123 Molecular Marker to Identify Two Stingless Bee Species: Tetragonisca angustula and Tetragonisca fiebrigi (Hymenoptera, Meliponinae) by Ana Lúcia Paz Barateiro Stuchi1, Vagner de Alencar Arnaut de Toledo2, Denise Alves Lopes1, Liriana Belizário Cantagalli1 & Maria Claudia Colla Ruvolo-Takasusuki1* ABSTRACT Tetragonisca angustula and T. fiebrigi esterases were biochemically character- ized by their inhibition pattern and thermostability. Workers of both species were collected from nests at the State University of Maringá. In T. fiebrigi three esterases were observed: EST-1 (β-esterase, cholinesterase I), EST-2 (α-esterase, cholinesterase II) and EST-4 (αβ-esterase, carboxylesterase). In T. angustula two esterases were detected: EST-3 (β-esterase, acetylesterase) e EST-4 (αβ-esterase, carboxylesterase). T. angustula EST-3 showed the highest thermostability, and it was not observed above 54°C, while in T. fiebrigi EST- 1 and EST-2 were not detected above 52°C. Through this characterization, it was observed that EST-4 of T. angustula and T. fiebrigi showed identical biochemical characteristics, and probably those esterases are encoded by the same gene in the two species. Together, the biochemical characterization and molecular markers show that the two species are differentiated and secondary contact between the populations can still be occurring. Key words: stingless bees, isoenzymes, inhibition, thermostability, tax- onomy INTRODUTION The stingless bees evolved from a group of wasps which at some point in evolution no longer transmitted to its descendants the genetic characteristics Universidade Estadual de Maringá, Departamento de Biologia Celular e Genética, Av. Colombo, 5790, Maringá – Paraná - CEP 87.020-900, (44) 3261-4678. 2Universidade Estadual de Maringá, Departamento de Zootecnia, Av. Colombo, 5790, Maringá – Paraná - CEP 87.020-900. *Corresponding author: mccrtakasusuki@uem.br 124 Sociobiolog y Vol. 59, No. 1, 2012 responsible for the sting formation (Alonso 1998). The explanation for the sting loss in this group of bees is probably related to the fact that their colony is not exposed when the bees swarm, and they usually nest in concealed and well protected places (Alonso & Paim 2001). The stingless bees are among the most common pollinators of tropical en- vironments, and in certain regions they are the dominant bee species, visiting various cultures (Macías-Macías et al. 2009). These insects are a diverse group, in which includes over 400 species which shows high variability in physiolog y, morpholog y and size, ranging from 0.2 mm in the genus Trigonisca to over 20 mm in some Melipona species (Michener 2000; Moure et al. 2007). The meliponines are though to have originated in the Gondwana western continent; this hypothesis is based on the fossil findings and by biogeography (Camargo & Menezes-Pedro 1992). They dwell mostly in regions of tropical and temperate subtropical weather of the world (Nogueira-Neto 1997). According to Moure (1961), there are two tribes in the Meliponinae sub- family: Meliponini and Trigonini. The Meliponini are characterized for not building real cells, therefore, queens, workers and males born and develop to adulthood in cells of the same size. The Trigonini consists of a highly diversi- fied group, with dozens of genera and often build real cells, bigger than the other, where queens-to-be emerge (Nogueira-Neto 1997). According to Castanheira & Contel (2005), in Tetragonisca angustula (Trigonini), there are two known subspecies, which are differentiated through the coloring of the mesepisterna. T. angustula angustula presents a black mese- pisterna, while T. angustula fiebrigi presents a yellow one. However, according to Camargo & Pedro (2007), they were considered as two taxonomically distinct species, T. angustula and T. fiebrigi. Considering that there is still disagreement regarding the classification of these bees in species or subspecies, the development of new studies to detect the occurrence of markers which enable an accurate classification is essential. The geographical distribution for T. angustula fiebrigi was firstly described by Schwarz (1938) and Nogueira-Neto (1970). This subspecies is present in Brazil in Mato Grosso state, in the Paraná river basin and also in Paraguay and Argentina. In 2005, Castanheira & Contel (2005) related the occurrence of T. angustula fiebrigi in North-western Paraná, Londrina and Maringá coun- ties. This subspecies was also collected in Altônia county, Paraná state (Ruiz 2006; Alves 2006). 125 Stuchi, A.L.P.B. et al. —Molecular Markers to Identify Two Tetragonisca Species The subspecies T. angustula angustula is distributed in most states through- out Brazil (Iwama & Melhem, 1979; Camargo & Posey, 1990), as well as in Panama (Roubik 1983), Venezuela (Vit et al. 1994) and Costa Rica (Veen & Sommeijer 2000a, b). Regarding the population genetics studies and the differentiation between these two species, Oliveira et al. (2004), by using the molecular marker RAPD, identified a marker for the Tetragonisca subspecies, the primer OPL-11. Later, Baitala et al. (2006) showed that with the use of RAPD markers, it is only possible to separate populations of Tetragonisca, not being able to detect a marker for subspecies, even using the primer mentioned above. Among the molecular markers, the esterase isoenzymes which present high multifunctional hydrolytic activity and catalyze the hydrolysis of a large number of esters can be highlighted (Walker & Mackeness 1983). Based on the sensibility to the synthetic substrate which those enzymes hydrolyze in vitro, two groups can be distinguished in insects, the α-esterases which hydrolyze preferably α-naphthyl-acetate, and β-esterases which hydrolyze preferably β-naphthyl-acetate (Oakeshott et al. 1993). Also as a classificatory criterion, according to the sensibility to different inhibitors of the enzymatic activity and to the amino acid residues in its active site, there are four esterase classes, the acetylesterases (E.C. 3.1.1.6), the arylesterases (E.C. 3.1.1.2), the carboxylesterases (E.C. 3.1.1.1) and the cholinesterases which includes the acetylcholinesterases (E.C. 3.1.1.7) and the pseudocholinesterases (E.C. 3.1.1.8) (Healy et al. 1991). Little is known about the esterases in T. angustula and T. fiebrigi, known as the jataí stingless bee. Ruvolo-Takasusuki et al. (2006) showed the esterase activity regions in T. angustula, and these authors have found two regions, which were characterized as EST-1 (β-esterase) and EST-2 (αβ-esterase). The objective in the present study is to identify isoenzyme esterase as a biochemical marker which differentiates the two species of jataí stingless bee, T. angustula and T. fiebrigi. MATERIALS AND METHODS Material Adult jataí stingless bee workers were collected from two natural nests located within the State University of Maringá (Universidade Estadual de 126 Sociobiolog y Vol. 59, No. 1, 2012 Maringá), Paraná (23o24’40’’ S; 51o56’23’’ W), one nest was of T. angustula species and the other of T. fiebrigi. After collection, the bees were euthanized and stored in properly labelled and numbered containers, at –20ºC. Preparing of the samples and electrophoresis PAGE Each bee worker had its head/thorax removed and homogenized individu- ally in propylene tubes 1.5 mL containing 35 µL of 0.1% 2-mercaptoetanol solution plus glycerol at 10%. The samples were centrifuged at 56.000G for 10 minutes at 4ºC. Vertical electrophoreses were performed, using PAGE gels at 8% concen- tration and stacking gel at 5% concentration. Tris-Glicine at 0.1 M pH 8.3 was used as buffer. The gels were submitted to electrophoresis at 200 V for 5 hours. The gel was incubated for 30 minutes in 50 mL of sodium phosphate buffer solution (0.1 M pH 6.2), for staining. Then the buffer was discarded and the staining solution was added - 50 mL of sodium phosphate buffer at 0.1 M pH 6.2, 0.03 g of α-naphthyl acetate; 0.03 g of β-naphthyl acetate, 0.06 g of Fast Blue RR Salt. The gel was incubated until the bands became visible. The gels remained in fixation solution (acetic acid at 75% and glycerol at 10%, dissolved in 1.000 mL of distilled water) for at least 24 hours. The gels were then soaked in gelatin at 5% and placed between two sheets of wet cellophane paper, stretched, pressed and kept in room temperature until completely dry (Ceron et al. 1992). Inhibition tests The head/thorax extracts of each worker were used twice in the same PAGE gel, the first trial as the control, and the second as the inhibition test. For the staining, first, the gel was cut and separated in two parts: control and inhibition. Each part, separately, was incubated for 30 minutes in 50 mL of sodium phosphate buffer solution (0.1 M pH 6.2). The inhibitor to be tested was the incubation buffer for the test gel (organophosphate – 60 µL, parachloromercuriobenzoate (ρ-CMB) – 0.01 g or eserine sulphate – 0.06 g ). After the incubation period, the buffer was discarded and the staining solution added as previously described. For the test, the inhibitor was added in the amounts described above. Bands in the control gel were compared with the inhibitor gel, and an inhibition table was produced. 127 Stuchi, A.L.P.B. et al. —Molecular Markers to Identify Two Tetragonisca Species Thermostability The thermostability test for the esterases was done through the pre incu- bation of the samples for 5 minutes at a temperature that ranged from 52oC to 58oC. After the incubation, 10 µL of the supernatant was applied in the PAGE gel and submitted to electrophoresis. As control, head/thorax extracts of T. angustula and T. fiebrigi which were not submitted to pre incubation were used. After the electrophoresis, the gels were stained for the esterase visualization as described above. RESULTS AND DISCUSSION Many differences were detected in the electrophoretic analysis, regarding the number of observed esterases, migration pattern, affinity to the substrate and thermostability, providing input that the Tetragonisca genus has two species, T. fiebrigi and T. angustula, as suggested by Camargo & Pedro (2007). The number of regions with esterase activity varied according to the spe- cies; in T. fiebrigi, three esterases were observed, which were called EST-1 (showing a most anodic migration), EST-2 (showing a intermediary migra- tion) and EST-4 (showing a least anodic migration), while in T. angustula, two esterase activity regions were observed: EST-3 (most anodic) and EST-4 (least anodic) (Fig. 1). Ruvolo-Takasusuki et al. (2006) and Stuchi et al. (2008) classified the T. angustula esterases according to their migration pattern as follows: EST-1 (most anodic) and EST-2 (least anodic). However, due to the results obtained in the present study, it has been suggested that these esterases could be classi- fied as EST-3 (most anodic) and EST-4 (least anodic), and EST-3 is a specific region of T. angustula (Fig. 1). According to the specificity to the substrates α-naphthyl-acetate and β-naphthyl-acetate in T. fiebrigi the EST-1 has been classified as β-esterase, EST-2 as α-esterase and EST-4 as αβ-esterase (Fig. 1). The biochemical characteristics described above identified the EST-1 and EST-2 as molecular markers for T. fiebrigi, and EST-3 as molecular marker for T. angustula. Insect esterases have been divided in four distinct classes based on their sensibility to three groups of inhibitors: organophosphates, eserine sulphate and sulphidril reagents (Healy et al. 1991). In this work, after the electro- 128 Sociobiolog y Vol. 59, No. 1, 2012 Fig. 1. Esterase isoenzymes profile in the PAGE system from head/thorax extracts of the Tetragonisca fiebrigi (A) and Tetragonisca angustula (B) workers. Table 1. Inhibition pattern for the characterization of esterases using the inhibitors Malathion, ρ-CMB, and eserine sulfate in Tetragonisca fiebrigi and Tetragonisca angustula. (+) inhibition, (-) no inhibition. Esterase Malathion ρ-CMB Eserine sulfate Classification T. fiebrigi EST-1 + - + Cholinesterase (I) EST-2 + + + Cholinesterase (II) EST-4 + + - Carboxylesterase T. angustula EST-3 - - - Acetylesterase EST-4 + + - Carboxylesterase 129 Stuchi, A.L.P.B. et al. —Molecular Markers to Identify Two Tetragonisca Species phoresis, head/thorax extracts from the worker bees were submitted to the three groups of inhibitors, the results concerning the inhibition pattern can be observed in Table 1. Due to their inhibition pattern against the used inhibitors, EST-1 and EST- 2 in T. fiebrigi can be classified as cholinesterases, EST-1 as a cholinesterase type I, while EST-2 is a cholinesterase type II. The EST-4 from both species is a carboxylesterase (Table 1). In T. angustula, EST-3 is an acetylesterase. The analyzed esterase inhibition pattern shows that T. fiebrigi presents two cholinesterases and a carboxylesterase and that T. angustula presents a carboxylesterase (Table 1). The carboxylesterases and the cholinesterases are common isoenzymes in insects, possibly due to their fundamental role in the process of detoxification of xenobiotic compounds, contributing to a resistance to insecticides. Regarding the organophosphates, these mechanisms involve the increase in the metabolic detoxification by hydrolysis or the kidnapping of these compounds (Hemingway 2000; Lee & Lees 2001; Cui et al. 2007) or structural changes in the acetylcholinesterase, the primary target for this insecticide class (Hsu et al. 2006). Therefore, bees from the genus Tetragonisca may be used in future studies as bio-indicators of the presence of pesticides in natural and cultivated areas. The thermostability tests have shown that there are differences in the es- terases inhibition depending on the temperature in which they are submitted. In T. fiebrigi, when the head/thorax extracts were submitted to a temperature of 52°C, the EST-4 presented a partial reduction of its activity, while the EST-1 and EST-2 presented total inhibition. Regarding T. angustula, partial inhibition was observed for EST-4, while EST-3 was not inhibited at the same temperature. However, from 54°C, every esterase lost its activity (Fig. 2 and Table 2). Concerning the thermostability it is possible to observe that the EST-4 from T. fiebrigi and EST-3 and EST-4 from T. angustula are more thermo- stable than the EST-1a from Apis mellifera. Ruvolo-Takasusuki et al. (1997) have observed that there was no activity of the EST-1a of A. mellifera when extracts from abdomen were previously incubated at 50°C or for over 4 minutes, while the T. angustula and T. fiebrigi ones were submitted to 52°C for 5 minutes and still remained active (Table 2). 130 Sociobiolog y Vol. 59, No. 1, 2012 EST-3 of T. angustula was the most thermostable esterase (Fig. 2), losing its activity at 54°C. Ruvolo-Takasusuki et al. (1998) observed a high thermo- stability for A. mellifera EST-2, which showed activity after pre-incubation at 60°C for 8 minutes. The esterase biochemical characterization showed that only EST-4 is common in the two species. The EST-1 (T. fiebrigi) and EST-3 (T. angus- tula) probably have differentiated by mutations along the evolution of both Fig. 2. Thermostability profile of the esterase isoenzymes in the PAGE system from head/thorax extracts of Tetragonisca fiebrigi and Tetragonisca angustula workers. Samples 1-2, 5-8 and 13-16 are head/thorax extracts of Tetragonisca angustula and samples 3-4, 9-12 and 17-20 are head/thorax extracts of Tetragonisca fiebrigi. Table 2. Inhibition patterns of esterases in Tetragonisca fiebrigi and Tetragonisca angustula at 52 °C and 54 °C. (++) total inhibition, (+) partial inhibition, (-) no inhibition. Temperature T. fiebrigi T. angustula EST-1 EST-2 EST-4 EST-3 EST-4 52°C ++ ++ + - + 54°C ++ ++ ++ ++ ++ 131 Stuchi, A.L.P.B. et al. —Molecular Markers to Identify Two Tetragonisca Species species, and EST-2 (T. fiebrigi) may have been originated by duplication and later mutations. The use of RAPD markers has shown that T. a. angustula and T. a. fiebrigi may be separated in two groups considered subspecies (Oliveira et al. 2004). On the other hand the use of RAPD by Baitala et al. (2006), in five T. a. angustula and T. a. fiebrigi populations from Junqueirópolis (SP), Maringá (PR) and Cianorte (PR), have not permitted the separation of the two subspe- cies; only the populations from São Paulo and Paraná states were separated by RAPD markers. Alves (2006) has applied RAPD for the analysis of three jataí populations in north-eastern Paraná state, Ivatuba, Umuarama and Altônia counties, and in Ivatuba collected only T. a. angustula and in Umuarama and Altônia only T. a. fiebrigi. The results have shown that the two bee species are separated with genetic distance values which justify their taxonomy, as between T. a. angustula and T. a. fiebrigi the genetic distance value according to Nei (1978) was 0.23 and between the two T. a. fiebrigi populations was 0. 0507. Castanheira & Contel (2005) have performed a morphometric analysis of the wings, mesepisterna staining and hexokinase polymorphism of T. a. angustula and T. a. fiebrigi bees from various regions within São Paulo, Paraná, Mato Grosso do Sul and Minas Gerais states of Brazil. The mesepisterna coloring and the allele HK88 frequency have enabled the authors to suggest that there is clinal distribution or race mixing between the two subspecies. A high correlation between the yellow coloring of the mesepisterna and the allele HK88 frequency has been observed. Diniz-Filho et al. (1998) have performed morphometric analysis of T. an- gustula from central and south-eastern Brazil. The obtained variations could be explained by secondary contact among previously isolated races. The controversy regarding the two species or subspecies of Tetragonisca seems to be nearly over, because the electrophoretic profile and the esterase biochemical characterization of these stingless bees have shown that they are two distinct species. The obtained results by the esterase biochemical characterization have shown that there are differences in these loci between the two species, which justifies their classification according Camargo & Pedro (2007). 132 Sociobiolog y Vol. 59, No. 1, 2012 The results show that the jataí stingless bees are an important group for speciation studies. The two species have probably originated from one that may have been differentiated due to geographical isolation. Anthropic action and a relatively short period of geographical isolation may have put the two species in touch again which could be causing the hybridization detected in the study performed by Castanheira & Contel (2005). 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