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.

The soft-shell clam Mya arenaria Linnaeus, 1758 is a commercially important fishery resource that occurs in boreal and temperate environments in the Northern Hemisphere. Whether the soft-shell clam is a single species with a circumboreal range or a species complex also comprising Mya japonica Jay, 1857 distributed in the north Pacific has long been debated by malacologists and palaeontologists based on slight differences in shell morphology. We used an integrative taxonomic approach incorporating available Mya spp. mitochondrial COI and 16S rRNA, and nuclear 28S rRNA gene sequences, as well as spermatozoan and shell morphological characters to test the validity of M. japonica and examine the range of soft-shell clam distribution. Although differences in shell morphology were minor, the results from tree topologies, pairwise uncorrected p-distances, Automatic Barcode Gap Discovery (ABGD) and spermatozoan ultramorphological data confirm the validity of M. japonica in both its endemic region in the northwest Pacific, and as here newly reported introduced populations in British Columbia in the northeast Pacific, and show that M. arenaria is distributed in the northeast Pacific, North Atlantic, Barents Sea (Arctic Ocean) and Mediterranean. We estimate these two closely related sister species diverged 4.1–12.5 Myr during early Pliocene to late Miocene, which is consistent with current evolutionary theory regarding M. arenaria. In addition, ABGD indicated the congener Mya truncata Linnaeus, 1758 may represent a species complex, but additional evidence is still needed to clarify its taxonomic status.

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.

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.

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.