A complementary DNA (cDNA) encoding eel atrial natriuretic peptide (ANP) precursor was specifically amplified from eel atrial mRNAs by rapid-amplification polymerase chain reaction. The sequence analysis of the cDNA using multiple clones revealed that the preproANP consists of 140 amino acid residues carrying a signal sequence at its N-terminus and a mature ANP at its C-terminus. An additional glycine residue was attached to the C-terminus of previously isolated eel ANP. The glycine residue may be used for amidation of the C-terminus or removed after processing. The cleavage site of a signal peptide with 22 amino acid residues was confirmed by isolation of proANP protein from eel atria. The proANP sequence deduced from the cDNA was also confirmed for 71% of the isolated protein. Sequence comparison with other natriuretic peptides revealed that eel ANP is more similar to mammalian ANP than to B-type natriuretic peptide (BNP) at both amino acid and nucleotide sequence levels. The eel ANP gene was a single copy gene as shown by Southern blot analysis. Northern blot analysis showed that eel ANP mRNA is approximately 0.8 kb in size and exclusively detected in the atrium. Thus, eel ANP is a true atrial hormone judging from both the sequence and the site of production. However, reverse transcription-polymerase chain reaction detected ANP message in the brain, gill, cardiac ventricle, red body of swim bladder (rete mirabilis), intestine, head kidney (including interrenal and chromaffin tissues) and kidney. Most of these tissues are involved in ion and/or gas exchange in fishes.
INTRODUCTION
A group of peptides, termed the natriuretic peptide family, consist of atrial (A-type), brain (B-type) and C-type natriuretic peptide (ANP, BNP and CNP) in tetrapods, and ANP, CNP and ventricular natriuretic peptide (VNP) in teleost fishes (Hagiwara et al., 1995). ANP, BNP and VNP are cardiac hormones circulating in blood (Saito et al., 1989; Takei et al., 1994a), but CNP is principally a paracrine factor in the brain and other peripheral tissues (Barr et al., 1996). An exception is elasmobranchs in which CNP is the sole natriuretic peptide acting as both an endocrine and paracrine factor (Suzuki et al., 1992). The physiological function of the NP family in mammals has been a target of intensive research for over a decade, and it is now evident that ANP exerts a spectrum of actions to decrease blood volume through inhibition of intake, and stimulation of output, of both water and sodium (Brenner et al., 1990). In the eel, however, it has become evident that ANP acts on various osmoregulatory organs to decrease, specifically, extracellular sodium ions (Takei and Bailment, 1993). Furthermore, ANP is secreted in response to an increase in blood volume in mammals (Roskoaho, 1992), whereas in the eel, ANP secretion is augmented more potently by an increase in plasma osmolality rather than an increase in blood volume (Kaiya and Takei, 1996). Although conflicting evidence exists in earlier fish studies using heterologous peptides (Lee and Malvin, 1987; Duff and Olson, 1986; see Evans, 1990 for review), it seems, at least in the eel, that ANP is a hormone secreted in response to increased plasma osmolality and promotes seawater adaptation by extruding sodium and chloride ions.
To elucidate further the role of ANP in eel osmoregulation, it is necessary to examine ANP synthesis in response to osmotic and volemic challenges. As a first step to achieve this aim, cDNA of eel ANP was cloned from eel atria and sequenced. The cloned cDNA was used as probes for Southern blot analysis of eel genomic DNA, and for Northern blot analysis to examine the expression of ANP mRNA in various eel osmoregulatory tissues. The mRNA expression in those tissues was further examined in greater detail by reverse-transcription polymerase chain reaction (RT-PCR). To date, cDNA cloning of the NP family in fish has been successfully performed for CNP of the spiny dogfish, Squalus acanthias (Schofield et al., 1991) and VNP of the eel (Takei et al., 1994b). Sequences of cloned cDNA and deduced proANP were compared with those of known ANP and other NPs sequenced to date. The cleavage site of the signal peptide and proANP sequence deduced from the cDNA were confirmed by isolating proANP protein from eel atria.
MATERIALS AND METHODS
cDNA cloning
Preparation of mRNA and cDNA
Cultured Japanese eels (Anguilla japonica) weighing approximately 200 g were purchased from a local dealer. After anesthesia in 0.1% (w/v) tricaine methanesulphonate (Sigma, St Louis, III, USA), the hearts were dissected out, atria and ventricles separated and immediately frozen in liquid nitrogen. Total RNA was extracted from 1.25 g atrial tissues from 40 eels by the guanidine thiocyanate method (Chomczynski and Sacchi, 1987). Poly (A)+ RNA was isolated with oligo(dT) coated latex beads (Oligotex-dT30; Takara Shuzo, Kyoto, Japan), and cDNA was synthesized by the use of a cDNA synthesis system plus (Stratagene, La Jolla, CA, USA).
Amplification of 3′ end of eel ANP cDNA
Degenerate oligonucleotides coding for eel VNP-(6-23), the most conserved region of VNP among all NPs, were synthesized with a 8700 DNA synthesizer (Millipore, New Bedford, MA, USA), and used as a mixed primer for PCR. Oligo(T) was used as the other primer. Double-strand cDNA (1 ng) and the primers (50 pmol each) in 50 μl reaction buffer containing 10 mM Tris-HCI, pH 8.3, 50 mM KCI, 1.5 mM MgCI2, 0.001% gelatin, 2.5 mM each of deoxy-NTP and 5 units AmpliTag DNA polymerase (Perkin-Elmer Cetus, Norwalk.CT, USA) were overlaid on a drop of mineral oil and subjected to amplification in a DNA cycler (Perkin-Elmer Cetus). The mixture was denatured at 94°C for 3 min, subjected to 4 cycles of 94°C (1 min)-37°C (1 min)-72°C (3 min), and then to 30 cycles of 94°C (1 min)-50°C (1 min)-72°C (3 min), and finally to 7-min extension at 72°C (Compton, 1990).
Amplification of 5′ end of eel ANP cDNA
The 5′ end of eel ANP cDNA was amplified by the rapid amplification of cDNA ends (RACE) method described by Frohman (1990). Based on the 3′ end sequence of the cDNA of eel ANP obtained by the previous PCR, an oligonucleotide primer was prepared with the sequence of 5′-TCGAGGTACAC-TGAGGGCTT-3′ (A-20). Eel atrial poly (A)+ RNA (1 μg) in 1.5 μl water was heated to 65°C for 3 min, chilled on ice, and added to 20 μl of reaction buffer containing 10 mM Tris-HCI, pH 8.3, 5 mM MgCI2, 50 mM KCI, 2.5 mM deoxy-NTP, and 150 pmol of the primer. Twenty units of ribonuclease inhibitor (Takara) and 24 units of avian myeloblastosis virus reverse transcriptase (Seikagaku Kogyo, Tokyo, Japan) were added, and the reaction mixture was incubated for 1 hr at 42°C and subsequently for 30 min at 52°C.
The mRNA-cDNA hybrids were precipitated using the cetyl trimethyl ammonium bromide (CTAB) method (Belyavsky et al., 1989). Briefly, the hybrids were added to 16 μl 0.1 M NaCI, 2.9 μl 0.5 M EDTA and 2 μl 10% CTAB, and centrifuged at 15,000 rpm for 15 min. The pellet was dissolved in 14 μl 1 M NaCI, 25 μl water and 1 μl 10% CTAB and centrifuged again. The pellet was dissolved again in the mixture of 20 μI 1 M NaCI and 54 ml ethanol. The DNA was precipitated at -30°C and collected by centrifugation. The pellet was finally dissolved in 20 μl tailing buffer containing 25 mM sodium cacodylate, 0.5 mM MnCI2, 25 mM dithiothreitol, 1 mM deoxy-CTP and 15 units terminal deoxynucleotidyl transferase (Takara). The mixture was incubated for 30 min at 37°C, and tailed cDNA was collected by CTAB precipitation as described above.
The tailed cDNA was dissolved in water, and one fourth of the solution was used for subsequent PCR reaction. The first reaction was carried out at 93°C (5 min)-42°C (1.5 min)-72°C (3 min) in a solution described above using only 75 pmol of Notl-G primer (5′-TAGCGGCCGC(G)15 -3′). Then, 25 pmol A-20 primer and 25 pmol Notl-G primer were added and subjected to the PCR reaction. The PCR parameters were 1 cycle of 93°C (3 min)-55°C (2 min)-72°C (3 min), 15 cycles of 93°C (1.5 min)-55°C (2 min)-72°C (3 min), and final extension at 72°C for 12 min.
cDNA cloning and sequencing
The amplified products were electrophoresed on 1.2% low melting-point agarose gel (Bio-Rad, Richmond, CA, USA), and the major band was excised and purified by phenol extraction. The cDNA inserts were subcloned into plasmid Bluescript II SK+ (Stratagene) T-vector (Marchuk et al., 1991) and transferred into E. coli JM109 competent cells (Toyobo, Osaka, Japan). Nucleotide sequencing was performed using an Auto Read sequencing kit with fluorescent primers in an A.F.L. sequencer (Pharmacia, Uppsala, Sweden). Multiple isolates were examined to guard against PCR errors.
Isolation of proANP
Crude acid extracts of atrial tissues (31.8 g) from 2500 eels were prepared as described previously (Takei et al., 1989). After desalting of the extracts by Sephadex G-25 chromatography (5 × 82 cm, Pharmacia), fractions with relaxant activity in the chick rectum were applied to a column of SP-Sephadex C-25 (1.6 × 15 cm, Pharmacia) and eluted with 1 M AcOH, 2 M pyridine, and 2 M pyridine/AcOH, pH 5.0. The fraction eluted with 2 M pyridine/AcOH contained mature ANP-(1-27) (Takei et al., 1989). Since the fraction eluted with 2 M pyridine showed a weak rectum-relaxant activity, it was subjected to cation-exchange high-performance liquid chromatography (HPLC) on an IEC CM column (7.5 × 75 mm, Jasco, Tokyo, Japan) with a linear-gradient elution from solvent A (10 mM CH3COONH4: CH3CN = 9:1) to solvent B (1 M CH3COONH4: CH3CN = 9:1) for 1 hr. Each bioactive fraction was subjected to reverse-phase HPLC on an ODS-120T column (4.6 × 250 mm, Tosoh, Tokyo, Japan) with a linear gradient of CH3CN concentration from 20 to 60% in 0.1 % trifluoroacetic acid (TFA) for 40 min. The bioactive peak fraction was further purified with the same column with a number of different gradients of CH3CN concentrations until a homogeneous peak was obtained.
The molecular weight of the purified protein was estimated by sodium dodesyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli (1970). The purified protein was sequenced in a protein sequencer (430A, Applied Biosystems, San Francisco, CA, USA) either directly or after digestion with lysine en-dopeptidase (Wako Pure Chemicals, Tokyo, Japan). For digestion, approximately 10 μg of the peptide was dissolved in 100 μl 10 mM Tris-HCI, pH 8.9 containing 8 M urea, mixed with 1 μg lysine endopeptidase in 100 μl Tris-HCI, and incubated for 4 hr at 30°C. The digests were applied onto an ODS-120T column and eluted with a linear gradient of CH3CN concentration from 2 to 40% in 0.1% TFA for 60 min.
Northern blot analysis
Total RNA was extracted from approximately 50 mg of atrium, ventricle, brain, stomach, anterior intestine, posterior intestine, kidney and skeletal muscle respectively as reported previously (Takei et al., 1994b). Oligonucleotides corresponding to eel ANP cDNA-(322-361) were synthesized, labeled with [α-32P] deoxyATP (222TBq/mmol, Amersham, Bucks, UK) using a 3′-end labeling kit, and used as a probe. This sequence has low (35%) identity with the corresponding region of eel VNP cDNA.
Aliquots containing 2 μg RNA were electrophoresed on a 1.5% formaldehyde agarose gel and transferred onto a nylon filter. The ANP mRNA was hybridized with the 32P-labeled probe in 6 × SSC (1 × SSC = 0.15 M NaCI, 0.015 M sodium citrate, pH 7.0)/0.1% SDS at 55°C for 20 hr and washed with 0.1 × SSC/0.1% SDS at 55°C for 1 hr. Autoradiography was performed by exposing Kodak X-Omat AR film to the filters at -80°C for 31 hr.
Southern blot analysis
Eel genomic DNA was prepared by the method of Blin and Stafford (1976). DNA (10 μg) was digested with 15 different restriction enzymes at 37°C overnight, purified by phenol extraction, and electrophoresed on a 0.7% agarose gel. The DNA transferred onto a nylon membrane was hybridized at 50°C for 25 hr with ANP cDNA labeled with [α-32P] dATP and dCTP using a Multiprime DNA labeling system (Amersham). The filters were washed in 0.2 × SSC/0.1% SDS at 65°C for 1 hr and autoradiographed for 27 hr at -80°C.
Reverse-Transcription Polymerase Chain Reaction (RT-PCR)
RT-PCR was performed by the method of Iwami et al. (1996) with some modifications. After anesthesia, brain, gill filament, cardiac atrium and ventricle, intestine, red body of swim bladder, head kidney and kidney were immediately removed from 3 freshwater adapted eels and 3 seawater adapted eels and frozen in liquid nitrogen. Total RNA was extracted with ISOGEN (Nippon Gene Co., Ltd., Toyama, Japan), and 1 μg of RNA from each was reverse-transcribed with 200 units of Superscript II (Life Technologies, Gaithersburg, MD, USA) in a 20 μl reaction mixture of 20 mM Tris-HCI (pH 8.4), 50 mM KCI, 2.5 mM MgCI2, and 10 mM dithiothreitol and 0.5 mM of all dNTPs in the presence of 0.1 mg Oligotex-dT30 Super (Takara). The mixture was incubated for 60 min at 42°C, boiled for 5 min, and chilled quickly on ice. The Oligotex-dT30 cDNA was separated by centrifugation at 12,000 × g for 5 min at 4°C and rinsed twice with 300 μl of 10 mM Tris-HCI (pH 8.0) containing 1 mM EDTA. The washed Oligotex dT30 -cDNA complex was suspended in a 50 μl solution of 10 mM Tris-HCI (pH 8.8), 50 mM KCI, 2.5 mM MgCI2, 0.2 mM of all dNTPs and 1.0 μM of primers (nucleotides 183-202 and 567-548 of eel ANP cDNA). Amplification was carried out using 2.5 units of DNA polymerase (Takara) for 40 cycles of 94°C (1 min)-60°C (1 min)-72°C (2 min). Ten μl of each reaction mixture was electrophoresed on a 2% agarose gel, stained in 0.1 jig/ml ethidium bromide for 30 min, and photographed under UV light.
RESULTS
Sequence analysis of ANP cDNA
Two different nucleotides with approximately 300 bp and 540 bp were amplified after the first PCR using the sequence encoding the intramolecular ring structure of eel VNP (PeV5) and oligo(T) as primers (Fig. 1 A). The 540 bp nucleotide was identified as the 3′ region of the previously cloned eel VNP cDNA. The 300 bp nucleotide had a sequence corresponding to Ser-Arg-Lys-Gly at its 5′ end. Since the Ser-Arg-Lys sequence was identical to the C-terminal sequence of eel ANP, the 300 bp sequence was considered as a 3′ region of eel ANP cDNA. Two clones were sequenced to confirm the identity.
Fig. 1
(A) Strategy for polymerase chain reaction (PCR) of eel ANP mRNA. PreproANP is boxed. Shaded portions at the 5′ and 3′ side are signal sequence and mature ANP, respectively. Three sets of primers show 3′ end PCR, 5′ end PCR and confirming PCR. For details, see text. (B) Nucleotide sequence of cDNA encoding eel ANP and its predicted amino acid sequence. Vertical arrow indicates cleavage site of signal peptide. Mature, circulating ANP-(1-27) is underlined. The polyadenylation signal is boxed. Amino acid residues are numbered beginning from the initiator methionine. Termination codon is indicated by asterisk.
The second PCR using A-20 and Notl-G primers produced nucleotides with various lengths (Fig. 1 A). Six clones containing the longest insert were sequenced. Five had the same 5′ end and the remaining one started from 18 bases upstream, whereas all six had the same 3′ end. The longest sequence consisted of 670 nucleotides excluding poly (A) tract. Since some nucleotide exchanges occurred, the sequence was confirmed as described below.
In order to confirm the sequence, the third PCR was performed with newly prepared atrial poly (A) RNA using 5′-end 20 nucleotide (N-20) and A-20 as primers (Fig. 1A). The sequencing of four clones gave consistent results except for 3 exchanges in one of the four clones. Thus the sequence of eel ANP cDNA was finally determined (Fig. 1B). The preproANP deduced from the cDNA sequence consisted of 140 amino acid residues. The mature eel ANP-(1-27) was located at the C-terminus, and an additional glycine residue was attached to its C-terminus just before the stop codon (Fig. 1B). The ATTAAA sequence was located 16 nucleotides upstream from the poly (A) tract. The first 22 amino acid residues, starting with an initiator methionine, were assumed to be a signal sequence based on the hydrophobicity of the amino acids, and analogy to other mammalian preproANP. There was no potential glycosylation site in the preproANP.
Sequence similarity was compared between eel preproANP and mammalian preproANP and BNP (Fig. 2). It is evident that eel ANP is more similar to ANP than to BNP. The homology was calculated at both cDNA and amino acid sequence levels, which confirmed the higher homology of eel ANP to mammalian ANP than to BNP (Table 1).
Fig. 2
Comparison of deduced amino acid sequence of eel preproANP with mammalian preproANP and BNP. Amino acid residues are expressed in one-letter code. Sequences are aligned with minimal gaps to give maximum homology. Amino acid residues identical to eel ANP are reversed.
Table 1
Nucleotide and amino acid sequence identity (%) of eel ANP to mammalian ANP and BNP sequenced to date
Isolation of proANP
After cation-exchange HPLC of a fraction eluted with 2 M pyridine from the Sephadex C-25 column, several fractions exhibited chick rectum relaxant activity (Fig. 3A). Reverse-phase HPLC of the bioactive fractions always resulted in three peaks as shown in Fig. 3B. Since rectum relaxant activity was too low to detect with one half of each peak, molecular weight (Mr) was estimated by SDS-PAGE. It was revealed that the material in the peak eluted at 41.5% CH3CN had a Mr of approximately 14 kDa, which was most similar to that predicted for eel proANP (data not shown). Thus the material in the peak was further purified by two steps of reverse-phase HPLC and finally, half was subjected to direct sequencing and the other half was digested by lysyl endopeptidase. Each peak of the digests was sequenced and compared with the sequence deduced from the cDNA (Fig. 4). The intact peptide started from the 23rd histidine residue of preproANP and could be read up to the 66th glutamate residue. The K-22 fragment covered from the 23rd histidine to the 38th lysine, K-18 from the 26th serine to the 38th lysine, and K-19 from the 115th serine to the 139th lysine. The K-33 fragment could be read from 39th serine to 79th arginine and the K-34 from the 39th serine to the 64th lysine. Although the total sequence was not determined by the digests, the cleavage site of the signal peptide and 71% of the proANP sequence was confirmed (Fig. 2).
Fig. 3
Purification of proANP from eel atrial extracts, (A) Cation exchange high-performance liquid chromatography (HPLC) of the fraction eluted from SP-Sephadex C-25 with 2 M pyridine. Solid bars represent relaxant activity in the chick rectum expressed as equivalents to nmol human AN P. Broken lines show gradients of solvent B (for details see Materials and Methods). (B) Reverse-phase HPLC of a fraction marked in (A) by arrows. The peak marked by an arrow was further purified by reverse-phase HPLC with reduced gradient of CH3CN and used for sequence analysis. Fraction was collected by each peak reflection.
Northern and Southern blot analysis
Northern blot analysis displayed ANP message only in the atrium and no signal was detected in other tissues such as kidney, gill, liver, digestive tracts, skeletal muscle and cardiac ventricle. The size of ANP mRNA was approximately 0.8 kb (Fig. 5), which was similar to the size predicted from the cloned cDNA. Among 15 restriction enzymes examined, EcoT22l, Hindlll, Hindi, Pstl, Sad and Xbal digests of eel genomic DNA resulted in a single positive band, showing that eel ANP is a single copy (data not shown).
Reverse-Transcription Polymerase Chain Reaction (RT-PCR)
RT-PCR of all tissues examined exhibited PCR products at approximately 400 bp, which were similar to the size (385 bp) amplified from eel ANP mRNA (Fig. 6). The positive tissues included the brain, cardiac ventricle, gill, intestine, red body of swim bladder, head kidney containing interrenal and chromaffin tissues, and kidney in addition to the atrium, although only the atrium was positive in the Northern blot analysis.
DISCUSSION
ANP is the first molecule to be identified in the NP family (de Bold et al., 1981). In nonmammalian species, however, ANP has been identified only in the eel (Takei et al., 1989) and two species of frogs (Sakata et al., 1988; Lazure et al., 1988). Furthermore, cDNA cloning of ANP has not yet been performed in nonmammalian species, although cDNAs of chicken BNP (Akizuki et al., 1991), bullfrog CNPII (Kojima et al., 1994), eel VNP (Takei et al., 1994b) and dogfish CNP (Schofield et al., 1991) have been already cloned. Thus, this is the first report of cDNA cloning of ANP in nonmammalian species.
In the previous study, we named the isolated peptide ANP because it is stored in large amounts in the atrium and is more similar to ANP than to BNP (Takei et al., 1989, 1992). However, we should be cautious to determine the type of NP peptides in nonmammalian species only with the homology of 20-30 amino acids and the site of storage. In fact, an NP peptide has recently been isolated from trout atria which is equally similar to mammalian ANP and BNP (Takei et al., 1997). Thus cDNA cloning is required for determining the molecular species of the trout atrial peptide. The present study showed that the eel atrial peptide is more similar to ANP than to BNP at the mRNA level, and that the message is almost exclusively expressed in the atrium, thereby confirming that the cloned NP is ANP of this species.
Another structural feature common to all ANP thus far identified is the presence of C-terminal 5 amino acid residues extending from the intramolecular ring (Hagiwara et al., 1995). BNP has a C-terminal extension of 6 amino acid residues except in the bullfrog (Fukuzawa et al., 1996), and CNP exclusively is without C-terminal extension. Since these features are well conserved among NPs sequenced to date, the presence of 5 amino acid residues at the C-terminus is one of the important structural criteria for identification of ANP. However, only 4 amino acid residues were detected at the C-terminus of eel ANP in the previous study (Takei et al., 1989) and in the current sequencing of enzymatic digests of purified proANP. The present cDNA cloning clearly showed that a glycine residue is present in addition to the C-terminal lysine of previously identified eel ANP. It is not yet determined whether the glycine residue is cleaved off during processing or it is used for amidation of the C-terminal lysine. Whichever the case, it is apparent that eel ANP has 5 amino acid residues at the C-terminus in the gene sequence.
Eel proANP was isolated from the pyridine fraction of Sephadex C-25 chromatography in this study. Human proANP was also eluted in this fraction although all mature NPs were eluted in pyridine-acetic acid fraction in this column (Kangawa and Matsuo, 1984). In the eel, proVNP and proCNP were not detected in the pyridine fraction in previous studies, although it is possible that these prohormones have only weak rectum relaxant activity. Judging from the recovery of purified protein, the major storage form of ANP in eel atria is not mature ANP-(1-27) but proANP. The whole proANP sequence was not determined from isolated protein, but the cleavage site for the signal sequence was confirmed. The signal cleavage site conformed to the rule of von Heijne (1986) and to those of other ANPs (Kangawa et al., 1984; Oikawa et al., 1984).
Northern blot analysis revealed that the expression of ANP message was detected only in the cardiac atria. However, RT-PCR detected the expression in the brain, cardiac ventricle, gill, intestine, red body of swim bladder, head kidney and kidney, although the expression in these tissues was smaller compared to that of atria. ANP has been implicated in osmoregulatory actions in some of these tissues such as inhibition of drinking (brain), stimulation of sodium excretion (gill), inhibition of sodium absorption (intestine), stimulation of mineralocorticoid secretion (interrenal tissues in head kidney), and antidiuresis and natriuresis (kidney) in the eel (see Takei and Balment, 1993). Furthermore, most of the tissues have been shown to possess ANP receptors (Mishina and Takei, 1997). In mammals, ANP synthesis and action have been reported in the brain (Komatsu et al., 1992), lung (functional homologue of the gill, Matsubara et al., 1988), intestine (Gerbes et al., 1994), adrenal gland (homologue of interrenal and chromaffin tissues in the head kidney, Ong et al., 1987), and kidney (see Vollmar, 1990 for review). In the eel, the red body of swim bladder is an important organ for gas exchange (Pelster and Schied, 1992), and it is shown to possess dense ANP receptors (Mishina and Takei, 1997). Thus it is possible that ANP synthesized in these tissues acts in a paracrine/autocrine fashion to regulate ion and/or gas exchange in the eel.
Acknowledgments
We thank Dr. Neil Hazon of University of St. Andrews for critical reading of the manuscript. This investigation was supported in part by grants from Ministry of Education, Science and Culture, Japan and from Fisheries Agency, Japan.
