Genome size-dependent pcna gene copy number in dinoflagellates and molecular evidence of retroposition as a major evolutionary mechanism.

Proliferating cell nuclear antigen (PCNA) plays critical roles in eukaryotic DNA replication and replication-associated processes. It is typically encoded by one or two gene copies (pcna) in eukaryotic genomes. Recently reported higher copy numbers of pcna in some dinoflagellates raised a question of how this gene has uniquely evolved in this phylum. Through real-time PCR quantification, we found a wide range of pcna copy number (2-287 copies) in 11 dinoflagellate species (n = 38), and a strong positive correlation between pcna copy number and genome size (log10 -log10 transformed). Intraspecific pcna diverged up to 21% and are dominated by nonsynonymous substitutions, indicating strong purifying selection pressure on and hence functional necessity of this gene. By surveying pcna copy numbers in eukaryotes, we observed a genome size threshold at 4 pg DNA, above which more than two pcna copies are found. To examine whether retrotransposition is a mechanism of pcna duplication, we measured the copy number of retroposed pcna, taking advantage of the 22-nt dinoflagellate-specific spliced leader (DinoSL) capping the 5' end of dinoflagellate nuclear-encoded mRNAs, which would exist in the upstream region of a retroposed gene copy. We found that retroposed pcna copy number increased with total pcna copy number and genome size. These results indicate co-evolution of dinoflagellate pcna copy number with genome size, and retroposition as a major mechanism of pcna duplication in dinoflagellates. Furthermore, we posit that the demand of faithful replication and maintenance of the large dinoflagellate genomes might have favored the preservation of the retroposed pcna as functional genes.

Metatranscriptome analysis reveals environmental and diel regulation of a Heterosigma akashiwo (raphidophyceae) bloom.

Despite numerous laboratory studies on physiologies of harmful algal bloom (HAB) species, physiologies of these algae during a natural bloom are understudied. Here, we investigated a bloom of the raphidophyte Heterosigma akashiwo in the East China Sea in 2014 using metabarcode (18S rDNA) and metatranscriptome sequencing. Based on 18S rDNA analyses, the phytoplankton community shifted from high diversity in the pre-bloom stage to H. akashiwo predominance during the bloom. A sharp decrease in ambient dissolved inorganic phosphate and strong up-regulation of phosphate and dissolved organic phosphorus (DOP) uptake genes, including the rarely documented (ppGpp)ase, in H. akashiwo from pre-bloom to bloom was indicative of rapid phosphorus uptake and efficient utilization of DOP that might be a driver of the H. akashiwo bloom. Furthermore, observed up-regulated expression of mixotrophy-related genes suggests potential contribution of mixotrophy to the bloom. Accelerating photosynthetic carbon fixation was also implied by the up-regulation of carbonic anhydrase genes during the bloom. Notably, we also observed a strong morning-to-afternoon shift in the expression of many genes. Our findings provide insights into metabolic processes likely important for H. akashiwo bloom formation, and suggest the need to consider timing of sampling in field studies on this alga.

Cloning and gene expression of allograft inflammatory factor-1 (AIF-1) provide new insights into injury and bacteria response of the sea cucumber Apostichopus japonicus (Selenka, 1867).

Allograft inflammatory factor-1 (AIF-1) is an interferon (IFN)-γ-inducible Ca(2+)-binding cytokine that associates with the immune defense and inflammatory response. In this study, we reported AIF-1 gene in sea cucumber Apostichopus japonicus (AjAIF-1). The full-length cDNA of AjAIF-1 is 1541 bp with an open reading frame (ORF) of 477 bp encoding 158 amino acids. Two EF-hand Ca(2+)-binding motifs were found in the deduced AjAIF-1. AjAIF-1 was widely expressed in all tested tissues (body wall, intestine, respiratory tree, tube feet, coelomocytes and longitudinal muscle), with the highest expression in respiratory tree. After Vibrio splendidus challenge and physical injury, AjAIF-1 transcripts were significantly upregulated in coelomocytes. The mRNA expression level of AjAIF-1 in coelomocytes reached to the highest value at 4 h (3.38-folds vs. the PBS control, P < 0.05) post injection. After papilla injury, the mRNA level of AjAIF-1 in coelomocytes was upregulated, and its peak value was found at 4 h (3.88-folds vs. the control, P < 0.05). These results indicated that 1) AjAIF-1 sensitively responds to pathogen infection; 2) AjAIF-1 is involved in acute inflammatory response. Our findings gain general information about the role of AjAIF-1 in the innate immunity of A. japonicus.

Complete mitochondrial genome of Striatobalanus tenuis Hoek, 1883 (Balanomorpha: Balanidae) and a novel molecular phylogeny within Cirripedia.

Barnacles are crustaceans that are critical model organisms in intertidal ecology and biofouling research. In this study, we present the first mitochondrial genome of Striatobalanus tenuis which is a circular molecule of 15,067 bp in length. Consistent with most barnacles, the mitochondrial genome of S. tenuis encodes 37 genes, including 13 PCGs, 22 tRNAs and 2 rRNAs. A novel insight into the phylogenetic analysis based on the nucleotide data of 13 PCGs showed that the S. tenuis clusters with Striatobalanus amaryllis (bootstrap value = 100) of the same genus, then groups with other Balanoidea species, the Chelonibiidae, Austrobalanidae and Tetraclitidae cluster together forming superfamily Coronuloidea. The result can help us to understand the novel classification within Balanomorpha.

The allelopathic effects of Heterosigma akashiwo on Skeletonema costatum: Insights from gene expression and metabolomics analysis.

The globally distributed harmful algal blooms (HAB) species, Heterosigma akashiwo, has been found to exhibit ichthyotoxicity. Previous studies have shown that H. akashiwo achieves a competitive edge during bloom occurrences by inhibiting the growth of a coexisting diatom, Skeletonema costatum, through allelopathy. However, the specific allelopathic mechanisms underlying the allelopathic effects of H. akashiwo on S. costatum remain unknown. To bridge this gap, our study utilized a combination of quantitative real-time PCR and metabolomics to examine the allelopathic processes of H. akashiwo on S. costatum. Our results demonstrate that the growth of S. costatum is hindered when co-cultured with H. akashiwo (initial cell concentration, 2 × 104 cell/mL). Gene expression investigation showed a substantial reduction in the mRNA levels of cytochrome b6, ribulose bisphosphate carboxylase large chain, and silicon transporter in S. costatum when grown in co-culture conditions. Furthermore, metabolic pathway analysis suggested that the allelopathic effects of H. akashiwo disrupted several vital metabolic pathways in S. costatum, including a reduction in purine and pyrimidine metabolism and an increase in fatty acid biosynthesis. Our investigation has revealed the intricate and substantial involvement of allelopathy in the formation of H. akashiwo blooms, demonstrating the complexity of the allelopathic interaction between H. akashiwo and S. costatum. These insights also contribute significantly to our understanding of the dynamics within HAB species.

Transcriptome analysis of the harmful alga Heterosigma akashiwo under a 24-hour light-dark cycle.

The photoperiod, which is defined as the period of time within a 24-hour time frame that light is available, is an important environmental regulator of several physiological processes in phytoplankton, including harmful bloom-forming phytoplankton. The ichthyotoxic raphidophyte Heterosigma akashiwo is a globally distributed bloom-forming phytoplankton. Despite extensive studies on the ecological impact of H. akashiwo, the influence of the photoperiod on crucial biological processes of this species remains unclear. In this study, gene expression in H. akashiwo was analyzed over a 24-hour light-dark (14:10) treatment period. Approximately 36 % of unigenes in H. akashiwo were differentially expressed during this 24-hour treatment period, which is indicative of their involvement in the response to light-dark variation. Notably, the number of differentially expressed genes exhibited an initial increase followed by a subsequent decrease as the sampling time progressed (T0 vs. other time points). Unigenes associated with photosynthesis and photoprotection reached their peak expression levels after 2-4 h of illumination (T12-T14). In contrast, the expression of unigenes associated with DNA replication peaked at the starting point of the dark period (T0). Furthermore, although several unigenes annotated to photoreceptors displayed potential diel periodicity, genes from various photoreceptor families (such as phytochrome and cryptochrome) showed unique expression patterns. Collectively, our findings offer novel perspectives on the response of H. akashiwo to the light-dark cycle, serving as a valuable resource for investigating the physiology and ecology of this species.

Full-Length Transcriptome Analysis of the Ichthyotoxic Harmful Alga Heterosigma akashiwo (Raphidophyceae) Using Single-Molecule Real-Time Sequencing.

The raphidophyte Heterosigma akashiwo is a harmful algal species. The bloom of this organism has been associated with the massive mortality of fish in many coastal waters. To investigate the molecular mechanism of H. akashiwo blooms, having a reliable reference transcriptome of this species is essential. Therefore, in this study, a full-length transcriptome of H. akashiwo was obtained by single-molecule real-time sequencing. In total, 45.44 Gb subread bases were generated, and 16,668 unigenes were obtained after the sequencing data processing. A total of 8666 (52.00%) unigenes were successfully annotated using seven public databases. Among them, mostly phosphorus and nitrogen metabolism genes were detected. Moreover, there were 300 putative transcription factors, 4392 putative long non-coding RNAs, and 7851 simple sequence repeats predicted. This study provides a valuable reference transcriptome for understanding how H. akashiwo blooms at a molecular level.

Isolation and characterization of an algicidal bacterium against the bloom-forming algae raphidophyte Heterosigma akashiwo.

Harmful algae blooms (HABs) have increased in intensity and frequency worldwide, causing negative effects on public health and marine ecosystems. This study isolated and identified the bloom causing species and its associated algicidal bacterium during a phytoplankton bloom in coastal waters of Lianyungang, China. Morphological observations and DNA barcoding analysis indicate that the studied phytoplankton bloom was caused by the raphidophyte Heterosigma akashiwo, and the algicidal bacterium, strain LD-B1, was identified as a species belonging to the genus Pseudoalteromonas. Furthermore, the algicidal effects of strain LD-B1 against H. akashiwo were characterized; revealing strain LD-B1 show strong algicidal activity against H. akashiwo. After 48 h of bacterium culture addition, the algicidal rate reached up to 98.8% with a 1% final volume rate. Moreover, our findings indicate strain LD-B1's extracellular compounds involved in algicidal activity are likely not proteinaceous. These findings indicate that the isolated strain, LD-B1, is a promising algicidal bacterium to control H. akashiwo blooms.

Changes in physiological activities are responsible for homoyessotoxin-induced toxicity in abalone Haliotis discus hannai.

Homoyessotoxin (homo-YTX) is a lipid-soluble toxin produced by toxic dinoflagellates. It is widely distributed in marine ecosystems worldwide, and it poses a threat to the survival of aquatic animals. The tissues of the abalone Haliotis discus hannai are easily damaged by homo-YTX during harmful algal blooms. In this study, H. discus hannai was exposed to homo-YTX (0, 2, 5, and 10 µg L-1) to evaluate the rates of survival (S) and death (D) and the antioxidative, metabolic, and digestive physiological responses in the gills and digestive gland of abalone. Homo-YTX decreased S and the activities of Na+/K+-adenosine triphosphatase, Ca2+/Mg2+-adenosine triphosphatase, superoxide dismutase, catalase, alkaline phosphatase, xanthine oxidase, lactate dehydrogenase, amylase, protease, and lipase. Meanwhile, D, the reactive oxygen species level, and the malondialdehyde content increased with increasing concentrations of homo-YTX. In addition, homo-YTX induced oxidative stress, enhanced the lipid peroxidation reaction, reduced the energy supply, and inhibited the metabolic and digestive physiological activities in the gills and digestive gland of abalone. Oxidative stress-mediated insufficient energy supply and physiological activity reduction caused the death of abalone.

Isolation and characterization of a high-efficiency algicidal bacterium Pseudoalteromonas sp. LD-B6 against the harmful dinoflagellate Noctiluca scintillans.

The dinoflagellate Noctiluca scintillans is a harmful algal species that is globally distributed and poses a certain threat to marine ecosystems. Recent research has shown that the application of algicidal bacteria is a promising method to prevent and control such harmful algal blooms (HABs), given its advantages of safety and efficiency. In this study, a strain of algicidal bacterium LD-B6 with high efficiency against N. scintillans was isolated from the coastal waters of Lianyungang, China. 16S rDNA sequence analysis showed that the strain LD-B6 belongs to the genus Pseudoalteromonas. Furthermore, the algicidal effect of LD-B6 on N. scintillans was investigated. The results showed that strain LD-B6 exerted strong algicidal activity against N. scintillans. After 12 h of bacterial culture addition to algal cultures at a 2% final volume rate, the algicidal activity reached 90.5%, and the algicidal activity of LD-B6 was influenced by the density of N. scintillans. In addition, the algicidal bacterium LD-B6 was found to indirectly lyse algal cells by secreting extracellular compounds. These algicidal compounds were stable, indicating that they are not proteins. Importantly, strain LD-B6 was broadly general, showing varying degrees of lysing effects against five of the six algal species tested. On the basis of the described studies above, the algicidal powder was also initially developed. In summary, the isolated bacterial strain LD-B6 shows the potent algicidal capability to serve as a candidate algicidal bacterium against N. scintillans blooms.

Transcriptomic response of the harmful algae Heterosigma akashiwo to polyphosphate utilization and phosphate stress.

Phosphorus (P) is one of the major macronutrients necessary for phytoplankton growth. In some parts of the ocean, however, P is frequently scarce, hence, there is limited phytoplankton growth. To cope with P deficiency, phytoplankton evolved a variety of strategies, including, utilization of different P sources. Polyphosphate (polyP) is ubiquitously present and serves an essential function in aquatic environments, but it is unclear if and how this polymer is utilized by phytoplankton. Here, we examined the physiological and molecular responses of the widely present harmful algal bloom (HAB) species, Heterosigma akashiwo in polyP utilization, and in coping with P-deficiency. Our results revealed that two forms of inorganic polyP, namely, sodium tripolyphosphate and sodium hexametaphosphate, support H. akashiwo growth as efficiently as orthophosphate. However, few genes involved in polyP utilization have been identified. Under P-deficient conditions, genes associated with P transport, dissolved organic P utilization, sulfolipid synthesis, and energy production, were markedly elevated. In summary, our results indicate that polyP is bioavailable to H. akashiwo, and this HAB species have evolved a comprehensive strategy to cope with P deficiency.

First record of a Takayama bloom in Haizhou Bay in response to dissolved organic nitrogen and phosphorus.

Since 1990s, harmful algal blooms (HABs) of Kareniaceae, primarily caused by species of Karenia and Karlodinium and rarely by Takayama species, have been substantially increasing in frequency and duration in the coastal waters of China. In this study, we recorded a bloom of high abundance of T. acrotrocha in the Haizhou Bay, the Yellow Sea in September 2020, which is the first record of a Takayama bloom in the temperate coastal waters of China. We found that high concentrations of DON and DOP accelerated the proliferation of T. acrotrocha in the Haizhou Bay. Intensive mariculture, and terrestrial nitrogen and phosphorus input may be responsible for the eutrophication in the Haizhou Bay featuring high concentrations of DON and DOP, and high DIN/DIP ratios. The results suggested that, under ocean warming, the HABs of Kareniaceae are becoming increasingly dominant in eutrophic temperate coasts with intensive mariculture activities.

The first mitochondrial genome of Tetraclita kuroshioensis (Crustacea: Sessilia) from China: insight into the phylogeny within Cirripedia.

We determined the first mitochondrial genome of Tetraclita kuroshioensis from China. The mitochondrial genome of T. kuroshioensis was found to be 15,175 bp in length and consisted of 13 protein-coding genes (PCGs), 22 tRNAs, and 2 rRNAs. The longest non-coding region was 425 bp in length. Phylogenetic analysis showed that T. kuroshioensis clustered with T. serrata and then clustered with T. squamosa squamosa with high bootstrap value (BP = 100). In the future, sequencing of additional mitochondrial genomes should provide additional insights into the deep phylogeny of Cirripedia.

The first mitochondrial genome of Balanus trigonus Darwin, 1854 (Sessilia: Balanidae) and molecular phylogeny within Cirripedia.

The triangle barnacle Balanus trigonus Darwin, 1854, a cosmopolitan inhabitant of tropical and warm temperate seas, is a member of robust system for the study of evolutionary processes in the intertidal zone. The first mitochondrial genome of B. trigonus is presented. The complete mitochondrial genome of B. trigonus is a circular molecule of 15,560 bp, which encodes 13 protein-coding genes (PCGs), 2 rRNA genes, and 22 tRNA genes. In comparison within Sessilia, the arrangement of the mitochondrial genome of B. trigonus is more similar to Megabalanus spp. than the congener Balanus balanus, which share a same inversion of a large gene block (P-nd4L-nd4-H-nd5-F). Phylogenetic analysis based on mitochondrial PCGs reveals that B. trigonus clusters with Acasta Sulcata (BP = 100), then grouped with Megabalanus volcano and Megabalanus ajax with high support (BP = 90). In further, more data and research are needed to reveal the phylogeny within Cirripedia.

The mitochondrial genome of Chthamalus malayensis (Sessilia: Chthamalidae) and its molecular phylogeny within Cirripedia.

Cirripedia is a lower crustacean that has an invaluable place in several aspects of intertidal ecology and anti-fouling research. In this study, we present the first mitochondrial genome of Chthamalus malayensis. The complete mitochondrial genome of C. malayensis is a circular molecule of 15,230 bp. In comparison to the pancrustacean ground pattern, the mitochondrial genome of C. malayensis has a deletion of the trnC gene. Phylogenetic analysis based on mitochondrial protein-coding genes showed that C. malayensis clusters with C. antennatus (BP = 98) and is grouped with C. challengeri, Octomeris sp. BKKC-2014, and Notochthamalus scabrosus. Further studies are needed to reveal the specific phylogenic relationships within Cirripedia.

The first mitochondrial genome of Fistulobalanus albicostatus (Crustacea: Maxillopoda: Sessilia) and phylogenetic consideration within the superfamily Balanoidea.

The first complete mitochondrial genome of the intertidal barnacle Fistulobalanus albicostatus Pilsbry, 1916 (Crustacea: Maxillopoda: Sessilia) is presented. The genome is a circular molecule of 15,665 bp, which encodes a set of 37 typical metazoan genes. All non-coding regions are 438 bp in length, with the longest one speculated as the control region (264 bp), which is located between srRNA and trnI. All protein-coding genes (PCGs) have an ATD (ATA, ATT, or ATG) start codon, except nad1, which is initiated with GTG. Remarkably, cox3, cob, nad1-5 have incomplete stop codons (T-- or TA-) and the remaining PCGs have the complete stop codon (TAA). Phylogenetic analysis based on 13 mitochondrial PCGs shows that the members of the Archaeobalanidae and Balanidae intermingle with species from Pyrgomatidae. The results supposed that Balanidae and Archaeobalanidae are non-monophyly.

The complete mitochondrial genome of Tetraclita japonica (Crustacea: Maxillopoda: Sessilia) from Zhejiang (China) and phylogeny within Sessilia.

The complete mitochondrial genome of Tetraclita japonica (Crustacea: Maxillopoda: Sessilia) from Zhejiang (China) was presented (T. japonica ZJ). The genome is a circular molecule of 15,192 bp, all non-coding regions are 694 bp in length, and the longest one (307 bp) is located between 12S rRNA and trnK. Phylogenetic analysis based on mitochondrial PCGs shows that T. japonica ZJ clusters with T. japonica CN, and they grouped with T. japonica (BP = 100), then grouped with Tesseropora rosea, Tetraclita serrata, Tetraclita rufotincta, Epopella plicata, and Tetraclitella divisa successively . The phylogenetic tree indicated that the Balanidae and Archaeobalanidae are polyphyletic, while the Chthamalidae is monophyletic. The result can help us to understand the phylogenetic history within Sessilia.

Utilization of various forms of nitrogen and expression regulation of transporters in the harmful alga Heterosigma akashiwo (Raphidophyceae).

Nitrogen (N) is an essential nutrient for phytoplankton growth. There is ample evidence that N enrichment promotes harmful algae blooms (HABs) but molecular mechanisms regulating N-nutrient uptake and metabolism are not so clear, especially for the raphidophyte Heterosigma akashiwo, which forms ichthyotoxic HABs in many coastal waters. In this study, the utilization of three different chemical forms of N (nitrate, ammonium, and urea) by H. akashiwo CCMA 369 was investigated in batch culture conditions. Results showed that H. akashiwo grew well on all three N compounds, and the highest cell yield occurred in the NH4+ culture group. Reverse transcription quantitative PCR analysis revealed that the expression of high-affinity NO3- transporter (NIT), NH4+ transporter (AMT) and high-affinity urea active transporter (DUR3), were significantly up-regulated under N-limitation compared to the N-replete control. The mRNA levels of AMT and DUR3 also displayed a clear diel rhythm, with high levels at midnight. In addition, NH4+ addition (5 µM) did not depress the transcript abundance of any of the three N transporters. Compared with the co-occurring immobile diatom Skeletonema costatum, the high expression of AMT in dark period in H. akashiwo is consistent with its diel vertical migration behavior, which may promote N-nutrient acquisition from deeper layers and give advantages for H. akashiwo to form blooms.

Identification and expression analysis of meiosis-related genes in the harmful alga Heterosigma akashiwo (Raphidophyceae).

Sexual reproduction (SR), hallmarked by meiosis, is widespread in eukaryotes. In phytoplankton, SR has been observed in many lineages, but molecular information on SR or meiosis of harmful algal bloom (HAB) species is scarce. The raphidophyte Heterosigma akashiwo is a globally distributed and devastating HAB species, but molecular evidence of its SR or meiosis is lacking. Here, to address the gap of knowledge, the presence of meiotic genes in H. akashiwo were examined. Interestingly, seven meiosis-specific or related genes (SPO11, MND, RAD21, RAD51, MSH2, MSH6 and MEI2) were identified from H. akashiwo transcriptomes. Furthermore, expression patterns of these genes except MSH6 (excluded due to primer failure) were investigated using quantitative reverse-transcription PCR. Results showed that the examined genes exhibited similar diel rhythms, typically, highest in early dark period and then gradually decreasing until mid-day. In addition, the expression of these six genes was not higher in the stationary growth stage than in the exponential stage, as would be expected if meiosis was to form cysts, and their elevated expression in response to colchicine treatment (arresting cells in the G2/M transition) indicated a potential role of these genes in cell division and population growth in H. akashiwo. Consistent with this, we also found a morning to afternoon shift in the expression of these genes during the bloom of H. akashiwo. This study documents a part of the typical SR gene repertoire and its potential role in regulating cell division in H. akashiwo, offering candidates for population growth markers for bloom monitoring although its linkage to meiosis and SR remain to be studied further in the future.

High throughput sequencing of 18S rRNA and its gene to characterize a Prorocentrum shikokuense (Dinophyceae) bloom.

Comparing phytoplankton non-bloom and bloom communities using rRNA and its coding gene can help understand the shift of dominant species and its driving processes (e.g., intrinsic growth or grazing). Here we conducted high-throughput sequencing of 18S rRNA and its coding gene for studying non-bloom and bloom plankton communities in East China Sea. The non-bloom community was dominated by diatoms whereas during the bloom it was dominated by the dinoflagellate Prorocentrum shikokuense (formerly P. donghaiense). P. shikokuense rRNA abundance and rRNA:rRNA gene ratio both increased markedly in the bloom, indicating that the bloom arose from active growth. In contrast, some non-bloom species showed low DNA abundances during the bloom albeit high rRNA:rRNA gene ratios, suggesting that cell loss processes such as grazing might have prevented these species from blooming or that these species might be at an early stage of bloom development. Furthermore, Pearson's correlation analysis showed that dinoflagellate abundance was positively correlated with temperature and negatively related to dissolved inorganic phosphate (DIP) concentrations, suggesting warm and DIP-poor environment as a niche space for P. shikokuense. Our results demonstrate the usefulness of combined analysis of rRNA and its gene in characterizing phytoplankton bloom development to shed light on the complex phytoplankton dynamics and regulating mechanisms in the course of bloom development.