- Research article
- Open Access
A new transcriptome and transcriptome profiling of adult and larval tissue in the box jellyfish Alatina alata: an emerging model for studying venom, vision and sex
© The Author(s). 2016
- Received: 18 May 2016
- Accepted: 18 July 2016
- Published: 17 August 2016
The Erratum to this article has been published in BMC Genomics 2016 17:980
Cubozoans (box jellyfish) are cnidarians that have evolved a number of distinguishing features. Many cubozoans have a particularly potent sting, effected by stinging structures called nematocysts; cubozoans have well-developed light sensation, possessing both image-forming lens eyes and light-sensitive eye spots; and some cubozoans have complex mating behaviors, including aggregations, copulation and internal fertilization. The cubozoan Alatina alata is emerging as a cnidarian model because it forms predictable monthly nearshore breeding aggregations in tropical to subtropical waters worldwide, making both adult and larval material reliably accessible. To develop resources for A. alata, this study generated a functionally annotated transcriptome of adult and larval tissue, applying preliminary differential expression analyses to identify candidate genes involved in nematogenesis and venom production, vision and extraocular sensory perception, and sexual reproduction, which for brevity we refer to as “venom”, “vision” and “sex”.
We assembled a transcriptome de novo from RNA-Seq data pooled from multiple body parts (gastric cirri, ovaries, tentacle (with pedalium base) and rhopalium) of an adult female A. alata medusa and larval planulae. Our transcriptome comprises ~32 K transcripts, after filtering, and provides a basis for analyzing patterns of gene expression in adult and larval box jellyfish tissues. Furthermore, we annotated a large set of candidate genes putatively involved in venom, vision and sex, providing an initial molecular characterization of these complex features in cubozoans. Expression profiles and gene tree reconstruction provided a number of preliminary insights into the putative sites of nematogenesis and venom production, regions of phototransduction activity and fertilization dynamics in A. alata.
Our Alatina alata transcriptome significantly adds to the genomic resources for this emerging cubozoan model. This study provides the first annotated transcriptome from multiple tissues of a cubozoan focusing on both the adult and larvae. Our approach of using multiple body parts and life stages to generate this transcriptome effectively identified a broad range of candidate genes for the further study of coordinated processes associated with venom, vision and sex. This new genomic resource and the candidate gene dataset are valuable for further investigating the evolution of distinctive features of cubozoans, and of cnidarians more broadly.
- Expression patterns
- Spawning aggregations
Cubozoa (box jellyfish) is a class of Cnidaria with a suite of distinct features including a cuboid bell, lens eyes and a typically highly potent sting. Like many cnidarians, cubozoan life history includes a swimming planula larva that ultimately settles onto a substrate to become an asexually reproducing polyp. Polyps then give rise to medusae (jellyfish), which have separate sexes and are the sexually reproductive stage. Some cubozoan taxa have evolved complex sexual behavior including synchronous spawning aggregations, mating and internal fertilization [1–3]. Cubozoan medusae vary widely in the potency of their sting; in humans, cubozoan stings range from being harmless to causing deadly envenomation [4–7]. A particularly note-worthy character of cubozoan medusae is their image-forming lens eyes, which have been implicated in visually-guided behavior [8–11].
Like all other cnidarians, cubozoans possess nematocysts (stinging organelles) essential for prey capture and defense. Nematocysts are remarkably complex subcellular structures that develop within specialized cells called nematocytes. Nematocysts are secreted from post-Golgi vesicles and consist of a double-walled capsule containing venom and a harpoon-like spiny tubule; and one to several different kinds can develop within a cnidarian throughout its life cycle . Nematocysts are of several forms, broadly divided into penetrant (e.g., euryteles) and adherent (e.g., isorhizas). Penetrant nematocysts are primarily concentrated in the tentacles of cubozoan medusae where they are used for prey capture. In some species, nematocysts are also found in body parts with putative digestive roles, such as the gastric cirri (in the stomach), where they may further immobilize prey items inserted into the cubozoan mouth (manubrium) . Adherent nematocysts are typically found on the exterior of the cubed-shaped bell and do not appear to function in predation there [14, 15]. The location of nematocyst development (nematogenesis) is poorly known in most cnidarians; having only been well-characterized in the model hydrozoan polyp Hydra, where morphology and molecular studies reveal clusters of developing nematocysts within the body . In contrast, molecular studies of another hydrozoan medusa Clytia, suggest that nematogenic regions are found in the tentacle bulb, proximal to the tentacles in which mature nematocysts are found . Transcriptomic and proteomic studies on the cubozoan Chironex fleckeri, the scyphozoans Chrysaora fuscescens and Stomolophus meleagris, and the hydrozoan Olindias sambaquiensis have focused on characterizing venom components from tentacle components [5, 18–21], but it is unknown whether nematogenesis and venom production occur solely in the medusa tentacles. In A. alata, tiny unidentified nematocysts have been documented within the tentacle base which is contiguous with the pedalium, but it is not clear if these represent an early developmental stage of the larger euryteles that are highly concentrated in the tentacle tips . Studies comparing expression of “venom implicated genes” across medusa body parts can help identify additional putative site(s) of venom production and regions of nematogenesis in cubozoans.
Unique among cnidarians, only cubozoan medusae possess image-forming eyes implicated in visual-guided behavior . Two complex eyes, complete with lens and retina, are located on special sensory structures called rhopalia on each of the four sides of the medusa bell. Each rhopalium also possesses a statocyst (balance organ), and two pairs of ocelli (light receptors) [8, 22] that lack a lens, like other simple animal eyes (having a single pigment cell and at least two photoreceptors [23–25]). Molecular components of the opsin-mediated phototransduction pathway have been identified in the rhopalium of the cubozoans Tripedalia cystophora and C. brevipedalia (as Carybdea rastonii) [26, 27], as well as in non-rhopalium medusa tissue, and planulae, which have simple eye spots [24, 27, 28]. Cubozoan planulae eye spots (ocelli) studied in T. cystophora are single cell structures containing a cup of pigment and photosensory microvilli, serving as rhabdomeric photoreceptors [24, 25]. Opsins have also been documented in other cnidarians without lens eyes [29–35] suggesting a role in light perception independent of image formation. Studies comparing expression of “vision implicated genes” across medusa body parts with and without eyes, and planula larvae with simple eyes, can help identify molecular components of the opsin-mediated phototransduction pathway in the rhopalium and aide in discovery of putative areas of extraocular photoreception in cubozoans.
Although most cnidarians reproduce sexually by simple broadcast spawning of their gametes (sperm and/or eggs), many cubozoan species engage in complex sexual behaviors including synchronous spawning aggregations, mating and internal fertilization [1–3]. In species with internal fertilization, such as Alatina alata  and Copula sivickisi , sperm are taken up by the female (as a spermatophore in the latter species), and following fertilization blastulae or planulae are released into the water [7, 36]. Histological studies have detected a gametogenic differentiation gradient within the gonads of two cubozoan taxa (Copula sivickisi and Carybdea xaymacana) [1, 22, 37], but it is unknown how widespread this process is in cubozoans. Equally elusive is the location of fertilization in cubozoans, although it has been hypothesized to occur in the gastrovascular cavity adjacent to the ovaries in a few species [1, 2]. Comparing expression patterns of “sex implicated genes” in different body parts can help determine whether a gametogenic differentiation gradient is present in additional cubozoan species, and might also aide in pinpointing more precisely the site of fertilization.
The goals of this study were to identify candidate genes in box jellyfish that may be involved in nematogenesis and venom production, vision and extraocular sensory perception, and sexual reproduction, which for brevity we refer to as “venom”, “vision” and “sex” implicated genes. We focused on the species Alatina alata, which provides a number of advantages for molecular investigation of these traits. The distribution of nematocysts has been well-documented in this species [2, 38, 39], and its sting is potent, causing serious human envenomation; like other cubozoans it has both simple and compound eyes on the medusa rhopalia as well as eye spots in planulae (ciliated swimming larvae); and mature medusae of this species form monthly nearshore spawning aggregations at predictable times (8–10 days after the full moon) in Indo-Pacific and Atlantic localities [2, 40, 41]. A. alata medusae have also been documented (as Carybdea alata) in the open ocean at great depths [2, 42]. The monthly predictability of mature medusae in nearshore waters [2, 43–45] and the ability to obtain planulae in vitro make A. alata a particularly favorable candidate for a cubozoan model.
RNA-Seq transcriptomics provides a reasonably unbiased method of profiling putative candidate genes in different body parts and life stages. This approach has been used successfully in other cnidarians to identify putative genes involved in different stages of a scyphozoan life cycle  and in different polyp types in a colony hydrozoan ; to identify candidate venom genes in anthozoans  and in the tentacles of venomous scyphozoans and cubozoans [5, 19]; and for reconstructing evolutionary relationships within Cnidaria . In the absence of a reference genome for A. alata, we generated a de novo transcriptome assembly pooled from RNA-Seq data from specific body parts (gastric cirri, tentacle—including the base of the adjoining pedalium, rhopalium, and ovaries) of a female medusa undergoing internal fertilization during a spermcast mating event. We compared these transcripts to known eukaryote gene and protein databases, and identified genes implicated in venom, vision and sex based on homology and tissue-specific gene expression profiles. We also investigated the expression of these candidate genes in planulae. Presented here is the first functionally annotated transcriptome of A. alata, which serves as a valuable resource for understanding the molecular underpinnings of cubozoan biological processes and their mediation of complex behaviors.
RNA-Seq and bioinformatics
De novo transcriptome assembly
A. alata pooled transcriptome assembly statistics
Filtered transcriptome (fpkm = 1.5)
No. of transcripts
No. of genes
Total assembled bases (bp)
Avg (mean) transcript length (bp)
Median transcript length (bp)
Max transcript length (bp)
GC content (%)
Percent proper pairs
Samtools percent mapped and paired
Our first objective was to annotate the A. alata transcriptome. The longest open reading frames (ORFs) were predicted for transcripts using TransDecoder  and subsequently annotated with Trinotate , which compiles results of homology searches of reliable databases (i.e., Uniprot; NCBI; eggNOG/GO; HMMER/PFAM, SingalP) to capture Basic Local Alignment Search Tool (BLAST) protein and gene homologies. The resulting Trinotate report for the ~32 K A. alata transcripts contained 12,317 BLASTP top hits from TrEMBL and 10,627 BLASTP top hits from SwissProt, from which 656 candidate genes were examined in this study for their putative roles in venom, vision and sex (see Candidate gene profiling below). In total 96 of the top 100 most abundant genes in the transcriptome (based on normalized counts) were assigned at least one Trinotate annotation category: 85 % of those had BLAST top hits; and 63 % corresponded to candidate genes we explored as implicated in either venom, vision or sex in this study (Additional file 2). The Trinotate report listed 14,551 transcripts corresponding to peptides based on TransDecoder predicted ORFs; 2098 transcripts with transmembrane protein domains (TMHMM database); 1610 transcripts containing the classical secretory signal peptide (SignalP database); and 5252 TrEMBL BLASTP top hits corresponded to cnidarian proteins (Additional file 3).
Gene expression patterns and profiles
We then sought to detect gene expression patterns across the five samples (gastric cirri, ovaries, tentacle with pedalium base, rhopalium, and planulae) with the aim of providing a descriptive analysis of the top expressed gene clusters by sample. In order to estimate transcript abundance we aligned each set of reads back to the A. alata transcriptome and generated an RNA-Seq fragment counts matrix for each sample using RSEM . We subsequently identified differentially expressed genes (see Methods) which we clustered according to their expression profiles using hierarchical clustering analyses within the framework of the EdgeR Bioconductor software package , a preferred methodology for studies lacking biological replicates [46, 56]. Of the ~32 K Trinity transcripts (~21 K Trinity genes) identified in the A. alata transcriptome ~10 K transcripts (6676 genes) were found to be differentially expressed, within a broad range, across the five samples (Additional file 4). EdgeR takes the normalized gene counts for all samples (generated from the initial RSEM counts matrix), and then clusters genes with similar mean expression rates across samples . Gene clusters are visualized in the form of a heatmap, permitting pinpointing of genes abundant in certain samples that might be of interest as candidate genes. The results of hierarchical clustering were consistent with our initial RSEM evaluation of abundant genes by sample.
Tissue-specific “core genes”
Candidate gene profiling
Putative venom implicated genes
Here we highlight our findings of the 450 transcripts we broadly refer to as “venom implicated genes” based on preliminary candidate gene profiling (above). This effort focused specifically on identifying genes highly expressed in the tentacle (used in prey capture) and gastric cirri (used in digestion). By comparing a body part with tissue abundant in penetrant nematocysts (tentacle and adjoining pedalium base) (Fig. 1e) with one lacking nematocysts (gastric cirri Fig. 1b) our aim was to identify putative site(s) of venom production and nematogenesis in A. alata. Although nematocysts are typically abundant in the gastric cirri of many box jellyfish species , only a single individual nematocyst has been documented in the gastric cirri of mature A. alata medusae  despite examination of hundreds of mature specimens in several independent studies [2, 15, 38, 40]. Conversely, nematocysts (Fig. 1e) are primarily concentrated in the tentacle (which is contiguous with the pedalium) of A. alata , as is the case with all cubozoans . Hierarchical gene cluster profiling (Fig. 6 a-k) revealed that many of the putative venom implicated genes were fittingly highly expressed in the tentacle (Figs. 5c and 6e-i), but were surprisingly also highly abundant in the gastric cirri (Figs. 5a and 6b,c; Additional file 7).
CaTX/CrTX toxin family genes
We report that an abundance of “cysteine-rich secretory protein family” (CRISPs) transcripts occurred almost exclusively in either the gastric cirri or the tentacle. Some examples include: “serine protease coagulation factor vii”, “chymotrypsin-like elastase family” homologs, and “serine protease inhibitor” (Figs. 5a and 6c). Likewise, multiple homologs of the “zinc metalloproteinase/astacin (peptidase family m12a)” (Figs. 5a, c and 6b, c, f, i, k) were primiarily abundant in the gastric cirri, but with high expression in the tentacle as well. Zinc metalloproteinases are peptidases with known roles in venom maturation in spiders and snakes, and were recently identified as tentacle venom components of some jellyfish taxa [5, 18, 58]. Conversely, homologs of well-known bilaterian venom proteins (e.g., pit viper (Croatulus)/zinc metalloproteinase nas-4/venom factor (Fig. 6i); scorpion (Lychas), venom protein 302 (Fig. 6h); the “venom prothrombin activator pseutarin-c non-catalytic subunit” from the eastern brown snake (Pseudonaja textilis) (Fig. 6i); and “alpha-2-macroglobulin family N-terminal region” (Fig. 6i) were most abundant in the gastric cirri and tentacle in this study, but were also expressed in the ovaries and rhopalium samples.
Nematocyst structural genes
Putative vision implicated genes
Here we highlight our findings of the 97 transcripts we broadly refer to as “vision implicated genes” based on preliminary candidate gene profiling (above). This effort focused specifically on genes expressed in the rhopalium of A. alata, which bears a pair of lens eyes with cornea and retina, two pairs of simple ocelli-comprising photoreceptors, and a statocyst (Fig. 1f). By comparing the rhopalium with its visual capabilities and planulae with its known eye-spot photoreceptors, against the medusa samples that lack known photoreceptors (gastric cirri, ovaries and tentacle), our aim was to identify the expression of opsins and other vision implicated genes in the rhopalium of A. alata, as well as in putative extraocular photoreceptors in A. alata. Hierarchical gene cluster profiling (Fig. 7a–k) revealed that most of the 97 putative vision implicated genes (see “gene-profiling” above) were abundant in the rhopalium (Fig. 5d), but in many cases they were more highly expressed in other samples, in particular in medusa samples (Fig. 7c, e-j; Additional file 8).
Among the top 10 most abundant genes in the rhopalium of A. alata was a homolog of the cubozoan lens-eye opsin for Carybdea rastonii (=C. brevipedalia) (Figs. 5d and 7i). The Carybdea lens eye opsin was also highly expressed in all A. alata samples, including planulae which have eye spots (Fig. 1h). Normalized counts revealed three additional opsin genes that were not differentially expressed across all samples, were almost exclusively found in the planulae sample (Fig. 11). Only two A. alata putative rhodopsin family homologs were expressed almost exclusively in the rhopalium (Trinotate top BLAST hits: “dopamine receptor 2” and “visual pigment-like receptor peropsin”) (Fig. 11). Among the putative “rhodopsin family” genes expressed in medusa samples, including those annotated in the Trinotate report as non-opsin based photoreceptors, were: “compound eye opsin bcrh1/d(1b) dopamine receptor” (Fig. 7e) and “opsin rh1/mu-type opioid receptor” (Fig. 7h), “blue-sensitive opsin” (Fig. 7k), “visual pigment-like receptor peropsin” (Fig. 7d), “melanopsin-b” (Figs. 7b and 8b; Additional files 8 and 9).
We found that several isozymes of cis-retinol dehydrogenase, members of the retinoic acid signaling pathway, which convert retinol to retinal (Vitamin A), were expressed in the rhopalium, and in other samples including planulae (Fig. 7c, g, k). In animals, retinal (i.e., 11-cis-retinal) is bound to opsin on the photoreceptors of the retina , and is thought to be a universal chromophore (light-activated pigment), though various chromophores are used across Metazoa . Carotenoid oxygenase beta, beta-carotene 15,15'-monooxygenase (BCDO1) (Fig. 7g, h) and beta, beta-carotene 9',10'-oxygenase (BCDO2), known to irreversibly cleave carotenoids to produce the essential visual pigments retinal and retinoic acid respectively , were expressed in the four A. alata medusa samples (Fig. 7e), suggesting that the catalytic components are present to make retinal. We also report the expression of a putative blue-sensitive photoreceptor protein and circadian clock regulator “cryptochrome-1” in the rhopalium, but with high expression in the gastric cirri (Fig. 8b). This suggests the presence of an additional putative chromophore in A. alata that functions in extraocular blue-light mediated behaviors (e.g., phototaxis), previously documented in coral and other metazoans [76–79].
We found transcripts showing similarity to all three known J-crystallin groups (J1, J2, J3), and all were highly expressed in the rhopalium (Figs. 5d and 7i, j). The J2 crystallin homolog was expressed in all samples including planulae (Fig. 7i); J3 crystallin was almost exclusively expressed in the rhopalium (Fig. 7j); as were all but a single J1 crystallin homolog that was also abundant in the ovaries (Figs. 6d and 7j). Crystallins are water-soluble stable structural proteins that provide transparency and increase the refractive index of eye lenses, though most also have roles unrelated to lens function. Numerous types of crystallins are found across Metazoa, and many are identical (or closely related) to commonly expressed metabolic enzymes or stress proteins [63, 80, 81]. J-crystallins are classified in three evolutionarily independent groups, J1, J2 and J3, and thus far have only been reported in cubozoans [63, 82, 83]. A study on T. cystophora showed that the promotors of all three J-crystallin genes can be activated by the paired domain transcription factor PaxB, but the promotor sites are non-homologous among the three J-crystallin types . We aligned all known cubozoan J-crystallins (J1, J2, J3) with the respective A. alata homologs identified in this study (Additional file 11). The resulting three alignments illustrate the similarity between A. alata transcripts identified in this study and homologs of the three distinct T. cystophora J-crystallin types. Additionally, Alpha-crystallin B chain” (vertebrate lens heat-shock proteins) (Fig. 7i) was abundant in the rhopalium, but expressed in all five samples. Conversely, we report transcripts annotated as S-type crystallin (cephalopod lens protein) variably expressed across samples: S-crystallin 2 abundant in the rhopalium and absent in ovaries (Fig. 7f), S-crystallin 3 abundant almost exclusively in the tentacle (Fig. 7d), and S-crystallin 4 most highly expressed in planulae and gastric cirri (Fig. 7k).
Homeobox genes and transcription factors
Expression of putative homeobox proteins “Six1b” and “Six4” and the “Six” transcriptional co-activator “eyes absent” (Eya) homolog occurred in all A. alata medusa samples, with the highest expression in the gastric cirri. Across Metazoa, the Six-Eya complex functions downstream from certain Pax homeobox genes in a diversity of developmental processes including early eye development . The putative “retinal homeobox proteins rx1b” and “rx3” were expressed in all samples, except for in the ovaries in the case of “rx1b”. Retinal homeobox proteins (“rax” or retina and anterior neural fold homeobox) are essential for early eye-development and in regulation of stem cell proliferation in vertebrates , but have not previously been reported in cnidarians.
Putative sex and development implicated genes
Here we highlight our findings of the 104 transcripts we broadly refer to as “sex implicated genes” based on preliminary candidate gene profiling (above). This effort focused mainly on genes expressed in the ovaries of A. alata during ovulation and internal fertilization. By definition, the ovaries are the site of oogenesis, and are situated within the gastrovascular cavity in A. alata . Microscopic examination of the gastrovascular cavity of the female A. alata medusa in this study revealed ovulation (Fig. 1c) and internal fertilization (Fig. 1d) occurring within this cavity. By comparing our ovaries sample, which also contained zygotes and embryos, with other body parts predicted to lack reproductive material (gastric cirri, tentacle, rhopalium, and planulae), our aim was to identify genes involved in gametogenesis as well as to determine more precisely the location of internal fertilization within A. alata, which we expected would occur adjacent to the ovaries, in the sperm-saturated gastrovascular cavity ). Hierarchical gene cluster profiling (Fig. 8a-k) revealed that the highest expression of the 104 putative sex and developmental genes (see “gene-profiling” above) occurred in the ovaries (Fig. 8d-i), but that many were also expressed across all samples (Additional file 9).
Oogenesis and embryogenesis
We report the abundance of multiple creatine kinase isozymes in the ovaries (Fig. 5b), all of which were expressed to some degree in all samples including planulae (Figs. 6d and 8h). Creatine kinase activity has a documented role in oogenesis and early embryogenesis in mammals . More broadly though, creatine kinase is important in cells with variable rates of energy turnover, such as muscle, neurons, photoreceptors, and primitive spermatozoa , which is consistent with its expression in all samples in this study.
Creatine kinase isozymes have also been documented in mediating high energy phosphate transport between sperm mitochondria and sperm flagellar tail [90, 91]. Among the most abundant genes in the ovaries (Fig. 5b) were homologs functioning in sperm tail development and motility: “parkin coregulated gene protein homolog” (Fig. 8i), “outer dense fiber protein 3/sperm-tail pg-rich repeat/shippo-rpt” (Fig. 8h), and multiple putative creatine kinase isoenzymes including “testis isozyme/protoflagellar creatine kinase” (Fig. 8j, h). Likewise multiple putative “serine/threonine-protein kinase” isoenzymes including “testis-specific serine/threonine-protein kinase 1” (Fig. 8g, h) were primarily abundant in the ovaries, but also variably expressed in all five samples. Expression in all samples of these sperm-related genes is consistent with the presence of ubiquitous sperm documented within the female gastrovascular cavity (where the ovaries are located) facilitating internal fertilization in this study (Fig. 1d). Sperm were also abundant in the surrounding seawater, and were undoubtedly adhered to the tentacles and exterior of the medusa bell when all tissue samples were excised from A. alata.
We report the exclusive upregulation in the ovaries of the putative sperm hyperactivation and acrosomal vesicle reaction promotor protein “cation channel sperm-associated protein” (CatSper2) (Fig. 8g). CatSper genes belong to the family of voltage-gated Ca2+ channels that are crucial for sperm fertility in mammals . In particular, capacitation, which typically occurs within the female reproductive tract, involves the destabilization of the acrosomal sperm head membrane allowing greater binding between sperm and oocyte during fertilization due to an increased permeability of Ca2+ . Recent studies have showed that sperm of several invertebrate species also undergo capacitation (for a review see ). However, until now the possibility of capacitation occurring in sperm of non-bilaterian invertebrates has not been investigated. Unexpectedly, the “CUB and zona pellucida-like domain-containing protein” was most highly abundant in the tentacle and gastric cirri (Fig. 6h, j), despite one of its known roles in ova of attracting sperm to eggs for fertilization in mammals . However, the CUB and zona pellucida-like domain-containing protein is also associated with trypsinogen activation and was previously found in box jellyfish tentacles . Overall, the abundance of transcripts related to sperm dynamics identified in the A. alata transcriptome permitted unforeseen profiling of molecular components involved in putative sperm capacitation and fertilization for the first time in a cubozoan.
This study has generated the first annotated transcriptome from multiple tissues of the cubozoan Alatina alata, focusing on both the adult (medusa) and larvae (planulae). Our transcriptome significantly adds to the genomic resources available for this emerging cubozoan model. This transcriptome, based primarily on multiple adult body tissues, complements a recently published transcriptome for the same species primarily from early developmental stages . Furthermore, in this study we annotated a large set of genes, allowing for an initial characterization of the molecular complexity of this cubozoan. We also compared transcript abundance across samples to identify genes putatively involved in several key features of cubozoans, namely nematogenesis and venom production, vision and sensory perception, and sexual reproduction. These quantitative data should be considered preliminary, due to lack of replication, but they are suggestive of interesting candidate genes that will be useful for future study. Below we highlight some of the major findings from this initial comparison across samples focusing specifically on genes relevant to i) prey capture and defense, ii) vision and the phototransduction pathway and iii) sexual reproduction and embryogenesis.
Prey capture and defense
In cubozoans, and more broadly in all cnidarians, prey capture and defense are based on nematocyst (stinging organelles) and associated venom. By comparing a body part abundant in penetrant nematocysts (tentacle and adjoining pedalium base) with one lacking nematocysts (gastric cirri) our aim was to identify putative site(s) of nematocyst development (nematogenesis) and venom production in A. alata.
We found that transcripts corresponding to a number of putative nematocyst structural proteins (minicollagens, nematogalectin, NOWA, chondroitin, and Dickkopf homologs) were abundant in the tentacle (and adjoining pedalium base). Although, putative nematogenic transcripts were detected primarily in the tentacle, some were also detected in non-tentacle medusa samples. We expect that this signal stems from the abundant adherent nematocysts covering the medusa bell. Together these findings are consistent with nematogenesis in A. alata occurring primarily, but not solely, in the region comprising the tentacle and adjacent pedalium base. Future in situ hybridization studies employing genes identified in nematogenesis in this study can help pinpoint more precisely nematogenic regions in A. alata.
Venom is a complex cocktail of bioactive compounds (e.g., protein and/or peptides called toxins, salts and neurotransmitters) secreted by one animal that is delivered to another animal by an infliction [57, 95]. Venom disrupts physiological and biochemical molecules of prey and predators, thus facilitating feeding and defense . Nematocysts have long been considered the sole secretory structure for venom deployment in cnidarians . However, we found preliminary evidence for venom production in the gastric cirri, where nematocsyts are lacking in mature A. alata. Futhermore, we found that the gastric cirri and tentacle express distinct groups of homologs of a major family of cnidarian venom proteins, the CaTX/CrTX toxin family [66, 67]. This suggests that venom plays an important, and possibly different role in the gastric cirri and tentacle. Venom components likley differ between the nematocyst-bearing tentacle, with a primary role in immobilizing prey and warding off predators, and the gastri cirri, with a primary role in killing and digesting prey .
Based on our findings, we hypothesize that A. alata has gland cells that secrete toxins associated with the gastric cirri. Evidence was recently presented for toxin-secreting gland cells in the ectoderm of the sea anemone Nematostella in regions containing nematocysts as well as areas that may lack nematocysts [97, 98], but our findings represent the first putative case in a cubozoan. Future morphological studies examining the ultrastructure of the stomach and gastric cirri, and venom gene candidate localization studies, will permit testing of the hypothesis of toxin-secreting gland cells associated with the gastric cirri of A. alata.
Although differences exist in the exact complement of putative bioactive toxins between the gastric cirri and tentacle sample, the venom cocktail in each body part includes transcripts from similar digestive enzyme families. A recent review of jellyfish toxins lists a number of toxin-like digestive enzymes that are deployed as components of nematocyst venom to disable homeostatic processes in prey or predators , as has been noted in animals possessing venom glands [57, 99, 100]. These bioactive proteins function in cytolytic, paralytic and hemolytic roles, thereby facilitating prey digestion [58, 64, 101]. Specifically we note the abundance of several enzyme groups primarily in either the tentacle or gastric cirri in A. alata that have been well studied in venomous animals [57, 99, 102, 103], namely astacin-like metalloproteinase and serine proteinase (and inhibitors), and more broadly cysteine-rich secretory proteins (CRISPs). Metalloproteinase and serine proteinase (and inhibitors) are a common component of the venom of animals with venom glands either activating toxins or acting as toxins themselves [18, 99, 100]. In particular, cysteine-rich secretory proteins identified in snake venoms are thought to inhibit smooth muscle contraction in bite victims . Both metalloproteinase and CRISPs have previously been characterized in the tentacles of cubozoan [5, 105] and other cnidarians [19, 20, 61, 68, 106]. The abundance of multiple isozymes of astacin-like metalloproteinase and serine proteinase (and inhibitors) and CRISPs in the gastric cirri and tentacle of A. alata suggest a dual role in venom and digestion. Further studies are required to test this hypothesis given the broad involvement of these bioactive proteins in other biological processes [5, 103].
Vision and the phototransduction pathway
Cubozoans are the earliest diverging animal clade to have image-forming lens eyes, which are part of specialized sensory organs called rhopalia. By comparing a medusa body part bearing conspicuous eyes (the rhopalium) and planulae with eye spots (rhabdomeric photoreceptors) against the medusa samples that lack documented photoreceptors (gastric cirri, ovaries and tentacle), our aim was to profile the molecular components of the opsin-regulated phototransduction pathway and identify additional regions of putative extraocular sensory perception in A. alata.
We found that many genes with conserved roles in vision (opsins and crystallins) were abundant in the rhopalium. Although transcripts with putative roles in light-mediated phototransduction pathway were detected primarily in the rhopalium where eyes are present, their expression was broadly detected across the medusa samples, and in some cases in planulae. We expect this signal stems from the presence of additional photoreceptors (yet undescribed) throughout the body of this cubozoan. Together these findings are consistent with a vision-related role for opsins and crystallins in the lens-eye of the rhopalium, as well as a role in putative photoreceptors within non-rhopalium tissues and in planulae eye spots.
The animal phototransduction pathway is mediated by photopigments in photoreceptors consisting of two parts: a membrane protein (apoprotein) “opsin” and a chromophore “retinal” (vitamin A derivative) . Opsins mediate light as phototypical G protein-coupled receptors in both visual and non-visual systems . Currently more than 1000 types of opsin are known across Metazoa, with three subfamilies recognized in bilaterians: rhabdomeric (r-opsins), Go-coupled plus retinochrome retinal G protein-coupled receptor (Go/RGR) and ciliary (c-opsins) . Studies characterizing opsins in cnidarians have raised the possibility that cnidarian opsins form a monophyletic clade referred to as “cnidops” that is sister to the c-opsins [26–28, 109, 110]. Other metazoan-wide analyses of opsins have categorized cnidarian opsins into three groups, A, B and C, in which each of these cnidarians opsin groups has been found, albeit with limited support, to be sister to each of the respective bilaterian opsin groups . One study has revealed support for r-opsins in cnidarians , which is consistent with the identification of planulae eye spots as rhabdomeric photoreceptors [24, 25]. Although a consensus is lacking on the relationships between cnidarian opsins and other metazoan opsins, our study identified a number of transcripts with molecular characters corresponding to diverse metazoan opsins (rhodopsin family) in A. alata, adding to the known diversity of this gene family within cnidarians.
Our opsin gene tree only included the known cnidarian opsins and thus does not address the question of cnidarian opsin monophyly. However, our analysis recovered homologs within two of the three previously identified cnidarian opsin groups, namely group A and B . Our analysis also recovered many opsin homologs within a large group that also contains cnidarian opsin group C, previously thought to only be present in anthozoans. The presence of this opsin group in both anthozoans and medusozoans suggests that it was present in the cnidarian ancestor.
We found several opsin genes in A. alata to be highly expressed in samples other than the rhopalium, and similar results have been reported for opsins in another cubozoan, T. cystophora [27, 28]. Based on these findings we hypothesize that cubozoans have opsin-mediated extraocular photoreception activity possibly related to phototaxis, circadian rhythm or light-mediated spawning, such as has been demonstrated in other animals, including anthozoans [29, 35, 76, 112]. These findings are also suggestive of extraocular photosensitivity [25, 108, 110, 113] that has a documented role in rhythmic behaviors and physiological processes in vertebrates and invertebrates, including nematocyst firing in cnidarians [28, 30, 32, 110, 111, 113]. Such suggested extraocular photoreceptor cells may also comprise anatomically dispersed light sensitive neurons, in addition to ciliary or rhabdomeric morphotypes, possibly functioning in dispersed photoreception, also called the “dermal light sense” (for a review see ). Future characterization of the absorbance spectra for different opsin types in cubozoans, and visualization of the precise locality of expression using in situ hybridization, will help elucidate their potential functions in different medusa body parts and planulae.
In this study the expression of some of the components of the retinal photoisomerization pathway in all samples including planulae suggests that A. alata metabolizes the universal chromophore retinal [35, 107]. However, transcripts for a putative blue-sensitive photoreceptor protein and circadian clock regulator cryptochrome homolog suggest an additional putative chromophore in A. alata that might function in non-rhopalium related blue-light mediated processes (e.g., phototaxis); such a function has previously been documented in other metazoans [35, 76]. Determining the precise chromophore utilized by A. alata must await future functional studies.
Crystallins are multifunctional proteins often related to stress or metabolic enzymes that serve as important lens components controlling optical properties . The dual role crystallins play in eye lens as well as non-eye related tissues is known as “gene sharing” . We found that all three types of J-crystallins previously reported in T. cystophora  were present in A. alata and that these were typically most highly expressed in the rhopalium, with J2 crystallins showing more variable expression across samples.
We also identified transcripts corresponding to the developmental transcription factors Six and eyes absent (Eya), representing the first homologs of these genes identified from cubozoans. Genes in the Six-Eya homolog complex have known functions in eye development, including during embryogenesis and regeneration, in both non-bilaterians and bilaterians [79, 84, 115]. Six-Eya complex genes have been shown to act downstream of Pax genes , and PaxB expression has been reported in both adult and larval eyes of T. cystophora, where it is inferred to promote J-crystallin expression . We also identified transcripts corresponding to the developmental transcription factors Six and eyes absent (Eya), representing the first homologs of these genes identified from cubozoans. Genes in the Six-Eya homolog complex have known functions in eye development, including during embryogenesis and regeneration, in both non-bilaterians and bilaterians [79, 84, 115]. Six-Eya complex genes have been shown to act downstream of Pax genes , and PaxB expression has been reported in both adult and larval eyes of T. cystophora, where it is inferred to promote J-crystallin expression . Conversely, in the scyphozoan Aurelia, development of simple eyes is mediated by Six-Eya complex genes independent of PaxB expression . Although the Trinotate report for the filtered A. alata transcriptome did not contain any transcripts annotated as PaxB, we identified two transcripts (comp95018 and comp20156) annotated as other homeobox genes that appear to be putative PaxB homologs based on sequence identity (tBLASTx) with Nematostella vectensis PaxB mRNA. Whether eye development and Six-Eya expression in A. alata are dependent or independent of PaxB expression remain open questions. Future studies determining the spatial localization of gene expression during eye development in A. alata may be useful for further elucidating the gene regulatory networks functioning in eye development in cubozoan rhopalia and planulae eyes spots.
Sexual reproduction and embryogenesis
Cubozoan lifecycles alternate between an asexually reproducing sessile polyp stage and a sexually reproducing motile medusa stage. By profiling the transcripts from an adult body part (ovaries) abundant in developing oocytes, our aim was to characterize the molecular components of oogenesis and early embryogenesis in A. alata. Additionally, because we found that sperm are internalized and interact with newly ovulated eggs within the gastrovascular cavity of A. alata females, our ovaries tissue sample also provided the opportunity to identify genes that might be involved in fertilization.
We identified several apparent homologs of Vitellogenin and Apolipoprotein, which have documented roles in oogenesis and embryogenesis, [86–89] and found these to be most abundant in the ovaries of A. alata. Vitellogenin is an animal egg yolk protein that is synthesized in somatic cell lineages and subsequently incorporated into developing oocytes (by receptor mediated endocytosis), eventually serving as a nutrition source during embryogenesis [86–88]. In medusozoans little is known about the characteristics of Vitellogenins as they have only been documented as egg yolk proteins in two coral species [88, 116] and the model sea anemone Nematostella vectensis . Vitellogenin proteins are expressed in both ovarian (or putative ovaries in anthozoans, e.g., ) and extra-ovarian somatic cells, consistent with their important roles in processing large lipoproteins in a broad range of complex biological processes among metazoans , including their distinct role as honey bee venom allergens . Consistent with this, we found that in A. alata, apparent homologs of Vitellogenin-2 were expressed most highly in the ovaries, yet they and other Vitellogenins and Apolipoprotein-like homologs were detected in all medusa samples. In this study we also found a number of creatine kinase genes to be most abundant in the ovaries, but many were also detected (though at much lower expression levels) in all of our samples. Creatine kinases play an important role in oogenesis and early embryogenesis in mammals , having a broad enzymatic function in yielding ATP by catalyzing the reversible transfer of phosphate from creatine phosphate to ADP in cells with high activity (e.g., photoreceptors, primitive-type spermatozoa) [90, 91].
We did not recover any genes characteristic of meiosis in the ovaries sample of A. alata, suggesting that the tissue was composed exclusively of mature ova at the time of sampling. It is also possible that the expression levels of putative meiosis transcripts were too low to be detected by our analyses, given our conservative transcriptome analysis protocol (see Methods). However, few studies exist that characterize the molecular aspects of sexual reproduction in cnidarians [88, 116], limiting the number of potential gametogenic candidate genes targeted in this study. Future transcriptome and proteome profiling studies of the gonads of A. alata and other cubozoans during medusa maturation are needed to shed light on the molecular underpinnings of the processes controlling gametogenesis in cubozoans.
In this study we also detected the expression of genes with putative roles in sperm flagella activation, pro-acrosomal vesicles and sperm capacitation, with many of these being most abundant in the ovaries of A. alata. These morphological and biochemical changes to the sperm are necessary for the sperm to reach and fertilize an oocyte, and their occurrence has been documented within the female reproductive tract in many animals . Sperm capacitation was previously thought to occur exclusively in mammals, but more recently it has been documented in several invertebrates . Our study is the first to suggest that sperm capacitation might occur within the gastrovascular cavity (putative female reproductive tract) of a cnidarian. We note that although sperm storage structures have been reported in a single family of cubozoans (Tripedaliidae) [1, 118], we do not expect sperm storage to occur in A. alata. Morphological observations during the course of this study as well as previous studies in A. alata have identified no structure(s) with a putative role in sperm storage in either male or female medusae [2, 38, 119]. Future histological studies of A. alata medusae undergoing internal fertilization should elucidate the ultrastructure of the female reproductive tract and provide further insight into fertilization dynamics in this species.
Based on our observations in this and a prior study , monthly spermcasting aggregations of A. alata medusae consist entirely of males and females with mature gonad morphology. We therefore hypothesize that gonad development occurs offshore in response to environmental and molecular cues related to the lunar cycle that may instigate inshore migrations. During these monthly nearshore aggregations, which span three to four consecutive days, both sexes exhaust their entire gamete reserves in a process known as “controlled gonad rupture” [2, 120]. Male gonads completely disintegrate over the course of several hours, and females simultaneously ingest massive quantities of sperm for internal fertilization. The interaction of sperm and eggs witnessed in the gastrovascular cavity, followed by release of blastulae into the surrounding water by females within hours, along with the abundance of sperm and fertilization-related transcripts detected in the ovaries sample, corroborate previous observations  that fertilization occurs immediately following sperm ingestion and ovulation, adjacent to the ovaries within the gastrovascular cavity. Future molecular studies characterizing expression in sperm and eggs prior to and during fertilization will provide further insight into the dynamics of fertilization in cubozoans.
Whereas most cubozoans are difficult to study in their natural settings, Alatina alata is becoming a useful model for evolutionary and molecular studies because mature adults can be found predictably in near-shore waters. In this study, we generated a new genomic resource for A. alata, a transcriptome of multiple adult tissues and larvae, and characterized patterns of expression of transcripts across several body parts of a female medusa and larval planulae. We identified a large suite of candidate genes implicated in predation and defense, vision and the phototransduction pathway, and sexual reproduction and embryogenesis. This new genomic resource and the candidate genes we have identified will be valuable for further investigating the evolution of distinctive features of cubozoans, and the evolution of cnidarians more broadly.
Male and female A. alata medusae were collected in Bonaire, The Netherlands. All proper collection and export permits were obtained. Medusae were kept within a glass aquarium in filtered seawater for several hours. Males shed sperm into the water that was taken up by the female manubrium. Using light microscopy (1000x) to observe the ovaries, which are located within the gastrovascular cavity, confirmation was made of ovulation and sperm and egg interaction (i.e., putative fertilization) (Fig. 1c, d). No prey items were present within the stomach and associated gastric cirri or attached to the tentacle. A live female A. alata medusa undergoing internal fertilization was placed on ice, and using a sterile RNase-free disposable scalpel tissue samples were quickly excised from the gastric cirri, ovaries, tentacle, rhopalium (Fig. 1). A fifth sample consisting of thousands of swimming planulae that had developed from blastulae released from different females in the lab was also collected. All samples were placed in 2 ml cryovials and flash frozen with liquid nitrogen. Frozen samples were shipped via Cryoport to the Smithsonian Biorepository. Additionally, a single spawning A. alata female medusa was collected at Oil Slick Leap, Kralendijk on April 22, 2014, relaxed in 7.5 % Magnesium chloride, fixed and preserved in 8 % formalin, and deposited into the collection of the National Museum of Natural History, Washington, D.C. as a morphological voucher (USNM 1248604). No specific permissions were required from an ethics committee to conduct the research described herein as no humans or protected species were used.
The five frozen tissue samples (gastric cirri, ovaries, tentacle (and adjoining pedalium base), rhopalium, and planulae) were sequenced at the University of Kansas Medical Center—Genomics Core (KUMC), where total RNA (0.5 ug) was used for library preparation for each sample. Illumina HiSeq 2500 Sequencing System was used to generate FASTQ files, which were de-multiplexed into individual sequences for further downstream analysis.
Transcriptome assembly and post-assembly analyses
The 278 M paired end (100 bp) raw reads from five samples were analyzed on the Smithsonian Institution High Performance Cluster, SI/HPC, and filtered using the program TrimGalore!  with the adaptor trimming tool Cutadapt  and FastQC  (--quality 30 --phred33 --length 25) to remove Illumina lane and multiplex adaptors (overlapping by 1 bp). ALLPATHSLG error correction software  was used on the 265 M trimmed paired end reads (PAIRED_SEP option was set to 100), and unpaired reads following trimming to predict and correct sequencing errors (see ) and mitigate potential errors in transcriptome assemblies. All five samples (i.e., gastric cirri, ovaries, tentacle, rhopalium, and planulae) were pooled and assembled de novo into a reference transcriptome (FASTA format) for A. alata using Trinity (version trinityrnaseq_r20131110) [50, 51], with the following additional flags: --no_bowtie --normalize_reads--path_reinforcement_distance 75.
Differential expression estimates and analyses
RNA-Seq by Expectation Maximization (RSEM) was run on each of the five samples separately to estimate transcript abundance (read counts). A single matrix was generated corresponding to expression values for all samples as normalized Trimmed Mean of M-values (TMM) . EdgeR was then used to identify differentially expressed genes in the counts matrix (--dispersion 0.1) ; followed by differential expression analysis to extract all genes most significantly expressed, i.e., with p-values < =0.005 and with at least a fourfold change of differential expression (--matrix iso_r123456.TMM.fpkm.matrix -P 1e-3 -C 2). This EdgeR step generated a single expression matrix of the results of all pairwise comparisons between the five samples. Further, hierarchical clustering generated a heatmap indicating clustering of similarly expressed genes (vertical axis) plotted by sample type (horizontal axis), while maintaining column order by sample (--order_columns_by_samples). This was done for all differentially expressed transcripts for all five samples (Additional file 4); just for the medusa samples (Fig. 3a); and for each of the three subsets of differentially expressed candidate genes (Figs. 6a, 7a and 8a). Color-coding on the vertical access of each heatmap indicates gene clusters with similar mean expression levels. Gene cluster patterns were further subdivided into 10 K-mean subclusters, which were visualized as subcluster profile plots (Figs. 3b-k, 6, 7 and 8b-k). In the absence of biological replicates in this study, the specific significance of fold-change expression levels of each of the differentially expressed genes was of limited value, and we therefore chose to not further filter transcripts based on additional statistical analyses. Instead, all differentially expressed genes were targeted as candidates for narrowing our search for genes of interest by sample type. Furthermore, redoing the hierarchical clustering analysis on just the three subsets of candidate genes (putative venom, vision and sex genes) allowed us to hone in on gene clusters that were relevant to transcriptome functional annotation and profiling of A. alata samples types.
Venn diagrams were constructed using Venny . Trinotate reports for each of the three sets of candidate genes investigated in this study (venom, vision and sex) were generated by filtering the original A. alata Trinotate report using this custom Python script: https://github.com/pbfrandsen/SI_scripts/blob/master/cheryl_trinotate.py.
Gene tree reconstruction
Amino acid sequences corresponding to predicted ORFs (TransDecoder), or translated nucleotide sequences, from the A. alata transcriptome were aligned using MUSCLE (default parameters with 5 iterations) against other cnidarian homologs from NCBI Genbank for the respective candidate genes of interest. ProtTest v. 3.2 was used to determine the most appropriate model of amino acid evolution (i.e., LG + G or WAG + G + F, or BLOSS62 I-G-F) for each alignment. Shimodaira-Hasegawa-like branch support indices  and are shown at each node of the ML topology. All ORF alignments (.nex files) predicted from TransDecoder, and the corresponding gene tree reconstructions (.tre files) are available at: https://figshare.com/articles/Supplemental_Information_for_A_new_transcriptome_and_transcriptome_profiling_of_adult_and_larval_tissue_in_the_box_jellyfish_Alatina_alata_an_emerging_modelfor_studying_venom_vision_and_sex/3471425.
We thank A. van Dorsten, J. van Blerk, A. Lin, R. Peachy and CIEE Bonaire staff for collection and lab assistance; S. Pirro, M. Shcheglovitova, M. Falconer, and D. Brinkman for assistance related to the study; Smithsonian Biorepository staff and Museum Support Center collections staff; KUMC Genome Core staff; Trinityrnaseq-users forum and B. Haas; Smithsonian Institution High Performance Computing Cluster and Laboratories of Analytical Biology staff: DJ Dajiang, M. Kweskin, V. Gonzalez, B. Bentlage and P. Frandsen for technical and bioinformatics support. We express our gratitude to two anonymous reviewers whose comments and suggestions helped improve the manuscript.
Funding for field work (CLA) was provided by University of Maryland Biological Sciences Eugenie Clark Scholarship and Smithsonian Peter Buck Predoctoral research grants, and for RNA-Seq by Paulyn Cartwright through NSF grant DEB–095357. JFR was supported by startup funds from the University of Florida DSP Research Strategic Initiatives #00114464 and University of Florida Office of the Provost Programs. AGC acknowledges the Mary & Robert Pew Public Education Fund, which supported the capture of some of the imagery in Fig. 1.
Availability of data and materials
The datasets supporting the conclusion of this article are available in the following repositories: Raw sequence data have been deposited into the NCBI Sequence Read Archive as BioProject PRJNA312373, as well as corresponding BioSamples each for gastric cirri, ovaries, tentacle, rhopalium, and planulae (SAMN4569893—SAMN 4569897). The Transcriptome Shotgun Assembly project has been deposited at DDBJ/ENA/GenBank under the accession GEUJ00000000. The filtered A. alata transcriptome described in this paper is the first version, GEUJ01000000. Additionally, BioProject PRJNA263637 and BioSample SAMN03418513 correspond to RNA-Seq data used for a cnidarian phylogenomic study  that we generated from the same A. alata medusa and cohort of planulae in the current study. Additional datasets supporting the conclusions of this article are included within the article and its additional files.
PC and CLA conceived of the study. All coauthors were involved in study design and data interpretation. CLA collected A. alata medusae and planulae and generated tissue samples. PC helped to generate RNA-Seq data. CLA and JFR contributed 80 % and 20 % respectively to bioinformatics. AGC and CLA contributed 80 % and 20 % respectively to gene tree reconstruction. CLA wrote the manuscript with substantial input by AEB. All coauthors have read and contributed to the final version of the manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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