AN INVESTIGATION OF SUNLIGHT STRESS RESPONSE GENES IN THE INTERTIDAL SEA ANEMONE, ACTINIA TENEBROSA
Jonathon Noel Muller Bachelor of Applied Science, Environmental Science (Honours)
Submitted in fulfilment of the requirements for the degree of
Master of Applied Science (Research)
School of Earth, Environment and Biological Sciences
Science and Engineering Faculty
Queensland University of Technology
2016
Keywords
Actinia tenebrosa, actiniarian, antioxidant, cellular stress response, cnidarian, DNA repair, intertidal, sunlight, UV, UV screening, sea anemone
Images and photographs
All photographs, diagrams, illustrations and other images in this thesis are the intellectual property of the author Jonathon Muller 2016, unless explicitly stated. Abstract
Organisms living in the intertidal zone are subjected to the impacts of climate change, and understanding their response to environmental conditions is becoming increasingly important. Direct exposure to sunlight can be hazardous to many organisms that inhabit the intertidal zone, and it is likely to interact synergistically with desiccation stress. Some intertidal sea anemone species (Phylum Cnidaria) withstand periodic sunlight exposure at low tide, yet lack the physical barriers and/or stratified epithelium that allow other sedentary intertidal organisms to survive in this environment. Sea anemones that inhabit intertidal areas may possess unique adaptations to withstand significant sunlight exposure, and the genetic basis of their stress response needs to be investigated. Sea anemones are important components of rocky intertidal ecosystems along Australian coastlines, yet virtually nothing is known about their response to sunlight stress or susceptibility to changing environmental conditions. The Waratah anemone, Actinia tenebrosa, is an excellent indicator species for understanding stress tolerances in intertidal sea anemones and may provide important insights into how other species will respond to a changing environment.
In chapter two, the sequenced (~7 Gbp) transcriptome of the sea anemone,
Actinia tenebrosa, was assembled and annotated, and genes involved in the UV response identified. From this data set, full-length sunlight stress response genes were identified and compared across existing sequenced cnidarian genomes.
Comparative and phylogenetic analysis of candidate UV stress response genes (i.e. blue chromoprotein, CPD photolyase and RAD23b) and one novel bioluminescent gene (photoprotein) were conducted across the cnidarians. Eight hundred and sixty two UV response genes were identified in A. tenebrosa and 179 were determined to have full length open reading frames. These 179 full length genes were found to be widely distributed across cnidarian species. This represents the first detailed investigation of the bioluminescent-associated photoprotein gene family in cnidarians. Both chromoprotein and photoprotein occurred as gene families in cnidarians, while CPD photolyase and UV excision repair RAD23 were present as single copy genes.
In chapter three, a detailed investigation of four candidate sunlight stress genes from A. tenebrosa was conducted, specifically, blue chromoprotein (UV and visible light screening/non-enzymatic antioxidant), CPD photolyase (DNA repair), and Cu-
Zn SOD and Fe/Mn SOD (enzymatic antioxidants) at the northern limit of its distribution, where temperature and UV are at their highest. Actinia tenebrosa individuals were subjected to four sunlight stress treatments (submerged dark, submerged light, exposed dark and exposed light) and qPCR was used to determine patterns of sunlight stress induced gene expression in the four candidate genes.
Comparative and phylogenetic analysis of these candidate UV stress response genes was then conducted across the sea anemones. CPD photolyase and Fe/Mn SOD expression significantly increased over time when submerged under sunlight, but not when exposed under sunlight, and that UV and photosynthetically active radiation
(PAR) were the best environmental predictors for the increase. It may be concluded that desiccation stress may invoke a different response, and that the up-regulation of
CPD photolyase and Fe/Mn SOD, and perhaps other sunlight stress response genes, may not be as important when the organism is aerially exposed. In addition, the first detailed comparison of the candidate gene families in sea anemones was conducted and two new full-length Fe/Mn SODs gene families identified. This thesis has provided many usefuls insights into the genes involved in A. tenebrosas sunlight stress resilience and provides a resource to better understand the molecular mechanisms that enable A. tenebrosa to survive periodic UV stress. The investigation of candidate stress genes in A. tenebrosa provides a first look at how an intertidal sea anemone tolerates sunlight stress, which can be used to better understand how other intertidal sea anemone species will respond to changing climactic conditions.
Table of Contents
Keywords ...... 2 Table of Contents ...... 6 List of Figures ...... 8 List of Tables ...... 10 Statement of Original Authorship ...... 11 Acknowledgements ...... 12 Chapter 1: Literature review ...... 1 Overview ...... 2 Introduction ...... 2 Sunlight stress ...... 2 UV and visible light screening ...... 6 DNA damage repair...... 10 Direct DNA damage repair ...... 10 Indirect DNA damage repair ...... 13 Antioxidants ...... 14 Enzymatic antioxidants ...... 14 Non-enzymatic antioxidants...... 15 Molecular chaperones ...... 17 Conclusions ...... 19 Project aims ...... 22 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species ...... 25 Abstract ...... 26 Introduction ...... 27 Materials and methods...... 30 Sample collection, RNA extraction and sequencing ...... 30 Data processing and assembly ...... 31 Comparative and phylogenetic analysis ...... 31 Results ...... 34 Sequencing, data processing and assembly ...... 34 Comparative and phylogenetic analysis of candidate genes ...... 34 Discussion ...... 43 Conclusions ...... 50 Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa ...... 53 Abstract ...... 54 Introduction ...... 55 Materials and methods ...... 58 Sample collection, stress experiment and RNA extraction ...... 58 Results ...... 64 The effects of sunlight stress on gene expression ...... 66 Comparative and phylogenetic comparison ...... 70 Discussion ...... 74 Physiological and gene expression response to sunlight stress ...... 74 Comparative and phylogenetic analysis of candidate genes in actiniarians ...... 77 Conclusion ...... 77 Chapter 4: General discussion ...... 79 Summary of results ...... 80 Potential research gaps and future direction ...... 81 Implications...... 84 Conclusion ...... 86 References ...... 87 Box 1. List of abbreviations and acronyms ...... 100 List of Figures
Figure 1.1 (A) Basic metazoan phylogeny shown in black with Phylum Cnidaria highlighted in purple. (B) Phylum Cnidaria with subclass Hexacorallia highlighted in blue, (C) Subclass Hexacorallia with order Actiniaria highlighted in green, (D) Order Actiniaria. Cladograms adapted from phylogenetic reconstructions by (Kayal et al. 2013; Rodríguez et al. 2014; Zapata et al. 2015)...... 5 Figure 1.2 (A) Side view of A. tenebrosa fully submerged, with tentacles expanded; (B) top view of A. tenebrosa fully exposed during low tide, with tentacles retracted; (C) A. tenebrosa distribution (adapted from Loh (2011))...... 21 Figure 2.1 Conceptual overview of select antioxidant, UV and visible light screening and DNA repair mechanisms and their possible associated candidate genes in the intertidal sea anemone, Actinia tenebrosa...... 30 Figure 2.2 Gene Ontology (GO) terms involved in UV response (left) and response to stimulus (right) from the A. tenebrosa transcriptome...... 35 Figure 2.3 (A) Cladogram showing the distribution of the A. tenebrosa transcriptome and the genomes of the three model cnidarian genomes, Nematostella vectensis, Acropora digitifera and Hydra magnipapilatta (B) Venn diagram illustrating how the 156 identified A. tenebrosa UV stress genes are shared amongst the three model cnidarian genomes. Cladogram adapted from Zapata et al. (2015)...... 35 Figure 2.4 Phylogenetic tree of blue chromoprotein gene from A.tenebrosa (shown in bold) and its homologues in other cnidarian taxa, with the bilaterian Pontella meadi as the outgroup using maximum-likelihood inference. Five independent lineages of cnidarian chromoproteins are highlighted. All genes shared the same domain structure as the canonical GFP domain from Aequoria victoria, shown in the top left hand corner...... 39 Figure 2.5 Location of blue colouration on: (A) C. polypus oral disk, (B) A. tenebrosa acrorhagi, (C) A. tenebrosa pedal disk...... 40 Figure 2.6 Phylogenetic tree of photoprotein-like gene from A.tenebrosa (shown in bold) and its homologues in other cnidarian taxa, with the ctenophore, Mnemiopsis leidyi as the outgroup using maximum- likelihood inference. Non-highlighted genes shared the same domain structure as the canonical aequorin domain from Aequoria victoria, shown in the top left hand corner. Highlighted genes show domain structures that differ from aequorin...... 41 Figure 3.1 (A) Aerial photograph of study site with 10m2 sampling grid highlighted in red; (B) field site facing east; (C) sun exposed treatment sample; (D) 50L housing tank...... 59 Figure 3.2 Comparison of volume change within different treatment types (i.e. control, submerged and dark, submerged and light, exposed and dark, exposed and light) over time. Error bars represent standard error...... 65 Figure 3.3 Comparison of blue chromoprotein gene expression ratio within different treatment types (i.e. control, submerged and dark, submerged and light, exposed and dark, exposed and light) over time. Error bars represent standard error...... 67 Figure 3.4 Comparison of CPD photolyase gene expression ratio within different treatment types (i.e. control, submerged and dark, submerged and light, exposed and dark, exposed and light) over time. Error bars represent standard error...... 68 Figure 3.5 Comparison of Cu-Zn SOD gene expression ratio within different treatment types (i.e. control, submerged and dark, submerged and light, exposed and dark, exposed and light) over time. Error bars represent standard error...... 69 Figure 3.6 Comparison of Fe/Mn SOD gene expression ratio within different treatment types (i.e. control, submerged and dark, submerged and light, exposed and dark, exposed and light) over time. Error bars represent standard error...... 70 Figure 3.7 Phylogenetic tree of Fe/Mn SOD gene from A.tenebrosa (shown in bold) and its homologues in other actiniarian taxa, with the scleractinian Acropora digitifera as the outgroup using maximum- likelihood inference...... 73 Figure 3.8 Phylogenetic tree of Cu-Zn SOD gene from A.tenebrosa (shown in bold) and its homologues in other actiniarian taxa, with the scleractinian Acropora digitifera as the outgroup using maximum- likelihood inference. Three independent lineages of actiniarian Cu- Zn SOD are highlighted...... 74
List of Tables
Table 2.1 Summary of full-length candidate sequences from the A. tenebrosa transcriptome ...... 36 Table 3.2 Results from an ANOVA conducted to assess the significance of time on volume change within each treatment type...... 65 Table 3.3 Results from an ANOVA conducted to assess the significance of time on candidate gene expression ratio within each treatment type...... 66 Statement of Original Authorship
The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other higher education institution. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made.
Signature: QUT Verified Signature
Date: August 2016 Acknowledgements
I wish to express my sincere thanks to my supervisor Dr Peter Prentis and my co-supervisors Dr Ana Pavasovic and Associate Professor Stephen Cameron for their guidance, suggestions, assistance and patience during the study period. I am grateful for the support I have received from all members of the PGL lab group, in particular
Shorash Amin for his help with various aspects of the Actinia tenebrosa transcriptome assembly and quality control, Joachim Surm and Hayden Smith for their help with RNA extractions and the sunlight stress experiment, Chloe van der
Berg for the transcriptome assemblies of Anthopleura buddemeieri, Aulactinia veratra, Calliactis polypus, Nemanthus sp. and Telmatactis sp., and Mathilde Klein for her help with qPCR.
I would like to thank all the technical support staff at EEBS, in particular, Will
Stearman, Linda Nothdurft and Brett Lewis for their help in the construction and maintenance of the marine housing tanks. My gratitude and thanks to Associate
Professor Jennifer Firn for letting me borrow her light meter and Dr Luke Nothdurft for lending me the underwater camera, which I used to take all the photos in this thesis.
Finally, my heartfelt thanks go to my family for always being there. To my beautiful wife Geunyoung, and my daughter Juni, thank you so much for your love, support and inspiration.
Chapter 1: Literature review
Chapter 1: Literature review 1
Overview
This review of the literature provides an introduction and background information on the effects of sunlight stress on intertidal cnidarians. In order to better understand sunlight stress resilience, the themes of UV and visible light screening,
DNA repair, antioxidants and molecular chaperones in cnidarians are be addressed.
Important knowledge gaps in these themes are highlighted, and additional studies identified. This review then focuses on the intertidal cnidarian, Actinia tenebrosa, as an appropriate system for examining gene expression in response to sunlight stress.
Introduction
How organisms tolerate environmental stress requires understanding the genetic basis of their stress response. Intertidal cnidarians, such as corals and sea anemones, are regularly exposed to elevated levels of sunlight (i.e. UV, visible and
IR wavelengths) when exposed at low tide. Sedentary intertidal anemones are remarkable as they survive long periods of direct sunlight at low tide despite having only a single layer of epithelial cells and no other anatomical features to provide physical protection. This research project examines the genes underlying sunlight stress tolerance through gene expression profiling of mRNA in a sessile intertidal cnidarian, the Waratah Anemone Actinia tenebrosa. Actinia tenebrosa is abundant and widely distributed along the Australian coastline, and understanding the genes involved in sunlight resilience of this species provides important information about the stress-response of these organisms.
Sunlight stress
Sunlight has been a ubiquitous challenge since life began around 3.5 billion years ago (Noffke et al. 2013). Electromagnetic radiation from the sun consists of
2 Chapter 1: Literature review
three wavelength categories: ultraviolet (UV), visible and infrared (IR), and the cellular damage caused by sunlight is due to their combined effects (Cadet et al.
1997; König et al. 1995; Pflaum et al. 1998). UV wavelengths range between 100 and 400 nanometres (nm), and are generally divided into three categories, UVA (λ =
400-315nm), UVB (λ = 315-280nm) and UVC (λ = 280-100nm). UVA and UVB, however, are the only wavelengths relevant to cellular damage as UVC is completely absorbed by stratospheric ozone (Parisi and Kimlin 1997). UVA and UVB are associated with photo-carcinogenesis, as these lower wavelengths can penetrate into living cells and tissues and damage molecules (World Health Organization 1995, 7-
19). The high energy of these lower wavelengths can break apart the chemical bonds holding cellular molecules together and lead to a wide range of deleterious effects such as mutagenesis and oxidative stress (Harm 1980). In comparison to UV, the effects of visible wavelengths to cells are poorly understood (Chiarelli-Neto et al.
2014; Kvam and Tyrrell 1997). Visible wavelengths range between 390 and 700 nm and constitutes the portion of the spectrum visible to the human eye. Visible light can be measured as photosynthetically active radiation (PAR) (400 – 700 nm) (Anderson
1964) and although considered the least harmful of the three major wavelength categories, extensive exposure to visible light has been shown to cause cellular damage by melanin photosensitization and oxidative stress (Chiarelli-Neto et al.
2014; Kvam and Tyrrell 1997). Infrared wavelengths range between 700nm to 1 mm, and referred to as “heat radiation” (Howell, Menguc and Siegel 2010). Although all wavelengths are converted into heat energy on contact with a surface, IR creates 49% of all heat on earth, with the remainder from absorbed visible light that is re-emitted at longer wavelengths (Howell, Menguc and Siegel 2010). Heat stress can directly and indirectly affect living organisms through the disruption of proteins and by
Chapter 1: Literature review 3
inducing oxidative stress (Grether‐Beck et al. 2014; Cho et al. 2009). It is important to consider all wavelengths when discussing the harmful effects sunlight stress has on living organisms.
Sunlight stress may be deleterious to animals (metazoans) dwelling in shallow marine environments such as the intertidal zone, where changes in the water column throughout tidal cycles can result in high levels of exposure. Intertidal animals have evolved various mechanisms to mitigate the effects of sunlight stress. Motile intertidal metazoans for example are able to avoid prolonged exposure by changing habitats, whereas more sedentary metazoans are usually equipped with protective barriers such as shells or carapaces. Intertidal sea anemones (order Actiniaria) lack these adaptations, yet are able to withstand prolonged exposure to sunlight stress at low tides. Like all cnidarians (jellyfish, hydroids, box jellyfish, sea anemones and corals, see Figure 1.1), sea anemones are diploblastic animals characterised by two layers of simple epithelium separated by an extracellular mesoglea (Fox 2001). This single layer of epithelial cells provides a poor physiological barrier to sunlight stress, and intertidal sea anemones must therefore cope with sunlight stress with physiological adaptations that include changing shape to reduce surface area and holding water to avoid desiccation (Ottaway 1973, 1978; Stotz 1979), and by secreting mucus (Banaszak 2003). The physiological adaptations of intertidal sea anemones work concurrently with the cellular stress response (CSR), a universal molecular mechanism largely conserved across metazoan lineages (Kültz 2005).
4 Chapter 1: Literature review
Figure 1.1 (A) Basic metazoan phylogeny shown in black with Phylum Cnidaria highlighted in purple. (B) Phylum Cnidaria with subclass Hexacorallia highlighted in blue, (C) Subclass Hexacorallia with order Actiniaria highlighted in green, (D) Order Actiniaria. Cladograms adapted from phylogenetic reconstructions by (Kayal et al. 2013; Rodríguez et al. 2014; Zapata et al. 2015).
The main function of the CSR is to protect macromolecules from damage caused by changes in extracellular conditions, and encompasses an extensive range of molecular changes. The most basic aspects of the CSR are conserved across many species, as many types of environmental stress are fundamental to all living organisms (Kültz 2003). Information about the stress response from one species may provide useful insights and be applied to a variety of other species, despite evolutionary distance. Understanding the cellular response to sunlight stress in intertidal sea anemones is therefore important, as it may hold important information about how these species will respond to global climate change.
To better understand the cellular basis for intertidal sea anemone sunlight stress resilience, this review explores four major components of the CSR relevant to
Chapter 1: Literature review 5
sunlight stress: UV and visible light screening, antioxidants, DNA repair and molecular chaperones. A focus of this review is to identify the functional products involved in these processes, and where available, their underlying gene expression.
UV and visible light screening
Photo-protective compounds found in the mucus and tissues of cnidarians are considered to play important functional roles in UV and visible light screening. Well studied examples in cnidarians include the diet-acquired mycosporine-like amino acids (MAAs) (Drollet, Glaziou and Martin 1993; Drollet et al. 1997; Cubillos et al.
2014; Hudson and Ferrier 2008), and melanin pigments (Palmer, Bythell and Willis
2010), both of which function to absorb and scatter incoming UV wavelengths.
There is also evidence to suggest that members of the green fluorescent protein
(GFP) family may play a role in cnidarian UV and visible light screening (Salih et al.
2006; Salih et al. 2000), however, information that summarizes how these proteins function in this role is lacking and very little is known about their distribution in sea anemones. In order to address this knowledge gap, this section of the literature has therefore focused on the UV and visible light screening role of cnidarian GFPs.
Fluorescent pigments
Fluorescent and non-fluorescent members of the GFP family are highly abundant in sun exposed anthozoans, being found in up to 97% of all Great Barrier
Reef corals (Salih et al. 2000). They are thought to be effective antioxidants, and provide a photoprotective function in cnidarians that compliments that of the MAAs by protecting against the effects of the longer wavelengths of UVA and visible blue light.
6 Chapter 1: Literature review
GFP-like proteins are a family of homologous 25–30 kDa polypeptides that constitute as much as 14% of total protein in some anthozoans (Leutenegger,
D'Angelo, et al. 2007), and are regarded as an important source of anthozoan colouration (Dove, Hoegh-Guldberg and Ranganathan 2001). The first fluorescent protein to be characterized was the green fluorescent protein (GFP), which, along with the calcium-activated photoprotein, aequorin, was first isolated from the bioluminescent jellyfish Aequorea victoria (Shimomura, Johnson and Saiga 1962).
GFP-like proteins have since been found in a variety of bioluminescent and nonluminescent cnidarians (Haddock, Mastroianni and Christianson 2010). All GFP- like proteins share a distinctive beta-can fold, and consist of eleven beta-strands that form an exterior beta-barrel shape, with a visible wavelength chromophore attached to an alpha-helix that runs through the centre of the barrel. The chromophore is formed from a sequence of 3 amino acids within the GFP sequence, and requires no accessory cofactors or substrates other than oxygen (Heim, Prasher and Tsien 1994).
The diverse range of colours and fluorescence is determined by the chromophores composition and neighboring amino acids within the chromophores microenvironment (Pakhomov and Martynov 2008). All GFPs share domain homology, and are encoded by a single gene (Hastings 1996).
GFPs can be divided into two major groups: the fluorescent proteins (FPs), and the non-fluorescent chromoproteins (CPs) (Alieva et al. 2008; D’Angelo et al. 2008;
Dove, Hoegh-Guldberg and Ranganathan 2001; Salih et al. 2006). Some fluorescent proteins are visible in ambient sunlight, whereas others are not (Matz et al. 1999;
Labas et al. 2002; Mazel et al. 2003). Naturally occurring anthozoan FPs can be cyan, green, yellow or red in colouration, and cover an excitation maxima ranging from 395-570 nm, emission maxima ranging from 420-620nm, and molar extinction
Chapter 1: Literature review 7
coefficient ranging from 27,000 – 126,000 (Alieva et al. 2008; Dove, Hoegh-
Guldberg and Ranganathan 2001; Kawaguti 1969; Labas et al. 2002; Matz et al.
1999; Mazel 1995). Fluorescent proteins have been found to occur in a wide variety of anthozoan cells and tissues, such as photocytes (Titushin et al. 2008), pigment granules (Schlichter, Fricke and Weber 1986; Salih et al. 2000), epithelial cells
(Kawaguti 1944; Logan, Halcrow and Tomascik 1990; Mazel 1995; Salih et al.
2000) tentacle tips and oral disks (Matz et al. 1999; Salih, Hoegh-Guldberg and Cox
1998), and occasionally, endodermal tissue (Schlichter et al. 1985; Salih et al. 1998).
In comparison to the FPs, CPs are essentially non-fluorescent and differ from
FPs by amino acids at only three positions in the peptide sequence (Lukyanov et al.
2000; Gurskaya et al. 2001a; Bulina et al. 2002; Chudakov et al. 2003). In contrast to
FPs, CPs are highly visible in ambient sunlight (Dove, Hoegh-Guldberg and
Ranganathan 2001; Gurskaya et al. 2001). CPs can be pink, purple or blue in colour
(Dove, Hoegh-Guldberg and Ranganathan 2001). CPs have greater absorption compared to the FPs (i.e. higher extinction coefficients), cover an excitation maxima ranging from 395-590 nm, and have no emission, but have a molar extinction coefficient ranging from 61,000 – 205,200 (Salih et al. 2006; Alieva et al. 2008).
Like anthozoan FPs, CPs are usually confined to extremities of the organism. For example in corals, this includes the branch, tentacle tips, or pedal disk in anemones
(Takabayashi and Hoegh-Guldberg 1995; Shkrob et al. 2005). A great diversity of fluorescent and non-fluorescent GFP homologs have been discovered within cnidarians but the functional differences of FPs and CPs in cnidarians is not yet fully understood.
It has been observed that corals exposed to higher amounts of sunlight exhibit greater increases in fluorescence and fluorescent protein concentrations than corals
8 Chapter 1: Literature review
exposed to lower amounts of sunlight, leading to the assumption that this protein family may be involved in UV stress response (Salih et al. 2006; Salih et al. 2000;
Takabayashi and Hoegh-Guldberg 1995; D’Angelo et al. 2008). It has been suggested that fluorescent proteins may serve a photoprotective function in cnidarians by light scattering and through radiant fluorescence energy transfer from shorter to longer wavelengths (Aranda et al. 2011; Bay et al. 2009; Leutenegger,
D'Angelo, et al. 2007; Roth et al. 2010; Salih et al. 2000). For example, many FPs extracted from shallow water coral reef species (acroporids, pocilloporids, poritiids and faviids) had peak excitation at 380nm, suggesting they are capable of UVA absorption (Salih et al. 2006). It has also been proposed that GFPs may serve a UV and visible light screening function in corals and sea anemones by absorbing or redistributing photons away from the major absorption bands of the photosynthetic pigments of algal symbionts (Salih et al. 2000).
Compared to the UVA screening function of FPs, CPs may function by absorbing mostly visible light. Like the fluorescent proteins, the purple-blue colouration caused by chromoproteins in corals was observed to be greater in individuals exposed to higher amounts of sunlight than individuals exposed to less sunlight (Takabayashi and Hoegh-Guldberg 1995). Smith et al. (2013)) found that high CP expression correlated with reduced photo-damage to coral algal symbionts under acute light stress, by screening a specific wavelength range (i.e. 562-586 nm) that would otherwise be absorbed by the algal symbionts photosynthetic pigments, causing photoinhibition. Chromoproteins are also likely to be more efficient in photoprotection than FPs owing to their higher molar extinction coefficient (Dove
2004), although this is only applicable to the visible wavelengths, as their excitation maxima falls outside the range of UV.
Chapter 1: Literature review 9
DNA damage repair
Direct DNA damage repair
UV absorption by DNA can result in the formation of DNA lesions, which if not repaired can result in apoptosis of the affected cells or uncontrolled cell division
(Sinha and Häder 2002). Ninety percent of all DNA lesions are cyclobutane pyrimidine dimers (CPDs), which are formed between the coupling of the C=C double bonds of two adjacent pyrimidine bases in radiation exposed DNA (Haddock,
Mastroianni and Christianson 2010). These lesions distort the shape of the DNA strand, blocking transcription or inducing mutations. Two major CPD repair mechanisms in cnidarians are photoreactivation and nucleotide excision repair
(NER).
Photoreactivation
Photolyase is the singular molecular catalyst of photoreactivation, a UVA/blue light activated response that involves the repair of UV-induced DNA damage. It is widely regarded as the most efficient DNA repair mechanism (Arbeloa et al. 2010;
Schul et al. 2002). This enzyme is able to recognise and bind to a UV-induced DNA dimer, and then break the dimer apart in a process that utilizes energy from an incoming photon. Photolyases are divided into two major groups based on the type of
UV-induced DNA damage they repair: the cyclobutane pyrimidine dimer (CPD), and the 6–4 pyrimidine–pyrimidone photoproducts (6–4PP) which make up 75 and 25%, respectively, of the total UV-mediated DNA damage products (Todo 1999; Sancar
2003; Arbeloa et al. 2010; Sinha and Häder 2002). Photolyase belongs to the
Cryptochrome/Photolyase Family (CPF), and are found to be widely distributed
10 Chapter 1: Literature review
throughout eukaryotes (Reef et al. 2009; Oliveri et al. 2014; Shick, Lesser and Jokiel
1996).
There is no direct evidence of CPD photolyase being differentially expressed in response to UV stress in any cnidarian, although this is not surprising given that UV- induced gene expression studies in cnidarians are limited (Haddock, Rivers and
Robison 2001; Richier et al. 2008; Tarrant et al. 2014). UV-induced gene expression studies focused on marine bilaterians suggests that light triggers the up-regulation of photolyase, and that this enzyme plays an important role in their UV resilience.
Oliveri et al. (2014)) for example, found photolyase mRNA levels increased in the presence of light in the larvae of the sea urchin, Stronglyocentrotus purpuratus.
These larvae were cultured in a 12h dark, 12h artificial light regime, and collected at
4h time points (4, 8, 12, 16 and 20 h). The authors found significant CPD photolyase expression occurring around the centre of the light cycle, which would approximately correspond to midday, where UV would be at its most intense.
Similarly, Isely et al. (2009) found that ambient UV light significantly induced the expression of CPD photolyase in Antarctic sea urchin larvae (Sterechinus neumayeri).
Despite a lack of cnidarian genomic information regarding photolyase, there is evidence from physiological and observational studies that photoreactiviation is an important UV DNA repair mechanism in cnidarians. For example, Siebeck (1981) observed physiological damage and mortality following UV exposure in the scleractinian coral species, Platygyra sinensis, Favia pallida, and Turbenaria mesenterina. The authors observed that these corals were able to repair UV induced
DNA damage more effectively in the presence of shortwave visible light, compared to samples maintained in darkness, suggesting that photoreactivation might play a
Chapter 1: Literature review 11
dominant role in UV induced DNA repair. Recent experiments support these findings; UV stress studies on coral planulae larvae of A. millepora involving UV exposure in light and dark treatments revealed DNA repair was much faster immediately following exposure to the light treatment, compared to the dark treatment, which suggests that photoreactivation plays a major role in DNA repair
(Reef et al. 2009).
Nucleotide excision repair
UV induced DNA lesions can be repaired in the absence of light by nucleotide excision repair (NER), a process which excises the damaged segment of one DNA strand before attaching complementary nucleotides to the undamaged template strand
(Dykens and Shick 1984). XPC and RAD23 proteins are crucial components of NER in metazoans, and are involved in damage recognition and the recruitment of repair factors (Dantuma, Heinen and Hoogstraten 2009; Bergink et al. 2012). NER is present and highly conserved in all eukaryotes (Sinha and Häder 2002), and while very little is known about the distribution of NER genes such as RAD23 in cnidarians, there is some evidence to suggest that it is important in this phylum.
Haddock, Rivers and Robison (2001) measured the transcriptional response of larvae from the scleractinian coral, Montastraea favelolata, that were exposed to natural UV levels at different time points during development (i.e. 12, 36, 60, 84 and
120 hours post-fertilization). The authors found that UV sensitivity varied, with only a few transcriptional changes in most time points, but a highly sensitive time point after 84 hours, which corresponded to the motile planula larval stage. Various stress response genes were significantly upregulated at this time point, including RAD23.
RAD23 was also found to be differentially expressed in response to chemical stress
(i.e. Benzo [a] pyrene), but not thermal stress, in a study involving adult colonies of
12 Chapter 1: Literature review
the alcyonacean soft coral, Scleronepthya gracillimum. This is an interesting result with respect to UV stress, as it suggests that the thermal stress caused by excessive sun exposure may not be an important driver in the upregulation of this gene, although this remains to be properly tested.
Observational studies by Hudson and Ferrier (2008) found Aiptasia pallida anemones were able to repair DNA damage after 12 hours of UV and visible light exposure. DNA repair was inferred by comparing levels of undamaged DNA to damaged DNA, and evidence for NER was suggested when DNA repair was observed during the first 2 hours of recovery in the dark.
Indirect DNA damage repair
Oxidative stress caused by sunlight exposure can result in reactive oxygen species (ROS) moving into the cell nucleus and causing DNA oxidation, which is the alteration of guanine residues by ROS, resulting in epigenetic alterations and mutagenesis (Sancar 1996a). To prevent DNA oxidation, oxidized bases are removed by enzymes operating within the base excision repair pathway, for example glycosylases, which recognize and remove damaged bases (Sancar 1996a).
Many glycosylase family genes have been reported in corals including NEIL1
(Putnam et al. 2007), MBD4 (Kortschak et al. 2003) and MutY (Downs et al. 2009;
Downs et al. 2006; Rougée et al. 2006). In a biochemical study of the hard coral,
Porites lobate under oxidative stress for example, the enzyme MutY was found to significantly increase (Downs et al. 2006). In a similar study, elevation of MutY was also observed in the coral Pocillopora damicornis under oxidative stress (Rougée et al. 2006). To date, there have been no studies on intertidal cnidarian species that have observed significant levels of expression of these genes under oxidative stress,
Chapter 1: Literature review 13
although there is evidence of this occurring in other intertidal marine invertebrates.
For example, increased expression of the glycosylase OGG1 was observed in surf clam Donax variabilis under oxidative stress (Joyner-Matos, Downs and Julian
2006). In addition, UVB-induced oxidative stress was also found to enhance the expression of OGG1 in the Intertidal benthic copepod, Tigriopus japonicus (Kim et al. 2012).
Antioxidants
Sunlight can indirectly damage cnidarian cells through the generation of excess
- free radicals and reactive oxygen species (ROS) such as singlet oxygen (O2 ) and hydroxyl radicals (H2O2) (Grabherr et al. 2011). Incoming sunlight or heat energy can break apart the chemical bonds of cellular molecules thereby releasing oxygen into the cell. These ROS cause damage to the key cellular macromolecules (lipids, proteins and DNA) when they accumulate to levels that exceed the capacity of a cell to remove them; a condition generally referred to as oxidative stress occurs.
Cnidarian cells are able to repair oxidative damage and neutralize ROS through highly conserved antioxidant enzymes such as superoxide dismutases (SODs)
(Hawkridge, Pipe and Brown 2000), catalases and ascorbate peroxidases (Kanehisa et al. 2014). SODs function by detoxifying singlet oxygen molecules whereas catalases and peroxidases detoxify hydroxyl radicals.
Enzymatic antioxidants
Evidence of antioxidant enzymes in cnidarians has been supported by a number of studies. The genome of the model sea anemone N. vectensis, was found to contain numerous SOD, catalase, and peroxidase genes (Reitzel et al. 2008). Genomic stress
14 Chapter 1: Literature review
studies on N. vectensis found that many antioxidant enzyme transcripts (i.e. catalase,
Cu-Zn SOD and MnSOD) were significantly upregulated following UV and/or chemical exposure, with expression patterns being most strongly affected by UV exposure (Tarrant et al. 2014). In the sea anemone Anthopleura elegantissima, higher
SOD activity was reported in UV exposed versus UV shielded individuals (Dykens and Shick 1984). Similarly, a catalase gene isolated from Hydra vulgaris was found to be up-regulated in response to oxidative stress (Dash and Phillips 2012).
Peroxidase has been reported in two intertidal dwelling sea anemone species,
Aulactinia marplatensis and Bunodosoma zamponii, although it remains unclear whether they increase in response to photo-oxidative stress (Nicosia et al. 2014).
Polyamine oxidase genes were found to be significantly upregulated in response to temperature stress in Anemonia viridis (Moya et al. 2012). In addition, Ferritin, an important iron-storage antioxidant was found to be significantly upregulated in response to temperature stress in Anthopleura elegantissima (Richier et al. 2008).
Non-enzymatic antioxidants
The non-enzymatic antioxidants present in cnidarians include MAAs, melanins, carotenoids and fluorescent proteins, all of which are thought to have a dual function in the sunlight stress response owing to their UV and visible light screening capacity. The MAAs mycosporine-glycine and shinorine in corals for example, have been shown to provide antioxidant functions by supressing singlet oxygen and hydroxyl radicals, even before the induced expression of oxidative stress response genes or antioxidant enzymes (Fink 1999; Daugaard, Rohde and Jäättelä
2007; Torres-Pérez and Armstrong 2012). There is also evidence to suggest that cnidarian pigments can have substantial ROS scavenging activity. It is well- documented in vertebrates that the pigment melanin plays an important role in ROS
Chapter 1: Literature review 15
scavenging, and melanin in corals has been shown to act as a scavenger of ROS and defend cells from the toxic effects of free radicals (Snyder and Rossi 2004). The genes that underlie melanin production in other cnidarians remain to be investigated.
Carotenoids and carotenoproteins are the most widespread class of pigments found in nature, and protect against UV effects indirectly as antioxidants, although they also have weak UV screening capabilities (Krinsky 1979). Carotenoids vary in colour from red to yellow, and are considered to be the predominant source of colouration in sea anemones (De Nicola and Goodwin 1954; Fox and Pantin 1944).
The carotenoid pigment actinioerythrin for example, was determined to be the principal source of pigmentation in the intertidal sea anemone Actinia equina
(Heilbron, Jackson and Jones 1935). Like MAAs, carotenoids and carotenoproteins cannot be synthesized de novo by animals, and must be acquired through diet, or through microbial association (Kayal et al. 2013). Studies on cnidarian carotenoids have so far been restricted to those species with endosymbiotic dinoflagellates, such as the corals A. millepora and Pocillopora damicomis, where it has been revealed increased concentrations of the carotenoid antioxidant b-carotene are induced following light stress (Tamura et al. 2013b). A number of genes have recently been found to be associated with total carotenoid content in molluscs (Li et al. 2014;
Zheng et al. 2012), implying that underlying genetic mechanisms play important roles in carotenoid function in metazoans, although detailed analysis of these genes needs to be conducted in other marine invertebrates such as cnidarians.
In addition to the melanins and carotenoids, GFPs are another group of non- enzymatic antioxidants. A positive correlation between GFP concentrations and ROS
(H2O2) scavenging both in vivo across multiple species and in vitro in purified proteins, in damaged coral tissue has been identified in seven different coral species
16 Chapter 1: Literature review
(Montastraea annularis, M. faveolata, M. cavernosa, Diploria strigosa, Porites astreoides, Dichocoenia stokseii and Sidastrea sidereal) (Lindquist 1986). The authors found that the up-regulation of GFPs was consistent with increases in tissue damage. The authors examined the scavenging rates of three pure FPs (green, cyan and red), and CP, along with their chromophore lacking mutants. Of the pure GFPs, chromoprotein was found to be more efficient than the FPs. The mutant GFPs were found to be more efficient at scavenging ROS than their naturally occurring counterparts, suggesting that GFPs have evolved other functions in addition to being effective antioxidants. These observations, taken together with the differing spectral properties of this group, suggests that CPs provide a different UV stress response function to FPs, with CPs perhaps evolving a role in colouration, in addition to being more efficient antioxidants and providing better protection from visible light damage, whereas the FPs may be better suited to a UVA protective function (D’Angelo et al.
2008; D’Angelo et al. 2012).
Molecular chaperones
Molecular chaperones are proteins that assist other proteins maintain a functional structure, by preventing or correcting damage caused by mis-folding (Fink
1999). Heat shock proteins (HSPs) are the most well-known and widely studied group of molecular chaperones, and virtually all life forms respond to heat stress by transcribing and expressing large numbers of HSPs (Kregel 2002). HSPs are assigned to different families based on their sequence homology and molecular weights which range from 10-110 kDa (Feder and Hofmann 1999), and may be either expressed constitutively under normal conditions, or expressed in response to protein denaturing stresses, such as heat stress (Feder and Hofmann 1999). The most well-known and widely studied HSPs are HSP60, 70 and 90. Of these, HSP 70 is the
Chapter 1: Literature review 17
most highly conserved HSP family (Daugaard, Rohde and Jäättelä 2007), and is the most abundant HSP following heat stress (Black and Bloom 1984; Lindquist 1986).
HSPs have been found across a diverse range of sea anemone species (Del Rio
2015), and various studies on sea anemones have shown that a range of HSPs are induced under sunlight stress. Under natural field conditions, it was discovered that
HSP70 expression was significantly higher following three hour exposure to aerial and sunlight exposure compared to submerged or foggy conditions in the intertidal anemone Anthopleura elegantissima (Snyder and Rossi 2004). In addition to this biochemical study, many genomic studies have also documented the upregulation of various HSPs under sunlight stress. In the Mediterranean sea anemone Anemonia viridis for example, small HSPs were found to be differentially expressed in response to six hours of extreme temperature increase (Nicosia et al. 2014). In the model sea anemone Nematostella vectensis, Tarrant et al. (2014) found HSP70 to be upregulated in response to high UV, and downregulated in response to low UV exposure. Similarly, two HSPs (68 and 72 kDa) were synthesized during temperature stress in the anemone Aiptasia pallida (Black, Voellmy and Szmant 1995). It is interesting to note that no evidence of differential expression of HSPs was found in the transcriptomic response to 24 hours of temperature and UV stress in A. elegantissima. The authors of this study suggest that the conflicting result might be due to fold change values being below the sequencing threshold, or that an inappropriate stress period was chosen. However, it is also possible that the experimental conditions of this study did not closely match natural sunlight (Richier et al. 2008).
18 Chapter 1: Literature review
Conclusions
The majority of cnidarian sunlight stress response studies are focused on scleractinian coral species and there is a general lack of studies on intertidal sea anemones that take into account all the components of sunlight stress. The function of GFPs as UV and visible light screening compounds is not well understood, and there is generally very little information about either the direct or indirect repair mechanisms of oxidative stress outside of the corals. In addition, no studies to date have looked at this response under natural sunlight. There is also a lack of information on the DNA repair genes involved in the cnidarian sunlight stress response. In summary, genomic studies that examine the UV and visible light screening, antioxidant and DNA repair stress response of intertidal sea anemone species regularly exposed to high amounts of sunlight could provide useful insights into how these organisms survive sunlight stress.
Many UV and visible light screening, DNA repair and antioxidant mechanisms are conserved in various phylogenetically distant metazoan species (including cnidarians and vertebrates), and it is likely, given the fundamental nature of sunlight stress, that many genes underlying these mechanisms acquired their sun protective roles early in their evolutionary history. Understanding the sunlight stress response in intertidal anemones may hold important information about the evolutionary origins of this ubiquitous mechanism, yet current knowledge is limited mainly to biochemical and physiological studies. Genomic studies can therefore provide useful additional and novel molecular evidence for these processes.
Experiments that have investigated the cnidarian sunlight stress response have been conducted in controlled laboratory situations, and have focused only on isolated components (i.e. UVA, UVB, or heat stress), and no studies have examined the
Chapter 1: Literature review 19
effects that visible wavelengths (measured as PAR) may have. In addition, no studies have explored changes in gene expression as a result of aerial desiccation in sea anemones. The natural stresses of the intertidal habitat of A. tenebrosa consists primarily of sunlight stress and aerial exposure (Ottaway 1973), and further studies should examine both of these natural stress categories.
The waratah anemone, Actinia tenebrosa (Farquhar 1898), is the most common intertidal sea anemone species along the Australian and New Zealand coastlines and offshore islands and is an excellent candidate for investigating the cellular mechanisms underpinning sunlight resilience (Figure 1.2). Individuals of A. tenebrosa are usually found attached to rocks or other hard substrates in the intertidal zone, and survive direct sunlight exposure during low tide (Farquhar 1898; Ottaway
1978, 1973). The genus Actinia is collectively one of only a few anemone groups to be completely exposed to sunlight during low tides in Australia, as other intertidal anemones are permanently submerged and sheltered under rock ledges or in crevices
(i.e. Anthopleura sp. and Aulactinia sp. (Fautin, Crowther and Wallace 2008)).
Important cellular response mechanisms in response to sunlight stress in A. tenebrosa can be assumed to involve UV and visible light screening, antioxidants and DNA repair, but the strategies that A. tenebrosa uses to reduce cellular damage and survive under these highly variable conditions are not well understood, and nothing is known about its gene expression response.
20 Chapter 1: Literature review
Figure 1.2 (A) Side view of A. tenebrosa fully submerged, with tentacles expanded; (B) top view of A. tenebrosa fully exposed during low tide, with tentacles retracted; (C) A. tenebrosa distribution (adapted from Loh (2011)).
Sea anemones are important components of rocky intertidal ecosystems along
Australian coastlines (Fautin, Crowther and Wallace 2008; Ottaway 1979), yet virtually nothing is known about their susceptibility to changing environmental conditions. Only recently have sea anemone species been comprehensively documented, and there are likely to be many species yet to be discovered. In the
Moreton Bay region in eastern Australia for example, Fautin, Crowther and Wallace
(2008) documented 19 species, three of which were not previously known to occur in
Australia, two that were not known to occur in the region, and one unidentified species that was the first recorded member of its genus in Australia. Actinia tenebrosa is an excellent indicator species for understanding stress tolerances along coastal Australia because it is abundant, easy to identify, and widespread (Fautin,
Crowther and Wallace 2008; Ottaway 1979; Loh 2011). These features also make A. tenebrosa useful for understanding the stress tolerances of other anemone species that may be more rare or cryptic.
Chapter 1: Literature review 21
Project aims
This research project investigated the effects of sunlight stress on A. tenebrosa by combining a range of molecular approaches with ecological, physiological and biochemical information. The first step involved sequencing and assembling the A. tenebrosa transcriptome to identify candidate genes involved in the physiological adaptation and stress response of A. tenebrosa to sunlight. To identify the genes that are differentially expressed in response to sunlight stress, controlled experimental studies were conducted. This information enabled us to infer if any candidate genes were differentially expressed under direct sunlight. Meaningful comparisons with other sequenced intertidal cnidarians species were then made to better understand the effects of sunlight across a wider range of species.
The aims of this research project were to:
1. Develop an A. tenebrosa transcriptome as a genomic reference to identify
genes involved in sunlight stress response, and compare them with other
cnidarian species.
2. Examine temporal patterns of gene expression under sunlight stress in A.
tenebrosa under controlled and natural environment experiments.
Aim 1.
Objective 1: Representative A. tenebrosa individuals were collected from a single sampling site near the edge of A. tenebrosa’s northern distribution limit as it had the highest average UV and temperature levels. Whole animals were snap-frozen in liquid nitrogen, followed by RNA extraction, mRNA isolation and transcriptome sequencing. Genes involved in the sunlight stress response were identified and compared to existing cnidarian genomes.
22 Chapter 1: Literature review
Objective 2: Candidate UV stress response genes from UV and visible light screening, antioxidant and DNA repair categories were identified and validated. The evolution of these genes in cnidarians was inferred through comparative and phylogenetic analyses.
Aim 2.
Objective 1: Representative A. tenebrosa individuals were collected from the same sampling site described in Aim 1. Organisms were transported to the marine lab and acclimated under standardized conditions. Actinia tenebrosa individuals were then be randomly subjected to one of four exposure categories (i.e. unshaded and submerged, shaded and submerged, unshaded and exposed, shaded and exposed) and relevant environmental conditions (e.g. PAR, UV, temperature) were recorded at various time intervals during exposure. Individuals were then snap-frozen in liquid nitrogen, RNA from each individual was extracted and cDNA synthesized. To assess levels of gene expression under the experimental conditions, qPCR using cDNA and candidate gene primers were used. Patterns of differential expression was determined using statistical analysis.
Objective 2: Candidate UV stress response genes were validated, and the evolution of these genes in actiniarians was inferred through comparative and phylogenetic analyses.
Chapter 1: Literature review 23
Chapter 2: Comparative analysis of
genes involved in UV response from
cnidarian species
Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species 25
Abstract
Ultraviolet radiation (UV) can be hazardous to many organisms that inhabit shallow marine environments such as the intertidal zone, where tidal cycles can influence levels of UV exposure. Some intertidal sea anemone species that inhabit intertidal areas may possess unique adaptations to withstand significant periodic UV exposure, and the cellular mechanisms underpinning this resilience need to be investigated. There is a lack of information on UV response in sea anemones and studies so far have focused on observational and physiological evidence. The identification and analysis of UV response genes can therefore provide additional and novel molecular insights. In this study, the deeply sequenced transcriptome (~7
Gbp) of the sea anemone, Actinia tenebrosa, was assembled and annotated, and genes involved in the UV response identified. Evolution of these genes in cnidarians was inferred through comparative and phylogenetic analyses. Eight hundred and sixty two UV response genes were identified in A. tenebrosa and 179 were determined to have full length open reading frames. These 179 full length genes were found to be widely distributed across cnidarian species. Four candidate genes, chromoprotein (a non-fluorescent member of fluorescent proteins), photoprotein,
CPD photolyase and UV excision repair RAD23 were examined in detail across 17 cnidarian transcriptomes and 3 genomes. Both chromoprotein and photoprotein occurred as gene families in cnidarians, while CPD photolyase and UV excision repair RAD23 were present as single copy genes. Multiple independent origins of non-fluorescent chromoproteins in cnidarians were found, and these were sister to fluorescent proteins in all cases. Both CPD photolyase and UV excision repair
RAD23 were highly conserved at the nucleotide level, but had patterns of nucleotide variation consistent with neutral evolution. Overall, this study has shown that many
26 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species
of the genes involved in UV stress response are conserved in cnidarians, and provides a resource to better understand the molecular mechanisms that enable A. tenebrosa to survive periodic UV stress.
Introduction
Ultraviolet radiation (UV, 200-400nm) has been a ubiquitous challenge for life on earth for over 3.8 billion years. Exposure to UV can be deleterious to many life- forms, causing UV induced DNA damage, as well as mutagenesis and apoptosis
(Oliveri et al. 2014). UV stress is particularly hazardous to sedentary cnidarians dwelling in shallow marine environments, such as the intertidal zone, where levels of
UV exposure may be increased at low tides. Cnidarians (jellyfish, hydroids, box jellyfish, sea anemones and corals) are diploblastic animals characterised by two layers of simple epithelium separated by an extracellular mesoglea (Rupert, Fox and
Barnes 2004). Cnidarians are often directly exposed to abiotic stresses due to an absence of physical protective barriers, other than mucus, with the exception of order
Scleractinia. Members of this particular order are afforded some protection from UV by retracting into their calcium carbonate exoskeleton. Intertidal sea anemones (order
Actiniaria), survive direct UV exposure with only a mucus barrier, which suggests that they may possess unique adaptations to withstand significant periodic UV exposure.
Sea anemones cope with UV exposure in part due to the cellular stress response (CSR), a universal molecular mechanism largely conserved across metazoan lineages (Kültz 2005). Three components of the CSR that protect cnidarian cells from UV stress are UV screening, antioxidants, and DNA repair (Figure 2.1).
UV screening compounds such as mycosporine-like amino acids (MAAs) and members of the green fluorescent protein family (GFPs) have been well-documented
Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species 27
in many cnidarians (Ayre, Read and Wishart 1991; Kawaguti 1944; Kortschak et al.
2003; Shick and Dunlap 2002).
MAAs are an abundant group of colourless, water soluble molecules, which play a role in absorption of both UVA and UVB (Svanfeldt et al. 2014), while GFPs transform the absorbed incoming short wavelengths (i.e. UV and near-UV) into higher wavelength visible fluorescence (Pakhomov and Martynov 2008). Recently, the role of GFPs has been redefined to include quenching and antioxidant roles, in addition to UV screening (Salih et al. 2000; Lindquist 1986; Salih et al. 2006). UV damage can also be mitigated by repairing damage to critical molecules such as DNA through nucleotide excision repair and photoreactivation (Sancar 1996b; Sancar and
Tang 1993). Despite the key roles that these mechanisms play, their role in cnidarians has largely been limited to observational and physiological studies, therefore, further studies of UV stress genes presents an opportunity to uncover novel responses, and further elucidate the molecular underpinnings of UV stress response in cnidarians.
Understanding the cellular stress response to UV stimulus in cnidarians may hold important information about the evolutionary origins of this ubiquitous mechanism. Studies that focus on phylum Cnidaria, the sister phylum to Bilateria can provide opportunities for uncovering novel genes that may contribute to this process.
The model cnidarian Nematostella vectensis for example, has numerous genes that have clear orthologs in humans (Putnam et al. 2007; Ziegler et al. 1993; Technau et al. 2005), many of which are not present in other model invertebrates such as
Drosophila melanogaster or Caenorhabditis elegans (Putnam et al. 2007).
Consequently, cnidarians present an excellent system for the discovery of genetic
28 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species
similarities with vertebrates while at the same time also allowing for novel genetic discoveries.
A growing amount of genomic information is available on existing model cnidarian species such as N. vectensis (Putnam et al. 2007), Hydra magnipapillata
(Chapman et al. 2010) and Acropora digitifera (Shinzato et al. 2011), however, these organisms are not well suited for UV stress studies. N. vectensis for example, is able to avoid UV by burrowing into soft substrate (Williams 1975), whereas H. magnipapillata is motile and not usually exposed to high levels of UV (Bergink et al.
2012). A. digitifera possess endosymbionts that influence host survival, in addition to having a calcium carbonate secreted exoskeleton which coral polyps retract into
(Dantuma, Heinen and Hoogstraten 2009). Studies that investigate the genomic basis for UV resilience in sedentary intertidal cnidarians (i.e. sea anemones from the order
Actiniaria), that are found in environments where UV is a constant factor can help to better understand how these organisms survive UV stress.
The Waratah sea anemone Actinia tenebrosa Farquhar (1898) is a common intertidal sea anemone found along the Australian and New Zealand coastlines and offshore islands (Ottaway 1979). This species is an excellent candidate to investigate the molecular underpinnings of UV resilience in intertidal sea anemones and has been used in physiological studies examining UV exposure (Cubillos et al. 2014).
Actinia tenebrosa are found attached to hard substrates in the intertidal zone and are distributed across rock crevices, shaded under-hangs and exposed tide pools, where they often survive direct exposure to UV during low tide. In this study, the transcriptome of A. tenebrosa was sequenced and analysed to identify genes involved in UV stress response of intertidal cnidarians. The following candidate UV stress response genes were then investigated in detail: The UV screening and antioxidant
Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species 29
blue chromoprotein and the DNA repair genes CPD photolyase and RAD23B (Figure
2.1). Finally, a comparative and phylogenetic analysis was conducted on these candidate genes to better understand their evolution.
Figure 2.1 Conceptual overview of select antioxidant, UV and visible light screening and DNA repair mechanisms and their possible associated candidate genes in the intertidal sea anemone, Actinia tenebrosa.
Materials and methods
Sample collection, RNA extraction and sequencing
Actinia tenebrosa individuals were collected from Point Cartwright, Australia
(26°32'9.83"S, 153° 5'45.12"E). A single individual animal was selected for sequencing and total RNA was extracted using a Trizol/chloroform protocol
(Invitrogen), followed by a silica membrane based column clean-up (Qiagen). RNA quantity and integrity was measured using a Bioanalyser 2100 and library preparation was undertaken as per Prentis and Pavasovic (2014). Sequencing was performed on an Illumina HiSeq 2000, using 91bp paired-end sequencing chemistry.
30 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species
Data processing and assembly
Raw sequence reads were converted to FastQ files and filtered for quality
(Q>20, N<1%). Sequence reads that met these quality criteria were assembled into contigs using CLC Genomics Workbench version 6.5 with a Kmer of 25 and bubble length of 100 and the Trinity short read de novo assembler using default settings
(Grabherr et al. 2011). The transcriptome assemblies were then merged and quality checked using CD-Hit-EST (Fu et al. 2012; Li and Godzik 2006), and sequences with > 95% similarity were merged into consensus sequences. In addition, CEGMA v.2.5 (Parra, Bradnam and Korf 2007) was used to check the completeness of the assembly by determining the percentage of full-length sequences from 248 core eukaryotic genes. The assembled contigs were then used as BLASTx queries against the non-redundant protein database (NCBI) using BLAST+ software (Camacho et al.
2009). A threshold stringency value for BLAST hits was set at E-value <10-6.
BLAST2GO (Conesa et al. 2005) software was used to assign gene ontology (GO) terms to contigs that received BLAST hits, and visualise the distribution of GOs across categories. Transdecoder (Haas et al. 2013) was used to batch extract, and convert to predicted protein sequences for open reading frames (ORFs) greater than
100 amino acids in length.
Comparative and phylogenetic analysis
To identify genes and gene families known to be involved in UV stress response, the annotated A. tenebrosa transcriptome was searched using GO terms
(Table S2.1); results from literature searches, and BLAST hit information. From this list, candidate genes were identified and selected from three UV protection categories: UV screening and quenching (blue chromoprotein) and DNA repair (CPD photolyase and RAD23b), and one identified novel gene (photoprotein) (Table S2.2).
Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species 31
Full-length candidates were determined using ORFs generated by Transdecoder and
ORF Finder (NCBI), followed by BLASTp analysis and alignment to other full length proteins. Functional domains for each candidate were determined using the
HMMER Pfam database (EMBL-EBI). Enzyme codes from each candidate were used to determine their presence in corresponding KEGG pathways (Kanehisa et al.
2014; Kanehisa and Goto 2000). The presence and absence of candidate genes in cnidarian species was determined using BLAST searches against publicly available transcriptome and proteome datasets, and hits with E-values <10-5 were retained for further comparative and phylogenetic analysis. Three unpublished sea anemone transcriptomes were also included in this analysis: Anthopleura buddemeieri,
Aulactinia veratra, and Calliactis polypus. Specifically, 17 cnidarian transcriptomes,
3 cnidarian genomes and 3 non-cnidarian data-sets (outgroups) were interrogated
(Table S2.12). Due to the amount of information available online for the GFP gene family, the chromoprotein candidate from A. tenebrosa was used as a BLASTn query against the NR database (NCBI), and naturally occurring GFP homologues with E- values <10-5 were retrieved and added. For phylogenetic analysis, only the top five hits for each candidate gene from each taxa was included.
Alignment of candidate genes was performed using a ClustalW codon alignment in MEGA v6 (Tamura et al. 2013b). Tests for positive selection were performed on aligned gene sequences by estimating the strength of selection operating on each individual codon in the alignment using HyPhy in MEGA v6. The appropriate model of evolution that was established for all candidate alignments was generalised time-reversible (GTR) with proportion of invariant sites (I) along with gamma-distributed among site rate variation (G) using jModel Test 2 (Darriba et al.
2012).
32 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species
Maximum likelihood (ML) analysis was performed using the program RAxML v8.1.24 (Stamatakis 2006), invoking a rapid bootstrap (1000 replicates) analysis and search for the best-scoring maximum likelihood tree with the general time-reversible model of DNA sequence evolution with gamma-distributed rate heterogeneity
(GTRGAMMA substitution model). Maximum-likelihood tree bootstrap support values were defined as follows: 70 as poor, 70-85 as average, and >85 as good.
Consensus trees from ML outputs were drawn and annotated with bootstrap values in
FigTree v1.4.2 (Rambaut 2014).
To validate the presence of candidate genes found in the A. tenebrosa transcriptome, six primer pairs were designed using Geneious version 8.0.4 (Kearse et al. 2012).
Validation was performed by first extracting RNA using the same protocol as for transcriptome sequencing, followed by cDNA synthesis using Sensi Mix (Bioline) as per manufacturer’s instructions. PCR amplification was performed using the
MyTaqTM (Bioline) protocol with the following modifications: 5 µL of 5x PCR buffer, 1µL of each primer (10µM concentration), 0.1 units of MyTaqTM DNA polymerase (Bioline), 16.9 µL ddH2O and 1µL of cDNA to a total of 25 µL. PCR conditions were as follows: 3 min denaturation at 95⁰C, followed by 30 cycles of 30 sec denaturation at 95⁰C, 30 sec annealing at 55⁰C, 45 sec extension at 72⁰C, then a final extension of 5 min at 72⁰C. Amplicons were purified using an Isolate II PCR
Kit (Bioline) and Sanger sequencing was carried out using BigDyeTM Terminator v3.1 Cycle Sequencing Kit (Life technologies) with the following specifications:
TM 13.5 µL ddH2O, 3.5 µL seq buffer, 1.0 µL BigDye , 1.0 µL primer (3.2 pmol) and
1.0 µL DNA template. Sequencing products were cleaned up using an
Ethanol/EDTA precipitation and run on an ABI 3500 Genetic analyser (Life
Technologies). All sequences were visualised and edited in Geneious version 8.0.4.
Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species 33
Sequences were then aligned to the original sequences from which primers were designed and also used as validation.
Results
Sequencing, data processing and assembly
Sequence reads (SRA accession number: PRJNA295912) from A. tenebrosa were assembled into 65,750 contigs using CLC Genomics software and 95,175 contigs using Trinity. When merged, the combined assembly consisted of 121,783 contigs, with a longest contig of 20,015bp, and an N50 value of 1,389bp. For a summary of assembly metrics, see Table S2.3. Over 90% of the ultra-conserved
CEGs (core eukaryote genes) were present in the merged assembly, of which > 70% were determined to be full-length. Of the 121,783 contigs from the combined assembly, 57,394 (47.13% BLASTx success) returned significant BLAST hits with a stringency of 1Ex10-6.
Comparative and phylogenetic analysis of candidate genes
Of the 57,394 contigs that received BLAST hits from NCBI, 28,991 were assigned GO terms, 425 of which fell under the response to stress category (Table
S2.1), which was the GO category most relevant to UV stress (Figure 2.2). In addition, 862 candidate contigs considered to be key components involved in UV stress response were identified. One hundred and fifty six of the candidate contigs contained full-length ORFs (Table S2.4). When compared against the proteomes of three cnidarian model species (Nematostella vectensis, Acropora digitifera, Hydra magnipapilatta), 128 (82.05%) were found in all four cnidarian species (Figure 2.3).
34 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species
Figure 2.2 Gene Ontology (GO) terms involved in UV response (left) and response to stimulus (right) from the A. tenebrosa transcriptome.
Figure 2.3 (A) Cladogram showing the distribution of the A. tenebrosa transcriptome and the genomes of the three model cnidarian genomes, Nematostella vectensis, Acropora digitifera and Hydra magnipapilatta (B) Venn diagram illustrating how the 156 identified A. tenebrosa UV stress genes are shared amongst the three model cnidarian genomes. Cladogram adapted from Zapata et al. (2015).
Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species 35
All candidate genes were successfully validated through cDNA synthesis and
Sanger sequencing before being submitted to GenBank (Table 2.1). The photoprotein
ORF was validated with 100% sequence identity, blue chromoprotein was validated with 99.5% sequence identity, CPD photolyase was validated with 99.5% sequence identity, half of the UV excision repair protein RAD23 homolog b was validated with 99.4% sequence identity, and carotenoid monoxygenase was validated with
99.8% sequence identity (Table S2.5). HyPhy analysis indicated no evidence of selection on codons in any of the candidate genes across the taxa that were used in this study (Tables S2.6-S2.11).
Table 2.1 Summary of full-length candidate sequences from the A. tenebrosa transcriptome
GenBank accession BLAST sequence Contig Protein length Sequence number description length (bp) (aa) length UV DNA repair KT779417 deoxyribodipyrimidine 2145 560 full photolyase-like
- deoxyribodipyrimidine 1444 329 partial photolyase-like
KT779418 UV excision repair protein 1562 387 full RAD23 homolog B - UV excision repair protein 2469 230 partial RAD23 homolog B
Bioluminescent and Fluorescent Pigments KT779416 photoprotein-like protein 912 188 full
KT779414 blue chromoprotein 1029 232 full
KT779415 blue chromoprotein 1768 232 full
- blue chromoprotein 887 170 full
36 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species
Chromoproteins
The full-length blue chromoprotein found in A. tenebrosa had an open reading frame 699 nucleotides (232 amino acid residues). Its domain structure consisted of a single GFP domain (Table S2.2). Homologs of the A. tenebrosa chromoprotein were found in all orders of hydrozoans and anthozoans (14 out of 21 taxa), although these were absent in scyphozoans (Table S2.12). Homologous proteins consisted of both fluorescent proteins and chromoprotein GFPs. Using BLASTp against the NCBI database, homologs in an additional 31 cnidarian taxa (28 anthozoans, 3 hydrozoans), were found and included in the analysis of this gene family. Of the 53 cnidarian taxa found to have GFP sequences, 46 had full-length sequences and 20 of these 46 taxa had more than one full-length GFP gene. All fluorescent proteins and chromoprotein GFPs had the same domain structure. The A. tenebrosa contig annotated as a blue CP shared 51.8% amino acid identity with its homolog found in
N. vectensis. From the combined list of 100 cnidarian GFPs, 24 were CPs, and 78 were FPs.
Phylogenetic analysis of the cnidarian GFPs using maximum-likelihood inference revealed two major clades, an anthozoan clade and a hydrozoan clade
(Figure 2.4). The sequences did not cluster according to taxonomic affinity (see
Figures S2.1 and S2.2 for a comparison), however, it did resemble the pattern of evolution seen in earlier cnidarian studies of cnidarian GFPs (see Alieva et al.
(2008)). Although tip branches were largely well-supported, the phylogenetic relationships within these clades remain largely unresolved due to poor branch support. A large number of GFPs were intermingled amongst distantly related taxa with high support. Five independent lineages of the chromoprotein-like proteins were
Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species 37
found across the cnidarian taxa with strong support. The first four independent origins of CP were all found in the anthozoan clade; lineage A was found in an actiniarian clade and was sister to Actinarian FP sequences. Lineage B, a second independent origin of CP was composed solely of Scleractinia species and this clade was sister to a Corallimorpharia lineage that contained a third independent origin of
CP. This independent origin of CP was found in Discosoma striata which was sister to a FP from the same species and these sequences were sister to other
Corallimorpharia FP sequences. Lineage D consisted of an independent actiniarian
CP sequence from Anemonia majano nested within a clade composed of FP sequences of Sclearctinian, Corallimorpharian and Zoanthidean species. Lineage E consisted of an independent CP origin in the Hydrozoan clade, Anhtomedusae sp., which was sister to all other Hydrozoan FP sequences.
38 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species
Figure 2.4 Phylogenetic tree of blue chromoprotein gene from A.tenebrosa (shown in bold) and its homologues in other cnidarian taxa, with the bilaterian Pontella meadi as the outgroup using maximum-likelihood inference. Five independent lineages of cnidarian chromoproteins are highlighted. All genes shared the same domain structure as the canonical GFP domain from Aequoria victoria, shown in the top left hand corner.
The blue chromoprotein from A. tenebrosa was found to be sister to a CP from
C. polypus and less similar to a CP from the other Actinia species, A. equina. Blue colouration was observed on C. polypus oral disk, and on A. tenebrosas pedal disk and acrorhagi (Figure 2.5). Actinia tenebrosa and C. polypus chromoproteins shared
Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species 39
100% similarity at the amino acid level, and differed by only four synonymous SNPs
(Figure S2.5).
Figure 2.5 Location of blue colouration on: (A) C. polypus oral disk, (B) A. tenebrosa acrorhagi, (C) A. tenebrosa pedal disk.
Photoproteins
The full-length photoprotein-like protein ORF from A. tenebrosa was 567 nucleotides (188 amino acid residues). Its domain structure consisted of EF-hand domain 5 and EF-hand domain 7 (Table S2.2). Most photoprotein homologs from cnidarians had a domain structure consistent with the canonical photoprotein, aequorin, with the exceptions highlighted in Figure 2.6. Photoprotein genes were found in 25 out of 26 taxa and all orders that were examined (Table S2.12). Of the 25 cnidarian taxa found to have photoprotein-like protein homologs, 16 had full-length sequences and 8 of these 16 taxa possessed multiple full-length photoprotein genes.
Alignment of the A. tenebrosa contig with its homolog from N. vectensis revealed
55.5% amino acid identity.
40 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species
Figure 2.6 Phylogenetic tree of photoprotein-like gene from A.tenebrosa (shown in bold) and its homologues in other cnidarian taxa, with the ctenophore, Mnemiopsis leidyi as the outgroup using maximum-likelihood inference. Non-highlighted genes shared the same domain structure as the canonical aequorin domain from Aequoria victoria, shown in the top left hand corner. Highlighted genes show domain structures that differ from aequorin.
Phylogenetic analysis revealed three well-supported clades (Figure 2.6). The sequences did not cluster according to taxonomic affinity (see Figures S2.1 and S2.2 for a comparison). The photoprotein-like clade consisted of anthozoan and
Hydrozoan taxa (Orders Actiniara, Scleractinia, Alcyonacea and Siphonophorae), with phylogenetic relationships remaining largely unresolved due to low branch support. The Hydrozoan, Nanomia bijuga, was the only species in this clade known to be bioluminescent. The Ca2+ triggered coelenterazine binding protein (CTCBP) clade consisted entirely of anthozoan species (all Order Alcyonacea), with well- supported phylogenetic relationships. The sea pansy, Renilla muelleri, was the only species in this clade known to be bioluminescent. The aequorin-like clade consisted
Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species 41
entirely of Hydrozoan taxa (all Order Leptothecata), with well-supported phylogenetic relationships. All species in this clade are known to produce bioluminescence.
CPD photolyase
The full-length CPD photolyase contig from A. tenebrosa had an open reading frame of 1683 nucleotides (560 amino acid residues). Its domain structure consisted of a DNA photolyase domain, followed by a Flavin Adenine Dinucleotide (FAD) binding domain (Table S2.2). Homologs of the CPD photolyase from A. tenebrosa were found in 20 out of the 22 cnidarian taxa, and were present in all classes analysed except for the Scyphozoa (Table S2.12). Of the 20 cnidarian taxa found to have CPD photolyase homologs, 10 had full-length sequences. None of the taxa in this study had more than one full-length CPD photolyase gene. All homologs of the
A. tenebrosa CPD photolyase had similar domain structures. Alignment of the A. tenebrosa contig with the same protein found in N. vectensis revealed that they shared 69.9% amino acid identity. Phylogenetic analysis of the CPD photolyase gene in cnidarians revealed that the sequences clustered according to taxonomic affinity
(see Figures S2.1 and S2.2 for a comparison). Two strongly supported clades were present, one representing Actiniaria and the other Scleractinia (Figure S2.6).
UV excision repair protein RAD23b
The full-length RAD23b open reading frame from A. tenebrosa was 1164 nucleotides (387 amino acid residues). Its domain structure consisted of sequential
UBQ, UBA, STI 1, and UBA domains (Table S2.2). Homologs of the RAD23b from
A. tenebrosa were found in 21 out of the 22 cnidarian taxa examined, and were present in all orders analysed except for Scyphozoa (Table S2.12). These predicted
42 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species
proteins were present in all species, with the exception of Platygyra daedalea.
RAD23b homologs were also found in the bilaterian Danio rerio, and the
Ctenophore, Mnemiopsis leidyi which were used as outgroups. Of the 21 cnidarian taxa found to have RAD23b, 17 had full-length sequences. None of the taxa in this study had more than one full-length RAD23b gene. The domain homology of this candidate was consistent across all taxa examined. This gene was found to be involved in the Eukaryotic NER KEGG pathway (Figure S2.6). Alignment of the A. tenebrosa contig with its homolog in N. vectensis showed that the two proteins shared 62.7 % pairwise identity. Anthozoan species formed a well-supported clade
(Figure S2.5), however, phylogenetic relationships in other cnidarian taxa were largely unresolved due to low branch support, and definitive conclusions cannot be established for the phylogenetic relationship of this gene. Nevertheless, the sequences did cluster according to taxonomic affinity (see Figures S2.1 and S2.2 for a comparison).
Discussion
In this study the genes responsible for UV resilience in the sedentary intertidal cnidarian, Actinia tenebrosa were investigated. The transcriptome of A. tenebrosa was sequenced and analysed, and used as a genomic reference to identify genes involved in UV stress response. A comparative analysis of A. tenebrosa UV stress response genes, with the predicted proteomes of three model cnidarian species
(Acropora digitifera, Hydra magnipapilata and Nematostella vectensis), demonstrated that the majority of these physiologically important genes were widely distributed across phylum Cnidaria. Analysis across current and publically available cnidarian transcriptome datasets using four candidate genes (i.e. blue chromoprotein, photoprotein, CPD photolyase and RAD23b) provided additional insight that these
Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species 43
genes are present in most cnidarian species, and in the case of the DNA repair genes, were highly conserved. Overall, these observations suggest that the UV candidate genes examined here likely play an important functional role in cnidarian species as a whole.
Blue chromoprotein The green fluorescent protein (GFP) family are a colourfully diverse group of pigments and a major component of the colouration of corals (Dove, Hoegh-
Guldberg and Ranganathan 2001). GFPs are also thought to play an important role in protecting scleractinian coral species (i.e. Acropora nobilis, Acropora palifera,
Goniastrea retiformis, Pocillopora damicornis) from UV stress (Salih et al. 2000;
Shick and Dunlap 2002). The two major classes of GFPs, the non-fluorescent chromoproteins (CPs), and the fluorescent proteins (FPs) have differing spectral properties which may give them different roles in the coral UV stress response. For example, the CPs can be pink, purple or blue in colouration (Dove, Hoegh-Guldberg and Ranganathan 2001) and are reported to be more efficient antioxidants (Lindquist
1986) and provide better protection from visible light damage due to their higher molar extinction coefficient (Dove 2004; Smith et al. 2013). In comparison, the FPs can be cyan, green, yellow or red in colouration (Dove, Hoegh-Guldberg and
Ranganathan 2001), and may be better suited to a UVA protective function based on their ability to absorb certain UVA wavelengths (Salih et al. 2006; Haddock, Rivers and Robison 2001; Takabayashi and Hoegh-Guldberg 1995; Camacho et al. 2009).
Knowledge of the GFP family in cnidarians is limited, and mostly restricted to coral species, and no studies have explicitly looked at their function or evolution in the sea anemones.
44 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species
Comparative and phylogenetic analysis of the GFP-like family across phylum
Cnidaria confirmed multiple independent origins of chromoproteins, and a second chromoprotein lineage in actiniarians was reported for the first time. Most cnidarian datasets that were examined contained GFP-like protein homologs while the majority of taxa also possessed multiple gene copies. This result indicates widespread gene duplication within the GFP-like gene family in cnidarians, and may provide some explanation for the diversity of fluorescent and non-fluorescent colours observed in this phylum. Furthermore, it is likely that there have been multiple independent origins of chromoproteins in actiniarians based on the support and placement of this
GFP in the phylogenetic analysis. This study supports early findings of independent origins of chromoproteins in anthozoans and hydrozoans (Dove, Hoegh-Guldberg and Ranganathan 2001). The pattern of repeated evolution of CPs from FPs suggests that the CPs have some significant and different function to their FP counterparts that remains to be explored in cnidarians. The diversification of duplicated GFP-like proteins may potentially lead to neofunctionalisation and generation of novel fluorescent proteins or alternatively converge on a similar functional protein product
(Ohno 2013). While the repeated evolution of CPs provides strong evidence for neofunctionalisation, molecular evidence for convergent evolution was also found.
The sequence most similar to the A. tenebrosa blue chromoprotein was not a member of the genus Actinia (family Actiniidae, superfamily Actinioidea), but was
Calliactis polypus (family Hormathiidae, superfamily Metridioidea). The blue chromoprotein genes from these different organisms differed by only four SNPs, which was remarkably low given the level of neutral divergence between them
(~30%), and the morphological and genetic similarity of A. tenebrosa and A. equina, when compared to C. polypus. While the exact source of blue chromoprotein in these
Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species 45
species has yet to be established, a pale blue colouration on the oral disk of the C. polypus individuals was observed, which appeared to match the coloration of the acrorhagi of A. tenebrosa, as well as the pedal disk of some individuals (Figure 2.6).
This may indicate convergent evolution although more data from A. equina would be needed to make a more definitive conclusion. Based on the information available, it appears as though distantly related cnidarian taxa are subjected to a similar yet unknown selective pressure.
The vivid blue pigmentation in both species are likely regions of blue chromoprotein concentration, and one explanation is that they function as a type of fluorescent “lure” to attract prey. It is possible that cnidarians have evolved under this prey capture constraint, giving rise to GFP genes with similar spectral properties appearing in distantly related taxa, such as A. tenebrosa and C. polypus.
Another explanation might be that the CPs in these species has evolved to deal with a set of common environmental stressors. While Calliactus polypus is reportedly a hermit crab commensal (Ross 1970), they are also known to colonize other objects such as pumice, which floats on the water surface. A C. polypus individual attached to a floating piece of pumice is essentially exposed to the same environmental stresses that A. tenebrosa faces in the intertidal zone: aerial exposure, and high UV and temperature as a result of sunlight exposure. Further investigations into the relationships between GFP expression, and UV stress in intertidal cnidarians such as sea anemones can provide a starting point to address this question.
Photoprotein A full-length photoprotein-like protein was discovered in the transcriptome of
A. tenebrosa. While these proteins are associated with bioluminescence, they were present in A. tenebrosa and other cnidarian species, many of which are not known to
46 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species
be bioluminescent. This study provides a first look at the photoprotein gene family evolution in phylum Cnidaria. Cnidarian photoprotein genes were found to be widespread and common in most cnidarian species, and formed three distinct and well-supported clades. The photoprotein-like clade shared a high degree of homology with photoprotein genes from the bioluminescent Mnemiopsis leidyii (Phylum
Ctenophora), and is therefore most likely ancestral to the other photoprotein clades.
While the majority of species with photoprotein genes are not known to be bioluminescent, bioluminescent species were found to be present in each of the three clades. These findings provide some insight into this gene family in cnidarians, and raise interesting questions about their evolution and function within this phylum.
Little is known about the evolutionary origins of cnidarian photoproteins although they most likely arose from a calcium-binding calmodulin-like ancestor
(Inouye et al. 1985; Schnitzler et al. 2012). All cnidarian taxa examined in study presented in this thesis possessed multiple photoprotein genes, which indicated that this gene family has undergone at least one round of gene duplication in its evolutionary history. Gene duplication followed by the evolution of a new function may help to explain why non-bioluminescent cnidarians possess photoprotein genes.
One possible explanation for the lack of luminescence in shallow water cnidarian species could be the absence of other molecular components required in the light reaction. In all cnidarians, bioluminescence involves the formation of a complex composed of a photoprotein enzyme, a dietary acquired substrate coelenterazine, and molecular oxygen (Daunert and Deo 2006; Shimomura, Johnson and Saiga 1962)
(Daunert and Deo 2006; Shimomura and Johnson 1978). In the presence of calcium ions, the prosthetic group coelenterazine, undergoes a conformational change into coelenteramide, resulting in the emission of CO2 and visible light as it
Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species 47
relaxes to the ground state. It is possible is that shallow water Cnidarians are in fact capable of bioluminescence, but lack the dietary acquired coelenterazine substrate required to carry out the reaction. So far coelenterazine has only been found to be synthesized de novo by deep sea copepods (Haddock, Rivers and Robison 2001; Oba et al. 2009; Thomson, Herring and Campbell 1995). Studies on the bioluminescent jellyfish Aequorea victoria for example, have shown that individuals with coelenterazine removed from their diets are no longer capable of bioluminescence
(Haddock, Rivers and Robison 2001). Similar experimentation could be performed on other cnidarian species that possess photoproteins in future studies.
Observational and experimental evidence suggests that the photoprotein/coelenterazine system first arose as an antioxidant system before diversifying and developing a bioluminescent function (Rees et al. 1998) . It has been suggested that lower oxygen levels at greater depth may have relaxed the need for antioxidant function in deep water cnidarians, and through the accumulation of mutations, allowed bioluminescence to evolve. Photoproteins work in unison with fluorescent proteins to produce light emission in deep sea cnidarians, and this light is thought to attract prey (Haddock et al. 2005). It is possible that the original antioxidant function of this system has been favoured in shallow water dwelling cnidarians exposed to UV stress, such as many actiniarian and scleractinian species, which may provide an explanation for their lack of luminescence. To support this explanation, the majority of non-bioluminescent cnidarians with photoproteins were sessile and shallow water dwelling species, although the ecology of each individual species was not explored fully in this study.
48 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species
DNA repair genes Both genes involved in DNA repair, CPD photolyase and RAD23b were found to be highly conserved single copy genes present in all metazoan phyla. The phylogenetic relationships based on these genes reflected the currently known taxonomic relationships within phylum Cnidaria, indicating they may be useful for future phylogenetic studies when used together with other species. This suggests the process of DNA repair in intertidal cnidarians may be largely carried out with single copy genes, with no redundancy.
RAD23B is a crucial component of nucleotide excision repair (NER) in metazoans. This process repairs DNA without the direct involvement of light through the excision of the damaged segment from one DNA strand and the attachment of complementary nucleotides to the undamaged template strand (Dykens and Shick
1984). In NER, RAD23 is involved in DNA damage recognition and the recruitment of repair factors (Dantuma, Heinen and Hoogstraten 2009; Bergink et al. 2012). The expression of RAD23b has been shown to be induced in response to UV stress in larvae of the scleractinian coral, Montastraea favelolata (Aranda et al. 2011), and evidence of NER has also been inferred in the sea anemone Aiptasia pallida following exposure to UV and visible light exposure (Hudson and Ferrier 2008).
Based on this limited information, it may be assumed that RAD23 plays an important role in the UV stress response of other cnidarian species such as sea anemones, however more studies are needed to test this.
CPD photolyase is the only molecule involved in photoreactivation, an important shortwave light-induced DNA repair mechanism. In planulae larvae of the scleractinian coral species, Acropora millipora, it has been shown that UV induced
DNA damage is more effectively repaired in the presence of shortwave visible light,
Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species 49
compared to samples maintained in darkness, and this suggests that photoreactivation might play a dominant role in the coral UV induced DNA repair system (Reef et al.
2009). This is the first known study to examine the distribution of photolyase genes across phylum Cnidaria. CPD photolyase was found to be highly conserved, and present in all cnidarian species investigated, and it is likely that this enzyme plays an important role in UV induced DNA repair in cnidarians. Whether CPD photolyase is differentially expressed in response to UV stress in cnidarians remains to be investigated.
Conclusions
Information on the UV stress response of Actinia tenebrosas is limited to only two studies; an observational and physiological study conducted in South Australia
(Ottaway, 1973), and a biochemical study in New Zealand (Cubillos et al., 2014).
The study presented here adds to this existing research by identifying and analysing the evolutionary history of candidate UV screening, antioxidant and DNA repair genes within cnidarians through comparative and phylogenetic analysis. UV stress genes were found to be widely distributed across phylum Cnidaria. The presence of multiple GFP copies in most cnidarian species was confirmed, as was the presence of multiple independent lineages of chromoproteins in cnidarians. A new chromoprotein lineage was uncovered, and a novel example of convergent evolution from the A. tenebrosa and C. polypus blue chromoprotein discovered. This study provides a first look at the photoprotein family evolution and found multiple copies of photoprotein genes in most cnidarian species examined. Three distinct photoprotein clades were discovered, and this bioluminescent-associated protein was present in the majority of cnidarian species, most of which are not known to bioluminesce. Finally, a first look at the evolution of the DNA repair genes, CPD
50 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species
photolyase and RAD23b was provided, and they were found to be single copy genes and highly conserved in cnidarians. Examining the expression of these genes under realistic UV stress conditions helps to elucidate their role in UV stress response.
The information from this study provides a foundation for more detailed examinations into A.tenebrosa’s survival in the intertidal zone. Future transcriptomic studies of A.tenebrosa can add to this information by examining differential expression of UV stress response genes between UV stressed and non-stressed individuals. For example, the relative importance of screening pigments such as chromoproteins can be examined. In addition, the genes and pathways involved DNA repair such as photoreactivation and nucleotide excision repair can be looked at more closely. By examining temporal patterns of gene expression under UV stress in A. tenebrosa, a more holistic understanding of its resilience to this ubiquitous stress can be established.
Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species 51
52 Chapter 2: Comparative analysis of genes involved in UV response from cnidarian species
Chapter 3: Gene expression changes in
response to sunlight stress in the
intertidal cnidarian, Actinia tenebrosa
Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa 53
Abstract
The sedentary intertidal sea anemone, Actinia tenebrosa, is able to survive direct and prolonged periods of sunlight despite having only a single layer of epithelial cells and no other anatomical features to provide physical protection. How
A. tenebrosa survives sunlight stress is poorly understood and limited to only a few physiological and biochemical studies and additional genomic information is needed.
Studies that measure gene expression changes in A. tenebrosa under sunlight stress are needed to better understand their sunlight stress resilience. The first detailed investigation of candidate sunlight stress genes was conducted in this study. This included blue chromoprotein (UV and visible light screening/antioxidant), CPD photolyase (DNA repair), and Cu-Zn SOD and Fe/Mn SOD (antioxidants) from the
A. tenebrosa, at the northern limit of its distribution, where sunlight stress is at its most extreme (Australian Government Bureau of Meterology 2015; World Health
Organization 1995). Actinia tenebrosa individuals were subjected to four sunlight stress treatments (submerged dark, submerged light, exposed dark and exposed light) and qPCR was used to determine patterns of sunlight stress induced gene expression in four candidate genes. Comparative and phylogenetic analysis across actiniarians was conducted to provide some background information about the candidate genes and to better understand their evolutionary history. It was found that CPD photolyase and Fe/Mn SOD expression significantly increased over time in the submerged light treatment, but not in the exposed light treatment, and that UV and PAR were the best environmental predictors for the increase. It can be concluded that desiccation stress may invoke a different response, and that the up-regulation of CPD photolyase and
Fe/Mn SOD may not be as important when the organism is aerially exposed. In addition, the first detailed comparison of the candidate gene families was conducted,
54 Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa
and two new full-length Fe/Mn SOD gene families that have not been previously identified in Cnidarians were identified.
Introduction
Intertidal sea anemones (order Actinaria), are regularly exposed to harmful levels of sunlight (i.e. UV radiation, visible light and temperature stress) when exposed at low tide. Sea anemones cope with UV exposure in part due to the cellular stress response (CSR), a universal molecular mechanism largely conserved across metazoan lineages (Kültz 2005). Understanding how intertidal sea anemones survive sunlight stress requires an understanding of the genes that underpin three critical
CSR components: UV and visible light screening, antioxidants, and DNA repair mechanisms.
Intertidal organisms are known to produce a variety of sunlight screening molecules, which function by intercepting and scattering, or by absorbing harmful incoming wavelengths of sunlight. The most well-known and widely studied sunlight molecules in marine invertebrates are mycosporine-like amino acids (MAAs) (Shick and Dunlap 2002), which are produced in sea anemones in response to UV stress
(Cubillos et al. 2014). The importance of other UV and visible light screening molecules in sea anemones such as the green fluorescent protein (GFP) family remains less clear. GFPs have been demonstrated to be upregulated in response to
UV stress in various intertidal scleractinian coral species (Roth et al. 2010; D’Angelo et al. 2008; Aranda et al. 2011), which suggests that this family of molecules is an important component of the cnidarian sunlight stress response. GFPs also have structures that enable them to absorb visible (PAR) and near UV wavelengths (Salih et al. 2000; Pakhomov and Martynov 2008). While GFPs have been identified in
Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa 55
numerous cnidarian species, it is unknown whether they play a similar role in intertidal anemone species.
Sunlight stress can indirectly damage intertidal organisms through the generation of ROS, which can cause oxidative stress damage to virtually all cellular component (Stochaj, Dunlap and Shick 1994; Friedberg 2003) . Cells are able to neutralize the effects of ROS through the actions of antioxidants such as SODs.
SODs are a family of ancient and highly conserved antioxidant enzymes found in all domains of life (Landis and Tower 2005), and function by catalysing superoxide oxidation to molecular oxygen, and by inducing the reduction of hydrogen peroxide free radicals. Two important SOD families include the cytosolic and/or extracellular copper/zinc containing SODs (Cu-Zn SODs), and the mitochondrial manganese/iron containing SODs (Fe/Mn SODs) (Landis and Tower 2005). It has been demonstrated that Cu-Zn SODs and Fe/Mn SODs are upregulated in response to UV stress in the estuarine anemone Nematostella vectensis, and it remains to be known whether these genes are similarly upregulated in sea anemone species that live in the intertidal zone.
DNA damage is arguably the most harmful cellular effect of sunlight stress and both UV and oxidative stress can induce the formation of cyclobutane pyrimidine dimers (CPDs), which can impair transcription and potentially lead to mutagenesis
(Ananthaswamy 1997). The most efficient form of UV damage repair is photoreactivation, a light activated DNA repair mechanism that involves a single enzyme, CPD photolyase (Sancar 1996b; Sancar and Tang 1993). Various coral species have been shown to repair UV induced DNA damage more effectively in the presence of shortwave visible light, compared to samples maintained in darkness, which suggests that photoreactivation might play a dominant role in the cnidarian
56 Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa
UV induced DNA repair system (Reef et al. 2009; Siebeck 1981). To date, no studies have investigated whether CPD photolyase is upregulated in response to sunlight stress in cnidarian species.
The Waratah anemone, Actinia tenebrosa, is an ideal candidate for examining the genes underlying sunlight stress tolerance. Actinia tenebrosa is the most abundant intertidal sea anemone along the Australian coastline, and is exposed to high temperatures and UV, especially at the northern limit of its distribution range
(Australian Government Bureau of Meterology 2015; World Health Organization
1995). The current understanding of sunlight tolerance of Actinia tenebrosa is limited to two studies; an observational and physiological study conducted in South
Australia (Ottaway, 1973), and a biochemical study in New Zealand (Cubillos et al.,
2014). While providing groundwork for understanding how A. tenebrosa survives sunlight stress, the genomic basis for A. tenebrosa’s sunlight stress response is yet to be established. Furthermore, there is a lack of comparative analysis of sunlight stress response genes within actiniarians, and this needs to be undertaken in order to better understand the evolutionary history of these genes. The response of A. tenebrosa to sunlight stress needs to be examined at the northern limits of their distribution range: previous studies were conducted only in the southern-most regions of A. tenebrosa’s distribution where UV levels and temperatures are at their lowest (Australian
Government Bureau of Meterology 2015; World Health Organization 1995).
This study extends the early observational, physiological and biochemical knowledge through the addition of new genomic information to provide a broader understanding of how A. tenebrosa and other anemone species survive sunlight stress. The aims of this study were to (i) develop an experimental design reflective of natural conditions at the northern limits of A. tenebrosa’s distribution (ii) examine
Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa 57
physiological differences of A. tenebrosa individuals to different sunlight stress conditions, (iii) measure expression changes of the candidate stress genes under these conditions and (iv) undertake a comparative analysis of candidate sunlight stress response genes across actiniarians.
Materials and methods
Sample collection, stress experiment and RNA extraction
Field sampling of Actinia tenebrosa took place at Point Cartwright, Australia
(26°32'9.83"S, 153° 5'45.12"E) (Figure 3.1A). Environmental variable measurements and observations were taken inside every 2m2 within a 10m2 grid (Figure 3.1A, 3.1B)
(Table S3.1). Individuals and colonies of A. tenebrosa were categorized based on aerial exposure and light (i.e. exposed and dark, exposed and light, submerged and dark, submerged and light) (Table S3.2). Other sea anemone species that were found within the 10m2 grid were also identified (Figure S3.7). Actinia tenebrosa individuals
(n=60) were collected and stored in 50ml containers of sea water before being transported to the marine facility at QUT Gardens Point campus.
58 Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa
Figure 3.1 (A) Aerial photograph of study site with 10m2 sampling grid highlighted in red; (B) field site facing east; (C) sun exposed treatment sample; (D) 50L housing tank.
Actinia tenebrosa individuals were acclimated for two weeks in 50L housing tanks (Figure 3.1D) under controlled laboratory conditions with a 8h:16h light: dark cycle with lighting provided by overhead fluorescent and UV lights, and the water was changed every two days. For more details on the water conditions of the housing tanks please refer to Table S3.3. To measure visible light, PAR was recorded using a
Li-1400 (LI-COR, Lincoln, USA). UV index and weather data was obtained from the
BOM website (Australian Government Bureau of Meteorology, 2015). Air temperature and humidity were recorded using a HOBO U23 Pro v2
Temperature/Relative humidity data logger (ONSET, Bourne, USA). Dissolved
Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa 59
oxygen, water temperature, salinity, and pH were recorded using a Multi 3430 IDS multi-parameter portable meter (WTW, Weilheim, Germany).
Following acclimation, A. tenebrosa individuals (n=48) were randomly allocated to experimental conditions. To provide an indication of the physiological effects of sunlight stress, the volume of all A. tenebrosa individuals was measured before and after treatments using the methods described by (Ottaway 1973). Three biological replicates were then subjected to one of four common, naturally observed exposure categories (i.e. unshaded and submerged, shaded and submerged, unshaded and exposed, shaded and exposed). This experiment occurred at QUT Gardens Point campus Q block (Figure 3.1C) across four time points: a control time point at
10:00am, and stress time points at 12:00pm, 2:00pm and 4:00pm. Environmental variable measurements and observations were taken at each time point with the same equipment used at the field sample site (Table S3.4). At the end of each time point, three animals (3 biological replicates) were removed and re-measured. The measurements recorded for each individual was used to establish volume changes following the stress treatments (Table S3.5). Immediately after measuring they were frozen in liquid nitrogen and stored at -80ºc. Individual animals were then homogenized in liquid nitrogen, before total RNA was extracted using a
Trizol/chloroform protocol (Invitrogen).
Selection of candidate genes and primer validation
Four candidate sunlight stress genes and the housekeeping gene 18S rRNA were selected from the A. tenebrosa transcriptome for qPCR analysis. The stress candidates were selected from the UV and visible light screening, DNA repair and antioxidant categories and contained domains consistent with their assigned function
(Table S3.6). Specifically, they included blue chromoprotein, CPD photolyase, Cu-
60 Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa
Zn Superoxide dismutase, Mn/Fe superoxide dismutase and 18s rRNA. To validate the presence of candidate genes found in the A. tenebrosa transcriptome, all qPCR primer pairs (blue chromoprotein, CPD photolyase, Cu-Zn superoxide dismutase,
Mn/Fe superoxide dismutase and 18s rRNA) (Table S3.7), and four RT PCR primer pairs (blue chromoprotein, CPD photolyase, SOD and Ferritin) were designed using
Geneious version 8.0.4 (Kearse et al. 2012), (Table S3.8).
Following RNA extraction, cDNA was synthesised using a Sensi Mix kit
(Bioline) as per manufacturer’s instructions. PCR amplification was conducted in accordance to the MyFiTM (Bioline) protocol with the following modifications: 12.5
µL of MyFi, 2µL of each primer (10µM concentration), 6 µL ddH2O and 2µL of cDNA to a total of 25 µL. PCR conditions were as follows: 3 min at 95⁰C, followed by 30 cycles of 30 sec at 95⁰C, 30 sec at 51⁰C, 45 sec at 72⁰C, then a final extension of 5 min at 72⁰C. Amplicons were purified using an Isolate II PCR Kit (Bioline) and cycle sequencing was carried out using BigDyeTM Terminator v3.1 Cycle Sequencing
Kit (Life technologies) with the following modifications: 13.5 µL ddH2O, 3.5 µL seq buffer, 1.0 µL BigDyeTM, 1.0 µL primer (3.2 pmol) and 1.0 µL DNA template.
Sequencing products were cleaned up using an Ethanol/EDTA precipitation and run on an ABI 3500 Genetic analyser (Life Technologies). All sequences were visualised and edited in Geneious version 8.0.4. Sequences were then aligned to the original sequences from which primers were designed and also used as validation.
qPCR quantification of gene expression
qPCR was performed on each sample using the Bioline One-Step Real Time
PCR kit. These reactions were performed using the Roche lightcycler machine, measuring specific fluorescence at each cycle, quantifying the initial levels of mRNA for each gene in each biological replicate. All quantitative PCR analyses were
Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa 61
repeated in three technical replicates to determine the validity of results along with negative controls for each gene in each sample and 18S as a reference gene for all samples. qPCR data was exported and viewed in Lightcylcer96 v1.1 software.
Normalization was then performed on each of the 24 samples for each tissue using
18S as the internal reference gene to determine fold-differences in expression of the five target genes. Following normalization of Cq values for all samples, a total of three outliers were removed from the dataset prior to ratio mean calculations for each gene in each condition. Relative quantification excluding efficiency was performed using the comparative 2−ΔΔCt method (Livak and Schmittgen 2001) to obtain relative expressions for each gene of interest.
Statistical analysis
To determine the significance of the experimental conditions on the change in volume of A. tenebrosa individuals, ANOVA was used with change in volume as the dependent variable, and time, exposure and light as fixed factors. Significant relationships were further explored using linear regressions between changes in volume with the corresponding environmental variables. To determine the significance of the experimental conditions on the expression of the candidate genes,
ANOVA was used with the gene expression ratio as the dependent variable, and time, exposure and light as fixed factors. Significant relationships were further explored using linear regressions between gene expression ratios with the corresponding environmental variables.
Comparative and phylogenetic analysis
Full-length candidate stress genes were determined using ORFs generated by
Transdecoder and ORF Finder (NCBI), followed by BLASTp analysis and alignment to other full length proteins. Functional domains for each candidate were determined
62 Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa
using the HMMER Pfam database (EMBL-EBI). Enzyme codes from each candidate were used to determine their presence in corresponding KEGG pathways (Kanehisa et al. 2014; Kanehisa and Goto 2000). The presence and absence of candidate genes in cnidarian species was determined using BLAST searches against publicly available transcriptome and proteome datasets, and hits with E-values <10-5 were retained for further comparative and phylogenetic analysis. Four unpublished sea anemone transcriptomes were also included in the analysis: Anthopleura buddemeieri, Aulactinia veratra, Calliactis polypus and an unidentified Telmatactis sp.. Specifically, 11 sea anemone transcriptomes, two sea anemone genomes
(Nematostella vectensis, Aiptasia pallida) and one scleractinian genome as an outgroup (Acropora digitifera) were interrogated (Table S3.20. Due to the amount of information available online for the GFP gene family, the chromoprotein candidate was used as a BLASTn query against the NR database (NCBI), and naturally occurring homologues with E-values <10-5 were retrieved and added. For phylogenetic analysis, only the top five hits for each candidate gene from each taxa was included.
Alignment of candidate genes was performed using a ClustalW codon alignment in MEGA v6 (Tamura et al. 2013a). Tests for positive selection were performed on aligned gene sequences by estimating the strength of selection operating on each individual codon in the alignment using HyPhy in MEGA v6. The appropriate model of evolution for all candidate alignments was generalised time- reversible (GTR) with proportion of invariant sites (I) along with gamma-distributed among site rate variation (G) using jModel Test 2 (Darriba et al. 2012). Maximum- likelihood (ML) analysis was performed using the program RAxML v8.1.24
(Stamatakis 2006), invoking a rapid bootstrap (1000 replicates) analysis and search
Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa 63
for the best-scoring maximum likelihood tree with the general time-reversible model of DNA sequence evolution with gamma-distributed rate heterogeneity
(GTRGAMMA substitution model). In maximum-likelihood trees bootstrap support values were defined as follows: < 70 as poor, 70-85/ as average, and > 85 as good
(modified from Hillis and Bull (1993)). Consensus trees from ML outputs were drawn in FigTree v1.4.2 (Rambaut 2014) then annotated with bootstrap values in
Adobe Illustrator CC 2014.1.0 (Adobe Systems Incorporated, San Jose, CA, USA).
Results
Comparison of environmental measurements between the field site, housing facilities and experimental site
The physical and chemical water variables measured at the housing facilities for the 14 day acclimation period fell within the range observed at the field site
(Tables S3.1, S3.3). The recorded environmental variables during the stress experiment fell within the range of measurements recorded at the field site (Tables
S3.3 and S3.4).
The effects of sunlight stress on animal volume
The volume of A. tenebrosa samples continually decreased over time in the two exposed treatments, but remained relatively unchanged in the two submerged treatments (Table S3.5) (Figure 3.2). Significant relationships were found in a 2-way interaction between volume change and light and exposure (F1,71=26.992, p=0.0001), as well as a 2-way interaction between volume change and time and exposure
(F2,71=3.504, p=0.035) (Table S3.9). There was statistically significant interactions between volume change and light (F1,71=20.321, p=0.0001) and between volume change and exposure (F1,71=127.128, p=0.0001) (Table S3.9).Volume changed
64 Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa
significantly over time within the exposed dark and the exposed light treatments
(Table 3.2). No significant relationship was found between loss of volume with air temperature, humidity, UV or PAR when examining the exposed dark treatment or exposed light treatments individually (Tables S3.10, S3.11). A significant relationship (R2=0.415, p=0.004) was found between loss of volume and air temperature (Figure S3.1) (Table S3.12) when exposed treatments were combined.
Table 3.2 Results from an ANOVA conducted to assess the significance of time on volume change within each treatment type.
Treatment type numDF denDF F-value p-value Submerged, Dark 3 17 3.589 .036 Submerged, Light 3 17 1.262 .319 Exposed, Dark 3 17 6.183 .005 Exposed Light 3 17 19.516 .000
Figure 3.2 Comparison of volume change within different treatment types (i.e. control, submerged and dark, submerged and light, exposed and dark, exposed and light) over time. Error bars represent standard error.
Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa 65
The effects of sunlight stress on gene expression
Overall, across the four candidate genes, the highest expression level was recorded in blue chromoprotein (Figure 3.3), followed by Cu-Zn SOD (Figure 3.4),
Fe/Mn SOD (Figure 3.5) and CPD photolyase (Figure 3.6).
Blue chromoprotein
Blue chromoprotein gene expression appeared to be lower than the control for each stress treatment, and did not appear to change over time in any of the stress treatments (Figure 3.3). The exposed dark treatment at time point 4 was one exception, being considerably higher than the other recorded measurements, although this sample had very high standard error. There was no significant interaction between blue chromoprotein gene expression and light, exposure and time, or any combination of these variables (Table S3.13). Blue chromoprotein gene expression did not change significantly over time within each treatment type (Table 3.3).
Table 3.3 Results from an ANOVA conducted to assess the significance of time on candidate gene expression ratio within each treatment type.
Candidate gene Treatment type numDF denDF F-value p-value Blue Submerged, Dark 3 17 0.544 0.659 Chromoprotein Submerged, Light 3 17 0.512 0.680 Exposed, Dark 3 17 0.535 0.664 Exposed Light 3 17 0.579 0.637 CPD Submerged, Dark 3 17 0.873 0.475 photolyase Submerged, Light 3 17 7.448 0.002 Exposed, Dark 3 17 0.907 0.458 Exposed Light 3 17 0.292 0.830 Cu-Zn SOD Submerged, Dark 3 17 0.934 0.446 Submerged, Light 3 17 0.888 0.467 Exposed, Dark 3 17 1.254 0.304 Exposed Light 3 17 0.904 0.460 Fe/Mn SOD Submerged, Dark 3 17 1.023 0.407 Submerged, Light 3 17 4.247 0.021 Exposed, Dark 3 17 0.164 0.919 Exposed Light 3 17 3.182 0.051
66 Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa
Figure 3.3 Comparison of blue chromoprotein gene expression ratio within different treatment types (i.e. control, submerged and dark, submerged and light, exposed and dark, exposed and light) over time. Error bars represent standard error.
CPD photolyase
CPD photolyase gene expression appeared to increase overtime only in the submerged light treatment (Figure 3.4). A significant relationship was found in a 3- way interactions between CPD photolyase gene expression and light, exposure and time (F2, 71=3.736, p=0.029), and also in a 2-way interaction with light and exposure
(F1, 71=4.322, p=0.041) (Table S3.14). CPD photolyase gene expression within each treatment changed significantly in submerged light treatment (F3, 17=7.448, p=0.002)
(Table 3.3). CPD photolyase gene expression correlated with UV in the submerged light treatment, and this relationship was significant (R2 =0.254, p=0.02) (Figure
S3.2). No significant relationship was found between CPD photolyase gene
Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa 67
expression with PAR, salinity, DO or water temperature in treatment 2 (Table
S3.15).
Figure 3.4 Comparison of CPD photolyase gene expression ratio within different treatment types (i.e. control, submerged and dark, submerged and light, exposed and dark, exposed and light) over time. Error bars represent standard error.
Cu-Zn SOD
Cu-Zn SOD gene expression did not appear to increase overtime in any of the treatments (Figure 3.5). There was no significant interactions between Cu-Zn SOD gene expression and exposure, light, or time (Table S3.16). Cu-Zn SOD gene expression did not significantly change over time in any of the stress treatments
(Table 3.3).
68 Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa
Figure 3.5 Comparison of Cu-Zn SOD gene expression ratio within different treatment types (i.e. control, submerged and dark, submerged and light, exposed and dark, exposed and light) over time. Error bars represent standard error.
Fe/Mn SOD
Gene expression appeared to increase overtime for the two light saturated treatments, although the standard error for time point 4 for treatment 4 was relatively high (Figure 3.6). Significant relationships were found in a 3-way interactions between Fe/Mn SOD gene expression and time, light and exposure (F2,71=4.929, p=0.010), and in a 2-way interactions between Fe/Mn SOD gene expression and time and exposure (F2,71=6.596, p=0.002) (Table S3.17). There was statistically significant interactions between Fe/Mn SOD gene expression and light (F1,71=4.204, p=0.044)
(Table S3.17). Fe/Mn SOD gene expression within each treatment changed significantly in the submerged light treatment (F3,17=4.247, p=0.021) (Table 3.3). In
Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa 69
treatment 2 higher Fe/Mn SOD gene expression correlated with higher UV and
PAR, and this relationship was significant for both UV (R2 =0.25, p=0.021) (Figure
S3.3) and PAR (R2 =0.2, p=0.042) (Figure S3.4). No significant relationship was found between Fe/Mn SOD gene expression with any other environmental variables
(Tables S3.18 and S3.19).
Figure 3.6 Comparison of Fe/Mn SOD gene expression ratio within different treatment types (i.e. control, submerged and dark, submerged and light, exposed and dark, exposed and light) over time. Error bars represent standard error.
Comparative and phylogenetic comparison
Blue chromoprotein
The full-length blue chromoprotein found in A. tenebrosa had an open reading frame 699 nucleotides (232 amino acid residues). Its domain structure consisted of a single GFP domain (Table S3.6). Homologs of the A. tenebrosa chromoprotein were
70 Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa
found in all actiniarian taxa, except for Nemanthus sp. (10 out of 11 taxa) (Table
S3.20). These homologues consisted of both fluorescent proteins and chromoprotein
GFPs. Using BLASTp against the NCBI database, homologs in an additional nine actiniarian taxa were found and included in the analysis of this gene family. Of the
20 actiniarian taxa found to have GFP sequences, 18 had full-length sequences and
11 of these 18 taxa had more than one full-length GFP gene. All fluorescent proteins and chromoprotein had the same domain structure. The A. tenebrosa contig annotated as a blue CP shared 51.8% amino acid identity with its closest homolog found in N. vectensis. From the combined list of 30 full-length actiniarian GFPs, 24 were CPs, and 14 were FPs. Phylogenetic analysis of the actiniarian CPs using maximum-likelihood inference were consistent with the chapter two investigation of the GFP gene family across cnidarians (Figure 2.4, Figure S3.5).
CPD photolyase
The full-length CPD photolyase found in A. tenebrosa had an open reading frame 1683 nucleotides (560 amino acid residues). Its domain structure consisted of a
DNA photolyase domain and a FAD binding 7 domain (Table S3.6). Homologs of the A. tenebrosa CPD photolyase were found in all actiniarians (11 out of 11 taxa)
(Table S3.20). Of the 11 actiniarian taxa found to have CPD photolyase sequences, eight had full-length sequences. There were no actiniarians with more than one full- length CPD photolyase gene. All CPD photolyase had the same domain structure.
The A. tenebrosa contig shared 80.8% amino acid identity with its homolog found in
A. pallida, and 75.4% amino acid identity with N. vectensis. Phylogenetic analysis of actiniarian CPD photolyase using maximum-likelihood inference revealed a species tree consistent with existing actiniarian species trees (Rodríguez et al. 2014) and was
Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa 71
consistent with the chapter two investigation (of this family across cnidarians (Figure
S2.6, Figure S3.6).
Fe/Mn SOD
The full-length Fe/Mn SOD ORF found in A. tenebrosa (contig ID: c60410_g1_i1) had an open reading frame 552 nucleotides (1183 amino acid residues). Its domain structure consisted of a Fe/Mn superoxide dismutase, alpha- hairpin domain and a Fe/Mn superoxide dismutase c-terminal domain (Table S3.6).
Homologs of the A. tenebrosa Fe/Mn SOD were found in all actiniarians (11 out of
11 taxa) (Table S3.20). Of the 11 actiniarian taxa found to have Fe/Mn SOD sequences, 10 had full-length sequences. All actiniarians had more than one full- length Fe/Mn SOD gene with the exceptions of Anthopleura elegantissima and
Nematostella vectensis.
The Fe/Mn SOD sequences in this study were different to the previously reported Fe/Mn SOD sequences from N. vectensis (Tarrant et al. 2014): the previously reported NvMnSOD1 and NvMnSOD2 shared 49.3% sequence homology with each other, whereas the N. vectensis contig (c30398_g1_i5) identified in this study shared only 37.8% sequence homology with NvMnSOD1 and 33.6% sequence homology with NvMnSOD2. The N. vectensis contig c30398_g1_i5 contained the same domain configuration as NvMnSOD1 and NvMnSOD2 (Table S3.21).
All actiniarian Fe/Mn SOD sequences examined in this study had the same domain structure. The two full-length A. tenebrosa Fe/Mn SOD contigs shared 30.9% amino acid identity with each other. The first A. tenebrosa contig (c60410_g1_i1) shared
74.5% amino acid identity with its closest homolog found in A. pallida, and 65.2% amino acid identity with N. vectensis. The second A. tenebrosa contig
72 Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa
(c90418_g1_i1) shared 87.1% amino acid identity with its closest homolog found in
A. pallida, and there was no significant match in N.vectensis. Phylogenetic analysis of the actiniarian Fe/Mn SOD using maximum-likelihood inference revealed two distinct and distantly-related clades (Figure 3.7).
Figure 3.7 Phylogenetic tree of Fe/Mn SOD gene from A.tenebrosa (shown in bold) and its homologues in other actiniarian taxa, with the scleractinian Acropora digitifera as the outgroup using maximum-likelihood inference.
Cu-Zn SOD
The full-length Cu-Zn SOD found in A. tenebrosa (contig ID: c62857_g1_i1) had an open reading frame 462 nucleotides (153 amino acid residues). Its domain structure consisted of a Copper/zinc superoxide dismutase (SODC) domain (Table
S3.6). All 11 actiniarian taxa were found to have full-length Cu/Zn SOD sequences.
All actiniarians had more than one full-length Cu/Zn SOD gene with the exception of
Aulactinia veratra. All Cu/Zn SOD had the same domain structure. The two full- length A. tenebrosa Cu/Zn SOD contigs shared 65.6% amino acid identity. The first
A. tenebrosa contig (c62857_g1_i1) shared 78.4% amino acid identity with A.
Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa 73
pallida, and 77.4% amino acid identity with N. vectensis. The second A. tenebrosa contig (c129237_g1_i1) shared 82.4% amino acid identity with A. pallida, and
69.9% amino acid identity with N. vectensis. Phylogenetic analysis of the actiniarian
Cu/Zn SOD gene family using maximum-likelihood inference revealed three distinct and strongly supported clades consistent with the findings of Tarrant et al.
(2014)(Figure 3.8). Relationships within each of these clades conformed to established actiniarian species trees (Rodríguez et al. 2014).
Figure 3.8 Phylogenetic tree of Cu-Zn SOD gene from A.tenebrosa (shown in bold) and its homologues in other actiniarian taxa, with the scleractinian Acropora digitifera as the outgroup using maximum-likelihood inference. Three independent lineages of actiniarian Cu-Zn SOD are highlighted.
Discussion
Physiological and gene expression response to sunlight stress
Intertidal organisms are frequently co-exposed to UV radiation, temperature stress and desiccation stress, all of which can induce the production of stress response mechanisms. While the effects of sunlight stress have been previously studied in corals, comparatively few studies have been conducted on other cnidarian species such as intertidal sea anemones species. There is a lack of knowledge
74 Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa
regarding gene expression in response to natural sunlight stress exposure in intertidal sea anemones that inhabit regions with high levels of UV and temperature.
This study represents the first detailed investigation of candidate sunlight stress genes (i.e. blue chromoprotein, CPD photolyase, Cu-Zn SOD and Fe/Mn SOD) from the transcriptome of the intertidal sea anemone Actinia tenebrosa, at the northern limit of its distribution, where sunlight stress is at its most extreme. Actinia tenebrosa individuals were subjected to four sunlight stress treatments (submerged dark, submerged light, exposed dark and exposed light) and qPCR was used to identify whether any of the candidate genes were be induced by intertidal zone stressors. The experiment also measured change in volume as a physiological effect of the stress treatments. Comparative and phylogenetic analysis across actiniarians was conducted to provide some background information about the candidate genes and to better understand their evolutionary history.
Aerial exposure and light were found to be strong predictors of anemone volume loss, with light exposed anemones losing more volume on average than shaded anemones. Air temperature was the only variable found to correlate with volume loss in the exposed treatments, which may explain why the light exposed anemones lost more volume than the shaded anemones. This finding was consistent with Ottaway (1973), who reported a similar result for A. tenebrosa near the southerly extreme of their distribution.
The expression of genes from two SOD gene families, Fe/Mn SOD and Cu-Zn
SOD have previously been reported to be upregulated in response to UV stress in sea anemones (Tarrant et al. 2014). An increase in Fe/Mn SOD expression in the submerged light treatment was observed and the most significant predictor was a 2- way interaction between time and exposure. UV and PAR were the best
Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa 75
environmental variable predictors of this increase. Interestingly, no increase was observed in the exposed light treatment. It is possible that desiccation stress induces a range of other cellular responses which may be a higher metabolic priority than sunlight damage. There was no evidence to suggest Cu-Zn SOD expression increased in any of the treatments.
CPD photolyase is an important light activated DNA repair enzyme that plays an important role in the UV stress response in many organisms, but its role in actiniarians has not been directly explored. In this study CPD photolyase expression increased in the submerged light treatment, and UV was the best predictor of this increase. This finding was similar to the pattern observed in Fe/Mn SOD, and a similar conclusion is made: that desiccation stress may override the importance of
DNA repair during aerial exposure.
Chromoproteins are thought to be effective antioxidants in some cnidarians
(Salih et al. 2000; Shick and Dunlap 2002), but their role in A. tenebrosa and other cnidarians is yet to be determined. There was no evidence of blue chromoprotein being differentially expressed in response to UV stress, which suggests that these molecules do not play a role in the sunlight stress response of A. tenebrosa, and may therefore have a different and unknown physiological function in sea anemones. The bioluminescent hydrozoan, Erenna sp. uses fluorescent filaments as lures to attract fish (Haddock et al. 2005), and it is possible that in sea anemones, the chromoproteins and fluorescent proteins may have evolved a similar function. For example, a blue chromoprotein in C. polypus is only found around its oral disk in combination with a fluorescent protein, which may act as a prey attractant.
76 Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa
Comparative and phylogenetic analysis of candidate genes in actiniarians
Comparative and phylogenetic analysis provided important information about the evolution and diversification of genes within actinarians. This study documents the first detailed comparison of the candidate gene families (i.e. BC, CPD, Cu-Zn and Fe/Mn SOD) across actiniarians. Two new full-length Fe/Mn SOD sequences were found that have not been previously identified in Cnidarians. Metazoans typically only have one Fe/Mn SOD gene, and previous studies from N. vectensis have demonstrated that sea anemones have two (Tarrant et al. 2014), however, the results of this study suggest that sea anemones have four members from this gene family. This study represents the first documentation of a cnidarian order (i.e.
Actiniaria) with four Fe/Mn SOD genes. Given the limited sequence identity between these genes, it probably arose via an ancient gene duplication event early in their evolutionary history. Cu-Zn SOD sequences formed three distinct clades within the actiniarians. This result was consistent with the established sea anemone phylogeny of (Rodríguez et al. 2014), and with the previously established Cu-Zn
SOD gene tree across metazoans (Tarrant et al. 2014). The BC and CPD photolyase gene trees were consistent with the findings of chapter two of this thesis, which looked at their distribution across cnidarians, and conformed to the relationships found in the most comprehensive and well resolved sea anemone phylogeny
(Rodríguez et al. 2014).
Conclusion
This investigation of candidate stress genes in A. tenebrosa provides a first look at how an intertidal sea anemone tolerates sunlight stress. Overall, the expression of two candidate stress genes, CPD photolyase and Fe/Mn SOD,
Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa 77
significantly increased in response to sunlight when submerged, but not when exposed to aerial desiccation.
This study was designed to emulate natural sunlight exposure conditions to provide an indication of the response of A. tenebrosa to sunlight stress in situ.
Information collected from the housing facilities and stress experiment where indicative of the conditions at the field collection site. Although the intention of the design was to match natural conditions as close as possible, there are a number of factors that may have influenced the results. For example, it was not possible to simulate tidal fluctuations or natural sunlight stress in the housing tanks, and this may have influenced gene expression in the aerially exposed treatments. Also, there were not an equal number of time points distributed across either side of the maximum noon sunlight exposure time point. The dramatic decrease in volume after only two hours of aerial exposure suggests that more time points within the first two hours are needed. In addition, the high standard error observed in many comparisons may have significantly influenced the results, and was most likely the result of under replication in the gene expression study.
This qPCR study provides a limited insight into the transcriptional response to sunlight stress in an intertidal sea anemone species. While it may indirectly show some gene expression levels corresponding to stress, it is important to consider that the genes in this study constitute only a tiny fraction of the sunlight stress response, and other important categories of stress response genes such as HSPs were not investigated here. Future research could involve transcriptome-wide measurements of changes in gene expression, which would provide a more comprehensive understanding of what is going on at the cellular level, and may also uncover novel mechanisms deployed by A. tenebrosa in response to sunlight stress.
78 Chapter 3: Gene expression changes in response to sunlight stress in the intertidal cnidarian, Actinia tenebrosa
Chapter 4: General discussion
Chapter 4: General discussion 79
The previous research chapters have focused on developing an A. tenebrosa transcriptome as a genomic resource for understanding its response to sunlight stress and the evolution of stress genes across actiniarians. In chapter two of this thesis, sunlight stress response genes were identified and four candidate genes from UV and visible light screening, antioxidant and DNA repair categories were analysed in more depth using comparative and phylogenetic methods. Following on from this, in chapter three a sunlight stress study was conducted and qPCR used to understand the expression of candidate genes in response to intertidal stressors. The combined results of these chapters helped to better understand A. tenebrosa’s sunlight stress resilience, and to provide useful information to infer the function of candidate sunlight stress response genes. In this final chapter, the results of the previous research chapters were summarized and direction for future sea anemone stress response studies provided. The wider implications of this thesis were then discussed.
Summary of results
The central focus of this thesis was to explore how an intertidal sea anemone,
A. tenebrosa, survives sunlight stress. The previous A. tenebrosa intertidal stress studies have focused on the physiological (Ottaway 1973) and biochemical (Cubillos et al. 2014) response to sunlight stress, but there was no molecular information about this species stress response. Consequently, new transcriptomic information was developed in this study to help address this knowledge gap.
In the first aim of this thesis the gene repertoire that potentially confers UV resilience in A. tenebrosa was investigated. The A. tenebrosa transcriptome was sequenced and assembled as a genomic reference in which the genes involved the
UV stress response were identified, and the majority of UV stress response genes in
A. tenebrosa were found to be widely distributed across phylum Cnidaria. A
80 Chapter 4: General discussion
comparative and phylogenetic analysis of candidate sunlight stress response genes was conducted and it was found that while the DNA repair genes CPD photolyase and RAD23b existed as single copy genes, the antioxidant blue chromoprotein and photoprotein were found to be multi-copy genes in the majority of cnidarian taxa examined.
In the second aim of this thesis the first investigation of expression of sunlight stress response genes in A. tenebrosa was conducted. Cyclobutane pyramindine photolyase and Fe/Mn SOD expression significantly increased over time in the submerged light treatment, and the best environmental predictors for the increase in expression for these genes was UV for CPD photolyase and both UV and PAR for
Fe/Mn SOD. The conclusion made in this study was that the combined effects of desiccation stress and sunlight stress may have invoked a different response to sunlight stress when individuals are submerged, and implied that other responses may be more critical to survival when the organism is aerially exposed.
Potential research gaps and future direction
In chapter two, most of the genes involved in sunlight stress were found to have a wide distribution in cnidarian species. This result is not surprising given that a number of the proteins encoded by these genes have multiple roles in the cellular stress response. The heat shock proteins (HSPs) for example, are known to be expressed in response to virtually any type of stress (Feder and Hofmann 1999). In fact, most of the proteins identified as important in screening, antioxidant and DNA repair in sunlight stress response also perform these functions in the cellular stress response. A possible exception to this rule is blue chromoprotein, a protein with potential functions in both a screening and antioxidant role under sunlight stress in cnidarians.
Chapter 4: General discussion 81
Five independent origins of chromoproteins in cnidarians were found. The repeated evolution of proteins of similar functions indicates that chromoproteins are likely to have a functionally important role in cnidarians. This pattern shows that the different clades of chromoproteins have evolved through convergence from an ancestral fluorescent protein. Within a large chromoprotein clade another potential example of convergent evolution was also found involving the blue chromoprotein of
A. tenebrosa and C. polypus. An interesting question that remains to be answered is whether the blue colouration of acrorhagi from A. tenebrosa, and the oral disk of C. polypus are the sources of blue chromoprotein in these two species. A good starting point for understanding this evolutionary novelty would be to determine if blue chromoproteins are in fact concentrated in these areas. Mass spectrometry and/or molecular methods of determining tissue specific expression of this gene and protein could be used to resolve this question.
P hotoprotein genes involved in the generation of bioluminescence were identified in the majority of cnidarian taxa, including A. tenebrosa. This was an unusual discovery given that the majority of these cnidarian species have not been observed to produce bioluminescence. One explanation is that the bioluminescent function of this protein has been lost in many species but retained in others. An alternative explanation is that cnidarian species with photoprotein, such as A. tenebrosa, are capable of producing bioluminescence but the catalyst required for this reaction to occur, coelenterazine, is absent. Coelenterazine cannot be synthesized de novo by cnidarians and must be acquired through their diet (Haddock, Rivers and
Robison 2001). It would be informative from an evolutionary perspective to test this experimentally by feeding A. tenebrosa and other non-bioluminescent species deep- sea copepods that are known to produce this enzyme de novo (Haddock, Rivers and
82 Chapter 4: General discussion
Robison 2001). This has already been demonstrated in the hydrozoan Aequorea victoria (Haddock, Rivers and Robison 2001), and similar studies on non- bioluminescent species could help to better understand the function and distribution of this protein.
In chapter three qPCR was used to provide insight into the transcriptional response of candidate genes involved in sunlight stress response. This approach was very selective and limited to only four genes. Future studies could use transcriptome- wide measurements of changes in gene expression under sunlight stress response conditions which would provide a more holistic understanding of the response of A. tenebrosa to sunlight stress, and in addition, provide a better opportunity for the identification of novel genes in this process. The experimental design implemented in chapter three could be improved through a number of modifications. To remove any potential acclimation effects, and to provide a more realistic estimate of sunlight stress response, future studies could take place in situ. Field collected individuals could be stored in RNAlater, and then transported back to the lab for RNA extraction and sequencing. Based on the high standard error of the qPCR results, the number of biological replicates should be increased to provide a more precise indication of expression. The aerial exposure stress treatment could be removed as the qPCR results suggest that it may be difficult or impossible to detect gene expression changes in response to sunlight stress in aerially exposed treatments. Finally, an equal number of time points could be distributed across either side of the maximum noon sunlight exposure time point to account for temperature and UV fluctuations over the course of the day.
Although this research project focused on various UV and visible light screening, antioxidant and DNA repair mechanisms, many other genomic
Chapter 4: General discussion 83
components of A. tenebrosas’ sunlight stress response were not explored. Most notably these include various genes involved in the production of melanin, the repair of indirect DNA damage, and molecular chaperones.
Melanin pigments are well-known for their role in intercepting and scattering
UV in animals (Cockell and Knowland 1999), and cnidarians are known to possess this pigment (Palmer, Bythell and Willis 2010). The expression of genes involved in the production of melanin was not examined in this study because the genomic basis of melanisation in cnidarians has never been examined, which represented a substantial body of work that was outside the scope of this Masters project.
Although this thesis examined genes involved in direct DNA repair, indirect
DNA repair was not a focus due to time and budget constraints. Indirect DNA repair gene expression has been shown to increase in corals under oxidative stress (Downs et al. 2009; Downs et al. 2006; Kortschak et al. 2003; Rougée et al. 2006), but this remains to be tested on sea anemones.
As described in chapter one, the up-regulation of heat shock proteins in sea anemones under sunlight stress has been extensively covered (for example, Del Rio
(2015); Black, Voellmy and Szmant (1995); Nicosia et al. (2014); Snyder and Rossi
(2004); Tarrant et al. (2014)), therefore HSPs were not included in the experimental component of this thesis. To more comprehensively understand A. tenebrosas sunlight stress response, genes involved in the production of melanin and the repair of indirect DNA damage components should be included in future investigations.
Implications
Intertidal shores provide a habitat that supports a multitude of diverse animal species that thrive in this extreme environment, yet little is known about their cellular
84 Chapter 4: General discussion
response to intertidal stressors. Sea anemones are an important group of invertebrates that inhabit rocky marine ecosystems yet are often overlooked. As a consequence, they are very poorly understood, both in isolation and in terms of their interactions in the marine environment. By investigating how A. tenebrosa copes with sunlight stress, reliable baseline data has been established that allows A. tenebrosa to be used as an indicator species for the stress tolerances of more rare, cryptic or difficult to study intertidal anemone species that share a similar habitat (e.g. Aulactinia verrucosa, Anthopleura handii, Actinia australianensis, Oulactis mucosa and
Anthopleura sp.) (Figure S3.7).
It is predicted that sea surface temperatures will rise as a result of global warming, and this will negatively impact the survival of corals with hard extracellular skeletons of the Great Barrier Reef (Hoegh-Guldberg 1999; Lesser et al.
1990; Glynn 1993; Lesser 2010). Furthermore, sunlight stress can work interactively with elevated water temperature to intensify the stress on coral species. In comparison, the temperature and sunlight stress tolerance of other Australian intertidal cnidarian species is poorly understood, and there are currently no cnidarian indicator species in Australia outside of corals. This research project has helped to establish A. tenebrosa as a non-coral intertidal cnidarian indicator species, as it is iconic, highly conspicuous, abundant, and widely distributed along the Australian coastline. Understanding the response of A. tenebrosa to sunlight stress, therefore, has broader implications for understanding the changing dynamics of intertidal communities, and their ecological response to global climate change, rising sea levels and temperatures.
Chapter 4: General discussion 85
Conclusion
In this thesis the genomic basis of the sunlight stress response in an intertidal sea anemone, Actinia tenebrosa was investigated. This thesis has provided many usefuls insights into the resilience of A. tenebrosa to sunlight stress near the northern limit of its distribution where UV and temperatures are at their highest, and enables future studies to use A. tenebrosa as an indicator species for other more rare/cryptic intertidal sea anemone species. The information presented here can more broadly be used to better understand how intertidal sea anemone species will respond to changing climactic conditions, an area of knowledge that is still poorly understood.
In this thesis important groundwork was been developed that future studies could build on, such as transcriptome-wide measurements of changes in gene expression, which would provide a more comprehensive understanding of the sunlight stress response in A. tenebrosa.
86 Chapter 4: General discussion
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Box 1. List of abbreviations and acronyms
CP Chromo protein
CPD Cyclobutane pyrimidine dimer
CSR Cellular stress response
FP Fluorescent protein
GFP Green fluorescent protein (family)
HSP Heat shock protein
IR Infrared
MAA Mycosporine-like amino acid
NER Nucleotide excision repair
PAR Photosynthetically active radiation
ROS Reactive oxygen species
SOD Superoxide dismutase
UV Ultraviolet
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