Shota Ohara, Goshi Araki, Takashi Kadono, Haruo Yamaguchi, Takeshi Tsumuraya, Kirsty F. Smith, Lesley L. Rhodes, Masao Adachi
The seafood illness, ciguatera poisoning (CP) is a significant public health concern in tropical and subtropical regions worldwide. In Japan, CP is particularly prevalent in Okinawa Prefecture, where is the subtropical region of Japan, due to the consumption of fish contaminated with Pacific ciguatoxins (P-CTXs). The benthic dinoflagellate Gambierdiscus polynesiensis is known to produce P-CTXs in the South Pacific, but P-CTX-producing microalgae have not been identified in Japanese waters. A practical method applicable to field samples containing diverse benthic microalgae is therefore required. To identify a P-CTX producer in Japan, an indirect immunofluorescence (IIF) assay using P-CTX-specific monoclonal antibodies that can be applied to various benthic microalgae such as diatoms and dinoflagellates was developed. The optimal conditions for cell membrane permeabilization were determined using the IIF assay with an anti-actin antibody on benthic diatoms and dinoflagellates treated with various concentrations of Triton X-100. Treatment of the cells with 5.0% (v/v) Triton X-100 resulted in the highest fluorescence and the lowest cell loss. When an IIF assay with an anti-P-CTX antibody (8H4) was performed on cells of Gambierdiscus australes, G. jejuensis, G. polynesiensis, and G. silvae treated with 5.0% Triton X-100, only G. polynesiensis showed fluorescence. The simultaneous application of two anti-P-CTX antibodies (8H4 and 10C9) doubled the fluorescence compared to 8H4 alone. Extending the antibody reaction time from one hour to 16 hours increased the fluorescence by about sevenfold. These optimized conditions enabled visualization of P-CTXs-derived fluorescence in G. polynesiensis cells under both not only a confocal laser scanning microscope but also a conventional inverted fluorescence microscope having deeper depths of focus. Combining the IIF assay with observing the cells under an inverted microscope provides a practical tool for detecting P-CTX producers in samples of various sizes of benthic microalgae collected from coastal waters. This will contribute to identifying unknown P-CTX-producing microalgae in Japanese and other coastal waters in the future.
Neogloboquadrina pachyderma is the dominant planktonic foraminiferal species in polar oceans and a major contributor to biogenic carbonate production in the Southern Ocean. Its calcite test serves as a key geochemical archive for reconstructing past ocean conditions through stable isotope and trace element proxies. As polar marine ecosystems undergo rapid environmental change, understanding the ecological associations and trophic dependencies of N. pachyderma within surrounding pelagic communities is increasingly important, particularly in extreme Antarctic environments. Here, we present the first molecular characterization of pelagic eukaryotic assemblages associated with individual N. pachyderma (genetic Type IV) specimens collected from Prydz Bay, East Antarctica, using a single-cell 18S rRNA gene metabarcoding approach. High-throughput sequencing revealed that the N. pachyderma eukaryome was dominated by dinoflagellates (Dinophyceae), diatoms (Bacillariophyceae), radiolarians, Syndiniales, and metazoans. In contrast, ambient seawater communities were enriched in cercozoans, followed by dinoflagellates, radiolarians, and ciliates. The distinct composition of eukaryotic assemblages associated with N. pachyderma relative to the surrounding seawater suggests selective incorporation of a subset of the local pelagic eukaryotic pool, potentially reflecting trophic interactions or transient associations with aggregate-associated prey. These results provide preliminary molecular insights into the trophic linkages or pelagic eukaryotic associations of Antarctic N. pachyderma Type IV, in Prydz Bay, East Antarctica, and establish an important baseline for future investigations. Our findings highlight the role of pelagic eukaryotes in shaping foraminiferal microbial interactions and underscore the relevance of such associations for interpreting palaeoceanographic proxies in a rapidly changing Southern Ocean.
Aimee Caye G. Chang, Mailor W. W. Amaral, Kyle G. Keepers, Megan Greenwood, Jingchun Li, Sarah E. Hamsher, Scott R. Miller, J. Patrick Kociolek
Despite their ecological significance and unique endosymbiotic capabilities, diatoms in the genus Rhopalodia remain poorly represented in genomic databases, particularly with respect to the availability of complete genomes from multiple organellar compartments. This study addresses that gap by presenting, for the first time, the complete chloroplast (133,086 bp), mitochondrial (36,786 bp), and spheroid body (3,024,495 bp) genomes of Rhopalodia sterrenburgii, a nitrogen-fixing diatom with a cyanobacterial endosymbiont. Phylogenetic reconstruction based on five genes from different cellular compartments (18S, 28S, rbcL, psbC, cob) placed R. sterrenburgii as a basal lineage relative to R. gibba (Rhopalodia sensu lato) and the Epithemia sensu stricto species E. argus, E. turgida, and E. sorex, providing new insights into evolutionary relationships within Rhopalodiales. Additionally, pathway analysis revealed the progressive loss of genes involved in vitamin B 12 and chlorophyll a biosynthesis in more recently diverged Rhopalodia and Epithemia lineages. Our findings support a pattern of genome reduction in symbiotic diazotrophic diatoms, potentially driven by coevolution with their endosymbionts, and an increasing reliance on integrated metabolic functions between the host and the endosymbiont.
Uranouchi Inlet, situated on the Pacific coast of southwestern Japan, has been a highly enclosed inlet known for yellowtail farming since 1959. Since the 1980s, harmful algal blooms (HABs) have repeatedly occurred, resulting in mass mortality of fish and shellfish. In the sediment at the inlet, the resulting cysts of the HAB species may be preserved, which reflects the history of HAB events. However, the vertical distributions of HAB species in sediment have not been elucidated. In this study, core sediment samples were analyzed by metabarcoding. The dating of each sample was cited from previous study dating the same samples. The findings revealed the presence of eleven HAB species, with notable shifts from approximately 1977–1988. The timing of the shifts corresponded to that of the development of aquaculture and the resulting eutrophication. Vertical core metabarcoding provides footprints of how HAB species composition may be influenced by anthropogenic environmental changes.
Linnéa M. V. Ström, Philip Ley, Eric A. Webb, David A. Hutchins, Mak Saito, Daniel Lundin, Rachel A. Foster
Decades of research has been devoted to understanding the occurrence, distribution, and activity of different N 2 -fixers in the oceans. Marine N 2 -fixing diatom symbioses involving the heterocyst-forming cyanobacterium Richelia are widespread, yet often go undetected. Some of the diatom- Richelia symbioses have the advantage to be identified by microscopy, however, observations alone cannot inform on the metabolic state of a cell. Here, we developed nine new specific quantitative PCR assays that detect three common symbiotic Richelia strains (ReuHH01, RintRC01, RrhiSC01) of diatoms based on molecular markers for: a high affinity phosphate transporter (pstS), an iron transporter (exbB), and a constitutively expressed gene (rnpA, protein component of ribonuclease P). We first tested the new assays in the lab, including a diel experiment to elucidate the temporal dynamics of gene expression for each molecular marker, then applied the new assays to field collected samples. In the field we also made microscopy observations of the diatom- Richelia symbioses and measured bulk N 2 and Carbon (C) fixation rates with stable isotope labelling incubations. The number of genes encoding the molecular markers varied; typically, fewer in the endobiont strains. Detection in field samples based on qPCR was consistent with microscopy observations. The environmental gene expression for all the new targets, and additionally nifH for N 2 fixation (nitrogenase) were low, and highest expression was detected in the upper water column (0-40 m) consistent with higher densities of the Richelia by qPCR and microscopy observations. Low in situ bulk rates of N 2 and C fixation corroborated the aforementioned low nifH expression. Expression of both nifH and pstS was temporally regulated in the lab experiment of the facultative symbiotic Richelia strain RrhiSC01 with higher expression in the early and late photoperiods, respectively. The nine new assays are an improvement over the previous assays based on the nifH gene as cross-reactivity between Richelia strains is minimum, expression can be normalized to rnpA, and expression is informative of the symbiont nutrient status.
