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Development of Species-Specific Edna-Based Test Systems For REPORT SNO 7544-2020 Development of species-specific eDNA-based test systems for monitoring of non-indigenous Decapoda in Danish marine waters © Henrik Carl, Natural History Museum, Denmark History © Henrik Carl, Natural NIVA Denmark Water Research REPORT Main Office NIVA Region South NIVA Region East NIVA Region West NIVA Denmark Gaustadalléen 21 Jon Lilletuns vei 3 Sandvikaveien 59 Thormøhlensgate 53 D Njalsgade 76, 4th floor NO-0349 Oslo, Norway NO-4879 Grimstad, Norway NO-2312 Ottestad, Norway NO-5006 Bergen Norway DK 2300 Copenhagen S, Denmark Phone (47) 22 18 51 00 Phone (47) 22 18 51 00 Phone (47) 22 18 51 00 Phone (47) 22 18 51 00 Phone (45) 39 17 97 33 Internet: www.niva.no Title Serial number Date Development of species-specific eDNA-based test systems for monitoring 7544-2020 22 October 2020 of non-indigenous Decapoda in Danish marine waters Author(s) Topic group Distribution Steen W. Knudsen and Jesper H. Andersen – NIVA Denmark Environmental monitor- Public Peter Rask Møller – Natural History Museum, University of Copenhagen ing Geographical area Pages Denmark 54 Client(s) Client's reference Danish Environmental Protection Agency (Miljøstyrelsen) UCB and CEKAN Printed NIVA Project number 180280 Summary We report the development of seven eDNA-based species-specific test systems for monitoring of marine Decapoda in Danish marine waters. The seven species are 1) Callinectes sapidus (blå svømmekrabbe), 2) Eriocheir sinensis (kinesisk uldhånds- krabbe), 3) Hemigrapsus sanguineus (stribet klippekrabbe), 4) Hemigrapsus takanoi (pensel-klippekrabbe), 5) Homarus ameri- canus (amerikansk hummer), 6) Paralithodes camtschaticus (Kamchatka-krabbe) and 7) Rhithropanopeus harrisii (østameri- kansk brakvandskrabbe). The following three are new developments: Callinectes sapidus, Hemigrapsus sanguineus and Hemi- grapsus takanoi, whilst the remaining four previously developed under earlier phases of the MONIS projects have been tested again. The additional tests on the four previously developed assays were performed with a broader diversity of Decapoda known from Danish seas, to re-evaluate the specificity of these assays. This additional testing revealed that two previously pub- lished assays were unspecific, and to accommodate this, two new assays are presented replacing those previously published. We recommend that the species-specific eDNA assays presented here will allow for continuous monitoring of these species as part of the NOVANA monitoring programme. Four keywords Fire emneord 1. Non-indigenous species 1. Ikke-hjemmehørende arter 2. eDNA 2. eDNA 3. Monitoring 3. Overvågning 4. Marine Strategy Framework Directive 4. Havstrategidirektivet This report is quality assured in accordance with NIVA's quality system and approved by: Jesper H. Andersen Jørgen Bendtsen Project Manager Office Manager ISBN 978-82-577-7279-6 NIVA-report ISSN 1894-7948 © Norsk institutt for vannforskning/Norwegian Institute for Water Research. The publication can be cited freely if the source is stated. MONIS 5 Development of species-specific eDNA-based test systems for monitoring of non-indigenous Decapoda in Danish marine waters Client: Danish Environmental Protection Agency NIVA 7544-2020 Preface We report the development of seven species-specific eDNA-based test systems for monitoring of non-indigenous marine Decapoda in Danish marine waters. The work has been funded by the Danish Environmental Protection Agency as an additional activity from the MONIS project (‘Monitoring of Non-Indigenous Species in Danish Marine Waters’) and been carried out collectively by NIVA Den- mark (lead partner) and the Natural History Museum of Denmark (NHMD). Thanks are due to: Kathe Jensen from the Natural History Museum of Denmark (NHMD), Jørgen Olsen (NHMD), Tom Schøitte (NHMD), Sune Agersnap (NHMD) and Danny Eibye Jacobsen (NHMD) for helping with taxonomical identification and loan of samples from NHMD, Ann Merete Hjelset (Havforskningsinstituttet, Norway) for providing samples of Paralithodes camchaticus, Kristian and Andreas at ‘Øresundsakvariet’ for providing access to various species of marine invertebrates from which we could obtain tissue samples, ‘Fiskeri- og Søfartsmuseet’ in Esbjerg for providing samples from Decapoda, and Henrik Carl at the Natural History Museum of Denmark for loan of photographs, including the photo of Eriocheir sinensis. More information about the MONIS project (phases 1-5) can be found in: ‘Steps toward nation-wide monitoring of non-indigenous species in Danish marine waters under the Marine Strategy Framework Directive’ (Andersen et al. 2016), ‘Development of species-specific eDNA-based test systems for monitoring of non-indigenous species in Danish marine waters’ (Andersen et al. 2018), ‘Tekniske anvisninger for eDNA-baseret overvågning af ikke-hjemmehørende marine arter’ (Knudsen et al. 2018), and ‘A baseline study of the occurrence of non-indigenous species in Danish harbours’ (Andersen et al. 2019). Copenhagen, 22 October 2020 Jesper H. Andersen Project Manager NIVA 7544-2020 Table of contents 1 Introduction ........................................................................................................................... 6 2 Methods................................................................................................................................. 7 2.1 Conditions of the specificity test – in silico testing ................................................................ 9 2.2 Laboratorial test of specificity – in vitro testing .................................................................. 10 3 Results ................................................................................................................................. 12 3.1 Development and testing of new assays ............................................................................. 12 Species-specific assay for detection of Callinectes sapidus ................................... 12 Species-specific assay for detection of Hemigrapsus sanguineus .......................... 17 Species-specific assay for detection of Hemigrapsus takanoi ................................ 22 3.2 Further development and testing of existing assays ........................................................... 26 Species-specific assay for detection of Eriocheir sinensis ...................................... 26 Species-specific assay for detection of Homarus americanus ................................ 30 Species-specific assay for detection of Paralithodes camtschaticus ...................... 38 Species-specific assay for detection of Rhithropanopeus harrisii .......................... 45 4 Conclusions .......................................................................................................................... 51 5 References ........................................................................................................................... 52 4 NIVA 7544-2020 Sammenfatning Titel: Udvikling af artsspecifikke eDNA-baserede testsystemer til overvågning af ikke-hjemmehørende krabber i de danske farvande År: 2020 Forfatter(e): Steen W. Knudsen, Jesper H. Andersen og Peter Rask Møller Udgiver: Norsk institutt for vannforskning, ISBN 978-82-577-7279-6 For at kunne spore ikke-hjemmehørende arter af marine invasive tibenede krebsdyr, ved hjælp af DNA-niveauer i filtrerede vandprøver, er der for denne rapport udviklet og testet tre nye artsspeci- fikke sporingssystemer, og for overblikkets skyld er fire tidligere udviklede og testede eDNA-sporings- systemer ligeledes efterkontrolleret, testet igen og enten inkluderet igen, eller erstattet af mere præcise eDNA-sporingssystemer. Ved brug af kvantitativ PCR (quantitative polymerase chain reaction) (qPCR) er det med disse systemer nu muligt at spore eDNA i vandprøver fra seks ikke-hjemmehørende tibenede krebsdyr i danske farvande, og det er muligt at vurdere niveauerne af miljø-DNA (environmental DNA, eDNA) i vandprøverne fra de enkelte arter. Alle syv sporingssystemer er blevet designet og testet både på DNA fra vævsprøver fra de eftersøgte arter, men også på DNA fra andre sameksisterende og hjemmehørende arter repræsenterende marine tibenede krebsdyr (Decapoda). De tre nye sporingssystemer er her eftervist som værende artsspecifikke og således i stand til at spore DNA fra ’pensel klippekrabbe’, ’den blå svømmekrabbe’ og ’stribet klippe-krabbe’. Specificiteten for hvert sporingssystem er eftervist først ud fra sammenligning af nukleotidsekvenser hvor de enkelte primere og en passende ’hydrolysis probe’ kan binde til et specifikt område i det mitokondrielle genom for den eftersøgte organisme. Primer og probe kombinationen er derpå testet under standardiserede temperaturomstændigheder i en qPCR opsætning, hvor den unikke sammen- sætning af nukleinsyrer i DNA sekvensen er med til at sikre at kun DNA fra den eftersøgte art regi- streres. Med qPCR-tests af forskelige kombinationer af primere og prober kan effektiviteten og specificiteten af kombinationerne så vurderes for at finde frem til den optimale kombination der senerehen bør bruges på indsamlede vandprøver. Sammenligningen med nukleotid-sekvenser fra andre arter af tibenede krebsdyr blev udført ved at identificere variable gen-regioner i de mitokon- drielle genregioner: cytochrome oxidase 1 (mtDNA-co1) og cytochrome b (mtDNA-cytb). Nukleotid sekvenser af mtDNA-co1 og mtDNA-cytb blev enten indhentet fra en genetisk database eller ind- hentet ved de novo-sekventering af DNA ekstraheret fra vævsprøver indsamlet fra de syv arter. Blandt de sporingssystemer
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