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.
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
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)
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.