara, C.A., Dórea, M.C. Santos, F.A.R., Silva, R.A. (2005) Estudo químico, análise palinológica e atividade antiradicalar do pólen apícola (Apis mellifera). Soc. Bras. Quim. http://sec.sbq.org.br/cd29ra/re- sumos/T1270-1.pdf Louveaux, J., Maurizio, A., Vorwohl, G. (1978). Methods of Melissopalynology. Bee World, 59: 139-157. Mantovani, A. & Morellato, L.P.C. (2000) Fenologia da flo- ração, frutificação, mudança foliar e aspectos da biologia floral do palmiteiro. In: Reis, M.S. & Reis, A. (Eds.), Euterpe edulis Martius – (Palmiteiro): biologia, conservação e manejo (pp. 283-301). Itajaí: Herbário Barbosa Rodrigues. Marchini, L.C., Reis, V.D.A. & Moreti, A.C.C.C. (2006) Composição físico-química de amostras de pólen coletado por abelhas africanizadas Apis mellífera (Hymenoptera: Api- dae) em Piracicaba, estado de São Paulo. Cienc. Rural, 36: 949-953. Modro, A.F.H. (2011) Influência do pólen sobre o desenvol- vimento de colônias de abelhas africanizadas (Apis mellifera L.). Tese (Doutorado) - Escola Superior de Agricultura “Luíz de Queiroz”, Universidade de São Paulo. Piracicaba, 2011, 98p. Modro, A.F.H., Message, D., Luz, C.F.P. da., Meira Neto, J.A.A. (2011) Flora de importância polinífera para Apis mel- lifera (L.) na região de Viçosa, MG. Rev. Árvore, 35: 1145- 1153. Moreti, A.C.C.C., Marchini, L.C., Rodrigues, R.R.; Souza, V.C. (2002) Atlas do pólen de plantas apícolas. Rio de Ja- neiro: Papel Virtual. Neves, L.C., Alencar, S.M., Carpes, S.T. (2009) Determina- ção da atividade antioxidante e do teor de compostos fenóli- cos e flavonoides totais em amostras de pólen apícola de Apis mellifera. Braz. J. Food Technol., 8: 107-110. Roubik, D.W., Moreno, J.E.P. (1991) Pollen and spore of barro Colorado Island. St. Louis: Missouri Botanical Garden, (Monographs in Systematics Botany, 36). 268 p. Santos, F.A.R.(Ed.) (2006). Apium plantae. Recife: Imsear. Sas Institute. (2001) SAS/STAT software: changes and en- hancements through release 8.02. Cary, 1167 p. Silveira, T.A.,Correia-Oliveira, M.E.,Moretti, C.C.C.A.,Otsuk, I.P., Marchini, L.M. (2012) Botanical origin of protein sourc- es used by honeybees (Apis mellifera L.) in Atlantic Forest. Sociobiology, 59(4): 1-10. Souza, V.C., Lorenzi, H. (2008) Botânica sistemática: guia ilustrado para identificação das famílias fanerógamas nati- vas e exóticas do Brasil, Baseado em APG II. (2. ed.) Nova Odessa: Intituto Plantarum. Tropicos.Org. (2011) Missouri Botanical Garden. . Venturieri, G. (2008) Floral biology and management of stingless bees to pollinate assai palm (Euterpeoleracea Mart., Arecaceae) in eastern amazon. In: Alvarez, C.A.B; Landeiro, M. Pollinators management in Brazil. Brasília: Ministério do MeioAmbiente, p. 10-13. Villanueva, G.R. & Roubik, D.W. (2004) Why are African honey bees and not European bees invasive? Pollen diet di- versity in community experiments. Apidologie, 35: 481–491. Webby, R. (2004) Floral origin and seasonal variation of bee- collected pollens from individual colonies in New Zealand. J. Apic. Res., 43: 83-92. Zerbo, A.C.,Moraes, R.L.M.S. & Brochetto-Braga, M.R. (2001) Protein requirements in larvae and adults of Scap- totrigona postica (Hymenoptera: Apidia, Meliponinae) midg- utproteolytic activity and pollen digestion. Comp. Biochem. Physiol., 129, p. 139 -147. DFD Araujo et al - Melissopalynology of A. mellifera in an Atlantic Forest440 Table 1. Mean rate (%) of pollen types in honey samples of Apis mellifera collected from fragments of the Atlantic Forest in the municipality of Piracicaba, São Paulo State, Brazil, from October 2011 to March 2012. Means followed by different uppercase letters in columns differ in the Tukey-Kramer test (P<0.05). 1 Means between parenthesis are converted to root (X+1). #statistics referring to converted data. Predominant Pollen (PP); Accessory Pollen (AP); Isolate Pollen (IP), subdivided into: Important Isolate Pollen (IIP) and Occasional Isolate Pollen (OIP). Botany Family/ Pollen type Sampling months Oct. Nov. Dec. Jan. Feb. Mar. Alismataceae/ Alismataceae Type 0,73±2,22 OIP (1.25±0.27)BC Amarantaceae/ Alternanthera ficoidea 4,17±1,82 IIP (2,11±0,22)BCD 0,l7±1,82 OIP (1,07±0,22)C Amarantaceae/ Amarantus sp. 0,40±1,99 OIP (1,16±0,24)C Anacardiaceae/ Anacardiaceae Type 0,20±1,99 OIP (1.08±0.24)CD 0,20±1,99 OIP (1.08±0.24)C Araceae/ Araceae Type 1.00±1,82 OIP (1.38±0.22)BC 16.83±1.82 AP (3.67±0.22)B 8.83±1.82 IIP (2.99±0.22)C Arecaceae/ Arecaceae Type 1.17±1.82 OIP (1.44±0.22)CD 37.83±1.82 AP (6.21±0.22)A 34.33±1.82 AP (5.71±0.22)A 21.67±1.82 AP (4.47±0.22)B 13.83±1.82 IIP (3.78±0.22)BC 12.00±1.82 IIP (3.49±0.22)B Bombacaceae/ Pachira aquatica 0.33±1.82 OIP (1.14±0.22)C 0.17±1.82 OIP (1.07±0.22)C Compositae/ Mikania laevigata 0.17±1.82 OIP (1.07±0.22)E Compositae/ Parthenium sp. 0.17±1.82 OIP (1.07±0.22)C Cruciferae/ Raphanus sp. 0.33±1.82 OIP (1.12±0.22)D 2.67±1.82 IIP (1.58±0.22)D Fabaceae/ Caesalpinia pelthophoroides 0.67±1.82 OIP (1.23±0.22)CD 0.17±1.82 OIP (1.07±0.22)C 0.17±1.82 OIP (1.07±0.22)C Fabaceae / Caesalpinia sp. 4.50±1.82 IIP (2.29±0.22)CD 2.50±1.82 OIP (1.71±0.22)CD 1.83±1.82 OIP (1.59±0.22)BC 0.17±1.82 OIP (1.07±0.22)C Fabaceae/ Cassia sp. 1.83±1.82 OIP (1.62±0.22)CD 0,17±1,82 OIP (1,07±0,22)C Fabaceae/ Centrosema sp. 3.17±1.82 IIP (1.96±0.22)CD 3.50±1.82 IIP (2.10±0.22)BCD 2.67±1.82 OIP (1.89±0.22)BC 0.50±1.82 OIP (1.21±0.22)C Fabaceae/ Anadenanthera sp. 17.50±1.82 AP (3.90±0.22)B 4.50±1.82 IIP (2.31±0.22)BCD 4.17±1.82 IIP (2.23±0.22)BC 3.67±1.82 IIP (2.05±0.22)C 0.17±1.82 OIP (1.07±0.22)D Fabaceae / Leucaena sp. 3.83±1.82 IIP (1.99±0.22)CD 5.67±1.82 IIP (2.53±0.22)BC 2.00±1.82 IIP (1.70±0.22)BC 1.50±1.82 OIP (1.57±0.22)C 0.50±1.82 OIP (1.21±0.22)D Fabaceae / Mimosa caesalpinifolia 1.33±1.82 OIP (1.44±0.22)C 55.00±1.82 PP (7.47±0.22)A 28.50±1.82 AP (5.37±0.22)A Fabaceae / Mimosa scabrella Type 5.00±1.82 IIP (1.96±0.22)CD 10.33±1.82 IIP (2.65±0.22)B 2.50±1.82 OIP (1.72±0.22)C 6.83±1.82 IIP (2.76±0.22)CD 1.67±1.82 OIP (1.53±0.22)D Fabaceae/ Macroptilium sp. 0.33±1.82 OIP (1.14±0.22)C Lamiaceae/ Hyptis sp. 0.67±1.82 OIP (1.26±0.22)D Malvaceae/ Dombeya sp. 0.40±1.99 OIP (1.15±0.24)CD Moraceae/ Morus sp. 0.17±1.82 OIP (1.07±0.22)D 0.17±1.82 OIP (1.07±0.22)D 0.17±1.82 OIP (1.07±0.22)C Myrtaceae/ Myrcia Type 54.17±1.82 PP (7.07±0.22)A 27.50±1.82 AP (5.31±0.22)A 34.67±1.82 AP (5.93±0.22)A 36.33±1.82 AP (6.03±0.22)A 2.00±1.82 OIP (1.68±0.22)DE 1.33±1.82 OIP (1.44±0.22)D Myrtaceae / Eucalyptus sp. 7.83±1.82 IIP (2.80±0.22)BC 9.17±1.82 IIP (3.15±0.22)B 6.83±1.82 IIP (2.72±0.22)B 13.50±1.82 IIP (3.78±0.22)B 21.83±1.82 AP (4.77±0.22)B 42.67±1.82 AP (6.58±0.22)A Rubiaceae/ Rubiaceae Type 0.50±2.22 OIP (1.21±0.27)CD Rutaceae/ Citrus sp. 0.17±1.82 OIP (1.08±0.22)D 0.17±1.82 OIP (1.07±0.22)D 0.50±1.82 OIP (1.19±0.22)C 0.33±1.82 OIP (1.14±0.22)C