Post provided by TILL CZYPIONKA, DANIEL GOEDBLOED, ARNE NOLTE and LEON BLAUSTEIN

Ecological Transcriptomics and Endangered Species

Friday was Endangered Species Day – so this is a good time to reflect on what science and scientists can do to support conservation efforts and to reduce the rate of species extinctions. One obvious answer is that we need to study endangered species to understand their habitat requirements as well as their potential for acclimatization and adaptation to changing environmental conditions. This information is crucial to for the design of informed conservation planning. However, for most endangered species the relevant phenotypes are not known a priori, which leaves the well-intentioned scientist asking “which traits should I measure?”. Transcriptome analysis is often a good way to answer to this question.

Transcriptome analysis measures the expression levels of thousands of genes in parallel. This amount of data circumvents the need to decide on a reduced number of traits of unknown relevance and allows for a relatively unbiased phenotypic screen of many traits. In particular, physiological changes, which often influence a species’ distributional range, can be studied using transcriptome analysis. Also, transcriptomics provide a direct connection to the genetic level. This is essential for in-depth analyses of aspects of evolution and might even be helpful for a new kind of conservation planning, which aims to foster endangered species by promoting (supposedly) beneficial hybridization. The integration of transcriptomic analysis with ecological studies is known as ‘Ecological transcriptomics’.

The Tiny Problem of Extracting RNA

Given the great potential and promises of ecological transcriptomics, there was a certain excitement in the room, when we (PIs and postdoctoral entourage of an Israeli-German research consortium) met for the first time after receiving a generous grant to study adaptation and acclimation to larval habitats in the European and Near Eastern Fire salamanders. Plans were made, study populations were selected, sampling strategies agreed upon. But eventually we realised that there was a problem.

Transcriptome analysis requires the extraction of RNA from the study organism. For smaller individuals, RNA is often extracted from the entire body. For larger animals, specific tissues (very often liver) are used. Obtaining these samples typically requires sacrificing animals, though. We wanted to avoid lethal sampling as the Near Eastern Fire salamander is endangered and because we wanted to comply with the general idea of avoiding the sacrifice of animals where possible.

Also, salamander larvae sometimes inhabit very small rockpools, with correspondingly few individuals. A sampling approach that involves killing numbers of larvae that are desirable from an experimental design standpoint could mean the end of a whole cohort in rockpool population, which would lead us to a situation like the old doctor’s joke “the operation was successful, but the patient died”.  For both ethical and practical reasons we needed to a better way to perform ecological transcriptomics on salamander larvae.

A Better Way to Perform Transcriptomics on Endangered Species

Our goal was to find a non-lethal method to sample RNA for transcriptome analysis. Non-lethal sampling of different tissues (e.g. tail-fin clips, blood, feathers and fur etc.) for DNA extraction is a common technique used for genetic analysis of many study organisms. Many of these tissues also contain RNA and could be used for transcriptome analysis. A recent study had shown that the sampling of a small piece of tail-tip did not cause lasting negative consequences for the Near Eastern fire salamander larvae, so we hoped that we could use this tissue for transcriptomics. However, the situation for transcriptome analysis differs from analyses at the DNA level, as gene expression can be highly dynamic and is often tissue-specific.

While transcriptome studies based on non-lethally sampled tissues have provided interesting results, there is the possibility that relevant gene expression changes do not manifest in peripheral tissues. Would non-lethal sampling of larval tail-fin tips allow us to infer transcriptome changes of relevance for the whole larvae? This is something you want to know before starting a large-scale ecological transcriptomics project. So, we tested it.

We raised larvae of European fire salamander at two different temperatures. The acclimatisation response was assessed at the transcriptome level based on (i) RNA extracted from tail-tips and (ii) RNA extracted from the entire body (minus of course the tail tip) of the same individuals. While there were large differences in gene expression between the two samples, expression in both the tail-fin tip and the entire body seemed equally responsive in the number of genes changing expression in response to temperature. Many of the responsive genes showed identical expression changes in both tissues. Most importantly, the functional analysis of the transcriptomic changes associated with temperature led to largely identical conclusions regarding the larvae’s acclimatization responses. Just what we were hoping for! We found a compassionate, conservation-friendly way to carry out meaningful transcriptome analysis of an endangered species without killing individuals.

Ecological Transcriptomics in Action

The use of tail-tip samples has now paved the way for ecological transcriptomics in European and Near Eastern fire salamander on a large scale.  We initiated a monitoring study where transcriptome patterns are associated with different larval habitats (e.g. lotic vs. lenthic, temporary vs. permanent) in the fire salamanders in Germany and in Israel. In terms of workflow, this is not so different from a typical population survey, where scientists collect animals to measure parameters like weight or length before releasing them back to nature. In addition to that, you just sample a tiny piece of the tail that the larva will regrow without any lasting damage. Some additional lab work then provides access to the expression levels of thousands genes specific to sampled larvae in its natural habitat.

We are using this strategy, to identify habitat specific expression patterns and to infer acclimatization responses to the different habitats at the functional level. Having the transcriptome data allows us to study how the use of larval habitat evolved between European and Middle Eastern fire salamanders that diverged approximately 4 million years ago.

For us, this non-lethal technique has proven to be a very useful tool to study ecological transcriptomics without compromising ethical concerns or endangering populations. We hope that our approach stimulates more research in natural populations and contributes to the acquisition of much needed data to understand details about the biology of threatened species.

To find out more about ecological transcriptomics, read the Methods in Ecology and Evolution article ‘Ecological transcriptomics – a non-lethal sampling approach for endangered fire salamanders’.

This article is part of our Virtual Issue on Endangered Species. All articles in this Virtual Issue are freely available for a limited time.