Free access
Issue
Apidologie
Volume 41, Number 4, July-August 2010
Page(s) 454 - 462
DOI http://dx.doi.org/10.1051/apido/2009082
Published online 14 January 2010

© INRA/DIB-AGIB/EDP Sciences, 2010

1. INTRODUCTION

The meliponini is a group of approximately 400 species of stingless bees that exhibit highly social behavior (Kerr et al., 1996; Velthuis, 1997; Michener, 2000). The stingless bees have a wide geographical distribution, the neotropical region is where most of the species occur (Camargo and Pedro, 1992). In Brazil, approximately 300 species were recorded (Kerr et al., 1999), being found in various ecosystems, such as the Atlantic Forest (Ramalho, 2003), Amazon Forest (Brown, 2001; Brown and Albrecht, 2001), Pantanal and Cerrado areas (Mittermeier et al., 2005).

The stingless bees play an important role in maintaining the ecological balance among several species of angiosperms and fauna of various ecosystems. (Janzen, 1980). According to the ecosystem, it is estimated that approximately 40 to 90% of angiosperms are dependent on this group of bees (Kerr et al., 1996). Some species of small size are responsible for the pollination of many species of angiosperms with small flowers (Nogueira-Neto et al., 1986; Nogueira-Neto, 1997; Coletto-Silva, 2005).

Tetragonisca angustula, popularly known in Brazil as ``Jataí'' is a meliponini bee that is characterized by its rusticity and important ecological role. It has been maintained in meliponiculture due to the excellent quality of honey and propolis produced (Miorin et al., 2003) and is also used in pollination of many cultivars (Heard, 1999).

Morphologically, based on the mesepisternum color (thoracic region), T. angustula is divided into two subspecies: T. angustula angustula and T. angustula fiebrigi characterized by the black and yellow mesepisternum, respectively (Schwarz, 1938). Both subspecies have different geographical distribution. While T. a. angustula is widely distributed in the Americas, ranging from Mexico to Argentina, T. a. fiebrigi is restricted to certain regions of Brazil (São Paulo, Paraná, western state of Santa Catarina), part of Argentina and Paraguay (Nogueira-Neto, 1970).

On the other hand, Castanheira and Contel (1995) found variation in the mesepisternum color of T. angustula samples collected in the states of Paraná and São Paulo. This variation suggested that the variability in the mesepisternum coloration is controlled by several genes and that the cross between individuals of basic color (black and yellow) may result in offspring with some degree of variation in their colors.

thumbnail Figure 1

Map of part South America showing Santa Catarina state (Brazil) with the municipalities where Tetragonisca angustula subspecies were sampled: Apiúna (1) Ascurra (2) Benedito Novo (3) Blumenau (4) Dona Emma (5) Indaial (6) Ituporaga (7) Lontras (8) Luiz Alves (9) Nova Trento (10) Pomerode (11) Pouso Redendo (12) Presidente Getúlio (13) Rio do Sul (14) Rio dos Cedros (15) Timbó (16) Trombudo Central (17) Vidal Ramos (18) Belmonte (21) Caçador (22) Caibi (23) Campos Novos (24) Iporã do Oeste (25) Iraceminha (26) Itapiranga (27) Joaçaba (28) Pinhalzinho (29) Santa Helena (30) São Domingos (31) São José do Cedro (32) São Miguel do Oeste (33) Treze Tilhas (34) Videira (35) Arvoredo (36) Águas Frias (37) Quilombo (38) Xaxim (39). Samples 1 to 18 = Vale do Itajaí, Samples 21 to 39 = Western.

Few molecular studies have been conducted in this species of bees. Castanheira and Contel (2005) found significant correlation between the mesepisternum color, with the hexokinase allele HK88 being found with great frequency in more yellow bees, indicating that it is an allele that has its origin in T. a. fiebrigi. Oliveira et al. (2004) using RAPD molecular markers, distinguished two T. angustula groups based in its natural geographic distribution. However, although this molecular marker showed great genetic variability among populations, it was not able to separate T. a. angustula and T. a. fiebrigi subspecies (Baitala et al., 2006). Therefore, this study was an effort to obtain genetic markers that could differentiate those two subspecies. Characterization of T. a. angustula and T. a. fiebrigi is here reported through the analysis of a mitochondrial DNA fragment (mtDNA) using the PCR + RFLP method.

2. MATERIAL AND METHODS

2.1. Worker samples

Worker bees from 210 T. angustula feral colonies, were collected from tree-trunks, stone walls and in rustic hives from two regions of Santa Catarina state. Individuals were collected from 138 colonies distributed among 18 municipalities from the Vale do Itajaí region (central to northern part of the state), and 72 colonies in 19 municipalities from the middle and far west of the state (Fig. 1 and Tab. I). Each sample was placed in 70% ethanol and stored at –20 °C.

Table I

Number of colonies (N) of Tetragonisca angustula sampled, their geographic coordinates origin in the Santa Catarina state and individual subspecies identification based on morphological and molecular analysis.

2.2. Morphological analysis

Each worker bee from every colony sampled, was morphologically separated into subspecies using the mesepisternum coloration classes from Castanheira and Contel (1995). Two to ten individuals were analyzed for each colony, resulting in 1368 bees from the Vale do Itajaí region and 482 bees from he Western region of Santa Catarina. Each collected bee was individually observed under a stereomicroscope and, according to a graded color scale ranging from black to yellow mesepisternum, were classified into following five classes: black (1), black to intermediate (2), intermediate (3), intermediate to yellow (4) and yellow (5). Individuals belonging to classes 1 and 2 and classes (4 and 5) were separated into T. a. angustula, and T. a. fiebrigi, respectively, while workers in class 3 were considered as intermediate and not included in either subspecies.

2.3. Molecular analysis

2.3.1. DNA extractions

The mtDNA analysis (PCR+RFLP) was conducted with one individual per colony. Total DNA was extracted from a leg of each T. angustula, using the rapid extraction method adapted by Anderson and Fuchs (1998). Each T. angustula worker bee, after being washed in 70% ethanol, was placed in a Petri dish and an anterior leg was removed with the aid of forceps. After dissection, the leg was transferred into a microcentrifuge tube containing 40 μL of 2 × lysis buffer (120 mg/mL Proteinase K, 0.1M KCl, 0.02M Tris-HCl pH 8.3, 5 mM MgCl2, 0.9% Tween 20, 0.9% NP40 and 0.02% of gelatin). The tube and contents were incubated first at 65 °C for 30 min, then at 97 °C for 10 minutes and then diluted into 20 μL of dH2O. The extracted DNA was stored at 20 °C for later use.

2.3.2. PCR+RFLP analysis

Mitochondrial ATPases 8, 6 and COIII genes were amplified using PCR and the following primers: mtD19 5′-GAA ATT AAT TGT GGA GCA CAT AG-3′ and mtD22 5′-AAG TGT TCA ACA CAG TAT CA -3′ (Simon et al., 1994). The PCR reactions were performed in a total volume of 25 μL for each sample, containing 1 × of standard buffer solution recommended by the manufacturer (Invitrogen), 2.5 mM MgCl2, 250 μM of dNTP, 1 μM of each primer (mtD19 and mtD22), 2.5 units of Taq DNA polymerase (Invitrogen) and 1 μL of total DNA. Each PCR reaction was submitted to initial denaturation at 94 °C/5 min, followed by 35 cycles of denaturation at 94 °C/1, annealing for 1 min and 20 s at 44 °C, elongation at 64 °C/2 min, and finally, an extension to the end at 64 °C for 10 minutes. The PCR products were analyzed in 0.8% agarose gel, stained with ethidium bromide and visualized under UV light, with the image being captured through a photodocumentation system.

To determine the presence of restriction sites, the ATPases 8, 6 and COIII fragment was digested for a minimum period of 6 h with the following restriction enzymes: Ase I, Dra I, EcoR I, EcoR V, Hae III, Hind III , and Hinf I. Each digestion was performed in a 20 μL reaction mixture using 2 μL of the PCR product and 2 units of the enzymes with appropriate buffer. The results of the digestion with restriction enzymes EcoR I, EcoRI, Hae III, HinfI, and Hind III, were analyzed in 1.5% agarose gel, then stained and visualized in the same manner as that used in the amplification process. The results obtained with the digestion performed with endonucleases Dra I and Ase I were analyzed in 12% polyacrylamide gel and visualized through silver staining.

3. RESULTS

3.1. Morphological analysis

The morphological analysis based on the mesepisternum coloration are shown in the table1. Some level of intracolonial variation was verified, especially among the bees collected in the western region, where 85% were classified as T. a. fiebrigi for belonging to classes 4 and 5 and 12% were considered as T. a. angustula, with almost all belonging to class 1. The other bees collected in this region (3%) were not included in any of the two subspecies, they were classified in class 3, which includes the group of bees with mesepisternum of intermediate coloration. In the Vale do Itajaí region, 98.7% of the samples belonged to classes 1 (black mesepisternum) and 2 (black to intermediate mesepisternum), which were classified as T. a. angustula. The others belonged to classes 4 and 5, being therefore morphologically classified as T. a. fiebrigi.

3.2. Molecular analysis

The amplified mtDNA segment containing the genes of ATPases 8, 6 and COIII enzymes was 1800 bp. No length polymorphism was observed in all individuals sampled in this study.

In the RFLP analysis endonucleases Hind III and Hae III showed no digestion product in any of the worker bees examined. The digestion of the PCR product with endonuclease EcoR V, resulted in two fragments of approximately 1200 and 600 bp, although it showed no polymorphism among bees collected. However, several polymorphic sites among bees from both geographic regions were identified with the other four restriction enzymes. The restriction enzyme EcoR I did not generate digestion product in T. angustula from the Vale do Itajaí region, but two fragments were observed in all samples from Western state of Santa Catarina (Fig. 2a). Digestion with Hinf I resulted in two and one specific fragments in bees from Western and Vale do Itajaí regions, respectively (Fig. 2b).

thumbnail Figure 2

Agarose (1.5%) gels showing different banding patterns observed between T. angustula bees from Santa Catarina state for the ATPases 8, 6 and COIII region after digestion with endonuclease EcoR I (A) and Hinf I (B). Columns 1and 5 = no digested fragment; 2, 3, 4 = Vale do Itajai; 6, 7, 8 = West region. M: molecular weight markers λ/Hind III and φx174/Hae III (in Kb).

Endonucleases Ase I and Dra I generated several fragments, although in some of them, the size could not be accurately estimated. However, several bands were specific in bees from the Vale do Itajaí and others for samples collected in the Western region. With the Ase I enzyme, we observed the presence of two bands (approximately 86 and 130 bp), exclusive to T. angustula from the Western region and one band of approximately 98 bp exclusive of bees from the Vale do Itajaí. With the use of Dra I, two bands were obtained (102 and 125 bp) exclusively in bees from the Vale do Itajaí. In the samples from the western region, part of the bees presented two bands sized 100 and 110 bp, while two bands (105 and 112 bp) were exclusively found in the other bees this geographic region. This enzyme also generated two major bands of 300 bp, both with specific sizes on bees from each geographic region.

4. DISCUSSION

The PCR+RFLP technique applied in this study showed that the mtDNA 19 and mtDNA 22 primers, although being designed to amplify the ATPases 8, 6 and COIII region of the mitochondrial genome of Apis mellifera (Simon et al., 1994), produced excellent amplification products in T. angustula when used at the annealing temperature of 44 °C. This methodology also made it possible to determine the size of the mtDNA fragment containing the genes of ATPases 8, 6 and COIII enzymes and several specific restriction sites in T. angustula, sampled in different geographical regions of Santa Catarina state. The size of 1800 bp of the amplified mtDNA fragment in this species of bees is approximately equal to that found for M. quadrifasciata (Moretto and Arias, 2005) M. mondury and M. rufiventris (Barni et al., 2007) and is approximately 100 bp larger than Plebeia species (Francisco et al., 2001).

The EcoR I restriction site in the ATPases 8, 6 and COIII region present in several Plebeia and Melipona species, (Francisco et al., 2001; Weinlich et al., 2004), has been reported as absent in the restriction map of T. angustula (Arias et al., 2006). In our study, it was absent from all bees collected in the Vale do Itajaí, but was present in all samples from the western region. The Vale do Itajaí region, where the EcoR I site was not verified, is part of the Atlantic forest ecosystem, where the natural dispersion of T. a. angustula occurs and the western region of Santa Catarina, characterized by the presence of the EcoR I site, is part of the distribution of T. a. fiebrigi.

In addition to the EcoR I site, Ase I, Dra I, and Hinf I also established different restriction patterns among T. angustula from both regions studied. Hinf I, which has been used to investigate the diversity of Apis cerana subspecies (Sihanuntavong et al., 1999), when used for the cleavage of the ATPases 8, 6 and COIII region, cut once in the bees from the Vale do Itajaí region but twice in samples from the Western region. A band of 1000 bp found in bees from both regions indicates the presence of a common site among them and the bands of 500 bp and 300 pb shown in bees from the western region is the result of another site restriction, Therefore, the fact that the last site has been found only in the region of natural distribution of T. a. fiebrigi may be considered as a specific marker of this subspecies.

The endonucleases Dra I and Ase I were used to assess the variability of the mitochondrial genome of Apis mellifera subspecies (Garnery et al., 1993; Franck et al., 1998) and Melipona quadrifaciasta subspecies (Moretto and Arias, 2005; Souza et al., 2008). In this study, although the bees sampled in both regions of Santa Catarina belong to the Tetragosnisca genus, we verified the occurrence of several restriction sites of Dra I and Ase I. This evidence suggests that the enzymes that recognize A + T restriction sites can also be used to detect polymorphism in T. angustula subspecies. Among all the bands that resulted from digestion with these two enzymes, some were specific for each of the geographical regions of natural distribution where T. a. angustula and T. a. fiebrigi subspecies were described.

Based on the mesepisternum color study of Schwarz (1938), it was considered that both T.angustula subspecies naturally occur in the state of Santa Catarina. T. a. fiebrigi would be dispersed throughout the western region of the state and T. a. angustula would be found exclusively in the Atlantic forest. All bees sampled in the Vale do Itajaí region showed molecular markers not found in bees colleted in the western region and almost 99% were morphologically identified as T. a. angustula and only around 1% were classified as T. a. fiebrigi. However, samples from western Santa Catarina also revealed several molecular markers specific of bees from that region, but great discrepancy was observed when compared with morphological makers. The morphological patterns typical of the fiebrigi subspecies (classes 4 and 5) was found in only 85% of the samples, with 12% exhibiting the morphological pattern (class 1) typical of T. a. angustula and 3% (with intermediate color), could not be included in either subspecies.

Variation in the measures of morphometric characters were found among T. angustula samples collected in several parts of Brazil. The study of morphological changes in fifteen characters in samples of bees from eight local populations conducted by Diniz-Filho et al. (1998), showed significant differences, with north-south clinal variation in body size. An east-west clinal variation in the size of the wing, explained as a possible racial mixing, was also found in T. angustula samples collected in the southeastern region of the country (Castanheira and Contel, 2005). However, despite the variation in mesepisternum color found in this study, mainly in bees from the western region, several markers obtained through the PCR+RFLP technique were specific to bees from each geographic region. Oliveira et al. (2004) also found similar results, 22% of the population of bees, initially identified as T. a. angustula based on the mesepisternum color, were T. a. fiebrigi, when analyzed with RAPD molecular markers. Thus, the presence of specific mtDNA markers, in each natural distribution regions for T. a. angustula and T. a. fiebrigi, suggests restricted gene flow between the two groups of bees analyzed in this work. According to Castanheira and Contel (2005), the polygenic inheritance of the mesepisternum color must be a cause of variability found in local T. angustula populations.

Traditionally, the identification of T. angustula subspecies was based on the mesepisternum color (Schwarz, 1938). However, the natural variability within each subspecies and the origin of hybrid colonies that may occur through contact of the two subspecies may lead to false identifications. Thus, the data from mtDNA verified in this study combined with the RAPD data reported in Oliveira et al. (2004), can be used as molecular markers for the analysis of T. a. angustula and T. a. fiebrigi populations.

According to Nogueira-Neto (1970), T. angustula is a bee widely used in meliponiculture and this encourages farmers and hobbyists to exchange bee colonies. This practice may promote contact and establish gene flow between the two subspecies and develop hybrid populations. Therefore, the mtDNA markers found in this work can be used to determine the maternal origin of commercial and natural populations.

Acknowledgments

This work was supported by FAPESC (Fundação de Apoio à Pesquisa Científica e Tecnológica do Estado de Santa Catarina). The authors thank two anonymous referees for suggestions that improved the paper.

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All Tables

Table I

Number of colonies (N) of Tetragonisca angustula sampled, their geographic coordinates origin in the Santa Catarina state and individual subspecies identification based on morphological and molecular analysis.

All Figures

thumbnail Figure 1

Map of part South America showing Santa Catarina state (Brazil) with the municipalities where Tetragonisca angustula subspecies were sampled: Apiúna (1) Ascurra (2) Benedito Novo (3) Blumenau (4) Dona Emma (5) Indaial (6) Ituporaga (7) Lontras (8) Luiz Alves (9) Nova Trento (10) Pomerode (11) Pouso Redendo (12) Presidente Getúlio (13) Rio do Sul (14) Rio dos Cedros (15) Timbó (16) Trombudo Central (17) Vidal Ramos (18) Belmonte (21) Caçador (22) Caibi (23) Campos Novos (24) Iporã do Oeste (25) Iraceminha (26) Itapiranga (27) Joaçaba (28) Pinhalzinho (29) Santa Helena (30) São Domingos (31) São José do Cedro (32) São Miguel do Oeste (33) Treze Tilhas (34) Videira (35) Arvoredo (36) Águas Frias (37) Quilombo (38) Xaxim (39). Samples 1 to 18 = Vale do Itajaí, Samples 21 to 39 = Western.

In the text
thumbnail Figure 2

Agarose (1.5%) gels showing different banding patterns observed between T. angustula bees from Santa Catarina state for the ATPases 8, 6 and COIII region after digestion with endonuclease EcoR I (A) and Hinf I (B). Columns 1and 5 = no digested fragment; 2, 3, 4 = Vale do Itajai; 6, 7, 8 = West region. M: molecular weight markers λ/Hind III and φx174/Hae III (in Kb).

In the text