BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions,research libraries, and research funders in the common goal of maximizing access to critical research.

Characterization of Alpha-Fetoprotein in Fetal Striped Dolphin (Stenellacoeruleoalba): Purification of Protein Product and Molecular Cloning of theCorresponding TranscriptAuthor(s): Yuka Morita, Naoshi Hiramatsu, Toshiaki Fujita, Haruna Amano, Takashi Todo andAkihiko HaraSource: Zoological Science, 28(3):215-224. 2011.Published By: Zoological Society of JapanDOI: http://dx.doi.org/10.2108/zsj.28.215URL: http://www.bioone.org/doi/full/10.2108/zsj.28.215

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological,and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and bookspublished by nonprofit societies, associations, museums, institutions, and presses.

Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance ofBioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use.

Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercialinquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.

2011 Zoological Society of JapanZOOLOGICAL SCIENCE 28: 215–224 (2011)

Characterization of Alpha-fetoprotein in Fetal Striped Dolphin

(Stenella coeruleoalba): Purification of Protein Product and

Molecular Cloning of the Corresponding Transcript

Yuka Morita1, Naoshi Hiramatsu2, Toshiaki Fujita2, Haruna Amano2,

Takashi Todo2 and Akihiko Hara2*

1Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato, Hakodate,

Hokkaido 041-8611, Japan2Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minato, Hakodate,

Hokkaido 041-8611, Japan

Alpha-fetoprotein (AFP) is a fetal glycoprotein that is known as a biomarker for monitoring preg-

nancy in many mammalian species. However, characterization of AFP has not yet been undertaken

in any cetacean species. Here, we purified AFP from the serum of fetal striped dolphin by chemical

precipitation followed by a combination of immunoadsorbent column chromatography and gel fil-

tration. The molecular masses of native and denatured dolphin AFP were estimated to be ~78,000

Da by gel filtration and ~68,000 Da by SDS-PAGE, respectively, representing typical masses reported

for mammalian AFPs. In fetal serum, only the AFP band (~68,000 Da) appeared to be immunoreac-

tive to an antiserum against purified dolphin AFP, indicating sufficient specificity for the develop-

ment of an AFP immunoassay. Full-length cDNA encoding for the dolphin AFP was cloned from

fetal liver and revealed an open reading frame comprising 610 amino acid residues, which included

a putative signal peptide of 18 amino acid residues. This was followed by a sequence identical to

the N-terminus of purified AFP. The deduced amino acid sequence of dolphin AFP showed more

than 80% identity to those of other mammalian AFPs. To our knowledge, the present report repre-

sents the first identification and characterization of AFP from any cetacean species.

Key words: alpha-fetoprotein, purification, cDNA cloning, cetaceans, Stenella coeruleoalba

INTRODUCTION

Many facilities maintaining cetacean species make spe-

cial efforts to breed and raise their animals in captivity.

Current captive programs for managing cetacean breeding

are satisfactory in general, but there remains significant

room for improvement.

As in other mammalian species, the first trimester of

pregnancy has been a particularly critical aspect of dolphin

breeding programs, and efforts to confirm early pregnancy

in cetacean species have been the focus of much attention

and priority (Ridgway and Benirschke, 1977; Jensen, 1999).

At present, controlled natural breeding, as well as the use of

advanced reproductive technologies, such as artificial

insemination and diagnostic ultrasound, have allowed man-

agers to maximize breeding success (Odell and Robeck,

2002).

However, artificial insemination also requires the devel-

opment of appropriate technology to accurately determine

pregnancy. This is important because the early detection of

pregnancy would allow managers to place pregnant individ-

uals in an appropriate social structure, provide necessary

nutrition, and control activity as needed for precautionary

reasons. Ultrasound has proven to be an effective means of

confirming pregnancy, and is utilized in such diagnoses.

However, the operation of ultrasound in this species

requires special skills, experience, and equipment.

In many facilities, serum progesterone levels are used to

detect cycling and pregnant female dolphins. However,

pseudopregnancy has been reported in several cetacean

species (Yoshioka et al., 1986; Atkinson et al., 1999), and it

remains clear that diagnoses based upon serum proge-

sterone may lead to false positives. Consequently, there is

a great need for the development of novel markers for the

detection and monitoring of pregnancy in cetacean species

in order to develop and manage breeding strategies effec-

tively.

Improving our understanding of mechanisms responsi-

ble for regulating pregnancy in cetaceans, and the accurate

detection of pregnancy and continuous monitoring of preg-

nancy, represent key factors in the further development of

successful breeding programs. The establishment and

maintenance of pregnancy in mammals requires the syn-

thesis and secretion of various proteins from the fetus and/

or placenta. These proteins often appear in the maternal

circulation and/or amniotic fluid as pregnancy-specific or

associated proteins. Several proteins, such as chorionic

* Corresponding author. Phone: +81-138-40-8878;

Fax : +81-138-40-8878;

E-mail: aki@fish.hokudai.ac.jp

doi:10.2108/zsj.28.215

Y. Morita et al.216

gonadotropin (CG), placental lactogen (PL) and pregnancy-

associated glycoprotein (PAG), have been used as markers

for detecting and/or monitoring pregnancies (i.e., prenatal

diagnosis) in several mammalian species, and may be

useful in cetaceans as well (Heap and Flint, 1984; Sinosich

et al.,1985; Reis et al., 2002; Sousa et al., 2008). However,

we currently know very little regarding the relative properties

and roles in reproduction of pregnancy-specific proteins in

cetacean species.

Alpha-fetoprotein (AFP) is a protein known to be asso-

ciated with pregnancy and is often referred to as carcino-

genesis-associated fetal protein, due to its involvement with

both ontogenic and oncogenic growth (Abelev et al., 1963;

Tatarinov, 1965). AFP is a major plasma protein synthesized

by both the yolk sac and the fetal liver. Once synthesized, it

is secreted into the blood stream. AFP synthesis may also

occur in the embryonic kidney, pancreas, and gastrointestinal

endoderm (Gitlin et al., 1972; Gitlin, 1975), while it is also

found, albeit in low levels, in the serum of normal healthy

adults. By contrast, during pregnancy, AFP originating from

the fetus can be found in relatively high levels in the amni-

otic fluid and maternal serum. Owing to this property, AFP

is often used in prenatal screening, especially for neural

tube defects and Down’s syndrome (Ross and Elias, 1997).

The biological role of this protein is not clearly understood,

but AFP may play a role in the maintenance of growth reg-

ulation, protection against maternal estrogen and regulation

of the immune system (Crandall, 1981; Deutsch, 1991;

Nunez, 1994; Ogata et al., 1995).

AFP has been purified and characterized in a variety of

mammalian species, including human, dog, rabbit, mouse,

rat, guinea pig, sheep, pig, and cattle (Nishi and Hirai, 1972;

Watabe, 1974; Lai et al., 1977; Aliau et al., 1978; Versée

and Barel, 1978; Gourdeau, and Bélanger, 1983; Fujimoto

et al., 1984; Karmali and Novo, 1990; Yamada et al., 1995).

Characterization of AFPs in these mammalian species

revealed that biochemical and antigenic properties were

largely similar, thus confirming its importance in pregnancy.

To date, AFP has not been identified or characterized in

cetaceans.

This study describes the detection, purification, bio-

chemical characterization, and molecular cloning of AFP in

the striped dolphin (Stenella coeruleoalba), a small ceta-

cean species commonly distributed around the world in both

tropical and temperate waters. As such, this particular spe-

cies represents a suitable model to aid in deepening our

understanding of cetacean reproduction.

MATERIALS AND METHODS

Experimental animals and tissue samples

Blood or tissue samples were collected from striped dolphin,

bottlenose dolphin, and Risso’s dolphin (Grampus griseus) caught

by drive fisheries off the Pacific coast of Taiji, Wakayama Prefec-

ture, Japan from December 2007 to February 2008. Adult and fetal

blood samples were taken from the abdominal cavity and stored in

plastic tubes. Samples were allowed to stand at 4°C overnight and

serum separated by centrifugation at 3,000 rpm for 20 min using a

Sepaclen tube (Eiken Kizai, Tokyo, Japan). Serum samples were

stored at –30°C until use. Placenta and fetal liver samples were

obtained from four pairs of mother and calf, respectively. Fetal body

lengths were 9.9, 35.4, 60.7 and 91.9 cm. Samples were dissected,

fixed in RNAlater (Ambion, Austin, TX, USA), and stored at –30°C

in preparation for total RNA extraction.

Antisera

Polyclonal antisera against fetal and male sera (anti-fetus and

anti-male respectively) were raised in rabbits by intra-dermal injec-

tion of each antigen. Rabbits received 0.5 ml of antigen emulsified

with an equal volume of Freund’s complete adjuvant (Iatron, Tokyo,

Japan). Injections were given eight times, at more than one-week

intervals. Blood was collected from the ear vein a week after the

fourth injection. and serum was collected for analysis. In addition,

an anti-fetus antibody, which had been pre-adsorbed with male

serum (termed hereafter as ‘ab.anti-fetus’) was raised for the detec-

tion of fetal-serum-specific protein(s). In addition, a specific antise-

rum against dolphin AFP (anti-dolphin AFP) was prepared by

immunizing rabbits with purified AFP via lymph node injection with

100 μg of antigen emulsified in Freund’s complete adjuvant, fol-

lowed by three additional injections (400 μg in total) into the dorsal

musculature. Blood was collected from the ear vein and serum was

collected as anti-dolphin AFP. Rabbit antiserum against purified pig

AFP (anti-pig AFP) and horse antiserum against human AFP (anti-

human AFP) were prepared as described in previous studies

(Fujimoto et al., 1984 and Nishi and Hirai, 1972, respectively).

Electrophoresis and immunological procedures

Immunoelectrophoresis (IEP) and double immunodiffusion

were carried out in 1% agarose gels prepared in 0.05 M sodium bar-

bital buffer (pH 8.6) and 0.9% NaCl containing 0.1% NaN3, respec-

tively, following the methods of Grabar and Williams (1953) and

Ouchterlony (1953), respectively. Gels were stained with Amido

black 10B. Discontinuous (DISC) polyacrylamide gel electro-

phoresis (PAGE) was carried out in 7.5% polyacrylamide gels using

the method of Davis (1964) and gels were stained with Amido black

10B. Sodium dodecyl sulfate-PAGE (SDS-PAGE) was performed

using a 10% stacking gel, following the method described by Laem-

mli (1970). Gels were stained with 0.1% Coomassie Brilliant Blue

R250 (CBB; Bio-Rad, Herculed, CA, USA). The molecular weight of

resultant separated protein bands was estimated using Precision

Plus Protein Standards (Bio-Rad, CA, USA). Western blotting was

carried out as described by Towbin et al. (1979) using the poly-

clonal rabbit antisera described above.

Column chromatography

Immunoadsorbent column chromatography was performed

using a Sepharose 4B (GE Healthcare UK Ltd., Buckinghamshire,

England) column (7.5 × 2.3 cm) coupled with anti-human AFP or

anti-male antibodies. Sepharose 4B was pre-activated by BrCN

according to the method of Nishi and Hirai (1972) modified by

Fujimoto et al. (1984) and coupled with the appropriate antibody.

Samples were first eluted by phosphate-buffered saline (pass-

through fractions) and then with 8.0 M urea at a flow rate of 20

ml/h. Eluted fractions were collected in a volume of 3.0 ml per

tube.

Gel filtration using a pre-packed Superdex200 column (GE

Healthcare UK Ltd.) was performed on an FPLC system (GE

Healthcare UK Ltd.). Samples were eluted with 0.02 M Tris-HCl

buffer (pH 8.0) containing 2% NaCl and 0.1% NaN3. The column

was run with a flow rate at 0.5 ml/min and fractions were collected

at a volume of 0.3 ml per tube. In order to calibrate the gel filtration

column, the following marker proteins were used: aprotinin (6.5

kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa), conal-

bumin (75 kDa), transferrin (81 kDa), immunoglobulin G (150 kDa),

aldolase (158 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa).

N-terminal amino acid sequencing

Purified dolphin AFP was separated by SDS-PAGE, electrob-

lotted onto polyvinylidene difluoride (PVDF) membrane (Immulilon-

PSQ; Millipore, Bedford, MA, USA) and stained with CBB. Bands

Alpha-fetoprotein in Striped Dolphin 217

were cut out from the membrane and subjected to N-terminal amino

acid (AA) sequencing on a Procise 492 HT system (Applied Biosys-

tems, CA, USA).

Isolation of cDNA clones

All procedures concerning the cloning of dolphin AFP cDNA

were performed following the manufacturer’s instructions, unless

otherwise stated below. Total RNA was prepared from fetal liver

(body length 60.7 cm) using Isogen (Nippon-GENE, Tokyo, Japan)

and utilized for the synthesis of first-strand cDNA using Super Script

III First-Strand Synthesis Super Mix Kit (Invitrogen, CA, USA). A

degenerate primer set (forward: 5′-TTCCAYATTRGATTCTTCCCA-

RTG-3′; reverse: 5′-TAASCAAYRAGRAACRSATTTTGTAAGTA-3′) was designed to amplify cDNAs encoding dolphin AFP sequences,

based on the published AFP sequences for Norway rat (Rattus

norvegicus ; Genbank accession number: BC097344), cattle (Bos

taurus; BC103123), house mouse (Mus musculus; BC066206), and

human (Homo sapiens; BC027881). Polymerase chain reaction

(PCR) amplification was performed using PCR Master Mix

(Promega, WI, USA) in a reaction volume of 20 μl containing each

degenerate primer (0.5 μM in the final concentration) and hepatic

cDNA template according to the following conditions: the amplifica-

tion was initiated by a denaturation step (95°C for 2 min), followed

by 35 cycles of an amplification step (95°C for 45 sec, 54°C for 30

sec, and 72°C for 2 min), followed by 10 min of elongation at 72°C.

Amplified cDNA fragments were separated by 1.5% agarose gel

electrophoresis and purified from the gel using GENECLEAN Turbo

Kit (MP Biomedicals Europe, Illkirch, France). The isolated cDNA

was ligated into pGEM-T Easy Vector (Promega, WI, USA) and

transformed into XL1-Blue competent cells (Strategene, CA, USA).

Recombinant clones were cultured overnight at 37°C following

selection of colonies by blue-white screening and antibiotic resis-

tance (ampicillin and tetracycline). Selected clones were then used

to extract and purify plasmid DNA using the Wizard Plus SV Mini-

preps DNA Purificaiton System (Promega, WI, USA). Purifed plas-

mid DNA was sequenced using the BigDye Terminator v3.1 Cycle

Sequencing Kit (Applied Biosystems, CA, USA) and primers target-

ing to either the T7 or SP6 priming site. The resulting product was

purified by the Big Dye X Terminator Purification Kit (Applied Bio-

systems) and sequenced on an ABI 3130 Genetic Analyzer (Applied

Biosystems).

Rapid amplification of cDNA ends (RACE) and full-length cDNA

sequence

In order to obtain 5′ and 3′ ends of AFP sequence, cDNA tem-

plates were synthesized from fetal liver total RNA using the SMART

RACE cDNA amplification Kit (Clontech, Takara Bio. Inc., Shiga,

Japan). Gene-specific primers (GSPs) were designed based on the

partial sequences of dolphin AFP as follows: AFP 5′-GCTGCCTTT-

GTTTGGAAGCATTCAAC-3′ (outer PCR primer used in 5′-RACE);

AFP 5′-AACTTGGAAAGGTGGGATGGATGCT-3′ (nested PCR

primer used in 5′-RACE); AFP 5′-CCGCACTTGAACTTGGTCAT-

TGC-3′ (outer PCR primer used in 3′-RACE); AFP 5′-GGAGAAAT-

GTTCACAGTCTGGAAACCCT-3′ (nested PCR primer used in 3′-RACE).

Following identification of the 5′ and 3′ ends of AFP sequences,

a GSP was newly designed at the 5′ end in order to obtain a con-

secutive full-length AFP sequence as follows: AFP-5′ end primer 5′-GAGGCATTGCTAGAGAAGACTATAAAAG-3′. This GSP was used

in the 3′-RACE reaction and resulting PCR products were sub-

cloned into the pGEM-T Easy vector for sequencing.

Observation of AFP expression by conventional RT-PCR

analysis

For conventional RT-PCR analysis of AFP gene expression,

total RNA was extracted from fetal liver and placenta and subjected

to reverse transcription, as described above. In addition to normal

reverse transcription, a reaction omitting the reverse transcriptase

but containing all other components was also performed in order to

produce a no-RT control (NRT) template for RT-PCR. A GSP primer

set for amplifying dolphin AFP (forward: 5′-GTATGGGCTTTCA-

GACTG-3′; reverse: 5′-TGTAAGCAACAAGAAACGC-3′) was

designed at positions flanking a 949 bp AFP fragment, while a

primer set for amplifying dolphin glyceraldehyde-3-phosphate dehy-

drogenase (GAPDH) (forward: 5′-GACAACCACCTCAAGATCGT-

3′; reverse: 5′-TCTCAAGTGTGTTGGAGGAC-3′) was designed at

positions flanking a fragment (651 bp) of dolphin GAPDH sequence

(DQ404538) for control purposes. Amplification was conducted as

described above, with the following modifications: 35 cycles of the

amplification step was performed at: 95°C for 45 sec, 55°C (AFP)

or 60°C (GAPDH) for 30 sec, and 72°C for 2 min. The resulting

PCR products were separated by 1.5% agarose gel electrophoresis

to determine AFP and GAPDH expression.

Alignment and phylogenetic analysis

Homology searches of the N-terminal amino acid sequence, as

well as nucleotide or deduced AA sequences of dolphin AFP, were

performed with the BLAST homology search tool (http://

blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analysis of mamma-

lian AFP was performed using the Neighbor Joining (NJ) method

using default settings and MEGA version 4.0.2 software (Tamura et

al., 2007) from CLUSTALW alignments (Thompson et al., 1994).

Statistical significance was evaluated by bootstrap analysis (1000

replicates).

RESULTS

Detection of dolphin AFP

In order to detect AFP, IEP was performed with dolphin

sera using anti-human AFP and anti-pig AFP antibodies.

The ab.anti-fetus antibody (pre-absorbed) was also utilized

in the IEP to detect fetal-serum-specific protein(s). Fetal

serum, but not adult male or female sera, showed immuno-

reactivity against both AFP antisera in IEP (Fig. 1A). In addi-

tion, ab.anti-fetus antibody gave rise to one precipitin line at

the position of AFP (Fig. 1B).

Purification of dolphin AFP

An outline of the procedure used to purify dolphin AFP

is presented in Fig. 2. Fetal serum (~30 ml) was precipitated

by adding ammonium sulfate at 40% saturation and the

resulting supernatant was collected. This fraction was

loaded onto the imunoadsorbent column chromatography

coupled with anti-human AFP antibody (Fig. 3A). Bound pro-

teins were eluted with 8 M urea; this fraction was rich in AFP

when assessed by IEP and SDS-PAGE (data not shown).

Although AFP was a major component in this fraction, other

serum components were present, indicating contamination.

Therefore, additional immunoabsorbent column chromatog-

raphy was performed using the anti-male antibody to

exclude contaminating serum components (Fig. 3B). The

pass-through fraction was rich in AFP and free of contami-

nation (data not shown). This fraction was then loaded onto

a Superdex 200 column (Fig. 3C). A major peak followed by

minor peaks was observed at a position corresponding to 78

kDa. This major peak was collected as purified dolphin AFP.

Biochemical and immunological characterization of

dolphin AFP

Purified AFP formed one precipitin arc in IEP using anti-

fetus, anti-human AFP, and the anti-fetus antibodies; all pre-

Y. Morita et al.218

ciptin arcs occurred in the same position (Fig. 4). No

reaction was found between purified AFP and anti-male

antibody, providing immunological evidence that AFP was

isolated specifically. Purified AFP was also analyzed by 10%

SDS-PAGE and Western blotting using anti-male and anti-

pig AFP antibodies (Fig. 5). In SDS-PAGE, purified AFP

appeared as a single band with a relative mass of ~68 kDa

under reducing condition. In Western blotting, this band was

immunoreactive to anti-pig AFP, but not to the anti-male

Fig. 1. Immunoelectrophoresis of male (M), female and fetal (Fe)

striped dolphin serum samples. Antisera were raised against (A)

alpha-fetoproteins of human (anti-human AFP) and pig (anti-pig AFP),

and (B) fetal serum (anti-fetus). The anti-fetus antibody was pre-

absorbed with male dolphin serum (ab. anti-fetus) prior to analysis.

Fig. 2. Outline of the procedure used to purify alpha-fetoprotein

(AFP) from striped dolphin fetal serum. See RESULTS for a detailed

description of the purification procedures. Ppt., precipitated fraction;

Sup., supernatant fractions; SAS, saturated ammonium sulfate;

Pass, Pass-through fraction; Urea, fractions eluted with 8 M urea.

Fig. 3. Elution profiles of crude alpha-fetoprotein (AFP) fractions

obtained from two steps of immunoadsorbent column chromatogra-

phy (A) Sepharose 4B coupled with antiserum against human AFP;

(B) antiserum against male dolphin serum; and, (C) gel filtration on

Superdex 200. The initial bound fractions were eluted with 8 M urea,

represented by shaded areas in (A), and subsequently pooled to be

applied onto the second immunoadsorbent column (B). The pass-

through fractions (shaded areas in B) were pooled and applied to

the gel filtration column; fractions eluted around the first major peak

(peak fraction number 49) were collected as purified AFP.

Alpha-fetoprotein in Striped Dolphin 219

antibody. In contrast, two

bands were observed when

purified AFP was subjected

to DISC-PAGE (Fig. 6).

N-terminal amino acid sequ-

ence analysis

Partial N-terminal AAs

obtained from purified dolphin

AFP and other mammals

were compared (Table 1).

The dolphin AFP sequence

showed considerably high

identity and similarity (70–

90%) against known mam-

malian AFP sequences.

Specificity of antiserum

against dolphin AFP

On IEP, anti-dolphin AFP

antibody reacted with both

purified AFP and fetal serum,

forming one precipitin line

(Fig. 7). Anti-human AFP

antibody also gave rise to a

precipitin line at the same

position as that of anti-

dolphin AFP antibody. In con-

trast, both antisera failed to

form any precipitin line with

male serum. The specificity

of anti-dolphin AFP was also

confirmed by Western blot-

ting. No immuno-reaction

was observed against male

dolphin serum, while one

band was detected at ~68

kDa in both fetal serum and

purified AFP. Fetal sera from

bottlenose dolphin and

Risso’s dolphin cross-reacted

with anti-dolphin AFP and

their precipitin lines fused

with fetal serum from striped

dolphin (Fig. 8).

Cloning and sequence

analysis of the dolphin AFP

A full-length cDNA

encoding AFP was cloned

from the striped dolphin fetal

liver by RT-PCR following

two RACE procedures. The

nucleotide sequence of the

AFP cDNA and its corre-

sponding deduced AA sequ-

ence are shown in Fig. 9

(Genbank accession number

GU359055) and its corre-

sponding deduced AA sequ-

ence (ADK77964). The AFP

Fig. 4. Immunoelectrophoresis (IEP) of striped dolphin sera and fractions obtained during purification.

Analyses were performed using antisera raised against male (M) and fetal (Fe) dolphin serum (i.e., anti-

male and anti-fetus), and purified human alpha-fetoprotein (anti-human AFP). The anti-fetus antibody was

used either directly (left) or pre-absorbed with male dolphin serum prior to use (ab.anti-fetus) (right) in the

IEP assay, as necessary. 2nd pass, pass-through fraction obtained during the second affinity chromatog-

raphy (see Fig. 3B); 49, the peak fraction (number 49) observed in Fig. 3C.

Fig. 5. (A) SDS-PAGE and Western blots using antisera raised against (B) male dolphin serum (anti-

male) and (C) purified pig alpha-fetoprotein (anti-pig AFP). M, male serum; Fe, fetal serum; 2nd Pass, the

pass-through fraction obtained during the second affinity chromatography (see Fig. 3B); 49, the peak frac-

tion (number 49) observed in Fig. 3C.

Table 1. Comparisons in N-terminal amino acid (AA) sequences and relative molecular weights deter-

mined for purified striped dolphin alpha-fetoprotein (AFP) and other mammalian AFPs. The N-terminus of purified dolphin AFP was aligned to deduced amino acid sequences of AFP for: cattle [GenBank Accession number, NP_001029434], dog [NP_001003027], pig [NP_999482], horse [NP_001075421], woodchuck [AAK55757], human [NP_00125] and Norway rat [NP_036625]. Bold and underlined letters represent AA residues identical to the dolphin AFP sequence. Molecular weights of AFPs are listed here for: cattle (Aliau et al., 1978), dog (Yamada et al., 1995), pig (Fujimoto et al., 1984), human (Karmali and

Novo, 1990), Norway rat (Versée and Barel, 1978).

Species N - terminal sequenceMolecular weight

SDS-PAGE Gel filtration

Striped dolphin (Stenella coeruleoalba) R T M Q K N A Y G I 68 kDa (2ME+) 78 kDa

Cattle (Bos taurus) R T M H K N A Y G I 67 kDa 75 kDa

Dog (Canis lupus familiaris) R T M H R N A Y G I 66 kDa (2ME-)

Pig (Sus scrofa) R T M H S N A Y G I 75 kDa 80 kDa

Horse (Equus caballus) R T M H S N A Y G I

Woodchuck (Marmota monax) R T L H N N A Y G I

Human (Homo sapiencs) R T L H R N E Y G I 72 kDa

Norway rat (Rattus norvegicus) R V L H T N E F G I 71 kDa

Y. Morita et al.220

cDNA clone (2089 bps in total length excluding poly-A tail)

consisted of a complete open reading fame (ORF; 1830

bps) encoding 610 AA residues and 5’ and 3’ untranslated

regions (94 and 165 bps, respectively). Within the ORF, the

partial deduced AA sequence (AA positions 19–28) was

identical to the sequence obtained from purified dolphin AFP

(RTMQKNAYGI); the ORF of dolphin AFP thus included a

putative signal peptide consisting of 18 AA residues. More-

over, three distinct domains were evident within the

sequence: domains I (170 AA, positions 40–209), II (182

AA, positions 221–402) and III (187 AA, positions 414–600).

These domains contained a total of 32 cysteine residues,

which are typically found in members of the albuminoid fam-

ily of genes. In addition, a valine residue was found at the

C-terminus, as in other mammalian AFPs. Sequence analy-

sis for N-linked glycosylation sites revealed that striped

dolphin AFP contains one potential site at residues 251–

Fig. 6. DISC-PAGE (7.5%) of purified dolphin alpha-fetoprotein.

Arrows represent two bands evident in the gel. An enlarged image

of these bands is shown in the right panel.

Fig. 7. Immunoelectrophoresis and Western blotting of dolphin

serum and purified alpha-fetoprotein (AFP) using antisera raised

against purified AFP (anti-dAFP) and human AFP (anti-human

AFP). M, male serum; Fe, fetal serum.

Fig. 8. Double immunodiffusion of fetal sera from striped dolphin

(1), bottlenose dolphin (2) and Risso’s dolphin (3) using antiserum

against purified dolphin AFP (anti-dAFP). The central well was

loaded with anti-dAFP, while peripheral wells were loaded with

serum samples.

Fig. 9. Nucleotide and deduced amino acid sequences of striped

dolphin alpha-fetoprotein (GENBANK accession number: dAFP;

GU359055 and ADK77964, respectively). The amino acid (AA)

sequence is shown below the nucleotide sequence. The N-terminal

sequence determined for the polypeptide of purified dolphin AFP

proteins was aligned to the deduced AA sequence and represented

as shaded letters. Sequences enclosed by solid lines and arrows

represent conserved albumin domains I, II and III. Csteine residues

are underlined and boldfaced. The dotted line indicates the potential

N-glycosylation site.

Alpha-fetoprotein in Striped Dolphin 221

253. The mass of dolphin AFP was estimated to be 68.3

kDa based upon the deduced AA sequence. Identities and

similarities of the deduced AA sequence of dolphin AFP

compared with other mammalian AFPs are as follows: 90

and 94% to pig, 88 and 92% to dog, 87 and 92% to cattle,

83 and 90% to horse, 83 and 90% to rhesus monkey, 82

and 89% to gorilla, 82 and 90% to human, respectively.

Thus, dolphin AFP exhibited more than 80% identity with AA

sequences from other mammalian AFPs.

Phylogenetic analysis was carried out using dolphin

AFP AA sequences along with sequences from mammalian

AFPs and other albuminoid gene family proteins (e.g., albu-

min, afamin, and vitamin D binding protein) sourced from the

NCBI Gen Bank. The phylogenetic tree generated by NJ

analysis using AA sequences of several discrete proteins

from the albumin superfamily of peptides is shown in Fig.

10. Albuminoid gene family proteins in mammals are cate-

gorized into four major families (i.e., AFP, albumin, afamin

and vitamin D binding protein). Phylogenetic analysis

showed that dolphin AFP was included in the AFP family

branch and grouped with AFPs from other cetartiodactyls

(cattle and pigs), as was predicted from mammalian phylo-

genetic relationships.

AFP gene expression in fetal liver and placenta

Expression of the AFP gene was investigated by RT-

PCR in two tissue types associated with pregnancy (fetal

liver and placenta). Amplification of GAPDH was used as an

internal reference control. RT-PCR targeting AFP (Fig. 11A)

produced an amplified product of approximately 990 bps in

fetal liver samples, but not in the placenta. The intensity of

visualized AFP bands appeared to decrease as pregnancy

progressed. The amplified GAPDH product (Fig. 11B)

appeared in a uniform manner at the predicted position of

approximately 690 bps in all fetal liver and placenta sam-

ples. No bands were detected in the NRT (data not shown)

for both genes.

DISCUSSION

Few dedicated studies of pregnancy-related proteins

have been conducted in cetacean species. In early work,

Hobson and Wide (1986) detected both bioactivity and

immuno-reactivity of CG in the placenta of bottlenose

dolphin. In the same species, Watanabe et al. (2007)

recently identified placental expression of luteinizing

hormone-like substances. However, such markers have not

yet been evaluated for practical use during prenatal dia-

gnosis in cetaceans. Other pregnancy-associated proteins

(e.g., PL, PAG, or other new markers) also need to be iden-

tified and evaluated for their significance in reproduction and

their potential as novel diagnostic markers. In the present

study, we detected, purified, characterized, and identified

AFP in striped dolphin. First, we confirmed the presence of

AFP in fetal serum by IEP analyses using anti-mammalian

AFPs and ab.anti-fetus antibodies. During purification, these

antisera were also utilized to probe and selectively bind

dolphin AFP.

One major problem typically associated with the purifi-

Fig. 10. Phylogenic relationships of the deduced amino acid

sequence for dolphin alpha-fetoportein (AFP) with those for various

members of the mammalian albuminoid gene family. The GenBank

accession numbers of sequences used in the analyses are as fol-

lows: dolphin AFP (dAFP; GU359055), cattle alpha-fetoprotein

(cAFP; NP_001029434), pig alpha-fetoprotein (pAFP; NP_999482),

dog alpha-fetoprotein (doAFP); NP_001003027), horse alpha-feto-

protein (eAFP; NP_001075421), rhesus monkey alpha-fetoprotein

(rmAFP; XP_001103873), chimpanzee alpha-fetoprotein (chAFP;

Q28789), gorilla alpha-fetoprotein (gAFP; P28050), human alpha-

fetoprotein (huAFP; NP_001125), woodchuck alpha-fetoprotein

(wAFP; AAK55757), house mouse alpha-fetoprotein (mAFP;

NP_031449), rat alpha-fetoprotein (rAFP; NP_036625), house

mouse afamin (mAfa; AAI00598), human afamin (hAfa; AAI09021),

house mouse albumin (mALB; AAH49971), cattle albumin (cALB;

AAH51411), human albumin (hALB; CAA00606), house mouse

vitamin D binding protein (mVDB; AAA37669), cattle vitamin D bind-

ing protein (cVDB; Q3MHN5), human vitamin D binding protein

(hVDB; AAA61704).

Fig. 11. Reverse transcription (RT) PCR analyses of alpha-

fetoprotein (AFP) expression in fetal liver and placenta. Total RNA

samples obtained from striped dolphins at different stages of devel-

opment (9.9, 35.4, 60.7 and 91.9 cm in fetal length) were subjected

to RT-PCR reactions targeting AFP (A) and glyceraldehyde-3-phos-

phate dehydrogenase (GAPDH) (B). Corresponding PCR products

were analyzed by agarose gel electrophoresis (lane 1, 2, 3, and 4,

respectively).

Y. Morita et al.222

cation of AFP is the elimination of albumin contamination.

Human AFP exhibits 39% overall amino acid sequence

identity to human albumin. Differences in the predicted sec-

ondary structure of certain areas of these molecules suggest

they have significantly different molecular configurations

(Morinaga et al., 1983). Although AFP and albumin are

structurally distinct from one other, they appear to be similar

in terms of a variety of physical and chemical properties,

including electrophoretic mobility, molecular weight (70 kDa

for human AFP, 68 kDa for human albumin), and isoelectric

point (4.7 for human AFP, 4.9 for human albumin) (Ruoslahti

and Seppälä, 1971; Hirai, 1982). The presence of carbohy-

drate compounds in AFP, along with significant differences

in antigenicity, permits the separation of these proteins by

means of affinity chromatography. The procedure described

herein was thus likely efficient at removing albumin contam-

ination.

Several different procedures have been reported for the

separation of AFP in a variety of animals. In the present

study, we utilized the purification procedure reported by

Fujimoto et al. (1984), which is characterized by the use of

an immunoadsorbent column. This method is simple and

typically gives satisfactory results when anti-AFP antibody is

available. Purity of the final AFP product was confirmed by

the use of three immunological and biochemical methods:

IEP, SDS-PAGE, and Western blotting. The purified product

reacted with two types of AFP antibodies (anti-pig AFP and

anti-human AFP), but did not react with the anti-male anti-

body in either IEP or Western blotting, indicating that there

was no contamination of the samples with either albumin or

other serum components. Thus, our purification yielded a

reasonable quantity of highly purified AFP (several hundred

μg), which appeared to be free of albumin and other pro-

teins, and thus sufficient for immunizing a rabbit.

We identified the purified protein as striped dolphin AFP

from the following characteristics: 1) the purified product

was immunoreactive to antisera against AFP from other

mammalian species, 2) the molecular masses of the purified

product in SDS-PAGE (68 kDa) and gel filtration (78 kDa)

resembled those of other mammalian AFPs (Table 1: Aliau

et al., 1978 for cattle; Versée and Barel, 1978 for Norway

rat; Fujimoto et al., 1984 for pig; Karmali and Novo, 1990 for

human; Yamada et al., 1995 for dog), 3) the N-terminal AA

sequence of the purified protein was nearly identical to that

of other mammalian AFPs (Table 1), and 4) the deduced

AA sequence of dolphin AFP exhibited extremely high

identity (> 80%) with that of other mammalian AFPs and

localized to the same cluster of mammalian AFP genes.

Consequently, the present study describes the first isolation

and identification of AFP (both protein and transcript) in any

cetacean species.

The deduced AA sequence of dolphin AFP exhibited

three distinct domains that commonly exist in sequences

from albuminoid gene family members (Mcleod and Cooke,

1989). In the present study, 32 cysteine residues were iden-

tified in dolphin AFP domains, as is the case in many other

mammalian AFP sequences. It has been reported that

human AFP is constructed by the formation of 15 disulfide

bridges between 30 out of the 32 cysteine residues identi-

fied in this molecule (Morinaga et al., 1983). This indicates

that dolphin AFP is likely to form a similar tertiary structure

to that of other mammalian AFPs, or other albuminoid gene

family members.

Although SDS-PAGE of purified dolphin AFP yielded a

single band, the purified product appeared as doublets on

analysis by DISC-PAGE. Such heterogeneity was also evi-

dent in purified rat AFP by DISC-PAGE or by isoelectric

focusing analysis (Watabe et al., 1974). Causes of this

heterogeneity might be attributed to differences in AA

composition, sugar chain composition, linkage position, or

branching pattern (Kerckaert et al., 1975). In fact, there is

one potential site in the deduced dolphin AFP AA sequence

at which N-glycosylation appears to occur. Furthermore,

molecular variants of mammalian AFP have been reported

and attributed to carbohydrate microheterogeneity and alter-

ations in isoelectric points, different lectin forms and genetic

variants (Mizejewski, 2001). However, as with other mam-

malian species, the cause of the potential heterogeneity of

dolphin AFP remains unknown, and its physiological signifi-

cance unverified.

In the present study, a specific antiserum against

dolphin AFP (anti-dolphin AFP) was prepared by immunizing

rabbits with purified AFP. Specificity of the anti-dolphin AFP

antibody was thoroughly verified by IEP and Western blot-

ting. This antiserum specifically recognized the AFP protein,

but did not cross-react with any other components of male

serum, indicating failure to cross-react with either albumin or

other serum proteins. In addition, our antiserum exhibited

cross-reactivity with fetal sera from two other dolphin spe-

cies (bottlenose dolphin and Risso’s dolphin). This indicates

common antigenicity among AFP proteins from different

cetacean species. Characterization of anti-dolphin AFP anti-

body indicated that this antiserum can be utilized to develop

specific immunoassays for the detection and quantification

of AFP in striped dolphin and possibly in other cetaceans.

We also examined the expression of the AFP gene in

striped dolphin fetal liver and placenta. The body size of

neonates from this species is approximately 100 cm

(Miyazaki, 1977), indicating that samples used in our RT-

PCR analysis covered early to late stages of pregnancy. RT-

PCR results indicated that significant AFP expression

occurred in fetal liver samples, but not in the placenta, sug-

gesting that the former tissue is likely to be a primary site of

AFP synthesis during pregnancy in cetaceans. We also

revealed that gene expression levels tend to be higher in

early pregnancy, decreasing as gestational age progresses.

In other mammals, levels of AFP in fetal serum appeared to

be high throughout the first trimester of pregnancy and

declined thereafter until term (Mizejewski, 2003). Results of

the durational change of fetal AFP gene expression in

striped dolphin might, hypothetically, reflect those in mater-

nal circulation, indicating that circulating AFP may potentially

be used as a reliable pregnancy-associated marker.

In summary, the present study described, for the first

time, the detection, characterization and identification of

AFP protein and its corresponding transcript in the striped

dolphin fetus¸ the first time AFP has been identified in any

cetacean. The present study provides a vital first step

towards the development of a specific AFP immunoassay,

which might enable us to utilize AFP as a practical bio-

marker for the diagnosis of pregnancy in cetacean species.

Quantitative characterization of the occurrence of such

Alpha-fetoprotein in Striped Dolphin 223

biomarkers during pregnancy will provide a better under-

standing of the mechanisms and physiology of reproductive

phenomena in cetaceans, which will in turn aid in the regu-

lation and manipulation of their reproduction in the future.

ACKNOWLEDGMENTS

We thank Dr. T. Iwasaki, the section leader of the Cetacean

Population Biology Section, National Research Institute of Far Sea

Fisheries, Fisheries Research Agency, for help in collecting sam-

ples and critically reading the manuscript. We also thank Mr. Y.

Mizutani, the president of Taiji Fisheries Association, and the drive

fishery team ‘Isana Kumiai’ for permitting the use of dolphin speci-

mens. We extend our gratitude to Mr. T. Hara and Mr. Y. Tomizawa

of the Cetacean Population Biology Section, National Research

Institute of Far Sea Fisheries, Fisheries Research Agency, for their

help in collecting samples. We also wish to express our thanks to

Dr. S. Nishi, Professor Emeritus of Hokkaido University, for provid-

ing antiserum for human AFP. We are also grateful to Dr. M.

Shimizu, Lecturer of the Faculty of Fisheries Sciences, Hokkaido

University, for critically reading the manuscript. We extend our

appreciation to Dr. M. Suzuki, Lecturer of the College of Bio-

resource Science, Nippon University, for collecting samples and

helpful discussion. Thanks also to Dr. M. Yoshioka, Professor of the

Faculty of Bioresources, Mie University, for helpful discussion. This

work was supported in part by the Grant-in Aid for 21st Century COE

program and the Grant-in-Aid for Research Fellows of the Japan

Society for the Promotion of Science (no 21·1649 to Y. Morita).

REFERENCES

Abelev GI, Perova SD, Khramkova NI, Postnikova ZA, Irlin IS (1963)

Production of embryonal α-globulin by transplantable mouse

hepatomas. Transplantation 1: 174–180

Aliau S, Marti J, Moretti J (1978) Bovine alpha-fetoprotein. Isolation

and characterization. Biochimie 60: 663–672

Atkinson S, Combells C, Vincent D, Nachtigall P, Pawloski J, Breese

M (1999) Monitoring of progesterone in captive female false

killer whales (Pseudorca crassidens). Gen Comp Endocrinol

115: 323–332

Crandall BF (1981) Alpha-fetoprotein: a review. Crit Rev Clin Lab

Sci 15: 127–185

Davis BJ (1964) Disc electrophoresis-II. Method and application to

human serum proteins. Ann NY Acad Sci 12: 404–427

Deutsch HF (1991) Chemistry and biology of alpha-fetoprotein. Adv

Cancer Res 56: 253–312

Fujimoto T, Hara A, Maede Y, Namioka S (1984) Serum concentra-

tion and properities of α-fetoprotein and serum level of albumin

in sucking piglets. Res Vet Sci 36: 212–216

Gitlin D (1975) Normal biology of alpha-fetoprotein. Ann NY Acad

Sci 259: 7–16

Gitlin D, Perricelli A, Gitlin GM (1972) Synthesis of alpha-fetoprotein

by liver, yolk sac, and gastrointestinal tract of the human con-

ceptus. Cancer Res 32: 979–982

Gourdeau H, Bélanger L (1983) Guinea-pig α1-fetoprotein: purifica-

tion, characterization, developmental and hormonal regulation,

and behavior in diethylnitrosamine hepatocarcinogenesis. Can

J Biochem Cell Biol 61: 1133–1146

Grabar P, Williams CA (1953) Méthode permettant l’étude conjugée

des propriétés électrophoreétique et immuno-chimiques d’un

mélange de protéines. Application en serum sanguine. Biochim

Biophys Acta 10: 193–194

Heap RB, Flint APF (1984) Pregnancy. In “Reproduction in mam-

mals 3: Hormonal control of reproduction, 2nd ed” Ed by CR

Austin, RV Short, Cambridge University Press, Cambridge, pp

153–194

Hirai H (1982) Alpha fetoprotein. In “Biochemical markers for

cancer” Ed by TM Chu, Marcel Dekker, New York, pp 25–59

Hobson BM, Wide L (1986) Gonadotropin in the term placenta of the

dolphin (Tursiops truncates), the Californian sea lion (Zalophus

californianus), the gray seal (Halichorus grypus) and man. J

Reprod Fertil 76: 637–644

Jensen ED (1999) Embryonic/early fetal loss in the Atlantic bottle-

nose dolphin (Tursiops truncatus). In “Bottlenose Dolphin

Reproduction Workshop Report” Ed by D Duffield, T Robeck,

AZA Marine Mammals Taxon Advisory Group, Silver Springs,

pp 273 –277

Karmali A, Novo C (1990) Human alpha-fetoprotein: isolation and

production of monoclonal antibodies. Biochimie 72: 369–374

Kerckaert JP, Bayard B, Quief S, Biserte G (1975) Electrophoretic

preparation of the two rat alpha-fetoprotein variants. FEBS Lett

53: 234–236

Laemmli UK (1970) Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature 227: 680–

685

Lai PCW, Hay DM, Peters EH, Lorscheider FL (1977) Immuno-

chemical purification and characterization of ovine α-fetopro-

tein. Biochim Biophys Acta 493: 201–209

Mcleod JF, Cooke NE (1989) The vitamin D-binding protein, alpha-

fetoprotein, albumin multigene family: Detection of transcripts in

multiple tissues. J Biol Chem 264: 21760–21769

Miyazaki N (1977) Growth and reproduction of Stenella coeruleoalba

off the Pacific coast of Japan. Sci Rep Whales Res Inst 29: 21–

48

Mizejewski GJ (2001) Alpha-fetoprotein structure and function: rele-

vance to isoforms, epitopes, and conformational variants. Exp

Biol Med 226: 377–408

Mizejewski GJ (2003) Levels of alpha-fetoprotein during pregnancy

and early infancy in normal and disease states. Obstet Gynecol

Surv 58: 804–826

Morinaga T, Sakai M, Wegmann TG, Tamaoki T (1983) Primary

structures of human α-fetoprotein and its mRNA. Proc Natl

Acad Sci USA 80: 4604–4608

Nishi S, Hirai H (1972) Purification of human, dog and rabbit α-

fetoprotein by immunoadsorbents of sepharose coupled with

anti-human α-fetoprotein. Biochim Biophys Acta 278: 293–298

Nunez EA (1994) Biological role of alpha-fetoprotein in the endocri-

nological field: data and hypotheses. Tumour Biol 15: 63–72

Odell DK, Robeck TR (2002) Captive breeding. In “Encyclopedia of

Marine Mammals” Ed by WF Perrin, B Würsig, JGM Thewissen,

Academic Press, San Diego, pp 188–192

Ogata A, Yamashita T, Koyama Y, Sakai M, Nishi S (1995) Sup-

pression of experimental antigen-induced arthritis in transgenic

mice producing human α-fetoprotein. Biochem Biophys Res

Commn 213: 362–366

Ouchterlony Ö (1953) Antigen-antibody reactions in gels. IV. Types

of reactions in coordinated systems of diffusion. Acta Path

Microbiol Scand 32: 231–240

Reis FM, D’Antona D, Petraglia F (2002) Predictive value of hor-

mone measurements in maternal and fetal complications of

pregnancy. Endocr Rev 23: 230–257

Ridgway SH, Benirschke K (1977) Breeding Dolphin : Present

Status, Suggestions for the Future. Report MNC 76/07. Marine

Mammal Commision, Washington DC

Ross HL, Elias S (1997) Maternal serum screening for fetal genetic

disorders. Obstet Gynecol Clin North Am 24: 33–47

Ruoslahti E, Seppälä M (1971) Studies of carcino-fetal proteins:

physical and chemical properties of human alpha-fetoprotein.

Int J Cancer 7: 218–225

Sinosich MJ, Grudzinskas JG, Saunders DM (1985) Placental pro-

teins in the diagnosis and evaluation of the “elusive” early preg-

nancy. Obstet Gynecol Surv 40: 273–282

Sousa NM, Beckers JF, Gajewski Z (2008) Current trends in follow-

up of trophoblastic function in ruminant species. J physiol phar-

macol 59 (Suppl 9): 65–74

Y. Morita et al.224

Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular

Evolutionary Genetics Analysis (MEGA) software version 4.0.

Mol Biol Evol 24: 1596–1599

Tatarinov YS (1965) Content of embryo-specific alpha-globulin in

the blood serum of the human fetus, newborn, and adult man in

primary cancer of the liver. Vopr Med Khim 11: 20–24

Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improv-

ing the sensitivity of progressive multiple sequence alignment

through sequence weighting, position-specific gap penalties

and weight matrix choice. Nucleic Acids Res 22: 4673–4680

Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of

proteins from polyacrylamide gels to nitrocellulose sheets: pro-

cedure and some applications. Proc Natl Acad Sci USA 76:

4350–4354

Versée V, Barel AO (1978) Rat α-foetoprotein. Purification, physico-

chemical characterization, oestrogen-binding properties and

chemical modification of the thiol group. Biochem J 175: 73–81

Watabe H (1974) Purification and chemical characterization of

alpha-fetoprotein from rat and mouse. Int J Cancer 13: 377–388

Watanabe N, Hatano J, Asahina K, Iwasaki T, Hayakawa S (2007)

Molecular cloning and histological localization of LH-like sub-

stances in a bottlenose dolphin (Tursiops truncates) placenta.

Com Biochem Physiol A Mol Inteqr Physiol 146: 105–118

Yamada T, Kakinoki M, Totsuka K, Ashida Y, Nishizomo K,

Tsuchiya R, Kobayashi K (1995) Purification of canine alpha-

fetoprotein and alpha-fetoprotein values in dogs. Vet Immunol

Immunopathol 47: 25–33

Yoshioka M, Mohri E, Tobayama T, Aida K, Hanyu I (1986) Annual

changes in serum reproductive hormone levels in the captive

bottlenosed dophins. Bull Jap Soc Sci Fish 52: 1939–1946

(Received June 2, 2010 / Accepted August 31, 2010)