Full-length transcriptome analysis of the bloom-forming dinoflagellate Akashiwo sanguinea by single-molecule real-time sequencing.

The dinoflagellate Akashiwo sanguinea is a harmful algal species and commonly observed in estuarine and coastal waters around the world. Harmful algal blooms (HABs) caused by this species lead to serious environmental impacts in the coastal waters of China since 1998 followed by huge economic losses. However, the full-length transcriptome information of A. sanguinea is still not fully explored, which hampers basic genetic and functional studies. Herein, single-molecule real-time (SMRT) sequencing technology was performed to characterize the full-length transcript in A. sanguinea. Totally, 83.03 Gb SMRT sequencing clean reads were generated, 983,960 circular consensus sequences (CCS) with average lengths of 3,061 bp were obtained, and 81.71% (804,016) of CCS were full-length non-chimeric reads (FLNC). Furthermore, 26,461 contigs were obtained after being corrected with Illumina library sequencing, with 20,037 (75.72%) successfully annotated in the five public databases. A total of 13,441 long non-coding RNA (lncRNA) transcripts, 3,137 alternative splicing (AS) events, 514 putative transcription factors (TFs) members from 23 TF families, and 4,397 simple sequence repeats (SSRs) were predicted, respectively. Our findings provided a sizable insights into gene sequence characteristics of A. sanguinea, which can be used as a reference sequence resource for A. sanguinea draft genome annotation, and will contribute to further molecular biology research on this harmful bloom algae.

A Sensitive and Portable Double-Layer Microfluidic Biochip for Harmful Algae Detection.

Harmful algal blooms (HABs) are common disastrous ecological anomalies in coastal waters. An effective algae monitoring approach is important for natural disaster warning and environmental governance. However, conducting rapid and sensitive detection of multiple algae is still challenging. Here, we designed an ultrasensitive, rapid and portable double-layer microfluidic biochip for the simultaneous quantitative detection of six species of algae. Specific DNA probes based on the 18S ribosomal DNA (18S rDNA) gene fragments of HABs were designed and labeled with the fluorescent molecule cyanine-3 (Cy3). The biochip had multiple graphene oxide (GO) nanosheets-based reaction units, in which GO nanosheets were applied to transfer target DNA to the fluorescence signal through a photoluminescence detection system. The entire detection process of multiple algae was completed within 45 min with the linear range of fluorescence recovery of 0.1 fM-100 nM, and the detection limit reached 108 aM. The proposed approach has a simple detection process and high detection performance and is feasible to conduct accurate detection with matched portable detection equipment. It will have promising applications in marine natural disaster monitoring and environmental care.

Spatiotemporal dynamics of marine microbial communities following a Phaeocystis bloom: biogeography and co-occurrence patterns.

Marine microbes play important roles in the development of phytoplankton blooms. The diversity and composition of free living (FL) and particle attached (PA) microbial communities have been well studied, while little is known about their geographic and co-occurrence patterns, especially during the subsiding process of Phaeocystis globosa blooms. Herein, the beta-diversity of FL and PA microbial communities in both the surface and bottom layers of different habitats were comprehensively examined during succession of a P. globosa bloom event. The results showed that microbial communities from bloom and non-bloom sites exhibited distinct community compositions. Among the different sampling sites, the community similarities decreased with spatial distance, in which the FL communities' similarity in bottom waters was more influenced by spatial variation. The variation of microbial communities was mostly attributed to environmental selection, spatial distance, and the abundance of P. globosa successively. The co-occurrence networks of microbial communities in bloom and non-bloom waters differed in terms of structure and composition, and the bloom network had more links and closer relationships between genera than the non-bloom network. The correlation among genera and modules suggested that the bloom microbes were likely driven by high environmental selection and low competitive effect between each other.

The parasitic dinoflagellate Hematodinium infects multiple crustaceans in the polyculture systems of Shandong Province, China.

The parasitic dinoflagellates of the Hematodinium genus have impacted wild and cultured stocks of commercial crustaceans worldwide. In the past decade, outbreaks of Hematodinium epizootics resulted in substantial mortalities in cultured Chinese swimming crabs Portunus trituberculatus in the polyculture ponds located in Shandong Peninsula, whereas the source and transmission of the parasite in the polyculture pond system remains to be determined. During April to December of 2018, 2034 crabs and 108 shrimps were collected from the polyculture pond systems in the highly endemic area of Hematodinium diseases in Qingdao, Shandong Province. Among those, 188 individuals of the 6 crab species were infected by the parasite, including 4 novel host species (Uca arcuate, Hemigrapsus penicillatus, Helice wuana and Macrophthalmus japonicas). No infection was identified in Penaeus monodon. Further phylogenetic analyses indicated that the Hematodinium isolate infecting the six crab hosts, together with other isolates reported from China, composed the genotype II of Hematodinium perezi. The parasite was more infectious to cultured Portunus trituberculatus and the dominant wild crab Helice tientsinensis dwelling in the waterways connecting to the polyculture ponds, even though it was found to be a host generalist pathogen. The prevalence of Hematodinium perezi infection in Helice tientsinensis was higher than that of other wild crabs and showed significant positive correlation with that of the cultured Portunus trituberculatus. The results indicated that the wild crabs, particularly Helice tientsinensis, were the important alternate hosts closely involved in transmission and spreading of the Hematodinium disease in the polyculture pond systems.

Population dynamics and interactions of Noctiluca scintillans and Mesodinium rubrum during their successive blooms in a subtropical coastal water.

A time series field survey were conducted in Port Shelter, a subtropical coastal water in NW Pacific, beginning before the onset of a chain of Noctiluca scintillans and/or Mesodinium rubrum blooms, and ending after the blooms had declined. At the first mixed bloom stage, seed of N. scintillans and the consequent outbreak of both N. scintillans and M. rubrum were largely due to the physical forcing. Plenty food supply and their different feeding habits supported N. scintillans and M. rubrum to bloom massively and concomitantly. Following that, there was a small N. scintillans bloom followed by a small crest of M. rubrum. Their initiation and scale were mainly affected by limited food supply and/or the inferior food source. Sudden change of wind from mild northeast wind to strong southeast wind might contribute to the termination of N. scintillans bloom. Finally, physical accumulation was the most important driving factors of the formation and dispersal of the third and largest bloom of N. scintillans. Formation of these bloom events may involve vertical migration and/or the concentrating mechanism of M. rubrum and N. scintillans. Meanwhile, biotic interactions such as mutual supportive relationship between N. scintillans and M. rubrum, and O. hongkongense fed on the progametes of N. scintillans, as well as other abiotic factors like seawater temperature and rainfall, also play important roles in this series of bloom events. Our findings have important implications for coastal zones worldwide, which are affected recurrently by these two ubiquitous red tide-forming species.

Distribution and genetic diversity of the parasitic dinoflagellate Amoebophrya in coastal waters of China.

Amoebophrya is an obligate endoparasite infecting wide ranges of marine organisms in coastal and oceanic waters. The parasitoid has received growing attention, due to its enormous genetic diversity in seawaters and suppressive effects on the growth of host dinoflagellates. Harmful algal blooms (HABs) caused by planktonic dinoflagellates have significantly impacted the coastal environment and mariculture in China. Series of studies have been conducted to reveal the occurrence mechanism and negative impacts of HABs in past decades, while the factors contributing to the recession of HABs have rarely been studied. Thus, the host range, prevalence and diversity of Amoebophrya along the coastline of China were systemically investigated to facilitate future studies on the ecological roles of the parasitoid. Overall, 10 dinoflagellate taxa were found to be infected by Amoebophrya spp., and the prevalence ranged from 0.03% to 2.50%. Sequencing of environmental genomic DNA revealed substantial diversity and significant regional heterogeneity of Amoebophryidae sequences derived from 12 coastal bays, while no significant correlation was observed among geographical locations. Phylogenetic analyses of 18S rDNA sequences derived from individual Amoebophrya-infected cells indicated the host divergence of the parasitoid and lend credence to the multiple species assumption. The results further revealed the broad host range, wide distribution and substantial diversity of Amoebophrya in the coastal waters of China, that should not be neglected in future studies on the succession of HABs, as well as the ecological significance of this parasitoid in marine microbial food webs.

Distribution of living coccolithophores in eastern Indian Ocean during spring intermonsoon.

We studied the biodiversity of autotrophic calcareous coccolithophore assemblages at 30 locations in the Eastern Equatorial Indian Ocean (EEIO) (80°-94°E, 6°N-5°S) and evaluated the importance of regional hydrology. We documented 26 species based on the identification of coccospheres and coccoliths, respectively. The coccolithophore community was dominated by Gephyrocapsa oceanica, Emiliania huxleyi, Florisphaera profunda, Umbilicosphaera sibogae, and Helicosphaera carteri. The abundance of coccoliths and coccospheres ranged from 0.2 × 103 to 160 × 103 coccoliths l-1 and 0.2 × 103 to 68 × 103 cells l-1, averaged 23 × 103 coccoliths l-1 and 9.4 × 103 cells l-1, respectively. Biogenic PIC, POC, and rain ratio mean values were 0.50 µgC l-1, 1.047 µgC l-1, and 0.10 respectively. High abundances of both coccoliths and coccospheres in the surface ocean layer occurred on the north of the equator. Vertically, the great majority of coccoliths and coccospheres were concentrated in water taken from depths of <75 m. The ratios between the number of coccospheres and free coccoliths indicated that coccoliths experience different levels of dissolution when transported to deep water. Abundant coccolithophores mainly occurred at the west of 90°E, which is in accordance with the presence of Wyrtki jets. Patterns of coccolithosphores and of coccoliths have been reflected in hydrological processes.

Characterization of the Parasitic Dinoflagellate Amoebophrya sp. Infecting Akashiwo sanguinea in Coastal Waters of China.

The endoparasitic dinoflagellate Amoebophrya infects a number of free-living marine dinoflagellates, including harmful algal bloom species. The parasitoid eventually kills its host and has been proposed to be a significant loss factor for dinoflagellate blooms in restricted coastal waters. For several decades, the difficulties of culturing host-parasitoid systems have been a great obstacle for further research on the biology of Amoebophrya. Here, we established an Akashiwo sanguinea-Amoebophrya sp. coculture from Chinese coastal waters and studied the parasitoid's generation time, dinospore survival and infectivity, as well as its host specificity. The lifespan of Amoebophrya sp. ex. A. sanguinea was approximately 58 h. The infective dinospores can survive up to 78 h in ambient waters but gradually lose their infectivity. The parasitoid was unable to infect other dinoflagellate species, its infection rate reached as high as 91% when the ratio of dinospores to host cells was 20:1. The high infectivity of dinospores suggests that the Amoebophrya strain was capable of removing a considerable fraction of host biomass within a short period, but that it is probably unable to maintain high infection levels under nonbloom conditions of its host, due to limited survival and time constraints in encountering host cells.

Population dynamics of Noctiluca scintillans during a bloom in a semi-enclosed bay in Hong Kong.

Noctiluca scintillans, a heterotrophic dinoflagellate responsible for most of the red tides in Hong Kong waters was investigated to determine the influence of biotic and abiotic factors in determining its population dynamics. N. scintillans first occurred in January when temperature was ~17°C, and reached a maximum of 1980cellsL-1 one month later. N. scintillans interacted with trophic compartments from picoplankton to mesozooplankton, but its population development was largely related to the availability of phytoplankton, especially diatoms. Growth rates in microcosms (with mainly biological factors) ranged from -0.38 to 1.11d-1 and were similar to the field in situ growth rates (physical and biological factors). Thus, optimum hydrographical conditions (temperature and water stability), and a rich food supply were necessary for N. scintillans to bloom. Its spatial distribution pattern was a result of biological and physical coupling, but mainly controlled by physical accumulation processes such as winds, tides and currents.

Molecular characterization of a novel nitric oxide synthase gene from Portunus trituberculatus and the roles of NO/O2(-)- generating and antioxidant systems in host immune responses to Hematodinium.

Increasing evidences have established that the nitric oxide synthase (NOS) and NADPH oxidase (NOX) play important roles in host defense system by catalyzing the production of nitrogen oxide (NO) and superoxide anions (O2(-)), respectively. While, there are limited studies to explore the roles of NOS/NOX enzymes in crustacean immunity, and no studies as yet were attempted to elucidate their functions in host immune responses to parasites. In the present study, we cloned a full-length cDNA of NOS and two partial cDNA fragments of NOX and GPx from the economic valuable crab Portunus trituberculatus. The full-length cDNA of NOS was 4002 bp in length that encoded 1203 amino acids containing motifs of the NOS protein and conserved domains. The phylogenetic analysis showed that the NOS protein sequence was clustered together with those of crustacean species in the phylogenetic tree. All of the three novel genes showed high mRNA transcripts in the immune-related tissues (e.g. hemocytes, hepatopancreas) of P. trituberculatus. Striking fluctuation in the transcripts of the critical NO/O2(-)- generating/scavenging related genes (NOS, NOX, CuZnSOD, CAT, GPx) as well as in the enzymatic activities of NOS, NOX, SOD, CAT and GPx were observed in the hemocytes and hepatopancreas of P. trituberculatus post challenged with the parasitic dinoflagellate Hematodinium, indicating that the NO/O2(-)- generating and the antioxidant systems played vital roles in the crustacean innate immunity against the parasitic intrusion. The results indicated a novel respect of the host-parasite interaction between the crab host and the parasitic dinoflagellate Hematodinium.

Early transcriptional response to the parasitic dinoflagellate Hematodinium in hepatopancreas of Portunus trituberculatus.

The swimming crab Portunus trituberculatus supports a large proportion of crab aquaculture in China. In the last decade, the sustainable culture of this crab was threatened by the parasitic dinoflagellate Hematodinuim, resulting in massive mortality (up to 95%) in severely impacted culture ponds. Previous studies of Hematodinium were mainly focused on histology, molecular characterization, epizootiology, etc., with limited studies conducted to explore this specific host-parasite interaction. Thus, to give a primary insight into the anti-parasitic immune response at the critical stage of infection, the expression levels of 8 immune-related genes together with enzyme activities of phenoloxidase (PO), alkaline phosphatase (AKP), and acid phosphatase (ACP), were evaluated in hepatopancreas during 3-192h (h) post inoculation. Hematoxylin-eosin (H&E) staining showed noticeable pathological changes in hepatopancreas. The enzyme activities of PO, AKP, and ACP were significantly induced after inoculation. The changes of the prophenoloxidase (proPO) transcripts and the constantly enhanced PO activity reflected the critical function of the proPO system in resisting against the parasites. The decreased expression levels of LGBP and PPAF implied an immunosuppressive mechanism of the parasites against the host proPO system. And the significant variations in transcriptional levels of two important proteinase inhibitors (serpin, α2m) and three P. trituberculatus clip-domain serine proteinases (PTcSPs) suggested that the parasites could affect proteinase cascade reactions associated with immune response by destroying the balance between serine proteinases and the inhibitors. Moreover, the results indicated that the hepatopancreas of P. trituberculatus was significantly affected by invasion of the parasite, and hepatopancreas played important roles in the crustacean innate immunity against the parasitic infection.

Molecular characterization and expression of a novel Toll gene from the swimming crab Portunus trituberculatus.

Tolls/Toll-like receptors (TLRs) are important cell-surface receptors serving as pattern recognition receptors (PRRs) in the Tolls/TLRs signaling pathway of innate immune responses. In the present study, we isolated and characterized a novel Toll gene (PtToll) from Portunus trituberculatus, and further investigated its expression in various tissues of crab hosts challenged with the parasitic dinoflagellate Hematodinium. The full-length cDNA of PtToll was 3745 bp, with a 3012 bp open reading frame (ORF) encoding 1003 amino acids. Conserved domains consist of 15 tandem leucine-rich repeats (LRRs), a single-pass transmembrane segment (TM) and a cytoplasmic Toll/interleukin-1R (TIR) domain. The PtToll protein shared high similarity to other crustacean Tolls and was clustered with the crustacean Tolls in the phylogenetic tree. The PtToll gene was constitutively expressed in various tissues of P. trituberculatus, with the highest expression in hemocytes. After being challenged with the parasite, the transcripts of PtToll reacted immediately with significant alterations in all the tested tissues, and decreased consistently in most of the detected tissues (e.g., hemocytes, gill, heart, and muscle) within 24h. Then the transcripts of PtToll were significantly up-regulated in hemocytes and heart at 48 h, and in hepatopancreas at 48 and 96 h post the parasitic challenge. By 192 h post challenge, the transcriptional level of PtToll indicated a significant suppression or a decreasing trend. The fluctuations of PtToll gene expression suggested that PtToll was closely associated with intrusion of the Hematodinium parasites, and may possess a vital and systematic function in the innate immunity of P. trituberculatus against the parasitic infection.

Immune response and gene expression in hemocytes of Portunus trituberculatus inoculated with the parasitic dinoflagellate Hematodinium.

The parasitic dinoflagellates in the genus of Hematodinium infect broad range of crustaceans around the world, causing fatal diseases in multiple species of wild and cultured crabs and subsequent economic loss. In order to explore this host-parasite interaction in the early development stage of infection, mRNA transcript levels of eight key immune-related genes, including LGBP, proPO, PPAF, serpin, α2m, and three PTcSPs were quantitatively assessed in Portunus trituberculatus artificially inoculated with the Hematodinium parasites. The fluctuation of proPO gene expression indicated that the host proPO system was disturbed overtly due to the intrusion of the parasites. And as manifested by the suppressed expression of LGBP and PPAF, an immunosuppressive mechanism was likely induced by the parasites against being entrapped or killed by the host proPO system. Furthermore, the significant variations of the transcript levels of serpin, α2m, and the three PTcSPs suggested that the parasites affected the proteinase cascade reactions associated with the immune response by destroying the balance between serine proteinases and the proteinase inhibitors. The hemocytes counts and PO activity varied accordingly over the time course of infection, showing that hemocytes were actively involved in the immune response against the parasitic invasion. This study primarily highlighted the anti-parasitic immune response of crab hosts, and presented the first report of the immune response of P. trituberculatus to the parasitic dinoflagellate Hematodinium.

[Size-fractionated biomass of phytoplankton in the Yangtze River estuary and its adjacent waters in winter and spring].

Based on the multi-discipline investigation in 28th February-10th March and 30th May-4th June, 2005, this paper studied the distribution of size-fractionated phytoplankton in the Yangtze River estuary and its adjacent waters, and the environmental factors that affected this distribution. The results showed that in winter, the average chlorophyll a concentration was 1.28 mg X m(-3), and the region with high concentration chlorophyll a was located near the river mouth. Small-sized phytoplankton (SSP, <20 microm) contributed 66.7% to the total phytoplankton biomass, but big-sized phytoplankton (BSP, >20 microm) was dominant in the plume system. In spring, the average chlorophyll a concentration in inner river and outer estuary was 0.67 and 6.03 mg x m(-3), respectively, and the maximum of chlorophyll a concentration appeared in the waters between 122.5 degrees E and 123.0 degrees E. The SSP dominated in the plume system, especially in the algal bloom frequently occurring area which located at south of survey area, contributed 83.5% to the total phytoplankton biomass. The vertical profile of size-fractionated chlorophyll a showed that with the increase of water depth, the SSP and BSP became equal in the offshores. Among the environmental factors in the Yangtze River estuary and its adjacent waters, salinity and nutrients were the main factors affecting the distribution and composition of size-fractioned phytoplankton biomass in winter and spring.

[Selective feeding of Calanus sinicus on harmful algal blooms species in East China Sea in spring].

From April 27th to June 5th 2005, the selective feeding of Calanus sinicus on harmful algal blooms (HABs) species was studied by in situ feeding experiments at six typical stations in a high frequency red tide occurring region of East China Sea. The results showed that the selective feeding behavior of C. sinicus was dependent on food density. It had an obvious food selectivity when the abundance of phytoplankton cell was low, but the selectivity changed from preference to discrimination at high phytoplankton cell abundance, with a threshold value of 329 cells ml. The selective feeding of C. sinicus was also dependent on food composition. It showed no selective feeding or discrimination on microzooplankton at most stations, but preferred to P. delicatissima and disliked Karenia mikimotoi at HABs occurring station, which induced the succession of phytoplankton community to K. mikimotoi blooming.

[Phytoplankton assemblage in Yangtze River estuary and its adjacent waters in winter time].

Water samples were collected from Yangtze River Estuary and its adjacent waters in 28th February-10th March, 2005, and the species composition of phytoplankton was analyzed by Utermöhl method. A total of 130 taxa (including 25 uncertain species) which belong to 67 genera of 5 phyla were identified. The phytoplankton community was mainly composed of Bacillariophyta, followed by Dinophyta. There were also a few species belonging to Chrysophyceae, Cyanophyceae and Chlorophyceae. The dominant species were Paralia sulcata, Skeletonema costatum, Thalassiosira rotula, Bleakeleya notata, Coscinodiscus radiatus and Thalassiosira excentrica. The cell abundance of phytoplankton ranged from 0.1 to 90.0 cells x ml(-1), with an average of 10.1 cells x ml(-1). Horizontally, the cell abundance was relatively high in inshore and low in offshore; while vertically, it was high in surface water and decreased slightly with increasing water depth. The cell abundance and chl a concentration of phytoplankton positively correlated with the concentrations of nitrate, nitrite, ammonium, phosphate and silicate in water, but negatively correlated with water salinity. The Shannon-Wiener diversity index and Pielou evenness index were higher in the center of survey area but lower in northeast part and inshore area.