Transcriptome of the Australian Mollusc Dicathais Orbita Provides Insights Into the Biosynthesis of Indoles and Choline Esters

marine drugs

Article Transcriptome of the Australian Mollusc Dicathais orbita Provides Insights into the Biosynthesis of Indoles and Choline Esters

Abdul Baten 1,†, Ajit Kumar Ngangbam 2,†, Daniel L. E. Waters 1 and Kirsten Benkendorff 2,*

1 Southern Cross Plant Science, Southern Cross University, Lismore NSW 2480, Australia; [email protected] (A.B.); [email protected] (D.L.E.W.) 2 Marine Ecology Research Centre, School of Environment, Science and Engineering, Southern Cross University, Lismore NSW 2480, Australia; [email protected] * Correspondence: [email protected]; Tel.: +61-2-6620-3755 † These authors contributed equally to this work.

Academic Editor: Paul Long Received: 16 May 2016; Accepted: 12 July 2016; Published: 20 July 2016

Abstract: Dicathais orbita is a mollusc of the Muricidae family and is well known for the production of the expensive dye Tyrian purple and its brominated precursors that have anticancer properties, in addition to choline esters with muscle-relaxing properties. However, the biosynthetic pathways that produce these secondary metabolites in D. orbita are not known. Illumina HiSeq 2000 transcriptome sequencing of hypobranchial glands, prostate glands, albumen glands, capsule glands, and mantle and foot tissues of D. orbita generated over 201 million high quality reads that were de novo assembled into 219,437 contigs. Annotation with reference to the Nr, Swiss-Prot and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases identiﬁed candidate-coding regions in 76,152 of these contigs, with transcripts for many enzymes in various metabolic pathways associated with secondary metabolite biosynthesis represented. This study revealed that D. orbita expresses a number of genes associated with indole, sulfur and histidine metabolism pathways that are relevant to Tyrian purple precursor biosynthesis, and many of which were not found in the fully annotated genomes of three other molluscs in the KEGG database. However, there were no matches to known bromoperoxidase enzymes within the D. orbita transcripts. These transcriptome data provide a signiﬁcant molecular resource for gastropod research in general and Tyrian purple producing Muricidae in particular.

Keywords: Muricidae; transcriptomics; Tyrian purple; tryptophan; metabolic pathways

1. Introduction Dicathais orbita is well known for the production of the dye Tyrian purple, which is a historically important colourant exclusively produced by the Muricidae family of marine molluscs [1,2]. Tyrian purple is not produced within the mollusc but is formed from oxidative and photolytic reactions from a precursor tyrindoxyl sulfate (Figure1A), which is stored as a salt of the choline ester murexine (Figure1B) [ 3,4]. Once the salt is liberated by an aryl sulfatase enzyme, a range of intermediate brominated indole precursors are produced, which have anticancer and antimicrobial properties [5–9]. The major dye component of Tyrian Purple, 6,61-dibromoindigo, was the ﬁrst marine natural product to be structurally elucidated [10]; however, a century later, limited information is available on the biosynthesis or gene regulation of this secondary metabolite.

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tissue or cell type [12,13]. Mollusc transcriptome studies have been instrumental in establishing the gene expression events associated with shell formation [14], host parasite interactions [15,16], nervous system function [17–19], immune defence [20,21], developmental processes [22,23] and cellular and physiological mechanisms [24,25]. Only a few transcriptome profiling studies have been undertaken on predatory marine neogastropods including the gonadal tissues of Reishia (Thais) clavigera [23], the mantle, foot, gills and gonadal tissues of Concholepas concholepas [26], the alimentary Mar.canal Drugs and2016 salivary, 14, 135 glands of Colubraia reticulata [27], the venom glands of Conus consors [28] and the2 of 16 venom ducts of C. tribblei, C. lenavati [29] and Lophiotoma olangoensis, a Turrid snail [30].

FigureFigure 1. 1.Tyrindoxyl Tyrindoxyl sulfate sulfate ( A(A),), the the ultimate ultimate Tyrian purple precursor precursor in in DictahaisDictahais orbita orbita, is, isheld held as asa a salt of the choline ester murexine (B). salt of the choline ester murexine (B).

Transcriptomics can identify genes involved in the biosynthesis of secondary metabolites [31,32]. The “post-genomics era” has seen an expansion in the application of bioinformatics to the The Australasian mollusc D. orbita is an ideal model species for gene expression studies of fields of transcriptomics, proteomics and metabolomics [11]. Transcriptomics provides information biosynthetic pathways that may be involved in the synthesis of biologically active secondary on the genes expressed by an organism under certain circumstances or stages of development, metabolites [3]. A preliminary transcriptome study of D. orbita used suppressive subtractive in a particular tissue or cell type [12,13]. Mollusc transcriptome studies have been instrumental hybridisation to identify genes that were upregulated in the hypobranchial gland, the biosynthetic inorgan establishing where Tyrian the gene purple expression is produced events [33]. This associated study confirmed with shell that formation the hypobranchial [14], host gland parasite is a interactionssignificant [site15, 16for], protein nervous synthesis system and function regulation, [17–19 but], immunethe only enzyme defence associated [20,21], developmental with Tyrian processespurple production [22,23] and identified cellular and was physiological arylsulphatase mechanisms [33]. However, [24,25 the]. Only study a was few transcriptomelimited by short profiling read studieslengths have and been a low undertaken total number on of predatory reads. Therefore, marine neogastropods the aim of this includingstudy was the to generate gonadal tissuesa more of Reishiacomprehensive(Thais) clavigera transcriptome[23], the mantle,from the foot, hypobranchial gills and gonadal glands, tissues prostate of Concholepas glands, capsule concholepas glands,[26 ], thealbumen alimentary glands, canal and mantle and salivary and foot tissues glands of of D. Colubraiaorbita (Muricidae reticulata Neogastropoda)[27], the venom and then glands search of Conusthese consors transcriptomes[28] and for the potential venom metabolic ducts of C.pathways tribblei, thatC. lenavaticould contribute[29] and toLophiotoma indole and olangoensis choline , a Turridester biosynthesis snail [30]. using the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAYS database.Transcriptomics can identify genes involved in the biosynthesis of secondary metabolites [31,32]. The Australasian mollusc D. orbita is an ideal model species for gene expression studies of biosynthetic pathways2. Results that and may Discussion be involved in the synthesis of biologically active secondary metabolites [3]. A preliminary transcriptome study of D. orbita used suppressive subtractive hybridisation to identify 2.1. De Novo Transcriptome Assembly genes that were upregulated in the hypobranchial gland, the biosynthetic organ where Tyrian purple is producedTranscripome [33]. This sequencing study confirmed across the that six the different hypobranchial tissue types gland (hypobranchial is a significant glands, site forprostate protein synthesisglands, andcapsule regulation, glands, but albumen the only glands, enzyme mantle associated and foot with tissues) Tyrian purpleresulted production in approximately identified 221 was arylsulphatasemillion sequencing [33]. However, reads (Table the 1). study Raw was sequencing limited by reads short in readFASTQ lengths format and were a low first total checked number for of reads.quality Therefore, using FASTQC the aim of[34] this followed study was by removal to generate of adapter a more comprehensivesequences, poly‐ transcriptomeN stretches and from low the hypobranchialquality (Phred glands, score < prostate 20) reads glands, using the capsule BBDuck glands, module albumen of the glands,BBMap software and mantle package and foot (version tissues 34_90, http://sourceforge.net/projects/bbmap), which resulted in 201 million high quality reads. Table of D. orbita (Muricidae Neogastropoda) and then search these transcriptomes for potential metabolic 1 shows the number of raw and quality controlled sequencing reads for all the tissues. pathways that could contribute to indole and choline ester biosynthesis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAYS database.

2. Results and Discussion

2.1. De Novo Transcriptome Assembly Transcripome sequencing across the six different tissue types (hypobranchial glands, prostate glands, capsule glands, albumen glands, mantle and foot tissues) resulted in approximately 221 million sequencing reads (Table1). Raw sequencing reads in FASTQ format were ﬁrst checked for quality using FASTQC [34] followed by removal of adapter sequences, poly-N stretches and low quality Mar. Drugs 2016, 14, 135 3 of 16

(Phred score < 20) reads using the BBDuck module of the BBMap software package (version 34_90, http://sourceforge.net/projects/bbmap), which resulted in 201 million high quality reads. Table1 shows the number of raw and quality controlled sequencing reads for all the tissues.

Table 1. Summary of the number of raw sequencing reads and the percent remaining after quality control from 14 tissue samples of Dicathais orbita.

High Quality Reads Snails (S) Description Raw Reads Number Percent S1 + S2 + S3 Female hypobranchial gland 1, August, Breeding season, 2014 15,531,322 15,100,466 97.23 S4 + S5 + S6 Female hypobranchial gland 2, August, Breeding season, 2014 15,693,385 15,258,671 97.23 S7 + S8 + S9 Female hypobranchial gland 1, January, 2015 15,835,271 15,425,533 97.41 S10 + S11 + S12 Female hypobranchial gland 2, January, 2015 16,457,635 15,990,724 97.16 S13 + S14 + S15 Male hypobranchial gland 1, January, 2015 16,142,317 15,684,926 97.17 S16 + S17 + S18 Male hypobranchial gland 2, January, 2015 17,461,007 16,997,497 97.35 S7 + S8 + S9 Female foot 1, January, 2015 16,015,535 15,595,463 97.38 S10 + S11 + S12 Female foot 2, January, 2015 17,057,433 16,653,222 91.40 S13 + S14 + S15 Male foot 1, January, 2015 14,241,690 13,885,327 97.50 S16 + S17 + S18 Male foot 2, January, 2015 15,813,363 15,406,030 97.42 S7 + S8 + S9 Capsule gland, January, 2015 15,805,867 15,291,498 96.75 S7 + S8 + S9 Albumen gland, January, 2015 14,442,864 14,011,099 97.01 S13 + S14 + S15 Prostate gland, January, 2015 15,600,688 15,113,842 96.88 S10 + S11 + S12 Mantle 1, January, 2015 16,273,556 15,804,247 97.12 - Total 222,371,933 216,218,545 -

High quality reads were de novo assembled into 219,437 contigs using CLC Genomics server (version 4.9, CLC Bio, Aarhus, Denmark) (Table2). Transdecoder (version 2.0.3, http://transdecoder.github.io/) identiﬁed 76,152 contigs that contained candidate-coding regions that were used for annotation and further downstream analysis.

Table 2. Summary statistics of the assembled contigs using CLC Genomics de novo assembler.

Contig Summary Statistics bp (Base Pair) Number of contigs 219,437 Total assembly length 117,767,308 N50 608 Mean contig length 537 Largest contig length 12,897 Number of contigs larger than 500 bp 59,144 Number of contigs larger than 1000 bp 22,818

2.2. Transcriptome Annotation Basic Local Alignment Search Tool (BLAST) analysis was performed using 76,152 contigs with Open Reading Frames (ORF)s against non-redundant protein database National Center for Biotechnology Information (NCBI) Nr (Mollusc specific proteins), Swiss-Prot and KEGG protein databases. Overall 28,364 contigs (~37%) had significant BLAST hits (e value 11 ˆ 10´5). The D. orbita contig BLAST hit rate was in a similar range to other whole mollusc genome/transcriptome studies with hits ranging from 25% to 40% [35]. A total of 24,996 contigs were assigned to various cellular components, molecular function and biological process gene ontology (GO) categories, as shown in Figure2. General cell and cell parts were the most frequent subcategories of the cellular components ontology category, while binding and cellular process was the most represented subcategory of molecular function and biological process. Analysis of KEGG pathways showed the largest number of contigs were involved in metabolic pathways (914 contigs), followed by biosynthesis of secondary metabolites (304 contigs) and microbial metabolism in diverse environment (173 contigs) (Supplementary Table S1). Previous studies have revealed a diversity of bacterial symbionts in D. orbita tissues [36,37]. However, we checked the overrepresented k-mers generated in the quality control phase of RNAseq reads and confirmed that these are mostly mitochondria RNA rather than bacterial (<0.1%). Mar. Drugs 2016, 14, 135 4 of 16 Mar. Drugs 2016, 14, x FOR PEER REVIEW 4 of 16

Figure 2. The proportion and number of Dicathais orbita contigs assigned to gene ontology (GO) terms from biological process, cellular component and molecular Figurefunction. 2. The Biological proportion process and number was the of most Dicathais highly orbita represented contigs assigned GO category to gene followed ontology by (GO) cellular terms component from biological and molecular process, function. cellular component and molecular function. Biological process was the most highly represented GO category followed by cellular component and molecular function.

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2.3. Tryptophan Metabolism and Phenylalanine, Tyrosine, Tryptophan Biosynthetic Pathways Speciﬁc searches in the KEGG PATHWAY database [38] were undertaken to identify genes potentially involved in the biosynthesis of Tyrian purple precursors and choline esters. Indoles like tyrindoxyl sulfate are thought to be derived from tryptophan metabolism, and we identiﬁed 28 enzyme contigs mapped to 35 reactions in the tryptophan metabolism pathway (Figure3; note that there are multiple KEGG enzyme (EC) numbers for some enzymes, and they can occur at different positions in the pathways, generating more matches to reactions than the number of matching contigs; this applies to all other pathways below). The list of 28 mapped contigs with the KEGG orthology assignment is provided in Supplementary Table S2. The annotated genomes of only three other molluscs are available for comparison in the KEGG PATHWAYS database, the gastropod Lottia gigantea, cephalopod Octopus bimaculoides and the bivalve Crassostrea gigas. Nearly all the genes involved in tryptophan metabolism found in D. orbita (Figure3) were identical to those found in the other three molluscs (Figure S1). However, an important point of difference is that unlike these other molluscs, the D. orbita transcriptome contained tryptophanase (4.1.99.1; Figure3), which converts tryptophan to indole. Consistent with the other molluscs such as L. gigantea and C. gigas, D. orbita expresses aromatic-L-amino-acid decarboxylase (4.1.1.28; Figure3) that converts tryptophan to tryptamine. However, we did not detect a transcript for tryptophan 5-monooxygenase (1.14.16.4), which converts tryptophan to 5-hydroxy-L-tryptophan, despite the presence of aromatic-L-amino-acid decarboxylase (4.1.1.28) involved in the production of serotonin. As the other three molluscs all contain matches to tryptophan 5-monoxygenase in their genomes (Figure S1), it is possible that this gene is also present in D. orbita but was not detected in our transcriptome due to low expression. Nevertheless, it appears likely that D. orbita diverts the conversion of tryptophan away from 5-hydroxy-L-tryptophan in favour of indoles, to facilitate Tyrian purple precursor production by higher expression of the tryptophanase gene. In the D. orbita transcriptome, we found no match in the reaction pathway for the conversion of indole to indoxyl, a precursor to indoxyl sulphate (Figure3, 1.14.16). However, in bacteria cytochrome P450 enzymes [39,40], and/or mono- or dioxygenases [41,42], are involved in the formation of indoxyl sulfate and indigo. We found matches to two cytochrome P450 enzymes, as well as a monoxidase and several dioxygenases in the D. orbita transcriptome (Supplementary Table S2). Our recent studies have also revealed numerous Vibrio sp. that have the ability to synthesize indoles in the Tyrian purple producing tissues of D. orbita [36,37] and these may provide a supplementary source of indoles for Tyrian purple production. Mar. Drugs 2016, 14, 135 6 of 16 Mar. Drugs 2016, 14, x FOR PEER REVIEW 6 of 16

FigureFigure 3. TryptophanTryptophan metabolism metabolism pathway pathway with with matches matches to Dicathais to Dicathais orbita orbita contigscontigs filled ﬁlled in green. in green. The Thematch match to a to tryptophanase a tryptophanase relevant relevant to indole to indole biosynthesis biosynthesis is ishighlighted highlighted by by the the red red box, box, whereas whereas the the tryptophantryptophan 5-monoxygenase5‐monoxygenase thatthat was not detected in our transcriptome is highlighted in in a blue box.

In the phenylalanine, tyrosine and tryptophan biosynthetic pathway (Figure 4), there was a In the phenylalanine, tyrosine and tryptophan biosynthetic pathway (Figure4), there was a match match in the D. orbita transcriptome to tryptophan synthase alpha chain (4.2.1.20; Figure 4), which in the D. orbita transcriptome to tryptophan synthase alpha chain (4.2.1.20; Figure4), which converts converts tryptophan to indoles and vice versa, the initial precursors for Tyrian purple biosynthesis tryptophan to indoles and vice versa, the initial precursors for Tyrian purple biosynthesis [3,43,44]. [3,43,44]. This tryptophan synthase was only found in the D. orbita transcriptome and not found in This tryptophan synthase was only found in the D. orbita transcriptome and not found in the annotated the annotated genomes of L. gigantea, O. bimaculoides and C. gigas. Most of the genes involved in genomes of L. gigantea, O. bimaculoides and C. gigas. Most of the genes involved in phenylalanine, phenylalanine, tyrosine and tryptophan biosynthesis in D. orbita were found to be different to the tyrosine and tryptophan biosynthesis in D. orbita were found to be different to the other three molluscs other three molluscs and this Neogastropoda had more matches to these aromatic amino acid and this Neogastropoda had more matches to these aromatic amino acid biosynthesis reactions (14 biosynthesis reactions (14 genes) compared to L. gigantea (five genes), O. bimaculoides (five genes) and genes) compared to L. gigantea (five genes), O. bimaculoides (five genes) and C. gigas (five genes) C. gigas (five genes) (Figure S2). (Figure S2).

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Figure 4. Phenylalanine, tyrosine and tryptophan biosynthetic pathways showing matches to Figure 4. Phenylalanine, tyrosine and tryptophan biosynthetic pathways showing matches to Dicathais orbita contigs highlighted in green, with tryptophan synthase highlighted in the red box. Dicathais orbita contigs highlighted in green, with tryptophan synthase highlighted in the red box.

2.4.2.4. Sulfur,Sulfur, CysteineCysteine andand MethionineMethionine Metabolisms Pathway in Dicathais orbita Tyrindoxyl sulphate contains a methane thiol group at the 2ʹ position of the indole ring (Figure Tyrindoxyl sulphate contains a methane thiol group at the 21 position of the indole ring (Figure1A). 1A). Examination of the D. orbita transcripts with reference to the sulfur metabolism pathway Examination of the D. orbita transcripts with reference to the sulfur metabolism pathway revealed revealed that most of the genes involved in D. orbita sulfur metabolism were found to be similar to that most of the genes involved in D. orbita sulfur metabolism were found to be similar to the other the other three molluscs. Enzymes like dimethyl‐sulfide monooxygenase (1.14.13.131; Figure 5) that three molluscs. Enzymes like dimethyl-sulﬁde monooxygenase (1.14.13.131; Figure5) that produces produces methyl mercaptan from dimethyl disulphide were not detected in any of the molluscs, methyl mercaptan from dimethyl disulphide were not detected in any of the molluscs, including including D. orbita. Overall, the number of genes detected in the D. orbita sulfur metabolism pathway D. orbita. Overall, the number of genes detected in the D. orbita sulfur metabolism pathway (15 genes, (15 genes, Figure 5) was only slightly higher than the number identified in the genome of L. gigantea Figure5) was only slightly higher than the number identiﬁed in the genome of L. gigantea (12 genes), (12 genes), O. bimaculoides (12 genes) and C. gigas (14 genes) (Figure S3). O. bimaculoides (12 genes) and C. gigas (14 genes) (Figure S3).

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FigureFigure 5. 5.Sulfur Sulfur metabolismmetabolism pathway with matches to to DicathaisDicathais orbita orbita contigscontigs highlighted highlighted in in green; green; therethere was was no no match match to to dimethyl-sulﬁde dimethyl‐sulfide monooxygenasemonooxygenase in our transcriptome ( (blue box).box).

The metabolism of sulfur containing amino acids provides another possible source of the The metabolism of sulfur containing amino acids provides another possible source of the methanethiol group in tyrindoxyl sulfate. Enzyme matches in the cysteine and methionine methanethiol group in tyrindoxyl sulfate. Enzyme matches in the cysteine and methionine metabolism metabolism pathways (Figure 6) indicate that D. orbita has the ability to produce 3‐ pathways (Figure6) indicate that D. orbita has the ability to produce 3-methylthioproprionate, methylthioproprionate, 3‐mercaptopyruvate and thiosulfate, but no match was found to methionine‐ 3-mercaptopyruvate and thiosulfate, but no match was found to methionine-gamma-lyase (4.4.1.11; gamma‐lyase (4.4.1.11; Figure 6), which converts the amino acid L‐Methionine directly into Figure6), which converts the amino acid L-Methionine directly into methanethiol. Nevertheless, we methanethiol. Nevertheless, we did detect a transcript for tyrosine aminotransferase (2.6.1.5; Figure did detect a transcript for tyrosine aminotransferase (2.6.1.5; Figure6), which may play a role in placing 6), which may play a role in placing the methane thiol onto the aromatic indole ring. Tyrosine the methane thiol onto the aromatic indole ring. Tyrosine aminotransferase genes were also found aminotransferase genes were also found in the Lottia, octopus and oyster genomes. Overall, the D. in the Lottia, octopus and oyster genomes. Overall, the D. orbita transcriptome had more matches orbita transcriptome had more matches to enzymes in the cysteine and methionine metabolism to enzymes in the cysteine and methionine metabolism pathway (41 genes) compared to L. gigantea pathway (41 genes) compared to L. gigantea (32 genes), O. bimaculoides (30 genes) and C. gigas (33 (32 genes), O. bimaculoides (30 genes) and C. gigas (33 genes) (Figure S4). This ability to metabolise genes) (Figure S4). This ability to metabolise sulfur from various sources is consistent with the sulfurbiosynthesis from various of indole sources mercaptans is consistent in Muricidae. with the biosynthesis of indole mercaptans in Muricidae.

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Figure 6. Cysteine and methionine metabolism pathways showing matches to Dicathais orbita contigs Figure 6. Cysteine and methionine metabolism pathways showing matches to Dicathais orbita contigs highlighted in green, including tyrosine aminotransferase (red box), but no match was found to highlighted in green, including tyrosine aminotransferase (red box), but no match was found to methionine‐gamma‐lyase (blue box). methionine-gamma-lyase (blue box). 2.5. Bromoperoxidase Enzymes 2.5. Bromoperoxidase Enzymes Tyridoxyl sulfate is a 6‐brominated indole derivative (Figure 1A), and bromoperoxidase activity Tyridoxyl sulfate is a 6-brominated indole derivative (Figure1A), and bromoperoxidase activity has been detected in the hypobranchial glands of D. orbita [45] and other Muricidae species [46]. has been detected in the hypobranchial glands of D. orbita [45] and other Muricidae species [46]. Consequently, a search was undertaken for bromoperoxidase genes by aligning D. orbita transcripts Consequently, a search was undertaken for bromoperoxidase genes by aligning D. orbita transcripts against known bromoperoxidase genes using BLAST (e value 1 × 10−3). However, no evidence of against known bromoperoxidase genes using BLAST (e value 1 ˆ 10´3). However, no evidence matches to bromoperoxidase genes was found using these sequences. This is consistent with a of matches to bromoperoxidase genes was found using these sequences. This is consistent with a previous study that examined the transcripts that were up‐regulated in the D. orbita hypobranchial previous study that examined the transcripts that were up-regulated in the D. orbita hypobranchial glands relative to other D. orbita tissues, which also found no matches to bromoperoxidase genes [33]. glands relative to other D. orbita tissues, which also found no matches to bromoperoxidase genes [33]. There appears to be no previous reports of bromoperoxidase coding genes or transcripts in any There appears to be no previous reports of bromoperoxidase coding genes or transcripts in any gastropods or other molluscs. However, recent metagenomic analyses of D. orbita hypobranchial gastropods or other molluscs. However, recent metagenomic analyses of D. orbita hypobranchial glands revealed the presence of bacterial symbionts known to produce bromoperoxidase enzymes glands revealed the presence of bacterial symbionts known to produce bromoperoxidase enzymes [36]. [36]. Our D. orbita transcriptome data therefore supports the suggestion that symbiotic bacteria play a role in the biosynthesis of Tyrian purple.

2.6. Dicathais Orbita Glycerophospholipid and Histidine Metabolism Pathway

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Our D. orbita transcriptome data therefore supports the suggestion that symbiotic bacteria play a role in the biosynthesis of Tyrian purple.

2.6. Dicathais Orbita Glycerophospholipid and Histidine Metabolism Pathway Mar. Drugs 2016, 14, x FOR PEER REVIEW 10 of 16 Tyrindoxyl sulfate is stored as a choline ester of murexine, which contains a choline ester group and imidazoleTyrindoxyl moiety sulfate is (Figure stored1 asB). a Thecholine glycerophospholipid ester of murexine, which metabolism contains pathwaya choline ester has agroup role in theand biosynthesis imidazole moiety of choline (Figure esters 1B). The [47] glycerophospholipid and more matches tometabolism reactions pathway in the glycerophospholipid has a role in the metabolismbiosynthesis pathway of choline (53 genes)esters [47] were and found more in thematchesD. orbita to transcriptomereactions in the compared glycerophospholipid to the annotated genomesmetabolism of other pathway molluscs (53 genes) (L. gigantea were found= 48, inO. the bimaculoides D. orbita transcriptome= 40 and C. compared gigas = 44 to genes) the annotated (Figure S5). ThegenomesD. orbita of othertranscriptome molluscs (L. included gigantea matches= 48, O. bimaculoides to choline/ethanol = 40 and C. amine gigas kinase= 44 genes) (2.7.1.32; (Figure Figure S5). 7), whichThe D. produces orbita transcriptome choline from included phosphocholine, matches to as wellcholine/ethanol as choline O-acetyltransferaseamine kinase (2.7.1.32; (2.3.1.6; Figure Figure 7), 7) which produces choline from phosphocholine, as well as choline O‐acetyltransferase (2.3.1.6; Figure and acetylcholinesterase (3.1.1.7; Figure7), which produce acetylcholine. Laffy et al. [ 33] found that 7) and acetylcholinesterase (3.1.1.7; Figure 7), which produce acetylcholine. Laffy et al. [33] found that acetylcholinesterase (3.1.1.7) transcripts were upregulated in the hypobranchial gland of D. orbita acetylcholinesterase (3.1.1.7) transcripts were upregulated in the hypobranchial gland of D. orbita relative to foot tissue. The octopus O. bimaculoides and gastropod L. gigantea genomes were also relative to foot tissue. The octopus O. bimaculoides and gastropod L. gigantea genomes were also found found to contain matches to these enzymes for acetylcholine biosynthesis, whereas the oyster C. gigas to contain matches to these enzymes for acetylcholine biosynthesis, whereas the oyster C. gigas lacks lacksphosphocholine phosphocholine phosphatase. phosphatase. In comparison In comparison to the togastropods the gastropods and the and bivalve, the bivalve, the octopus the octopus O. O.bimaculoides bimaculoides waswas found found to tolack lack any any matches matches to genes to genes in the in the phosphothanolamine phosphothanolamine N‐methyltransferaseN-methyltransferase pathwaypathway for for the the productionproduction of phosphocholine phosphocholine from from phosphoethanolamine. phosphoethanolamine.

Figure 7. Glycerophospholipid metabolism pathway with matches to D. orbita contigs highlighted in Figure 7. Glycerophospholipid metabolism pathway with matches to D. orbita contigs highlighted in green including choline kinase, choline O‐acetyltransferase and acetylcholinesterase (red boxes) used green including choline kinase, choline O-acetyltransferase and acetylcholinesterase (red boxes) used to generate the acetyl choline moiety found in murexine. to generate the acetyl choline moiety found in murexine. Choline or acetyl choline combines with imidazole to produce the muscle relaxant murexine [48] (FigureCholine 1B). Imidazole or acetyl cholineis a derivative combines of the with amino imidazole acid histidine to produce [49], and the musclethere were relaxant several murexine matches [48] (Figureto the histidine1B). Imidazole metabolism is a derivative pathway in of the the D. amino orbita acid transcriptome histidine [ (Figure49], and 8). there These were include, several diamine matches tooxidase the histidine (1.4.3.22; metabolism Figure 8), pathway aldehyde in dehydrogenase the D. orbita transcriptome (NAD+) (1.2.1.3; (Figure Figure8). These 8) and include, monoamine diamine oxidaseoxidase (1.4.3.22;(1.4.3.4; Figure Figure 8),8), which aldehyde convert dehydrogenase histamine into (NAD+) imidazole. (1.2.1.3; There Figure was8 )also and a monoaminematch to histidine ammonia‐lyase (4.3.1.3; Figure 8), which converts L‐histidine to urocanate, which could feasibly combine with choline ester to form murexine. All these biosynthetic enzymes are also found in the L. gigantea, O. bimaculoides and C. gigas genomes. Previous studies of choline esters in molluscs have focused on the predatory neogastropods, and there is no record of murexine or similar derivatives being isolated from limpets or oysters. Roseghini

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et al. [48] found no evidence for imidazole or acryl choline esters in 27 species from eight families of herbivorous and scavenging gastropods, including three Patellidae limpets, while at least one of these compounds was found in 53 of 55 species of the predatory Muricoidae superfamily. This implies the Mar.Neogastropoda Drugs 2016, 14, have 135 evolved a specific murexine biosynthesis pathway and, consistent with this,11 ofthe 16 D. orbita transcriptome had more matches to enzymes in the histidine metabolism pathway (19 genes) when compared to L. gigantea (10 genes), O. bimaculoides (11 genes) and C. gigas (13 genes) (Figure oxidaseS6). Specifically, (1.4.3.4; Figure the 8enzyme), which convertinvolved histamine in imidazole into imidazole. biosynthesis There imidazoleglycerol was also a match‐ tophosphate histidine ammonia-lyasedehydratase (4.2.1.19; (4.3.1.3; Figure Figure 8) 8was), which only found converts in theL -histidineD. orbita transcriptome. to urocanate, Overall, which couldit appears feasibly the combineneogastropod with cholineD. orbita ester has evolved to form a murexine. complex suite All these of metabolic biosynthetic capabilities enzymes that are are also not foundrepresented in the L.in gigantea the more, O. primitive bimaculoides orthogastropodand C. gigas orgenomes. bivalve, for which complete genome sequences are available.

Figure 8. Histidine metabolism pathway showing matches to Dicathais orbita contigs highlighted in Figure 8. Histidine metabolism pathway showing matches to Dicathais orbita contigs highlighted green, including several enzymes that convert histidine into imidazole (red boxes) and in green, including several enzymes that convert histidine into imidazole (red boxes) and imidazoleglycerol-phosphateimidazoleglycerol‐phosphate dehydratase ((red box top pathway).

3. Materials and Methods Previous studies of choline esters in molluscs have focused on the predatory neogastropods, and there is no record of murexine or similar derivatives being isolated from limpets or oysters. 3.1. Specimen Collection Roseghini et al. [48] found no evidence for imidazole or acryl choline esters in 27 species from eight familiesEighteen of herbivorous adult specimens and scavenging of D. orbita gastropods, (Table 1) were including collected three during Patellidae low tide limpets, from whilethe sub at‐tidal least oneand ofintertidal these compounds rocky reefs of was Flat found rock, inBallina 53 of (28 5584 species′ S and of 153 the60 predatory′ E), NSW, Muricoidae Australia. Six superfamily. spawning Thisfemales implies were thecollected Neogastropoda during the havebreeding evolved season a August speciﬁc 2014 murexine and a biosynthesisfurther six females pathway and and, six consistentmales were with collected this, after the D. breeding orbita transcriptome season in January had 2015, more under matches the topermit enzymes number in theF89/1171 histidine‐6.0 metabolismissued by the pathway Department (19 genes) of Primary when compared Industries, to NSWL. gigantea Government,(10 genes), Australia.O. bimaculoides Total RNA(11 genes) was andextractedC. gigas from(13 the genes) hypobranchial (Figure S6). glands Speciﬁcally, of the females the enzyme collected involved in August in imidazole 2014 and biosynthesis from five imidazoleglycerol-phosphatedifferent tissues from female and dehydratase three tissues (4.2.1.19; from male Figure D. orbita8) was (Figure only 9) collected found in in the JanuaryD. orbita2015 transcriptome.(Table 1). Overall, it appears the neogastropod D. orbita has evolved a complex suite of metabolic capabilities that are not represented in the more primitive orthogastropod or bivalve, for which complete genome sequences are available.

Mar. Drugs 2016, 14, 135 12 of 16

3. Materials and Methods

3.1. Specimen Collection Eighteen adult specimens of D. orbita (Table1) were collected during low tide from the sub-tidal and intertidal rocky reefs of Flat rock, Ballina (28˝841 S and 153˝601 E), NSW, Australia. Six spawning females were collected during the breeding season August 2014 and a further six females and six males were collected after breeding season in January 2015, under the permit number F89/1171-6.0 issued by the Department of Primary Industries, NSW Government, Australia. Total RNA was extracted from the hypobranchial glands of the females collected in August 2014 and from ﬁve different tissues from

Mar.female Drugs and 2016 three, 14, x tissues FOR PEER from REVIEW male D. orbita (Figure9) collected in January 2015 (Table1). 12 of 16

Figure 9. Dicathais orbita male (A) and female (B) tissues used for RNA extraction to generate the Figure 9. Dicathais orbita male (A) and female (B) tissues used for RNA extraction to generate transcriptome. the transcriptome.

3.2. Transcriptome Sequencing Three independent independent replicate replicate snails snails were were used used for for each each tissue tissue sample sample summarised summarised in Table in Table 1. The1. tissuesThe tissues were were stabilized stabilized prior prior to RNA to RNA extraction extraction in inRNase RNase free free 2 2ml ml Eppendorf Eppendorf tube tube using using 600 600 µLL of RNAlater RNARNA stabilizationstabilization reagent reagent (Qiagen, (Qiagen, Chadstone, Chadstone, Victoria, Victoria, Australia). Australia). The The stabilized stabilized tissue tissue was wasincubated incubated at 4 ˝atC 4 overnight °C overnight and storedand stored at ´80 at˝ −C,80 prior °C, prior to extracting to extracting the total the RNA.total RNA. The total The RNAtotal RNAwas extracted was extracted from the from RNA thelater RNAstabilizedlater stabilized tissue usingtissue the using E.Z.N.A. the E.Z.N.A. Mollusc Mollusc RNA Kit RNA (Omega Kit (Omega Bio-tek, BioNorcross,‐tek, Norcross, GA, USA.) GA, following USA.) thefollowing manufacturer’s the manufacturer’s instructions. instructions. The concentration The concentration and quality (purity and qualityand integrity) (purity ofand total integrity) RNA was of total assessed RNA bywas NanoDrop assessed by and NanoDrop the Agilent and Bioanalyzer the Agilent 2100 Bioanalyzer System 2100(Agilent System Technologies, (Agilent SantaTechnologies, Clara, CA, Santa USA). Clara, The totalCA, RNAUSA). extracted The total from RNA three extracted biological from replicates three biologicalof each tissue replicates type (hypobranchial of each tissue type gland, (hypobranchial prostate gland, gland, albumen prostate gland, gland, capsule albumen gland, gland, mantle capsule and gland,foot) was mantle pooled and within foot) was the pooled same tube within for the each same tissue tube in for equal each masses. tissue in The equal pooled masses. extracted The pooled RNA extractedwas stored RNA at ´ was80 ˝ storedC until at further −80 °C used. until further The RNA used. samples The RNA were samples shipped were to Macrogen shipped to Inc. Macrogen (Seoul, Inc.Korea) (Seoul, for high Korea) throughput for high sequencing. throughput Prior sequencing. to shipping, Prior each to RNAshipping, sample each was RNA precipitated sample was in a precipitatedmixture of 2ˆ inethanol a mixture (96%) of and2× ethanol 0.1ˆ sodium (96%) acetateand 0.1× (3 M).sodium mRNA acetate isolation (3 M). and mRNA library isolation construction and librarywere performed construction by Macrogen. were performed The libraries by Macrogen. were sequenced The libraries using were the sequenced Illumina HiSeq using 2000 the platformIllumina HiSeq(HCS2.2.38 2000 version,platform Illumina, (HCS2.2.38 Seoul, version, Korea). Illumina, Seoul, Korea).

3.3. De Novo Transcriptome Assembly and Annotation FASTQ format raw sequencing reads were checked for quality using using FASTQC FASTQC (version 0.10.4, 0.10.4, http://www.bioinformatics.babhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc)raham.ac.uk/projects/fastqc) [34]. [34 ].The The adapter adapter sequences, sequences, poly-Npoly‐N stretches and low quality reads (Phred score < 20) were removed using the BBDuck module of the BBMap software package (version 34_90, http://sourceforge.net/projects/bbmap) using command “bbduk.sh in1 = in1.fastq in2 = in2.fastq out1 = out1.fastq out2 = out2.fastq ref = adapters.fa qtrim = rl trimq = 20 ktrim = rl k = 28 hdist = 1 minlength = 20”. BLAST search of over‐represented k‐mers against NCBI confirmed these were mostly mitochondrial and ribosomal Dicathais obita genes, and no evidence of bacterial contamination was found. CLC Genomics Workbench, version 4.9 (CLC Bio, Aarhus, Denmark; www.clcbio.com) with the option to map reads back to contigs, automatic word size and automatic bubble size was used to de novo assemble the high quality reads. All the contigs were clustered using CD‐hit‐est (version v4.6.1, http://weizhongli‐lab.org/cd‐hit) [50], and Transdecoder (version 2.0.3, http://transdecoder.github.io/) was used to identify candidate coding regions within transcript sequences. BLAST analysis was done against non‐redundant protein database Nr (Mollusc specific proteins), Swiss‐Prot and KEGG protein databases. Gene ontology analysis was performed with

Mar. Drugs 2016, 14, 135 13 of 16 stretches and low quality reads (Phred score < 20) were removed using the BBDuck module of the BBMap software package (version 34_90, http://sourceforge.net/projects/bbmap) using command “bbduk.sh in1 = in1.fastq in2 = in2.fastq out1 = out1.fastq out2 = out2.fastq ref = adapters.fa qtrim = rl trimq = 20 ktrim = rl k = 28 hdist = 1 minlength = 20”. BLAST search of over-represented k-mers against NCBI conﬁrmed these were mostly mitochondrial and ribosomal Dicathais obita genes, and no evidence of bacterial contamination was found. CLC Genomics Workbench, version 4.9 (CLC Bio, Aarhus, Denmark; www.clcbio.com) with the option to map reads back to contigs, automatic word size and automatic bubble size was used to de novo assemble the high quality reads. All the contigs were clustered using CD-hit-est (version v4.6.1, http://weizhongli-lab.org/cd-hit) [50], and Transdecoder (version 2.0.3, http://transdecoder.github.io/) was used to identify candidate coding regions within transcript sequences. BLAST analysis was done against non-redundant protein database Nr (Mollusc speciﬁc proteins), Swiss-Prot and KEGG protein databases. Gene ontology analysis was performed with Interproscan (version 5.10.50, https://code.google.com/archive/p/interproscan) [51] using command “interproscan.sh -appl ProDom,PfamA,PANTHER -i longest_orfs.pep.fa -o out.txt -f TSV -goterms -iprlookup -pa” and plotted using WEGO (http://wego.genomics.org.cn/cgi-bin/wego/index.p) [52]. D. orbita transcripts were searched for bromoperoxidase gene by aligning against known bromoperoxidase genes available in the NCBI GenBank using BLAST (e value 1 ˆ 10´3) (Supplementary Table S3).

3.4. Nucleotide Sequence Accession Number All raw sequence data were deposited in the European nucleotide archive (ENA) with the accession numbers PRJEB12262. Assembled contigs are available from the authors upon request.

4. Conclusions This transcriptome study of D. orbita generated over 216 million high quality reads that were de novo assembled into 219,437 contigs, of which 76,152 contigs contained candidate-coding regions that were annotated with Nr, Swiss-Prot and KEGG databases. This provides a signiﬁcant new molecular resource for neogastropod molluscs, and adds to pool of genomic data for molluscs in general. Several genes that are potentially associated with Tyrian purple precursor biosynthesis in D. orbita were identiﬁed. It appears the neogastropod D. orbita has evolved a complex suite of metabolic capabilities that are not represented in the more primitive orthogastropods or bivalves, for which complete genome sequences are available.

Supplementary Materials: The following are available online at www.mdpi.com/1660-3397/14/7/135/s1, Figure S1: Tryptophan metabolism pathways for (A) Crassostrea gigas; (B) Lottia gigantean; and (C) Octopus bimaculoides showing enzyme matches in green including tryptophan 5-monoxygenase (red box), which was missing form Dicathais orbita, but no match to tryptophanase (blue box), Figure S2: Phenylalanine, tyrosine and tryptophan biosynthetic pathways for (A) Crassostrea gigas; (B) Lottia gigantean; and (C) Octopus bimaculoides with enzyme matches in green, but with no match to with tryptophan synthase highlighted in the blue box, Figure S3: Sulfur metabolism pathways for (A) Crassostrea gigas; (B) Lottia gigantea; and (C) Octopus bimaculoides, Figure S4: Cysteine and methionine metabolism pathway of (A) Crassostrea gigas; (B) Lottia gigantea; and (C) Octopus bimaculoides, Figure S5: Glycerophospholipid metabolism pathway of (A) Crassostrea gigas; (B) Lottia gigantea; and (C) Octopus bimaculoides showing enzyme matches in green with those relevant to choline ester synthesis highlighted in red (present) and blue (absent), Figure S6: Histidine metabolism pathway of (A) Crassostrea gigas; (B) Lottia gigantea; and (C) Octopus bimaculoides showing matching enzymes in green, including several enzymes that convert histidine into imidazole (red boxes) but not imidazoleglycerol-phosphate dehydratase (blue box), Table S1: Analysis of KEGG pathway showing the top 20 metabolic pathway involving the largest number of contigs in D. orbita trancriptome, Table S2: List of the 28 mapped contigs and with the KEGG orthology assignment in D. orbita trancriptome for tryptophan metabolism, Table S3: List of known bromoperoxidsae genes available in NCBI GenBank used for BLAST against D. orbita. Acknowledgments: This work was supported by the Southern Cross University International Postgraduate Research Scholarship and School of Environment, Science and Engineering Postgraduate support for A.K.N. and a philanthropic grant to K.B. Mar. Drugs 2016, 14, 135 14 of 16

Author Contributions: K.B. initiated the study and all authors contributed to the experimental design. A.K.N. prepared the samples for sequencing and A.B. assembled and annotated the transcriptome. All authors contributed to the data analysis and interpretation. A.B., A.K.N. and K.B. wrote the paper with editorial input from D.L.E.W. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations The following abbreviations are used in this manuscript: ENA European Nucleotide Archive NCBI National Center for Biotechnology Information BLAST Basic Local Alignment Search Tool ORFs Open Reading Frames KEGG Kyoto Encyclopedia of Genes and Genomes

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