Proceedings of the 8th International Symposium of Terrestrial Isopod Biology ISTIB 2011, Bled, Slovenia
Wolbachia DIVERSITY WITHIN Porcellionides SPECIES COMPLEX F. Grandjean1, S. Verne1, E. Séchet2, S. Taiti3, F. Vilisics4, S. Sfenthourakis5 & M. Johnson1 1
LEES “Ecologie, Evolution, Symbiose” UMR 6556, Poitiers, France. E-mail:
[email protected] 2 20 rue de la résistance, F-49125 Cheffes, France. E-mail :
[email protected] 3 Istituto per lo Studio degli Ecosistemi, Consiglio Nazionale delle Ricerche Via Madonna del Piano 10 50019 Sesto Fiorentino (Firenze), Italy E-mail:
[email protected] 4 Helsinki University, Fact. Biol. And Env. Sci., Department of Environmental Sciences 00014 Helsinki, Viikkinkari 1, Finland. E-mail:
[email protected] 5 Section of Animal Biology Department of Biology University of Patras GR-26500 Patra Greece. E-mail:
[email protected] ABSTRACT: Wolbachia are maternally inherited endosymbiotic -Proteobacteria infecting a wide range of arthropods. Wolbachia induce feminization in many terrestrial isopod species such as Porcellionides pruinosus (Crustacea, Oniscidea). The diversity of Wolbachia strains infecting P. pruinosus was examined from 23 populations sampled mainly in Europe. Results reveal that natural populations of P. pruinosus contain five different Wolbachia strains (PruI to PruV). The polymorphism of infection is different among strains. PruI infects only females (at a rate of 60%), PruII infects both sexes (with 30% rate in males and 40% rate in females), while PruIII, IV and V infect both sexes at 100% rate.Host divergence from 16S RNA gene is high between some specimens probably due to the fact that P. pruinosus is actually a ‘species complex’. Keywords: Wolbachia, Porcellionides pruinosus species complex, genome plasticity, symbiosis. 1. Introduction Wolbachia is an alpha-proteobacterium occuring in 15-75% of all insects and present also in crustaceans, spiders and nematodes. It causes a number of reproductive alterations in their hosts, including induction of thelytokous parthenogenesis, male-killing and cytoplasmic incompatibility (reviewed in Stouthamer et al. 1999). In most of terrestrial isopods (woodlice), Wolbachia bacteria are mainly responsible for sex reversion and female-biased sex ratios, changing genetic males into functional neo-females. Males are generally free of Wolbachia. Compared to other isopods, the pattern of Wolbachia infection in Porcellionides pruinosus is rather atypical, since infection affects both sexes, with infection ratio in natural populations up to 100% of inspected individuals (Marcadé et al. 1999). Michel-Salzat et al. (2001) and Lefebvre & Marcadé (2005) reported the existence of two sibling species of P. pruinosus, one in western Europe called the French group and the other in Greece, Réunion island and Tunisia. Sibling species showed two different patterns of infection. The Type 1 of infection was found in the four French populations and in a Spanish one, where Wolbachia infected both sexes with a high prevalence, ranging between 80 to 100% of individuals. Type 2 infections has been described in three populations from Tunisia, Greece and Réunion island, where Wolbachia was found in females only, at a rate of approximately 60% of individuals. The aims of this study were: (i) To describe the diversity of Wolbachia strains infecting P.
pruinosus using the 7 genes commonly present in Wolbachia genome; (ii) To check the infection status of animals according to sex; (iii) To verify the taxonomic status of the host by a molecular approach based on the sequencing of the mitochondrial 16S RNA gene; (iv) To show the recombination between strains of the wsp gene coding for an outer membrane protein. 2. Materials & Methods 2.1 Presence of Wolbachia and sequencing 23 isopod populations have been sampled. PCR assays were performed to test for the presence of Wolbachia, by amplification of wsp genes. Positive and negative controls using known infected and uninfected isopods (Armadillidium vulgare, laboratory strains) were performed with each set of PCR reactions. We also sequenced six other Wolbachia genes, namely groE operon and 5 genes widely distributed across the Wolbachia genome, which were used in one of the two available multilocus sequence typing approaches (Baldo et al. 2006; Paraskevopoulos et al. 2006), named: ftsZ, gatB, coxA, hcpA and fbpA. Sequenced individuals testing positive for Wolbachia were used for both host mitochondrial and Wolbachia gene sequencing. The “wsp” region of the Wolbachia DNA was amplified using the specific primers wsp81F and wsp691R. After purification, the both strins of PCR products were sequenced with the amplification primer set, using the ABI Prism BigDye terminator cycle sequencing kit (PE Applied Biosystems, Foster City).
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Proceedings of the 8th International Symposium of Terrestrial Isopod Biology ISTIB 2011, Bled, Slovenia Sequences were analysed on ABI 310 automated sequencer. 2.2 Taxonomic status of host The 3’-half end of the SSU rDNA region of the host's mitochondrial DNA was amplified using the specific primers 16Sar and 16Sbr. 3. Results & Discussion 3.1 Wolbachia presence Wolbachia infection was confirmed in all analysed populations, in which five strains of Wolbachia, named wPruI to wPruV, have been characterized from 250 positive specimens tested. Three types of infection were detected. Type 1, where infection occurs in 100% of individuals of both sexes was detected in all French, two Italian, one Spanish and two Hungarian populations (Strains wPruIII, IV and V). The two Tuscanian populations harboured wPruIII and wPruV. Type 2 includes only infection by wPruI, occurring in females only, at a rate around 60%. It was detected in populations from Athens (GR), Tunis (TU), Haïfa (ISR) and SaintPaul (La Réunion FR). Type 3 has been caused by wPruII strain and infected males and females at a rate that ranged between 30% and 40% respectively. It was detected from Manzanares (ES) and Camarade (FR). No relationship exists between the genetic characteristics of strains considering the wsp gene and the infection status, because wPruI and wPruIII is genetically closely related but their infection status are different. The effects on host of these strains are unknown for most of them. wPrIII is a feminizing bacterium (Marcadé et al. 1999) even if it is present in males. Preliminary studies based on quantitative PCR have showed that Wolbachia density is lowest in males than in females. The mitochondrial 16S RNA sequences showed a high divergence between isopod specimens from different localities, probably due to the presence of different species (see for example P. percanus in Lefebvre and Marcadé 2005). In P. pruinosus, the same haplotype was shared between French and Spanish populations but with two different strains of Wolbachia. This is a good opportunity to test the effect of Wolbachia in host reproduction. Crossing experiments are in progress to test the role of Wolbachia in reproductive isolation. The sequences of different Wolbachia from the wsp gene showed recombination events, probably due to horizontal transfers of Wolbachia which seems frequent in isopods (Verne et al. 2007)
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4. References Baldo, L., et al., 2006. Multilocus sequence typing system for the endosymbiont Wolbachia pipientis. Applied Environmental Microbiology 72, 7098–7110. Lefebvre, F., Marcadé, I. (2005) New insights in the Porcellionides pruinosus complex (Isopoda, Oniscidea): Biological, behavioural, and morphological approaches. Crustaceana, 78: 465480. Marcadé I., Souty-Grosset C., Bouchon D., Rigaud T. & Raimond R. (1999) Mitochondrial DNA variability and Wolbachia infection in two sibling woodlice species. Heredity, 83: 71-78. Paraskevopoulos, C., Bordenstein, S.R., Wernegreen, J.J., Werren, J.H., Bourtzis, K., 2006. Toward a Wolbachia multilocus sequence typing system: discrimination of Wolbachia strains present in Drosophila species. Current Microbiology 53, 388–395. Michel-Salzat, A., Cordaux, R., Bouchon, D., 2001. Wolbachia diversity in the Porcellionides pruinosus complex of species (Crustacea, Oniscidea): evidence for host-dependant patterns of infection. Heredity 87, 428–434. Stouthamer, R., Breeuwer, J.A., Hurst, G.D.D., 1999. Wolbachia pipientis: microbial manipulator of arthropod reproduction. Annual of Review of Microbiology 53, 71–102 Verne, S., M. Johnson, D. Bouchon, and F. Grandjean. 2007. “Evidence for recombination between feminizing Wolbachia in the isopod genus Armadillidium.” Gene 397 (1-2): 58–66.
Molecular biology and microbiology
