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魚類環境DNAの性質および動態に関する研究

Jo, Toshiaki 神戸大学

2021.03.25

概要

DNA molecules are released into and present in every terrestrial and aquatic environment, which is termed environmental DNA (eDNA). The analysis of eDNA has recently been developed to estimate the current distribution and abundance of macro-organisms such as fish and amphibians, and its non-invasiveness, cost-efficiency, and high detection sensitivity has been reported for various taxa and in natural environments. However, there are some challenges in biological monitoring via eDNA analysis; first, although eDNA analysis has a higher detection sensitivity than traditional methods, the detection is not necessarily perfect. Second, despite the positive correlation between eDNA concentration and species abundance, it is highly challenging to establish the method quantifying species biomass/abundance via eDNA analysis with high level of accuracy and reliability. Third, the range of spatiotemporal eDNA signal at a given sampling location and time cannot be fully understood. These uncertainties relating to eDNA detection and quantification is a big question for all eDNA researchers, and should be solved for the establishment of eDNA analysis as a more refined tool to monitor biodiversity and fishery resources.

These uncertainties can ultimately originate from the lack of information on the characteristics and dynamics of eDNA; (a) characteristics: physiological and ecological sources of eDNA production, and its physiochemical and molecular states, and (b) dynamics: the processes of eDNA production, transport, and persistence, and environmental biotic/abiotic factors affecting such eDNA dynamics. The characteristics of eDNA can multifacetedly influence its vertical/horizontal transport and persistence, which eventually determines the spatiotemporal scale of eDNA signal. Therefore, understanding the characteristics of eDNA can assist to understand the dynamics of eDNA, which will refine the knowledge on spatiotemporal scale of eDNA signal, improve the performance of eDNA analysis, and fill a gap between eDNA detection/quantification and species presence/abundance in the field.

The aim of my doctoral thesis is to comprehensively refine the relationship between the characteristics and dynamics of eDNA from macro-organisms, and to obtain the clue to mitigate and eliminate the uncertainties relating to eDNA detection and quantification. First, in Chapters 2 to 4, by performing tank experiments using Japanese jack mackerel (Trachurus japonicus) as a model species,1 comprehensively analyzed the effects of biotic/abiotic and molecular factors on eDNA shedding and degradation. Especially, with regard to a molecular factor, I focused on the characteristics and dynamics of eDNA derived from mitochondria and nuclei (mt-eDNA and nu-eDNA, respectively). Most eDNA studies have targeted mt-eDNA, while some have examined the applicability of nu-eDNA, targeting multi-copy ribosomal RNA (rRNA) gene, and reported its higher detection sensitivity and potential usefulness in eDNA analyses. I compared eDNA shedding and degradation rates, its particle size distributions, and the effects of various factors on them between mt- and nu-eDNA. The results showed that (i) eDNA decay rates were generally higher in nuclear than mitochondrial DNA, (ii) the ratio of mitochondrial to nuclear eDNA shedding rates were lower in larger fish body sizes and older fish individuals, and (iii) degradation of mt- and nu-eDNA was suppressed in smaller size fractions, which might be owing to its inflow from larger to smaller size fractions.

Second, in Chapter 5,1 analyzed the effect of DNA fragment size on eDNA degradation and quantification. DNA fragment size (PCR amplification length) is known to negatively correlate with detected DNA copy number/detection rate in fecal samples. Given it, eDNA degradation can be caused by the decrease in DNA fragment length owing to base cutting and deletion. I thus conducted a tank experiment using Japanese jack mackerels, verifying a hypothesis that eDNA with longer DNA fragment degrades faster. In addition, if the hypothesis is true, detection of longer DNA fragment in environmental samples might indicate more recent biological information, despite lower copy number, contrary to that of shorter DNA fragment studied in most eDNA studies (<200 bp). Therefore, I compared correlations between fish biomass based on echo intensity and eDNA concentration between different eDNA fragment sizes, and showed that (i) eDNA was degraded faster in longer DNA fragments than shorter ones and (ii) eDNA concentrations were correlated more clearly with fish biomass in longer DNA fragments.

Third, in Chapters 6 and 7,1 integrated the understanding of eDNA characteristics and dynamics obtained above. In the former chapter, I tested the applicability to selectively collect the eDNA with specific particle size. Aqueous eDNA can exist in water with various sizes and states. Among them, relative to extra-cellular DNA, intra-cellular DNA such as cell and tissue fragments can mainly be detected at larger size fractions, and may be protected from enzymatic DNA degradation processes. I investigated the relationship between filter pore size and DNA fragment size, and verified whether selective collection of such large-sized eDNA increased the collection efficiency of longer DNA fragments from water samples. Moreover, in the latter chapter (Chapter 7), I conducted meta-analyses targeting previous eDNA studies to assess how the factors relating to eDNA characteristics such as filter pore size, DNA fragment size, and target genetic region influenced the persistence and degradation of aqueous eDNA. The results of meta-analyses showed the multiple and complex interactions between filter pore size, target gene, water temperature, and water source on first-order eDNA decay rate constants.

Throughout the thesis, I studied the characteristics and dynamics of eDNA released from macro-organisms, unveiled *the ecology of eDNA* based on complex interactions between eDNA characteristics and dynamics, and provided the perspectives for the innovation of eDNA analysis based on these eDNA basic information.

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