Genetic tools for shark fishery management Mahmood S. Shivji Save Our Seas Foundation Shark Research Consortium & Guy Harvey Research Institute Nova Southeastern University Oceanographic Center Florida, USA
Presentation Outline
Background on shark fin trade and U.S. fishery regulations
Existing DNA forensic tools (currently in use) Case studies using DNA tools in fishery
regulation enforcement
The future: new DNA forensic tools under development
Shark stock structure
High demand for shark products worldwide
Unprocessed shark fin imports (Hong Kong market: 1992-2002)
Source: S. Clarke, 2003
Shark population declines globally IUCN Status of Globally Assessed Chondrythians
591 species assessed
40%
Dulvy et al. 2008. Aquatic Conserv: Mar. Freshw. Ecosyst.
Large declines in some species
IUCN Status of Pelagic Chondrythians
21 pelagic species assessed 75%
Dulvy et al. 2008. Aquatic Conserv: Mar. Freshw. Ecosyst.
Critical needs for improved shark management
• Information on landings by individual species
• Regulation and enforcement of catch limits by species
U.S. ATLANTIC FISHERIES MANAGEMENT PLAN REGULATIONS: 他 19 shark species prohibited from fishery
他 shark finning illegal 他 quotas, time/area closures for legal species ENFORCEMENT RESPONSIBILITIES: NOAA Office for Law Enforcement (NOAA OLE) U.S. Coast Guard Coastal State Fisheries Agencies
but catch monitoring and regulation enforcement difficult due to species identification problems
Carcharhinus altimus
Carcharhinus obscurus
Carcharhinus altimus
Carcharhinus falciformis
Carcharhinus plumbeus
species identification even more difficult with shark body parts
species?
Management requires much better identification tools and capacity to: 1. Monitor shark landings on a species-specific basis
2. Improve enforcement of species-specific fishing regulations (e.g. detect protected / CITES species)
Streamlined DNA forensic tool for shark identification is now available Rapid & easy PCR only test – DNA sequencing not required (lowers costs) DNA test is globally applicable Likely portable (current development)
Single-tube multiplex PCR test ~ 4hrs
sample
Diagnostic primers + PCR reagents
PCR
RUN GEL TO GET SPECIES FINGERPRINT
Single species DNA test: e.g. shortfin mako SF MAKO
Atl
+ sf mako
NON-TARGET SHARKS
Pac
+
Distinguishing multiple shark species simultaneously
Single tube PCR Test
J
A I
B C D
E F
G
H
SPECIES ID OBTAINED
At Pa
+
blacknose
bull bull
blacktip blacktip
finetooth
lemon
spinner
Simultaneous, multiple species PCR test (single-tube reaction) non-targets
At Pa At Pa
+
Species-specific DNA markers (primers) available for 30 species Sandbar Blue Silky SF Mako Tiger Porbeagle Bull Common thresher Spinner Java Blacktip Salmon Lemon Pelagic thresher Blacknose Finetooth Gray reef Great hammerhead Scalloped Hammerhead Smooth hammerhead bonnethead
PROHIBITED SPECIES (U.S.) White - CITES II Basking - CITES II Sand tiger LF mako Dusky Bignose Bigeye thesher Caribbean reef Night
Global distribution
Practical law enforcement case studies US NOAA Office for Law Enforcement (28 cases; ~ $1.2M in fines) US Coast Guard Govt. of South Africa Govt. of Palau Govt. of Singapore
A few examples of NOAA and U.S. Coast Guard Cases Case
Name
Prohibited Species Identified
Legal Species Identified
050053
M/V Lung Soon
dusky-1 longfin mako – 1
blue - 25 silky - 1
063016
US Trader
white shark - 10
silky - 8 blacktip - 4
063016
Swordfish/tuna longliner
dusky - 9 night - 11
shortfin mako - 3 tiger - 1 shortfin mako - 4 common thresher - 1
New York Seafood Dealer (Agar case)
basking sharks – 2 white sharks - 21 sandtigers – 19 bignose - 1 bigeye threshers - 19 night - 7 dusky – 17 blacktip - 1 spinner - 1 dusky – 1
0
033055
USCGC Valiant
Commercial vessel
Season closed
MAIN MESSAGE FOR SHARK FISHERY MANAGERS:
1. DNA technology exists currently to rapidly identify shark body parts
2. DNA technology is being used in practical shark management (law enforcement)
But will this DNA technology work in the field? Is it portable? Is it easy to use without extensive training?
Mobile DNA labs being used in the field for bird-flu detection
Vietnam inaugurates mobile bird flu lab BANGKOK, Aug 23 (TNA)
Thailand's Public Health Ministry has introduced a state-of-the art bird flu mobile laboratory to be dispatched to the bird flu prone province of Phichit.
Portable lab could revolutionize bird flu detection Faster test results would speed disease containment efforts 19 March 2007, Roma/Vienna
Automation of DNA analysis for field use (currently in prototype testing)
Result
Automated DNA extraction ~20 mins
PCR ~60 mins
Automated electrophoretic analysis ~ 10 mins
The
International Barcode of Life Project
What is DNA barcoding? Barcoding is a standardized approach to identifying species by the sequence of short fragment of their DNA
Barcodes
Products and Life
THE GOAL: Developing a Reference DNA Barcode Library for All Known Species •Master identification key •ID all life stages (eggs, larvae, adults) • ID all wildlife body parts •IDs cheap & fast •Residual taxonomic uncertainty low
Technology Development – 5-7 Years
Automated hand-held DNA sequencers
Sphyrna lewini Western Atlantic Stock Structure
USGoM vs USAtlantic: Φst = n.s.d from 0
USA vs C Amer: Φst = 0.61* (* P<0.0001)
· 3 DISTINCT MANAGEMENT UNITS (MUs). · ALL HAPLOTYPES ARE ENDEMIC TO WESTERN ATLANTIC.
USA vs Brazil Φst = 0.90* C Amer. vs Brazil Φst = 0.43*
WHERE DO THE FINS COME FROM? N=59 FINS 11 Hong Kong traders
W. ATL INDO-PAC
30% MARKET FINS ORIGINATED FROM W. ATL POPULATIONS SIGNIFICANT FRACTION OF LANDINGS?
Assessing porbeagle shark global population structure (complete mitochondrial CR sequences; N=280 individuals)
NW Atlantic n=101
Chile n=49
Denmark n=30
South Africa n=31 Falkland Islands n=13
New Zealand n=51 Tasmania n=5
WHAT DO THE DNA SEQUENCES TELL US?
Lnas74 Lnas13 Lnas49 Lnas73 Lnas23 Lnas46 Lnas44 Lnas40 Lnas68 Lnas27 Lnas03 Lnas24 Lnas42 Lnas09 Lnas16 Lnas02 Lnas66 Lnas38 Lnas76 Lnas47 Lnas48 Lnas41 Lnas10 Lnas20 Lnas21 Lnas30 Lnas69 Lnas65 Lnas25 Lnas55 Lnas52 Lnas67 Lnas08 Lnas50 Lnas32 Lnas12 Lnas54 Lnas57 Lnas11 Lnas53 Lnas75 Lnas15 Lnas39 Lnas64 Lnas60
Lnas72
Lnas33
Lnas14
Lnas35 Lnas58 Lnas71 Lnas59
Lnas63
Lnas01 Lnas70 Lnas06 Lnas45 Lnas43 Lnas56 Lnas61 Lnas29 Lnas04 Lnas17 Lnas62 Lnas28 Lnas05 Lnas07 Lnas26 Lnas37 Lnas34 Lnas51 Lnas36 Lnas22 Lnas19 Lnas18 Lnas31
0.01
• North Atlantic Porbeagle mitochondrial DNA sequence evolutionary relationships Lnas91
Lnas122 Lnas96 Lnas115 Lnas114 Lnas89 Lnas93 Lnas119 Lnas109 Lnas78 Lnas107 Lnas84 Lnas102 Lnas99 Lnas86 Lnas106 Lnas117 Lnas88 Lnas94 Lnas112 Lnas101 Lnas80 Lnas77 Lnas104 Lnas116 Lnas85 Lnas87 Lnas110 Lnas121 Lnas105 Lnas79 Lnas83 Lnas92 Lnas113 Lnas108 Lnas100 Lnas103 Lnas97 Lnas111 Lnas95 Lnas118 Lnas90 Lnas120 Lnas82 Lnas81 Lnas98
• Southern Hemisphere
SPECIES & POPULATION IDENTICATION TEST FOR PORBEAGLES
ALL PORBEAGLE
NORTH ATLANTIC PORBEAGLE
SOUTHERN HEMISPHERE PORBEAGLE
THANKS TO: THE RESEARCH TEAM: R. Horn A. Bernard V. Richards C. Testerman L. Murphy J. Magnussen J. Hester D. Chapman D. Abercrombie M. Henning J. Neilsen
FUNDING Save Our Seas Foundation NOAA Hai Stiftung GHRI
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. 2005 Cons Genet; Clarke et al. 2006 Cons Biol; Clarke et al. 2006 Ecology Lett; Magnussen et al. 2007 Animal Cons)
NOAA Office for Law Enforcement Cases Case
Name
Prohibited Species Identified Legal Species Identified
020262 F/V Rhonda M
0
blacknose - 6 nurse sharks - 4
030086 F/V Bluefin
dusky - 47 night - 4
tiger â&#x20AC;&#x201C; 3 sandbar - 1
030107 F/V Gulf Mariner
0
blacktip - 12 blacknose - 10 tiger - 5 scalloped hammerhead- 5
030103 F/V Sea Nymph III
dusky - 16
scalloped hammerhead- 12 tiger â&#x20AC;&#x201C; 12 sandbar - 1
030105 F/V Janice Ann
dusky - 2
silky - 4
0
scalloped hammerhead - 16 blacktip - 1
night - 11
silky - 6 sandbar - 4
F/V Osprey 040130 F/V Janice Ann
NOAA Office of Law Enforcement Cases Case
Name
Prohibited Species Identified
Legal Species Identified
033055
US Seafood Dealer
basking sharks – 2 white sharks - 21 sandtigers – 19 bignose - 1 bigeye threshers - 19 night - 7 dusky – 17
shortfin mako - 4 common thresher - 1
dusky - 10 night - 4
silky - 1
**Right pectoral fins only analyzed
050217
Commercial Vessel
NOAA OLE and USCG Cases Case
Name
Prohibited Species Identified
Legal Species Identified
050053
M/V Lung Soon
dusky-1 longfin mako â&#x20AC;&#x201C; 1
blue - 25 silky - 1
F/V Bluefin
dusky -1
0
063016
US Trader
white shark - 10
silky - 8 blacktip - 4
063016
Swordfish/tuna longliner
dusky - 9 night - 11
shortfin mako - 3 tiger - 1
050207
Processed fins from T & L Trading
dusky - 1
blue - 2 bull - 1 blacktip - 1 silky - 3
USCGC Valiant
Commercial vessel
blacktip - 1 spinner - 1 dusky â&#x20AC;&#x201C; 1 *** Season closed
0
OF 18 U.S. ATLANTIC NOAA OLE CASES ANALYZED BY DNA:
Dusky sharks – 12 cases PROHIBITED
Night sharks – 5 cases Tiger sharks – 5 cases Scalloped Hammerheads – 5 cases
LOW VALUE FLESH, VERY HIGH VALUE FINS
STOCK STATUS OF U.S. ATLANTIC SHARK SPECIES Dusky sharks: 2006 Stock Assessment NW Atlantic: 62-80% depletion compared to unexploited levels Scalloped hammerhead sharks: 2007 Stock Assessment NW Atlantic: 70-85% depletion compared to unexploited levels IUCN Endangered Listing Tiger sharks: ? Night Sharks: ?
LAW ENFORCEMENT CASES: A “RANDOM”/OPPORTUNISTIC “SURVEY”
ANY INSIGHTS FROM THESE CASES? 1. A REASONABLE REFLECTION OF THE UNRECORDED SPECIES COMPOSITION OF THE SHARK FISHERY? 2. HIGH FREQUENCY OF PROHIBITED DUSKY AND NIGHT SHARKS – ARE REGULATIONS INEFFECTIVE DUE TO ID PROBLEMS? 3. DOES THE HIGH FREQUENCY OF TIGER AND ScHH FINS INDICATE FINNING IS COMMON DESPITE REGULATIONS?
EXTRA STUFF: BILLFISH DNA FORENSICS
BILLFISH MULTIPLEX PCR DNA FORENSICS 5 Species Multiplex
Roundscale
univ
tRNA
ND4
ND4L White marlin
Longbill
Blue marlin
Sailfish
univ
Single Tube Billfish “Septaplex” PCR Assay Distinguishing among 5 species simultaneously M
M
sailfish blue marlin Roundscale spf Longbill spf white marlin
NOAA Office of Law Enforcement Cases Case
Name
Prohibited Species Identified
Legal Species Identified
053017
M/V MGB
0
spinner - 35 blacktip - 1 blacknose - 5 scalloped hammerhead - 5
Guam finning case Seafood Shoppe (meat) ** White sharks suspected based on informant tip
0
0
blue shark - 119 silky - 7 blacktip - 22 spinner - 2 sandbar - 18 tiger - 4 blacknose - 9
INTERNATIONAL CASES Case
Name
Prohibited Species Identified
Legal Species Identified
Govt. of Palau via Coral Reef Research Foundation
Commercial Vessel
0
shortfin mako - 1 gray reef shark - 1 pelagic thresher - 2 silky - 2 blue - 1
DEAT South Africa
Various fin trading companies
0
scalloped hammerhead - 2 smooth hammerhead - 3 bigeye thresher - 2 oceanic whitetip - 5 tiger - 2 shortfin mako - 24 porbeagle - 27 blue - 39
DEAT South Africa
Trader
0
shortfin mako - 3 common thresher -
** White sharks suspected
MORE STREAMLINED ASSAY COMBINING MANY SPECIES-SPECIFIC PRIMERS IN A SINGLE “HIGH-DENSITY” MULTIPLEX PCR (ONE TUBE REACTION)
HIGH DENSITY MULTIPLEXING LARGE & SMALL COASTAL NON-RIDGEBACK SHARK PRIMER SUITE
UNIV-R
SPINNER-F
LEMON-F
FINETOOTH-F
BLACKTIP-F
BULL-F
UNIV-F
28S rDNA
ITS2 BLACKNOSE-F
5.8S rDNA
TECHNIQUE: MULTIPLEX PCR WITH SPECIESSPECIFIC PRIMERS POSITIVE CONTROL AMPLICON
+ UNIV-R
5.8S rDNA
28S rDNA
ITS2
Species-Specific- F (e.g. SF mako)
UNIV-F
SPECIES-SPECIFIC AMPLICON
IF SF MAKO
GEL RESULTS
+
IF NOT SF MAKO
+
VARIOUS PRIMER COMBINATIONS POSSIBLE PELAGIC SHARK PRIMER SUITE (10 species: silky, blue, dusky, SF mako, LF mako, porbeagle, thresher, bigeye thresher)
COASTAL NON-RIDGEBACK SUITE (6 species: blacktip, bull, lemon, finetooth, blacknose, spinner)
COASTAL RIDGEBACK SUITE (7 species: sandbar, dusky, silky, bignose, Carib. Reef, tiger, night)
PROHIBITED SPECIES SUITE (U.S.) (8 species: white, sand tiger, dusky, bignose, LF mako, bigeye thresher, night, Carib. Reef)
HAMMERHEAD SUITE (4 species: great HH, scalloped HH, smooth HH, bonnethead)
“SUPERPLEX” SUITE (15 species: various - depending on amplicon size)