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  • Background

    • Environmental DNA (eDNA), which has become useful tool in biological survey in recent years, is particularly effective for research on rare and endangered species because it does not capture living organisms.

      Environmental DNA is also has been introduced in the investigation of Japanese eel, which was registered as an endangered species (EN) by the International Union for Conservation of Nature (IUCN) in 2014, but various things must be concerned before applying this new technique on study of Japanese eel.

      Japanese eel is migratory fish with a complicated life history. Also it has large variations in morphology and physiological condition depending on the growth stage.

    • 网页地址 图标
      Life history of Japanese eel 网页地址

    • Takeuchi et al., 2019 reported that the eDNA concentration increased as the grew and upper developmental stages, while the body weight ratio concentration decreased, in Japanese eel.

    • 网页地址 图标
      [Open Access] Takeuchi et al., 2019, Scientific Reports 网页地址

    • In addition, it is known that Japanese eel has large individual differences in size even at the same growth stage, and its behavior affected by environment (e.g. Japanese eel stop eating and rarely moving when the water temperature decreased in winter). These can cause the eDNA concentration as DNA emission to water changes.

      In this study, variations of eDNA under sequentially changing rearing conditions was investigated using the same individuals of Japanese eel.

  • Rearing experiment of Japanese eel

    • Three eels were kept in a 40-L water tank and aerated for water quality control and water circulation. 

      (To make sure that the eDNA changed by rearing condition, not by individual differences, the same individual was used throughout the rearing experiment period.)


      Eels were fasted for a month before rearing experiment started and acclimatized in a water tank with a set temperature of  15℃(measured water temperature 16-17℃).

      → Non-Feeding & Low temperature : NFL

      After water sampling, the water temperature was set to 25℃ (measured water temperature 22-23℃) while fasting, and acclimatize for 2 weeks.

      → Non-Feeding & High temperature : NFH

      After water sampling, feeding was started, the water temperature was returned to a low water temperature (measured water temperature 16-17℃), and acclimatized for 2 weeks.

      → Feeding & Low temperature : FL

      After water sampling, the water temperature was raised (measured water temperature 22-23℃) while feeding, and acclimatize for 2 weeks.

      → Feeding & High temperature : FH


    • Water sampling was carried out after two weeks of acclimatization of every experimental condition, and water was filtered immediately after sampling.

      (During the experiment period, eels were fed twice a week (Tuesday, Friday), and rearing tank was cleaned twice a week (Wednesday, Saturday) regardless feeding or non-feeding condition. On Tuesday of sampling day, eels were fed after water sampling.)

       

      Triplicate samples were taken at 50 mL, 100 mL, and 200 mL, respectively (9 samples in total), to evaluate the relationship between the amount of filtration and eDNA concentration.


  • Quantitative analysis of eDNA

    • Environmental DNA from filter cartridge was extracted and quantitative PCR was performed using specific primers.

      The expected result was that the concentration increased in the order of NFL, NFH, FL and FH as metabolic rate increased. However, it showed the highest value at FL.


    • The result that eDNA concentration under non-feeding condition was higher at high water temperature is quite predicable because the amount of secretion increased as metabolism changed by high water temperature.

      On the contrary, during feeding condition, eDNA concentration was higher at low temperature. This might be explained by feeding after the long-term fasting period causing a steep increase in eDNA emission.

      It has been reported that starvation causes an increase in nutrient carrier (peptide transporter 1) and digestive enzyme (trypsinogen) in Japanese eels (Ahn et al., 2013)

    • 网页地址 图标
      Ahn et al., 2013, Comparative Biochemistry and Physiology, Part B 网页地址


    • Generalized linear model (GLM) suggested that both “feeding” and “temperature” affected eDNA concentrations significantly.

      There was also an interaction between “feeding” and “temperature” (P < 0.001).


    • On the other hand, the volume of filtration did not affect the concentration of eDNA.

      From the data of qPCR below, the deviations among samples were large in some experimental treatments, even with the same individuals, in the same environment, and filtered in the same amount of water.


    • Unlike water temperature or salinity, eDNA in water is uneven. Therefore, it is important to take multiple samples to avoid incorrect analysis.

    • 网页地址 图标
      Uneven distribution of eDNA in water 网页地址

    • In nature, more complex factors are involved in survey of eDNA than in controlled aquariums.

      DNA released from living organisms has different degradation rates due to biological (microbial community, extracellular enzymes, etc.) and abiotic (water temperature, salinity, pH, light, oxygen, etc.) effects. The concentration also degraded by time distance from the release source (Barnes et al., 2014; Deiner & Altermatt, 2014).

      Careful approach (e.g. understanding of the physiology of target species, environmental features of their habitats, water chemistry) is needed if quantification of eDNA is to be used to estimate the biomass, especially when comparing biomass across the region or season.

    • 网页地址 图标
      Barnes et al., 2014, Environmental Science & Technology 网页地址
    • 网页地址 图标
      [Open Access] Deiner & Altermatt, 2014, PLoS ONE 网页地址

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