Molecular Biology in Tardigrades
- 583 Downloads
Molecular biology, a term first coined in the 1930s, can be viewed as a set of techniques and approaches, as well as a subdiscipline within biology. Molecular approaches have and continue to be used in nearly every area of biological study today, including genetics, biochemistry, biophysics, cell and developmental biology, physiology, and evolutionary biology. The adoption of molecular techniques to tardigrade research has helped to propel these fascinating animals from obscure biological novelties to important emerging models.
While the unique biology of tardigrades has fascinated scientists for centuries, at the most basic level, the molecular building blocks, nucleic acids, proteins, lipids, and carbohydrates that make up these animals are no different than other organisms. This makes tardigrades amenable to study using classic molecular techniques. The use and adaptation of molecular techniques for understanding the basic biology of tardigrades has risen dramatically within the past two decades, increasing the utility of tardigrades as emerging model organisms. As in other model systems, the use of molecular techniques in tardigrades often crosses over with other classically defined areas of study, such as genetics, cell biology, developmental biology, ecology, physiology, and evolutionary biology. For example, DNA extraction and sequencing in tardigrades was pioneered by researchers interested in tardigrade systematics and phylogeny (see Chap. 3 of this book). Molecular techniques have been adapted for tardigrades that allow researchers to isolate and study the building blocks listed above. This chapter presents an overview of techniques that have been adapted to and employed in the study of tardigrades and the importance of these and new techniques to the field. The details of these studies will not be discussed extensively here, because other chapters in this book highlight many of the pertinent findings. The concluding section of this chapter highlights existing molecular techniques and approaches that have yet to be employed, but show promise, for studying tardigrades.
13.2 Isolation of Biomolecules from Tardigrades
Classical molecular techniques such as the polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), and western blotting require the isolation of nucleic acids or proteins from biological samples. In addition, the extraction of lipids and carbohydrates from tardigrades is of interest to researchers, especially those studying stress tolerance (Westh and Ramløv 1991; Ingemar Jönsson et al. 2005; Hengherr et al. 2008; Jönsson and Persson 2010; Rizzo et al. 2010, 2015; Hashimoto et al. 2016; Hygum et al. 2017). Researchers have adapted a variety of methods for isolating biological macromolecules from tardigrades, including DNA, RNA, and proteins from both bulk multi-animal samples and individual specimens, to provide insight into tardigrade biology.
13.2.1 Bulk DNA and RNA Isolation
13.2.2 Extracting DNA and RNA from Single Specimens
For some applications, it is not absolutely necessary to purify large amounts of DNA or RNA, and simple lysates will suffice. Additionally, in some instances limited access to specimens or experimental constraints or design may require the use of a few or even a single specimen. Unpurified extracts from single or small pools of tardigrades can be used for simple amplification procedures such as PCR and RT-PCR. A variety of protocols have been adapted from other invertebrate systems (mostly nematodes) for this purpose (Blaxter et al. 2004; Blaxter et al. 2005; Schill 2007; Bertolani et al. 2011; Tenlen et al. 2013; Levin et al. 2016).
13.2.3 Protein Extraction
13.2.4 Lipid and Carbohydrate Extraction
Along with proteins and nucleic acids, lipids and carbohydrates have also been extracted from tardigrades. The ability to obtain high-quality lipid and carbohydrate extracts is of great importance to researchers interested in the cryptobiotic survival abilities, as changes in the content of these molecules have been linked to stress tolerance in other cryptobiotic organisms (Crowe et al. 1984; Hoekstra et al. 1989; Holmstrup et al. 2002; Erkut et al. 2011; Tapia and Koshland 2014).
Two recent studies report the extraction and quantification of fatty acid composition from tardigrades. The first study (Rizzo et al. 2010) examined changes in fatty acid composition in hydrated and desiccated tardigrades. As with protein and DNA extraction, the authors adopted an existing chloroform/methanol technique (Folch et al. 1957). The results indicate that the percent of polyunsaturated fatty acids, and specifically arachidonic acid, increases in desiccated animals relative to controls (Rizzo et al. 2010). The second study examined fatty acid composition in two different tardigrade species exposed to the stresses of space flight (Rizzo et al. 2015). The results indicate that differences in fatty acid composition exist between tardigrade species and are subject to change under stress conditions (Rizzo et al. 2015).
Carbohydrates have also been extracted from tardigrades (Westh and Ramløv 1991; Hengherr et al. 2008; Jönsson and Persson 2010). The primary focus of this work was on assessing the trehalose content of hydrated and desiccated specimens, because this disaccharide is known to or is implicated in mediating desiccation tolerance in several other anhydrobiotic organisms (Crowe et al. 1984; Erkut et al. 2011; Tapia and Koshland 2014). However, carbohydrates in addition to trehalose are suspected to play roles in stress tolerance, e.g., sucrose in plants (Hoekstra et al. 1989), and the field would benefit from more extensive surveys of carbohydrate content in tardigrades.
Combined, these studies show that tardigrades are amenable to detailed study using existing lipid and carbohydrate extraction protocols.
13.3 Tardigrades in the Omics Era
The ability to extract and purify various macromolecules from tardigrades has led to an explosion of omics studies. Researchers have sequenced the genomes, transcriptomes, and proteomes of several tardigrade species (Mali et al. 2010; Schokraie et al. 2010, 2011, 2012; Yamaguchi et al. 2012; Wang et al. 2014; Sarkies et al. 2015; Boothby et al. 2015, 2017; Smith et al. 2016; Levin et al. 2016; Koutsovoulos et al. 2016; Hashimoto et al. 2016). These studies have mainly, but not exclusively, been used to contribute to our understanding of tardigrade physiology, evolution, and phylogeny, and such omics data will continue to inform nearly every subdiscipline of tardigrade research.
It is important to remember and recognize the work of researchers using sequencing, nucleic acid, and protein techniques to study tardigrades prior to the adoption of next-generation and high-throughput approaches. Expressed sequences tags (EST) and barcoding projects gave the first glimpses of tardigrade genomic and transcript sequences, while targeted protein-based studies were used to identify, localize, and quantify levels of specific proteins (Blaxter et al. 2004, 2005; Rebecchi et al. 2009; Mali et al. 2010; Rizzo et al. 2010; Bertolani et al. 2011; Tanaka et al. 2015; Hering et al. 2016). Only the handful of tardigrade species routinely kept in laboratory culture are amenable to omics approaches requiring large nucleic acid or protein inputs. The advent of ultralow input techniques for next-generation sequencing and proteomics means that these technologies are or soon will be used, for the study of individual specimens (Hashimshony et al. 2012; Hughes et al. 2014; Levin et al. 2016).
13.3.1 Next-Generation Sequencing
This approach offers researchers access to vast amounts of data at dramatically reduced cost per base pair compared with older sequencing methods (Wetterstrand 2016). Researchers can now get high-quality data, numbering in the billions of bases, in less than 2 weeks for a few thousand dollars. Next-generation sequencing has and will continue to allow researchers to rapidly sequence complete tardigrade genomes and transcriptomes, biological replicates for comparative studies, and different conditions or developmental stages (Wang et al. 2014; Sarkies et al. 2015; Boothby et al. 2015, 2017; Levin et al. 2016; Koutsovoulos et al. 2016; Hashimoto et al. 2016).
A good example of the completeness of transcriptomes that can be generated with next-generation sequencing versus traditional EST sequencing is highlighted by two studies of the tardigrade Milnesium tardigradum (Mali et al. 2010; Wang et al. 2014). The first study (Mali et al. 2010) sequenced ~10,000 ESTs from tardigrades in active and inactive (desiccated) states, which were assembled into 3283 unique transcripts. The second study (Wang et al. 2014) used a combination of Sanger, 454, and Illumina technology to sequence RNA from hydrated, desiccating, desiccated, as well as desiccated and then rehydrated specimens, which resulted in a transcriptome assembly containing 79,064 transcripts. One method for assessing the completeness of a transcriptome or genome, beyond simply assessing the raw number of transcripts/genes assembled, is to assess the representation of core eukaryotic genes (CEGs) in the assembly (Parra et al. 2007). Comparing the percent of CEGs represented in the EST-based transcriptome (21.77%) to the next-generation transcriptome (93.55%), one can easily see the degree to which the completeness differs (Percentages were estimated by TCB for this chapter. Percentages include partial gene sequences).
Besides being able to generate large amounts of sequencing data at relatively low costs, next-generation sequencing (as well as other omics approaches) offers an unbiased means of identifying molecular mediators of different biological processes. With regard to tardigrades, this may be particularly pertinent to the study of stress tolerance. For example, the disaccharide trehalose has long been considered a key molecular player in desiccation tolerance. While this sugar is indeed essential for desiccation tolerance in some systems (Crowe et al. 1984; Erkut et al. 2011; Tapia and Koshland 2014), other anhydrobiotic organisms, such as rotifers, do not appear to make this sugar, even in response to drying (Lapinski and Tunnacliffe 2003).
Because trehalose promotes desiccation tolerance in other organisms, numerous studies have been conducted to examine levels of trehalose in both hydrated and desiccated tardigrades (Westh and Ramløv 1991; Hengherr et al. 2008; Jönsson and Persson 2010). The picture that emerges is complicated, with some studies detecting trehalose in certain species, but not in others. Even studies examining the same species differ on the presence or absence of trehalose. One point of interest is that in all cases where the sugar is detected, it is detected at low levels relative to other animals that rely on trehalose to survive desiccation (Westh and Ramløv 1991; Hengherr et al. 2008; Jönsson and Persson 2010). The question as to the functional significance of trehalose to tardigrade desiccation tolerance remains unanswered. Several omics (both next-generation sequencing and proteomics) studies have examined changes in gene expression and protein abundance in tardigrades exposed to drying. None of these reports noted increases in the expression of genes involved in trehalose synthesis (Mali et al. 2010; Wang et al. 2014; Boothby et al. 2017). Instead they note increases in fatty acid-binding proteins, cytochrome c oxidase subunit 1, protease inhibitors, as well as heat shock proteins, antioxidant enzymes, and aquaporins or the abundance of intrinsically disordered proteins (Mali et al. 2010; Schokraie et al. 2011; Yamaguchi et al. 2012; Wang et al. 2014; Boothby et al. 2017). These studies do not rule out the possibility that trehalose plays a functional role in tardigrade desiccation tolerance, but they do indicate that there are likely other important mediators of desiccation tolerance. Thus, while there is clear value in targeted studies examining the presence or role of specific genes or gene products, omics style approaches can be used for the unbiased detection of known or novel candidates for additional in-depth studies.
In addition to transcriptome studies of gene expression during stress, next-generation sequencing has been used to sequence small RNAs and detailed embryonic time course transcriptomes of H. dujardini (Sarkies et al. 2015; Levin et al. 2016) as well as multiple H. dujardini genome assemblies (Boothby et al. 2015; Koutsovoulos et al. 2016). Additionally, the genome of Ramazzottius varieornatus has been published (Hashimoto et al. 2016). Transcriptome and whole-genome assemblies provide researchers with in-depth views of which genes tardigrades have gained and lost over the course of their evolution and how the expression of these genes varies between developmental stages and under different environmental conditions.
13.3.2 Shotgun and Targeted Proteomics
The central dogma of molecular biology states that the flow of genetic information in cells goes from DNA to RNA to proteins. While various nonprotein-based molecules (e.g., ribozymes) also carryout cellular functions, proteins are predominantly viewed as the functional product of most genes. Advances in proteomics and next-generation sequencing have shed light on the fact that there are a number of different posttranscriptional and posttranslational mechanisms that influence the abundance, localization, and function of proteins within cells (Vogel and Marcotte 2012). Because of this, mRNA abundance estimates generated from next-generation RNA sequencing may not always correlate with protein abundance or activity (Taniguchi et al. 2010; Vogel and Marcotte 2012). In this light, proteomics offers an important and additional level of insight into gene expression and regulation.
Proteomic techniques have been applied to tardigrades mainly with the goal of identifying potential mediators of stress tolerance (Schokraie et al. 2010, 2011, 2012; Yamaguchi et al. 2012). These studies have highlighted the putative role of conserved heat shock proteins (Schokraie et al. 2011) as well as novel phylum-specific heat soluble proteins (Yamaguchi et al. 2012; Tanaka et al. 2015) in tardigrade stress tolerance.
13.3.3 Ultralow Input Omics Techniques
In recent years, there has been a decrease in both the cost of next-generation sequencing and other omics approaches as well as in the amount of input material (DNA, RNA, protein, etc.) required for these techniques. While early next-generation protocols called for microgram levels of nucleic acids as input, the advent of ultralow input sequencing has spawned protocols and technologies that require only picogram inputs, allowing researchers to sequence minuscule amounts of starting material, including single cells (for review: Kolodziejczyk et al. 2015; Gawad et al. 2016).
Researchers have already begun to apply such techniques to tardigrades. Recently Levin et al., performed RNAseq on libraries derived from single H. dujardini embryos spanning different developmental stages (Levin et al. 2016) using the single-cell sequencing technique, CEL-seq (Hashimshony et al. 2012). In addition to examining H. dujardini embryos, the authors looked at temporal transcriptomes from nine additional species from different phyla. They found that early and late phases of embryonic development display conserved gene expression across phyla and that these phases are bridged by a “mid-developmental transition,” when species-specific genes are upregulated. The mid-developmental transition overlaps the phylotypic period for several of the species. Thus, the authors propose the exciting possibility that phyla can be defined as groups of species whose gene expression at the mid-developmental transition diverges from other species but is conserved within the group (Levin et al. 2016).
As omics technologies utilizing ultralow DNA, RNA, and protein inputs improve, sequencing and proteomic analysis of individual tardigrades or even individual cells will become more accessible and commonplace. Much as next-generation sequencing has, for the most part, replaced EST sequencing or microarrays for expression studies, ultralow input omics promises to overtake techniques requiring large inputs, which will greatly expand the range of biological questions accessible using tardigrades.
13.4 Disrupting Gene Function
With the expansion of omics techniques in tardigrades has come the identification of vast numbers of conserved and novel genes. Additionally, the use of antibodies and in situ hybridization probes allow localization of gene products at both the protein and transcript level (Gabriel and Goldstein 2007; Smith and Jockusch 2014; Tanaka et al. 2015; Hering et al. 2016; Smith et al. 2016). While these approaches have and will continue to provide researchers with insight into the basic biology of tardigrades, these techniques can only hint at the functional role(s) and biological significance of genes and their products.
To gain insight into gene function, researchers often turn to either forward or reverse genetic approaches. Forward approaches consist of using natural or induced mutations to identify the genetic basis of a phenotype. To date, no forward screens have been conducted in tardigrades. However, the availability of tardigrade sequencing data and the amenability of these animals to (re)sequencing and cryopreservation make forward genetic screens a possible and likely productive approach (Gabriel et al. 2007).
While forward genetic screens are employed in many fields of biology, the development of reverse genetic approaches makes targeted studies of gene function possible. As opposed to forward genetic approaches, which examine a phenotype to uncover the underlying genetic basis of a mutation, reverse genetic approaches target specific known genes to examine the phenotypic effect of perturbing that gene’s function. Reverse genetic techniques include directed point mutations, deletions, or insertions in an organism’s genome or the disruption of a gene’s function through the targeted destruction or silencing of gene products using techniques such as RNA interference (RNAi), morpholinos, or through ectopic expression of a gene.
To date the only reverse genetic technique employed in tardigrades is RNAi (Tenlen et al. 2013). RNAi was first demonstrated by Fire and Mellow who showed that double-stranded RNA injected into the nematode worm, Caenorhabditis elegans, resulted in the potent and specific destruction, or “knockdown,” of targeted transcripts, thus interfering with the function of a specific gene (Fire et al. 1998). In tardigrades, as in other organisms, RNAi works by introducing double-stranded RNA (dsRNA) into cells, where endogenous RNAi machinery processes the dsRNA and ultimately uses the processed dsRNA to destroy complimentary transcripts within the cell (for review: Hannon 2002). Some organisms, including tardigrades, display a systemic RNAi response, where dsRNA is transported into and between cells, facilitating the spread of the RNAi effect to additional cells or the whole organism (Winston et al. 2002; Shannon et al. 2008; Tenlen et al. 2013).
Using dsRNA injected into the gut of H. dujardini specimens, Tenlen et al. (2013) demonstrated that RNAi works in tardigrades and is specific, since targeting different genes produced different phenotypes and resulted in reduced levels of target but not control transcripts (Tenlen et al. 2013). RNAi has also been used to assess the functional significance of specific genes in tardigrade stress tolerance (Boothby et al. 2017).
Once one has high-quality dsRNA, the next step is to introduce this dsRNA into the tardigrade. Currently the only published method for introducing dsRNA in tardigrades is microinjection, but other methods such as soaking, feeding, chemical transfection, or electroporation maybe be more efficient. Using microinjection, Tenlen et al. (2013) found that introduction of dsRNA into the gut of the tardigrade H. dujardini leads to the spread of the RNAi effect to embryos, suggesting that tardigrades, like C. elegans, have a systemic RNAi response. While microinjection is by no means a high-throughput technique, a proficient injectionist can inject ~10–12 tardigrades in an hour. The physical disruption of injection has little or no effect on the tardigrade survival (Tenlen et al. 2013).
Care should be taken to insure that phenotypes resulting from RNAi are specific. Introducing large amounts of dsRNA into cells could have toxic or unanticipated off-target effects that might be confused with specific effects of knocking down a target transcript (Jackson et al. 2003; Grimm et al. 2006). To control non-specific toxicity, a control dsRNA targeting a non-tardigrade or nonessential gene should be used at the same concentration as experimental dsRNA to insure effects are not the result of dsRNA toxicity.
The sequence of the dsRNA should be carefully chosen to reduce the risk of knocking down transcripts with similar sequences. In addition, it is optimal to test at least two non-overlapping dsRNAs targeting the same transcript. Knockdowns using non-overlapping dsRNAs should produce similar phenotypes at similar concentrations. If they do not, one or both of the dsRNAs could be targeting unintended transcripts, increasing or decreasing the severity and variety of phenotypes.
Since RNAi does not knockout or remove a gene, its effects are both concentration and time dependent. Enough time should be given to allow RNAi to take effect before phenotypes are scored. Likewise, the RNAi effect will dissipate with time, so phenotypes should be assessed before the dsRNA is completely degraded and the animals recover their initial levels of endogenous transcripts. Factors influencing the timing and duration of RNAi include the amount of endogenous transcript, the range of endogenous transcript level needed to maintain the normal phenotype, the concentration of dsRNA introduced, as well as the redundancy of the target transcript with transcripts from other genes.
The efficacy of an RNAi experiment depends on the level of knockdown. This level will likely vary by target transcript and will be subject to the concentration of dsRNA used and effectiveness of introduction (injection). The efficacy of a knockdown can be assessed by examining relative levels of target RNA and/or protein in experimental and control animals. Performing RT-PCR to assess relative levels of the target transcript can assess efficacy. Primers for RT-PCR should be chosen such that they do not amplify a region of the transcript that is also contained within the dsRNA, or else amplification of both the transcript and the dsRNA could make detection of a knockdown difficult or impossible. Other methods such as qRT-PCR or northern blotting could also be used to quantify levels of RNA after treatment. Assessment of RNAi efficiency can also be carried-out by examining relative levels of protein encoded by the target RNA using western blotting or similar approaches.
One final note is that RNAi can produce different levels of knockdown within an experimental group of animals resulting in different phenotypes. It is therefore important to establish a good scoring system for reporting both the effects of RNAi and the variability of phenotypes.
To date, RNAi remains the only reverse genetic approach adapted for use in tardigrades. Despite its limitations, RNAi provides researchers with an established method for assessing the function of specific genes in tardigrades. Coupled with the vast amount of new transcriptomic and genomic data, this technique promises to substantially increase the utility of tardigrades as model systems.
13.5 Future Perspectives
Tardigrade research has seen the adaptation of a number of molecular techniques that have facilitated research in many areas of study including evolutionary biology, developmental and cell biology, physiology, genetics, taxonomy, and ecology, positioning tardigrades as a valuable emerging model system.
With advances in sequencing technology has come a plethora of information, including whole genomes from multiple species with thousands of genes homologous to those of other organisms as well as novel, tardigrade-specific genes. Researchers will be tasked with uncovering the function of these genes in tardigrades. To this end, both forward and reverse genetics hold significant promise.
Given that one can obtain sufficient material to perform next-generation sequencing and resequencing from a single tardigrade, forward genetics could be an efficient method for generating stable lines with known mutations. Forward genetics have been used to great effect in other model systems, such as D. melanogaster, C. elegans, and S. cerevisiae, and the applicability of forward screens for studying the biology of tardigrades has been previously suggested (Gabriel et al. 2007). Given that many tardigrade species can remain viable when frozen, as well as their relatively short generation times, producing and maintaining stocks of mutant lines appear to be a technical possibility (Gabriel et al. 2007).
An alternate to forward genetics for generating mutant lines is to perform targeted genome editing. This involves making a specific genetic deletion, insertion, or point mutation in the genome of germline cells, so that the mutation is passed on to subsequent generations. A variety of methods have been established in other systems toward this end, such as clustered regulatory interspaced short palindromic repeats (CRISPR)-, zinc finger nucleases (ZFNs)-, and transcription-activator like-effector nucleases (TALENs)-based approaches (Miller et al. 2007; Urnov et al. 2010; Mussolino et al. 2011; Sander and Joung 2014). The recent explosion in the use of CRISPR/Cas technology highlights the efficiency, reprogramability, and lower overall cost of this technique. CRISPR/Cas strategies allow the insertion or deletion of essentially any sequence from or into any portion of a genome, a significant advantage over random insertional methods and has been used in a wide variety of organisms, including fungi, plants, and both vertebrate and invertebrate animals (Doudna and Charpentier 2014). Given the relative ease, widespread applicability, and power of CRISPR/Cas genome editing technology, attempts to adapt this technique for use in tardigrades should be made.
A key point in tardigrades regarding the development of CRISPR/Cas genome editing is the need to make heritable changes to the genome of germ cells. In tardigrades, the cuticle surrounding eggs is extremely hard, making microinjection of embryos difficult (Boothby, personal observation). In C. elegans, injections for CRISPR genome editing can be performed by injecting into the germline of worms (Dickinson et al. 2013), which is syncytial. It is not clear whether the germline of tardigrades is also syncytial, but if it is, microinjection or electroporation may be an effective means of getting the necessary reagents into the germline and ultimately oocytes, before eggshell deposition.
Through the hard work and dedication of researchers worldwide, the use and adaptation of molecular approaches to tardigrades have increased dramatically within the past few decades. These techniques have and will continue to promote the use of tardigrades as important model systems and provide novel avenues for studying the basic biology of these fascinating animals.
- Bertolani R, Rebecchi L, Giovannini I, Cesari M (2011) DNA barcoding and integrative taxonomy of Macrobiotus hufelandi CAS Schultze 1834, the first tardigrade species to be described, and some related species. Zootaxa 2997:e36Google Scholar
- Nayak S Single worm PCR. http://genetics.wustl.edu/tslab/protocols/genomic-stuff/single-worm-pcr/. Accessed 12 May 2016
- Schokraie E, Hotz-Wagenblatt A, Warnken U et al (2011) Investigating heat shock proteins of tardigrades in active versus anhydrobiotic state using shotgun proteomics: investigating heat shock proteins of tardigrades. J Zool Syst Evol Res 49:111–119. https://doi.org/10.1111/j.1439-0469.2010.00608.x CrossRefGoogle Scholar
- Wetterstrand KA (2016) DNA sequencing costs: data from the NHGRI Genome Sequencing Program (GSP). www.genome.gov/sequencingcosts. Accessed 23 May 2016