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Authors: Chiesa, S., Lucentini, L., Marzano, F.N., Breda, S., Figueira, E., Filonzi, L., Vaghi, M., Caill-Milly, N., Herbert, R.J.H. and Soares, A.

Start date: 14 October 2014

GENOTYPING OF MANILA CLAM VENERUPIS PHILIPPINARUM: SUCCESSES AND PITFALLS S. Chiesa*1, L. Lucentini2, R. Freitas1, F. Nonnis Marzano3, S. Breda4, E. Figueira1, L. Filonzi3, M. Vaghi3, N. Caill-Milly5, R. J. H. Herbert6, A. Soares1 and E. Argese4.

1Department of Biology & CESAM, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal) e-mail:

2Department of Chemistry, Biology and Biotechnologies, University of Perugia, Via Elce di Sotto, 06123 Perugia (Italy)

3Department of Life Sciences, University of Parma, Viale delle Scienze 11/a, 43124 Parma (Italy)

4Department of Molecular Sciences and Nanosystems, Ca’ Foscari University of Venice, Dorsoduro 2137, 30123 Venice (Italy)

5Ifremer, Laboratory Halieutic Resources from Aquitaine, UFR Sciences and Technics, 1 allées du Parc Montaury, 64600 Anglet (France)

6Centre for Conservation Ecology and Environmental Change, Faculty of Science and Technology, Bournemouth University, Christchurch House, Fern Barrow, Poole, Dorset, BH12 5BB (UK)

Introduction Manila clam Venerupis philippinarum - synonym Ruditapes philippinarum (Adams and Reeve, 1850) is a bivalve mollusc belonging to the family Veneridae. Originally from the Indo-Pacific region (Gosling, 2003), it has been introduced worldwide for aquaculture and has become one of the most successful marine invaders. In Europe, this species was originally introduced in ’70 and ’80 for commercial cultivation in Italy, England, Ireland, France, Spain and Portugal, yet self-sustaining, naturalised populations are now also commercially exploited as a fisheries resource. Nevertheless, the biology of the Manila clam is still poorly known, particularly the species genetics within invaded environments. This aspect is important to accurately determine the origin of stocks and traceability of clam products, concerning seafood safety. Consumer’s safety and food traceability are also some of objectives of the EC Horizon 2020 programme concerning “Sustainable and competitive agri-food sector for a safe and healthy diet; informed consumer choices; healthy and safe foods and diets for all”.

Materials and Methods Samples of manila clam were collected in 15 different productive areas in both Mediterranean (Northern Adriatic Sea, Italy) and Atlantic (England, France, Spain and Portugal) coasts of Europe. Twenty adults were collected in each area. Native clam Venerupis decussata (Linnaeus, 1758) was included as one of the out-groups for both mitochondrial and microsatellite analyses. High molecular weight genomic DNAs were extracted and purified from ethanol-fixed mantle tissue to avoid co-extraction of male type mtDNA (see Plazzi and Passamonti, 2010). Both mtDNA and nuclear DNA were analysed to compare different molecular markers: 16S rDNA and COI gene fragment were sequenced to be compared with sequences available in genomic databases. Samples were also genotyped for seven microsatellite loci: Asari 16, Asari 23, Asari 24, Asari 54 (Yasuda et al., 2007), Ktp5, Ktp8, Ktp22 (An et al., 2009). For detailed protocols, see Chiesa et al., 2011.

Results The 16S rDNA fragments were successfully amplified in most of the samples (80%) and compared with those available in GenBank. Eleven haplotypes were identified in European populations (GenBank A.N. KF736199-211), with haplotype 1 as the most frequent. Other haplotypes were rare and distributed at local scale, as for example only in Northern Adriatic Sea, Spain or Portugal. Limited reference sequences were available for comparison with native populations.

Due to the high variability observed in 16SrDNA gene, COI gene analyses are now currently ongoing to investigate the genetic structure of invasive populations with an additional marker, and sequences obtained will be then compared with more than 100 haplotypes already deposited in Genbank from native populations.

As for microsatellites, all loci were affected by missing amplifications. These observations are in accordance with Micro-checker results, showing that each locus could be globally affected by null-alleles, except for locus Asari 16 and Asari 54. The results of null-alleles analyses for single populations showed that all populations are affected by null alleles at almost one locus.

Discussion and conclusion The biogeographic and phylogenetic analyses of introduced Manila clam populations based on 16S rDNA showed a complex scenario, shaped by multiple introductions of mixed source populations, as a consequence of multiple founder effects. The mtDNA gave valuable results in terms of biogeographic structure and genetic diversity of the investigated populations. COI gene fragment analyses will be soon completed to compare the data obtained by 16S rDNA with an additional mitochondrial marker, and to provide more extensive comparisons with native Manila clam populations.

Yet, microsatellite results were strongly influenced by null alleles occurrence and heterozygosity deficits, as frequently observed in other bivalves, but not communicated in Manila clam literature. Microsatellites did not provide reliable data for investigated populations. These problems are due to the high number of generations and the occurrence of point mutations in the flanking regions. As a consequence, population genetics and stock identification of this species on a large scale could be difficult to achieve with only microsatellites, and should be assessed using new powerful markers, such as those obtained by NGS (Next Generation Sequencing) methods.

References An, H.S., Kim, E.M., Park, J.Y., 2009. Isolation and characterization of microsatellite markers for the clam Ruditapes philippinarum and cross-species amplification with the clam Ruditapes variegata. Conservation Genetics, 10: 1821-1823.

Chiesa, S., Nonnis Marzano, F., Minervini, G., De, Lucrezia D., Baccarani, G., et al., 2011. The invasive manila clam Ruditapes philippinarum (Adams & Reeve, 1850) in Northern Adriatic Sea: population genetics assessed by an integrated molecular approach. Fisheries Research, 110: 259-267.

Gosling, E., 2003. Bivalve Molluscs: Biology, Ecology and Culture. Fishing News Books, Blackwell Publishing, Oxford.

Plazzi, F., Passamonti, M., 2010. Towards a molecular phylogeny of mollusks: bivalves’ early evolution as revealed by mitochondrial genes. Molecular Phylogenetics and Evolution, 57: 641-657.

Yasuda, N., Nagai, S., Yamaguchi, S., Lian, C.L., Hamaguchi, M., 2007. Development of microsatellite markers for the manila clam Ruditapes philippinarum. Molecular Ecology Resources, 7: 43-45.

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