A baseline study of the occurence of non-indigenous species in - PowerPoint PPT Presentation

A baseline study of the occurence of non-indigenous species in Danish harbours. TG BALLAST 13-14. december 2018 Ulrik Chr. Berggreen Kim Larsen

Introduction First nation-wide study of non- indigenous species in Danish harbours. Aim of the study: • Monitor, assess and report occurence of non-indigenous species in 16 selected Danish harbours mainly by eDNA. • Utilise both conventional methods (JHP) and biomolecular methods (eDNA). • Provide a proof-of-concept regarding the Danish strategy of combining conventional and biomolecular methods. DEPA Page 2

Methodology - introduction Sampling: • General at 16 selected Danish harbours Biomolecular (eDNA) 16 harbours: • Environmental DNA (eDNA) identified through species specific qPCR. • 20 target species. • Supplemented by dive transets (snorkeling) at night (fish, jellyfish, epifauna) and fish nets. • Sampling early summer (May-July) and autumn (September-November) 2017. Conventional at comparison harbours Aarhus (1) and Esbjerg (2): • Joint Harmonized Procedure (JHP). • Expanded JHP - dive transets (snorkeling) day and night (fish, jellyfish, epifauna) and fish nets. • Monitoring spring and autumn (May- November) 2017. DEPA Page 3

Methodology - biomolecular Environmental DNA (eDNA). 1. Water sampling. 2. Filtration and storage. 3. eDNA extraction and amplification with qPCR. eDNA extraction, amplification Water sampling . Filtration and storage . • >1,5l. and analysis . • Amphiltrator - 22µm Millipore ”Sterivex filter”. • eDNA extraction and amplification with • Short term storage - dry ice and - qPCR. 20 ºC freezing facility. • Species specific identification system • Long term storage - -80 ºC freezing (primers and probes) for 20 species. facility (potential fixation buffer). • Analysis for presence/absence of eDNA from target species. • Three replicates for each target species. DEPA Page 4

Methodology - biomolecular qPCR – ”quantitative PCR” Estimation of amount of DNA in water samples by a standard curve. • Concentrations of 100 mio copies/µL, 10 mio copies/µL, etc. • DNA from water samples. • The amplification curves for the water samples are compared to known concentrations. • Amount of eDNA in the water sample can be calculated by the standard curve (if the eDNA concentration in the sample is above LoQ). DEPA Page 5

Methodology - biomolecular Species specific detektion systems (primers and probes) . DEPA Page 6

Methodology - biomolecular Water samples analysed with qPCR are categorised: (Ct=Critical threshold, LoD=Limit of Detection, LoQ=Limit of Quantification) 1. Negative (white): No Ct observed in any of the triplicates – no amplification at all. 2. Weak possible positive (yellow): amplification observed in at least one triplicate but amplification below LoD 3. Possible positive (orange): Ct observed in at least one triplicate, and amplifiation detected aboved LoD but below LoQ. 4. Positive (red): Ct observed in at least one triplicate, with levels above LoQ. 5. Positive, possible to quantify (black): all triplicates with Ct amplification levels above LoQ. DEPA Page 7

Results - eDNA qPCR points related to number of positive replicates Spring Autumn eDNA resultat Interpretation No Ct No 'target eDNA' in water sample Below LOD Possible weak trace of eDNA from 'target species' Above LOD below LOQ Weak trace of eDNA from 'target species' 1Above LOQ eDNA from 'target species' 3Above LOQ Solid eDNA fra 'target species' DEPA Page 8

Methodology - conventional JHP well established platform: • Physical parameters (temperature, salinity, oxygen) and secchi disc. • Grab samples for water and sediment (benthic infauna, epifauna). • Plankton nets (phyto- and zooplankton). • Traps (mobile epifauna). • Scrape poles and fouling plates (fouling organisms). • Fish nets. Expanded JHP: • Diver transects – night and day. DEPA Page 9

Results – conventional monitoring KalundborgStatiol Aalborgportland Frederikshavn AalborgHavn Koebenhavn Ass Id Kalundborg Species_name Helsingoer Fredericia No Hirtshals Esbjerg Grenaa Roedby Aarhus Odense Gedser Koege Havn 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Bonnemaisonia hamifera 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 Prorocentrum minimum 2 0 0 1 2 1 1 0 1 0 1 1 1 0 0 1 0 Pseudochattonella farcimen 3 0 0 1 2 1 1 0 1 0 1 1 1 0 0 1 0 Pseudochattonella verruculosa 4 Karenia mikimotoi 5 1 1 1 2 1 1 0 1 1 1 1 1 1 0 1 0 Carassius auratus 6 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 Cyprinus carpio 7 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 1 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 Colpomenia peregrine 8 0 0 0 0 0 0 2 0 0 0 2 2 2 2 0 2 Neogobius melanostomus 09A 1 1 1 0 0 0 0 1 1 0 1 1 1 1 1 0 Oncorhynchus mykiss 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acipenser gueldenstaedtii 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acipenser ruthenus 12 Oncorhyncus gorbuscha 13 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Crassostrea gigas 14 2 2 0 2 0 0 0 0 2 0 1 0 2 0 0 0 Mya arenaria 15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16 0 0 0 0 0 0 1 0 0 0 0 0 1 1 0 1 Rhithropanopeus harrisii 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Paralithodes camtschaticus 17 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Eriocheir sinensis 18 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Homarus americanus 19 2 2 2 2 2 2 2 2 2 1 2 2 2 2 1 2 Mnemiopsis leidyi 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acipenser baerii 21 Cordylophora caspia 22 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Found before this project: 1 Found during this project: 2 DEPA Page 10

Results – comparison between eDNA and conventional results Spring Autumn Table 8. Table for interpretation of colors in validation table 5 and 6. Abbreviations are based on eDNA results and conventional monitoring. CONVENTIONAL RESULT Found before, but Found during eDNA RESULT: Not found ever not during Monis 4 field Monis4 field work work No Ct NF_NoCt FB_NoCt FM4_NoCt Below LOD NF_BeLOD FB_BeLOD FM4_BeLOD Above LOD below NF_AbLOD FB_AbLOD FM4_AbLOD LOQ 1Above LOQ NF_1AbLOQ FB_1AbLOQ FM4_1AbLOQ 3Above LOQ NF_3AbLOQ FB_3AbLOQ FM4_3AbLOQ DEPA Page 11

Conclusion First nation-wide study of non-indigenous species in 16 Danish harbours completed. The conventional methodology detected non-indigenous species • Higher diversity in Esbjerg harbour (2) compared to Aarhus harbour (1). • NIS found are generally known in Danish waters. • New NIS include two marine bristle worms from Esbjerg harbour: - Streblospio benedicti - Eteone heteropoda Streblospio benedicti . Photo: David Johnson Eteone heteropoda . Photo: Dean Janiak DEPA Page 12

Conclusion The eDNA methodology detected non-indigenous species. Overall agreement between eDNA monitoring and conventional monitoring. • Difference in timing of eDNA and conventional monitoring. • eDNA is expected to reflect the species presence at the time of sampling due to the fast deterioation of eDNA in the water (1-7 days). • For comparison, the conventional included previous knowledge on the distribution of the 20 non-indigenous target species (past recordings, assumed occurence, suspected distribution). Maintain Danish monitoring strategy. Evaluate JHP scope in hot spots to suit danish conditions. DEPA Page 13