Detection of Wolbachia (Alphaproteobacteria: rickettsiales) in three species of terrestrial isopods (crustacea: isopoda: oniscidea) in Brazil.

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.

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.

Table 1

Total of individuals of each species tested for the presence of Wolbachia. FT: females tested; MT: males tested; FI: females infected; MI: males infected; RS: Rio Grande do Sul; MG: Minas Gerais.

Species	FT	MT	FI	MI	Locality	Geographical Coordinates
Atlantoscia floridana	45	3	25	0	Porto Alegre/RS	30°04'76''S/51°07'28''W
Burmoniscus meeusei	2	0	2	0	Porto Alegre/RS	30°04'49''S/51°07'31''W
Circoniscus bezzii	7	0	3	0	Presidente Olegário/MG	18°24'02''S/46°25'50''W

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.

Figure 1

Alignment of Wolbachia 16S rDNA sequences found in A. floridana (wFlo), B. meeusei (wMee) and C. bezzii (wBez1 and wBez2).

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.