Two species were used for assessing the persistence of detectable amounts of DNA. Bullfrog tadpoles were studied to validate the possibility of integrating the approach of Ficetola et al. [5] in the species eradication strategy. Due to the risk of invasion and/or pathogen transmission to native populations (e.g. Batrachochytrium dendrobatidis[12] and Ranavirus [13] (link)) bullfrog tadpoles cannot be used outdoors, and the experiments were performed in aquariums (as in Ficetola et al. [5] ). The siberian sturgeon was used as DNA source species to test field conditions and placed in artificial ponds created about 20 years ago on the University campus, where this species has never been present.
For the bullfrog experiment, 3 different densities of tadpoles were used. One, 5 and 10 tadpoles were reared in 900 mL glass beakers for 5 days and each density was replicated 5 times. A 900 mL glass beaker without tadpoles was used as control. At the fifth day, the tadpoles were removed. At this time and every 24 h during 20 days, 15 mL of water were sampled from each glass beaker. Room temperature was maintained constant throughout the experimental period and the water temperature measured in the glass beakers was 17±1°C.
For the sturgeon experiment, three ponds of dimensions 12 m2 and 0.40 m deep were used. In each pond, a sturgeon (20 cm long) was housed for 10 days (from November the 04th to 13th 2009). On the tenth day, the sturgeons were removed and 15 mL of water were sampled from each pond. Water samples were collected every 24 h during 14 days. Water temperature fluctuated from 8 to 11°C during this period.
The duration of each experiment was determined after a preliminary test on the same condition without replication. In both experiments, water samples were added to a solution composed of 1.5 mL of sodium acetate 3 M, and 33 mL absolute ethanol immediately after collection, and then stored at −20°C until the DNA extraction.
For the bullfrog experiment, 3 different densities of tadpoles were used. One, 5 and 10 tadpoles were reared in 900 mL glass beakers for 5 days and each density was replicated 5 times. A 900 mL glass beaker without tadpoles was used as control. At the fifth day, the tadpoles were removed. At this time and every 24 h during 20 days, 15 mL of water were sampled from each glass beaker. Room temperature was maintained constant throughout the experimental period and the water temperature measured in the glass beakers was 17±1°C.
For the sturgeon experiment, three ponds of dimensions 12 m2 and 0.40 m deep were used. In each pond, a sturgeon (20 cm long) was housed for 10 days (from November the 04th to 13th 2009). On the tenth day, the sturgeons were removed and 15 mL of water were sampled from each pond. Water samples were collected every 24 h during 14 days. Water temperature fluctuated from 8 to 11°C during this period.
The duration of each experiment was determined after a preliminary test on the same condition without replication. In both experiments, water samples were added to a solution composed of 1.5 mL of sodium acetate 3 M, and 33 mL absolute ethanol immediately after collection, and then stored at −20°C until the DNA extraction.
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