Transcriptome sequencing and annotation of the polychaete Hermodice carunculata (Annelida, Amphinomidae)
© Mehr et al. 2015
Published: 10 June 2015
The amphinomid polychaete Hermodice carunculata is a cosmopolitan and ecologically important omnivore in coral reef ecosystems, preying on a diverse suite of reef organisms and potentially acting as a vector for coral disease. While amphinomids are a key group for determining the root of the Annelida, their phylogenetic position has been difficult to resolve, and their publically available genomic data was scarce.
We performed deep transcriptome sequencing (Illumina HiSeq) and profiling on Hermodice carunculata collected in the Western Atlantic Ocean. We focused this study on 58,454 predicted Open Reading Frames (ORFs) of genes longer than 200 amino acids for our homology search, and Gene Ontology (GO) terms and InterPro IDs were assigned to 32,500 of these ORFs. We used this de novo assembled transcriptome to recover major signaling pathways and housekeeping genes. We also identify a suite of H. carunculata genes related to reproduction and immune response.
We provide a comprehensive catalogue of annotated genes for Hermodice carunculata and expand the knowledge of reproduction and immune response genes in annelids, in general. Overall, this study vastly expands the available genomic data for H. carunculata, of which previously consisted of only 279 nucleotide sequences in NCBI. This underscores the utility of Illumina sequencing for de novo transcriptome assembly in non-model organisms as a cost-effective and efficient tool for gene discovery and downstream applications, such as phylogenetic analysis and gene expression profiling.
The amphinomid polychaete Hermodice carunculata (Annelida, Amphinomidae) is a cosmopolitan and ecologically important omnivore inhabiting coral reefs and other habitats throughout the Atlantic Ocean, including the Gulf of Mexico and the Caribbean Sea, as well as the Mediterranean and Red seas . It is known to prey on a diverse suite of reef organisms such as zoanthids [2,3], scleractinian corals [4-7], milleporid hydrocorals [5,8], anemones  and gorgonians . Hermodice carunculata is also a winter reservoir and spring-summer vector for the coral-bleaching pathogen Vibrio shiloi  and plays a complex and potentially ecologically important role in coral reef ecosystem health.
Amphinomidae is a well-delineated clade within aciculate polychaetes and it comprises approximately 200 described species from 25 genera [11-13]. Amphinomids are distributed worldwide and are known to inhabit intertidal, continental shelf and shallow reef communities, with a few species also recorded from the deep-sea . The clade is primarily identified by a series of morphological apomorphies including nuchal organs situated on a caruncle, a ventral muscular eversible proboscis with thickened cuticle on circular lamellae, and calcareous chaetae [12,14]. Due to the lack of knowledge regarding their morphological variability (particularly within closely related genera), previous studies based mainly on morphology have failed to clarify the evolutionary history of the group, leading to taxonomic problems. In fact, several nominal species have been regarded as conspecifics, often without evaluation of molecular data, which might explain the common occurrence of cosmopolitan species within the clade . Consequently, detailed revisions of species and even genera are needed , which incorporate molecular phylogenetic studies to clarify the affinities within the family [11,16]. Additionally, amphinomids are group with unclear phylogenetic position within Annelida as different studies find different evolutionary affinities for the group [16,17], but regarded as morphologically primitive and considered of prime interest for determining the root of the annelid Tree of Life . However, the availability of genomic data in public databases for Hermodice carunculata and other amphinomid species is particularly scarce. Previous to this study, only 279 sequences were accessible in NCBI for H. carunculata.
Furthermore, the annelid Hermodice carunculata is a representative of the Lophotrochozoa, a clade of protostome bilaterian animals that comprises about half of the extant animal phyla, including Mollusca, the second most diverse phylum . Annelids, in general, are of interest within lophotrochozoans because they are among the first coelomates  and polychaetes in particular, exhibit ancestral traits in body plan and embryonic development [20,21]. Nevertheless, polychaete annelids and lophotrochozoans have been heavily underrepresented in sequencing efforts, therefore, genomic resources for this key bilaterian clade are still relatively poor compared to the other two major bilaterian clades (Ecdysozoa and Deuterostomia) . A more complete representation of taxa in the genomic databases is needed to better understand animal evolution and unravel the origins of organismal diversity, especially of crucial clades such as the Lophotrochozoa [21,22].
Here, we provide a de novo transcriptome assembly of Hermodice carunculata, a cosmopolitan Lophotrochozoan polychaete that inhabits coral reefs throughout the Atlantic Ocean. In this study we use the Illumina HiSeq platform to generate a cDNA library for H. carunculata. These Next-Generation Sequencing (NGS) libraries have an enormous sequencing depth and better effectiveness, producing at least 100 to 10,000 times higher throughput than classical Sanger sequencing . This allows for the examination of thousands of transcripts from uncharacterized species and renders it useful for a wide range of biological applications including phylogenomics , regulatory gene discovery [25-28], molecular marker development , single nucleotide polymorphism (SNP) identification for trait adaptation [30,31], haplotype detection [32,33], and differential gene expression profiling [25,32]. In this study we provide a reference set of mRNA sequences for H. carunculata, which will facilitate annotation of the genome and future studies of polychaete evolution, systematics and functional genomics. We specifically focused on major signaling pathways and housekeeping genes, as well as genes related to reproduction and immune response, and we provide a comprehensive list of genes related to these key processes in the annelid H. carunculata.
Results and discussion
Sequencing and de novo assembly
Summary Statistics for individual and merged assemblies
Number of transcripts > 200 bp
Mean length bp
Max length bp
Total number of bp
Generated ORFs from Assembly
Number of ORFs >200 AA
Mean length AA
Max length AA
Total number of AA
ORFs > 200AA
A six frame translation (ORFs) from stop to stop for each assembled contig was generated using the EMBOSS package, version: 22.214.171.124 . This file contained 58,454 predicted ORFs longer than 200 AA, with the N50 of 490 AA, and mean length of 443.92 AA.
Comparative sequence similarity with other annelids
Functional annotation and characterization
One of the important aspects of mining the transcriptomic data is assigning function to individual transcripts. Functional annotation is an effective way to categorize genes into physiological classes to assist in understanding the large quantity of transcripts and for evaluating functional differences between subgroups of sequences. These data provide a tool for designing custom microarray experiments related to annotated functions . Gene ontology (GO, http://www.geneontology.org) [42,43] is an extensive scheme for this purpose. This framework covers a wide biological scope, and with its directed acyclic graph (DAG) structure, it accounts for biological dependencies. In addition, programs such as InterProScan [44,45] provide an integrated platform for domain-based searches against databases such as PROSITE , PRINTS , Pfam , and SMART , in addition to others. Over the past few years, resources have been developed for automatic GO term and InterPro ID assignment to unknown sequences. Blast2GO  was utilized for functional annotation, visualization and its associated statistics.
As part of the Blast2GO pipeline, ORFs longer than 200 AA (58,454) were subjected to sequence homology search against the non-redundant protein database (NR) at NCBI, using BlastP (E 10–10, cutoff =55, GO weight = 5, HSP coverage = 0). Followed by mapping to collect GO terms, and assigning reliable information to each query sequence. Default values of Blast2GO annotation parameters were chosen to optimize the ratio between annotation accuracy and coverage . This provided a framework for categorizing genes into functional annotation groups, namely biological process (sets of molecular events or operations with a defined beginning and end), molecular function (the primary activities of gene product at the molecular level, such as catalysis or binding), and cellular compartment. Furthermore, InterPro IDs (protein domain IDs) were assigned to sequences by running InterProScan (part of the Blast2Go pipeline).
Identification of candidate genes and potential phylogenetic markers
Signaling pathway and housekeeping genes
We identified 21 homologs of housekeeping genes belonging to CAT, MAT, PFK, ATP Synthase and 4,450 homologs of signaling pathways belonging to Activin, Deltex, DPP, Fringe, Jagged, Notch, Notch2, SMAD, TGF- β; (Additional file 2: Table S1). Riesgo and colleagues , in their analysis of ten transcriptomes of newly sequenced invertebrates, found similar homologs in mollusk and annelid transcriptomes.
Immune response genes
We identified 172 orthologous sequences of 37 genes involved in immune response (Additional file 2: Table S1), including caspase, interleukin, toll-like receptors, IRF genes, ficolin, antistasin and angiopoietin among others.
We identified 46 homologous sequences to 17 genes in>volved in reproduction, including attractin, vasa, germ cell-less, piwi, smaug, nanos, zona pellucida, spermatogenesis-associated proteins and zonadhesin (Additional file 2: Table S1).
Potential phylogenetic markers
Using reciprocal BLAST searches between the Hermodice carunculata transcriptome and publicly available sequences, we have identified putative H. carunculata homologues of genes that have been previously used as phylogenetic markers in Annelida but were unavailable for H. carunculata and amphinomids in general, with a few exceptions. We identified 900 homologous sequences of EF-1α, 101 homologous to H3, 7 homologous to CytB, and 400 homologous to U2 snRNA. We chose the longest sequence in each category for downstream phylogenetic analysis. The alignment of each of these sequences, along with the five best hits retrieved by BLAST from the NCBI database, are available in the supplementary materials (Additional files 3, 4, 5 and 6). Sequences were deposited in GeneBank.
Light production genes
In silico quantification of the hermodice carunculata transcriptome
Summary statistics of read counts and coverage
Total number of reads
Number of read used reads for assembly
Number of unused reads
Number of non-redundant transcripts (>200 bp)
Number of non-redundant trasncripts with back-aligned reads (>200 bp)
Number of transcripts with coverage fpkm >1
Number of transcripts with coverage fpkm >5
Average coverage for contigs from filtered dataset 2 (fpmk)
Average number of reads mapped per contig (with coverage fpkm >5)
Relying on Next Generation Sequencing techniques and a thorough bioinformatics pipeline we have generated a comprehensive list of major signaling pathways, housekeeping genes, and genes related to reproduction and immune response in a representative of the Lophotrochozoa, the polychaete annelid Hermodice carunculata, whose phylogenetic placement within Annelida has been difficult to resolve. Major signaling pathways are highly evolutionarily conserved across Metazoa and play an important role during embryonic and adult development, regulating many fundamental cellular processes such as proliferation, stem cell maintenance, differentiation, migration or apoptosis . In addition, some genes such as those involved in Notch signaling might have a role in segment formation and adult regeneration in polychaetes . Housekeeping genes are required for the maintenance of essential basal cellular functions and consequently, under normal conditions, they are expressed in all cells regardless of tissue type or developmental stage . They are especially interesting because they represent the minimal set of genes required to sustain life and they can be used as comparative controls for experimental and computational studies , for example, to assess the suitability of transcriptome datasets for gene discovery . Immune response genes are also of great concern especially among invertebrates because they represent an early model of the more highly evolved innate immune system of vertebrates . Knowledge of the invertebrate immune system is based mainly in two ecdysozoan model organisms, Drosophila melanogaster and Caenorabditis elegans, and although Lophotrochozoan systems show some distinct differences , studies focusing on this group are very limited. Lastly, characterization of the reproductive genes of polychaetes is of interest as they exhibit an astonishing diversity of reproductive strategies, including both sexual and asexual reproduction, and range from spawning and external fertilization to brooding or viviparism, often involving marked morphological, physiological and behavioral modifications . For example, some amphinomids such as Eurythoe complanata or Cryptonome conclava exhibit both sexual and asexual reproduction, the latter accomplished by architomic scissiparity: the body fragments in two or more parts which regenerate head, tail or both [13,59].
Several so-called cosmopolitan species within amphinomids have proven to comprise various cryptic species . Hermodice carunculata has a widespread distribution and has been reported throughout the Atlantic Ocean, Caribbean, Mediterranean and Red Sea [63,64]. Despite its widespread distribution, its representation in NCBI consisted of only 359 nucleotide sequences and only a handful of studies have examined genetic aspects of H. carunculata. For example, in a species delineation study, two mitochondrial genes (COI and 16S rDNA) and the internal transcribed spacer 1 (ITS1) were used to test for cryptic speciation in H. carunculata . This analysis showed that genetic divergence is low among samples across the Atlantic Ocean, and these particular three genes do not reflect any genetic basis for the observed morphological differences (e.g., variable filament abundance) among populations. Therefore, identification of informative loci for phylogeographic application is necessary. However, a different study using COI sequences has found that Eurythoe complanata represents a complex of three genetically distinct and morphologically indistinguishable lineages inhabiting the Atlantic and Pacific Oceans. Also, the deep-sea genus Archinome has been shown to comprise four genetically distinct lineages with no apparent morphological differences . Therefore, the de novo assembled transcriptome presented herein for Hermodice carunculata, can also be used to develop additional molecular phylogenetic markers to aid forthcoming studies of species boundaries and evolutionary relationships within Amphinomidae. Furthermore, amphinomids are a morphologically plesiomorphic group of annelids, considered as a highly important taxon for reconstructing relationships at the base of the annelid tree . Thus, the vast amount of molecular data provided herein can also help to elucidate the basal relationships of Annelida.
An additional recent approach in estimating more accurate intergeneric and intrageneric level relationships utilizes conserved blocks of homologous sequences shared between genomic regions of multiple species . Our data provides a complementary resource for this kind of application in the future. Also, the annotation of the genomes is reliant on transcriptome data for the exon intron boundary delimitation. Our data provide a base for future genomic and ecological research on Hermodice carunculata, as well as a resource to understand the natural history of polychaetes and the evolution of annelids in general.
Research, collecting and export permits were obtained from the government of the Bahamas while working out of the Perry Institute for Marine Science on Lee Stocking Island during a December 2011 expedition. The sample was collected by scientific divers D. Gruber, J. Sparks and M. Lombardi from Norman’s Pond Cay Cave, Norman’s Pond Cay, Exumas, Bahamas (GPS N 23 47.181, W 076 08.428). The cave’s entrance is a 2 m by 8 m sinkhole located just above high tide level and the cave is approximately 50 m linear and to a depth of 40 m. Divers explored the walls of the cavern zone using compact LED lights for cryptic invertebrate specimens. The Hermodice carunculata specimen was collected 30 m within the cave, transported back to the field station where it was frozen in liquid nitrogen less than two hours following collection.
RNA extraction and transcriptome sequencing
Total RNA was extracted from dissected tail muscles. The muscle tissue was homogenized in TriZol reagent (Life Technologies, NY) and the total RNA was precipitated with isopropanol and dissolved in ddH2O. The quality of RNA was assessed on a 2100 Bioanalyzer and with agarose gel electrophoresis. The total RNA was pooled for Library preparation. Libraries were prepared using a HiSeq RNA sample preparation kit (Illumina Inc, San Diego, CA) according to the manufacturer’s instructions. One lane was multiplexed for four samples and was sequenced as 80-bp PE reads. FASTQ file generation was performed by CASAVA version 1.8.2 (Illumina).
De novo assembly
All the assemblies were performed on a server with 50 cores and 250 GB random access memory. Obtained reads were de novo assembled, using ABySS  followed by Blat version: 34x12 , according to the proposed pipeline for merge and redundancy removal  in contigs generated by ABySS. In order to recover high and low expressed transcripts, a range of k-mers (21–55) was used prior to merge with Blat.
Sequences for the sex pheromone attractin were downloaded from GenBank (accession number generation in progress) and aligned with the Hermodice carunculata translated sequence using MUSCLE in SEAVIEW 4.3.0 . A phylogenetic analysis using amino acid sequences was conducted with RAxML ver. 7.7.1  using the maximum likelihood optimality criterion with a JTT amino acid substitution model. Support values were estimated using a rapid bootstrap algorithm with 1,000 replicates. The protozoan symbiont Capsaspora owczarzaki was specified as the outgroup.
Gene ontology (GO) terms and InterPro IDs were assigned to ORF sequences longer than 200 AA, using Blast2GO .
Availability of supporting data
Hermodice carunculata paired-end reads and assembled contigs can be downloaded at the NCBI Sequence Read Archive: http://www.ncbi.nlm.nih.gov/sra/SRX194586%5Baccn%5D. We have also made available at LabArchives (https://mynotebook.labarchives.com/share/smehr/MjAuOHw4NTE4MS8xNi9UcmVlTm9kZS8yNzE4MjI2NjQ1fDUyLjg=): 1) a Fasta file of homologous of contigs shared between Capitella teleta, Helobdella robusta and Hermodice carunculata; 2) a Fasta file of homologous contigs shared between Eurythoe complanata, Paramphinome jeffreysii and Hermodice carunculata; and 3) the functionally annotated Open Reading Frames generated from the Hermodice carunculata transcriptome.
We thank Ana Reisgo and Jean Gaffney for helpful comments, Zhou Han for laboratory assistance, Mike Lombardi for assistance with sample collection assistance and the staff of the John H. Perry Caribbean Research Center for hosting our field visit to the Bahamas. This work was supported by City University of New York Collaborative Incentive Research Grant #2064, PSC-CUNY Research Award # 66474–00 44, National Geographic Society/Waitt Grant W101-10 and National Science Foundation Grant to DFG, to the Sackler Institute for Comparative Genomics and the Korein Foundation to RD and the American Museum of Natural History to JSS.
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