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Advances in DNA Barcoding of Toxic Marine Organisms

There are more than 200,000 marine species worldwide. These include many important economic species, such as large yellow croaker, ribbonfish, tuna, and salmon, but also many potentially toxic species, such as blue-green algae, diatoms, cnidarians, ctenophores, Nassarius spp., and pufferfish. However, some edible and toxic species may look similar, and the correct identification of marine species is thus a major issue. The failure of traditional classification methods in certain species has promoted the use of DNA barcoding, which uses short, standard DNA fragments to assist with species identification. In this review, we summarize recent advances in DNA barcoding of toxic marine species such as jellyfish and pufferfish, using genes including cytochrome oxidase I gene (COI), cytochrome b gene (cytb), 16S rDNA, internal transcribed spacer (ITS), and Ribulose-1,5-bisphosphate carboxylase oxygenase gene (rbcL). We also discuss the application of this technique for improving the identification of marine species. The use of DNA barcoding can benefit the studies of biological diversity, biogeography, food safety, and the detection of both invasive and new species. However, the technique has limitations, particularly for the analysis of complex objects and the selection of standard DNA barcodes. The development of high-throughput methods may offer solutions to some of these issues.


Introduction
Toxic marine organisms are traditionally considered as those produce biotoxins or concentrate biotoxins from other organisms or the marine environment in their life periods. Toxic algae, such as dinoflagellates, diatoms and cyanobacteria, are associated with the production of many marine toxins, which can cause harmful algal blooms (HAB), paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), neurotoxic shellfish poisoning (NSP), and ciguatera fish poisoning (CFP). Some seafood species can adapt to tolerate high levels of certain algal toxins. For example, softshell clams (Mya arenaria) from areas exposed to red tides can accumulate more PSP toxins and are more resistant to toxins, compared with sensitive clams from areas not exposed [1]. These kinds of toxic marine organisms are always highlighted in daily life, because toxins might lead to greater toxin resistance in seafood species and increased risk of toxins in humans, even resulting in long-term changes to communities and ecosystems.
Some marine organisms can still be health hazards, even though they do not produce or concentrate biotoxins in their life periods. Scombroid food poisoning is a foodborne illness resulting from eating spoiled (decayed) fish [2]. In fact, not only Scombroid fish, mackerel, tuna and bonito, Acc.species represents the number of accepted marine species within the specific rank in World Register of Marine Species (WoRMS); Barcoded represents the number of barcoded species in Barcode of Life Data System (BOLD); Rate represents the barcoding rate that calculated by divide Barcoded by Acc.species.

Figure 1.
Histogram of marine species' barcoding rates. The marine species' barcoding rates were calculated using the data in Table 1. The percentages of the abscissa indicate the percentage of the number of species, and the percentage of the ordinate indicates the frequency of the barcoding rate.
As the histogram shows, most (65%) marine species' barcoding rate is less than 30%. Almost half (43%) of marine species' barcoding rates fall in the range of 0-10%. The DNA barcoding rates are still relatively low. Meanwhile, 20% of marine species' barcoding rates are larger than 100% because the data from BOLD are not focus on marine species. Histogram of marine species' barcoding rates. The marine species' barcoding rates were calculated using the data in Table 1. The percentages of the abscissa indicate the percentage of the number of species, and the percentage of the ordinate indicates the frequency of the barcoding rate.
As the histogram shows, most (65%) marine species' barcoding rate is less than 30%. Almost half (43%) of marine species' barcoding rates fall in the range of 0-10%. The DNA barcoding rates are still relatively low. Meanwhile, 20% of marine species' barcoding rates are larger than 100% because the data from BOLD are not focus on marine species.    Figure 2 represents the difference of 10 times. Marine Acc.species represents the number of accepted marine species within the specific rank in WoRMS; BOLD species represents the number of species in BOLD; Barcoded represents the number of barcoded species in BOLD.

Survey of Methodology
The data in this review were collected from notable databases. World Register of Marine Species (http://www.marinespecies.org/) is a common database that provides an authoritative and comprehensive list of marine organism names, including information on synonymy. Barcode of Life Data System (http://www.barcodinglife.org) is not only a database but also an analysis platform, which consists of data portal, registry of Brcode Index Numbers-BINs (putative species), data collection and analysis workbench. This review investigates basic papers, case studies and some frontier research.

DNA Barcoding Is widely Used in Toxic Marine Algae and Metazoans
A DNA barcode can be derived from the nucleus, mitochondrion and chloroplast. The most commonly used nuclear DNA barcodes are 18S rDNA, 28S rDNA, and internal transcribed spacer (ITS) [16][17][18]. Common mitochondrial DNA barcodes widely used in the animal kingdom include the genes for COI, cytochrome b gene (cytb), and control region (or displacement-loop, d-loop) [19] [20,21]. Common chloroplast DNA barcodes widely used in the plant kingdom include Maturase K gene (matK), Ribulose-1,5-bisphosphate carboxylase oxygenase gene (rbcL), and non-coding trnH-psbA spacer region [22][23][24]. Different DNA barcodes are suitable for different species, according to their specific sequence characteristics. This review focuses on the identification of several toxic marine species (diatoms, Cnidaria, Ctenophora, Mollusca, pufferfish, and tuna) and their commonly used DNA barcodes (Tables 2-6).

Toxic Marine Algae
Marine biotoxins are one kind of secondary metabolites produced by marine photosynthetic organisms including dinoflagellates, diatoms and cyanobacteria [44]. Under an appropriate environment, these microorganisms can grow and produce a large amount of biotoxins rapidly. Consequently, poisoning can occur when people swim, take bath or farm economic species in contaminated water. Furthermore, because of global climate change, eutrophication, urbanization and modern agriculture, the proliferation, frequency, and persistence of harmful algal blooms (HAB) are increasing in many parts of the world over the past decades [45].
Diatoms comprise one of the highest biomasses of phytoplankton in the ocean. They are widely distributed in both marine and fresh water, with an estimated 200,000 species [46]. Pseudo-nitzschia can be responsible for one of the most harmful algal blooms in coastal ecosystems, causing amnesic shellfish poisoning in humans as a result of the production of domoic acid [47].
Evans et al. [25] studied 34 diatom individuals from four genera and assessed the effectiveness of several genes (COI, rbcL, 18S rDNA and ITS) for distinguishing cryptic species. They showed that the COI gene was best suited for DNA barcoding of most diatoms, with a greater divergence than rbcL and greater variability than 18S rDNA. However, Moniz and Kaczmarska [27] found that COI had a low efficiency of amplification and that the 5.8S + ITS2 fragment had a higher success rate, making it a better candidate for diatom DNA barcoding.
Hamsher et al. [28] used Sellaphora isolates to test the ability of several markers (~1400 bp rbcL, 748 bp at the 3 end of rbcL, LSU D2/D3, and UPA) to discriminate among closely related diatom species. The results suggested that rbcL-3P should be used as the primary marker for diatom DNA barcodes, as supported by Hamsher and Saunders [29]. However, after comparing several genes, MacGillivary and Kaczmarska [20] did not support the use of rbcL alone for DNA barcoding, and they recommended using rbcL-3P together with the 5.8S + ITS2 fragment, which has a higher mutation rate. Based on these studies, the rbcL-3P should be used as a primary marker and 5.8S + ITS2 fragment can also be used as a potential DNA barcodes due to its higher degree of variation.

Mollusca
Harm of HAB is not limited to the organisms that generate HAB because their biotoxins can accumulate in other marine organisms (i.e., fish or shellfish) and end up in the human food web [48]. For example, some species of Nassarius are toxic at some growth seasons [49,50] and can pollute non-toxic marine species, such as softshell clams (Mya arenaria), an edible shellfish that is used in a variety of dishes. The claims from areas exposed to red tides are usually more tolerant to PSP toxins and can accumulate toxins at greater rates than those sensitive clams from unexposed areas [1]. The prevention of contaminated seafood reaching the markets is currently an effective way to ensure human health [51]. Legislative requirements are implemented to monitor shellfish to control the risk of shellfish poisoning to human consumers [52].
Zou et al. [30] showed that COI was better than 16S rDNA when using distance-based methods for identifying 40 species of Neogastropoda. However, both genes showed 100% success rate in identifying all the samples when using the character-based method. In a different study, Zou et al. [31] analyzed different Nassarius species and found four cryptic species and one pair of synonyms using the COI gene. Couceiro et al. [17] used the COI gene to distinguish between the two morphologically similar species, Nassarius nitidus and Nassarius reticulatus, which showed a clear barcoding gap. Using COI and 28S rDNA, Galindo et al. [32] analyzed the Nassarius pauperus complex from the eastern Indian Ocean and western Pacific Ocean, including a revised concept of Nassa paupera Gould, 1850, type species of the genus Reticunassa Iredale, 1936, and discovered six new species. Lobo et al. [53] pointed out that some of the so-called "universal primers" still failed to amplify COI-5P in some marine animal groups. Therefore, they designed a new pair of enhanced primers for the COI-5P region for a wide range of marine organisms, including Mollusca and Cnidaria. Thus, COI appears to be the most widely used DNA barcoding gene in Mollusca.

Cnidaria
In addition to the shellfish mentioned above, there are other toxic marine invertebrates including jellyfish, which can cause skin damage to human, one of the most common poisoning incidents for treatment in clinics and hospitals in highly industrialized countries [54]. The phylum Cnidaria consists of four categories including Anthozoa, Cubozoa, Hydrozoa and Scyphozoa, the last three of which are collectively known as Medusozoa. Jellyfish use compounds with neurotoxic and cardiotoxic effects for hunting and for defense against predators or other potential threats [55]. Jellyfish toxins cause toxic effects in diverse organisms and can trigger local and systemic reactions [56].
Since Cnidaria shows far lower COI divergences than any other phylum, efforts have been taken to find better DNA barcodes for Cnidaria. Moura et al. [33] studied 56 sequences from the Hydrozoan families Eudendriidae, Lafoeidae, Haleciidae, Sertulariidae, Plumulariidae, and Aglaopheniidae, and found that 16S rDNA was a useful DNA barcoding tool for Hydrozoa. Armani et al. [34] used five sets of primers for the COI gene to identify jellyfish products and found that 100% of ready-to-eat jellyfish products were mislabeled. McInnes et al. [35] used 18S rDNA to study the occurrence of jellyfish predation by black-browed and Campbell albatross. McFadden et al. [57] reviewed the limitation of mitochondrial DNA barcoding in Octocorallia. Their recent studies revised Genus Ovabunda in the Red Sea and divided four clades using three mitochondrial (mtMutS, COI, and ND2) and four nuclear (ITS, 28S rDNA, ATPSα, and ATPSβ) genes [36]. Miranda et al. [37] conducted a comprehensive molecular phylogenetic analysis of Staurozoa with a set of DNA barcodes (mitochondrial markers COI and 16S rDNA, and nuclear markers ITS, 18S rDNA and 28S rDNA) using three methods (Parsimony phylogenetic hypothesis, Maximum likelihood phylogenetic hypothesis and Bayesian phylogenetic hypothesis). The comprehensive comparison of these five DNA barcodes showed that only 28S rDNA supported each main group observed in the three methods. Therefore, nuclear genes (ITS, 18S rDNA and 28S rDNA) and their combination with mitochondrial genes are increasingly used for DNA barcoding in Cnidaria.

Toxic Marine Fish
Fishes are the largest and most diverse vertebrates, with the value of many commercial fisheries exceeding US$ 200 billion [5]. Pufferfishes are well-known to be toxic and are thus the subject of a unique type of commercial fishery. The high rate of mislabeling of marine fish in markets makes it highly urgent to review the research and development of toxic commercial fisheries [34].

Pufferfish
Most members of the family Tetraodontidae carry tetrodotoxin (TTX), which is typically concentrated in the liver, but also in the ovaries, intestines, and skin. Tetrodotoxin is a heat-stable neurotoxin that can cause weakness or paralysis and death [58]. There were 28 cases of pufferfish poisoning in Florida, New Jersey, Virginia, and New York from January 2002 to May 2004 [59].
Amaral et al. [34] used the COI gene to analyze the pufferfish in Tocantins River and identified a new species of the genus Colomesus, which was formerly thought to contain only two species, C. asellus and C. psittacus. Huang et al. [38] used the cytb gene as the basis for identifying pufferfish. This system was tested in several specimens of pufferfish, as well as in simulated products and commercial samples. They reported that 3.92% of the tested samples were from toxic species using the cytb gene as a barcoding marker. A reported study used Full and mini COI DNA barcodes to identify 68 ethnic seafood products from an Italian market [39] and showed that two poisonous pufferfish samples forbidden in the European Union were wrongly labelled as squid. Tuney [40] used 16S rDNA and cytb to identify Lagocephalus sceleratus and Lagocephalus spadiceus, which are well-known invasive marine species from the Red Sea, and concluded that cytb was more useful than 16S rDNA in this study. Thus, COI and cytb are the most accepted DNA barcodes in Pufferfish.

Scombridae
Although studies of toxic marine fishes often focus on fishes that produce and accumulate specific toxins, other fishes, such as the genus Scombermorus and tuna, do not contain toxins but do have high levels of vitamins and histamine that can also cause harmful effects, even though they are often not considered as poisoning incidents. Scombridae fishes, which contain 15 genera and 51 species, have a worldwide importance for their economic and ecological value [16].
Vinas and Tudela [41] found that the mitochondria control region or displacement-loop (d-loop) combined with ITS1 could fully distinguish the eight Thunnus species in different kinds of processed tissue. Pedrosa-Gerasmio et al. [42] and Kumar et al. [19] used only d-loop to identified Scombridae fish. Using COI in larval fishes, Seyhan and Turan [43] studied nine Scombrid species in Turkey and authenticated the efficacy of COI in identifying the Scombrid species with designated barcodes. COI and d-loop are the common DNA barcodes for identifying Scombridae fish.

Disadvantages of DNA Barcoding
Species identification using DNA barcoding-based methods have advantages over conventional morphological methods, but they also have limitations. A major issue with DNA barcoding from the very beginning of its application is the failure to find the universal primer or universal DNA barcode for a specific target organism. The evolution rates of nuclear and mitochondrial DNA are different. COI may be useful to some species, but the evolution rates of mitochondrial DNA are not uniform for all species' evolution. The same is true for the nuclear DNA barcodes. Furthermore, pseudogenes and heteroplasmy can complicate the DNA barcoding research. Pseudogenes can lead to false division of one species into several species by mistake [60]. Pseudogenes can cause heteroplasmy, resulting in the coexistence of more than one type of mtDNA in the same individual, which can significantly decrease the reliability of species identification by DNA barcoding and increase the complexity of the database [61]. Differences in evolutionary rates provide various DNA barcoding options but make it difficult to find a universal DNA barcode for all species. Most studies using DNA barcoding use Sanger sequencing, which determines the sequence of only one sample. If DNA samples contain more than one template, the determined sequence will produce misleading results. Pseudogenes can have similar problems [62]. Batovska et al. [63] reported that Sanger sequencing is not an appropriate method for characterizing ITS2 in the majority of mosquito species for the variety of polymorphisms in the gene.

Real-Time Fluorescence PCR
Unlike traditional PCR method, real-time fluorescence PCR (RT-PCR, qPCR) allows rapid determination of target gene in samples with higher sensitivity. By using the fluorescent signal, the PCR procedure also has an advantage for monitoring target sequences with very low DNA concentrations. Smith et al. [64] developed qPCR assays to determine the presence of Gambierdiscus/Fukuyoa species in environmental samples, which contains potentially toxic species. Farrell et al. [8] used DNA barcodes ITS, 5.8S rDNA and the sxtA gene specific to the saxitoxin synthesis pathway of Alexandrium minutum to detect saxitoxin-producing microalgae in shellfish. Both kinds of genes showed reliable results. Mullet roe is commonly adulterated by the addition of other species, such as escolar and oilfish. Kuo et al. [65] developed real-time PCR methods that could be used to verify the labelling of actual mullet roe products. Further tests on a random survey of commercial fish roe products demonstrated the efficacy of the technique in the detection of mullet DNA. Water, seabream Pagrus auratus and seabass Dicentrarchus labrax samples were collected from Abu Qir, Alexandria to evaluate the concentrations of dioxin. RT-PCR assays were conducted to verify the expression of certain immune genes in the fish species resulting from water pollution [66]. DNA barcoding has also been used to assess the genomic identity of the microalga species Scenedesmus sp. Barcode markers rbcL and ITS1-5.8S-ITS2 were sequenced and the obtained genomic information was used to design a quantitative PCR assay to precisely quantify the S. almeriensis concentrations in microalgal cultures of industrial interest [67]. Park et al. [68] used COI gene as DNA barcode to identify 14 species of microalgae from the South Sea of Korea, and found that species-specific PCR of the COI gene could be used to monitor the seasonal dynamics of microalgae in the South Sea of Korea.

High Resolution Melting
DNA barcoding, usually mini-barcoding, can be combined with high resolution melting (HRM) for the authentication of many commercial species from fake products such as Gadidae fish [69], Monofloral honeys [70] as well as herbal medicines from toxic species, such as Crotalaria spectabilis Roth. in Thunbergia laurifolia Lindl. [71], Armeniacae semen amarum in Persicae semen [72].

DNA Metabarcoding
As discussed above, many toxic species are associated with toxic algae and biotoxins. Marine biotoxins regularly occur along the coast, with serious consequences for the environment as well as the food industry. Monitoring of these compounds in seawater is important to assure the safety of human consumers. However, early determination of marine biotoxins in seawater to prevent seafood contamination events has not been explored [44]. By using high-throughput methods of DNA barcoding, it is feasible to develop a reliable taxonomic identification tool. The development of metabarcoding approaches was aided by the advancement of next-generation sequencing (NGS) [73]. DNA metabarcoding is a high-throughput method of taxon identification based on very short (usually <100 bp) but informative DNA fragments [74]. In this respect, metabarcoding differs from normal DNA barcoding, because classic DNA barcoding aims to identify complete genomic DNA up to species level, and metabarcoding aims to identify degraded DNA samples (eDNA) up to the family or higher levels.
Lallias et al. [75] investigated the richness of marine nematode species by high-throughput sequencing using 18S as a DNA barcode. The abundances of certain species can be used to investigate hunters' diets. Salvitti et al. [76] used DNA metabarcoding to conduct diet analysis to verify the source of TTX. Certain applications, such as the identification of gastric contents, present additional problems. Gastric contents can be analyzed by DNA barcoding for drugs and poisons [77], but can be very complex depending on the species, feeding habits, and environment. A high-throughput method would be better for analysis of such complex systems. Furthermore, DNA metabarcoding can be used in the identification of amphibians and bony fish. Evans et al. [78] conducted a DNA metabarcoding analysis using environmental DNA (eDNA) sampling to measure species diversity in aquatic. Similarly, Valentini et al. [79] used eDNA barcoding extracted from water samples to explore the rich of amphibians and bony fish. They argued that the DNA metabarcoding has the potential to become the next-generation tool for ecological studies and biodiversity monitoring in aquatic ecosystems.

Microarray
Microarray analysis represents another high-throughput method for identifying species. Microarray, also known as gene chip or biochip technology, can be used to analyze a large number of genes simultaneously. DNA barcodes can be used to design probe sequences in microarray analysis. Kochzius et al. [65] used COI probes to identify 30 fish species. Therefore, microarray-based identification methods will play a larger role in molecular species identification in the near future, especially for complex mixtures [80]. Increased use of DNA chips will help develop new methods for DNA barcoding [81].

Other Methods Used with DNA Barcoding
Besides the methods mentioned above, there are other techniques that can be combined with DNA barcoding. DNA barcoding can combine with nanotechnology. NanoTracer developed by Valentini et al. [82] simplified the analytical steps with standard DNA barcoding analysis and making it sequencing-free and portable outside specialized laboratories. The design of the specifically labeled primers involves their linkage through an antigen-antibody reaction to gold nanoparticles. Taboada et al. [83] developed species-specific lateral flow dipstick (LFD) assays for species identification in food products, in which gold nanoparticles enabled visual detection with good sensitivity even for processed samples. In addition, nanobiosensors can achieve on site, in situ and online measurements, and exhibit an unprecedented level of performance and the ability to "nano-tune" various properties to achieve the desired levels of sensitivity and detection limit. Their applications include a barcode assay for genetically modified organisms (GMO) using Surface Enhanced Raman Spectroscopy (SERS), and a mobile barcode enzymatic assay [84].
The loop-mediated isothermal amplification (LAMP) method is a highly sensitive method based on the use of a set of four specially designed primers that recognize a total of six distinct sequences of the target DNA [85]. LAMP has been used in detection of toxic and non-toxic species [86][87][88]. Su et al. [89] successfully developed the one-step RT-LAMP technique, which is a rapid and reliable method to detect HuNoV in stool samples and oysters with high sensitivity. Furthermore, LAMP methods have already been successfully developed for the detection of foodborne bacteria and fungi [85,90].
Digital PCR (dPCR) is a method that allows for absolute quantitation of nucleic acids, which has been widely used in cancer mutation studies, environmental monitoring, low-level pathogens and rare genetic sequences. dPCR is more sensitive than traditional qPCR and has been used to estimate eDNA concentration, fish abundance and biomass [91]. Multiplex digital PCR was used to co-amplify 16S rDNA and a metabolic gene from single bacterial cells [92,93]. Singapore grouper iridovirus (SGIV) is one of the major causative agents of fish diseases and has caused significant economic losses in the aquaculture industry. Droplet digital PCR (ddPCR) confirmed ribavirin, harringtonine, and 2-hydroxytetradecanoic acid (2-HOM) were effective at inhibiting SGIV infection [94]. In addition, ddPCR was also considered more suitable for the detection of Z. marina DNA from marine sediments [95].

Summary and Conclusions
Through the rapid development over the past 15 years or so, DNA barcoding has represented a well-proven molecular tool on taxonomic research. It relies on sequence variation within a short and standardized region of the genome to provide accurate species identification [13]. DNA barcoding lends aspiration to the assessment of biodiversity in a more accurate as well as inexpensive manner. Several DNA barcodes including mitochondrial COI gene, rbcL, matK, trnH-psbA, and ITS (nuclear internal transcribed spacer) have been extensively used as a global bio-identification system for detecting the alien species that invade different ecosystems [4]. Currently, the mitochondrial genes coding COI and cytb are considered reliable DNA barcodes for the identification of toxic marine species and seafood products [34,38,39,69]. Species adulteration is common for China's roasted Xue Yu fillet products. Xiong et al. [96] applied DNA and mini-DNA barcoding for the species identification of 153 roasted Xue Yu fillet products from 30 brands. Giusti et al. [97] selected cytb gene as the molecular target to identify sixteen mislabeled commercial products containing pufferfish with degraded DNA. Cytb dataset's phylogenetic analysis supported the most recent species classification of the Lagocephalus genus and highlighted the presence of toxic L. spadiceus in the products.
However, DNA barcoding has limitations. For example, species-specific universal primers or universal DNA barcodes are hard to find. Differences in evolutionary rates provide various DNA barcoding options but make it difficult to find a universal DNA barcode for all species [98,99]. Therefore, techniques of DNA barcoding for species identification are rapidly evolving as well. Traditional methods such as PCR-RFLP, PCR-SSCP and species-specific PCR are reliable but cannot meet the demands for high throughput, high speed, high sensitivity, standardization and automation. Fluorescence methods and high-throughput methods of DNA barcoding show high potential for characterizing samples to species-level. New molecular techniques such as LAMP and dPCR can be combined with DNA barcoding with high sensitivity and high speed. All these methods shall play an important role in species identification, specimen identification, biodiversity investigation, HAB forecast, detection of pathogens and seafood spoilage, and assessment of food authenticity. In particular, the use of molecular authentication methods has become one of the prospective standards to ensure food safety in the future [100].
In view of the current development, Sanger sequencing methods and fluorescence methods are rapid, cost saving but low throughput while high-throughput methods are suitable for large scale, and complex systems but are expensive and require long turn-around time. With the development of sequencing technologies, MinION-based DNA barcoding methods has been used in biodiversity research which are cost-effective and portable [35,101,102]. Furthermore, nanosensors can achieve on site, in situ and online measurements, and exhibit an unprecedented level of performance and the ability to "nano-tune" various properties to achieve the desired levels of sensitivity and detection limit. Nanobiosensors are used for the monitoring of food additives, toxins and mycotoxins, microbial contamination, food allergens, nutritional constituents, pesticides, environmental parameters, plant diseases, and genetically modified organisms [84]. We anticipate that nanotechnology will be widely used in conjunction with DNA barcodes, and standardized and automated high-throughput methods will become the mainstream of DNA barcode research in the future.