1 Introduction

The sea potato (Acaudina spp.), a species of sea cucumber, widely distributes in the waters along some Asian counties including Philippine and China. Most of these species live in the intertidal zone, and only a few of them live in the coastal sediment. The sea potato has simple body structure with a fast reproductive ability. Every summer, it becomes blooming in the East Sea of China, causing serious problems on the maintenance and operation of marine facilities. For example, large numbers of adults and larvae of sea potato caused the blockage of the pipes for seawater intake and drainage for nuclear power plants. It is necessary to carry out species identification and establish the risk evaluation system for this species. The classic species identification is mainly based on morphology observation. However, this approach is hard to identify the larvae of sea potato from a large number of plankton. The molecular methods based on the mitochondria genome have become an effective way to solve this problem.

Mitochondria are subcellular organelles unique to eukaryotes, playing an important role in a series of cytological processes, such as energy metabolism (Brand, 1997; Morenosánchez et al., 2010), cellular aging and diseases (Wallace et al., 1995). Mitochondrial genome data have been utilized to study phylogeographic (Scribner et al., 2003), evolution and phylogenetic relationships of the animals in the metazoans (Boore et al., 2005). Mitochondrial DNA (mtDNA) is a double-stranded circular DNA molecule that is independent of the chromosome. Its size is about 16 kb (Boore, 1999). Except for a few species, most of animals' mitochondrial genomes consist of 37 genes: 13 protein-coding genes, 2 ribosomal RNAs (rRNAs), 22 transfers RNAs (tRNAs) (Boore and Brown, 1994; Peregrino-Uriarte et al., 2009), and a non-coding putative control region, in which signal sequences exist for transcription and replication (Takata et al., 2001). mtDNA is essential for the process of protein synthesis, transcription, and translation. In addition, it has the characteristics of the small genome (Behera et al., 2018), compact structure, high copy number (Ingman et al., 2000) and no rearrangement during cell meiosis (Fan et al., 2011). It is widely used in evolution analysis, genetic diversity study and species identification (Curole and Kocher, 1999). The full mitochondrial sequences of most species of the sea cucumber have been determined (Shen et al., 2009; Perseke et al., 2010).

In our previous study, the mitochondrial DNA sequence of Acaudina spp. has been detected through polymerase chain reaction (PCR) amplification and Sanger sequencing (Wang et al., 2019). In this study, based on the complete mitochondrial genome sequences, we screened and identified the partial sequence of nd1 gene as a specific molecular marker for the identification of Acaudina spp. from the collected plankton samples. With the molecular marker, we developed an effective method to identify and evaluate the population dynamic of Acaudina spp. through PCR.

2 Materials and Methods 2.1 Animal Collection

Four adults of Acaudina spp. (Fig. 1A) were collected from the coast in Fujian Province, China. Species were identified by morphology (Xiao, 2015) and 16S rRNA sequencing based on the published sequences (Wen et al., 2011). The samples were then fixed in 75% ethanol and stored at 4℃ until DNA extraction.

The larvae of Acaudina spp. were collected using a type Ⅱ plankton collection net (diameter 31.6 cm, length 140 cm, mesh size 0.16 mm, rope length 10 m) from 13 stations. The plankton samples were concentrated in a 500 mL plastic sampling bottle and then fixed with 75% ethanol. The positions of sampling stations were indicated in Fig. 3A.

2.2 DNA Isolation

Total genomic DNA was extracted from the gonad of adult Acaudina spp. and the planktonic samples, respectively, using a modified phenol/chloroform/isoamyl alcohol method (Wei et al., 2020). Firstly, 45 mL STE buffer (100 mmol L^-1 NaCl, 10 mmol L^-1 Tris-HCl, 100 mmol L^-1 EDTA, pH 8.0) and 4.5 mL 10% SDS (dissolved in ddH₂O) were mixed in a 50 mL tube to make the lysis buffer. The gonads were dissected from the animal and were put into a 1.5 mL tube. Then 700 μL lysis buffer and 2.8 μL Proteinase K (Merck, dissolve in ddH₂O with the concentration of 50 mg mL^-1) were added into the tube to make a final concentration of 200 μg mL^-1 proteinase K. The mixture was shaken gently and incubated in a water bath at 58℃ for 3 h. Finally, DNA was extracted with phenol-chloroform-isoamyl alcohol and chloroform-isoamyl alcohol, respectively, and then precipitated by alcohol and dissolved in ddH₂O. The dissolved DNA was stored at -20℃.

2.3 PCR Amplification

PCR reaction was carried out using PfuS DNA polymerase (gift from Dr. Zhiyi Lv) in a 50 μL volume. The reaction solution includes 10 μL 5× Phusion HF Buffer (Thermo fisher Catalog number: F518L), 1 μL 10 mmol L^-1 dNTP, 2.5 μL 10 μmol L^-1 Primer F, 2.5 μL 10 μmol L^-1 Primer R, 1 μL template DNA (200 ng μL), 0.5 μL PfuS DNA polymerase, and 32.5 μL ddH₂O. The PCR was performed as follows: Pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s; annealing at 55℃ for 15 s, followed with elongation at 72℃ for 1 min, and totally for 35 cycles; and a final extension at 72℃for 5 min. The PCR products were purified by Gene JET Gel Extraction Kit (Thermo Fisher Scientific, Lithuanian).

The primers for the amplification of nd1 from Acaudina spp. were designed based on the published mitogenomes (Wang et al., 2019). The primer sequences and annealing temperatures of PCR were shown in Table 1.

2.4 TOPO Clone, Sequencing, and Sequence Alignment

TOPO cloning was performed using pEasy-Blunt3 kit (Transgen, Beijing, China). Firstly, 4 μL purified PCR product was mixed with 1 μL pEasy-Blunt3 vector and incubated at 25℃ for 15 min. Then they were transformed into Trans1-T1 competent cells and spray on Luria-Bertani (LB) agar plates. The LB plates were cultured overnight at 37℃. Monoclonal colonies were picked up for sequencing. Sequencing was performed by Genwiz company (Tianjin, China). Multiple sequence alignments were carried out using the ClustalW program in BioEdit software (version 7.0.4.1) with default parameters.

2.5 Image Analysis and Statistical Analyses

Totally 116 plankton samples were examined by PCR to determine whether the larvae of Acaudina spp. were in the samples. PCR products were detected on a 1% agarose gel. The electrophoresis bands of 116 samples were analyzed using ImageJ software to calculate the gray value, which can reflect the content of DNA. 18S rDNA (also known as 18S rRNA gene) was used as a reference. The distribution of Acaudina spp. larvae at 13 detected stations were evaluated based on molecular data.

3 Results 3.1 Morphological and Molecular Identification of Acaudina spp.

One adult sample was collected from the coast of Fujian province. It was about eight cm long and its body presented the light brown color with dark brown spots (Fig. 1A). They were morphologically similar to sea potato. To identify the species, we designed primers 16S rRNA-F and co1-R to amplify a mitochondrial fragment including two genes 16S rRNA and cox based on previously published sequences (FJ971405 and FJ971380) of Acaudina spp. (Wen et al., 2011). A 1500 bp DNA fragment was amplified from the total DNA (Fig. 1B). After sequencing and alignment, both COI and 16S rRNA genes from our samples showed high identify with the ones from Acaudina spp.. However, the identity of COI sequence from our samples with FJ971405 was only 85% (Fig. 1C), and the 16S rRNA sequence from our samples showed 90% identity with FJ971380 (Fig. 1D). Based on these data, we identified our samples as Acaudina spp..

3.2 The Fragment of Mitochondrial nd1 Gene Was an Effective Species-Specific Molecular Marker

Mitochondrial genes are widely used in population identification and genetic studies, such as atp6 (Das et al., 2018), co1 (Wang et al., 2018), cytb (Behera et al., 2015). In our previous work, we got the mitochondrial genome sequence of Acaudina spp.. Based on this data, we designed primers to amplify different fragments from the total DNA of Acaudina spp. and other marine species to identify the species-specific marker. The results showed that a 229 bp DNA fragment of nd1 gene could be effectively amplified through a primers pair, Am-mt-nd1-F2 (Fig. 2A) and Am-mt-nd1-R (Fig. 2B) from the total DNA of Acaudina spp., but could not from other marine species including ascidians (Ciona robusta, Styela clava), clam (Mactra quadrangularis) and scallop (Patinopecten yessoensis) (Fig. 2C). We then tested the validity of this pair of primers using total DNA extracted from the collected plankton samples. The results showed that one specific fragment could be successfully amplified from 9 of 13 samples (Fig. 2D), suggesting that the fragment of nd1 gene is an effective species-specific molecular marker for species identification and larva detection from the plankton samples.

3.3 Quantitative Evaluation of Larval Population of Sea Potato

To evaluate the population of sea potato, we developed a relative quantitative analysis method based on the species-specific nd1 marker. The quality of DNA from each sample was verified by 18S rDNA with universal primers. The optical density ratio of nd1 and 18S rDNA PCR bands were used to represent the relative quantity of sea potato larvae, and compare the population variation among different samples. The 116 samples were collected from 13 stations distributed along the coast region of Fujian province (Fig. 3A). We divided all the sampling stations into four groups by the distance to the S01 station: group Ⅰ included S01, S02, and S03, which were off S01 station less than 1 km; group Ⅱ included S04, S05, S06 and S07, off S01 station 1 to 2 km; Group Ⅲ included S08, S09, and S10, off S01 station 4 to 6 km; group Ⅳ included S11, S12, and S13, off S01 station more than 9 km. The total DNA was extracted from the samples, respectively. All DNA samples were diluted to 20 ng μL^-1, and 10 μL was utilized for PCR. The DNA quality of most samples (107/ 116) were sufficiently good for the amplification of the fragments of nd1 and 18S rDNA. The results showed obvious differences in the relative larval density among the samples and the groups (Fig. 3B). It clearly showed that the high-density sea potato was presented in group Ⅰ station in spring and summer near the land, suggesting that distribution of sea potato is associated to the marine sediment environment.

3.4 Season Variation of Acaudina spp. Larvae

The samples were collected in four seasons. The data showed that the highest larval density appeared in June and July. For stations in group Ⅰ, there was a significant increase from May to July, and then a rapid decline after September (Fig. 4A). For stations in groups Ⅱ and Ⅲ, they showed similar patterns: the lowest density in Winter, and other seasons showed the moderate density (Figs. 4B and 4C). For group Ⅳ, the relative density was globally low and two small peaks were observed in January and June (Fig. 4D).

4 Discussion

In this study, we successfully identified a 229 bp fragment of gene nd1 as Acaudina spp. specific molecular marker, which provides an effective molecular tool for species identification and population dynamic evaluation. By analyzing the relative quantity of this fragment with PCR, we quantitatively detected the larval density of Acaudina spp. in a marine coast, and found that their density decreased gradually from offshore to deeper waters. In the samples that were collected from about 20 m deep of the seawaters, the larval density is quite low. In addition, the higher density of larvae was presented in the samples collected in May, June, and July, suggesting that the larvae of Acaudina spp. might propagate from April to July.

It is worthy to note that the method that we developed in this study cannot distinguish the species within one genus. CRISPR-based specific high sensitivity enzymatic reporter unlocking technology (Gootenberg et al., 2017; Gootenberg et al., 2018) and DNA endonuclease targeted CRISPR trans reporter technology (Chen et al., 2018) can be the alternative ways to accurately quantify target molecules. Loop-mediated isothermal amplification is another potentially useful technology with high specificity and efficiency under isothermal conditions (Notomi et al., 2015).

Our results showed that the larval density of Acaudina spp. is dynamic and tightly correlates with the temperature and the sediment in the offshore seawaters. These results therefore provide useful information to understand the seasonal and regional distribution of Acaudina spp. larvae. Based on this information, population dynamics can be effectively evaluated, early warning mechanisms can be established, and the population density of Acaudina spp. can be regulated in the specific sea region.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (No. 2018YFD090 0705), the Key Laboratory of Integrated Marine Monitoring and Applied Technologies for Harmful Algal Blooms Funds (No. MATHAB201706), and the Fundamental Research Funds for the Central Universities (No. 201822016).