Species identification of Anguilla japonica by real-time PCR based on a sequence detection system: a practical application to eggs and larvae

ICES Journal of Marine Science, Oct 2009

Minegishi, Y., Yoshinaga, T., Aoyama, J., and Tsukamoto, K. 2009. Species identification of Anguilla japonica by real-time PCR based on a sequence detection system: a practical application to eggs and larvae. ā€“ ICES Journal of Marine Science, 66: 1915ā€“1918.To develop a practical method for identifying Japanese eel Anguilla japonica eggs and larvae to species by a sequence detection system using a real-time polymerase chain reaction (PCR), we examined (i) the sensitivity of the system using samples at various developmental stages, and (ii) influences of intra- and interspecific DNA sequence variations in the PCR target region. PCR amplifications with extracted DNA solution at 7.0 ng Āµlāˆ’1 or lower were efficient at distinguishing A. japonica from other anguillids. A single egg at the gastrula or later developmental stages could also be identified. Two sequence variations in the PCR target region were observed in 2 out of 35 A. japonica collected from three localities, and from four year classes at a single locality. These mutations, however, did not affect the result of species identification achieved by A. japonica-specific PCR primers and probe. The accuracy of this PCR-based method of species identification will help in field surveys of the species.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://icesjms.oxfordjournals.org/content/66/9/1915.full.pdf

Species identification of Anguilla japonica by real-time PCR based on a sequence detection system: a practical application to eggs and larvae

Yuki Minegishi 0 Tatsuki Yoshinaga 0 Jun Aoyama 0 Katsumi Tsukamoto 0 0 Y. Minegishi, J. Aoyama, and K. Tsukamoto: Ocean Research Institute, University of Tokyo , 1-15-1 Minamidai, Nakano-ku, Tokyo 164-8639, Japan. 6512; fax: To develop a practical method for identifying Japanese eel Anguilla japonica eggs and larvae to species by a sequence detection system using a real-time polymerase chain reaction (PCR), we examined (i) the sensitivity of the system using samples at various developmental stages, and (ii) influences of intra- and interspecific DNA sequence variations in the PCR target region. PCR amplifications with extracted DNA solution at 7.0 ng ml21 or lower were efficient at distinguishing A. japonica from other anguillids. A single egg at the gastrula or later developmental stages could also be identified. Two sequence variations in the PCR target region were observed in 2 out of 35 A. japonica collected from three localities, and from four year classes at a single locality. These mutations, however, did not affect the result of species identification achieved by A. japonica-specific PCR primers and probe. The accuracy of this PCR-based method of species identification will help in field surveys of the species. Introduction The Japanese eel Anguilla japonica is commercially very important in eastern Asia. The stock, however, has been decreasing for the past three decades (Dekker, 2003). To compensate for the shortage of eels, artificial fertilization has been attempted since the 1960s, but it has not yet been achieved practically. A problem in the current culture technique is the lack of information on the reproductive ecology of eels. The spawning area of Japanese eels was recently pinpointed as at the West Mariana Ridge in the western North Pacific (Tsukamoto, 2006), but eggs have not yet been collected, so the spawning ecology of eels remains elusive. Field surveys of spawning eels require rapid and accurate species identification. The eel egg, however, is difficult to identify morphologically because of the few diagnostic characters. During a research cruise in 1998, three morphologically eel-like eggs were collected. However, these were found to be probably Serrivomer, an anguilliform fish, but not Anguilla, by DNA sequences determined in the laboratory after the cruise (Aoyama et al., 2001). Morphological characters change with developmental stage, so molecular characters have emerged as crucial in identifying eel eggs. The basis of the genetic species identification method for Japanese eels using a real-time polymerase chain reaction (PCR) has been developed by Watanabe et al. (2004). A PCR-based identification detects a species-specific DNA nucleotide sequence with designed primers and probe (sequence detection system, SDS). When the method is employed, however, other factors such as template DNA concentrations and sequence variations in the target DNA region by PCR need to be considered because they can affect PCR amplification efficiency, so may cause errors in species identification. The aim of this study was to develop a practical species identification method of A. japonica at an early life stage, e.g. eggs and larvae. Therefore, we investigated (i) the sensitivity (a range of template DNA concentrations) of the system, (ii) system appropriateness for single eggs at various stages of development, and (iii) the effect of intra- and interspecific sequence variations in the SDS target region of the 16S ribosomal RNA (rRNA) gene of mitochondrial DNA. Finally, we determined a practical criterion to identify A. japonica by SDS with real-time PCR. Material and methods To examine the sensitivity of PCR in the system, we used DNA samples from A. japonica, Anguilla marmorata, and Anguilla bicolor pacifica which had been stored in TE buffer (Aoyama et al., 1999; Minegishi et al., 2005). The last two species were analysed because their larvae have been collected around the spawning area of A. japonica (Aoyama et al., 1999; Miller et al., 2002; Kuroki et al., 2006). To test the appropriateness of PCR for a single egg, material obtained by artificial fertilization (Irago Institute, Aichi, Japan) was sampled at five developmental stages (unfertilized, 4, 12, 27, and 39 h after fertilization) and immediately stored in 99% ethanol until DNA extraction. Egg developmental stages after fertilization roughly correspond to the morula, gastrula to embryonic body formation, eye vesicles and heart formation, and hatching periods, respectively (Yamamoto et al., 1975; Yamamoto, 1981). Sequence variations in the PCR target region were investigated as follows. A total of 15 A. japonica from three localities (Miyagi and Ibaraki in Japan, and Taiwan, n 5 each) was analysed for geographic variation. Temporal variations among different year classes of A. japonica were examined using 20 eels that had recruited to Tanegashima Island, Japan, in 1991, 1992, 1995, and 1998 (n 5 each). Interspecific variations were characterized with 22 A. marmorata from six localities (Japan, Taiwan, Indonesia, Fiji, Tahiti, and Madagascar), and four A. bicolor pacifica (from the Indonesian Sea). Sensitivity of PCR To examine the sensitivity of PCR in the species-identification system, a real-time PCR was performed with a wide range of extracted DNA solutions, from 2.0 pg ml21 to 7.0 ng ml21, as template DNA in the three species of A. japonica, A. marmorata, and A. bicolor pacifica. Real-time PCRs were carried out on an Applied Biosystems 7300 real-time system, with a total of 20 ml reaction volume containing 10 ml of 2 TaqMan MasterMix (Applied Biosystems), 900 nM each of forward (Aja16S-L3, 50-AAT CAG TAA TAA GAG GGC CCA AGC-30) and reverse primers (Aja16S-H3, 50-TGT TGG GTT AAC GGT TTG TGG TA-30), 200 nM of TaqMan probe (50-CAC ATG TGT AAG TCA GAA CGG ACC GAC C-30), and 3 ml of template DNA at various concentrations. Except for the PCR cycle number (50), amplification parameters, primers, and probe sequences were the same as used by Watanabe et al. (2004). Appropriateness of PCR for a single egg DNA extraction from a single egg was performed with three volumes (50, 100, and 300 ml) of 5% w/v Chelex resin solution (BioRad). After homogenization of a single egg in a certain volume of solution, genomic DNA was prepared by incubation at 958C for 15 min. PCRs were conducted as described above. Intra- and interspecific variation For a total of 35 A. japonica, 22 A. marmorata, and 4 A. bicolor pacifica, partial DNA nucleotide sequences of 16S rRNA gene of mitochondrial DNA were determined using a pair of PCR primers L1854 and H3058 (Inoue et al., 2001). PCR and sequencing methods are described by Minegishi et al. (2005). For specimens with a mutation in the PCR target region, a real-time PCR was carried out to evaluate the effect of such mutation on species identification. Results Sensitivity of PCR Anguilla japonica was clearly discriminated from the other congeners at a wide range of template DNA concentrations. In A. japonica, a PCR with 7.0 ng ml21 of extracted DNA as template DNA yielded cycles at threshold (Ct) values ranging from 22.0 to 24.3 Figure 1. Relationship between concentrations of template DNA and cycles at threshold (Ct) by real-time PCR. White and black circles indicate A. japonica and other congeners, respectively. The grey underlay shows the template DNA concentration recommended by the manufacturer (1.33 133 ng ml21; Applied Biosystems). (Figure 1). Ct values from 25.2 to 32.4 were observed when the PCR was carried out with less template DNA, at 0.7 and 0.07 ng ml21. Even a reaction with 2.0 pg ml21 of template DNA of A. japonica yielded 37.3 of Ct. In contrast, PCR with 7.0 ng ml21 of template DNA yielded Ct values of 40.0 for the two anguillids other than A. japonica. Moreover, amplifications were not detected with template DNA at 0.7 ng ml21 in either of A. marmorata or of A. bicolor pacifica (Figure 1). Appropriateness of PCR for a single egg With DNA samples from an unfertilized egg and material 4 h after fertilization, PCR amplifications were not detected regardless of the volume of Chelex extracting solution. In contrast, amplifications were observed in eggs 12 h or more after fertilization (Ct 21.1 35.3), except when DNA was extracted with 100 ml of solution for an egg 12 h after fertilization. DNA concentrations extracted from a single egg 39 h after fertilization with 50 ml of Chelex resin solution were 38.2 77.3 ng ml21. Intra- and interspecific variations About 700 bp of partial nucleotide sequences of 16S rRNA gene were determined for the three anguillids. In all, five haplotypes were found among 15 A. japonica collected from Miyagi, Ibaraki (Japan), and Taiwan. Four had mutations outside the target region of the species identification. One eel from Ibaraki had four continuous guanines (G) from the position of the 15th nucleotide from the 50 end of the priming site of the forward primer, whereas the others had three guanines (Figure 2). Among 20 glass eel stages of A. japonica collected at Tanegashima Island in different years, five haplotypes were found. Four contained mutations outside the target region. One eel collected in 1998 had a nucleotide substitution from thymine (T) to cytosine (C) at the fifth position from the 50 end of the probe hybridize region (Figure 2). These two types of mutation in A. japonica were not observed in the other two congeners. Species identification of Anguilla japonica by real-time PCR In all, nine haplotypes were found among 22 A. marmorata, and four A. bicolor pacifica had an identical sequence. Nucleotide variations occurred outside the target region. Subsequently, a real-time PCR was performed using two A. japonica from Ibaraki and Tanegashima with a mutation in the hybridized region. With various concentrations of extracted DNA as template DNA (1.0 57.0 ng ml21), these eels yielded positive results, with Ct values of 18.9 27.0. Discussion We tested experimental conditions to optimize the PCR-based species identification protocol of Watanabe et al. (2004) for practical application to eggs and larvae of Japanese eels. According to the relationship between template DNA concentration and PCR amplification efficiency, there were obvious differences in Ct values between Japanese eels and its congeners at 7.0 ng ml21 of extracted DNA as template DNA (Figure 1). Moreover, PCR amplifications were detected in Japanese eels (Ct 37.3) even with a DNA solution of 2.0 pg ml21, considerably lower than the concentrations recommended by the manufacturer (1.33 133 ng ml21; Applied Biosystems). In contrast, amplification was not observed in the other anguillids with DNA solutions at 0.7 ng ml21. Therefore, to distinguish A. japonica from the other anguillids, the template DNA solution needs to be adjusted to 7.0 ng ml21. We also confirmed the suitability of the method for single eel eggs at the gastrula or later stages. Indeed, the DNA concentration from a single egg was sufficiently high to be applied as template DNA in this system (38.2 77.3 ng ml21), so, to prepare them for optimum DNA concentration (7.0 ng ml21), the extracted DNA solution using 50 ml of Chelex resin solution should be diluted ten times. DNA samples from unfertilized eggs or those at the morula stage were not feasible for use by the current method. This may be due to the low DNA availability in the eggs at such early developmental stages, so undeveloped eggs need to be incubated before DNA extraction. Further, greater sensitivity than in the current system will be necessary to identify unfertilized eggs. Subsequently, we examined the influences of DNA nucleotide sequence variations on the results of the PCR. Only 2 out of 35 A. japonica had mutations within the hybridized region (Figure 2), and these did not affect species identification by PCR. On the other hand, there was no mutation within the hybridized region in either A. marmorata or A. bicolor pacifica. Therefore, the nucleotide sequence of the target region in the present protocol is likely to be conservative at an intraspecific level, but variable at an interspecific level, suggesting that this region is suitable for species identification of Japanese eels based on SDS. Considering the sensitivity of this PCR-based species-identification technique, it is unlikely that A. japonica would not be detected (pseudo-negative) and the other congeners incorrectly identified as A. japonica (pseudo-positive). The method described here has, in fact, contributed to the recent discovery of the spawning area of the Japanese eel in a seamount area of the southern West Mariana Ridge (Tsukamoto, 2006). Additionally, the method works for boiled and baked eels (TY, unpublished data). The decline of the Japanese eel stock and its relatively high commercial value in Japan have incidentally resulted in an escalation of imports of eels from elsewhere and the camouflage of commercial products. Therefore, the method can be used not only for ecological surveys but also to monitor products for conservation reasons, and to identify the international source. Acknowledgements We thank Yoshiaki Yamada of Irago Institute for providing the eggs. The study was partly supported by Grants-in-Aid for Creative Scientific Research No. 12NP0201 (DOBIS) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Eel Research Foundation from Nobori-kai.


This is a preview of a remote PDF: https://icesjms.oxfordjournals.org/content/66/9/1915.full.pdf

Yuki Minegishi, Tatsuki Yoshinaga, Jun Aoyama, Katsumi Tsukamoto. Species identification of Anguilla japonica by real-time PCR based on a sequence detection system: a practical application to eggs and larvae, ICES Journal of Marine Science, 2009, 1915-1918, DOI: 10.1093/icesjms/fsp158