dna identification of pacific bluefin tuna {thunnus orientalis) in

New Zealand Journal of Marine and Freshwater Research, 2001, Vol. 35: 843-8500028-8330/01/3504-0843 $7.00 © The Royal Society of New Zealand 2001

843

DNA identification of Pacific bluefin tuna{Thunnus orientalis) in the New Zealand fishery

P. J. SMITH

L. GRIGGSNational Institute of Water & Atmospheric

Research LtdP.O.Box 14 901,KilbirnieWellington, New Zealandemail: [email protected]

S. CHOWDivision of Pelagic Fish ResourcesNational Research Institute of Far Seas FisheriesOrido 5-7-1, SmimizuShizuoka 424-8633, Japan

Abstract Muscle samples were collected from 69specimens identified as Pacific bluefin tuna (Thunnusorientalis) (Temminck and Schlegel, 1844) in the NewZealand Exclusive Economic Zone (EEZ) between1990 and 2000. Identifications before 1996 were basedon body size and colour of the caudal keel; later iden-tifications were mostly based on the shape of abdomi-nal cavity. The tissue samples were tested with adiagnostic mitochondrial DNA marker that distin-guishes southern bluefin Thunnus maccoyii(Castelnau, 1872) and Pacific bluefin tuna T. orientalis;59 specimens were confirmed as T. orientalis and 10as T. maccoyii. Specimens recorded as Pacific bluefintuna by the shape of the abdominal cavity were cor-rectly identified as T. orientalis, and this character canbe used to identify large specimens landed on tunavessels. Some specimens recorded as Pacific bluefintuna on the basis of colour and size were T. maccoyii;and early records of T. orientalis in New Zealandwaters, based on these characters, are unreliable. Unu-sual colour patterns were reported in some specimensof T. orientalis but not T. maccoyii. The Pacific bluefintuna T. orientalis accounted for less than 0.3% of thebluefin tuna catch in the New Zealand EEZ during the1990s.

M01022Received 5 March 2001; accepted 5 April 2001

Keywords bluefin tuna; Thunnus maccoyii;Thunnus orientalis; mitochondrial DNA; PCR

INTRODUCTION

Southern bluefin tuna Thunnus maccoyii (Castelnau,1872) are widely distributed in all oceans south ofc. 30°S. Commercial fishing of southern bluefin tunastarted in the 1950s and was initially focused on thespawning grounds south of Indonesia. The fisheryexpanded rapidly during the 1960s and 1970s,moving into Australian waters and eventually intoNew Zealand waters. The stock is now in a depletedstatus at only 5-8% of the 1960 parental biomass(Anon. 1998). Around New Zealand bluefin tuna arecaught by domestic and charter vessels by longliningand handling, and occasionally trolling. This is asmall fishery of c. 420 t/annum, but with an exportvalue of nearly NZ$19 million in the 1999-2000fishing year. The highest price paid for a fish fromthe New Zealand domestic fishery was NZ$90 000in Tokyo in 1999.

The New Zealand southern bluefin tuna fishingregulations define southern bluefin tuna as "fish withthe scientific name Thunnus maccoyi; and includesthe fish with the scientific name Thunnus thynnus"(Ministry of Fisheries 2000). Each year a number ofbluefin tuna caught in the New Zealand fishery arerecorded as Pacific bluefin tuna Thunnus orientalis(Temminck and Schlegel, 1844) by Ministry ofFisheries observers and skippers on both domesticand charter vessels. The New Zealand southernbluefin tuna fishing regulations dictate that Pacificbluefin tuna be recorded against the southern bluefintuna quota, and that the fishery be managed as asingle species fishery, with no allowance for thepresence of a second, closely related species. Pacificand southern bluefin tuna are separate species,originally distinguished by the position of the firstventrally directed parapophysis on the 9th (T.maccoyii) and 8th (T. orientalis) vertebrae, and thecolour of the caudal keel (Gibbs & Collette 1966;Collette & Nauen 1983), although the colour of the

844 New Zealand Journal of Marine and Freshwater Research, 2001, Vol. 35

caudal keel was not reliable in large specimens(Gibbs & Collette 1966). In the northern hemispheretwo subspecies of bluefin tuna Thunnus thynnusthynnus, in the Atlantic and Indian Oceans and theMediterranean Sea, and T. t. orientalis, in the PacificOcean, were recognised, but now are considered asfull species, Atlantic bluefin tuna T. thynnus andPacific bluefin tuna T. orientalis, based onmorphological and molecular data (Collette 1999).Given the high unit value of the New Zealand fisheryand the limited quota, it is important that individualfish, in particular specimens of T. orientalis, arecorrectly identified.

The bluefin tuna species have differentdistributions in the Pacific Ocean with T. maccoyiisolely in the southern hemisphere and T. orientalisprimarily in the northern hemisphere, althoughspecimens of T. orientalis have been reported fromAustralia, the Galapagos Islands, and New Zealand(Collette & Smith 1981; Collette & Nauen 1983;Bayliff 1994). Different characters have been usedto identify specimens as Pacific bluefin tuna in theNew Zealand fishery. Before 1996 most specimensrecorded as Pacific bluefin tuna, by New Zealandobservers, were identified by large size and colourof the caudal keel.

Use of external characters to distinguish bluefintuna has been questioned (Anon. 1994) and genetictests, based on allozymes, indicated that size andbody colour alone are unreliable for correct species'identification (Smith et al. 1994). Similarly DNAtests on Australian specimens of Pacific bluefin tunahave shown that some specimens have beenincorrectly identified (Ward et al. 1995). Japanesefishing skippers have identified Pacific bluefin tunaby the presence of a "bust", a muscular protrusionin the dorsal abdominal cavity, that is present in T.maccoyii and T. thynnus but not T. orientalis (Iwaiet al. 1965; Gibbs & Collette 1966).

Molecular techniques are increasingly being usedto identify fish product and specimens, and severalgenetic methods have been applied to theidentification of tuna species (Bartlett & Davidson1991; Chow & Inoue 1993; Smith et al. 1994; Wardet al. 1995). Diagnostic DNA markers have beendeveloped for the identification of Pacific andsouthern bluefin tuna and more than 200 specimenshave been tested for variation in the mitochondrialgenome (Chow & Inoue 1993; Chow & Kishino1995). Here we use the DNA markers that have beendeveloped to distinguish the major commercial tunaspecies (Chow & Inoue 1993).

The aim of this project was to apply a diagnosticDNA marker to identify specimens reported asPacific bluefin tuna from New Zealand waters, andto determine if there are reliable field characters foridentification of Pacific bluefin tuna on commercialfishing vessels.

METHODS

Sample collectionMuscle samples were collected from specimens ofT. orientalis off Japan in 1990 and 1993, and fromT. maccoyii off New Zealand in 1990, 1998, and1999 (Table 1). Specimens were identified fromgeographical location and colour of the caudal keel(Japan with black caudal keel for T. orientalis andNew Zealand with yellow caudal keel for T.maccoyii (Collette & Nauen 1983)). Muscle sampleswere collected from 69 fish in the New ZealandExclusive Economic Zone (EEZ) between 1990 and2000 that had been recorded as northern (= Pacific)bluefin tuna, and from a further three specimensrecorded as southern bluefin tuna with black caudalkeels (Table 2). Early samples (pre-1994) of Pacificbluefin tuna were identified by New Zealand

Table 1 Summary of the number of suspect Pacific bluefin tuna (NTU) fromthe New Zealand Exclusive Economic Zone identified as Thunnus orientaliswith a diagnostic DNA marker.

Year

19901991199319961997199819992000

No. suspect NTU

1011

101822

34

No. T. orientalis by DNA

3019

1821

34

Smith et al.—DNA identification of bluefin tuna 845

Ministry of Fisheries observers on the basis of largesize and a dark caudal keel. Samples collected fromPacific bluefin tuna specimens between 1990 and1992 were the same as those used by Smith et al.(1994) for allozyme identification. Most specimensrecorded as Pacific bluefin tuna from 1996 onwards

were identified by the shape of the dorsal wall of thebody cavity.

For each specimen recorded as Pacific bluefintuna, and for control samples, a small piece(c.10 g) of muscle tissue was removed and frozenin individual plastic bags -60 to -70°C at sea.

Table 2 Summary of identification characters recorded by observers, and DNA identification results, for bluefintuna Thunnus orientalis and Thunnus maccoyii in the New Zealand Exclusive Economic Zone. (B, black caudal keel;D, dark body colour; E, elongated body shape; M, ventral mottling; N, Pacific northern bluefin tuna; P, prominentbody wall; R, reduced body wall; S, southern bluefin tuna; S*, southern bluefin tuna with black caudal keels; Se,relatively small eye; Sp, speckled body patterns; Y, yellow caudal keel). A space in the table indicates no informationrecorded by observer.

Year

1990

Control199119931996

1997

1998

Control1999

2000

No. offish

371211111611811113211233121124111011121111

Obs.ID

NNSNNNNNNNNNNNNNNNNNNNNNNNNNNNS*sNNNS*NNNN

DNA

NS

ssNNNNNSNNNNNNNNNNNNNNNNNNNS

ssNNNSNNNN

Keelcolour

BBY

BBBBBBBBBBBB

BBBBB

BB

B

BBB

BB

Bodycolour

D,SpSpD

M

MD

DD,M

MM

D,M

DSp

MM

Sp

Bodyshape

EEEE

EE

E

E

E

EE

Eyesize

Se

Se

SeSe

SeSeSe

Se

Se

Se

Abdominalwall shape

RRRR

R

RRRR

RRRRRRR

P

RR

RR


Tissue samples were stored at -70°C in thelaboratory.

DNA amplification and digestionDNA was extracted from muscle tissue of T.maccoyii and T. orientalis and the suspect Pacificbluefin tuna with a proteinase K extraction, followedby chloroform-isoamyl alcohol clean-up and ethanolprecipitation after Chow & Inoue (1993). The DNApellet was air dried and resuspended in 40 ul sterilewater.

The primer pair flanking the region between themitochondrial ATPase and cytochrome oxidaesubunit III genes, designated ATCO (Chow & Inoue1993), was used to amplify DNA in all bluefin tunaspecimens. Amplifications were carried out in a finalvolume of 20 |ll of polymerase chain reaction (PCR)reaction mixture as described in Chow & Inoue(1993). PCR-RFLP (restriction fragment lengthpolymorphism) analysis of mtDNA has been usedin population (Cronin et al. 1993; Chow & Ushiama1995) and taxonomic (Chow et al. 1993) fisheriesstudies, including identification of tuna species(Chow & Inoue 1993). All Thunnus species couldbe identified from species-specific restrictionprofiles by the restriction enzymes Alu I, Mse I andHinfl or Hinc II (Chow & Inoue 1993), and thesespecific restriction profiles have been substantiatedby nucleotide sequence and RFLP analyses with alarge number of individuals (Takeyamaet al. 2001).Two restriction enzymes, Alu I and Mse I, produceddiagnostic restriction profiles in 82 specimens of T.maccoyii and 122 specimens of T. orientalis,although T. thunnus and T. maccoyii shared Mse Irestriction profiles (Takeyama et al. 2001). Asspecimens used in this study were either T. maccoyiior T. orientalis, the two diagnostic restrictionenzymes, Alu I and Mse I, were used to digest theamplified product. Digested PCR products wereseparated in 1.4% agarose gels in a TBE buffer (25vaM Tris, 0.5 vaM EDTA, and 25 mM boric acid) andstained with ethidium bromide. DNA fragmentswere viewed under an ultraviolet (UV) light sourceand photographed.

Field charactersObserver records on all tuna identified as northern(= Pacific) bluefin tuna were extracted from the NewZealand Ministry of Fisheries database, and therecords matched with the DNA results. The length-weight relationships of T. orientalis and T. maccoyiiwere tested in fish of comparable size, becausefishery observers had reported that T. orientalis

Alu I Mse I

100 •

Fig. 1 Restriction profiles of the mitochondrial DNAATCO segment of Thunnus orientalis (lanes 2 and 4) andT. maccoyii (lanes 3 and 5) digested with the diagnosticrestriction enzymes Alu I (lanes 2 and 3) and Mse I (lanes4 and 5). Lanes 1 and 6 are molecular size markers (100bp DNA ladder, New England BioLabs), and sizes "inbase pairs" are indicated along the left margin.

appeared to be more elongated than T. maccoyii.Likewise the size frequencies of T. orientalis, andspecimens identified as T. maccoyii by the diagnosticDNA marker, were plotted, because fisheryobservers reported that Pacific bluefin tuna werelarger than T. maccoyii.

The shape of the dorsal wall of the body cavitydiffers among the three species of bluefin tuna(Godsil & Holmberg 1950; Gibbs & Collette 1966).In T. thynnus and T. maccoyii there is "a wideanterior bulge without lateral concavity, but a deep,narrow trough lateral to the bulge" (Gibbs & Collette1966). In T. orientalis "the anterior bulge is narrowwith a lateral concavity, and a wide trough lateral tothe bulge" (Gibbs & Collette 1966). This characterhas been referred to as the bust by Japanese fishersand can be observed as a muscular protrusion in T.maccoyii when the gills are removed as part ofstandard on-board processing. The muscularprotrusion is small or absent in T. orientalis. Mostspecimens recorded as Pacific bluefin tuna by NewZealand observers since 1996 have been checked forthe absence or reduced size of the dorsal bulge.


Fig. 2 Distribution of bluefintuna specimens identified asThunnus orientalis by DNA mark-ers, and the observed bluefin tunasets, in the New Zealand ExclusiveEconomic Zone from 1990 to2000.

30 °S

165 °E 170 °E 175 °E

35 °S

40 ° S

45 ° S

60 ° S

?00m O = observed sets

+ = Thunnus orientalis

RESULTS

DNA analysesDNA, extracted from bluefin tuna muscle tissuesamples collected between 1990 and 2000, wassuccessfully amplified for the ATCO region ofmitochondrial DNA. The resultant DNA fragments,cut with the restriction enzyme Alu I or Mse I,revealed diagnostic restriction profiles in the controlsamples from T. orientalis and T. maccoyii (Fig. 1).The suspect Pacific bluefin tuna had either a T.orientalis or T. maccoyii restriction profile. Resultsare summarised by year of collection in Table 1.Sixty-nine suspect Pacific bluefin tuna were testedand 59 were identified as T. orientalis. Threespecimens landed in 1998 and 1999, and recordedas southern bluefin tuna (T. maccoyii) with blackcaudal keels, were identified as T. maccoyii by the

diagnostic DNA marker (Table 2). The locations ofT. orientalis specimens caught in the New ZealandEEZ, and identified by DNA markers, are given inFig. 2, along with the positions of observed tunalongline sets.

Field charactersThe field characters recorded by Ministry of Fish-eries observers and the DNA results are summarisedin Table 2. Most of the specimens recorded as north-ern (= Pacific) bluefin tuna in New Zealand watersbefore 1994 were T. maccoyii. These specimens hadbeen identified by large body size and dark colourof the caudal keel, although the observer records areincomplete (Table 2). Most of the specimens re-corded as Pacific bluefin tuna since 1996 had beencorrectly identified (Table 1). All specimens of Pa-cific bluefin tuna identified by the absence/reduced


1800

1600

1400

5" 1200

<5 1000

O" 800

£ 600

400

200

0

20 -

18 -

16 -

s-14

si

12

10

6 -4 -2 -0

Thunnus maccoyiiFig. 3 Length frequency distribu-tions of Thunnus orientalis and T.maccoyii caught on observed ves-sels in the New Zealand ExclusiveEconomic Zone.

n=17 543

80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250

Fork length (cm)

Thunnus orientalis

n = 53

n80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250

Fork length (cm)

size of the dorsal bulge were confirmed as T.orientalis by the DNA test (33 specimens, Table 2).

Some observers noted specimens with unusualcolour patterns with blue flecks or speckling abovethe pectoral fins and sometimes on the head (Table2). Other unusual colour patterns were a bluishmottled pattern, posterior to the anal fin, and blue-grey flanks. Specimens of T. orientalis displayedeither the speckling or the mottling pattern, but nospecimens were reported with both colour patterns(Table 2). Bluefin tuna specimens that displayed thespeckling or mottling colour pattern were always T.orientalis when tested for DNA. Observers recordedfour specimens with blue flecks or speckling, and 13with the ventral mottling pattern; for several otherspecimens the colour patterns were not recorded(Table 2). In addition, observers recorded 10specimens with darker body coloration, all of whichwere confirmed as T. orientalis with the DNAmarker (Table 2).

The length-weight relationship of T. maccoyii wascompared with that of T. orientalis, using fish ofcomparable size (127 cm fork length (FL) or larger,

36 T. orientalis and 14 458 T. maccoyii). A f-test ofequivalent slopes showed a significant difference (P< 0.05, d.f. = 8, n = 14 494), confirming that thereis a quantifiable difference in the length-weightrelationship of the two species, that is consistent withthe observers' descriptions of elongated shape in T.orientalis. Fig. 3 shows the length frequencydistributions for T. maccoyii and T. orientalis.Fork lengths were recorded for 17 543 T.maccoyii from observed tuna longline vesselsbetween 1987 and 1999, and from 53 of theconfirmed T. orientalis. The T. orientalismeasured by observers in the New Zealand EEZare generally larger (mean FL 190 cm, range 127—250 cm) than T. maccoyii (mean FL 151 cm,range 82-215 cm). Ninety-percent of T. maccoyiiwere less than 180 cm, whereas 67% of T.orientalis were greater than 180 cm in FL.

Thunnus orientalis are found throughout the NewZealand EEZ (Fig. 2) but appear to be more commonaround the North Island with 44 out of 54 recordsfrom the North Island, despite c. two-thirds of theobserved sets around the South Island.


DISCUSSION

Specimens of Pacific bluefin tuna T. orientalis fromJapan and southern bluefin tuna T. maccoyii fromNew Zealand have different mitochondrial DNAhaplotypes; amplified fragments of the ATCO regionproduce species-specific fragments when cut withthe diagnostic restriction enzyme Alu I or Mse I(Chow & Inoue 1993; Chow & Kishino 1995).

Most of the specimens recorded as northern (=Pacific) bluefin tuna in New Zealand waters before1994 were T. maccoyii. These specimens had beenidentified by large body size and dark colour of thecaudal keel. The four specimens identified as T.orientalis with the DNA restriction enzyme digestswere the same specimens identified as T. orientaliswith allozyme markers (Smith et al. 1994). Theallozyme data (Smith et al. 1994) and the DNA datapresented here indicate that most specimens recordedas Pacific bluefin tuna before 1994 had beenmisidentified and that T. orientalis are rare in NewZealand waters. The external characters of caudalkeel colour and body colour alone are unreliable fieldcharacters for identifying T. orientalis in NewZealand waters. Large specimens with a yellowcaudal keel are T. maccoyii (Gibbs & Collette 1966),but large fish with a dark caudal keel can be eitherT. maccoyii or T. orientalis.

Field identification based on the presence of themuscular dorsal bulge in the body cavity is a reli-able character in large fish. All 33 specimens iden-tified by this character from 1996 onwards wereconfirmed by the diagnostic DNA marker (Table 2).However, differences in the shape of the dorsal wallof the body cavity may not be apparent in specimensless than c. 130 cm (Gibbs & Collette 1966), and sothis character is only useful for distinguishing largespecimens of T. maccoyii and T. orientalis. In smallspecimens the colour of the caudal keel may be amore reliable character, but a DNA test would con-firm identification. One small Pacific bluefin tuna(127 cm FL) was reported from the New Zealandfishery. The specimen was correctly identified by thecrew and confirmed as T. orientalis by the DNA test,but unfortunately the identification characters werenot recorded.

Dark body coloration was only recorded inspecimens of T. orientalis. Japanese crew on tunalongline vessels refer to both T. orientalis and T.thynnus as kuro maguro (= black tuna). Speckled ormottled colour patterns were also restricted to T.orientalis. Specimens of bluefin tuna with speckledor mottled colour patterns have not been reported in

the scientific literature (Gibbs & Collette 1966;Collette & Nauen 1983). It is possible that thesecolour patterns fade after death and are lost by thetime that frozen specimens are examined in port.Any specimen of bluefin tuna in New Zealand waterswith unusual coloration (dark body colour, speckledor mottled pattern) and/or dark caudal keels shouldbe considered as a possible T. orientalis. Identity canbe confirmed by the presence/absence of the dorsalbulge in large (>130 cm) specimens. A small pieceof muscle tissue should be fixed in ethanol or frozenfor DNA confirmation of small specimens. Recently,Takeyama et al. (2000) developed species-specificluminescent DNA probes, for the ATCO region ofmitochondrial DNA that differs in just fournucleotides between T. thynnus and T. orientalis.Future application of this technology will provide atool for the rapid identification of large numbers oftuna specimens.

The definition of southern bluefin tuna as "fishwith the scientific name Thunnus maccoyi; andincludes the fish with the scientific name Thunnusthynnus" that appears in the southern bluefin tunaquota regulations (Ministry of Fisheries 2000) isincorrect. The definition of southern bluefin tunashould be modified to exclude Atlantic bluefin tunaThunnus thynnus.

T. orientalis occur in New Zealand waters but areuncommon. In the New Zealand fishery more than17 500 specimens of T. maccoyii were measuredbetween 1990 and 2000. Over the same time periodonly 59 T. orientalis were confirmed, and thusaccount for less than 0.3% of all bluefin tunameasured in New Zealand waters.

ACKNOWLEDGMENTS

We thank the Ministry of Fisheries observers RichardWin, Eric Prattley, Dave Wrightson, Alan Knox, AndrewSealey, Jacqui Mclntosh, Peter Benson, Chris Petyt, RobCoy, Simon Anderson, and P. J. Kenney, who returnedtissue samples and descriptions of the bluefin tuna speci-mens. In particular, we acknowledge Dave Wrightsonwho first used the dorsal bulge method for identificationof northern bluefin tuna on commercial tuna vessels inthe New Zealand EEZ. We also thank Peter Benson(NIWA) for technical assistance and Talbot Murray(NTWA) for advice on tuna identification and construc-tive comments on the manuscript. The project was sup-ported by the Ministry of Fisheries project MOF2000/02B.


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dna identification of pacific bluefin tuna {thunnus orientalis) in

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