Among different pufferfish species, members of the genus Takifugu are most prominent and the species belonging to this genus are closely related. This is especially true for two pufferfish species such as torafugu T. rubripes, the most prominent member of this genus and also the most expensive among pufferfish, and karasu T. chinensis, both of which share their morphological characters and occupy the same habitation around the Yellow Sea and the East China Sea. However, the two species have different external color patterns. T. rubripes has some irregular black marks on a dorsal half of the posterior body part with white or pinkish anal fin. On the other hand, T. chinensis has no black marks on that body part with black anal fin. Despite such differences, the availability of hybrid-like individuals having intermediate color patterns between the two species leads to disputes arising in the Japanese fish markets during trade on species identification and classification. It is, therefore, necessary to provide definitive information about the two species by obtaining species-specific data and based on the analysis of genetic variation. Furthermore, the detection of genetic variation at the species level as well as genetic relationships among different Takifugu species is of great importance for sustainable aquaculture practices to reply on the expansion of aquaculture of T. rubripes in Japan, China and other countries. The present study was, therefore, undertaken to elucidate genetic relationship of T. rubripes and T. chinensis by sequence analyses of several mitochondrial (mt) and nuclear genes. Efforts were also made to identify the gene(s) responsible for the evident color-pattern differences between T. rubripes and T. chinensis by using two differential analysis methods, mRNA arbitrarily primed reverse transcription-polymerase chain reaction (RAP RT-PCR) and suppression subtractive hybridization (SSH). Finally genetic diversity was analyzed at the population level using microsatellite markers and mt control region (CR).

1. Studies on mitochondrial and nuclear genes of Takifugu rubripes and T. chinensis

Based on the mt genome data of T. rubripes reported by Elmerot et al. (2002), mt genes including those encoding 16S ribosomal RNA (rRNA), adenosine triphosphatase 6 (ATPase 6), nicotinamide adenine dinucleotide dehydrogenase subunit 4 (ND4), ND5 and cytochrome b (cyt b) were compared between T. rubripes and T. chinensis and several hybrid-like individuals having skin and anal fin color patterns of both species. CR was selected because noncoding regions have generally evolved faster than coding regions. In addition, the nuclear genes such as internal transcribed spacer 1 (ITS1) and ITS2 were used to compare their genetic elements.

Primers were designed for mt genes based on the reported sequence of T. rubripes (DDBJ/EMBL/GenBank accession no. AJ421455) to amplify the segments of 640 bp in 16S rRNA, 308 bp in ATPase 6, 344 bp in ND4, 554 bp in ND5, 697 bp in cyt b and 1020 bp in CR. The sequences determined covered 20% in mtDNA. Informative sites were composed of one for 16S rRNA (position 1886), two each for ATPase 6 (8427 and 8559), ND4 (11 346 and 11 394) and ND5 (12 219 and 12 298), and three for cyt b (14 762, 14 858 and 14 978) among 24 wild T. rubripes, 24 wild T. chinensis and six hybrid-like samples. Among 24 wild T. rubripes identified by external color patterns, 15% possessed nucleotide sequences consistent with T. chinensis registered in the DDBJ/EMBL/GenBank databases for T. chinensis (AP009534), whereas the sequences of 60% T. chinensis individuals were consistent with those registered for T. rubripes (AJ421455). As for the hybrid-like samples, two possessed T. chinensis-specific sequences in some base positions and T. rubripes-specific sequences in others. The remaining hybrid-like samples possessed T. rubripes-specific sequences. CR showed the intra-specific nucleotide sequence identity of 89 - 90% which was the lowest level among the mtDNA fragments analyzed for the three groups distinguished with their external color patterns. The rest of the mt genes showed 99 - 100% sequence identity with the respective gene fragments.

The nuclear genes consisting of linearized partial 18S rRNA, complete internal transcribed spacer 1 (ITS1), 5.8S rRNA and partial ITS2 showed the same levels of sequence identity (99 - 100%) between T. rubripes and T. chinensis. These results indicate a very low level of variation between the two Takifugu species.

2. Studies on the genes responsible for color differentiation between Takifugu rubripes and T. chinensis

To shed light on the events on how these two pufferfish species show differentiation in their skin and anal fin colors, the nuclear genes encoding melanocortin receptor (MC1R and MC4R) and pro-opiomelanocortin (POMC) were analyzed for their sequences, since these genes have been reported to be very closely associated with pigmentation and related functions in human and other vertebrates. The determined complete nucleotide sequences of 903 bp and 969 bp for MC1R and MC4R, respectively, and a partial sequence of 374 bp for POMC showed 99 - 100% inter- and intraspecific sequence identity, declining their roles in the observed color pattern differences.

Then two differential analysis techniques, RAP RT-PCR and SSH, were employed by using skin and anal fin tissues which show different color patterns between T. rubripes and T. chinensis. RAP RT-PCR was performed with cDNAs from anal fin tissues of the two target species using an arbitrarily chosen primer A-5 (5'-AATCTAGAGCTCCCTCCA-3') from the RAP-PCR kit (Stratagene, La Jolla, CA, USA). SSH, on the other hand, was performed for skin tissues from two different locations and anal fin tissues, and three SSH libraries enriched in differentially expressed transcripts were constructed using PCR-select cDNA subtraction kit (Clontech, Palo Alto, CA, USA). Reverse transcription-PCR confirmed species-specific expression for serine pamitoyl transferase subunit 2 gene (sptlc2) obtained by RAP RT-PCR and 17 different genes obtained by SSH which include those encoding secreted frizzled-related protein 4 (sfrp4), vomeronasal 1 receptor F4 (v1rf4), DEAH (Asp-Glu-Ala-His) box peptide 16 (dhx16), E74-like factor 2 (elf2), protocadherin2 (pcdh2), LRRGT00033 protein, immunoglobin super family 21 (ifsf21), kelch repeat and BTB (POZ) domain containing 3 (btb3), c15orf24 protein, h2A histone family member Y (h2afy), s100 calcium binding protein a11 (s100a11), mKIAA1506 protein (mll3), annexin 2a (anxa2a), c1q-like adipose specific protein, IgM heavy chain constant region (ighm), MGC108117 protein and 40S ribosomal protein S17 (rps17).

Sptlc2 notified by RAP RT-PCR in the present study has been reported to act as an epidermal barrier in skin and up-regulated by UVB (λ, 290 - 320 nm) irradiation in human keratinocytes. The significantly higher levels of sptlc2 expression in T. chinensis anal fin tissues compared to that of T. rubripes indicates its possible role in controlling the movement of melanosomes to maintain melanocytes confined in the anal fin region in T. chinensis. Among the differentially expressed SSH transcripts, calcium ion binding proteins including s100a11 and anx2a, cell adhesion molecules including pcdh2, proteolytic proteins including c15orf24 and LRRGT00033, and Wnt signaling pathway related proteins including sfrp4 and v1fr4 could play vital roles for creating the observed color pattern differences between T. rubripes and T. chinensis.

Functional analysis was performed with zebrafish using morpholino oligonucleotide (MO) for two genes among the differentially expressed ones obtained by SSH including dhx16 and s100a11 since these two genes are thought to be closely related to coloration events in fish. Zebrafish embryos injected with dhx16 MO showed little difference in the coloration until 48 hours post fertilization (hpf) in comparison with the control batch. Several larvae injected with MO showed chromatophore diffusion on the lateral side of the body at 72 hpf. Furthermore, there were some deformities in the body shape with hatched larvae at 48 hpf. Dhx16 is involved in pre-mRNA splicing and thought to possess a complex role in various physiological pathways. As for zebrafish embryos injected with s100a11 MO, no evident differences in the color pattern were observed when compared with those in the control batch up to 8 days post fertilization, indicating the formation of pigment granules in zebrafish to be independent of s100a11. A comprehensive study of all differentially expressed genes is required to elucidate the event of color differentiation between T. rubripes and T. chinensis.

3. Genetic diversity analysis between Takifugu rubripes and T. chinensis

The discovery of overlapping mt gene sequences in partial 16S rRNA, ATPase 6, ND4, ND5 and cyt b between T. rubripes and T. chinensis rendered it essential to estimate the degree of relatedness of the two species at the population level. Genetic variation was, therefore, surveyed with four microsatellite loci at the nuclear gene level and with CR (561 bp) at the mt gene level among two wild T. rubripes populations [collected at Tsushima Island in 2003 (TT) and Soneshinden, Kitakyushu in 2008 (ST)] and one wild T. chinensis population [collected on the east coast of Korea in 2004 (KK)].

Microsatellite genotyping, among which three were reported by Furukawa et al. (2004) and one unpublished, was carried out by PCR for the total genomic DNA from 150 pufferfish individuals, and the amplified PCR products were analyzed using QIAxcel system (QIAGEN, Irvine, CA, USA). The sample size was 50 each for the three populations. Data analyses were performed using Genepop v3.4 (Raymond and Rousset, 1995), TFPGA (Miller, 1997) and Fstat softwares (Goudet, 1995). All four microsatellite loci were polymorphic and yielded a total of 138 different alleles. Total number of alleles per locus ranged from 25 to 47. The genetic diversity index values of TT, ST and KK populations were 0.9505, 0.9350 and 0.9335, respectively. The values of genetic distance and genetic differentiation (G(ST)) between TT and KK (0.0543 and 0.0189, respectively) were smaller than those between TT and ST (0.0857 and 0.0194, respectively). UPGMA dendrogram using microsatellite data also showed that TT formed one clade with KK, whereas ST was separated from this clade.

Population dynamics study using CR for the same 150 pufferfish specimens was performed by direct sequencing of this gene fragment which yielded 161 variable sites (28.87%) and resulted in 106 haplotypes. Haplotype diversity (h) was highest in ST population (0.99837 ± 0.004) and lowest in KK (0.91184 ± 0.026). Sequence diversity (π) also showed a similar trend among the three Takifugu populations (0.02914 ± 0.019175 for ST and 0.00546 ± 0.004993 for KK). Overall h and π values were 0.98192 ± 0.018 and 0.01756 ± 0.00130, respectively. The genetic distance using CR among the three Takifugu populations was consistent with those obtained using microsatellite loci where the distance between TT and KK populations was smaller (0.0821) than those between TT and ST populations (0.1473). Average pairwise difference also showed a similar trend of genetic relationship where the value was lowest between TT and KK (7.49040) and largest between TT and ST populations (17.76480). These results suggest that T. rubripes and T. chinensis are indeed very closely related and probably regarded as the same species.

Conclusion

A very close genetic relationship was found between T. rubripes and T. chinensis in the present study, showing the possibility that these two species are the same species. Their evident difference in color may be due to regional or site variations where samples were collected, although it is uncertain whether or not the flesh texture and taste would be different between the two species. In addition, the differentially expressed genes obtained in the present study are also important as they are potential candidates for color pattern variation between the two species. From the data obtained so far, a scheme on the phylogeography as well as admixture of these two Takifugu species around the Sea of Japan can be concluded which may contribute to the studies on the mechanisms involved in speciation of the members of Takifugu genus.

フグ類の中ではトラフグ属が最も重要で本属には近縁な種が多くある。とくにトラフグTakifugu rubripesは市場価値が高く産業的にも大変重要であるが、形態形質が酷似する上、東シナ海を生息域にするなど、きわめて類縁のカラスT. chinensisが存在する。両種は表皮の紋様が異なるほか、尻鰭はトラフグで白、カラスで黒と、外見的な特徴は明白であるものの、これらの形質は判然としない場合、すなわち両種の中間の外見を示す魚体も市場では少なからずみられ、雑種の疑いがもたれている。このような生物学的な問題のほか、市場でもカラスはトラフグに比べて廉価で、雑種様の魚体については商品価値が定まりにくい状況にある。そこで本研究ではトラフグとカラスのミトコンドリアおよび核DNAを分析して比較した。次に、両種で紋様の異なる表皮および尻鰭からmRNAを調製して、arbitrarily primed reverse transcription-polymerase chain reaction (RAP-PCR)およびsuppression subtractive hybridization (SSH)により両種で発現量の異なる遺伝子を探索した。さらに、トラフグおよびカラスの異なる集団間について遺伝的距離を調べた。

ミトコンドリアDNAの比較に当たっては、16S rRNA, ATPase6, ND4, ND5およびシトクロムb (cyt b)をコードする遺伝子の一部領域の塩基配列を分析した。試料は紋様から判別した天然トラフグ成魚24尾、天然カラス24尾、天然および養殖の雑種様魚体6尾を対象に鰭から抽出した全DNAを鋳型に、各遺伝子に特異的なプライマーを設計してPCRで遺伝子増幅断片を得た。なお、いくつかの試料につき脊椎骨数や鰭の棘数からも判定を試みたが、既報のように両種で明確な差は認められなかった。分析結果を比較したところ、16S rRNA, ATPase6, ND4, ND5およびcyt b各遺伝子でそれぞれ、1, 2, 2, 2および3カ所の計10カ所のinformative siteが見つかった。データベースに登録されているトラフグおよびカラスのミトコンドリア遺伝子全塩基配列と比較したところ、トラフグ試料の15%はカラスの登録配列と同じであり、カラス試料の60%はトラフグの登録配列と一致した。一方、雑種様試料はトラフグおよびカラス登録配列を混在して含むことが示された。さらに、各遺伝子の18S rRNAの一部, internal transcribed spacer 1 (ITS1), 5.8S rRNAの一部を含む連続した領域の配列や、ITS2の配列でもトラフグとカラスを識別する特異な塩基置換は認められなかった。

次に、色素胞の発現に関与していることが報告されているmelanocortin receptor (MC1RおよびMC4R)およびpro-opinomelanocortin (POMC)をコードする核遺伝子を調べたところ、両遺伝子ともにトラフグとカラスで良く類似し、異なる紋様や色調への関与は不明であった。そこで次に、トラフグとカラスで異なる紋様を示す同じ表皮の位置、および尻鰭からmRNAを調製して遺伝子発現の違いをRAP-PCRおよびSSHで調べた。その結果、前者からはserine palmitoyl transferase subunit 2, 後者からはsecreted frizzled-related protein 4, vomeronasal 1 receptor F4 (v1rf4), DEAH (Asp-Glu-Ala-His) box peptide 16 (dhx16), E74-like factor 2, protocadherin2, LRRGT00033 protein, immunoglobin super family 21, kelch repeat and BTB (POZ) domain containing 3, c15orf24 protein, h2A histone family member Y, s100 calcium binding protein a11 (s100a11), mKIAA1506 protein, annexin 2a, c1q-like adipose specific protein, IgM heavy chain constant region, MGC108117 protein and 40S ribosomal protein S17をコードする遺伝子の計17遺伝子が得られた。そこで、色調の発現に関与していることが推定されるdhx16およびs100a11遺伝子を特異的にノックダウンするモルフォリノオリゴヌクレオチド(MO)を作成し、ゼブラフィッシュの受精卵に遺伝子導入して影響を調べた。その結果、前者のMOでは受精後18時間目までは体表の色調に大きな変化は認められなかった。72時間後では、いくつかの胚で色素胞の乱れが、また、ふ化後48時間では体型の異常が観察された。一方、s100a11遺伝子のMOではふ化後8日目においても体表の紋様や骨格の異常は認められなかった。

最後に対馬沖で漁獲したトラフグ(TT)、北九州市の沖合で漁獲したトラフグ(ST)、韓国の東海岸沖で漁獲したカラス(KK)各50尾を対象に、核遺伝子由来のマイクロサテライト(MS)マーカー、ミトコンドリア遺伝子の調節領域を分析した。MS解析による遺伝的多様性指数(genetic diversity index)はTT, ST, KK集団でそれぞれ、0.9505, 0.9350, 0.9335であった。一方、遺伝距離および遺伝子分化指数(Gst: genetic differentiation)は、TTおよびKK間でそれぞれ0.0543, 0.0194、TTおよびST間でそれぞれ0.0857, 0.0189と、トラフグおよびカラス間の方がむしろ小さかった。また、ミトコンドリア遺伝子調節領域の分析から求めた遺伝距離でもMSマーカーによる解析と同様の傾向がみられた。

以上、本研究はトラフグおよびカラスの遺伝的類似性につき、ミトコンドリアおよび核遺伝子の解析できわめて高いことを明らかにした。また、両種の体表の紋様および尻鰭の色調の違いについても分子生物学的な解析を試み、有用な知見を得たもので、学術上、応用上貢献するところが少なくない。よって審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。