NL2033411B1 - Construction method for gene editing system of apostichopus japonicus - Google Patents
Construction method for gene editing system of apostichopus japonicus Download PDFInfo
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- apostichopus japonicus
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Abstract
The present invention belongs to the field of aquatic genetic breeding, and 5 particularly relates to a construction method for a gene editing system of Aposlichopus japonicus. Sperms and ova of Aposlichopus japonicus are acquired, the ova are immobilized and arranged into a single cell row and fertilized, a prepared mixed solution containing the ngNA of target genes of Aposiichopus japonicus is injected into the fertilized ova of Aposlichopus japonicus arranged into a single cell 10 row, the injected dose is 8%-12% of the volume of a Aposiichopus japonicus embryo, and embryos after microinjection are incubated at 23 °C, so as to obtain a gene editing system of Aposiichopus japonicus. With the construction method of the present invention, embryos With expected phenotype can be obtained after development to a target stage, and the success rate is more than 90%. The application of the present 15 invention can significantly enhance the breeding efficiency, realize site-directed and accurate alteration of DNA sequences of Aposiichopus japonicus, obtain a new variety of Aposiichopus japonicus with target traits, and improve the product quality and economic benefits of Aposiichopus japonicus.
Description
CONSTRUCTION METHOD FOR GENE EDITING SYSTEM OF
APOSTICHOPUS JAPONICUS
The present invention belongs to the field of aquatic genetic breeding, and particularly relates to a construction method for a gene editing system of
Apostichopus japonicus.
Gene editing is a new genetic engineering technology that can modify specific target genes in the genome of an organism accurately. In the breeding process, the gene editing technology can realize the modification and alteration of genetic loci of target traits in varieties, so as to accelerate the improvement of varieties. The gene editing technology is still in the initial development stage in animal breeding, but has a broad prospect in genetic breeding with genome selection as the core, and can achieve efficient and accurate improvement of target traits of target species. Zinc
Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENS) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 are three different gene editing tools. The CRISPR/Cas system is essentially an
RNA-guided nuclease. Unlike ZEN and TALEN nucleases which recognize target sequences through protein-DNA interaction, the CRISPR/Cas nuclease recognizes target sequences through RNA and DNA base pairing. The CRISPR/Cas9 provides a low-cost, efficient and easy-to-use gene editing system. Compared with TALENS,
CRISPR adopts small proteins that are easy to deliver, so gene editing 1s faster; and compared with ZEN, CRISPR can target a specific locus without pairs of proteins, so
CRISPR is more convenient to use. CRISPR/Cas has the ability of promoting multiple genomic modifications at one time, and is much easier to design than the other two DNA nucleases.
Compared with other marine economic animals, Apostichopus japonicus will take a longer time to reach sexual maturity, which is usually more than 3 years.
Therefore, the breeding cycle of a new variety is at least 12-13 years, and the breeding is limited by the factors such as long breeding cycle and low breeding efficiency. In addition, as the key quantitative traits such as length, weight, number of parapodia, length of parapodia and color are difficult to be determined quantitatively and qualitatively, the analysis of basic quantitative genetic characteristics is not clear.
Therefore, the traditional breeding technologies represented by hybrid breeding and selective breeding can no longer meet the current technical requirements on timeliness and reliability of breeding good varieties of Apostichopus japonicus. As a result, it is an urgent need to explore molecular markers with breeding value, carry out the optimal design and breeding of new color varieties (species) of Apostichopus japonicus with excellent target traits, establish a gene editing and breeding technology for Apostichopus japonicus, and realize an accurate breeding system of new color varieties of Apostichopus japonicus, thus to achieve the healthy and sustainable development of the Apostichopus japonicus industry.
With the improvement and development of the CRISPR technology, gene editing has become more and more widely used in aquatic animals. Aquatic products are the third largest source of animal protein in the world, and aquatic animals provide an economical and high-quality animal protein. Therefore, the application of the gene editing technology in aquatic animals enables us to obtain more fishery resources with high quality, and contributes to the healthy and sustainable development of fisheries. At present, CRISPR/Cas9 has been successfully applied to fish such as zebra fish, tilapia, Atlantic salmon, carp, grass carp and tonguefish and to shellfish such as Crepidula fornicata, Lymnaea stagnalis, cuttlefish and Crassostrea gigas.
However, for Echinodermata, CRISPR/Cas9 has only been successfully applied to sea urchins. Apostichopus japonicus is an important representative species of
Echinodermata, but no gene editing system has been established yet, and the research on microinjection of Apostichopus japonicus 1s urgently needed.
Taking zebra fish with relatively mature application of gene editing as an example, the preliminary preparation process of microinjection is generally as follows: on the day before injection, adult zebra fish are placed in breeding tanks with a female to male ratio of 1:1 or 2:1, and each breeding tank is separated by a partition board. On the morning the day of injection, the partition boards are removed, and the female and male zebra fish begin to mate. Generally, the parent fish will oviposit and make ova fertilized after about more than 10 minutes. Generally, the partition boards of two tanks are removed at the first time, and then the partition boards of the other tanks are removed in due time according to the progress of the experiment. To save the time for cleaning embryos, an inner tank together with parent fish can be transferred to another clean outer tank before a partition board is removed. After parent fish oviposit, ova on the bottom of the outer tank are collected by a filter screen with a bore diameter of 180 um and washed with aquaculture water for 2-3 times. Fertilized ova shall be collected within 20 minutes as far as possible (i.e., for the parent fish in one tank, the time interval between two collections of fertilized ova shall not exceed 20 minutes, so as to ensure the consistency of development stages).
After the fertilized ova are sorted and cleaned, the collected ova are placed in a plate petri dish through a pipette, foreign matters in the dish and water around embryos are removed, and the embryos are treated by dry injection.
Due to species specificity, the method for acquiring sperms and ova of zebra fish and the like is not suitable for Apostichopus japonicus, and even the method for acquiring sperms and ova of a closest relative of Apostichopus japonicus, sea urchins, through injection of KCL is also not suitable for Apostichopus japonicus. In addition, zebra fish and the like are treated by dry injection, but Apostichopus japonicus cannot be treated by dry injection, as Apostichopus japonicus will die in large numbers during dry injection. Therefore, it is extremely urgent to establish a special gene editing system suitable for Apostichopus japonicus.
In order to realize the construction of an accurate breeding system of a new variety of Apostichopus japonicus body color and solve the difficulties of the site-directed precision technology in the genetic breeding of Apostichopus japonicus, the present invention aims to provide a construction method for a gene editing system of Apostichopus japonicus, so as to obtain a new variety of Apostichopus japonicus with excellent target traits, thus providing support for the healthy development of the
Apostichopus japonicus industry.
To achieve the above purpose, the present invention adopts the following technical solution:
A construction method for a gene editing system of Apostichopus japonicus, comprising the steps of acquisition of sperms and ova, immobilization for microinjection, sgRNA design of target genes, preparation of an injection system, and microinjection, wherein sperms and ova of Apostichopus japonicus are acquired, the ova are immobilized and arranged into a single cell row and fertilized, a prepared microinjection mixed solution containing sgRNA of target genes of Apostichopus japonicus 1s injected into the fertilized ova of Apostichopus japonicus arranged into a single cell row, the injected dose is 8%-12% of the volume of a Apostichopus japonicus embryo, and embryos after microinjection are incubated at 23°C, so as to obtain a gene editing system of Apostichopus japonicus.
The obtained ova of Apostichopus japonicus are immobilized in a petri dish containing a protamine sulfate solution and arranged in a single cell row, and then semen is added to the petri dish to fertilize the ova; and the immobilized fertilized ova of Apostichopus japonicus are injected with a microinjection mixed solution containing the sgRNA of target genes of Apostichopus japonicus under a microscope.
The immobilization is realized by drawing a line on the back surface of the bottom of the petri dish for microinjection, rinsing the inner part of the petri dish with a 0.1%-1% protamine sulfate solution, and then using a sucking mouth part to orderly arrange the ova in the position with the line on the surface of an injection dish coated with protamine sulfate.
The rinsing is realized by applying a 0.1%-1% protamine sulfate solution to the petri dish, draining, and bleaching with deionized water immediately, or drying, and bleaching with deionized water, for use.
The acquisition of ova of Apostichopus japonicus is realized by promoting maturation of Apostichopus japonicus by using a neuropeptide NGIWY-NH2 to obtain ova.
NGIWY-NH2 (10 uM) is injected into Apostichopus japonicus body cavities, the injected dose is about 0.1% (v/w) of Apostichopus japonicus, Apostichopus japonicus discharges ova 15-20 minutes after shaking, and the ova of Apostichopus japonicus are collected.
The mixed solution containing the sgRNA of target genes of Apostichopus japonicus comprises 500 ng of Cas9 albumen, 300 ng of the sgRNA of target genes, 0.2 ug of 20% glycerine, 0.2 pl of FITC and 0.16 ul of RNase-free water per microliter.
The concentration ratio of Cas9 albumen to sgRNA in the microinjection mixed solution containing the sgRNA of target genes of Apostichopus japonicus is 30:1 to 1:1.
The sgRNA of Apostichopus japonicus for gene editing related to growth and development is acquired by designing gRNA sequences according to CRISPRscan (http://www.crisprscan.org/?page=sequence) and selecting gRNAs with high scores according to CRISPRscan score, and when the microinjection mixed solution is 5 prepared, the sequences can be used individually or in a mixed manner or in a combined manner.
Further, the steps of constructing a gene editing system of Apostichopus japonicus are as follows: 1) Collecting and acquiring breeding Apostichopus japonicus in Laizhou sea area of Yantai in Shandong province in May. Sperms of Apostichopus japonicus are obtained by dissection, and ova of Apostichopus japonicus are obtained by promoting maturation by injecting a neuropeptide NGIWY-NH2. NGIWY-NH2 (10 uM) is injected into Apostichopus japonicus body cavities, and the injected dose is about 0.1% (v/w) of Apostichopus japonicus. After the polypeptide is injected,
Apostichopus japonicus begins to shake, and discharges sperms or ova after about 15 minutes. 2) Rinsing a petri dish for microinjection with a 0.1%-1% protamine sulfate solution, drawing a 1/3 line from the edge on the back surface of the bottom of the petri dish, and scratching behind this line. Applying protamine sulfate to the petri dish, draining, and bleaching with deionized water immediately, or drying, and bleaching with deionized water, and then storing in a dust-free container for later use.
Using a sucking mouth part to orderly arrange the ova on the surface of an injection dish coated with protamine sulfate. Arranging the ova in a row greatly facilitates rapid and successful microinjection. At the beginning of microinjection, injecting 1 ul of diluted semen into the petri dish with a pipettor to finish fertilization.
The 0.1%-1% protamine sulfate solution is prepared as follows: adding 0.4-0.5 g of protamine sulfate to 40-400 ml of distilled water, and preparing a 0.1%-1% protamine sulfate solution in a 50 ml bacteria-free taper pipe until the protamine sulfate is dissolved completely (> 1 h). The solution can be used for at least three months if stored at 4°C. Some protamine sulfate will precipitate after being stored at 4°C, and the solution shall be heated to room temperature for the protamine sulfate to be dissolved completely in use every time. 3) Designing gRNA for gene editing of Apostichopus japonicus by using
CRISPRscan (http://www .crisprscan.org/?page=sequence). A target locus contains two guanine nucleotides at the 5' end for initial gRNA transcription using T7 RNA polymerase, while the 3' end is adjacent to an NGG motif (PAM) in Endo16 Module
A. For preliminary evaluation, gRNAs with high CRISPRscan scores are considered first. CRISPRscan provides a guide sequence with a T7 sequence and a tail sequence.
Ordering selected gRNA sequences provided by CRISPRscan and 80-nucleotide tail primer sequences from Eton, and annealing and extending gRNA and tail primers through PCR by using Phusion Master mix (Phusion High-Fidelity PCR Master Mix with HF Buffer). Then purifying gRNA by a QIAquick PCR purification kit.
Carrying out in vitro transcription by using a MEGAshortscript™ T7 Transcription
Kit, and carrying out purification through alcohol precipitation. 4) Preparing a microinjection mixture containing Cas9 albumen, sgRNA, 20% glycerine, FITC and RNase-free water, with the final volume of 5 ul. The concentration range of the Cas9 albumen is 250 ng/ul-750 ng/ul. The concentration range of sgRNA is 100 ng/ul-400 ng/ul. Before microinjection, placing the mixture on ice. 5) Injecting the solution into the fertilized ova of Apostichopus japonicus immobilized in the petri dish. The diameter of the injected solution is about 1/3-1/4 of an embryo (less than 25% of the volume of an embryo). Experimental controls shall include Cas9 protein injection alone, without sgRNA, to evaluate the effects of exogenous protein injection and expression. Incubating injected embryos and control embryos at 23°C. After the embryos reach the desired stage, extracting genomic DNA for genotyping, and extracting RNA for gene expression level assessment and imaging. 6) If sgRNA/Cas9 injected embryos have no detectable phenotype, increasing the injection concentration of sgRNA/Cas9. However, a high dose of Cas9 protein is toxic to Apostichopus japonicus embryos. On the other hand, if the injected embryos exhibit a severe nonspecific phenotype, reducing the injection concentration. The proportion of the Cas9 albumen amount to sgRNA can be adjusted (from 30:1 to 1:1).
Through tests, the gene editing efficiency of Apostichopus japonicus can reach more than 90%.
The injection site in step 1) is the abdomen of breeding Apostichopus japonicus.
The petri dish for microinjection in step 2) is rinsed with 0.1%-1% protamine sulfate, the protamine sulfate will produce a solution with positive charges, and the negatively charged surface of an embryo will adhere to the solution. Immobilizing embryos in this manner is conductive to rapid injection without affecting development.
The present invention has the advantages and positive effects that: 1. The present invention can efficiently and stably acquire ova of Apostichopus japonicus in the breeding season for microinjection; and the petri dish rinsed with protamine sulfate can be used to immobilize ova of Apostichopus japonicus without affecting fertilization and development of the ova. 2. The microinjection system and injection method for gene editing of
Apostichopus japonicus explored in the present invention can avoid the damage of
Apostichopus japonicus embryos, and the editing efficiency can reach more than 90%. 3. The application of the present invention can significantly enhance the breeding efficiency, realize site-directed and accurate alteration of DNA sequences of
Apostichopus japonicus, obtain a new variety of Apostichopus japonicus with target traits, and improve the product quality and economic benefits of Apostichopus japonicus.
Fig. 1 is a schematic diagram of ova immobilized for microinjection provided in an embodiment of the present invention;
Fig. 2 is a schematic diagram of arranging ova with a sucking mouth part provided in an embodiment of the present invention.
Detailed description of the present invention is further illustrated below in combination with examples. It shall be noted that the detailed description described herein is only used to illustrate and explain the present invention, not limited to the present invention.
Breeding Apostichopus japonicus used for the following experiments is obtained from Laizhou sea area of Yantai in Shandong province.
Embodiment 1
In the embodiment, a gene editing system of Apostichopus japonicus is constructed for the key gene ALXI in the skeleton of Apostichopus japonicus embryos based on the CRISPR/Cas9 technology: 1) Design of gRNA sequences for ALX1 gene of Apostichopus japonicus and synthesis of sgRNA:
Designing gRNA sequences of the ALXI gene of Apostichopus japonicus by using CRISPRscan (http://www.crisprscan.org/?page=sequence). Entering the DNA sequences of the ALX1 gene of Apostichopus japonicus in the "Submit sequence" page of the CRISPRscan website, selecting "Sea urchin - Strongylocentrotus purpuratus", "No mismatch", "Cas9-NGG" and "In vitro T7 promoter”, and then clicking the "Get sgRNAs" button to obtain a list of gRNA sequences.
A target locus contains two guanine nucleotides at the 5' end for initial gRNA transcription using T7 RNA polymerase, while the 3' end is adjacent to an NGG motif (PAM) in ALXI1. According to CRISPRscan score (Moreno-Mateos et al, 2015) provided in the CRISPRscan website and blast alignment of the gRNA sequences with the Apostichopus japonicus genome, four gRNA sequences with high
CRISPRscan scores and fewer identical sequences after alignment are selected for subsequent experiments (see Table 1). Ordering target gRNA sequences provided by
CRISPRscan and tail primer sequences (GTTTTAGAGCTAGAA), and annealing and extending gRNA and tail primers through PCR by using Phusion Master mix (Phusion High-Fidelity PCR Master Mix with HF Buffer). Then purifying gRNA by a
QIAquick PCR purification kit. Carrying out in vitro transcription by using a
MEGAshortscriptTM T7 Transcription Kit, and carrying out purification through alcohol precipitation to obtain sgRNA.
Table 1 gRNA Sequences of Alx1 Gene of Apostichopus japonicus Designed in
This Experiment
Name Sequemce(5'to3) gRNAI GGGGCTTATCGAGGTGGCGT gRNA2 GGGTTGGCGCCGCCCGGCTC gRNA3 GGGTTCGACTCTCGCCGCAT gRNA4 GGAGGCGGCTAACTCGTGTA 2) Preparation of microinjection mixed solution:
Preparing a microinjection mixture containing Cas9 albumen (purchased from
GenScript), sgRNA, glycerine with the mass concentration of 20%, FITC fluorescent dye (purchased from Molecular Probes) and RNase-free water, with the final volume of 5 ul. Before preparation, the concentration of the Cas9 albumen is 2.5 1 g/ul, and the concentration of sgRNA is 1.25 u g/ul. Before microinjection, placing the mixture on ice of 4°C. 5 ul of microinjection mixture in experiment group 1 comprises 1 ul of Cas9 albumen, 1 ul of 20% glycerine, 1 ul of dye (FITC), 1.2 ul of sgRNA (0.3 ul of sgRNA |I, 0.3 ul of sgRNA2, 0.3 ul of sgRNA3 and 0.3 ul of sgRNA4), and 0.8 ul of
RNase-free water. Finally, the injection concentration of the Cas9 albumen in the experiment group 1 is 500 ng/ul, and the concentration of sgRNA is 300 ng/ul. 5 ul of microinjection mixture in experiment group 2 comprises 1.5 ul of Cas9 albumen, 0.8 ul of 20% glycerine, 1 ul of dye (FITC), 1.5 ul of sgRNA (0.375 ul of sgRNA 1, 0.375 ul of sgRNA2, 0.375 ul of sgRNA3 and 0.375 ul of sgRNA4), and 0.2 ul of RNase-free water. Finally, the injection concentration of the Cas9 albumen in the experiment group 2 is 750 ng/ul, and the concentration of sgRNA is 375 ng/ul. 5 ul of microinjection mixture in experiment group 3 comprises 0.5 ul of Cas9 albumen, 1 ul of 20% glycerine, 1 ul of dye (FITC), 1.0 ul of sgRNA (0.25 ul of sgRNA 1, 0.25 ul of sgRNA2, 0.25 ul of sgRNA3 and 0.25 ul of sgRNA4), and 1.5 ul of RNase-free water. Finally, the injection concentration of the Cas9 albumen in the experiment group 3 is 250 ng/ul, and the concentration of sgRNA is 250 ng/ul. 5 ul of microinjection mixture in experiment group 4 is the same as that in the experiment group 1, except the injected dose is different, which is used to test the influence of the injected dose on the gene editing effects of Apostichopus japonicus. 5 ul of microinjection mixture in a control group comprises 1 ul of Cas9 albumen, 1 ul of 20% glycerine, 1 ul of dye (FITC), and 2 ul of RNase-free water.
Finally, the injection concentration of the Cas9 albumen in the control group is 500 ng/ul, and the concentration of sgRNA is 0. 3) Acquisition of sperms and ova of Apostichopus japonicus:
Obtaining sperms by dissecting Apostichopus japonicus from the above region; and obtaining ova of Apostichopus japonicus by promoting maturation by injecting a neuropeptide NGIWY-NH2, specifically as follows: NGIWY-NH2 (10 uM) is injected into Apostichopus japonicus body cavities, and the injected dose is about 0.1% (v/iw) of Apostichopus japonicus. After the polypeptide is injected,
Apostichopus japonicus begins to shake, and discharges sperms or ova after about 15 minutes. 4) Immobilization and fertilization of ova of Apostichopus japonicus:
Immobilizing the ova obtained above in a petri dish for microinjection, specifically as follows: adding 0.4 g of protamine sulfate to 40 ml of distilled water, and preparing a 1% protamine sulfate solution in a 50 ml bacteria-free taper pipe until the protamine sulfate is dissolved completely (> 1 h) for use. Drawing a line parallel to the diameter of the petri dish in the 1/3 position of the area of the petri dish on the back surface of the petri dish, then adding a 1% protamine sulfate solution to the petri dish to rinse the petri dish, draining, and bleaching with deionized water immediately, or drying, and bleaching with deionized water, for use; and storing in a dust-free container for later use. Finally, using a sucking mouth part to orderly arrange and immobilize the ova in the position with the marked line of the petri dish coated with protamine sulfate (see Fig. 1 and Fig. 2). At the beginning of microinjection, injecting 1 ul of diluted semen into the petri dish with a pipettor to finish fertilization. 5) Microinjection:
Placing the immobilized embryos under a stereomicroscope, focusing the embryos with a low power objective, gently lowering a needle tip, pushing the injection needle tip into the center of the field of view, and adjusting the position of the injection needle through fine adjustment of a micro operation system until the needle tip can be seen clearly. Further adjusting the focal distance of the microscope and the positions of the injection needle and embryos to achieve the best clarity of the embryos and the injection needle tip. Pushing a control level, and carefully inserting the needle to make the injection needle tip enter the embryos. Pushing a foot switch to inject a sample into the embryos. The injected dose of the experiment groups 1-3 and the control group is 10% of the volume of a Apostichopus japonicus embryo, generally 1 nL. The injected dose of the experiment group 4 is 15% of the volume of a Apostichopus japonicus embryo, generally 1.5 nL. Each of the four experimental groups and one control group is injected with 500 Apostichopus japonicus embryos.
After injection, the success of the injection is judged according to the fluorescence in the embryos under a fluorescence microscope. According to the microscopy results, 488 embryos are successfully injected in the experiment group 1, 478 embryos are successfully injected in the experiment group 2, 490 embryos are successfully injected in the experiment group 3, 450 embryos are successfully injected in the experiment group 4, and 489 embryos are successfully injected in the control group. 6) Embryo culture after injection:
Incubating the injected embryos in a 23 °C incubator until the stage of developing into auricularia larva. 7) Gene editing success rate
For normally developing Apostichopus japonicus embryos, each larva has only one solid skeleton at the stage of auricular larva, and the position of the skeleton is fixed, only on the right side of the back. However, at the early stage of the development of doliolaria, the number and positions of skeletons of the embryos begin to change, and some embryos begin to have two skeletal elements in different positions.
For the injected embryos in the experiment group 1, 450 living embryos develop to the stage of auricular larva, among which 439 embryos have skeletons disappeared, and the gene editing success rate is 97.5%; for the injected embryos in the experiment group 2, 419 living embryos develop to the stage of auricular larva, among which 365 embryos have skeletons disappeared, and the gene editing success rate is 87.1%; for the injected embryos in the experiment group 3, 446 living embryos develop to the stage of auricular larva, among which 387 embryos have skeletons disappeared, and the gene editing success rate is 86.7%; for the injected embryos in the experiment group 4, only 42 living embryos develop to the stage of auricular larva, the other injected embryos become malformed or die, 35 of 42 normal embryos have skeletons disappeared, and the gene editing success rate is 83.3%; and for the injected embryos in the control group, 459 living embryos develop to the stage of auricular larva, among which no embryo has skeletons disappeared, and the gene editing success rate is 0. The experiment proves that the gene editing system of Apostichopus japonicus is successfully constructed.
It can be seen from the above embodiment that the present invention constructs a gene editing breeding system of Apostichopus japonicus based on the CRISPR/Cas9 technology, solving (1) the problem that ova and sperms of Apostichopus japonicus for gene editing cannot be acquired in time; and meanwhile, the fixation and efficient microinjection of ova of Apostichopus japonicus of the present invention can be used to complete the injection of a large number of embryos in a short time, significantly improving the experimental efficiency, so as to improve the gene editing efficiency according to the reagent ratio and the injected dose of the optimized microinjection system of Apostichopus japonicus. The present invention can realize site-directed and accurate genetic transformation of Apostichopus japonicus, be applied to different genes according to the target traits for breeding of Apostichopus japonicus, and realize the editing of a plurality of target genes and multiple target traits by constructing a gene editing system, so as to obtain an excellent variety of
Apostichopus japonicus with target traits, thus providing a new way and method for breeding of Apostichopus japonicus.
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