PCR-based assay for Mya arenaria detection from marine environmental samples and tracking its invasion in coastal ecosystems

Abstract The introduction of invasive species into a new environment is one of the most important factors implicated in loss of native biodiversity. Their early detection is indispensable to efficient control and management response, because after their incursion and establishment the eradication is nearly impossible. The Mediterranean Sea is one of the world´s hotspots of biological invasions and existing monitoring programs are often insufficient for effective surveillance of the established invasive populations or early detection of new incursions. The aim of the current study was to design a specific molecular marker for detecting and monitoring the soft shell clam Mya arenaria, listed among 100 Mediterranean Worst Invasive Species, from environmental samples. The 16S marker was designed in silico and validated in vitro on serial dilutions of M. arenaria DNA and other closely related mollusk species; its performance was also tested on complex environmental samples collected from locations in the Baltic Sea with known established M. arenaria population. The new marker allows amplifying small fragments of 279 bp and detecting as low as 0.00103 ng/μl of M. arenaria DNA with no cross-amplification detected. We suggest the use of this and other species-specific markers for targeted surveillance of invasive species and prompt detection of the prospective incursions. Such monitoring approach has the potential to be adopted by citizen science programs due to its easy implementation, minimal technical skills requirements and reasonable amount of associated expenditures.

[1]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[2]  Jeffrey A. Crooks,et al.  Characterizing ecosystem‐level consequences of biological invasions: the role of ecosystem engineers , 2002 .

[3]  Jennifer L. Molnar,et al.  Assessing the global threat of invasive species to marine biodiversity , 2008 .

[4]  James T. Carlton,et al.  Remarkable invasion of San Francisco Bay (California, USA), by the Asian clam Potamocorbula amurensis. I. Introduction and dispersal , 1990 .

[5]  F. Briand.  CIESM atlas of exotic species in the Mediterranean , 2002 .

[6]  T. Notomi,et al.  Loop-mediated isothermal amplification of DNA. , 2000, Nucleic acids research.

[7]  P. Castro-Díez,et al.  What explains the invading success of the aquatic mud snail Potamopyrgus antipodarum (Hydrobiidae, Mollusca)? , 2008, Hydrobiologia.

[8]  C. Wiuf,et al.  Monitoring endangered freshwater biodiversity using environmental DNA. , 2012, Molecular ecology.

[9]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Matthew A. Barnes,et al.  The ecology of environmental DNA and implications for conservation genetics , 2016, Conservation Genetics.

[11]  Barrie M. Forrest,et al.  Population control to mitigate the spread of marine pests: insights from management of the Asian kelp Undaria pinnatifida and colonial ascidian Didemnum vexillum , 2013 .

[12]  S. Pierce,et al.  The effects of salinity stress on the electrophysiological properties ofMya arenaria neurons , 1981, Journal of comparative physiology.

[13]  David L. Strayer,et al.  Mollusks as ecosystem engineers: the role of shell production in aquatic habitats , 2003 .

[14]  Michael J. Keough,et al.  Introduced and cryptogenic species in Port Phillip Bay, Victoria, Australia , 2004 .

[15]  Katsanevakis Stylianos Marios,et al.  Invading the Mediterranean Sea: human-shaped biodiversity patterns , 2014 .

[16]  Michael Elliott,et al.  Recommendations on methods for the detection and control of biological pollution in marine coastal waters. , 2011, Marine pollution bulletin.

[17]  Anastasija Zaiko,et al.  New specific molecular marker detects Ficopomatus enigmaticus from water eDNA before positive results of conventional sampling , 2017, Journal for Nature Conservation.

[18]  E. Garcia-Vazquez,et al.  An extremely sensitive nested PCR-RFLP mitochondrial marker for detection and identification of salmonids in eDNA from water samples , 2017, PeerJ.

[19]  Xavier Pochon,et al.  Assessing the effects of salmon farming seabed enrichment using bacterial community diversity and high-throughput sequencing. , 2015, FEMS microbiology ecology.

[20]  W. L. Chadderton,et al.  “Sight‐unseen” detection of rare aquatic species using environmental DNA , 2011 .

[21]  G. Stora,et al.  Time and spatial dynamics of Mediterranean lagoon macrobenthos during an exceptionally prolonged interruption of freshwater inputs , 1995, Hydrobiologia.

[22]  C. Goldberg,et al.  Characterizing the distribution of an endangered salmonid using environmental DNA analysis , 2015 .

[23]  Anastasija Zaiko,et al.  Early detection of eukaryotic communities from marine biofilm using high-throughput sequencing: an assessment of different sampling devices , 2015, Biofouling.

[24]  John A Darling,et al.  From molecules to management: adopting DNA-based methods for monitoring biological invasions in aquatic environments. , 2011, Environmental research.

[25]  A. Sfriso,et al.  Current status and trends of biological invasions in the Lagoon of Venice, a hotspot of marine NIS introductions in the Mediterranean Sea , 2015, Biological Invasions.

[26]  Keith R Hayes,et al.  Biological invasions: recommendations for U.S. policy and management. , 2006, Ecological applications : a publication of the Ecological Society of America.

[27]  M. Miya,et al.  Environmental DNA metabarcoding reveals local fish communities in a species-rich coastal sea , 2017, Scientific Reports.

[28]  Anastasija Zaiko,et al.  eDNA and specific primers for early detection of invasive species--A case study on the bivalve Rangia cuneata, currently spreading in Europe. , 2015, Marine environmental research.

[29]  D. Lodge,et al.  Environmental DNA (eDNA) detects the invasive rusty crayfish Orconectes rusticus at low abundances , 2016, The Journal of applied ecology.

[30]  M. Coll,et al.  Invading the Mediterranean Sea: biodiversity patterns shaped by human activities , 2014, Front. Mar. Sci..

[31]  R. Freckleton,et al.  Declines in the numbers of amateur and professional taxonomists: implications for conservation , 2002 .

[32]  S. Monari.  Phylogeny and Biogeography of Pholadid Bivalve Barnea (Anchomasa) with Considerations on the Phylogeny of Pholadoidea , 2009 .

[33]  E. Garcia-Vazquez,et al.  Detection and characterisation of the biopollutant Xenostrobus securis (Lamarck 1819) Asturian population from DNA Barcoding and eBarcoding. , 2016, Marine pollution bulletin.

[34]  Douglas W. Yu,et al.  Environmental DNA for wildlife biology and biodiversity monitoring. , 2014, Trends in ecology & evolution.

[35]  Á. Borja,et al.  Environmental Status Assessment Using DNA Metabarcoding: Towards a Genetics Based Marine Biotic Index (gAMBI) , 2014, PloS one.

[36]  F. Andaloro,et al.  Annotated list of marine alien species in the Mediterranean with records of the worst invasive species , 2005 .

[37]  D. Lercari,et al.  Impacts of two invasive mollusks, Rapana venosa (Gastropoda) and Corbicula fluminea (Bivalvia), on the food web structure of the Río de la Plata estuary and nearshore oceanic ecosystem , 2011, Biological Invasions.

[38]  Ryo Sakurai,et al.  Citizen science: a new approach to advance ecology, education, and conservation , 2015, Ecological Research.

[39]  C. Günther.  Settlement and recruitment of Mya arenaria L. in the Wadden Sea , 1992 .

[40]  S. Ball,et al.  DNA barcodes for biosecurity: invasive species identification , 2005, Philosophical Transactions of the Royal Society B: Biological Sciences.

[41]  D. Lodge,et al.  An ounce of prevention or a pound of cure: bioeconomic risk analysis of invasive species , 2002, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[42]  S. Focardi,et al.  Potential role of cholinesterases in the invasive capacity of the freshwater bivalve, Anodonta woodiana (Bivalvia: Unionacea): a comparative study with the indigenous species of the genus, Anodonta sp. , 2007, Comparative biochemistry and physiology. Toxicology & pharmacology : CBP.

[43]  Louis A. Tremblay,et al.  Molecular genetic tools for environmental monitoring of New Zealand's aquatic habitats, past, present and the future , 2013 .

[44]  Margaret R. Caldwell,et al.  Harnessing DNA to improve environmental management , 2014, Science.

[45]  Anastasija Zaiko,et al.  Metabarcoding approach for the ballast water surveillance--an advantageous solution or an awkward challenge? , 2015, Marine pollution bulletin.

[46]  E. Garcia-Vazquez,et al.  Food control and a citizen science approach for improving teaching of Genetics in universities , 2016, Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology.

[47]  X. Pochon,et al.  Evaluating Detection Limits of Next-Generation Sequencing for the Surveillance and Monitoring of International Marine Pests , 2013, PloS one.

[48]  Cohen,et al.  Accelerating invasion rate in a highly invaded estuary , 1998, Science.

[49]  E. Garcia-Vazquez,et al.  Controlling populations of invasive pygmy mussel (Xenostrobus securis) through citizen science and environmental DNA. , 2016, Marine pollution bulletin.

[50]  D. Strayer.  Alien species in fresh waters: ecological effects, interactions with other stressors, and prospects for the future , 2010 .

[51]  Herdina,et al.  Toward routine, DNA-based detection methods for marine pests. , 2010, Biotechnology advances.

[52]  E. Papastergiadou,et al.  Aquatic alien species in Greece (2009): tracking sources, patterns and effects on the ecosystem. , 2009 .

[53]  J. Geller,et al.  Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all‐taxa biotic surveys , 2013, Molecular ecology resources.

[54]  S. Palumbi,et al.  Nucleic acids II: the polymerase chain reaction , 1996 .

[55]  Barrie M. Forrest,et al.  Successful eradication of a non‐indigenous marine bivalve from a subtidal soft‐sediment environment , 2011 .

[56]  F. Crocetta,et al.  Mya arenaria Linné, 1758 (Mollusca: Bivalvia) in the Mediterranean Sea: its distribution revisited , 2011 .

[57]  Susan L. Williams,et al.  Eradication of the invasive seaweed Caulerpa taxifolia by chlorine bleach , 2004 .

[58]  M. Çınar,et al.  An updated review of alien species on the coasts of Turkey , 2011 .

[59]  E. Garcia-Vazquez,et al.  Learning gains in lab practices: teach science doing science , 2014 .

[60]  S. Katsanevakis,et al.  Impacts of invasive alien marine species on ecosystem services and biodiversity: a pan-European review. , 2014 .

[61]  Amy J. Benson,et al.  Chronological history of zebra and quagga mussels (Dreissenidae) in North America, 1988-2010 , 2013 .

[62]  P. Taberlet,et al.  Species detection using environmental DNA from water samples , 2008, Biology Letters.

[63]  Marinko Učur,et al.  International convention for the control and management of ships’ ballast water and sediments (imo, 2004) , 2011 .

[64]  Sergej Olenin,et al.  Vulnerability of benthic habitats to the aquatic invasive species , 2007, Biological Invasions.

[65]  R. Rosenberg,et al.  Recruitment, abundance and production of Mya arenaria and Cardium edule in marine shallow waters, Western Sweden , 1983 .

[66]  H. MacIsaac,et al.  Are genetic databases sufficiently populated to detect non-indigenous species? , 2016, Biological Invasions.

[67]  E. Garcia-Vazquez,et al.  Novel tools for early detection of a global aquatic invasive, the zebra mussel Dreissena polymorpha , 2017 .

[68]  H. Mooney,et al.  Effect of aquaculture on world fish supplies , 2000, Nature.

[69]  E. Leppäkoski.  Introduced species-resource or threat in brackish-water seas ? Examples from the Baltic and the Black Sea , 1991 .

[70]  E. Garcia-Vazquez,et al.  An Easy Phylogenetically Informative Method to Trace the Globally Invasive Potamopyrgus Mud Snail from River’s eDNA , 2016, PloS one.

[71]  A. Zenetos,et al.  Alien Marine Species in the Mediterranean - the 100 'Worst Invasives' and their Impact , 2006 .

[72]  Yasuyoshi Mori,et al.  Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products , 2008, Nature Protocols.

[73]  Richard F. Piola,et al.  Marine biosecurity: management options and response tools , 2009 .

[74]  R. May,et al.  Can We Name Earth's Species Before They Go Extinct? , 2013, Science.

[75]  T. A. Hall,et al.  BIOEDIT: A USER-FRIENDLY BIOLOGICAL SEQUENCE ALIGNMENT EDITOR AND ANALYSIS PROGRAM FOR WINDOWS 95/98/ NT , 1999 .

[76]  Anastasija Zaiko,et al.  Metabarcoding approach for nonindigenous species surveillance in marine coastal waters. , 2015, Marine pollution bulletin.

[77]  A. Ricciardi.  Global range expansion of the Asian mussel Limnoperna fortunei (Mytilidae): Another fouling threat to freshwater systems , 1998 .

[78]  Matej David,et al.  Dose of truth—Monitoring marine non-indigenous species to serve legislative requirements , 2015 .

[79]  A. Zenetos,et al.  Globalisation in marine ecosystems: the story of non-indigenous marine species across European seas , 2005 .