Fish stock surveys using genetic methods

Background and Objective

Sustainable marine fisheries require a regular assessment of the state of fish stocks. This assessment is based on various data bases, including the evaluation of commercial and scientific catches, as well as biological observations.

Disadvantages of this traditional methodology include high costs and complex logistics. In addition there is the scarce data availability in space and time, and the often associated lack of accuracy of stock estimates and other key parameters for sustainable stock management.

DNA high-throughput sequencing based on Next Generation Sequencing (NGS)  methods now offer the opportunity to complement traditional methods or even to replace parts of it to support fisheries management in the long term. Environmental DNA analysis (eDNA analysis) uses the genetic traces left by organisms in the environment, e.g. in seawater. It is a promising, non-invasive method that can significantly improve the spatial and temporal monitoring of aquatic ecosystems. Although several studies have already shown that this approach is effective for the inventory of fish in different environments, some important methodological challenges need to be addressed before it can be applied in fisheries research.

Approach

Together with the University of the Balearic Islands (Spain), the Thünen-Institute is researching eDNA analysis. Within the framework of an International Bottom Trawl Survey (IBTS) on board the research vessel Walther Herwig III, water and sediment samples will be taken. DNA will be extracted in the laboratory and analysed qualitatively and quantitatively. After bioinformatic and statistical evaluation, the data obtained will be compared with the catches obtained by trawling in the same areas from which the water and sediment samples originate. In addition to a pure assessment of fish diversity, a quantitative approach will be tested to find out whether the frequency data of the fish correlate with the eDNA levels, using cod (Gadus morhua) as the test species.

In addition, different tissue samples of cod and hake (Merluccius merluccius) will be taken and DNA will be extracted to allow our project partners to test further genetic methods. The results of all project partners will be summarised in standard protocols and the entire data collection process will finally be subjected to a cost-benefit analysis in order to assess the potential of the genetic methods to support fisheries management.

Data and Methods

By simply taking water or sediment samples, followed by DNA extraction and DNA analysis, the presence or absence of species in a particular area can be detected. This is usually done using metabarcoding, in which certain gene regions are first amplified using polymerase chain reaction (PCR) and then sequenced using NGS. The determined DNA sequences can be assigned to individual fish species by suitable bioinformatic processing. Using quantitative PCR methods (real-time PCR), statements can also be made about the biomass or abundance of fish species.

In addition to eDNA analysis, Close-Kin-Mark-Recapture-Analysis (CKMR) is another promising method for fish population estimation. Through the analysis of parent-offspring relationships, the CKMR methodology can theoretically answer three crucial parameters for fisheries management: the absolute abundance of fish within a population, their fertility and their survival rates over time.

Epigenetic age determination methods (EAD) offer a further alternative to traditional techniques in fisheries research, in this case classical age determination by annual ring counting of earstones (otoliths). The DNA methylation (DNAm) of vertebrates is used as a promising alternative biomarker for age. In fish, such studies have so far only been carried out on zebrafish as a model species, where some genes have shown a gradual and significant methylation loss with age. For wild fish, there is so far only limited DNAm age data. Their potential use as biomarkers for ageing needs to be evaluated.

Our Research Questions

Our focus is on questions concerning eDNA analysis. We want to find answers to the following questions: 

Which fish species can be detected by eDNA analysis? 

We will use the metabarcoding approach to determine the biodiversity of the fish and compare the results with catch data from the respective trawls. 

Is it possible to derive fish abundance quantitatively or at least semi-quantitatively from eDNA data?

We will use quantitative real-time PCR methods to correlate the amount of DNA in the water sample with the frequency of cod in the sample area.

orrelate DNA copy numbers in the water sample with the frequency of cod (Gadus morhua) in the respective sample area.

Results

As part of the project, various methods for extracting eDNA from sediments and seawater were compared and optimized.

When recording the overall biodiversity of fish, the metabarcoding analysis of eDNA from water samples revealed a larger species spectrum than the trawling carried out in parallel. Due to the lack of numerous fish barcodes in international reference databases, not all sequences found could be clearly assigned to the respective species.

In addition, a cod (Gadus morhua) specific qPCR assay for abundance estimates was developed. In the first tests, cod could be detected at very low densities of less than 1 kg / ha with a high specificity of up to 100% at the same time.

The application of this test to water samples from the North Sea (voyage WH428) enabled the detection of cod eDNA in all those stations where it also appeared in the accompanying trawl catches. In addition, there was a surprisingly high correlation (98%) between the number of detected eDNA copies and the catch per unit of effort (CPUE) in the trawls.

The results of the study suggest that eDNA-based methods are well suited to substantially complement conventional, invasive methods in marine biodiversity research and possibly even to replace them for specific research questions in the future.