The first chromosome-level cubozoan genome differentiates unique and common cnidarian features

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Law, Yiqian Li, Chade Li, Wenyan Nong, Kam Ling Chan, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7573227/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract Cnidarians are aquatic invertebrates that can be found in both freshwater and marine habitats, including the corals, sea anemones, hydroids, true jellyfish (scyphozoans), and box jellyfish (cubozoans). Despite most cnidarian groups already having representative genomic resources, a high-quality genome of the cubozoan remains lacking. Here, we obtained the first chromosomal-level genome of the box jellyfish Tripedalia maipoensis , having a genome assembly size of 637.8 Mb and a scaffold N50 of 47.1 Mb. By comparing the cubozoan genome to other cnidarian lineages, unique and common features of cnidarians, including cnidarian-specific opsins, toxins, neuropeptides, sesquiterpenoid hormones, and microRNAs were revealed. The high-quality genome of a cubozoan presented in this study reveals distinct cnidarian features and provides a crucial missing foundation for further understanding cnidarian evolution more broadly. box jellyfish sesquiterpenoid hormones neuropeptides microRNAs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The Cnidaria is a diverse phylum of invertebrates characterized by unique stinging cells used for predation and defense. It is estimated that approximately 10,000 species of cnidarians inhabit freshwater and marine environments worldwide, playing important roles in the ecosystem and human society. Cnidarians are broadly divided into several major groups, including Anthozoa (corals and sea anemones), Cubozoa (box jellyfishes), Hydrozoa (hydroids), Myxozoa, Scyphozoa (true jellyfishes), and Staurozoa (stalked jellyfishes). Genomics has emerged as a critical field for unraveling the biology and evolution of various animals. With advancements in sequencing technologies, a wealth of genomic resources has been amassed over the last decade for different cnidarian taxa, including the corals [ 1 – 3 ], sea anemones [ 4 – 7 ], hydroids [ 8 , 9 ], myxozoans [ 10 , 11 ], true jellyfishes/scyphozoans[ 12 ], and stalked jellyfishes/staurozoans [ 13 ]. However, genomic resources for box jellyfishes (Cubozoa) remain limited and are primarily available at draft-quality level [ 14 , 15 ] (Fig. 1 D), which hinders our understanding of their evolutionary history. Cubozoa includes approximately 50 described species of box jellyfish species, which can be further divided into two orders: Carybdeida (characterized by one tentacle per pedalium) and Chirodropida (characterized by multiple tentacles per pedalium) [ 16 ]. Box jellyfishes are notable for their potent venom; they are primarily found in the Indo-Pacific and Atlantic Oceans [ 17 ]. In this study, we present a high-quality genome of the box jellyfish Tripedalia maipoensis and compare it with other published cnidarian genomes, offering novel insights into the genomic evolution of the Cnidaria. Results and Discussion The first high-quality cubozoan genome Here, we present the first high-quality genome assembly of T. maipoensis , which is 637.8 Mb in size with sequence continuity of scaffold N50 = 47.1 Mb, and 96.76% of the sequences are anchored to 18 chromosomes (Fig. 1 A-C; Supplementary Tables 1 and 2). The genome size is comparable to the other published cubozoan genomes (Fig. 1 D; Supplementary Table 3). Utilising the Benchmarking Universal Single-Copy Orthologs (BUSCO, v5.5.0) as an estimation of completeness, 82.6% and 90.0% were respectively found on the genome and predicted gene models (Fig. 1 D). Macrosynteny analyses further revealed T. maipoensis shares a 1-to-1 relationship with other chromosomal-level cnidarian genomes, with a higher number of orthologous chromosomes compared to the scyphozoan genomes than to the anthozoan and hydrozoan genomes (Fig. 2 , Supplementary Figs. 1 and 2). Cnidopsin and toxin gene gains in cubozoans To understand the patterns of gene gains in the cubozoan lineage, we compared predicted genes across ten cnidarian genomes and two non-cnidarians (Fig. 3 A; Supplementary Table 4). Gene enrichment analyses identified gene gains in different KEGG pathways in T. maipoensis , including retinol metabolism (ko00830), steroid hormone biosynthesis (ko00140), metabolism of xenobiotics by cytochrome P450 (ko00980), serotonergic synapse (ko04726), and neuroactive ligand-receptor interaction (ko04080) (Fig. 3 B, Supplementary Table 5). Specifically, we found that gene families including cytochrome P450 (Pfam00067), UDP-glucuronosyltransferase (Pfam00201), trypsin (Pfam00089) and G protein-coupled receptors (Pfam00001) were expanded in the cubozoan lineage (Supplementary Fig. 3). Contrary to the hydrozoans and scyphozoans, cubozoans possess a special visual system that involves four different eye types located in each of the four rhopalia, namely one upper lens-eye, one lower lens-eye, two pit eyes and two slit eyes, adding up to a total set of 24 eyes [ 18 – 20 ]. Previous studies have also found that certain opsin gene family members are specific to cnidarians namely cnidopsins [ 21 – 24 ], including 19 cnidopsins that have been identified from the transcriptomes of box jellyfish Tripedalia cystophora (Tcop1-18 in [ 22 ] and TcGEO in [ 25 ]). Using sequence homology searches, we have identified a total of 108 and 123 opsin gene candidates in T. maipoensis and Morbakka virulenta genomes, respectively. Phylogenetic analyses further confirmed that 31 and 14 cnidarian specific opsins could be respectively identified in T. maipoensis and M. virulenta (Fig. 3 C-D; Supplementary Fig. 4–8; Supplementary Table 6). High level of sequence conservation in functional sites could also be observed between most cubozoan cnidopsin orthologues, including the lysine residue (K296) for chromophore binding and the cysteine pair (C110 and C187) for the disulphide bridge that stabilises the opsin structure [ 26 , 27 ] (Fig. 3 E). In sum, the first set of cubozoan cnidopsins revealed in this study could provide a valuable baseline for further understanding the functions of different cnidopsins. The cubozoan or box jellyfish are also well known for their potent and rapid-acting venom, and previous studies have suggested that jellyfish toxins (JFTs) are cnidarian-specific pore-forming toxins that contribute to the potency of cubozoan venoms [ 28 – 30 ]. In the genomes of T. maipoensis and M. virulenta , 29 and 31 JFTs were identified, respectively (Fig. 4 A; Supplementary Table 7). Phylogenetic analysis with putative JFTs [ 28 ] revealed that a greater number of gene copies in the JFT-1 clade (n = 18 in T. maipoensis and n = 26 in M. virulenta ) than the JFT-2 clade (n = 7 in T. maipoensis and n = 5 in M. virulenta ) (Supplementary Fig. 9). Many of the JFT genes are genomically located next to each other, suggesting that gene duplication events occurred in these lineages (24 in T. maipoensis = 24, and 22 in M. virulenta ) (Fig. 4 B; Supplementary Table 7). Conservation of neuropeptides and sesquiterpenoid farnesoic acid hormones in cnidarians On the other hand, neuropeptides are important signaling molecules in the nervous system that regulate various physiological activities in animals. A previous study has shown that neuropeptides in cnidarians and bilaterians do not share structural similarities [ 31 ]. In T. maipoensis , nine neuropeptide genes including FRamide, VWamide, RFamide, LWamide, RAamide, OKamide, RRFamide, and RYamide could be identified, and in agreement to previous studies [ 25 ] (Fig. 4 C; Supplementary Table 8; Supplementary Fig. 10). The sesquiterpenoid hormones are well known to regulate the development and reproduction in insects, and are now found in different invertebrates including the cnidarians [ 6 , 12 , 32 ]. In T. maipoensis , all genes involved in sesquiterpenoid hormone biosynthetic pathways can be identified for producing farnesoic acid (Fig. 4 D-E; Supplementary Table 9). Given these genes can also be identified in other investigated cnidarian lineages including scyphozoans [ 12 ], anthozoans [ 2 ], and hydrozoan [ 32 ], this suggested that the production of sesquiterpenoid hormone farnesoic acid is a conserved feature throughout cnidarians. These studies shown that cnidarians shared similar sesquiterpenoid farneosoic acid hormone but different neuropeptides to those in the bilaterians. Conserved microRNAs between cnidarians but not to bilaterians MicroRNAs act as crucial post-transcriptional regulators in animals, and they showed similarities and differences between cnidarians and bilaterians [ 33 ]. For instance, the targeting properties and regulation between microRNAs of cnidarians and bilaterians differ, and there is only one shared microRNA miR-100 between cnidarians and bilaterians [ 34 – 37 ]. Previous studies have identified lineage-specific microRNAs shared among scyphozoans and among anthozoans [ 6 , 12 , 38 ]. By sequencing the small RNAs in T. maipoensis and various scyphozoans, we are able to identify a total of 152, 201, 70, 214, 118, and 68 microRNAs in the cubozoan T. maipoensis and scyphozoans Sanderia malayensis , Chrysaora quinquecirrha , Aurelia coerulea , Rhopilema esculentum and Mastigias papua , respectively (Fig. 5 A; Supplementary Tables 10–15). Similar to previous studies, miR-100, miR-2022, and miR-2030 could be identified in most cnidarian genomes including the cubozoan [ 12 , 35 , 39 ]. Nevertheless, miR-2036 which was previously thought to be an anthozoan-specific microRNA, can now also be identified in the cubozoan lineage (Fig. 5 B, Supplementary Fig. 11). Furthermore, we have identified nine novel microRNAs (namely miR-CC1 to CC9) that exhibit sequence conservation across cubozoan, scyphozoans, and anthozoans; seven novel microRNAs (namely miR-MC1 to MC7) to be conserved between cubozoan and scyphozoans; and nine novel microRNAs (namely miR-SC1 to SC9) to be conserved only in scyphozoans (Fig. 5 B, Supplementary Figs. 12–14). These findings suggested that there are indeed deeply conserved microRNAs between different cnidarian lineages, despite there is only one microRNA shared between cnidarians and bilaterians. Conclusion In summary, this study presents the first high-quality genome of a cubozoan, filling a crucial gap in our understanding of cnidarian genome evolution. Our findings highlight genomic features that are uniquely acquired in cubozoans, such as cnidopsins and jellyfish toxins, as well as conserved features shared among cnidarians, including the sesquiterpenoid hormone farnesoic acid and various neuropeptides. Additionally, we identify divergences between cnidarians and bilaterians, particularly in the realm of microRNAs. Methodology High molecular weight DNA extraction High molecular weight (HMW) genomic DNA was extracted from ~ 500 mg tissue of a mature T. maipoensis medusa individual using CTAB [ 40 ] with modification of adding 1/3 volume 3M potassium acetate (Buffer P3, Qiagen) to the supernatant after the first chloroform wash. The isolated HMW DNA was subject to quality assessment using the NanoDrop™ One Microvolume UV–Vis Spectrophotometer (Thermo Scientific), Qubit Fluorometer, and overnight pulse-field gel electrophoresis. PacBio library preparation and long read sequencing DNA shearing was carried out using 5.2 µg HMW DNA in 120 µL elution buffer in a g-tube (Covaris) for 6 passes of centrifugation at 2,000 × g for 2 min, followed by a DNA purification step using SMRTbell® cleanup beads (PacBio). A SMRTbell library was subsequently prepared using the SMRTbell® prep kit 3.0 (PacBio) following the manufacturer’s instructions. 2 µL of the resulting library was subject to quality assessment using Qubit Fluorometer and pulse-field gel electrophoresis. The final library preparation for sequencing was carried out with Sequel® II binding kit 3.2 (PacBio) which allows SMRT bell structures to be annealed with Sequel II® primer 3.2 and bound with Sequel II® DNA polymerase 2.2 with the addition of Sequel II® DNA Internal Control Complex. The final library was sequenced on the Pacific Biosciences SEQUEL IIe System with the diffusion loading mode set at an on-plate concentration of 90 pM for 30-hour movies with 120 min pre-extension. Two SMRT cells were used for the generation of HiFi reads (Supplementary Table 1). Omni-C library preparation and sequencing ~ 100 mg tissue of the same individual used in HMW DNA extraction was used for Omni-C library construction using the Dovetail® Omni-C® Library Preparation Kit (Cantata Bio) following the manufacturer’s protocol. The concentration and fragment size of the lysate in the intermediate step and the final library were assessed by Qubit Fluorometer and TapeStation D5000 HS ScreenTape (Agilent), respectively. The resulting library was sent to Novogene (Hong Kong) and sequenced on an Illumina HiSeq-PE150 platform (Supplementary Table 1). RNA extraction, transcriptome and small RNA sequencing RNA from an 8-arm juvenile and a mature medusa individual of T. maipoensis ; the arm, bell, tentacle and gonad tissues of Chrysaora quinquecirrha; and the bell of Mastigia pupua were extracted using the mirVana miRNA Isolation Kit (Ambion) according to the manufacturer’s instructions. For Mastigia pupua , the tissues were pre-treated with an additional step using a CTAB solution (1M Tris-HCl, 0.2M EDTA, 5M NaCl, CTAB, 1% PVP and 1% beta-mercaptoethanol) for removal of insoluble materials from the samples. The isolated RNA was subject to quality control with gel electrophoresis and NanoDrop™ One Microvolume UV–Vis Spectrophotometer (Thermo Scientific). RNA samples were sent to Novogene (Hong Kong) for total RNA transcriptome library construction and sequencing on the Illumina Novaseq PE150 platform, and for small RNA library construction and sequencing on the Illumina Novaseq 50SE platform (Supplementary Table 1). Genome assembly and Gene model prediction De novo genome assembly of T. maipoensis was first performed using Hifiasm (version 0.19.8-r603)[ 41 ] and then searched against the NT database using blastn (version 2.15.0+)[ 42 ] to remove possible contamination using BlobTools (version 1.1.1)[ 43 ]. Subsequently, haplotypic duplications were removed according to the depth of the HiFi reads using “purge_dups” (version 0.0.3) [ 44 ]. Proximity ligation data from Omni-C were used to scaffold the assembly using YaHS (version 1.2a.2) [ 45 ] and manual checking using Juicebox (version 1.11.08)[ 46 ]. Briefly, Omni-C reads were mapped and aligned by BWA (version 0.7.18-r1243-dirty)[ 47 ] with parameters "mem − 5SP -T0", the parsing module of the pairtools pipeline [ 48 ] was used to find ligation junctions with parameters "--min-mapq 40 --walks-policy 5unique --max-inter-align-gap 30 --nproc-in 8 --nproc-out 8". The parsed pairs were then sorted using pairtools sort under default parameters. PCR duplicate pairs were removed using pairtools dedup with parameters "--nproc-in 8 --nproc-out 8 --mark-dups". The pairs file was split using pairtools split with default parameters and used to generate the contact matrix using juicertools and Juicebox. For the genomic characteristics of the assembly, the k-mer count and histogram were generated at k = 21 from the Omni-C reads using Jellyfish (version 2.3.0)[ 49 ] with the parameters "count -C -m 21 -s 1000000000 -t 10", and the reads.histo was uploaded to GenomeScope to estimate genome heterozygosity, repeat content and size using default parameters (version 2.0) ( http://qb.cshl.edu/genomescope/genomescope2.0/ )[ 50 ]. The resulting GenomeScope plots are shown in Fig. 1 C. For gene model prediction, the genomes were soft-masked using redmask (version 0.0.2)[ 51 ]. RNA sequencing data were first processed using Trimmomatic (version 0.39)[ 52 ] with parameters "TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:4:5 LEADING:5 TRAILING:5 MINLEN:25" and kraken2 (v2. 0.8 with kraken2 database k2_standard_20210517)[ 53 ] to remove the low quality and contaminated reads, and then aligned to the soft-masked repeat genome using hisat2 (version 2.2.1)[ 54 ] to generate the bam file. A total of 429,757 Cnidaria reference protein sequences were downloaded from NCBI on 27 May 2024 as protein hits, along with the RNA bam file, to perform genome annotation using Braker (version 3.0.8)[ 55 ] with default parameters. The same pipeline was used to perform genome annotation on the other three cubozoan genomes for comparison purposes. Two RNA-seq datasets (SRR7983770 and SRR7983771) were downloaded from the National Center for Biotechnology Information (NCBI) to provide transcriptome information for the Morbakka virulenta genome (GCA_003991215.1), and four RNA-seq datasets (SRR8101947, SRR8101944, SRR8101946 and SRR8101945) were downloaded to provide transcriptome information for the Chironex yamaguchii genome (GCA_024741275.1). However, the Braker pipeline could not be run for the Alatina alata (GCA_008930755.2), Carybdea cf. marsupialis (Linnaeus, 1758) (GCA_010016065.2) and Tamoya ohboya (GCA_028566775.1) genomes, as these contain highly fragmented assemblies (see Supplementary Table 3). Macrosynteny analyses Macrosynteny analyses were conducted by comparing the T. maipoensis genome with other chromosome-level assemblies using SyntenyFinder[ 56 ] (Supplementary Table 4). Briefly, single-copy orthologues were first identified by OrthoFinder v2.5.5[ 57 ] and were then further used to generate a data set of orthologous genes linked with the genomic position among the genomes. The gene linkage between each pair of genomes was analyzed in the SyntenyFinder pipeline and visualized using RIdeogram [ 58 ]. Furthermore, a synteny mixing score was generated between each genome pair to reflect the degree of chromosomal rearrangement [ 56 ]. Oxford dot plots of the genome pair were also generated as previously described[ 59 ]. Gene gain and functional enrichment analyses Gene orthology was inferred from the longest transcripts of the gene set extracted from 12 genomes, including 10 cnidarian genomes, and a placazoan and a ctenophoran genomes as outgroups, using OrthoFinder v2.5.5 [ 57 ] (Supplementary Table 4). The species tree and orthologue assignment generated from OrthoFinder were used as the input for CAFE 5[ 60 ], where three separate lambdas were set to infer the birth-death rate of orthologs for metazoans, anthozoans and outgroups, respectively. Functional annotations were performed in each gene set using eggnog [ 61 ] for the assignment of annotation terms, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and KEGG Orthology (KO). Enrichment analysis on gene gain was performed with the function ‘compareCluster()’ in the R package ‘clusterProfiler’ v.3.16.1[ 62 ] with p-value adjusted using Benjamini and Hochberg (BH) method and p-value cutoff at 0.05. For the validation of gene family gains, protein family candidates were searched in the gene set using HMMER[ 63 ] (version 3.3.1; cut-off E-value < 10 − 5) and were further screened using Reverse Position-Specific BLAST (RPS-BLAST) against the Conserved Domain Database [ 64 ]. The domain sequence was extracted and aligned using MAFFT v7.271[ 65 ]. A phylogenetic tree was constructed for each protein family using FastTree[ 66 ], which was then visualized in evolview3 [ 67 ]. Opsin gene family analyses Putative candidates of opsin genes in the T. maipoensis and Morbakka virulenta genomes were identified using 18 opsin protein sequences from the cubozoan jellyfish Tripedalia cystophora [ 22 ] as a query for sequence homology search with tblastn (E-value cut-off at 1e-05). Putative opsin candidates were further validated with phylogenetic tree construction. Briefly, the opsin candidates were aligned with other documented opsins in Cnidaria [ 21 , 24 , 25 , 68 , 69 ] and other non-opsin G-protein coupled receptors sequences as outgroup[ 69 ]. The aligned sequences were trimmed as described in [ 24 ] using TrimAl[ 70 ], which included removal of positions with a gap threshold of 90% and spurious sequences that do not possess an overlap of 50% with other sequences in 65% of their residues. Maximum-likelihood phylogenetic tree was constructed in IQ-TREE2[ 71 ] with 1,000 under the LG + F + R10 model, and visualised in evolview v3[ 67 ]. Gene positions on chromosomes were visualised with TBtools-II[ 72 ]. To reveal the cnidopsin orthology, the trimmed sequence alignment was visualized with Jalview v2[ 73 ]. Conserved structural and functional opsin motifs corresponding to the bovine rhodopsin numbering system were inspected as previously described [ 69 ]. Pairwise sequence identity was computed with the option “—percent-id” and output in a matrix format in Clustal Omega (v1.2.4)[ 74 ]. Jellyfish toxin gene identification Pore-forming jellyfish toxin (JFT) sequences extracted from previous studies[ 28 , 75 ] were used as a query in tblastn search against the T. maipoensis and Morbakka virulenta genomes (E-value cut-off at 1e-05). The JFT gene candidates were aligned with reference sequences together with the bacterial Cry toxins as outgroups as described in [ 28 ] using MAFFT v7.271[ 65 ]. The aligned sequences were trimmed with TrimAl [ 70 ]. A maximum-likelihood phylogenetic tree was constructed with Fasttree [ 66 ], followed by visualization in iTOL [ 76 ]. The locations of T. maipoensis JFTs gene were visualized in TBtools-II[ 72 ]. Sesquiterpenoid hormonal pathway genes and neuropeptide gene identification Sesquiterpenoid hormonal pathway genes of various cnidarian species were retrieved from KEGG and previous studies[ 6 ]for gene annotation in T. maipoensis . Retrieved gene sequences were used as query and carried out blastp (TBtools-II v2.310)[ 72 ] and tblastn (e-value of 1e-4) to identify potential candidates in predicted gene models and genomes, respectively. Potential candidates were also used to carry out reciprocal blastp in the NCBI ClusteredNR database with default settings. Annotated neuropeptide genes of Tripedalia cystophora [ 25 ] were retrieved from NCBI as a query for carrying out blastp and tblastn against gene model and assembled genome, respectively, to identify potential neuropeptide candidates in T. maipoensis with an e-value of 1e-4 through TBtools-II (v2.330) (Chen et al., 2023). Reciprocal blastp was conducted for potential candidates against the NCBI ClusteredNR database to identify confident candidates with default settings. Identified neuropeptide candidates were further submitted to the DeepNeuroPred function of NeuroPep2.0[ 77 ] to predict signal sequences and cleavage sites. microRNA analyses For the small RNA data in Tripedalia maipoensis , Chrysaora quinquecirrha , Mastigia pupua , Aurelia coerulea , Sanderia malayensis and Rhopilema esculentum , small RNA sequencing reads with adaptor sequences were trimmed, and Phred quality scores of less than 20 were deleted. Processed readings ranging in size from 18 to 27 bp were then mapped to their respective genomes using mapper.pl module of the mirDeep2 package [ 78 ]. miRDeep2 was used to identify novel microRNAs, which were then manually checked to ensure they fulfilled the criteria of MirGeneDB ( http://mirgenedb.org/information )[ 79 ]. The main criteria of MirgeneDB for determining authentic miRNAs include: the abundance of sequencing reads (both 5p and 3p read should have expression); and base pairs in at least 16 of the ~ 22 nucleotides; the 5p and 3p reads are offset by two nucleotides; the length of the loop is at least eight nucleotides long. Small RNA sequencing data from each species were examined independently for the presence of novel microRNAs. Only the candidate who fulfilled all MirGeneDB requirements was considered confidential microRNAs. MicroRNA annotations for Exaiptasia pallida , Anemonia viridis , Acropora millepora , Acropora digitifera , Stylophora pistillata and Catalaphyllia jardinei , Nematostella vectensis and Hydra magnipapillata were obtained from previous studies [ 12 , 35 , 38 ]. The mature arm and precursor sequences of all cnidarian microRNAs were used as BLASTn queries against genomes, while mature miRNA sequences were also used as query sequences with MapMi to identify potential conserved miRNA loci in each cnidarian genome (MapMi scorer cutoff = 15)[ 80 ], followed by checking the hairpin structure with CentroidFold[ 81 ]. In this study, only conserved microRNAs found in at least three species were regarded as confident, and sequence alignments were further carried out with MUSCLE in MEGA7[ 82 ] and visualized in Jalview[ 73 ]. Data availability This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number JBDZFW000000000. The version described in this paper is version JBDZFW010000000. The raw reads generated in this study, including transcriptome, Omni-C and PacBio HiFi data, have been deposited in the NCBI database under the BioProject accession number PRJNA1117645. The genome and repeat annotation files have been deposited and are publicly available in Figshare ( https://figshare.com/s/f212c65b74aa6318c8ae ) and CUHK Research Data Repository ( https://doi.org/10.48668/QSFWRO ). Declarations Conflict of interest The authors declare no conflict of interests. Funding This project was supported by the Hong Kong Research Grant Council Collaborative Research Grant (C4015-20EF) and General Research Fund (14103823, 14102224), The Chinese University of Hong Kong Direct Grant (4053618, 4053687), and The TUYF Charitable Trust. Author Contribution Jerome H.L. Hui supervised the study. Jian-Wen Qiu and Jerome H.L. Hui conceived the study. Sean T.S. Law carried out the synteny, toxin, gene gain analyses. 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As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7573227","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":515816033,"identity":"df8d8790-2f2e-47b7-af7a-cf93dfa9dbce","order_by":0,"name":"Sean T.S. 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The coloured lines represent connected orthologous genes from the 18 respective chromosomes of \u003cem\u003eT\u003c/em\u003e. \u003cem\u003emaipoensis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7573227/v1/8a601862c66f1c64266894eb.png"},{"id":91630402,"identity":"aa573fd0-d663-456d-a634-2a9228ad5637","added_by":"auto","created_at":"2025-09-18 12:54:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1398884,"visible":true,"origin":"","legend":"\u003cp\u003eA) Gene family expansion and contraction of the orthologous groups are indicated in green and red, respectively; B) Enriched KEGG pathways of gene family gains. The gene ratio indicates the number of gene gains divided by the total number of genes in the respective gene pathway; C) Maximum-likelihood tree of cnidopsins genes. Cnidopsins identified from \u003cem\u003eT. maipoensis\u003c/em\u003e and \u003cem\u003eMorbakka virulenta \u003c/em\u003eare highlighted in lightblue and blue, respectively, while the reference sequences from \u003cem\u003eTripedalia cystophora\u003c/em\u003e are highlighted in yellow. The internal nodes of opsins other than cnidopsins are collapsed for better visualisation (see Supplementary Figure 4 for the full tree); D) Gene locations of 31 cnidopsins in the genome of \u003cem\u003eT. maipoensis\u003c/em\u003e. Group 1a, 1b, 2 and 3 cnidopsins were labelled in purple, blue and orange and green, respectively; E) Summary of the 31 cubozoan orthologues.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7573227/v1/3828d9f96224c16138f67d89.png"},{"id":91630403,"identity":"4ce4c8be-181c-4106-b1c6-90039dc9f8fa","added_by":"auto","created_at":"2025-09-18 12:54:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":586316,"visible":true,"origin":"","legend":"\u003cp\u003eA) Jellyfish toxins (JFTs) identified in \u003cem\u003eTripedalia maipoensis\u003c/em\u003eand \u003cem\u003eMorbakka virulenta\u003c/em\u003e; B) Locations of JFTs in \u003cem\u003eT. maipoensis \u003c/em\u003egenome; C) Neuropeptide genes identified in \u003cem\u003eT. maipoensis\u003c/em\u003e; D) Schematic diagram showing the biosynthetic pathway of sesquiterpenoid hormones. Abbreviation: ACAT: Acetyl-CoA acetyltransferase; HMGS: 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR: 3-hydroxy-3-methylglutaryl-CoA reductase; MvK: Mevalonate kinase; MevPK: Phosphomevalonate kinase; MevPPD: Mevalonate (Diphospho) decarboxylase; FPPS: farnesyl diphosphate synthase; IPPI: Isopentenyl-diphosphate delta isomerase; FNTA: Farnesyltransferase, CAAX box, alpha; FNTB: Farnesyltransferase, CAAX box, beta; ZMPSTE24: STE24 endopeptidase; RCE1: Prenyl protein protease; ICMT: Isoprenylcysteine carboxyl methyltransferase; PCYOX1L: Prenylcysteine oxidase 1; ALDH: Aldehyde dehydrogenase III; E) Number of gene copies of sesquiterpenoid biosynthetic pathway genes in different cnidarian genomes.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7573227/v1/dbcee5b47324775d9266e391.png"},{"id":91630404,"identity":"3837e406-c508-482a-8648-fdcb8d96c0f7","added_by":"auto","created_at":"2025-09-18 12:54:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1110955,"visible":true,"origin":"","legend":"\u003cp\u003eA) Gains of microRNAs in different cnidarian lineages. The numbers inside the circles represent the total number of identified microRNAs in each species. B) Conservation of microRNAs shared between three or more species. “+” indicates the presence of respective microRNA, whereas “-” refers to the absence of respective microRNA. Abbreviation: \u003cem\u003eTmai\u003c/em\u003e, \u003cem\u003eTripedalia maipoensis\u003c/em\u003e;\u003cem\u003e Smal, Sanderia malayensis\u003c/em\u003e;\u003cem\u003e Cqui\u003c/em\u003e, \u003cem\u003eChrysaora quinquecirrha\u003c/em\u003e; \u003cem\u003eAcoe\u003c/em\u003e, \u003cem\u003eAurelia coerulea\u003c/em\u003e; \u003cem\u003eResc\u003c/em\u003e,\u003cem\u003e Rhopilema esculentum\u003c/em\u003e;\u003cem\u003e Mpap\u003c/em\u003e,\u003cem\u003e Mastigia pupua\u003c/em\u003e; \u003cem\u003eHmal\u003c/em\u003e,\u003cem\u003e Hydra magnipapillata\u003c/em\u003e;\u003cem\u003e Nvec\u003c/em\u003e,\u003cem\u003e Nematostella vectensis\u003c/em\u003e; \u003cem\u003eEpal\u003c/em\u003e, \u003cem\u003eExaiptasia pallida\u003c/em\u003e; \u003cem\u003eAvir, Anemonia viridis\u003c/em\u003e;\u003cem\u003e Amil\u003c/em\u003e, \u003cem\u003eAcropora millepora\u003c/em\u003e;\u003cem\u003e Adig\u003c/em\u003e,\u003cem\u003e Acropora digitifera\u003c/em\u003e;\u003cem\u003e Cjar\u003c/em\u003e, \u003cem\u003eCatalaphyllia jardinei\u003c/em\u003e; \u003cem\u003eSpis\u003c/em\u003e,\u003cem\u003e Stylophora pistillata\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7573227/v1/c6a4b6560ad51bfde0b481cf.png"},{"id":91633212,"identity":"9ff59fc5-1913-4b1e-b314-c3e5959cb84f","added_by":"auto","created_at":"2025-09-18 13:26:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8979076,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7573227/v1/cb5d865b-9019-4558-8884-68922bc7a16e.pdf"},{"id":91632349,"identity":"85978f1c-9a25-489a-ad9c-f32d0f022a2f","added_by":"auto","created_at":"2025-09-18 13:18:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":7588490,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7573227/v1/36b378cd0aca0607b13cd000.pdf"},{"id":91630407,"identity":"120e27bb-7091-40a4-bcea-c92d9bad85b0","added_by":"auto","created_at":"2025-09-18 12:54:33","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":193506,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7573227/v1/1ded79fe3d6d195f6e12182f.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The first chromosome-level cubozoan genome differentiates unique and common cnidarian features","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Cnidaria is a diverse phylum of invertebrates characterized by unique stinging cells used for predation and defense. It is estimated that approximately 10,000 species of cnidarians inhabit freshwater and marine environments worldwide, playing important roles in the ecosystem and human society. Cnidarians are broadly divided into several major groups, including Anthozoa (corals and sea anemones), Cubozoa (box jellyfishes), Hydrozoa (hydroids), Myxozoa, Scyphozoa (true jellyfishes), and Staurozoa (stalked jellyfishes).\u003c/p\u003e\u003cp\u003eGenomics has emerged as a critical field for unraveling the biology and evolution of various animals. With advancements in sequencing technologies, a wealth of genomic resources has been amassed over the last decade for different cnidarian taxa, including the corals [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], sea anemones [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], hydroids [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], myxozoans [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], true jellyfishes/scyphozoans[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and stalked jellyfishes/staurozoans [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, genomic resources for box jellyfishes (Cubozoa) remain limited and are primarily available at draft-quality level [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), which hinders our understanding of their evolutionary history.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCubozoa includes approximately 50 described species of box jellyfish species, which can be further divided into two orders: Carybdeida (characterized by one tentacle per pedalium) and Chirodropida (characterized by multiple tentacles per pedalium) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Box jellyfishes are notable for their potent venom; they are primarily found in the Indo-Pacific and Atlantic Oceans [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In this study, we present a high-quality genome of the box jellyfish \u003cem\u003eTripedalia maipoensis\u003c/em\u003e and compare it with other published cnidarian genomes, offering novel insights into the genomic evolution of the Cnidaria.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eThe first high-quality cubozoan genome\u003c/h2\u003e\u003cp\u003eHere, we present the first high-quality genome assembly of \u003cem\u003eT. maipoensis\u003c/em\u003e, which is 637.8 Mb in size with sequence continuity of scaffold N50\u0026thinsp;=\u0026thinsp;47.1 Mb, and 96.76% of the sequences are anchored to 18 chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C; Supplementary Tables\u0026nbsp;1 and 2). The genome size is comparable to the other published cubozoan genomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD; Supplementary Table\u0026nbsp;3). Utilising the Benchmarking Universal Single-Copy Orthologs (BUSCO, v5.5.0) as an estimation of completeness, 82.6% and 90.0% were respectively found on the genome and predicted gene models (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Macrosynteny analyses further revealed \u003cem\u003eT. maipoensis\u003c/em\u003e shares a 1-to-1 relationship with other chromosomal-level cnidarian genomes, with a higher number of orthologous chromosomes compared to the scyphozoan genomes than to the anthozoan and hydrozoan genomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Figs.\u0026nbsp;1 and 2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCnidopsin and toxin gene gains in cubozoans\u003c/h3\u003e\n\u003cp\u003eTo understand the patterns of gene gains in the cubozoan lineage, we compared predicted genes across ten cnidarian genomes and two non-cnidarians (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; Supplementary Table\u0026nbsp;4). Gene enrichment analyses identified gene gains in different KEGG pathways in \u003cem\u003eT. maipoensis\u003c/em\u003e, including retinol metabolism (ko00830), steroid hormone biosynthesis (ko00140), metabolism of xenobiotics by cytochrome P450 (ko00980), serotonergic synapse (ko04726), and neuroactive ligand-receptor interaction (ko04080) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Supplementary Table\u0026nbsp;5). Specifically, we found that gene families including cytochrome P450 (Pfam00067), UDP-glucuronosyltransferase (Pfam00201), trypsin (Pfam00089) and G protein-coupled receptors (Pfam00001) were expanded in the cubozoan lineage (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eContrary to the hydrozoans and scyphozoans, cubozoans possess a special visual system that involves four different eye types located in each of the four rhopalia, namely one upper lens-eye, one lower lens-eye, two pit eyes and two slit eyes, adding up to a total set of 24 eyes [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Previous studies have also found that certain opsin gene family members are specific to cnidarians namely cnidopsins [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], including 19 cnidopsins that have been identified from the transcriptomes of box jellyfish \u003cem\u003eTripedalia cystophora\u003c/em\u003e (Tcop1-18 in [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and \u003cem\u003eTcGEO\u003c/em\u003e in [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]). Using sequence homology searches, we have identified a total of 108 and 123 opsin gene candidates in \u003cem\u003eT. maipoensis\u003c/em\u003e and \u003cem\u003eMorbakka virulenta\u003c/em\u003e genomes, respectively. Phylogenetic analyses further confirmed that 31 and 14 cnidarian specific opsins could be respectively identified in \u003cem\u003eT. maipoensis\u003c/em\u003e and \u003cem\u003eM. virulenta\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D; Supplementary Fig.\u0026nbsp;4\u0026ndash;8; Supplementary Table\u0026nbsp;6). High level of sequence conservation in functional sites could also be observed between most cubozoan cnidopsin orthologues, including the lysine residue (K296) for chromophore binding and the cysteine pair (C110 and C187) for the disulphide bridge that stabilises the opsin structure [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In sum, the first set of cubozoan cnidopsins revealed in this study could provide a valuable baseline for further understanding the functions of different cnidopsins.\u003c/p\u003e\u003cp\u003eThe cubozoan or box jellyfish are also well known for their potent and rapid-acting venom, and previous studies have suggested that jellyfish toxins (JFTs) are cnidarian-specific pore-forming toxins that contribute to the potency of cubozoan venoms [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In the genomes of \u003cem\u003eT. maipoensis\u003c/em\u003e and \u003cem\u003eM. virulenta\u003c/em\u003e, 29 and 31 JFTs were identified, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; Supplementary Table\u0026nbsp;7). Phylogenetic analysis with putative JFTs [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] revealed that a greater number of gene copies in the JFT-1 clade (n\u0026thinsp;=\u0026thinsp;18 in \u003cem\u003eT. maipoensis\u003c/em\u003e and n\u0026thinsp;=\u0026thinsp;26 in \u003cem\u003eM. virulenta\u003c/em\u003e) than the JFT-2 clade (n\u0026thinsp;=\u0026thinsp;7 in \u003cem\u003eT. maipoensis\u003c/em\u003e and n\u0026thinsp;=\u0026thinsp;5 in \u003cem\u003eM. virulenta\u003c/em\u003e) (Supplementary Fig.\u0026nbsp;9). Many of the JFT genes are genomically located next to each other, suggesting that gene duplication events occurred in these lineages (24 in \u003cem\u003eT. maipoensis\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24, and 22 in \u003cem\u003eM. virulenta\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; Supplementary Table\u0026nbsp;7).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eConservation of neuropeptides and sesquiterpenoid farnesoic acid hormones in cnidarians\u003c/h3\u003e\n\u003cp\u003eOn the other hand, neuropeptides are important signaling molecules in the nervous system that regulate various physiological activities in animals. A previous study has shown that neuropeptides in cnidarians and bilaterians do not share structural similarities [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In \u003cem\u003eT. maipoensis\u003c/em\u003e, nine neuropeptide genes including FRamide, VWamide, RFamide, LWamide, RAamide, OKamide, RRFamide, and RYamide could be identified, and in agreement to previous studies [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; Supplementary Table\u0026nbsp;8; Supplementary Fig.\u0026nbsp;10).\u003c/p\u003e\u003cp\u003eThe sesquiterpenoid hormones are well known to regulate the development and reproduction in insects, and are now found in different invertebrates including the cnidarians [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In \u003cem\u003eT. maipoensis\u003c/em\u003e, all genes involved in sesquiterpenoid hormone biosynthetic pathways can be identified for producing farnesoic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E; Supplementary Table\u0026nbsp;9). Given these genes can also be identified in other investigated cnidarian lineages including scyphozoans [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], anthozoans [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and hydrozoan [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], this suggested that the production of sesquiterpenoid hormone farnesoic acid is a conserved feature throughout cnidarians. These studies shown that cnidarians shared similar sesquiterpenoid farneosoic acid hormone but different neuropeptides to those in the bilaterians.\u003c/p\u003e\n\u003ch3\u003eConserved microRNAs between cnidarians but not to bilaterians\u003c/h3\u003e\n\u003cp\u003eMicroRNAs act as crucial post-transcriptional regulators in animals, and they showed similarities and differences between cnidarians and bilaterians [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. For instance, the targeting properties and regulation between microRNAs of cnidarians and bilaterians differ, and there is only one shared microRNA miR-100 between cnidarians and bilaterians [\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Previous studies have identified lineage-specific microRNAs shared among scyphozoans and among anthozoans [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. By sequencing the small RNAs in \u003cem\u003eT. maipoensis\u003c/em\u003e and various scyphozoans, we are able to identify a total of 152, 201, 70, 214, 118, and 68 microRNAs in the cubozoan \u003cem\u003eT. maipoensis\u003c/em\u003e and scyphozoans \u003cem\u003eSanderia malayensis\u003c/em\u003e, \u003cem\u003eChrysaora quinquecirrha\u003c/em\u003e, \u003cem\u003eAurelia coerulea\u003c/em\u003e, \u003cem\u003eRhopilema esculentum\u003c/em\u003e and \u003cem\u003eMastigias papua\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; Supplementary Tables\u0026nbsp;10\u0026ndash;15). Similar to previous studies, miR-100, miR-2022, and miR-2030 could be identified in most cnidarian genomes including the cubozoan [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Nevertheless, miR-2036 which was previously thought to be an anthozoan-specific microRNA, can now also be identified in the cubozoan lineage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, Supplementary Fig.\u0026nbsp;11). Furthermore, we have identified nine novel microRNAs (namely miR-CC1 to CC9) that exhibit sequence conservation across cubozoan, scyphozoans, and anthozoans; seven novel microRNAs (namely miR-MC1 to MC7) to be conserved between cubozoan and scyphozoans; and nine novel microRNAs (namely miR-SC1 to SC9) to be conserved only in scyphozoans (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, Supplementary Figs.\u0026nbsp;12\u0026ndash;14). These findings suggested that there are indeed deeply conserved microRNAs between different cnidarian lineages, despite there is only one microRNA shared between cnidarians and bilaterians.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study presents the first high-quality genome of a cubozoan, filling a crucial gap in our understanding of cnidarian genome evolution. Our findings highlight genomic features that are uniquely acquired in cubozoans, such as cnidopsins and jellyfish toxins, as well as conserved features shared among cnidarians, including the sesquiterpenoid hormone farnesoic acid and various neuropeptides. Additionally, we identify divergences between cnidarians and bilaterians, particularly in the realm of microRNAs.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003eHigh molecular weight DNA extraction\u003c/h2\u003e\u003cp\u003eHigh molecular weight (HMW) genomic DNA was extracted from ~\u0026thinsp;500 mg tissue of a mature \u003cem\u003eT. maipoensis\u003c/em\u003e medusa individual using CTAB [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] with modification of adding 1/3 volume 3M potassium acetate (Buffer P3, Qiagen) to the supernatant after the first chloroform wash. The isolated HMW DNA was subject to quality assessment using the NanoDrop\u0026trade; One Microvolume UV\u0026ndash;Vis Spectrophotometer (Thermo Scientific), Qubit Fluorometer, and overnight pulse-field gel electrophoresis.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003ePacBio library preparation and long read sequencing\u003c/h3\u003e\n\u003cp\u003eDNA shearing was carried out using 5.2 \u0026micro;g HMW DNA in 120 \u0026micro;L elution buffer in a g-tube (Covaris) for 6 passes of centrifugation at 2,000 \u0026times; g for 2 min, followed by a DNA purification step using SMRTbell\u0026reg; cleanup beads (PacBio). A SMRTbell library was subsequently prepared using the SMRTbell\u0026reg; prep kit 3.0 (PacBio) following the manufacturer\u0026rsquo;s instructions. 2 \u0026micro;L of the resulting library was subject to quality assessment using Qubit Fluorometer and pulse-field gel electrophoresis. The final library preparation for sequencing was carried out with Sequel\u0026reg; II binding kit 3.2 (PacBio) which allows SMRT bell structures to be annealed with Sequel II\u0026reg; primer 3.2 and bound with Sequel II\u0026reg; DNA polymerase 2.2 with the addition of Sequel II\u0026reg; DNA Internal Control Complex. The final library was sequenced on the Pacific Biosciences SEQUEL IIe System with the diffusion loading mode set at an on-plate concentration of 90 pM for 30-hour movies with 120 min pre-extension. Two SMRT cells were used for the generation of HiFi reads (Supplementary Table\u0026nbsp;1).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eOmni-C library preparation and sequencing\u003c/h2\u003e\u003cp\u003e~\u0026thinsp;100 mg tissue of the same individual used in HMW DNA extraction was used for Omni-C library construction using the Dovetail\u0026reg; Omni-C\u0026reg; Library Preparation Kit (Cantata Bio) following the manufacturer\u0026rsquo;s protocol. The concentration and fragment size of the lysate in the intermediate step and the final library were assessed by Qubit Fluorometer and TapeStation D5000 HS ScreenTape (Agilent), respectively. The resulting library was sent to Novogene (Hong Kong) and sequenced on an Illumina HiSeq-PE150 platform (Supplementary Table\u0026nbsp;1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eRNA extraction, transcriptome and small RNA sequencing\u003c/h2\u003e\u003cp\u003eRNA from an 8-arm juvenile and a mature medusa individual of \u003cem\u003eT. maipoensis\u003c/em\u003e; the arm, bell, tentacle and gonad tissues of \u003cem\u003eChrysaora quinquecirrha;\u003c/em\u003e and the bell of \u003cem\u003eMastigia pupua\u003c/em\u003e were extracted using the mirVana miRNA Isolation Kit (Ambion) according to the manufacturer\u0026rsquo;s instructions. For \u003cem\u003eMastigia pupua\u003c/em\u003e, the tissues were pre-treated with an additional step using a CTAB solution (1M Tris-HCl, 0.2M EDTA, 5M NaCl, CTAB, 1% PVP and 1% beta-mercaptoethanol) for removal of insoluble materials from the samples. The isolated RNA was subject to quality control with gel electrophoresis and NanoDrop\u0026trade; One Microvolume UV\u0026ndash;Vis Spectrophotometer (Thermo Scientific). RNA samples were sent to Novogene (Hong Kong) for total RNA transcriptome library construction and sequencing on the Illumina Novaseq PE150 platform, and for small RNA library construction and sequencing on the Illumina Novaseq 50SE platform (Supplementary Table\u0026nbsp;1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eGenome assembly and Gene model prediction\u003c/h2\u003e\u003cp\u003e\u003cem\u003eDe novo\u003c/em\u003e genome assembly of \u003cem\u003eT. maipoensis\u003c/em\u003e was first performed using Hifiasm (version 0.19.8-r603)[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and then searched against the NT database using blastn (version 2.15.0+)[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] to remove possible contamination using BlobTools (version 1.1.1)[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Subsequently, haplotypic duplications were removed according to the depth of the HiFi reads using \u0026ldquo;purge_dups\u0026rdquo; (version 0.0.3) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Proximity ligation data from Omni-C were used to scaffold the assembly using YaHS (version 1.2a.2) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and manual checking using Juicebox (version 1.11.08)[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Briefly, Omni-C reads were mapped and aligned by BWA (version 0.7.18-r1243-dirty)[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] with parameters \"mem \u0026minus;\u0026thinsp;5SP -T0\", the parsing module of the pairtools pipeline [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] was used to find ligation junctions with parameters \"--min-mapq 40 --walks-policy 5unique --max-inter-align-gap 30 --nproc-in 8 --nproc-out 8\". The parsed pairs were then sorted using pairtools sort under default parameters. PCR duplicate pairs were removed using pairtools dedup with parameters \"--nproc-in 8 --nproc-out 8 --mark-dups\". The pairs file was split using pairtools split with default parameters and used to generate the contact matrix using juicertools and Juicebox. For the genomic characteristics of the assembly, the k-mer count and histogram were generated at k\u0026thinsp;=\u0026thinsp;21 from the Omni-C reads using Jellyfish (version 2.3.0)[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] with the parameters \"count -C -m 21 -s 1000000000 -t 10\", and the reads.histo was uploaded to GenomeScope to estimate genome heterozygosity, repeat content and size using default parameters (version 2.0) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://qb.cshl.edu/genomescope/genomescope2.0/\u003c/span\u003e\u003cspan address=\"http://qb.cshl.edu/genomescope/genomescope2.0/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The resulting GenomeScope plots are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. For gene model prediction, the genomes were soft-masked using redmask (version 0.0.2)[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. RNA sequencing data were first processed using Trimmomatic (version 0.39)[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] with parameters \"TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:4:5 LEADING:5 TRAILING:5 MINLEN:25\" and kraken2 (v2. 0.8 with kraken2 database k2_standard_20210517)[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] to remove the low quality and contaminated reads, and then aligned to the soft-masked repeat genome using hisat2 (version 2.2.1)[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] to generate the bam file. A total of 429,757 Cnidaria reference protein sequences were downloaded from NCBI on 27 May 2024 as protein hits, along with the RNA bam file, to perform genome annotation using Braker (version 3.0.8)[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] with default parameters. The same pipeline was used to perform genome annotation on the other three cubozoan genomes for comparison purposes. Two RNA-seq datasets (SRR7983770 and SRR7983771) were downloaded from the National Center for Biotechnology Information (NCBI) to provide transcriptome information for the \u003cem\u003eMorbakka virulenta\u003c/em\u003e genome (GCA_003991215.1), and four RNA-seq datasets (SRR8101947, SRR8101944, SRR8101946 and SRR8101945) were downloaded to provide transcriptome information for the \u003cem\u003eChironex yamaguchii\u003c/em\u003e genome (GCA_024741275.1). However, the Braker pipeline could not be run for the \u003cem\u003eAlatina alata\u003c/em\u003e (GCA_008930755.2), \u003cem\u003eCarybdea cf. marsupialis\u003c/em\u003e (Linnaeus, 1758) (GCA_010016065.2) and \u003cem\u003eTamoya ohboya\u003c/em\u003e (GCA_028566775.1) genomes, as these contain highly fragmented assemblies (see Supplementary Table\u0026nbsp;3).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMacrosynteny analyses\u003c/h2\u003e\u003cp\u003eMacrosynteny analyses were conducted by comparing the \u003cem\u003eT. maipoensis\u003c/em\u003e genome with other chromosome-level assemblies using SyntenyFinder[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] (Supplementary Table\u0026nbsp;4). Briefly, single-copy orthologues were first identified by OrthoFinder v2.5.5[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] and were then further used to generate a data set of orthologous genes linked with the genomic position among the genomes. The gene linkage between each pair of genomes was analyzed in the SyntenyFinder pipeline and visualized using RIdeogram [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Furthermore, a synteny mixing score was generated between each genome pair to reflect the degree of chromosomal rearrangement [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Oxford dot plots of the genome pair were also generated as previously described[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eGene gain and functional enrichment analyses\u003c/h2\u003e\u003cp\u003eGene orthology was inferred from the longest transcripts of the gene set extracted from 12 genomes, including 10 cnidarian genomes, and a placazoan and a ctenophoran genomes as outgroups, using OrthoFinder v2.5.5 [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] (Supplementary Table\u0026nbsp;4). The species tree and orthologue assignment generated from OrthoFinder were used as the input for CAFE 5[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], where three separate lambdas were set to infer the birth-death rate of orthologs for metazoans, anthozoans and outgroups, respectively. Functional annotations were performed in each gene set using eggnog [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] for the assignment of annotation terms, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and KEGG Orthology (KO). Enrichment analysis on gene gain was performed with the function \u0026lsquo;compareCluster()\u0026rsquo; in the \u003cem\u003eR\u003c/em\u003e package \u0026lsquo;clusterProfiler\u0026rsquo; v.3.16.1[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] with p-value adjusted using Benjamini and Hochberg (BH) method and p-value cutoff at 0.05. For the validation of gene family gains, protein family candidates were searched in the gene set using HMMER[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] (version 3.3.1; cut-off E-value\u0026thinsp;\u0026lt;\u0026thinsp;10\u0026thinsp;\u0026minus;\u0026thinsp;5) and were further screened using Reverse Position-Specific BLAST (RPS-BLAST) against the Conserved Domain Database [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The domain sequence was extracted and aligned using MAFFT v7.271[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. A phylogenetic tree was constructed for each protein family using FastTree[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], which was then visualized in evolview3 [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eOpsin gene family analyses\u003c/h2\u003e\u003cp\u003ePutative candidates of opsin genes in the \u003cem\u003eT. maipoensis\u003c/em\u003e and \u003cem\u003eMorbakka virulenta\u003c/em\u003e genomes were identified using 18 opsin protein sequences from the cubozoan jellyfish \u003cem\u003eTripedalia cystophora\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] as a query for sequence homology search with tblastn (E-value cut-off at 1e-05). Putative opsin candidates were further validated with phylogenetic tree construction. Briefly, the opsin candidates were aligned with other documented opsins in Cnidaria [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] and other non-opsin G-protein coupled receptors sequences as outgroup[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. The aligned sequences were trimmed as described in [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] using TrimAl[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e], which included removal of positions with a gap threshold of 90% and spurious sequences that do not possess an overlap of 50% with other sequences in 65% of their residues. Maximum-likelihood phylogenetic tree was constructed in IQ-TREE2[\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e] with 1,000 under the LG\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;R10 model, and visualised in evolview v3[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Gene positions on chromosomes were visualised with TBtools-II[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. To reveal the cnidopsin orthology, the trimmed sequence alignment was visualized with Jalview v2[\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Conserved structural and functional opsin motifs corresponding to the bovine rhodopsin numbering system were inspected as previously described [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Pairwise sequence identity was computed with the option \u0026ldquo;\u0026mdash;percent-id\u0026rdquo; and output in a matrix format in Clustal Omega (v1.2.4)[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eJellyfish toxin gene identification\u003c/h2\u003e\u003cp\u003ePore-forming jellyfish toxin (JFT) sequences extracted from previous studies[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e] were used as a query in tblastn search against the \u003cem\u003eT. maipoensis\u003c/em\u003e and \u003cem\u003eMorbakka virulenta\u003c/em\u003e genomes (E-value cut-off at 1e-05). The JFT gene candidates were aligned with reference sequences together with the bacterial Cry toxins as outgroups as described in [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] using MAFFT v7.271[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The aligned sequences were trimmed with TrimAl [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. A maximum-likelihood phylogenetic tree was constructed with Fasttree [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], followed by visualization in iTOL [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. The locations of \u003cem\u003eT. maipoensis\u003c/em\u003e JFTs gene were visualized in TBtools-II[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eSesquiterpenoid hormonal pathway genes and neuropeptide gene identification\u003c/h2\u003e\u003cp\u003eSesquiterpenoid hormonal pathway genes of various cnidarian species were retrieved from KEGG and previous studies[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]for gene annotation in \u003cem\u003eT. maipoensis\u003c/em\u003e. Retrieved gene sequences were used as query and carried out blastp (TBtools-II v2.310)[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] and tblastn (e-value of 1e-4) to identify potential candidates in predicted gene models and genomes, respectively. Potential candidates were also used to carry out reciprocal blastp in the NCBI ClusteredNR database with default settings. Annotated neuropeptide genes of \u003cem\u003eTripedalia cystophora\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] were retrieved from NCBI as a query for carrying out blastp and tblastn against gene model and assembled genome, respectively, to identify potential neuropeptide candidates in \u003cem\u003eT. maipoensis\u003c/em\u003e with an e-value of 1e-4 through TBtools-II (v2.330) (Chen et al., 2023). Reciprocal blastp was conducted for potential candidates against the NCBI ClusteredNR database to identify confident candidates with default settings. Identified neuropeptide candidates were further submitted to the DeepNeuroPred function of NeuroPep2.0[\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e] to predict signal sequences and cleavage sites.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003emicroRNA analyses\u003c/h2\u003e\u003cp\u003eFor the small RNA data in \u003cem\u003eTripedalia maipoensis\u003c/em\u003e, \u003cem\u003eChrysaora quinquecirrha\u003c/em\u003e, \u003cem\u003eMastigia pupua\u003c/em\u003e, \u003cem\u003eAurelia coerulea\u003c/em\u003e, \u003cem\u003eSanderia malayensis\u003c/em\u003e and \u003cem\u003eRhopilema esculentum\u003c/em\u003e, small RNA sequencing reads with adaptor sequences were trimmed, and Phred quality scores of less than 20 were deleted. Processed readings ranging in size from 18 to 27 bp were then mapped to their respective genomes using mapper.pl module of the mirDeep2 package [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. miRDeep2 was used to identify novel microRNAs, which were then manually checked to ensure they fulfilled the criteria of MirGeneDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mirgenedb.org/information\u003c/span\u003e\u003cspan address=\"http://mirgenedb.org/information\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. The main criteria of MirgeneDB for determining authentic miRNAs include: the abundance of sequencing reads (both 5p and 3p read should have expression); and base pairs in at least 16 of the ~\u0026thinsp;22 nucleotides; the 5p and 3p reads are offset by two nucleotides; the length of the loop is at least eight nucleotides long. Small RNA sequencing data from each species were examined independently for the presence of novel microRNAs. Only the candidate who fulfilled all MirGeneDB requirements was considered confidential microRNAs. MicroRNA annotations for \u003cem\u003eExaiptasia pallida\u003c/em\u003e, \u003cem\u003eAnemonia viridis\u003c/em\u003e, \u003cem\u003eAcropora millepora\u003c/em\u003e, \u003cem\u003eAcropora digitifera\u003c/em\u003e, \u003cem\u003eStylophora pistillata\u003c/em\u003e and \u003cem\u003eCatalaphyllia jardinei\u003c/em\u003e, \u003cem\u003eNematostella vectensis\u003c/em\u003e and \u003cem\u003eHydra magnipapillata\u003c/em\u003e were obtained from previous studies [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The mature arm and precursor sequences of all cnidarian microRNAs were used as BLASTn queries against genomes, while mature miRNA sequences were also used as query sequences with MapMi to identify potential conserved miRNA loci in each cnidarian genome (MapMi scorer cutoff\u0026thinsp;=\u0026thinsp;15)[\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e], followed by checking the hairpin structure with CentroidFold[\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. In this study, only conserved microRNAs found in at least three species were regarded as confident, and sequence alignments were further carried out with MUSCLE in MEGA7[\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e] and visualized in Jalview[\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThis Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number JBDZFW000000000. The version described in this paper is version JBDZFW010000000. The raw reads generated in this study, including transcriptome, Omni-C and PacBio HiFi data, have been deposited in the NCBI database under the BioProject accession number PRJNA1117645. The genome and repeat annotation files have been deposited and are publicly available in Figshare (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://figshare.com/s/f212c65b74aa6318c8ae\u003c/span\u003e\u003cspan address=\"https://figshare.com/s/f212c65b74aa6318c8ae\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e and CUHK Research Data Repository (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.48668/QSFWRO\u003c/span\u003e\u003cspan address=\"10.48668/QSFWRO\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis project was supported by the Hong Kong Research Grant Council Collaborative Research Grant (C4015-20EF) and General Research Fund (14103823, 14102224), The Chinese University of Hong Kong Direct Grant (4053618, 4053687), and The TUYF Charitable Trust.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eJerome H.L. Hui supervised the study. Jian-Wen Qiu and Jerome H.L. Hui conceived the study. Sean T.S. Law carried out the synteny, toxin, gene gain analyses. Yiqian Li carried out the microRNA analyses. Chade Li carried out the neuropeptide analyses. Wenyan Nong assembled the genome. Chade Li and Kam Ling Chan carried out the sesquiterpenoid hormone analyses. Carmen Or carried out the field collection. Sean T.S. Law, Yiqian Li, Chade Li, Wenyan Nong, Jerome H.L. Hui wrote the first draft of manuscript. All authors approved the final version of manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThis project was supported by the Hong Kong Research Grant Council Collaborative Research Grant (C4015-20EF) and General Research Fund (14103823, 14102224), The Chinese University of Hong Kong Direct Grant (4053618, 4053687), and The TUYF Charitable Trust.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShinzato C, Khalturin K, Inoue J, Zayasu Y, Kanda M, Kawamitsu M, et al. Eighteen coral genomes reveal the evolutionary origin of Acropora strategies to accommodate environmental changes. 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Mol Biol Evol. 2016;33:1870\u0026ndash;4. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/molbev/msw054\u003c/span\u003e\u003cspan address=\"10.1093/molbev/msw054\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Biology](https://bmcbiol.biomedcentral.com/)","snPcode":"12915","submissionUrl":"https://submission.springernature.com/new-submission/12915/3","title":"BMC Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"box jellyfish, sesquiterpenoid hormones, neuropeptides, microRNAs","lastPublishedDoi":"10.21203/rs.3.rs-7573227/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7573227/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCnidarians are aquatic invertebrates that can be found in both freshwater and marine habitats, including the corals, sea anemones, hydroids, true jellyfish (scyphozoans), and box jellyfish (cubozoans). Despite most cnidarian groups already having representative genomic resources, a high-quality genome of the cubozoan remains lacking. Here, we obtained the first chromosomal-level genome of the box jellyfish \u003cem\u003eTripedalia maipoensis\u003c/em\u003e, having a genome assembly size of 637.8 Mb and a scaffold N50 of 47.1 Mb. By comparing the cubozoan genome to other cnidarian lineages, unique and common features of cnidarians, including cnidarian-specific opsins, toxins, neuropeptides, sesquiterpenoid hormones, and microRNAs were revealed. The high-quality genome of a cubozoan presented in this study reveals distinct cnidarian features and provides a crucial missing foundation for further understanding cnidarian evolution more broadly.\u003c/p\u003e","manuscriptTitle":"The first chromosome-level cubozoan genome differentiates unique and common cnidarian features","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-18 12:54:28","doi":"10.21203/rs.3.rs-7573227/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-24T08:37:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-14T10:30:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-11T16:26:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-30T16:56:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-26T12:49:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-20T22:11:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200415943643029131342214450292327683450","date":"2025-09-16T09:03:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99618603842097587593686087001116091821","date":"2025-09-15T07:34:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66373615522147039738008506381961325701","date":"2025-09-11T08:08:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273235146013343081099009725717733599890","date":"2025-09-11T07:58:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"29342054669704850222805388294452018953","date":"2025-09-11T07:02:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-10T18:56:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-10T16:19:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-10T07:08:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Biology","date":"2025-09-09T11:15:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Biology](https://bmcbiol.biomedcentral.com/)","snPcode":"12915","submissionUrl":"https://submission.springernature.com/new-submission/12915/3","title":"BMC Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"29a935d8-2acd-463c-9599-24705167b341","owner":[],"postedDate":"September 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-19T14:55:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-18 12:54:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7573227","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7573227","identity":"rs-7573227","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}