A molecular survey based on eDNA to assess the presence of a clown featherback (Chitala ornata) in a confined environment

Introduction

The clown featherback (Chitala ornata, Notopteridae) is a freshwater fish, native to tropical Asia, mainly found in the Mekong and Chao Phraya basins of Indochina and Thailand (Roberts, 1992) and Laos (Baird et al., 1999). Large individuals of this range of variety are often seen for sale for consumption at local markets. The clown featherback also features prominently in the aquarium trade. Fish is the primary source of animal protein for most people in Thailand. In 2016, fish consumption was 33.73 kg/capita/year, the highest consumed protein among all other animal protein sources such as pork, beef, and chicken. The clown featherback is popularly consumed and is one of the economically important fish in Thailand which, it is often used in food products such as fish balls and fish cakes (Vidthayanon, 2012; Uk-katawewat, 2004; Department of Fisheries, 2017).

Thai fisheries are characterized by their diversity of species and habitats, as well as their diverse inland fisheries. The importance of the inland fisheries sector in food security is that it provides much-needed protein and is an income supplier. This has been highlighted especially in poor rural communities of developing countries. (Oopatham, 2003). Inland capture fisheries in Thailand are carried out nationwide in rivers, lakes, swamps and reservoirs. These fisheries are an important sector of many local economies and are considered to be important in sustaining the livelihoods of many rural communities. Kwan Phayao or Phayao Lake is one of major lakes for inland fisheries in Thailand. In a total area of 19.8 km², around 50 fish species (17 families) were reported in the Phayao Lake (Rattanadaeng, Panboon & Soe-been, 2015). Along with other fish species, the clown featherback is one of the main fish of inland fisheries at Phayao Lake. The Inland Aquaculture Research and Development Division (IARDD), Thailand routinely carries out surveys of fish species in the Phayao Lake. A combination of netting and electrofishing is the current method used to monitor fish species. The main limitations of this method are the varying efficiency of its tools (netting or electrofishing), incomplete samplings, not to mention the fact that it is time-consuming. Seasonal activity or behavior has had an influence on detection probabilities in traditional sampling approaches to measure the abundance or presence/absence of organisms (MacKenzie et al., 2002; Canessa et al., 2012). The survey team of IARDD reported that fish species are caught during different months of the year and their sampling sizes are different (Inland Fisheries Research and Development Division, 2019). Notable, none of them can be found in some years of the surveys based on traditional approaches despite the fact that locals reported their presence in the lake. Traditional samplings are labor intensive and limited in their reliability. The efficiency of each technique depends on the targeted species size and developmental stage (Jackson & Harvey, 1997; Lintermans, 2015) and different fishing tools may introduce different degrees of reliability in fish surveys (e.g., Porreca et al., 2013; Tate et al., 2003; Hanchin, Willis & St. Sauver, 2002). Thus, a more sensitive and reliable survey method is needed.

Advanced molecular techniques facilitate the estimating and monitoring of biodiversity, especially the increasing application of environmental DNA (eDNA). eDNA can be defined as short DNA fragments that organisms leave behind in environments (i.e., water, soil, or air). It holds great promise for monitoring both invasive and imperiled species and has proven to be a sensitive, effective and convenient method with increased speed. Recently the use of eDNA was proved to be a powerful tool in biodiversity science and conservation action, especially for fish species. For example, eDNA was used to evaluate the reintroduction program and to monitor the reintroduced fish species (e.g., Hempel et al., 2020; Riaz et al., 2020), to track distribution of endangered fish or those in difficult to access habitats (e.g., Laramie, Pilliod & Goldberg, 2015; Itakura et al., 2019), to gain more understanding about various ecological aspects such as breeding season, spawning activity, reproductive migration, and habitat (e.g., Takeuchi et al., 2019; Sakata et al., 2017; Antognazza et al., 2019; Thalinger et al., 2019), to detect invasive species (e.g., Nevers et al., 2018; Balasingham et al., 2018; Bellemain et al., 2016; Boothroyd et al., 2016) and design management strategies (e.g., Levi et al., 2019; Lacoursière-Roussel et al., 2016). Here, we developed an eDNA-based method for detecting the clown featherback in Phayao Lake as an alternative tool fore more effective, sensitive and less time-consuming survey which could be useful for fishery management.

Materials & Methods

eDNA field collection

Water samples were collected at six points: within Payao Lake in September 2018; at the end of the rainy season; in February 2019; the middle of the dry season; and again at the beginning of rainy season in June 2019 (Fig. 1). Water samples were collected from the surface water of the lake. Each site was sampled in triplicate and 300 mL samples of water were collected and filtered on GF/F filter (0.7 µm Whatman International Ltd., Maidstone, UK). For every sampling day, deionized water (300 mL) was filtrated as a negative control. The water samples and real-time PCR were processed as described above. Statistical analyzes (Factorial ANOVA) were performed with R (R Core Team, 2017). Boxplots were produced to visualize the data using the ‘ggplot2’ package (Wickham, 2016).

Results

Here, we designed primers that amplify short target regions which is 183 bp specific the clown featherback (C. ornata). To determine specificity, the designed primers were tested in silico. The specificity of the generated primers was checked against the NCBI database by comparing of similarity of primers to Chitala and related species. The homology of Chitala sequences to the specific forward and reverse primers of C. ornata is 82.6%–87.0% with at least five mismatches on the forward and four mismatches on the reverse primers (Table 2). When compared with other fish species commonly found in Phayao Lake, the homology was found to be lower (69.6%–78.3%). In addition, all the qPCR tests for the primers’ specificity of DNA extracted from other fish and water collected from the tank with mix fish species (without the clown featherback) were negative.

Sensitivity of the qPCR assay is expressed as the limit of detection (LOD) with 95% of confidence and the limit of quantification (LOQ) with a threshold of ≤ 35%. The qPCR assay of the clown featherback had a LOD = 9.8 copies per µL and LOQ = 9.8 copies per µL. We also used an aquarium experiment to test the extent to which the qPCR of water samples can detect the clown featherback. We were able to detect the eDNA of the clown featherback from water samples collected at time 0, 3, 6, 12, 24, 48, 72, 96, 120, 144, and 168 h, after removing the fishes. Thus, the eDNA qPCR assay resulted in positive DNA signals in 100% of the aquarium experiment even 7 days after the removal of the fishes from the tanks.

Previous traditional surveys of fish in Phayao Lake failed to recognise the presence of the clown featherback in some seasons and years even though reported by locals who had found and caught them from the lake.. We collected water samples from six sites at the same locations where the teams from the Inland Fisheries Research and Development Division, Department of Fisheries conducted the traditional surveys (Fig. 1). Three different sampling months (February, June and September) were compared. The eDNA of the clown featherback were detected in water samples from all collecting sites in Phayao Lake of all three sampling months (Table 3 and Fig. 2). The efficiency for the qPCR assay was 91% (y =  −3.549x + 40.397; R² = 0.98). These regression equations were used to convert the quantification cycle (Cq) data from the qPCR product (i.e., the PCR cycle at which the target is considered positively amplified in a given sample) to the concentration of DNA in a given sample (copies of DNA per unit volume).

Concentration of the clown featherback found in Prayao Lake ranges from around 0.05 –0.94 copies/mL in February, 0.06–0.50 copies/mL in June, and 0.08–0.92 copies/mL in September with no difference over sampling month (P = 0.42). In contrast, we found a significant difference (p = 0.005) in measured eDNA concentrations between the collecting sites, an average eDNA concentration of 0.23 copies/mL at Kwan1 (range: 0.05–0.50), 0.18 copies/mL at Kwan2 (range: 0.05–0.55), 0.22 copies/mL at Kwan3 (range: 0.06–0.75), 0.25 copies/mL at Kwan4 (range: 0.06–0.58), 0.51 copies/mL at Kwan5 (range: 0.25–0.93), and 0.40 copies/mL at Kwan6 (range: 0.09–0.94). The findings show that eDNA based survey is one of a few powerful tools for monitoring and surveying the clown featherback at any time of the year.

Discussion

Overexploitation of a fishery may not simply be marked by species being undetected during sampling in which may resulting from insensitivity and unreliable of traditional survey methods. Recently, several studies have already successfully shown eDNA sampling can be more sensitive and required lower sampling effort than traditional approaches using physical capture in several instances (e.g., Jerde et al., 2016; Takahara, Minamoto & Doi, 2013; Laramie, Pilliod & Goldberg, 2015; Smart et al., 2015; Bista et al., 2017; Hinlo et al., 2017; Evans et al., 2017). eDNA method holds great potential of overcoming many limitations of traditional sampling methods, although there are some challenges. Breeding and migratory seasons are factors that influence eDNA detectability and should be considered in eDNA surveys (Spear et al., 2015; Barnes & Turner, 2016; Buxton et al., 2017). However, we did not find seasonal dynamics in eDNA concentration of the clown featherback in this study. The average eDNA copies detected from all collecting sites did not significantly different over the sampling months. On the other hand, eDNA signals of each sampling site (Kwan1-Kwan6) were found to be different. The highest average eDNA copies of the clown featherback were found at Kwan5. Similarly, Kwan5 is the sampling station where fish abundance is the highest in Phayao Lake (Rattanadaeng, Panboon & Soe-been, 2015). The variation of eDNA detection between sampling sites within the lake could be due to an uneven (patchy) distribution of organisms which is commonly found in the lentic system, thus leading to higher concentrations of eDNA in areas where organisms were present (Takahara et al., 2012; Eichmiller, Bajer & Sorensen, 2014).

In this study, although, experimentation on the relationship between organismal density and eDNA concentration was not carried out, there are several previous studies which indicated positive correlations between biomass or abundance and eDNA quantities (e.g., Takahara et al., 2012; Maruyama et al., 2015; Lacoursière-Roussel, Rosabal & Bernatchez, 2019; Itakura et al., 2019). Also, eDNA detection probability was observed to increase with specimen density (Stoeckle et al., 2017).

In addition, we cannot rule out that environmental conditions such as temperature, light and pH impact eDNA concentration of target species (Review in Stewart, 2019; Harrison, Sunday & Rogers, 2019).

Conclusions

Recently, the use of environmental DNA (eDNA) has proved to be a powerful tool for fish survey or sampling. eDNA surveying is likely to have a major impact on the ability of species detection. The ability to rapidly and sensitively detect the presence of a target species through eDNA analysis has enabled a wide range of scientific discoveries and technological advancements. To the best of our knowledge, this is the first report of eDNA detection of the clown featherback in Thailand. Taken together, these findings suggest the eDNA method is an effective tool for targeted species detection to complement traditional sampling/survey approaches and is one of molecular methods which useful for assessment the presence of a fish species in a confined environment such as a lake.

Supplemental Information