Ann Microbiol (2010) 60:43–50 DOI 10.1007/s13213-009-0001-z ORIGINAL ARTICLE Detection and phylogeny of the bacteria Wolbachia in the terrestrial isopod Porcellio laevis Latr. (Crustacea, Oniscoidea), isolated from Lebna and Bizerte stations, Tunisia Sarra Ben Nasr & Maher Gtari & Atf Azzouna Received: 11 May 2009 / Accepted: 18 November 2009 / Published online: 5 January 2010 # Springer-Verlag and the University of Milan 2009 Abstract Wolbachia Hertig, 1936, is an intracytoplasmic bacterium that infects several species of arthropods by causing deterioration of host reproduction. In terrestrial isopods, Wolbachia infection can generate cytoplasmic incompatibility and feminization. This work examined two samples of Wolbachia from two different stations in Tunisia (Lebna in the North-East, and Bizerte in the North), and the study is limited to the host species Porcellio laevis Latreille, 1804. Enumeration of males and females revealed a very strong imbalance in the sex ratio in favour of females in the former population, and intersexual females in the latter. Dissection of the genital apparatus of various phenotypically normal male specimens allowed some malformations to be noted. These males were marked by the presence of an oviduct joined to the vas deferens, and one to three hypertrophied androgenic glands at the top of the testicles. Further molecular study confirmed the presence of the endosymbiont in the two populations. Sequencing of the P. laevis wsp gene from the Bizerte population revealed a high degree of identity with the Wolbachia endosymbiont of Armadillidium vulgare. S. Ben Nasr (*) : A. Azzouna Laboratory of Reproduction and Development, Faculty of Sciences, University Tunis El Manar, 2092 El Manar II, Tunis, Tunisia e-mail: sarra.bennasr@gmail.com M. Gtari Laboratory of Microorganisms and Actives Biomolecules, Faculty of Sciences, University Tunis El Manar, 2092 El Manar II, Tunis, Tunisia Keywords Wolbachia . Terrestrial isopods . Porcellio laevis . Intersexual . Sequencing Introduction Wolbachia Hertig, 1936 is an endosymbiotic Gram-negative bacteria (Masui et al. 2001), belonging to the β-supergroup (Wu et al. 2004), the class α-Proteobacteria and the order Rickettsiales (Lo et al. 2002). It infects several species of Arthropods and causes various sexual alterations in their hosts: cytoplasmic incompatibility for several orders of Insects (Rousset et al. 1992; Masui et al. 2000); thelytokous parthenogenesis in the haplo-diploid wasps, Thysanoptera and acariens (Juchault et al. 1994; Werren 1997; Sun et al. 2001; McGraw and O’Neil 2004); male-killing in Lepidoptera, Coleoptera and the Diptera Drosophila bifasciata (Felix 2004); and feminization of genetic males in the Lepidoptera Eurema hecabe (Hiroki et al. 2002; Narita et al. 2007) and the Hemiptera Zyginidia pullula (Negri et al. 2006). In the terrestrial isopods or Oniscoidea characterised by a heterogametic (ZW) female (Juchault and Legrand 1979) and a natural, genetically determined amphogeny (Beaumont et al. 2006), the bacterium may cause cytoplasmic incompatibility in some species of the Porcellionidae family, e.g. Porcellio dilatatus petiti Vandel, 1951 (Legrand and Juchault 1986) and Cylisticus convexus De Geer, 1778 (Moret et al. 2001); this effect can become pathogenic and even lethal for the species Porcellio laevis Latreille, 1804 and Porcellio dilatatus Brandt, 1833 (Juchault et al. 1974), and involves a slowing down of growth, a reduction in vitality, and a reduction in fertility in strongly contaminated genetic females (Juchault and Mocquard 1989). Because of the strong penetration of the microorganisms, other species belonging to the Armadillidae family can undergo a more 44 or less advanced feminizing effect in genotypic (ZZ) males, leading quickly to a catastrophic situation due to the appearance of functional neo-females at a very high rate (Juchault and Mocquard 1989; Moreau et al. 2001). This feminizing effect is probably caused by dysfunctional androgenic glands (Juchault et al. 1980; Martin et al. 1990), as described in Armadillidium vulgare Latreille, 1804 (Martin et al. 1974; Juchault and Legrand 1981; Vandekerckhove-Tom et al. 2003; Cordaux et al. 2004; Rigaud and Moreau 2004; Verne et al. 2006; Johnson et al. 2007), Armadillidium nasatum Budde-Lund, 1885 (Juchault and Legrand 1979), Porcellionides pruinosus Brandt, 1833 (Juchault et al. 1994; Braquart-Varnier et al. 2005), Oniscus asellus Linnaeus, 1758 (Rigaud et al. 1999) and Porcellio scaber Latreille, 1804 (Juchault et al. 1994). In Tunisia, Wolbachia has been detected in Armadillidium vulgare species (Cordaux et al. 2004) and, in insects, it has been detected in the two mosquito species Phlebotomus papatasi and Phlebotomus perniciosus (Benlarbi-Ben Khedher 2006). When this endosymbiotic infects a population of terrestrial isopods, it can generate four principal types of intersexes (Legrand and Juchault 1986): 1. Functional males with female genital apertures redescribed on fga males by Azzouna et al. (2004), who demonstrated that this phenotype is associated with a lack of bacteria in its cytoplasm. It is characterised by the presence of a pair of remnant oviducts—sometimes only one—attached to the base of the seminal vesicle and exhibiting female genital apertures; moreover, one or more hypertrophied androgenic glands are attached to the end of the testicular segments (utricles). 2. Functional females (iF) hosting the bacterium; sexual differentiation is inversed and infected specimens present a mixture of male and female organs, which has led Legrand and Juchault (1986) to class them as protandric hermaphrodites whose male phase is not functional. 3. Sterile intersexual males (iM) hosting the bacterium; their genital apparatus is characterised by testicles producing spermatozoa, remnant oviducts connected to the seminal vesicle and succeeded by female genital apertures; androgenic glands are extensively hypertrophied and are observed at the extremity of each testicular segment, outside a flanked genital apophysis of reduced gonopods. 4. Functional intersexual females (f): sexual differentiation very similar to the hermaphrodite case, differing only by the fact that masculinisation can be restored by androgenic gland implantation. It was shown recently that “factor f” (Juchault and Legrand 1972) is derived from a segment of only 7.1 kb DNA of the Wolbachia chromosome inserted into the genome of the host (G. Martin et al. unpublished); this phenotype never hosts the bacterium itself. Ann Microbiol (2010) 60:43–50 Wolbachia has been the subject of several molecular studies, which aimed not only to identify the bacterial genome, but also to gain knowledge of its phylogeny and its mode of transmission in several of its Arthropod hosts. The genome of Wolbachia has been sequenced from several Arthropods hosts. To mention only Oniscoidea, the list includes Armadillidium vulgare (Werren et al. 1995), Porcellio dilatatus petiti (Cordaux et al. 2001), Oniscus asellus (Cordaux et al. 2001) and Porcellionides pruinosus (Michel-Salzat et al. 2001). The use of rRNA and ftsZ primers revealed that the sequences of the symbionts of Armadillidiidae (Armadillidium vulgare, Armadillidium album Dollfus, 1877) and of Philosciidae Kinahan, 1857 (Chaetophiloscia elongata Dollfus, 1884) are identical: Wolbachia of Porcellio spinicornis Say, 1818, Philoscia muscorum (Scopoli, 1763), Porcellio scaber Latreille, 1804 and Oniscus asellus form a monophyletic group. On the other hand, Porcellio dilatatus petiti is more distant (Bouchon et al. 1998). Sequencing using a wsp primer shows that the bacteria associated with Armadillidium vulgare, Armadillidium album, Armadillidium nasatum and Cylisticus convexus form a monophyletic group, as is also the case for Oniscus asellus, Porcellio scaber, Porcellionides pruinosus and Ligia oceanica Linné, 1767. These two groups are close to each other, but are distant from stocks infesting Porcellio spinicornis and Helleria brevicornis Ebner, 1868 (Cordaux et al. 2001). Sequencing using wsp and 16S rRNA primers shows that stocks infesting the Armadillidiidae (A. vulgare, A. album and A. nasatum) are distant from those infesting Porcellio dilatatus petiti. This is also the case for Porcellio spinicornis, which is more distant (Cordaux et al. 2004). This is the first study to focus on Wolbachia bacterium of Porcellio laevis species in two Tunisian populations. The aim of this research was to sequence the genomes of this population, with a view to establishing its phylogeny within the groups mentioned in the literature. Materials and methods Collection of animals Populations of terrestrial isopods belonging to Porcellio laevis species were collected in Bizerte and Lebna stations (Tunisia). The sex ratio (SR) was defined as SR = number of males (♂)/number of females (♀). DNA extraction and PCR amplification DNA from gonads and nervous tissue was extracted and purified using a Wizard RSV Genomic DNA Purification System (Promega, Madison, WI). The wsp (Wolbachia Ann Microbiol (2010) 60:43–50 45 Fig. 1a,b Anomalies of the male genital apparatus observed in Porcellio laevis species from Bizerte. a Remnant oviduct (O) leaving the intersection of the vas deferens (VD), seminal vesicle (SV). b Hypertrophied androgenic gland (AG) at the extremity of the testicular segments (T) in a phenotypically normal male Surface Protein) gene was amplified using the specific wsp primers 81F (5′ TGG TCC AAT AAG TGA TGA AGA AAC 3′) and 691R (5′ AAA AAT TAA ACG CTA CTC CA 3′) (Azzouna et al. 2004), in the following reaction mixture: 1X Promega buffer (50 mM KCl, 10 mM TrisHCl, pH 9; 0.1% X-100 Triton), 1.5 M MgCl2, 0.5µM dNTPs, 0.25µM forward primer, 0.25µM reverse primer, 0.5 U Taq polymerase, 1µl DNA, adjusted to a final volume of 50µl with H2O. PCR was performed in a thermal cycler (MyCycler Thermal Cycler, Bio-Rad, Hercules, CA) according to the following program: (1) initial denaturation at 95°C for 1 min; (2) 40 cycles of denaturation at 95°C for 1 min, annealing step at 55°C for 1 min, extension at 72°C for 2 min; and (3) a final extension at 72°C for 1 min. DNA fragments were resolved by electrophoresis on horizontal agarose gels whose Fig. 2 Amplification profiles of the wsp gene (on 1.5 % agarose gel) of four individuals: three Porcellio laevis (male, female and intersex female) and one Armadillidium vulgare. Lanes: M1 Molecular marker 50 bp, 1 negative control, 2 P. laevis normal ♂ from Lebna, 3 P. laevis infested ♀ from Lebna, 4 P. laevis infested ♂ from Bizerte, 5 P. laevis infested intersex ♀ from Bizerte, 6 A. vulgare infested ♀ from Bizerte, M2 molecular marker 1 Kb concentration was adjusted according to the size of the DNA fragments and the precision of separation required: 0.8% for genomic DNA, 1.5% for PCR wsp gene products. Samples were mixed with 6X loading buffer. Gels were prepared in 0.5X TBE buffer. Sequencing and phylogenetic analysis Sequencing of amplified wsp PCR products was carried out with incorporation of dideoxynucleotides (ddNTPs) using the chain termination method (Sanger et al. 1977). The nucleotide wsp gene sequence was aligned using ClustalX 1 (Thompson et al. 1997) and compared to reference sequences retrieved from public databases. Phylogenetic trees were constructed using TREECON for Windows software version 1.2 (Van de Peer and De Wachter 1994) and 46 the neighbour-joining algorithm (Saitou and Nei 1987). Bootstrap values were determined from 1,000 replicates (Felsentein 1985). Results Composition and dissection of the two populations Examination of the various specimens from the two populations revealed that the Lebna population consisted of 42 individuals composed of 9 males and 33 females, therefore with a female-biased SR (0.272), whereas the population of Bizerte consisted of 202 individuals showing three different phenotypes: 96 males with well developed copulator appendix, 45 females without copulator appendix, and 61 intersexual females characterised by a reduced copulator appendix; of these females, 34 were ovigerous. We noted that, during breeding, all the intersexual females have become ovigerous. Taking into account both the female and intersexual female phenotypes, the SR favours females. Fig. 3 Phylogenetic tree of Wolbachia microsymbionts based on wsp (Wolbachia surface protein) gene nucleotide sequences (algorithm: ClustalX 1) Ann Microbiol (2010) 60:43–50 The dissection of some individuals of the Lebna population showed no anomalies at the level of their genital apertures. On the contrary, the Bizerte population was confirmed to be composed of four phenotypes: normal males, normal females, functional intersexual females and functional males with female genital apertures characterised by a hypertrophied androgenic gland and a remnant oviduct at the base of the seminal vesicle (Fig. 1). Wolbachia infection and phylogeny Amplification of the wsp gene results in a single band of approximately 635 base pairs (bp) in size. Detection of this band in specimens from the two stations, i.e. in males and intersexual females in Bizerte, and in normal females in Lebna, confirmed the presence of the bacterium (Fig. 2). To consolidate the results, one amplified wsp gene obtained from a P. laevis individual from the Bizerte station was selected for sequencing. The reconstituted phylogeny based on the nucleotide (Fig. 3) and predicted protein sequences (Fig. 4) revealed an identity of 98% out of Ann Microbiol (2010) 60:43–50 47 Fig. 4 Phylogenetic tree of Wolbachia microsymbionts based on predicted WSP protein sequences (algorithm: ClustalX 1) 600 bp and 100% out of 190 amino acids compared with A. vulgare Wolbachia (DQ778096). Discussion Macroscopic examination of the SR suggested that the imbalance observed in the Lebna population (SR=0.27: female-biased sex ratios) and the strong intersexuality in the Bizerte population (61 individuals/202 total) may be due to the presence of Wolbachia—a bacterium reputed for its feminizing effect and disruption of male sexual differentiation. The population with strong thelygeny observed in Lebna led us to conclude that it contains females of phenotype f—meaning integration of the bacterial genome (G. Martin et al. unpublished data). Sex ratio fluctuations in favour of females were also reported in Tunisia by other authors in the species Porcellionides sexfasciatus Budde-Lund, 1885 in Garat Nâam station (Achouri and Charfi-Cheikhrouha 2002), Porcellio variabilis Lucas, 1846 (Medini-Bouaziz 2002) and Armadillidium pelagicum Arcangeli, 1955 (Hmaied and CharfiChekhrouha 2004). The first two authors proposed the hypothesis that this phenomenon could be due to the disappearance of a certain number of males after copulation, whereas the latter author suggested the presence of the feminizing bacterium as the underlying cause. However, neither dissection nor molecular studies were carried out prior to the present study. Within the genus Porcellio Latreille, 1804, the presence of this intersexuality phenomenon was reported in Porcellio dilatatus Brandt, 1833 (Juchault 1966) and for the first time in Porcellio laevis by Ben Nasr et al. (2009). In the Armadillidiidae family, this phenomenon was described in Armadillidium vulgare (Juchault and Legrand 1981; Legrand and Juchault 1986; Juchault and Mocquard 1989; Vandekerckhove-Tom et al. 2003; Azzouna et al. 2004) and Armadillidium nasatum (Juchault and Legrand 1979). Moreover, this intersexuality was observed in other isopod species such as Ligia oceanica (Martin et al. 1974) and Sphaeroma rugicauda Leach, 1814 (Martin et al. 1994). The high fertility (high frequency of ovigerous females in the population) and the presence of normal ovaries in the intersexes in individuals from Bizerte station prove that they are functional females. They are iF females according to the nomenclature of Legrand and Juchault (1986). They cannot be f females because the population is not strictly or strongly thelygenic (Legrand and Juchault 1986). 48 According to Azzouna et al. (2004), the anomaly revealed at the level of the gonads of phenotypically normal males— where the presence of remnant oviduct at the level of the vas deferens and hypertrophied androgenic glands at the extremity of the testicle segments were noted—suggests that this phenotype can be classified as ♂fga (functional male with female genital apertures). Porcellio laevis of the Bizerte population thus proved to be composed of four different phenotypes: normal males, ♂fga, normal females and intersex females iF. These various phenotypes do not have different degrees of intersexuality. This study also shows for the first time, and in P. laevis species, that intersexuality is due to disturbances of sexual differentiation caused by the bacterial endosymbiont Wolbachia—a bacterium recognised to have a very large geographical distribution (Bordenstein and Wernegreen 2004). This disturbing effect can be explained by an anomaly in the development of the androgenic gland that causes a reduction in the amount of circulating androgenic hormone (Azzouna et al. 2004)—the hormone responsible for male differentiation (Juchault et al. 1980). This explanation is based on observations of the abnormally hypertrophied androgenic glands. Wolbachia could also inhibit the receptors for these hormones, with the blockage in feedback generating development of gonadic outlines in ovaries (Felix 2004). The detection of a gene from the Wolbachia bacterium by PCR in normal females from Lebna station and in males and intersexes of the Bizerte population confirmed that the imbalance observed at the population from the former and the intersexuality revealed at the latter is due to the presence of the endosymbiont Wolbachia. The suggestion that the Lebna population is composed of female f, according to G. Martin et al. (unpublished), is advanced. It can be concluded from this that the bacterium causes more or less advanced degrees of sexual inversion: little apparent sexual inversion producing a male with female genital apertures (♂fga); more advanced inversion leads to iM sterile males; extreme inversion produces iF females, and total sexual inversion f females. These observations show that normal phenotypes can carry the bacterium, which explains the various bands observed on the gel shown in Fig. 2. The males of Bizerte station could only be ♂fga, and the females of Lebna station could be females f. There is some data in the literature suggesting that the Porcellionidae reaction to Wolbachia is a cytoplasmic histoincompatibility causing a high death rate (Martin et al. 1973). In contrast, the specimens studied, although they belong to Porcellionidae family, did not show this reaction but rather a functional feminization as observed in Armadillidiidae. The sequencing data and reconstitution of the phylogenetic tree reveal the close relationship with Wolbachia Ann Microbiol (2010) 60:43–50 infesting A. vulgare. This relationship can explain the feminizing effect of the bacterial stock on the studied species. There have been no previous reports in the literature concerning sequencing of the genome of Wolbachia infesting Porcellio laevis species. Sequencing of the Wolbachia genome for species close to Porcellio laevis (P. spinicornis, P. muscorum, P. scaber, Oniscus asellus, Helleria brevicornis and P. dilatatus petiti) showed that these species are more distant from the symbionts of Armadillidiidae (A. vulgare, A. album and A. nasatum; Bouchon et al. 1998; Cordaux et al. 2001, 2004). These results are in contradiction to those reported here. Further studies are required to resolve these apparent contradictions, and to further understand how this bacterial mechanism evolved, in particular development of the f female phenotype. Acknowledgements We thank Mr. Abdellatif Boudabous, Professor and Head of the Active Microorganisms and Bimolecular Laboratory of the Scientific Faculty of Tunis, for accommodating us in his laboratory; Hayet Ketfi, Faten Godbane, Chadlya Hamdi for their valuable assistance and Kouakou Anatole for his collaboration. 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