Print version ISSN 1517-8382
Braz. J. Microbiol. vol.43 no.2 São Paulo Apr./June 2012
GENETICS AND MOLECULAR MICROBIOLOGY
Bianca Laís ZimmermannI, *; Maurício Pereira AlmerãoI; Didier BouchonII; Paula Beatriz AraujoI
IDepartamento de Zoologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil
IILaboratoire Ecologie, Evolution, Symbiose, Université de Poitiers, Poitiers, France
Terrestrial isopods are widely infected with Wolbachia. However, little is known about the presence of bacteria in the Neotropical species. The objective of this study was to test the hypothesis of presence of Wolbachia infection in the native species of terrestrial isopods, Atlantoscia floridana and Circoniscus bezzii, and in the introduced species Burmoniscus meeusei.
Key words: Wolbachia, terrestrial isopods, 16S rDNA, Brazil.
Wolbachia are Alphaproteobacteria that infect a wide variety of arthropods and nematodes (26). These bacteria use different strategies of symbiosis in their hosts, acting as reproductive parasites in arthropods and as mutualists in nematodes (6). Wolbachia has attracted considerable interest, not only because of its pandemic nature (13), but also because of the reproductive phenotypes observed as a consequence of its reproductive parasitism (26). Moreover, the potential of Wolbachia as a biocontrol agent has been explored in recent years, with studies that suggest their use in controlling pests and disease vectors (5, 9, 16).
Terrestrial isopods are important representatives of the soil fauna, because they take part in soil formation and also in nutrient recycling, in addition to being a food source for a variety of animals (22, 23, 25). This group has developed structural, physiological and behavioral characteristics in order to become fully independent of the aquatic medium for reproduction, which has enabled them to occupy a variety of environments, from the coastal zone to deserts (21).
Terrestrial isopods are widely infected with Wolbachia; however, the majority of studies of these associations have been conducted in Europe (8). The first molecular identification of Wolbachia was in 1992, in populations of Armadillidium vulgare and Porcellio dilatatus from France (20). The presence of the bacterium was detected in Chaetophiloscia elongata and Porcellionides pruinosus, also in France, in 1994 (14). Later, Wolbachia was found in more than 14 European species of terrestrial isopods (7) and in Hyloniscus riparius, Trachelipus rathkii and T. ratzeburgii in Hungary (17). More recently, the infection was detected in 11 species from Tunisia (4) and in two species of Philoscia sp. occurring in Thailand (28). Little is known about the presence of bacteria in terrestrial isopods in Brazil, which has a rich isopod fauna of more than 120 species (21).
The objective of the present study was to test the hypothesis of presence of Wolbachia infection in the native species Atlantoscia floridana and Circoniscus bezzii, and in the introduced species Burmoniscus meeusei. Atlantoscia floridana occurs in the United States, Ascension Island and St. Helena, and from northern Brazil to La Plata in Argentina (2). It is a generalist species in terms of habitat (19), occurring in diverse environments, often in abundance (1, 3). Circoniscus bezzii is a little-studied species that occurs in Brazil (in the states of Minas Gerais and Pará) and Paraguay (15). Burmoniscus meeusei is an introduced species from Asia, with records in England, Hawaii, Brazil and Taiwan (21).
The specimens of A. floridana and B. meeusei examined were collected in Porto Alegre in Rio Grande do Sul, and the individuals of C. bezzii were collected in the municipality of Presidente Olegário in Minas Gerais. The specimens were collected by hand, fixed in absolute ethanol, and transported to the Carcinology Laboratory at Federal University of Rio Grande do Sul, where they were stored in a freezer at -20°C for subsequent DNA extraction.
The terrestrial isopods were dissected according to the methodology proposed by Bouchon et al. (7). The DNA extractions were performed from reproductive tissue (ovaries of females and utricles of males), part of the nerve cord, and the muscle at the base of the pereiopods, using the Chelex (Bio-Rad) protocol. The PCR assays to detect the presence of Wolbachia were performed under conditions adapted from Bouchon et al. (7), targeting the 16S rDNA gene (18). The use of this gene is due to the fact that it has proven to be the most efficient to detect Wolbachia in Neotropical terrestrial isopods (data not shown). Part of 16S rDNA gene was amplified using the Wolbachia specific primers 99F 5'- TTG TAG CCT GCT ATG GTA TAA CT - 3' and 994R 5' - GAA TAG GTA TGA TTT TCA TGT - 3', which produced fragments of approximately 900 bp (18).
The PCRs were carried out in volumes of 25 µl, using 1.0 µl of DNA, 0.16 µl of Platinum® Taq (5 U/µl) (Invitrogen), 2.5 µl of 10X buffer (Invitrogen), 1.6 µl MgCl2 (50 mM) (Invitrogen), 0.5 µl of forward primer (20 µM), 0.5 µl of reverse primer (20 µM), 0.5 µl of dNTPs (10 mM) (Invitrogen) and 18.18 µl of ultrapure water. The amplifications were carried out under the following conditions: 35 cycles (1 min at 95°C, 1 min at 50.6°C, 1 min at 72°C), including an initial denaturation step of 95°C for 2 min and a final extension step of 72°C for 5 min. As a positive control for the PCR reaction, DNA extracted from an individual of the terrestrial isopod Balloniscus glaber was used, in which infection by Wolbachia was previously detected (unpublished data).
PCRs were confirmed by electrophoresis in 1% agarose gels. Possible failures in the amplifications with the 16S rDNA primers could occur for the following reasons: (i) absence of Wolbachia; (ii) a flaw in the DNA extraction process; and/or (iii) an incorrect concentration of DNA solution (27). In order to control the last two possibilities, we tested samples assumed to be negative, with primers of subunit I of the Cytochrome Oxidase mitochondrial gene (COI) (11). Samples that generated a product of the expected size were considered to be truly negative for the presence of Wolbachia.
The amplified DNA fragments of positive samples were sent to the company MACROGEN Advancing through Genomics for purification and sequencing. This company uses the BigDyeTM Terminator protocol, and sequencing was conducted on a 3730xl DNA analyzer. The resulted sequences were compared by the Blastn algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with sequences deposited in the GenBank database. All sequences were subsequently submitted in the Genbank-EMBL database (http://www.ncbi.nlm.nih.gov/) under accession numbers: JF799948, JF799949, JF799950 and JF799951. Alignment of sequences was performed using the BioEdit program (12). The Wolbachia strain names were based on the nomenclature system proposed by Zhou et al. (29) and specified by Charlat et al. (10). Each strain's name is defined by w (in italics) denoting Wolbachia. This is followed by three letters coming from the first three letters of the species name. Multiple strains present in a given species are distinguished by numbers added at the end.
Positive samples were detected in the three species A. floridana, B. meeusei and C. bezzii, representing the first record of Wolbachia infection in these species. The infection was observed in 30 individuals: 25 females of A. floridana, 2 females of B. meeusei and 3 females of C. bezzii (Table 1). A greater number of individuals of A. floridana were tested because this species usually occurs in abundance in nature (1), which facilitates their collection. However, B. meeusei and C. bezzi are difficult to collect. In addition, this is the first record of the presence of B. meeusei in the state of Rio Grande do Sul; until now, this species has been known only from Santa Catarina.
A single 16S rDNA sequence from Wolbachia was identified for A. floridana (wFlo) and B. meeusei (wMee). For C. bezzii, two very different 16S rDNA sequences (wBez1 and wBez2) were found in two different individuals (Figure 1). Thus, multiple infections were not detected. Natural multiple infections in a single host have never been demonstrated in terrestrial isopods species (24), and this work supports this statement.
This study reports one of the first records of Wolbachia infection in species of terrestrial isopods in Brazil, in particular in the native species A. floridana and C. bezzii and the introduced species B. meeusei. Although these bacteria are widely studied, very few investigations of their interactions with terrestrial isopods and other organisms have been carried out in the Neotropical region. It is hoped that further studies will be undertaken to expand knowledge of the Wolbachia bacteria and their Neotropical hosts.
To CAPES for the doctoral fellowship for BLZ, and to CNPq for financial support (Projects Number: 479412/2008-1 and 472381/2009-1) and for the research productivity grant to PBA.
1. Almerão, M.P.; Mendonça Jr, M.C.; Quadros, A.F.; Pedó, E.; Silva, L.G.R.; Araujo, P.B. (2006). Terrestrial isopod diversity in the subtropical neotropics: Itapuã State Park, Southern Brazil. Iheringia Ser. Zool. 96, 473-477. [ Links ]
2. Araujo, P.B. (1999). Subordem Oniscidea (isópodos terrestres, "tatuzinhos"). In: Buckup, L., Bond-Buckup, G. (eds). Os Crustáceos do Rio Grande do Sul. Ed. Universidade/UFRGS, Porto Alegre, Brasil, p.237-256. [ Links ]
3. Araujo, P.B.; Bond-Buckup, G. (2005). Population structure and reproductive biology of Atlantoscia floridana (van Name, 1960) (Crustacea, Isopoda, Oniscidea) in southern Brazil. Acta. Oecol. 28, 289-298. [ Links ]
4. Ben Afia Hatira, H.; Charfi Cheikhrouha, F.; Bouchon, D. (2008). Wolbachia in terrestrial isopods in Tunisia. In: Zimmer, M., Charfi-Cheikhrouha, F., Taiti, S.S. (eds). Proceedings of the international symposium on terrestrial isopod biology. Verlag, Aachen, p.117-124. [ Links ]
5. Bian, G.; Xu, Y.; Lu, P.; Xie, Y.; Xi, Z. (2010). The Endosymbiotic Bacterium Wolbachia Induces Resistance to Dengue Virus in Aedes aegypti. PLoS Pathog. 6 (4), 1-10. [ Links ]
6. Bordenstein, S.R.; Paraskevopoulos, C.; Hotopp, J.C.; Sapountzis, P.; Lo, N.; Bandi, C.; Tettelin, H.; Werren, J.H.; Bourtzis, K. (2009). Parasitism and mutualism in Wolbachia: what the phylogenomic trees can and cannot say. Mol. Biol. Evol. 26 (1), 231-241. [ Links ]
7. Bouchon, D.; Rigaud, T.; Juchault, P. (1998). Evidence for widespread Wolbachia infection in isopod crustaceans: molecular identification and host feminization. Proc. R. Soc. Lond., B, Biol. Sci. 265, 1081-1090. [ Links ]
8. Bouchon, D.; Cordaux, R.; Grève, P. (2008). Feminizing Wolbachia and the evolution of sex determination in isopods. In: Bourtzis, K., Miller, T. (eds). Insect Symbiosis. CRC Press, p.273-294. [ Links ]
9. Bourtzis, K. (2008). Wolbachia-based technologies for insect pest population control. Adv. Exp. Med. Biol. 627, 104-113. [ Links ]
10. Charlat, S.; Bourtzis, K.; Merçot, H. (2002). Wolbachia-induced cytoplasmic incompatibility. In: Seckbach, J., (ed). Symbiosis, Mechanisms and Model Systems (Cellular Origin and Life in Extreme Habitats, Vol. 4). Kluwer, Dordrecht, The Netherlands, p. 623-644. [ Links ]
11. Folmer, O.; Black, M.; Hoeh, W.; Lutz, R.; Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294-299. [ Links ]
12. Hall, T.A. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic. Acids. Symp. Ser. 41, 95-98. [ Links ]
13. Hilgenboecker, K.; Hammerstein, P.; Schlattmann, P.; Telschow, A.; Werren, J.H. (2008). How many species are infected with Wolbachia? a statistical analysis of current data. FEMS Microbiol. Lett. 281, 215-220. [ Links ]
14. Juchault, P.; Frelon, M.; Bouchon, D.; Rigaud, T. (1994). New evidence for feminizing bacteria in terrestrial isopods evolutionary implications. C. R. Acad. Sci. Paris III 317, 225-230. [ Links ]
15. Leistikow, A.; Wägele, J.W. (1999). Checklist of the terrestrial isopods of the new world (Crustacea, Isopoda, Oniscidea). Rev. Bras. Zool. 16 (1), 1-72. [ Links ]
16. Moreira, L.A.; Iturbe-Ormaetxe, I.; Jeffery, J.A.; Lu, G.; Pyke, A.T.; Hedges, L.M.; Rocha, B.C.; Hall-Mendelin, H.; Day, A.; Riegler, M.; Hugo, L.E.; Johnson, K.N.; Kay, B.H.; McGraw, E.A.; van den Hurk, A.F.; Ryan, P.A.; O'Neill, S.L. (2009). A Wolbachia symbiont in Aedes aegypti limits infection with dengue, chikungunya, and Plasmodium. Cell 139 (7), 1268-1278. [ Links ]
17. Nyirõ, G.; Oravecz, O.; Márialigeti, K. (2002). Detection of Wolbachia pipientis infection in arthropods in Hungary. Eur. J. Soil Biol. 38, 63-66. [ Links ]
18. O'Neill, S.L.; Giordano, R.; Colbert, A.M.E.; Karr, T.L.; Robertson, H.M. (1992). 16S rDNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects. Proc. Natl. Acad. Sci. USA 94, 2699-2702. [ Links ]
19. Quadros, A.F.; Caubet, Y.; Araujo, P.B. (2009). Life history comparison of two terrestrial isopods in relation to habitat specialization. Acta. Oecol. 35, 243-249. [ Links ]
20. Rousset, F.; Bouchon, D.; Pintureau, B.; Juchault, P.; Solignac, M. (1992). Wolbachia endosymbionts responsible for various alterations of sexuality in arthropods. Proc. R. Soc. Lond. B Biol. Sci. 250, 91-98. [ Links ]
21. Schmalfuss, H. (2003). World catalog of terrestrial isopods (Isopoda, Oniscidea). Palaeodiversity (Stuttg) 654, 1-341. [ Links ]
22. Sunderland, K.D.; Sutton, S.L. (1980). A serological study of arthropod predation on woodlice in a dune grassland ecosystem. J. Anim. Ecol. 49 (3), 987-1004. [ Links ]
23. Van Sluys, M.; Rocha, C.F.D.; Souza, M.B. (2001). Diet, reproduction and density of the Leptodactylid litter frog Zachaenus parvulus in a atlantic of southeastern Brazil. J. Herpetol. 35 (2), 322-325. [ Links ]
24. Verne, S.; Johnson, M.; Bouchon, D.; Grandjean, F. (2007). Evidence for recombination between feminizing Wolbachia in the isopod genus Armadillidium. Gene 397, 58-66. [ Links ]
25. Vitt, L.J.; Souza, R.A.; Sartorius, S.S.; Avila-Pires, T.C.S.; Espósito, M.C. (2000). Comparative ecology of sympatric Gonatodes (Squamata: Gekkonidae) in the western Amazon of Brazil. Copela 1, 83-95. [ Links ]
26. Werren, J.H.; Baldo, L.; Clark, M.E. (2008). Wolbachia: master manipulators of invertebrate biology. Nat. Rev. Microbiol. 6, 741-751. [ Links ]
27. Werren, J.H.; Windsor, D.; Guo, L.R. (1995). Distribution of Wolbachia among neotropical arthropods. Proc. R. Soc. Lond. B Biol. Sci. 262, 197-204. [ Links ]
28. Wiwatanaratanabutr, I.; Kittayapong, P.; Caubet, Y.; Bouchon, D. (2009). Molecular phylogeny of Wolbachia strains in arthropod hosts Based on groE-homologous gene sequences. Zool. Sci. 26, 171-177. [ Links ]
29. Zhou, W.; Rousset, F.; O'Neill, S.L. (1998). Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proc. R. Soc. Lond., B, Biol. Sci. 265, 509-515. [ Links ]
Submitted: April 29, 2011
Returned to authors for corrections: September 20, 2011
Approved: January 16, 2012
* Corresponding Author. Mailing address: Avenida Bento Gonçalves, nº 9500, Bairro Agronomia, Porto Alegre, RS, Brasil. Cep.: 91501-970.; Tel.: (+5551) 3308-7698 Fax: (+5551) 3308-7696.; E-mail: firstname.lastname@example.org