1. Introduction
Seafood generally refers to a diverse range of aquatic organisms utilized for food and has been one of the major traded food commodities in the world [
1]. Globally, there is a growing demand for seafood since human consumption continues to increase annually by 3.1% [
2]. Over the past few decades, a rapid growth in aquaculture production has certainly enabled a substantial increase in the supply of seafood and is projected to outnumber capture fisheries production by 2030 [
3]. However, there is still a considerable amount of seafood from capture fisheries [
2,
3]. Furthermore, the species diversity of seafood from capture fisheries could be more complex due to the nature of aquatic biodiversity.
Seafood identification has increasingly received attention since seafood mislabeling has been reported, raising public awareness of the safety and sustainability of seafood [
4]. Moreover, seafood mislabeling could lead to seafood fraud when a low commercial value seafood is intentionally labeled as a high commercial value one. In general, mislabeling of seafood could be attributable to the inaccurate identity and origin of aquatic organisms used for seafood [
5]. Misidentification of seafood species is perhaps one of the most common problem for seafood mislabeling. To address this issue, it requires accurate and reliable methods for the identification of seafood species.
Conventionally, morphological characters are routinely used for the taxonomical identification of seafood species [
6]. While visual discrimination of seafood species is certainly simple and cheap, it also requires experts with well-trained experience. Moreover, visual discrimination of seafood species could be difficult or impossible since morphological characters are often removed, altered, or destroyed following the process, storage, and transport of seafood [
6]. Alternatively, DNA-based methods have proven to be effective for accurate seafood species identification that have been frequently used for the authentication of seafood products [
7,
8,
9,
10,
11,
12]. However, conventional DNA-based approach often requires DNA samples from seafood products, and it could be challenging when specimens are precious and/or required to keep alive. Moreover, a large scale of DNA-based seafood species identification also requires a considerable number of DNA samples. In this case, it could be laborious and expensive for species identification using conventional DNA-based approach.
Environmental DNA (eDNA) is generally considered as DNA present in a variety of environmental samples, including soil, air, or water [
13,
14]. Recently, eDNA-based sampling has emerged as a promising tool to monitor the presence of species within an environment. With the aid of high throughput sequencing technology, eDNA metabarcoding has further allowed the simultaneous identification of multiple species present in environments [
15,
16]. Hence, eDNA sampling has been extensively applied to explore the biodiversity, distribution, and habitat of aquatic organisms [
15,
17,
18,
19,
20].
Taiwan is a relatively small island (36,197 km
2) with rich marine biodiversity and resources since it is geographically located in the Western Indo-Pacific region, a hotspot of marine biodiversity [
21]. More than 3000 finfish species have been recorded in the water of Taiwan, accounting for 9% of the fish species in the world [
22]. Coastal fisheries in Taiwan are highly active with more than 20 fishery sectors in this region [
23]. Consequently, seafood has been one of major food resources in Taiwan. However, seafood in Taiwan, especially in fish markets and seafood restaurants, is rarely sourced from a fixed species and routinely prepared with similar species depending on their availability. Moreover, the oversimple and inconsistent names of seafood also raise the concerns on the exact identities of seafood. Hence, the species diversity of seafood could be potentially complicated in Taiwan.
In the present study, we aim to determine whether it is feasible to identify fish species potentially consumed in fish markets and seafood restaurants using eDNA metabarcoding. Unlike the conventional DNA-based approach, we have collected and analyzed eDNA samples in a local fish market and nearby seafood restaurants to provide a reference list of fish species potentially consumed, particularly those threatened, commercially important, nutrition valued species. Our findings are also expected to provide a comprehensive understanding of the species diversity in seafood for the conservation and management of marine resources.
4. Discussion
In the present study, we have demonstrated a novel approach for a large scale of seafood authentication using eDNA metabarcoding. Compared to the conventional DNA-based approach, it does not require a considerable number of DNA samples from seafood visible and accessible in the fish markets and/or seafood restaurants. Furthermore, it could also allow the simultaneous detection of multiple species. Despite the disruption of COVID19 pandemic for this work, we have still identified 153 fish species based on a limited number of eDNA samplings in a local fish market and its nearby seafood restaurants. Thus, we have shown the first evidence that it is feasible to provide a more comprehensive understanding of fish species potentially consumed in fish markets and restaurants using this novel approach.
The number of fish species identified from eDNA samplings in the present study were found to vary with different molecular markers. A total of 153 species were identified from eDNA samples in the present study, 112 species were identified by 12S and the rest of fish species were identified using COI and ND5. Indeed, 12S have proved to be the effective molecular marker for the detection of fish species from eDNA samples [
25,
34] and have been extensively utilized for the study of fish biodiversity using eDNA metabarcoding [
36,
37,
38,
39]. However, some studies also have shown the limitation of 12S for the detection of fish species from eDNA samples [
40,
41,
42]. To maximize the number of fish species detected from eDNA samples in the present study, we have selected 19 universal primers targeted for three mitochondrial gene fragments (12S, ND5, COI). Additionally, the amplicons of these gene fragments from eDNA samples were further processed with relatively higher sequencing depth (6,995,762–11,021,896 reads per sample) in the present study. Inappropriate sequencing depth of eDNA samples might fail to detect those species with low abundance in the environments [
43].
The number of fish species were also found to vary with sampling dates and sites in the present study. The differences in the number of fish species between different sampling dates might be explained by the availability of fish species since fishing seasons varied with fish species in Taiwan [
23]. As for the differences in the number of fish species between two sampling sites in the present study might be explained by the nature of eDNA samples sourced from fish market and seafood restaurants. In the fish market, most fish species were still alive and/or unprocessed whereas most fish species in seafood restaurants are highly processed. The concentration of eDNA decays over time and its degradation rate also varies with different environmental conditions, including salinity, temperature, and pH [
44].
Accumulatively, Chondrichthyan, Anguilliformes, and Serranidae species accounted for approximately one third of fish species identified from the fish market and its nearby seafood restaurants in the present study. These species are routinely used for the traditional seafood in Taiwan, including smoked sharks, braised moray eels and grouper soups (
Figure 2F–H). Chondrichthyan species (sharks, rays, and chimeras) have continuously received much attention due to their declined abundance [
45] and vulnerable life history traits (e.g., slow growth rates and long generation time) [
46]. DNA barcoding analysis of Chondrichthyan species have revealed approximately 20 to 24 species involved in the seafood consumption in Taiwan [
10,
47]. In the present study, we have identified a total of 22 chondrichthyan species based on eDNA samplings from a limited area compared to previous studies in Taiwan [
10,
47]. It also worth mentioning that 17 species are now considered as IUCN threatened species. Anguilliformes are a group of eel-like shape species comprising 13 families and 206 species in Taiwan [
48]. In the present study, 14 species were identified in the present study. Of these species, three species are rarely found, and one species is endemic species of Taiwan. These findings also highlight the potential use of eDNA metabarcoding for the conservation and managements of Anguilliformes in the future studies. Serranidae species, especially groupers, are globally popular seafood species with relatively higher economical value and many of them have been supplied by the aquaculture [
49]. In the present study, 12 Serranidae species were identified, including seven wild and five aquaculture species. Notably, there is a huge difference in the market price between the wild and farmed groupers in Taiwan. However, these species are often simply named as the grouper in the fish markets and seafood restaurants in Taiwan. Our findings further imply the Serranidae species from both capture fisheries and aquaculture were routinely consumed in the fish markets and seafood restaurants in Taiwan.
Over the past decades, the global marine biodiversity is declining rapidly due to climate change, habitat deconstruction, and overfishing [
50]. Seafood consumption also plays a crucial role in the maintenance of marine biodiversity. Our findings have unveiled the underestimation of species diversity in seafood of Taiwan, especially those routinely consumed in fish markets and seafood restaurants. However, further improvements and optimizations of eDNA metabarcoding in fish markets and seafood restaurants would be required for the future studies, including the increase of sampling times and sites, the development and employment of molecular markers effective for the other seafood species (e.g., cephalopods, decapod crustaceans) to provide more comprehensive understandings of species diversity in seafood routinely consumed in fish markets and seafood restaurants.