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Received Date : 23-Aug-2014 Revised Date : 04-Dec-2014 Accepted Date : 08-Dec-2014 Article type : Resource Article

Title: De novo assembly of the transcriptome of Acanthaster planci testes

Authors: Michael James Stewart1*, Praphaporn Stewart1, and Jairo Rivera-Posada2,3 1

Genecology Research Centre, Faculty of Science, Health, Education and Engineering,

University of the Sunshine Coast, Maroochydore DC, Queensland, Australia, 4558 2

ARC Centre of Excellence for Coral Reefs Studies, James Cook University, Townsville,

Queensland 4812 3

Australian Institute of Marine Science, PMB No. 3, Townsville, Queensland 4810, Australia

Corresponding Author: *Dr. Michael Stewart, Genecology Research Centre, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore DC, Queensland, Australia, 4558, [email protected]

Key words: testis, COTS, Illumina sequencing, spermatogenesis, bindin

Abstract A key strategy to reduce coral loss is the development of effective control method for the corallivorous crown-of-thorns sea star (Acanthaster planci), an omnipresent scourge and threat to the biodiversity of reefs in the Indo-Pacific region. Limited genetic resources are available for this highly fecund species. In the present study, we explored one aspect at the This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1755-0998.12360 This article is protected by copyright. All rights reserved.

heart of A. planci outbreaks, the male reproductive system. Using high-throughput

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sequencing technology, we report for first time the production of a comprehensive transcriptomic dataset for the testes of A. placni that can aid in understanding the molecular mechanisms involved in A. planci spermatogenesis and fertilisation. Through de novo transcriptome sequencing, we produced 52,965,998 raw reads corresponding to 4.76 Gb clean read data. From this, 243,870 contigs were assembled with Trinity and used to construct 92,792 unigenes. Distinct genes were then annotated with Blastx yielding 30,810 unigenes above the cut-off e-value set at 10-5, with ESTscan database query analyses yielding up to 5,366 unigenes to known hits. The identification of genes directly involved in sperm development

(DEAD-box

family

proteins),

motility,

fertilisation

and

signalling

(Bindin/Speract receptor) are also discussed.

Introduction Outbreaks of the coral feeding asteroid, crown-of-thorns sea star (COTS), Acanthaster planci are currently the single biggest biological threat to tropical coral reefs (Kayal et al. 2012; Pratchett et al. 2009). During an outbreak of COTS, coral cover, species diversity, and coral composition are affected (Bos et al. 2013; Kayal et al. 2012; Souter & Linden 2000), leading to increases in benthic algae, loss of coral-feeding assemblages, and a decline in biodiversity and productivity (Lane 2012; Pratchett et al. 2009). Numerous theories for the cause of these outbreaks have been put forward, with some suggesting that predator removal through overfishing increases the survival of larvae and near undetectable juveniles (Sweatman 2008). Birkeland (1982) though proposed that it is the influx of nutrient loads in waterways, during periods of high rainfall along mountainous coastlines that support the survival of COTS larvae leading to successful outbreaks (Birkeland 1982; Brodie et al. 2005; Fabricius et al. 2010). Nevertheless, despite pointing to a single cause, it is clear the frequency and

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intensity of outbreak events has increased during recent decades, due to a combination of

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natural and anthropogenic disturbances (Bos et al. 2013; Lane 2012; Mendonça et al. 2010). Management options for A. planci include the manual removal of COTS specimens

from reefs with sticks and hooks, but this is disadvantageous due to the inflicted damage to the reef matrix, as well as injury to collectors caused by the COTS venomous spines (Lin et al. 2008; Sato et al. 2008). Manual extraction of the sea star to correctly administer chemicals has been considered the most successful method for proper eradication (Johnson et al. 1990), but alas it is a painstaking process. In some instances, it can take up to 15 injections (in the oral disc, stomach and each of its limbs) to kill an individual COTS. Further hampering this method is that the chemicals employed include ammonia, sodium bisulfate and copper sulfate and once injected, left over decomposing sea stars can leech these chemicals into the surrounding water harming other reef organisms (Bos et al. 2013; Johnson et al. 1990). A safer alternative to curtailing COTS populations, are bile salts. Bile salts at very low concentrations can effectively kill the sea stars, administered via a single 10ml injection anywhere on the animal (Rivera-Posada et al. 2014). Importantly due to their relative ease of delivery, bile salts are now the preferred method for culling COTS on the Great Barrier Reef (GBR), and has become rapidly adopted in many other Pacific countries with active outbreaks (Vanuatu, Fiji, Samoa) (Rivera-Posada 2014). However combined, these manual removal initiatives are only not just ineffective (useful in very small areas), they are overly costly and labor intensive (Johnson et al. 1990; Kayal et al. 2012). Thus, relying on manual control of individual sea stars is not a future management option. To increase management options to control COTS and alleviate the ineffectiveness of

removal methods, it seems of utmost importance that their reproduction processes are given careful consideration as a single large female sea star can produce up to 65 million eggs (Conand 1984). If at least half of these eggs were fertilized, and survived through adulthood,

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it would ensue for the development of another successful outbreak. There are a wealth of

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studies in sea urchins about sperm chemotaxis and sperm-egg binding proteins (Lessios 2007; Sunday & Hart 2013; Zigler et al. 2005), but limited studies have focused the COTS male reproductive system, despite a clear advantage in understanding egg-sperm relationships even just at an evolutionary level (Hart 2013). Recently a study addressing the fertilization dynamics of this species, demonstrated how increasing ocean acidification conditions can impede sperm motility and fertilization rates (Uthicke et al. 2013). Whilst this study potentially highlights a slight benefit of anthropogenic disturbances (i.e. retarding sperm motility through ocean acidification), and adds more insight to the population genetic studies out there (Benzie 1999; Nash et al. 1988; Vogler et al. 2013; Yasuda et al. 2009) we are still no closer in understanding the male reproductive toolkit for this highly fecund animal. It is clear genomic information and next generation sequencing is becoming a

valuable for aiding ongoing efforts to understand and control the ecological and economic impacts of invasive species (Prentis et al. 2010; Wang et al. 2010). This technology has been proven to be an invaluable addition to ecological research for non-model organisms, for example the Asian bivalve Limnoperna fortune and Whitefly Bemisia tabaci (Uliano-Silva et al. 2014; Wang et al. 2010). These like many other species often lack reference genomes that impede their analyses (Feldmesser et al. 2014). The present study seeks to add a new dimension to the list of data that goes toward managing and preventing future outbreaks of COTS. Herein we report the identification gene transcripts in the male gonad of A. planci specifically involved in spermatogenesis, germ cell formation, and spermatozoon motility. We also provide insight into a select list of reproduction-related genes that provide insight into sperm-egg interactions.

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Materials and Methods

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Ethics statement All experiments were conducted in accordance with Australian laws, and specimen collections were approved by the GBR Maritime Park Authority (Permit No. G12/35336.1). All specimens were collected at Lizard Island, 14°40’S, 145°27’E on the northern section of the GBR, Queensland, Australia.

Animal preservation and maturity confirmation Healthy sexually mature male A. planci (~36 cm in diameter, n = 6) that had reached the stage of rapid testis development and possessed mature sperm were used (Fig. 1A-C). Firstly, the testes removed surgically, washed with SMT solution (250mM sucrose, 2mM MgCl2, 10mM Tris–HCl, pH 7.4), and fixed in 4% glutaraldehyde/2% paraformaldehyde in Millonig’s buffer pH7.2 at 40C for 1 h. After post fixation in 1% osmium tetroxide, testes specimens were dehydrated in increasing concentrations of ethyl alcohol. The testes specimens were then dried in a Quorum K850 critical-point drier (Quorum Technologies, Deben, UK), using liquid CO2 as a transitional medium. They were coated with gold in a Quorum Q150TS ion sputtering apparatus for 2 min and then examined by a JSM 6010LA Analytical Scanning Electron Microscope (Jeol, Massachusetts, USA) at 15 kV.

Transcriptome sequencing and assembly Testes (n = 6) were removed surgically and were stored in RNAlater (invitrogen) and held at -800C until required. Testes (100 mg/ animal) were homogenized in TRIzol (Invitrogen, Victoria, Australia), and processed following the manufacturers protocols. The quantity and quality RNA was assessed through a Bioanalyser. Approximately 5μg of total RNA was then used for transcriptome analysis using the Illumina sequencing platform. Following

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construction, double-stranded cDNA libraries (≈ 5 Gigabases, normalised) were sequenced

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on an Illumina HiSeq 2000 (BGI, Shenzhen, China). Prior to assembly and mapping the raw reads were first preprocessed by discarding reads with adaptors, reads with unknown nucleotides larger than 5%, and low quality reads (reads containing more than 50% bases with Q-value ≤ 20). Transcriptome de novo assembly was carried out with the short read assembling program Trinity with the k-mer size set to the default 25 (Grabherr et al. 2011). That is reads of a certain length with overlapping areas were first joined to form longer fragments, called contigs without gaps. Then the reads were mapped back to contigs; with paired-end reads. Next, Trinity connects the contigs and obtains sequences that can no longer be extended. These sequences are termed unigenes. Assembled sequence data is deposited in the

National

Center

for

Biotechnology

Information

(NCBI,

USA,

http://www.ncbi.nlm.nih.gov/) in the Sequence Read Archive (SRA); accession number SRR1197243.

Transcriptome annotation and protein translation The unigene sequences from A. planci were searched against protein databases (NCBI nr and Swiss- Prot) using BLASTx (threshold: 1e-5). Unigenes that could not be aligned to the above databases were examined by ESTScan to predict their coding region of transcripts (5′– 3′) and translated into amino sequences (Iseli et al. 1999). Both BLASTx results and ESTScan generated protein sequences, and served as a database for protein identification. To determine which transcribed proteins in A. placni testes may be important for spermatogenesis, fertilization, sperm motility and acrosome formation, annotated unigenes were screened against the Gene Ontology (GO) database, using the Blast2GO program (Conesa et al. 2005) and WEGO software (Ye et al. 2006). Using EC (Enzyme Commission number) terms, information on inner-cell metabolic pathways was collected by downloading

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relevant maps from the KEGG database (http://www.genome.jp/kegg/) (Kanehisa & Goto

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2000). The Clusters of orthologous groups (COG) database was then used to classify putative functions based on known orthologous gene products (Tatusov et al. 2000). Lastly to further define putative orthologs of A. planci to an asteroidean, a reciprocal BLASTp comparison between the annotated gene transcripts of A. planci testes to the annotated genes in the testes of the bat star Patiria miniata (Hart & Foster 2013) was performed using Protein ortho and default values E-value< 10-10, 30 percent identity and coverage of query sequence 40% (Lechner et al. 2011). Putative orthologs were defined only when reciprocal top scoring hit (at unigene/gene level) between both searches were the same and were unique. Once genes were identified, the amino acid sequences were aligned using

CLUSTALW (Larkin et al. 2007), and are represented as Text logo, or single open reading frames (ORF) using the LaTeX TexShade package (Beitz 2000). ORF and pfam domain schematics were also constructed using DOG 2.0 (Ren et al. 2009) and the simple modular architecture research tool (SMART) (Schultz et al. 1998). All reference sequences for phylogenetic analyses were retrieved from GenBank, and trees constructed using MEGA v6.0 (Tamura et al. 2011) using the Maximum likelihood using the Jones-Taylor-Thornton (JTT) method-model (Jones et al. 1992; Saitou & Nei 1987). Confidence levels for the groups defined in the topology were assessed by bootstrap and interior branch tests (1000 replicates). Signal peptides were predicted by SignalP 4.0 (Petersen et al. 2011) and three-dimensional homology models were built using the SWISS-MODEL Workspace (Arnold et al. 2006; Bordoli et al. 2008; Schwede et al. 2003). The quality of the resulting three-dimensional models were assessed using QMEAN Z-score (Benkert et al. 2011) and viewed using SwissPDB Viewer 4.04 (Swiss Institute of Bioinformatics). Rendering was undertaken with POVRay 3.6 (Persistence of Vision Raytracer Pty Ltd).

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Results and Discussion

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Illumina sequencing and de novo assembly Our transcriptome sequencing project produced a total of 52,965,998 of 90 bp raw reads from Illumina paired-end sequencing. After a stringent quality filtering process, 4.76 Gb of clean data (90% of raw data) was retained (GC content 45.15%). Of note, the Q20 data percentage (an error probability < 1%) of the final sequence generated in this study (96.25%) indicated that sequencing throughput and quality was acceptable for further analysis. De novo assembly of all clean reads produced 243,870 contigs with a mean contig size of 297 bp and N50 of 461 bp (Fig. 2A). After, the assembled scaffolds further clustered into 92,792 unigenes using TGICL software (Pertea et al. 2003). Of all 92,792 unigenes verified 36,065 (38.7%) were ≥500 bp and 17,405 (18.76%) were ≥1,000 bp respectively. In determining annotations, a total of 30,565 (38.9% of all distinct sequences) unigenes had a BLASTx result above the cut-off E-value of 10-5, whereas 25,213 unigenes (33.2% of all unigenes) returned annotated protein coding sequences (CDs) compared to 5,366 (5.78%) returned matches to ESTs respectively (Fig. 2B). As shown in Figure 2C, the e-value distribution of the top hits in the nr database showed that 36.1% of the mapped sequences have good similarity (smaller than 1.0E-45) with 63.9% of sequences ranging in e-values of 1.0E-5 to 1.0E-45. Furthermore, 6.2% of the query sequences have strong similarity to nr databases which is higher than 80%, while 67.6% of hits have a similarity ranging from 40% to 80% (Fig. 2D). Among species, 54.2% of A. planci testes sequences have strong matches with sequences from the purple sea urchin Strongylocentrotus purpuratus which is expected, while only 13.2%, 6.7%, and 1.8%, of COTs sequences matched those of the acorn worm, the lancet and the pacific oyster, respectively. Since no genome or limited EST information exists for A. planci, approximately 20% of the unigenes could not be matched (Fig. 2E). On an orthologous protein level, a reciprocal BLASTp analysis yielded 5,053 A. planci unigenes orthologous with P. miniata

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genes. The overlap between two gene sets by using reciprocal BLASTp searches with

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relatively stringent criteria for comparisons between pairs of long and highly similar sequences (i.e. expectation values of 0 and bit scores >1000), further revealed 208 A. planci gene transcripts which had 100 percent identity matches to that of the testis of P. miniata (Table S1).

Gene ontology (GO), clusters of orthologous groups (COG) and Kyoto Encyclopedia of genes and genomes (KEGG) ontology (KO) classification A total of 92,792 unigenes from A. planci were assigned for GO analysis. Based on the sequence similarity, 63,447 sequences were further categorized into three main categories (biological process, cellular component and molecular function) and 57 subcategories. Sequences specifically involved in reproduction included 806 individual genes (1.27%), and 702 genes involved in reproductive processes; or 1.1% of all annotated unigenes (Fig. 3). To evaluate further the completeness of our transcriptome library and effectiveness of our annotation process, we screened the annotated sequences to COG classifications and KEGG metabolic pathways. In total, 30,565 unigene nr hits, have COG classification (Fig. 4). The highest represented biological processes included ‘‘replication, recombination and repair’’ (1,687; 8.46%), followed by ‘‘transcription’’ (1,502; 7.53%), ‘‘translation, ribosomal structure and biogenesis’’ (1,445; 7.24%), ‘‘cell cycle control, cell division, chromosome partitioning’’ (1,143; 5.73%), and ‘‘post-translational modification, protein turnover, and chaperones’’ (1,093; 5.48%). Of the 30,565 unigene nr hits, a total of 21,143 unigenes were associated directly with

257 predicted KEGG metabolic pathways, and the number of unigenes in different pathways ranged from 1 to 3011. The most important pathways that may be relevant to reproduction are highlighted in Fig. S1 and spermatogenesis S3. KEGG analyses of our transcriptome

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dataset indicate also that COTs sperm can uptake glucose, and metabolise mannose which is

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consistent with the ability of mammalian sperm to metabolize this sugar as a primary energy source (Mukai & Okuno 2004; Urner & Sakkas 2005). Transcripts of proteins detected as members of the solute carrier family 2 glucose transporters i.e. Slc2a3/GLUT3 (CL1794.Contig1) and Slc2a8/GLUT8 (Unigene47413); thought to be key regulators in this role (Schürmann et al. 2002). However, Slc2a5 which directs metabolism of fructose was not among the Slc family proteins identified (Gawlik et al. 2008; Schürmann et al. 2002).

Functional genes involved in protein folding, spermatogenesis cell cycle, signalling, sperm motility and development. A multitude of genes central to A. planci spermatogenesis and developmental processes were identified. This includes 11 important functional genes directly involved in acrosome vesicle formation (GO:0001669), 7 genes for fertilization (GO:0009566) and 10 genes, involved in intraflagellar transport i.e. motor (Dyein) proteins (GO:0005930). We have also identified 114 genes that regulate spermatogenesis, as well as nine transcripts encoding DEAD (AspGlu-Ala-Asp)-box family proteins (Fig. 5, Table S2). Protein folding- The COTS testes transcriptome contained 57 genes relating to protein

folding. This list includes various members of the heat shock protein family (Hsp) i.e. hsp60, hsp70, hsp90, as well as endoplasmin, calreticulin, and calnexin families (Table S2). Several Hsps have been identified with potentially important functions in the male testis (Huang et al. 2005; Neuer et al. 2000). For example, hsp70 is strongly expressed in spermatogenic cells in normal testicular tissue with lower expression noted in tissues showing spermatogenic arrest around the spermatid stage (Dix 1997). Expression of hsp90 too is found in the cytoplasm of all male germ cells and is detected mainly in spermatogonia and elongated spermatids (Ecroyd et al. 2003). However, while it appears that Hsps serve a role in fertilization

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processes, published evidence suggests they are not critical, as none of the chaperones (i.e.

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hspE1, DnaJB1, hspD1, hspA1A, hspA5 and Tra1) are associated with human spermatozoa or are expressed on the sperm surface (Mitchell et al. 2007). On the basis of those findings, it is unlikely that Hsp homologs identified herein play an active role in the remodelling of the sperm during capacitation in COTs, despite the expression of genes that underpin the mechanisms that drive sperm–egg recognition. Nevertheless as COTs often do and will encounter a wide variety of environmental stressors (i.e. storms, salinity changes) Hsps will undoubtedly play a part in protein folding, and integrity to the organism as a whole. Their identification may assist as markers in understanding these processes (Neuer et al. 2000). Cell cycle proteins - Spermatogenesis is a highly ordered developmental process of

continuous germ cell maturation, and significant structural and biochemical changes (Stewart et al. 2010). Key genes involved in these processes are the serine/threonine protein kinase complexes, which are composed of a regulatory subunit, cyclin and a catalytic subunit, cyclin dependent kinase (Cdk). Here we report 23 cyclin related unigenes in A. planci (Table S2) and in addition report a polo-like kinase gene (Ap-PlK Unigene15503) that enables cell cycle progression through G2 into mitosis. Sperm Signalling Proteins - Signalling pathways are important for post testicular

sperm function, capacitation, and fertilization. Analysis of proteins associated with the GO term for ‘‘acrosome vesicle ’’ (GO:0001669) and fertilization revealed 16 sperm proteins in these categories (Table S2). This group included a calcitonin peptide-receptor component (CL252), which is documented in the presence of the ligand calcitonin (high concentration in vivo) acts as an endogenous regulator of sperm fertilization (Pondel 2000). We also report a transcripts encoding for t-complex polypeptide 1 (Unigene52727), which is a heterooligomeric complex that is upregulated during spermatogenesis and is central to male germ cell development (Rivenzon-Segal et al. 2005).

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Sperm Motility - Both axonemal and cytoplasmic forms of dynein proteins were

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found. The axonemal dyneins are involved in producing flagellar motion, whereas the cytoplasmic dyneins act as molecular motors moving membrane proteins, vesicles, and other cargo in the cell (Inaba 2011; Roberts et al. 2013). The most abundant dyneins in the COTS transcriptome were the heavy chain motor domain types 2, 3, 6 and 7. Two other intraflagellar transport protein homologs 27 and 88 were also identified. As the latter are important for cilia formation in multiple organisms, it is of no surprise they are found in A. planci (Fig. 6). As are the existence of transcripts encoding members of the FUcosyl transferase family (Fut) (i.e. Fut3-like [Fig. 6]). This family has implicated roles in acrosome formation, and spermatozoa-zona pellucida binding in vertebrates (Costache et al. 1997). Other intracellular signalling molecules detected included are shown in Fig. 5 and Table S2. Development – In the current study, we report nine DEAD (Asp-Glu-Ala-Asp)-box

family of ATP-dependent RNA helicases. This list includes DDX51 (CL2943.Contig2), DDX55 (CL6030.Contig1), and homolog of eukaryotic initiation factor 4A (eIF4A [CL7581.Contig1]). Other DEAD box proteins responsible for embryonic patterning and for primordial germ cell (PGC) development were also identified (Table S2). Specifically, this includes a partial Ap-Piwi-like protein (Unigene16033), Ap-Nanos (Unigene48560), and ApVasa (CL185.Contig1). To date few studies have shown the existence of these genes in echinoderms. However, in the sea urchin S. purpuratus, two genes Sp-Seawi and Sp-Piwilike1 encoding Piwi proteins have been reported, as well as transcripts for Sp-Vasa and SpNanos (Yajima et al. 2014). Functionally the activity of the Vasa is characterized by the presence of zinc finger motifs, a DEXDc/DEAD/DEAH box helicase domain, and a helicase conserved C-terminal domain (HELICc) (Raz 2000). The gene for Ap-Vasa encodes three ZnF Cys(2) His(2) (C2H2) domains (Fig. 6A) as well as both DEXDc and the helicase HELICc c-terminal domains. In the phylogenetic analysis, the predicted Ap-Vasa proteins

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clustered with the rest of Vasa proteins from other echinoderms, being the closest proteins

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those of the bat star P. miniata (Fig. 6B). Of note all echinoderm Vasa contained the DEXDc and HELICc domains (Fig 6C). However each species contained either 3 or more repeats of the ZnF C2H2 domain, aside from the green sea urchin Lytechinus variegatus which possessed none. Nevertheless, despite the absence of, or repeats of the ZnF C2H2 domain, conservation of both DEXDc and HELICc domains in Vasa within all species identified, suggests its helicase activity is required for echinoderm germ cell migration and development (Raz 2000; Yajima & Wessel 2011). Nanos is essential for germline pole plasm formation, and necessary for early

development (Agee et al. 2006; Kobayashi et al. 1996). The Nanos protein itself is a translational repressor characterized by a nanos RNA-binding domain with two conserved Cys–Cys–His–Cys zinc finger motifs (Oulhen & Wessel 2014). Nanos orthologs have been isolated from various vertebrates (Köprunner et al. 2001) and invertebrates (Leclère et al. 2012) including in the sea urchin S. purpuratus (Yajima et al. 2014). Here we report a full length transcript for Ap-Nanos (Unigene48560) (Fig. 7A) verified by BLAST and phylogeny assessment (Fig. 7B). We observed Ap-Nanos had the highest similarity to nanos in the purple sea urchins, S. purpuratus and Paracentrotus lividus, as well as nanos identified in the sea urchin Hemicentrotus pulcherrimus [e value = 2e−24, identities 39/53 (73%) without gaps respectively]. Furthermore, multiple sequence alignment indicated Ap-Nanos has high conservation of the CCHC zinc finger motifs to other orthologs of nanos found within echinoderms i.e. the blue bat star Patiria pectinifera nanos-like protein [e value = 8e−20, identities 35/53 (66%), without gaps] (Fig. 7C). But this degree of similarity was not retained when compared to the nanos-like gene encoding PoNos found in porifera.

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Reproduction-related Genes

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Speract/Resact receptors - Here we report partial precursor sequences for A. planci resact receptor (103 residues, Unigene 52331) and speract receptor (912 residues, Unigene 52332) (refer to Supporting Fasta file). Vacquier and colleagues (1985) demonstrated that resact, a 14-residue peptide with the sequence CVTGAPGCVGGGRL-NH2 found on eggjelly coats of sea urchin Arbica punctulata, can induce sperm directed chemotaxis (Ward et al. 1985). When receptors (resact receptors) on the surface of the sperm bind resact, a signal transduction cascade involving cyclic GMP is set in motion, triggering faster sperm swimming toward eggs (Kaupp et al. 2008; Strünker et al. 2006). Speract on the other hand, a small peptide derived from eggs of the sea urchin S. purpuratus evokes similar biochemical reactions as resact, although only resact has been established as a chemoattractant (Hansbrough & Garbers 1981). Here we performed a multiple sequence alignment of the A. planci speract receptor to other speract receptors (Fig. S3). This alignment highlights its conservation of a three transmembrane spans that divides the molecule into a large extracellular domain for ligand binding and an intracellular region harbouring a kinase and a cyclase-homology domain, which carries the catalytic site (Kaupp et al. 2008). Compared, A. planci speract receptor shows the highest homology to urchins i.e. A. punctulata [e value = 0.0, identities 584/943 (62%), without gaps], and S. purpuratus [e value = 0.0, identities 567/960 (60%), without gaps] respectively. There was also strong homology of A. planci speract receptor to the predicted speract receptor identified in the acorn worm Saccoglossus kowalevskii [e value = 0.0, identities 484/965 (49%), without gaps] and to the guanyl cyclase receptor in drosophila [e value = 0.0, identities 385/915 (42%), without gaps]. This degree of identity, perhaps suggests the putative A. planci speract receptor reported herein belongs to the GC receptor-type family. Taken together the identification of these two receptors (speract/resact) in COTS demonstrates that sea stars possess a suite of sperm-egg

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receptor/molecules like urchins which are required for sperm motility and fertilization

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success. Bindin – Although the molecular evolution of bindin and its role in gamete

compatibility and fertilization success (Zigler et al. 2005) has been carefully analyzed in studies in sea urchins, the absence of evidence for bindin expression in other taxa has led to the suggestion that bindin might be a sea urchin specific molecule (Lessios 2007; Zigler 2008). However recently, Hart and colleagues (Patiño et al. 2009) have reported a full length cDNA for bindin in the broadcast-spawning asterinid sea star P. miniata (Hart 2013). To add to the list, for the first time we also present a preprobindin sequence (Unigene49477) in A. planci which is homologous to the mature bindin protein found in P. miniata [67% identity] (Fig. S4). While this is a partial sequence (204 residues) it supports the observation that bindin is not just unique to urchins but is also in asteroidea (Hart 2013). This is also supported by the identification of a 1563 AA residue bindin receptor (EBR1) in A. planci (Unigene 52664) (Fig. S5A). To date the EBR1 gene displays strong protein domain similarity between echinoidea, especially between the two sea urchins Strongylocentrotus franciscanus and S. purpuratus where they share the same structure of EBR1 at the NH2 end of the predicted protein, reprolysin (Hart 2013). Aside from this, in general all EBR1’s reported in echinoderms share highly conserved EBR repeat domains (CUB and thrombospondin type 1 [TSP-1]) [Fig. S5B]. However, it appears that the last 6 EBR repeats of the carboxy end are species-specific and are highly divergent (Hart 2013). For example in S. franciscanus (and P. miniata) EBR protein domain repeats are replaced by a number of shorter hyalin-like repeats in S. purpuratus (Kamei & Glabe 2003). While the comparison of A. planci to P. miniata (and urchins) suggests a well conserved structure of EBR repeats, further and more in depth functional studies (which includes isolation of the full length EBR1 gene in A. planci) are needed to assess its role in species-specific sperm binding by eggs.

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Epididymal protease inhibitor – The epididymal protease inhibitor (Eppin) has been

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demonstrated to be both antimicrobial (Yenugu et al. 2004) and a central cog for the sperm surface protein-protein network that binds semen coagulating protein semenogelin I (SEMG1) (Wang et al. 2005). Since 2004, Eppin has been a prime target in contraceptive development (Wang et al. 2006). Experiments headed by O’rand and colleagues, demonstrated that nonhuman primates immunized with recombinant human Eppin antibodies at a high serum titer (1:1000) could inhibit sperm motility (sustained over several months) resulting in an effective level of contraception (100%) (O'rand et al. 2004). Based on Swissprot annotation, we reveal herein a partial 85 residue Eppin-like protein (Unigene18275) from the A. planci testes transcriptome (Fig 8A). This transcript possessed the Kunitz-type protease inhibitor motif including residues C56 Y61 and F71 of the Eppin Cterminus which have been identified as containing the major binding sites for SEMG1 within humans (Silva et al. 2012). Comparatively, within humans (and nonhuman primates) residues Cys102, Phe117 along with Tyr107, play key roles in these interactions (Silva et al. 2012). Using multiple sequence alignment and phylogeny, we observe A. planci Eppin has a moderate probability of matching with human Eppin (accession NP065131, e value = 2e−13, identities 28/56 [51.85%], without gaps), including moderate homology to other vertebrate species i.e. the brown rat Rattus norvegicus (accession NP001102927, e value = 8e−14, identities 29/54 [54%], without gaps). It does though show stronger homology and identity to Eppin found in invertebrates i.e. the buff-tailed bumblebee, Bombus terrestris (accession X003396032, e value = 6e-17, identities 31/52 [60%] without gaps) and abalone Haliotis asinina (accession P86733, e value = 2e-16, identities 29/54 [54%] without gaps) respectively (Fig. 8B). To further characterize the putative Ap-Eppin, we developed a homology model of the Eppin C-terminus (residues G1-Q85; Fig. 8C). We selected the Kunitz-type protease inhibitor 1 (SMLT 1yc0.1.B) as the reference structure because it

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provided a model with a strong QMEAN Z-score (-0.09), and provided the best

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representative model to which it could be cross referenced to the predictive model of human Eppin (Silva et al. 2012). The Ap-Eppin C-terminus three-dimensional model contained the three-disulfide bridges and secondary-structure arrays (α-helix, turn, beta-strand and random coil) typical of the Kunitz domain (Silva et al. 2012). The aromatic side chains of residues Tyr71 and Phe61 were located at the N- and C-terminus, respectively. This segment loop is conserved in part to the epitope of contraceptive antibodies made from infertile nonhuman primates (O'rand et al. 2004).

Conclusion Through our Illumina dataset we observe COTs like echinoidea posses a well-defined

set of genes involved in spermatogenesis (i.e. Hsps), sperm development (i.e. Vasa, Nanos) and motility (i.e. dyneins), including a large repertoire of receptors (speract/resact/EBR1) chemoattractant molecules (bindin) and genes required for egg fertilisation (Kaupp et al. 2008). Taken together our study not only provides a new molecular ecology recourse that provides greater insight into the COTS reproductive toolkit, but greater knowledge that we hope may bring forward new advances to technologies, and approaches to stop this pest.

Acknowledgements We wish to thank Ciemon Caballes for supplying images of Acanthaster planci and Mr. Daniel Powell for technical assistance with reciprocal BLASTp analysis. Funds for this project were provided by the Australian Institute of Marine Science, the ARC Centre of Excellence for Coral reef Studies, James Cook University and the John and Laurie Proud 2013 Fellowship to Dr. Jairo Rivera-Posada, to conduct part of this research at Lizard Island Research Station.

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Authors’ contributions

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MJS conceived the idea, performed field work and experiments, analysed results and drafted the manuscript. PS analysed results and assisted with the manuscript JRP conceived the idea, performed field work and drafted the manuscript and provided funding for the project.

Data Accessibility Raw Illumina reads: NCBI SRA: SRR1197243. Alternatively raw data can be sourced from Dr. Michael Stewart [email protected] on request.

Figure legends Figure1: Male reproductive system of Acanthaster planci. A) a COTS arm from proximal (P) to distal (D) shows the testis (tes) connected to the radial canal (rc); a white branching mass ventral to the hepatopancreas (hep) and intestine (int), and the ambulacral ridge (ar) made up of diminutive bulb-shaped ampullae juxtaposed to the testis. B) A spawning mature male COTS; sperm (S) thorns (th). C) SEM of a single upright sperm displaying a short arrow like head, midpiece and long tail.

Figure 2: Statistics of the Acanthaster planci testes transcriptome assembly. A) Summary of the assembly. B) The length distribution, quality and nr homology of de novo assemblies. X-axes indicate sequence size (nt) and Y-axes indicates number of assembled contigs, unigenes, CDSs and ESTs respectively. C) E-value distribution of the top BLAST hits for each unigenes against the nr dataase. D) Similarity distribution of the best hits for each

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unigene. E) Distribution of BLAST results by species as shown as the percentage of total

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homologous sequences (with an E-value ≤1.0E-5).

Figure 3: Distribution of Gene Ontology classifications. Transcripts classified into biological process, cellular component and molecular function, and 57 subcategories. The right y-axis indicates the number of genes per category. The left y-axis indicates the percentage of genes in the main category.

Figure 4: Histogram of clusters of orthologous groups (COG). Homology (E-value ≤1.0E5) of unigenes to genes within any one of the 25 categories (A-W, Y and Z) found in the COGs database at NCBI.

Figure 5: Proteins associated with spermatogenesis or germ cell function. Schematic overlay of a single spermatozoon released in a spawning event with GO annotated proteins associated with fertilization (capacitation), acrosome formation and motility shown. * Full list of spermatogenesis related proteins with GOs can be found in table S3.

Figure 6: Characterisation of Acanthaster planci Vasa. A) Amino acid sequence of the A. planci Vasa-like gene. Zinc finger C2H2 domains are boxed in blue and the DEAD-like helicase superfamily (DEXDc) protein family boxed in orange. The c-terminal HELICc is also boxed in red. B) Phylogeny of the Vasa proteins using the maximum-likelihood method. Numbers after the internal branches indicate the bootstrap value (value/1000) for each group. C) Comparison of the A. planci Vasa protein architecture to Vasa proteins and motifs found in echinoidea. Low complexity regions of the schematics (pink) are not shown in echinoidea

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schematics; however conserved ZnF C2H2, DEXDc and HELICc domains are indicated

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using colour codes for A. planci Vasa.

Figure 7: Characterisation of Acanthaster planci Nanos. A) Amino acid sequence and protein architecture of A. planci Nanos. The zinc-finger motif is shaded in gray. B) Phylogenetic relationships of A. planci Nanos and other Nanos proteins. C) Amino acid alignment of the zinc-finger domain of echinoidea Nanos. Identical residues are in black, conservative substitutions are in gray. The height of each letter in the text logo is proportional to the observed frequency of the corresponding amino acid.

Figure 8: Identification and characterization of Acanthaster planci Eppin. A) Amino acid sequence of the putative A. planci Eppin precursor and protein domain schematic. Kunitz domain is highlighted in aqua blue and cysteine residues in orange. Conserved residues C56, Y61, and F71 critically involved in Eppin’s interaction with SEMG1 and LTF are highlighted by *. B) Phylogenetic analysis and comparative sequence alignment of partial and full length precursors for Eppin found in protein and EST databases. C) Protein model of Eppin rendered with POV-Ray 3.6 to compare the overall shape and lipophilic potentials, and the ligand-binding sites. Hydrophobic surfaces are indicated by cyan-yellow and the hydrophilic surface by cyan-dark blue. Loops (green-yellow) are hydrophobic. Two of the three disulfide bonds are shown in orange, whilst the third was not highlighted to visualise the aromatic side chains of the residues Tyr61 and Phe71.

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Supporting Information

Additional Supporting Information may be found in the online version of this article:

Supporting Fasta file Compilation of unigenes-contigs used for comparative analysis Table S1 List of orthologous genes across each species Table S2 GOs of genes related to reproduction Table S3 Additional spermatogenesis with significant BLAST matches against Nr databases.

Fig. S1 Pathways relevant to spermatogenesis. A) Biological protein synthesis and DNA regulation. B) Cellular organisation and behaviour. C) Enzymatic processes. D) Extracellular and intracellular signalling. E) Carbohydrate fatty acid metabolism. X-axis equals the number of unigenes involved in each function.

Fig. S2 Components and pathways directly involved in spermatogenesis, reproduction and fertilization determined by the KEGG Orthology. In order these include enzymes of the glycolysis pathway, TCA cycle fructose and mannose metabolism pathways, pentose phosphate pathway, fatty acid metabolism pathway, fatty acid biosynthesis pathway, glutathione metabolism pathway, components of the ribosome, genes involved with amino acyl-tRNA biosynthesis, component of the proteosome, components of the ubiquitinmediated proteolysis pathway, components of the GnRH signalling pathway and genes associated with the oxidative phosphorylation pathway. Red stars designate genes identified in the pathway present in the transcriptome, whereas mapped objects are boxed in blue. All illustrations are used with permission http://www.genome.jp/kegg/kegg1.html.

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Fig. S3 Multiple sequence alignments of the Acanthaster planci speract receptor.

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Comparisons were performed with ClustalW and printed as text logo. Aside from A. planci speract

receptor

(Unigene52332),

sequences

were

obtained

from

Genbank

-

Strongylocentrotus purpuratus (NP_999705), Saccoglossus kowalevskii (XP_006821919), Drosophila melanogaster (Q07553) and Arbacia punctulata (P11528).

Fig. S4 Multiple sequence alignments of echinoidea bindin. A. planci bindin was obtained from the testes transcriptome (Unigene49477), other echinoidea sequences were obtained from Genbank - Patiria miniata (ACJ70121), Pisaster ochraceus (AHX71678), Pisaster giganteus (AHX71679) and Pisaster brevispinus (AHX71680).

Fig. S5 Acanthaster planci partial egg bindin (EBR1) receptor coding sequence structure. A) A. planci EBR1 partial precursor encodes 6 tandem pairs of TSP1+CUB domains form EBR repeats. B) EBR1 protein architecture of EBR1 in other echinoidea. All other echinoidea encode for the M12B-type propeptide (Pro) is shown in black; and nonrepetitive domains for a zinc-dependent metalloprotease (Reprolysin). The structure of the variable carboxy ends of the predicted protein domain structures consist of TSP1 and CUB domains (in S. franciscanus, A. planci and Patiria miniata) or hyalin-like domains (in S. purpuratus; black trapezoids). Repetitive domains TSP1 and CUB are shown as diamonds and ovals respectively. Genbank accessions: S. franciscanus (AAP44488), Patiria miniata (A03256a) and S. purpuratus (NP_999830); A. planci (Unigene52664).

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