DNA approaches to marine wildlife fishery monitoring and law enforcement. Mahmood S. Shivji

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1 DNA approaches to marine wildlife fishery monitoring and law enforcement Mahmood S. Shivji Presentation given at FIU Forensics Workshop - July 26, 2010

2 Major forensics questions for marine wildlife 1. What species is the body part from? - is it from a legal or illegal to harvest/trade species? 2. What regulatory jurisdiction does it come from? - some marine species protected in territorial waters of some countries but not others.

3 SPECIES IDENTIFICATION I. Traded seafood mislabeling (Grocery store and Restaurant CSI ) II. Shark fisheries management and law enforcement

4 Techniques 1. DNA sequencing: Mitochondrial genes (Barcode COI and 16S rrna) Nuclear ribosomal Internal Transcribed Spacer 2 (ITS2) - NCBI BLAST comparisons - Phylogenetic tree building (clustering) 2. SNP Typing: Allele - specific PCR (= Species-specific PCR primers) - mitochondrial and nuclear loci

5 SEAFOOD MISLABELLING DNA Barcoding (COI gene) showed ~ 25% of samples from 96 grocery stores and restaurants in NE US and Canada mislabeled

6 Pacific halibut: Healthy stocks Atlantic halibut: Severely overfished

7 Mt 16S rrna gene sequencing showed 50% of 174 samples from seafood wholesalers and restaurants in South African cities mislabeled

8 Fish labeled monkfish in Chicago (fishmonger) was actually pufferfish (contains neurotoxin) pufferfish importation highly restricted

9 MORE SURVEYS REVEAL: Restaurant fish mislabeling common in US - low-price fish mislabeled as high-price fish (e.g., grouper, red snapper) npr_dnadetective (5).mp3

10 DNA sequences as a species identity marker Caution! Intra-specific sequence divergence ( up to 2%) can exist depending on taxa and locus used. Inter-species sequence divergence can be very small (0.5%) depending taxa and locus used In NCBI BLAST matches, how close (% match) is OK to conclude identity of sample? - depends on taxonomic group and purpose of test - what the sample is NOT easier to confirm

11 Q) Is the fish sample a grouper? Legally: 159 species in family Serranidae, subfamily Epinephelinae Mitochondrial 16S rrna sequence BLAST search Fish Sample 1 Acce ssion DQ AY AY DQ Description Max score Total score Query coverage E value Max ident Epinephelus bleekeri 16S ribosomal RNA gene, partial sequence; mitochondrial % 0 99% Epinephelus bleekeri 16S ribosomal RNA gene, partial sequence; mitochondrial % 0 97% Epinephelus miliaris 16S ribosomal RNA gene, partial sequence; mitochondrial % 0 96% Epinephelus areolatus 16S ribosomal RNA gene, partial sequence; mitochondrial % 0 96% EU Epinephelus akaara mitochondrion, complete genome % 0 95%

12 Mitochondrial 16S rrna sequence BLAST search Fish Sample 2 Acc essi on AY AY AY DQ AY AY Description Max score Total score Query coverage E value Max ident Epinephelus tauvina 16S ribosomal RNA gene, partial sequence; mitochondrial % 0 97% Epinephelus maculatus 16S ribosomal RNA gene, partial sequence; mitochondrial % 0 97% Epinephelus melanostigma 16S ribosomal RNA gene, partial sequence; mitochondrial % 0 97% Epinephelus spilotoceps voucher epne S ribosomal RNA gene, partial sequence; mitochondrial % 0 97% Epinephelus spilotoceps 16S ribosomal RNA gene, partial sequence; mitochondrial % 0 97% Epinephelus macrospilos 16S ribosomal RNA gene, partial sequence; mitochondrial % 0 96%

13 Is it a grouper? Phylogenetic clustering Confirmation (NJ tree) Subfamily Epinephelinae

14 Determining sushi (sashimi) identity and tracing individual whales to market

15 Japanese whale individual exported to S. Korea (illegal) (multilocus STR genotyping) Survey of whale meat sashimi in Los Angeles and Seoul restaurants Mitochondrial cyt b gene and Control Region sequencing and BLAST/phylogenetic matching LA (4 products) Seoul (13 products) 2 whale labeled: Sei whale All whale labeled 2 horse labeled: domestic cow 7 minke whale 4 Sei whale Illegal in US 1 Fin whale ** 1 Risso s dolphin

16 DNA tools for shark fishery management, conservation and law enforcement

17 High demand for shark products worldwide

18 Global shark fin trade > US $ 550m/yr

19 NOAA FISHERIES U.S. ATLANTIC FISHERIES MANAGEMENT PLAN REGULATIONS: 20 SHARK SPECIES PROHIBITED FROM FISHERY SHARK FINNING ILLEGAL QUOTAS, TIME/AREA CLOSURES FOR LEGAL SPECIES ENFORCEMENT RESPONSIBILITIES: NOAA Office for Law Enforcement (NOAA OLE) U.S. Coast Guard Coastal State Fisheries Agencies

20 BUT ENFORCEMENT PROBLEM DUE TO SPECIES IDENTIFICATION DIFFICULTIES Not from same animal Illegal

21 DEVELOPING & USING STREAMLINED DNA FORENSIC TOOLS PCR ONLY TEST DNA SEQUENCING NOT REQUIRED Global scale applications Potential field portability

22 TECHNIQUE: ALLELE-SPECIFIC PCR MULTIPLEX PCR WITH SPECIES-SPECIFIC PRIMERS POSITIVE CONTROL AMPLICON + UNIV-R 5.8S rdna ITS2 28S rdna UNIV-F Species-Specific- F (e.g. SF mako) SPECIES-SPECIFIC AMPLICON GEL RESULTS + IF SF MAKO IF NOT SF MAKO +

23 SF MAKO PRIMER (TRIPLEX PCR) SF MAKO NON-TARGET SHARKS Atl Pac sf mako + +

24 SPECIES-SPECIFIC PRIMERS DEVELOPED FOR 30 SPECIES Silky Tiger Bull Spinner Blacktip Lemon Blacknose Finetooth Gray reef Great hammerhead Scalloped Hammerhead Smooth hammerhead bonnethead Blue SF Mako Porbeagle Common thresher Java Salmon Pelagic thresher CITES II? PROHIBITED SPECIES (U.S.) White - CITES II Basking - CITES II Sand tiger Sandbar LF mako Dusky Bignose Bigeye thesher Caribbean reef Night Global distribution

25 MORE STREAMLINED ASSAY COMBINING MANY SPECIES-SPECIFIC PRIMERS IN A SINGLE HIGH-DENSITY MULTIPLEX PCR (ONE TUBE REACTION)

26 HIGH DENSITY MULTIPLEXING LARGE & SMALL COASTAL NON-RIDGEBACK SHARK PRIMER SUITE UNIV-R 5.8S rdna ITS2 28S rdna UNIV-F BLACKNOSE-F BULL-F BLACKTIP-F FINETOOTH-F LEMON-F SPINNER-F

27 6 SPECIES MULTIPLEX PCR LARGE & SMALL COASTAL NON-RIDGEBACK SHARKS non-targets spinner lemon finetooth blacktip blacktip bull bull blacknose At Pa At Pa At Pa + +

28 VARIOUS SPECIES-SPECIFIC PRIMER COMBINATIONS POSSIBLE (up to 14 species-specific primers/pcr)

29 CHARACTERIZATION OF GLOBAL FIN TRADE DNA forensics + Hong Kong fin trader auction records Determine Hong Kong fin trade species composition Establish relationship between Chinese trade names and species identity Estimate of number of sharks utilized in the global fin trade annually = 38 million (median) Abercrombie et al Cons Genet; Clarke et al Cons Biol; Clarke et al Ecology Lett; Magnussen et al Animal Cons)

30 LAW ENFORCEMENT CASES NOAA Office for Law Enforcement US Coast Guard South Africa Dept. Environ. Affairs and Tourism (DEAT) Govt. of Palau SEAFDEC (Singapore Govt)

31 BILLFISH DNA FORENSICS

32 BILLFISH MULTIPLEX PCR DNA FORENSICS 5 Species Multiplex univ Roundscale ND4L ND4 trna White marlin Longbill Blue marlin Sailfish univ

33 Single Tube Billfish Septaplex PCR Assay Distinguishing among 5 species simultaneously M M sailfish blue marlin Roundscale spf Longbill spf white marlin