Welcome to the second phase of our investigations into the dynamics of eDNA release rate by Proteus!
Since the results of the two analyses carried out in 2017 and 2018 were promising, but inconclusive (for more details check out my previous lab notes), we took a step backward and turned to the captive animals at the Tular Cave Laboratory. This is a privately owned subterranean laboratory located in a natural cave under a beautiful town nested in the Slovenian Alps, where the olms and other smaller subterranean organisms have been studied in a semi-natural environment since 1960 (http://www.tular.si/index.php/tular). The results of these experiments are summarized on the poster reproduced below and were presented to the expert community at the 4th SOS Proteus meeting held in Trieste on May 21 and 22 2022 (at the time of writing this note): https://www.sosproteus2022.news/home. A more detailed report, which will include all the methodological particulars and statistical information will be published, as has been the standard practice, sometime after the conference in the journal of conference proceedings.
![](https://d3t9s8cdqyboc5.cloudfront.net/images?path=61182/y0E2PIiTyOwfkQa88wwE_eDNA poster SOS 2022.jpg&width=650&height=)
So let’s take a look.
First we sampled water from two large permanent pools housing white Proteus animals to determine how differences in sample volume, filter pore size, and animal density affect observed eDNA concentrations. After preliminary results (additional sampling parallels have yet to be analyzed) the observed eDNA concentrations in standing water most prominently reflected filter pore sizes but did not correlate with animal densities. It was not possible to determine if this was the result of a lower release- or higher decomposition rate in the smaller, more densely populated pool, or simply a methodological artefact. The good news is, the isolated eDNA stored at -80ºC appeared to be very stable and no losses were detected after over a year and a half of storage.
![](https://d3t9s8cdqyboc5.cloudfront.net/images?path=61182/5tG4YDo3T7GxrjgQ2Fny_IMG_20191207_164554675.jpg&width=650&height=)
Next, we transferred two animals separately into two plastic barrels filled with fresh water, and subjected them to a stream of water simulating water current velocities measured during our field work in different segments of the Pivka channel in the Postojna-Planina Cave System. The most promising finding of the running water simulation was that a single animal in 100 L of water released detectable quantities of eDNA in less than a minute of sampling time. On the down side, the observed eDNA concentrations were very prone to DNA extraction losses.
![](https://d3t9s8cdqyboc5.cloudfront.net/images?path=61182/wk3qE47vSL2MGFAglJyq_IMG_20191207_155138741.jpg&width=650&height=)
So the obvious next step is to develop and introduce two DNA standards. This will increase the cost of the method considerably, but without them it is simply impossible to reliably monitor Proteus population sizes using this indirect method. First, a known quantity of an appropriate DNA standard must be added to the samples prior to filtration to monitor filtration and extraction error. Second, a method to detect and quantify a stable reference (microbial) DNA inherently present in any given natural site populated with Proteus will have to be developed to control for fluctuations in Proteus eDNA that would be due to rainfall and other extraneous factors. This way, we hope to be able to finally develop a method that we can use at least for monitoring relative changes in eDNA release rate of Proteus in selected, most critically imperilled locations.
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