Gen-2018-0021.Pdf

Genome Considerations for incorporating real-time PCR assays into routine marine biosecurity surveillance programmes: a case study targeting the Mediterranean fanworm (Sabella spallanzanii) and club tunicate (Styela clava) Journal: Genome Manuscript ID gen-2018-0021.R3 Manuscript Type: Article Date Submitted by the 19-Jun-2018 Author: Complete List of Authors: WOOD, Susanna; Cawthron Institute, Pochon, Xavier;Draft Cawthron Institute; University of Auckland Ming, Witold; Cawthron Institute von Ammon, Ulla; Cawthron Institute; University of Auckland Woods, Chris; National Institute of Water & Atmospheric Research Ltd Carter, Megan; National Institute of Water & Atmospheric Research Ltd Smith, Matt; National Institute of Water & Atmospheric Research Ltd Inglis , Graeme ; National Institute of Water & Atmospheric Research Ltd Zaiko, Anastasija ; Cawthron Institute; University of Auckland Environmental DNA and RNA, Non-indigenous species, Occupancy Keyword: models, Real-time Polymerase Chain Reaction, Surveillance Is the invited manuscript for consideration in a Special 7th International Barcode of Life Issue? : Note: The following files were submitted by the author for peer review, but cannot be converted to PDF. You must view these files (e.g. movies) online. Fig 1 - compressed.tif https://mc06.manuscriptcentral.com/genome-pubs Page 1 of 38 Genome 1 Considerations for incorporating real-time PCR assays into routine marine biosecurity 2 surveillance programmes: a case study targeting the Mediterranean fanworm (Sabella 3 spallanzanii) and club tunicate (Styela clava) 4 5 6 Susanna A Wood1*, Xavier Pochon1,3, Witold Ming1, Ulla von Ammon1,2, Chris Woods4, Megan 7 Carter4, Matt Smith4, Graeme Inglis4, Anastasija Zaiko1,2 8 1 Coastal and Freshwater Group, Cawthron Institute, Nelson, New Zealand 9 2 School of Biological Sciences, University of Auckland, Auckland, New Zealand 10 3 Institute of Marine Science, University of Auckland, Auckland, New Zealand 11 4 National Institute of Water & Atmospheric Research Ltd, New Zealand 12 13 *corresponding authors: Coastal and Freshwater Group, Cawthron Institute, 98 Halifax Street East, 14 7010, Nelson, New Zealand. [email protected] 15 16 17 18 19 20 21 22 23 24 25 26 27 1 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 2 of 38 28 29 30 31 32 Abstract 33 Molecular techniques may provide effective tools to enhance marine biosecurity surveillance. 34 Prior to routine implementation, evidence-based consideration of their benefits and limitations is 35 needed. In this study, we assessed the efficiency and practicality of visual diver surveys and real-time PCR 36 assays (targeting DNA and RNA) for detecting two marine invasive species whose infestation levels varied 37 between species and location; Sabella spallanzanii and Styela clava. Filtered water samples (n=171) 38 were collected in parallel with dive surveys atDraft two locations as part of the New Zealand Marine High Risk Site 39 Surveillance programme: Nelson Harbour (27 sites) and Waitemata Harbour (30 sites). Diver surveys 40 resulted in a greater number of detections compared to real-time PCR: S. clava – 21 versus 5 sites 41 in Nelson, 6 versus 1 in Auckland; S. spallanzanii – 18 versus 10 in Auckland, no detections in 42 Nelson. Occupancy modelling derived detection probabilities for the real-time PCR for S. clava 43 were low (14%), compared to S. spallanzanii (66%). This could be related to abundances, or 44 species-specific differences in DNA shedding. Only one RNA sample was positive, suggesting 45 that most detections were from extracellular DNA or non-viable fragments. While molecular 46 methods cannot yet replace visual observations, this study shows they provide useful 47 complementary information. 48 49 Key words: Environmental DNA and RNA, Non-indigenous species, Occupancy models, Real- 50 time Polymerase Chain Reaction, Surveillance 2 https://mc06.manuscriptcentral.com/genome-pubs Page 3 of 38 Genome 51 Draft 3 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 4 of 38 52 Introduction 53 The arrival and establishment of marine non-indigenous species can have dramatic effects on the 54 structure and functioning of coastal ecosystems (Galil 2007; Wallentinus and Nyberg 2007; 55 Ehrenfeld 2010). Early detection and monitoring of marine non-indigenous species has become a 56 priority in many countries. Surveillance programmes commonly rely on techniques that use 57 morphological identification of organisms detected in samples or in situ, e.g., visual surveys by 58 divers (Cohen et al. 2001; Hewitt and Martin 2001; Inglis et al. 2006). Divers can detect moderate- 59 sized organisms even when present at relatively low densities, with limited water visibility (> 0.8 60 m Secchi depth) (Inglis et al. 2006). However, smaller individuals, especially when they are 61 amongst complex biofouling assemblages, may be overlooked. Utilization of divers to search for 62 marine non-indigenous species may alsoDraft not be possible at certain times/locations due to health 63 and safety concerns. 64 65 Molecular approaches are being advocated to circumvent some of the limitations of 66 morphological-based detection and monitoring methods in aquatic systems (e.g., Comtet et al. 67 2015). A suite of different techniques has been developed and applied in the last decade, with the 68 most commonly employed now being barcoding, real-time (or quantitative) polymerase chain 69 reaction (PCR) and metabarcoding using high-throughput sequencing (Ficetola et al. 2008; 70 Thomsen et al. 2012a; Wood et al. 2013). In aquatic systems, these techniques rely on collecting 71 and detecting either: (i) entire organisms (particularly in the case of micro-organism such as 72 bacteria, micro-algae, zooplankton), (ii) cells of organisms which may originate from various 73 sources, including scales, faeces, epidermal mucus, urine, saliva and gametes (Barnes et al. 2014), 74 or (iii) extracellular DNA which can be free-floating or particle-bound. These techniques have 4 https://mc06.manuscriptcentral.com/genome-pubs Page 5 of 38 Genome 75 been applied to determine aquatic biodiversity or the presence (and in some cases abundance) of 76 specific taxa in a wide range of habitats (e.g., Thomsen et al. 2012b; Dowles et al. 2016; Laroche 77 et al. 2016; Ulibarri et al. 2017; Keeley et al. 2018). Studies have shown that in some situations 78 molecular methods can be more efficient for detecting species than traditional approaches (Dejean 79 et al. 2012; Keskin 2014; Zaiko et al. 2016), making them a promising tool for the early detection 80 of newly introduced species and for monitoring the dispersal of established taxa (Blanchet 2012; 81 Piaggio et al. 2014; Rees et al. 2014; Comtet et al. 2015). 82 83 To date, most studies in aquatic environments investigating molecular techniques, DNA stability, 84 and appropriate sampling methods have targeted fish (Thomsen et al. 2012a; Takahara et al. 2013), 85 crustaceans (Forsströma and VasemägiDraft 2016), amphibians (Dejean et al. 2012), and 86 macroinvertebrates (Mächler et al. 2014). While sensitive and specific molecular assays have been 87 developed to detect sessile marine non-indigenous species (Gillium et al. 2014; Simpson et al. 88 2017; Wood et al. 2017), experimental and field studies to assess applicability of using these in 89 routine marine biosecurity programmes are lacking. Few studies have directly compared molecular 90 techniques with traditional visual survey methods (Ulibarri et al. 2017). Before molecular methods 91 can be routinely applied for surveillance, more information is required on achievable detection 92 rates and optimal sampling methods. Better understanding of what the assays are detecting (i.e., 93 free-floating DNA, non-viable fragments of organisms, or living organisms (propagules)) is also 94 needed for providing robust advice to effectively guide management decisions. 95 96 In the present study, we focus on two marine non-indigenous species that are already established 97 in New Zealand coastal waters; Sabella spallanzanii and Styela clava. The Mediterranean fanworm 5 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 6 of 38 98 S. spallanzanii (Gmelin, 1791) (Polychaeta: Sabellidae) is a large, tube-dwelling polychaete worm 99 native to the Mediterranean Sea and Atlantic coast of Europe (Patti and Gambi 2001). Once 100 established, it can form dense populations (100’s to 1000’s per m2) covering a variety of marine 101 habitats (e.g., Holloway and Keough 2002). It was first detected in New Zealand in 2008 (Read et 102 al. 2011) and has been detected at multiple locations, including several at which it is now well 103 established (for specific distribution data please refer to https://www.marinebiosecurity.org.nz). 104 The club tunicate S. clava, Herdman, 1881 (Ascidiacea: Styelidae) is thought to be native to the 105 northwest Pacific (Japan, Korea, Northern China, and Siberia). It too can form dense populations 106 (100’s to 1000’s per m2) covering a variety of marine habitats once established (e.g., Clarke and 107 Therriault 2007; Davis and Davis 2010). It was first detected in New Zealand in 2005 (Davis and 108 Davis 2006), and is nowDraft relatively widespread and established 109 (https://www.marinebiosecurity.org.nz). Both these non-indigenous species present significant 110 ecological, economic and societal values risk to New Zealand (e.g., Soliman and Inglis 2018), and 111 are part of a suite of non-indigenous species subject to targeted surveillance

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