Chromosome-level genome assembly of a deep-sea Venus flytrap sea anemone sheds light upon adaptations to an extremely oligotrophic environment.

The Venus flytrap sea anemone Actinoscyphia liui inhabits the nutrient-limited deep ocean in the tropical western Pacific. Compared with most other sea anemones, it has undergone a distinct modification of body shape similar to that of the botanic flytrap. However, the molecular mechanism by which such a peculiar sea anemone adapts to a deep-sea oligotrophic environment is unknown. Here, we report the chromosomal-level genome of A. liui constructed from PacBio and Hi-C data. The assembled genome is 522 Mb in size and exhibits a continuous scaffold N50 of 58.4 Mb. Different from most other sea anemones, which typically possess 14-18 chromosomes per haplotype, A. liui has only 11. The reduced number of chromosomes is associated with chromosome fusion, which likely represents an adaptive strategy to economize energy in oligotrophic deep-sea environments. Comparative analysis with other deep-sea sea anemones revealed adaptive evolution in genes related to cellular autophagy (TMBIM6, SESN1, SCOCB and RPTOR) and mitochondrial energy metabolism (MDH1B and KAD2), which may aid in A. liui coping with severe food scarcity. Meanwhile, the genome has undergone at least two rounds of expansion in gene families associated with fast synaptic transmission, facilitating rapid responses to water currents and prey. Positive selection was detected on putative phosphorylation sites of muscle contraction-related proteins, possibly further improving feeding efficiency. Overall, the present study provides insights into the molecular adaptation to deep-sea oligotrophic environments and sheds light upon potential effects of a novel morphology on the evolution of Cnidaria.

Detection and Quantification of Two Parasitic Ciliates Boveria labialis and Boveria subcylindrica (Ciliophora: Scuticociliatia) by Fluorescence in situ Hybridization.

The thigmotrich scuticociliates Boveria labialis and Boveria subcylindrica are obligate parasites that may cause high mortality in cultured sea cucumbers and bivalves. Morphological methods can identify these organisms in active state, but are unable to discern them in resting stages. In aquaculture practice, these parasitic ciliates are hard to eradicate when massive infection occurs in sea cucumbers. Thus, early detection and precaution are crucial for the control of these pathogens. Under such circumstances, fluorescence in situ hybridization (FISH) will serve as a fast way to detect and monitor the occurrence of these parasites. We designed two SSU-rDNA targeted oligonucleotide probes labeled with fluorochromes, and optimized the FISH protocols for the detection of B. labialis and B. subcylindrica from the host sea cucumber Apostichopus japonicus and the bivalve Atrina pectinata, respectively. The assays resulted in a clear differentiation of the two similar species by strong fluorescence signals from the oligonucleotide probes. Moreover, we successfully used the FISH protocol to detect the cysts of B. labialis and variation in abundance of active parasites to evaluate the efficacy of chemical treatments. This is the first report and detection of the cysts of B. labialis from the host sea cucumber A. japonicus.

Systematics and Molecular Phylogeny of the Ciliate Genus Pseudokeronopsis (Ciliophora, Hypotrichia).

A still challenging study on ciliate systematics is to clarify the taxonomic and phylogenetic confusions of the fascinating pigmented Pseudokeronopsis species because of their high interspecific similarities. We evaluated the identities and internal relationships within Pseudokeronopsis based on morphological descriptions in combination with the 18S and ITS-5.8S rDNA genetic distances, ITS2 secondary structures and phylogenetic analyses. Results showed that (1) Pseudokeronopsis pararubra, a species ever synonymized as Pseudokeronopsis carnea, is a valid species; (2) most Chinese populations identified as Pseudokeronopsis rubra represent a new species namely Pseudokeronopsis songi sp. nov.; (3) within Pseudokeronopsis, P. carnea branched early, while P. songi sp. nov., P. flava, and P. erythrina showed close relationships and formed a sister clade with P. pararubra; (4) compared to 18S and ITS2 rDNA, ITS-5.8S rDNA is more suitable for separating Pseudokeronopsis species, with pairwise distances of 0-0.0441 at the intraspecific level and 0.0635-0.1150 at the interspecific level; and (5) the core structure of Pseudokeronopsis ITS2 includes three helices, with helix III being the longest and showing an identical pattern in conspecific populations and small differences among species. Based on the evaluation, we clarify all misidentified and dubious 18S and ITS-5.8S rDNA sequences of Pseudokeronopsis in GenBank.

Miamiensis avidus, a Novel Scuticociliate Pathogen Isolated and Identified from Cultured Large Yellow Croaker (Larimichthys crocea).

Scuticociliates are recognized as the causative agents of mass mortalities in certain cultured marine fishes, resulting in enormous economic losses. This study aimed to investigate a fatal infection caused by scuticociliates in farmed large yellow croaker (Larimichthys crocea) in Fujian province, China. Microscopic examinations of focal organs, including the brain, eyes, gills, and skin, revealed the presence of parasites. Active masses of scuticociliates were observed in these organs, and the ciliates were subsequently isolated and maintained in vitro. An immersion challenge experiment revealed that L. crocea experienced cumulative mortalities reaching 73% within 7 d upon exposure to 1.0 × 104 ciliates mL-1. Based on the microscopic and PCR testing of infected fishes, the brain was comprehensively inferred as the main infection organ for the isolated strain. Microscopic and submicroscopic observations of the isolated scuticociliate, coupled with cortical ciliature patterns revealed through α-tubulin indirect immunofluorescence techniques, identified these scuticociliates as Miamiensis avidus. The sequencing of two genetic markers (small subunit ribosomal RNA, SSU rRNA and cytochrome c oxidase subunit I, COI) further confirmed that the isolated strains exhibited the highest sequence similarity to most M. avidus sequences in GenBank. However, significant differences in SSU sequences compared to the M. avidus strain Ma/2, and the lack of published COI and ITS (internal transcribed spacer) sequences for Ma/2, indicate the need for further molecular data to resolve whether there are potential cryptic species within the M. avidus complex.

Transoceanic ships as a source of alien dinoflagellate invasions of inland freshwater ecosystems.

Ships' ballast water and sediments have long been linked to the global transport and expansion of invasive species and thus have become a hot research topic and administrative challenge in the past decades. The relevant concerns, however, have been mainly about the ocean-to-ocean invasion and sampling practices have been almost exclusively conducted onboard. We examined and compared the dinoflagellate cysts assemblages in 49 sediment samples collected from ballast tanks of international and domestic routes ships, washing basins associated with a ship-repair yard, Jiangyin Port (PS), and the nearby area of Yangtze River (YR) during 2017-2018. A total of 43 dinoflagellates were fully identified to species level by metabarcoding, single-cyst PCR-based sequencing, cyst germination and phylogenetic analyses, including 12 species never reported from waters of China, 14 HABs-causing, 9 toxic, and 10 not strictly marine species. Our metabarcoding and single-cyst sequencing also detected many OTUs and cysts of dinoflagellates that could not be fully identified, indicating ballast tank sediments being a risky repository of currently unrecognizable invasive species. Particularly important, 10 brackish and fresh water species of dinoflagellate cysts (such as Tyrannodinium edax) were detected from the transoceanic ships, indicating these species may function as alien species potentially invading the inland rivers and adjacent lakes if these ships conduct deballast and other practices in fresh waterbodies. Significantly higher numbers of reads and OTUs of dinoflagellates in the ballast tanks and washing basins than that in PS and YR indicate a risk of releasing cysts by ships and the associated ship-repair yards to the surrounding waters. Phylogenetic analyses revealed high intra-species genetic diversity for multiple cyst species from different ballast tanks. Our work provides novel insights into the risk of bio-invasion to fresh waters conveyed in ship's ballast tank sediments and washing basins of shipyards.

Studies on western Pacific gorgonians (Anthozoa: Octocorallia, Chrysogorgiidae). Part 1: a review of the genus Chrysogorgia, with description of a new genus and three new species.

Members of Chrysogorgia Duchassaing & Michelotti, 1864, known as golden corals, are distributed in the deep sea worldwide. Based on specimens obtained from the tropical western Pacific and an integrated morphological-molecular approach, we established a new genus Parachrysogorgia, which includes 13 species separated from Chrysogorgia, and described nine species of Chrysogorgia including three new species C. arboriformis sp. nov., C. cylindrata sp. nov. and C. tenuis sp. nov., and two new combinations of Parachrysogorgia. The genus differs from Chrysogorgia by possessing a bare, sclerite-free area at the base of each tentacle and eight distinct projections beneath tentacles. Chrysogorgia cylindrata sp. nov. is distinguished by a slender bottlebrush-shaped colony in adults, elongate and lobed scales in coenenchyme, and warty and thick sclerites near the polyp mouth area. Chrysogorgia arboriformis sp. nov. is characterized by a tree-shaped colony with a long unbranched stem, slender and thin scales usually with large warts in coenenchyme. Chrysogorgia tenuis sp. nov. can be separated by a broad bottlebrush-shaped colony, long interbranch distance, elongate scales with broad lobes in polyp body wall. In contrast to the intraspecific variation of colony form, branch internodes, branching sequence and polyps, the sclerite forms and arrangement in different parts can be viewed as the most important character to separate Chrysogorgia species. For convenient identification, we divided the Chrysogorgia species into 12 morphological groups and divided Parachrysogorgia into three groups based on their sclerite forms and arrangement.

The complete mitochondrial genome of an economic sea anemone (Paracondylactis sinensis) in the East China Sea.

Paracondylactis sinensis Carlgren, 1934 (Actiniidae, Actiniaria) is an edible sea anemone in China. Their wild population has intensively decreased in recent years due to overharvesting. In this study, the complete mitochondrial genome of this economic species collected in the coast of Zhejiang, China is sequenced and obtained using high throughput methods. The total length of this circular molecule is 20,786 bp. Thirteen protein coding genes, two ribosomal RNA genes, two transfer RNA (tRNATrp, tRNAMet) genes and a putative ORF are annotated in it. Phylogenetic analysis based on the amino acids of mitochondrial genomes indicates that this species belongs to the family of Actiniidae. This result is consistent with the previous work that identified the edible sea anemone as Paracondylactis sinensis although it has always been recognized as Calliactis sinensis (of family Hormathiidae) in most Chinese reports. Overall, the mitochondrial genome produced in this study assists in clarifying the phylogenetic status of this sea anemone and provides a molecular foundation for future protection and breeding work.

Morphological and Molecular Characterization of Five Species Including Three New Species of Golden Gorgonians (Cnidaria: Octocorallia) from Seamounts in the Western Pacific.

Members of genus Iridogorgia Verrill, 1883 are the typical deep-sea megabenthos with only seven species reported. Based on an integrated morphological-molecular approach, eight sampled specimens of Iridogorgia from seamounts in the tropical Western Pacific are identified as three new species, and two known species I. magnispiralis Watling, 2007 and I. densispicula Xu, Zhan, Li and Xu, 2020. Iridogorgia flexilis sp. nov. is unique in having a very broad polyp body base with stout and thick scales. Iridogorgia densispiralis sp. nov. can be distinguished by rods present in both polyps and coenenchyme, and I. verrucosa sp. nov. is characterized by having numerous verrucae in coenenchyme and irregular spindles and scales in the polyp body wall. Phylogenetic analysis based on the nuclear 28S rDNA indicated that I. densispiralis sp. nov. showed close relationships with I. splendens Watling, 2007 and I. verrucosa sp. nov., and I. flexilis sp. nov. formed a sister clade with I. magnispiralis. In addition, due to Rhodaniridogorgia fragilis Watling, 2007 nested into the Iridogorgia clade in mtMutS-COI trees and shared highly similar morphology to the latter, we propose to eliminate the genus Rhodaniridogorgia by establishing a new combination Iridogorgia fragilis (Watling, 2007) comb. nov. and resurrecting I. superba Nutting, 1908.

Alpha-tubulin and small subunit rRNA phylogenies of peritrichs are congruent and do not support the clustering of mobilids and sessilids (Ciliophora, Oligohymenophorea).

Peritrich ciliates have been traditionally subdivided into two orders, Sessilida and Mobilida within the subclass Peritrichia. However, all the existing small subunit (SSU) rRNA phylogenetic trees showed that the sessilids and mobilids did not branch together. To shed some light on this disagreement, we tested whether or not the classic Peritrichia is a monophyletic group by assessing the reliability of the SSU rRNA phylogeny in terms of congruency with alpha-tubulin phylogeny. For this purpose, we obtained 10 partial alpha-tubulin sequences from peritrichs and built phylogenetic trees based on alpha-tubulin nucleotide and amino acid data. A phylogenetic tree from the alpha-tubulin and SSU rRNA genes in combination was also constructed and compared with that from the SSU rRNA gene using a similar species sampling. Our results show that the mobilids and sessilids are consistently separated in all trees, which reinforces the idea that the peritrichs do not constitute a monophyletic group. However, in all alpha-tubulin gene trees, the urceolariids and trichodiniids do not group together, suggested mobilids may not be a monophyletic group.

Morphology and molecular phylogeny of three new deep-sea species of Chrysogorgia (Cnidaria, Octocorallia) from seamounts in the tropical Western Pacific Ocean.

Three new species of Chrysogorgia were discovered from seamounts in the tropical Western Pacific Ocean. Chrysogorgia dendritica sp. nov. and Chrysogorgia fragilis sp. nov. were collected from the Kocebu Guyot of the Magellan Seamount chain with the water depth of 1,821 m and 1,279-1,321 m, respectively, and Chrysogorgia gracilis sp. nov. was collected from a seamount adjacent to the Mariana Trench with the water depth of 298 m. They all belong to the Chrysogorgia "group A, Spiculosae" with rods distributed in body wall and tentacles, and differ from all congeners except C. abludo Pante & Watling, 2012 by having a tree-shaped colony (vs. bottlebrush-shaped, planar or biflabellate). Chrysogorgia dendritica sp. nov. is unique in having a monopodial stem, the 1/3L branching sequence and the amoeba-shaped sclerites (sclerites branched toward to many directions) at the body bases of polyps. Chrysogorgia fragilis sp. nov. is most similar to C. abludo, but differs by the regular 1/3L branching sequence and elongate flat scales in coenenchyme. Chrysogorgia gracilis sp. nov. is easily separated from congeners by the 1/4L branching sequence, the absence of sclerites in the basal body wall, and the very sparse scales in coenenchyme. Based on the phylogenetic and genetic distance analyses of mtMutS gene, all the available Chrysogorgia species were separated into two main groups: one includes C. binata, C. cf. stellata and C. chryseis, which have two or more fans emerging from a short main stem (bi- or multi-flabellate colony); the other one includes all the species with the branching patterns as a single ascending spiral (clockwise or counterclockwise, bottlebrush-shaped colony), a fan (planar colony) and a bush of branches perched on top of a long straight stem (tree-shaped colony). Additionally, the tree-shaped colony represents a new branching pattern in Chrysogorgia, and therefore we extend the generic diagnosis.

Morphology and phylogenetic analysis of five deep-sea golden gorgonians (Cnidaria, Octocorallia, Chrysogorgiidae) in the Western Pacific Ocean, with the description of a new species.

Explorations of seamounts in the Western Pacific Ocean and South China Sea resulted in collecting 18 specimens of golden gorgonians. Based on the morphology and the genetic analysis of mtMutS, they are described as one new species, Chrysogorgia carolinensis sp. nov., and four known species, including Chrysogorgia dendritica Xu, Zhan & Xu, 2020, Metallogorgia melanotrichos (Wright & Studer, 1889), Metallogorgia macrospina Kükenthal, 1919, and Pseudochrysogorgia bellona Pante & France, 2010. Chrysogorgia carolinensis belongs to the Chrysogorgia "group A, Spiculosae" with rods or spindles distributed in the polyp-body wall and tentacles, and differs from all of its congeners except C. dendritica by the 1/3L branching sequence and amoeba-shaped sclerites at the basal polyp body. The mtMutS sequence of C. carolinensis sp. nov. has six deletion mutations compared to those of its congeners, supporting the establishment of the new species. Although no genetic variability was observed between the closely related species C. dendritica and C. abludo Pante & Watling, 2012, the former is different from the latter by the apparently irregular sclerites in the polyp body wall. The two specimens of Metallogorgia melanotrichos match well with the original description except for relatively larger polyps, while the M. macrospina specimens have slightly smaller polyps than the holotype. The juvenile of Metallogorgia has an obvious morphological difference with the adults in the colony shape and branches, but they can be unified by the same polyps and sclerites as well as mitochondrial MutS sequences. Thus, the generic diagnosis of Metallogorgia is slightly extended to include the morphology of juveniles. The morphology of Pseudochrysogorgia bellona Pante & France, 2010, as a new record for the South China Sea, matches well with that of the original description. In the phylogenetic trees, the Chrysogorgia species are separated into two clades, and while Metallogorgia and Pseudochrysogorgia formed a sister clade.

Morphology and phylogenetic analysis of two new species of deep-sea golden gorgonians (Cnidaria: Octocorallia: Chrysogorgiidae) from seamounts in the Western Pacific Ocean.

Two new species of deep-sea chrysogorgiid gorgonians are described from the tropical Western Pacific: Iridogorgia densispicula n. sp. from an unnamed seamount in the Caroline Plate with water depth of 1204 m and Iridogorgia squarrosa n. sp. from an unnamed seamount near the Mariana Trench with water depth of 1458 m. Iridogorgia densispicula n. sp. has a slender stem with bud-like polyps, dense sclerites in polyps and branches, spindle-like scales in polyp bases, and long rods in tentacles. Iridogorgia squarrosa n. sp. has a brown to nearly black axis, large polyps with eight obvious columns, elongated scales in the back of tentacles, and special scales at base of polyps with many sculptures. By the combination of these features, the two new species differ distinctly from each other and all other congeners. Phylogenetic analyses indicate that I. densispicula n. sp. is sister to all other Iridogorgia Verrill, 1883, while I. squarrosa n. sp. showed close relationship with I. splendens Watling, 2007.

Reconsideration of phylogenetic relationships of the subclass Peritrichia (Ciliophora, Oligohymenophorea) based on small subunit ribosomal RNA gene sequences, with the establishment of a new subclass Mobilia Kahl, 1933.

ABSTRACT. Based on its characteristic oral apparatus, the ciliate subclass Peritrichia has long been recognized as a monophyletic assemblage composed of the orders Mobilida and Sessilida. Following the application of molecular methods, the monophyly of Peritrichia has recently been questioned. We investigated the phylogenetic relationships of the peritrichous ciliates based on four further complete small subunit ribosomal RNA sequences of mobilids, namely Urceolaria urechi, Trichodina meretricis, Trichodina sinonovaculae, and Trichodina ruditapicis. In all phylogenetic trees, the mobilids never clustered with the sessilids, but instead formed a monophyletic assemblage related to the peniculines. By contrast, the sessilids formed a sister clade with the hymenostomes at a terminal position within the Oligohymenophorea. We therefore formally separate the mobilids from the sessilids (Peritrichia sensu stricto) and establish a new subclass, Mobilia Kahl, 1933, which contains the order Mobilida Kahl, 1933. We argue that the oral apparatus in the mobilians and sessilid peritrichs is a homoplasy, probably due to convergent evolution driven by their similar life-styles and feeding strategies. Morphologically, the mobilians are distinguished from all other oligohymenophoreans by the presence of the adhesive disc, this character being a synapomorphy for the Mobilia.

Ciliate Environmental Diversity Can Be Underestimated by the V4 Region of SSU rDNA: Insights from Species Delimitation and Multilocus Phylogeny of Pseudokeronopsis (Protist, Ciliophora).

Metabarcoding and high-throughput sequencing methods have greatly improved our understanding of protist diversity. Although the V4 region of small subunit ribosomal DNA (SSU-V4 rDNA) is the most widely used marker in DNA metabarcoding of eukaryotic microorganisms, doubts have recently been raised about its suitability. Here, using the widely distributed ciliate genus Pseudokeronopsis as an example, we assessed the potential of SSU-V4 rDNA and four other nuclear and mitochondrial markers for species delimitation and phylogenetic reconstruction. Our studies revealed that SSU-V4 rDNA is too conservative to distinguish species, and a threshold of 97% and 99% sequence similarity detected only one and three OTUs, respectively, from seven species. On the basis of the comparative analysis of the present and previously published data, we proposed the multilocus marker including the nuclear 5.8S rDNA combining the internal transcribed spacer regions (ITS1-5.8S-ITS2) and the hypervariable D2 region of large subunit rDNA (LSU-D2) as an ideal barcode rather than the mitochondrial cytochrome c oxidase subunit 1 gene, and the ITS1-5.8S-ITS2 as a candidate metabarcoding marker for ciliates. Furthermore, the compensating base change and tree-based criteria of ITS2 and LSU-D2 were useful in complementing the DNA barcoding and metabarcoding methods by giving second structure and phylogenetic evidence.

Morphology and phylogenetic analysis of two new deep-sea species of Chrysogorgia (Cnidaria, Octocorallia, Chrysogorgiidae) from Kocebu Guyot (Magellan seamounts) in the Pacific Ocean.

Two new species of Chrysogorgia Duchassaing & Michelotti, 1864 collected from Kocebu Guyot in the Magellan seamounts of the Pacific Ocean are described and illustrated: Chrysogorgia ramificans sp. nov. collected from a depth of 1831 m and Chrysogorgia binata sp. nov. collected from a depth of 1669 m. Chrysogorgia ramificans sp. nov. belongs to the Chrysogorgia "group A, Spiculosae" with rods distributed in body wall and tentacles, and C. binata sp. nov. belongs to the "group C, Squamosae typicae" with rods and/or spindles not present but only scales. Chrysogorgia ramificans sp. nov. differs from congeners by its main stem with 2/5R branching sequence at the bottom forming two large bottlebrush-shaped branches with 1/3R branching sequence at the top. Chrysogorgia binata sp. nov. is similar to C. scintillans Bayer & Stefani, 1988, but differs by its larger polyps, larger sclerites in the body wall, and different scales in the upper part of polyps. The mtMutS genetic distances between C. ramificans sp. nov. and C. binata sp. nov. and congeners are in the range of 0.33%-2.28% and 0.33%-2.94%, respectively, while the intraspecific distances are in the range of 0-0.16%. Molecular phylogenetic analysis indicates that C. ramificans sp. nov. is clustered with C. monticola Cairns, 2007 and C. binata sp. nov. is clustered with C. chryseis Bayer & Stefani, 1988, both with high support indicating close relationships.

Metagenomic Sequencing Identifies Highly Diverse Assemblages of Dinoflagellate Cysts in Sediments from Ships' Ballast Tanks.

Ships' ballast tanks have long been known as vectors for the introduction of organisms. We applied next-generation sequencing to detect dinoflagellates (mainly as cysts) in 32 ballast tank sediments collected during 2001-2003 from ships entering the Great Lakes or Chesapeake Bay and subsequently archived. Seventy-three dinoflagellates were fully identified to species level by this metagenomic approach and single-cell polymerase chain reaction (PCR)-based sequencing, including 19 toxic species, 36 harmful algal bloom (HAB) forming species, 22 previously unreported as producing cysts, and 55 reported from ballast tank sediments for the first time (including 13 freshwater species), plus 545 operational taxonomic units (OTUs) not fully identified due to a lack of reference sequences, indicating tank sediments are repositories of many previously undocumented taxa. Analyses indicated great heterogeneity of species composition among samples from different sources. Light and scanning electron microscopy and single-cell PCR sequencing supported and confirmed results of the metagenomic approach. This study increases the number of fully identified dinoflagellate species from ballast tank sediments to 142 (> 50% increase). From the perspective of ballast water management, the high diversity and spatiotemporal heterogeneity of dinoflagellates in ballast tanks argues for continuing research and stringent adherence to procedures intended to prevent unintended introduction of non-indigenous toxic and HAB-forming species.

Identification of the pathogenic ciliate Pseudocohnilembus persalinus (Oligohymenophorea: Scuticociliatia) by fluorescence in situ hybridization.

Many scuticociliatid ciliates are regarded as devastating pathogens in aquaculture. Among these, Pseudocohnilembus persalinus is a facultative pathogen that often results in refractory diseases of mariculture fish. Although traditional silver staining methods have been successfully used to identify these ciliates, their identification is hampered by their small size and their morphological similarity to closely related species. We designed an alternative method of identification of P. persalinus using an SSU-rDNA targeted oligonucleotide probe labeled with a fluorochrome, and optimized in a fluorescence in situ hybridization (FISH) protocol. The assay results in a clear identification by strong fluorescence signals from the oligonucleotide probe. The method can be used for quick and early detection of P. persalinus infections on host fish, as well as other susceptible organisms in aquiculture water. It may also be used in studies of the geographical distribution of this scuticociliate.