Comparative Transcriptome Analysis of Shell-Matrix-Protein Genes and Observation of the Shell Microstructure in the Deep-Sea Clam Calyptogena marissinica

The deep-sea clam Calyptogena marissinica is widely distributed in the Haima cold seep ecosystem on the northwes-tern slope of the South China Sea with low pH values,low temperature and high pressure.Limited information is available on the biomineralization of this species.In this research,we generated a comprehensive transcript dataset of C.marissinica’s mantle tissue,and a total of 19821 unigenes were assembled.Fourteen shell matrix proteins(SMP)-related genes were identified.The qPCR results showed that four out of six prismatic matrix genes(MSP2,MSP5,prisilkin-39,and shematrin),four out of the six nacreous matrix genes(perlucin,pif,pif97,and papilin),and two extrapallial fluid proteins(SPARC and calmodulin)were significantly expressed in the mantle.Both the nacreous and the prismatic layers are chrysanthemum-shaped,which are stacked on the top of each other to form a laminated nacreous structure.The alignment and phylogenetic analysis of MSP-5,Prisilkin-39,Perlucin,and Pif homologues showed that some amino acids of C.marissinica that differed from those detected in other molluscs may cause the different shape of the nacreous and prismatic layers,but do not lead to a change in the species’evolutionary status.These results indicated the conservation of the functions of SMP-related genes in C.marissinica,and the specific shape of the prismatic and nacreous layers of this deep-sea mollusc,which will contribute to the research on the molecular regulation mechanisms of biomineralization in C.marissinica and provide a new perspective to investigate biomineralization in deep-sea clams in general.

Transcriptome Analysis of Mantle Tissues Reveals Potential Shell-Matrix-Protein Genes in Gigantidas haimaensis

Haima cold seep ecosystem is on the northwestern slope of the South China Sea,which is characterised by high pressure,low temperature,hypoxia,and low pH value.The deep-sea mussel Gigantidas haimaensis is distributed in this ecosystem.Previous studies have focused on its adaptation to abiotic stress,biogeography,ecotoxicology,genomes,immunity and symbiosis,but knowledge on biomineralisation remains lacking.Herein,we generated a comprehensive transcript dataset from G.haimaensis mantle tissue,and 30255 unigenes were assembled.The top 20 most highly expressed genes are related to energy supply,such as mitochondrial genes,suggesting they may mediate the adaptability of this deep-sea mussel to the high pressure and hypoxia environment.Eleven shell matrix protein(SMP)-related genes were identified from the transcriptome data.Quantitative PCR analyses showed that five of ten acidic SMPs and nacreous-layer-matrix-protein genes(nacrein,perlucin,perlwapin,pif and mantle protein)were highly expressed in mantle tissue,while the expressions of other five genes(chitinase,SPARC,TRY,papilin and calmodulin)were low.Scanning electron microscopy showed that the shell was composed of a prismatic layer and a nacreous layer,and every nacreous layer was made of the whole pieces of aragonite that stacked on top of each other.These results indicated the conservation of the structure and functions of nacreous matrix genes in G.haimaensis.Moreover,the nacreous layer was made of whole pieces of aragonite that were not quadrilateral or polygonal pieces.Studying these genes will likely reveal the molecular mechanisms of biomineralisation in G.haimaensis and other deep-sea mussels.

Identification and Expression of the Conotoxin Homologous Genes in the Giant Triton Snail (Charonia tritonis)

Conotoxin(CTX)is a small peptide toxin,and plays crucial role in anesthetizes prey,preys and defends competitors.Charonia tritonis is the natural predator of the crown of thorns starfish(CoTS)and the protector of coral reefs.It plays an important role in the coral reef ecosystem.However,the types of toxins produced by tritons and the molecular mechanisms of toxin secretion of C.tritonis are unknown.In the present study,the four conotoxin homologous genes(CTXs)from C.tritonis were identified.Species and conotoxin superfamily phylogenetic tree indicated that CTX-1(CL2216.Contig2)and CTX-4(Unigene 58438_All)belong to the C superfamily,CTX-2(Unigene 66414_All)belong to the V superfamily,and CTX-3(Unigene 21408_All)belong to the B1 superfamily.CTXs were highly expressed in salivary gland,liver and digestive gland.The predation control experiment revealed that the expressions of CTXs were significantly different in salivary gland,liver and digestive gland.The results indicated that CTXs may participate in the process of C.tritonis predating CoTs,and provided a scientific basis for further studying the toxins secretion mechanism of C.tritonis.

Molecular quantification of copepod Acartia erythraea feeding on different algae preys

Quantitative evaluation of the copepod feeding process is critical for understanding the functioning of marine food webs, as this provides a major link between primary producers and higher trophic levels. In this study, a molecular protocol based on quantitative polymerase chain reaction(qPCR) targeting 18S rDNA was developed and used to investigate the feeding and digestion rates of the copepod Acartia erythraea in a laboratory experiment using microalgae Thalassiosira weissflogii, Prorocentrum shikokuense, and Alexandrium catenella as prey. Although offered an equal encounter rate based on biovolume, prey uptake varied substantially among the three algal species, with the ingestion rate(IR) and digestion rate(DR) of A. erythraea differing significantly(P <0.001) based on both cell counting and qPCR detection. Acartia erythraea showed the highest IR(2.79×10~4 cells/(ind.·h)) and DR(2.43×10~4 cells/(ind.·h)) on T. weissflogii, and the lowest amounts of ingested P. shikokuense were detected. The highest assimilation rate(~90.64%, IR/DR) was observed in copepods fed with P. shikokuense. The qPCR method used here can help determine the digestion rate and assimilation rate of copepods by detecting cells remaining in the gut hence providing the possibility to examine trophic links involving key species in the marine ecosystem. Our results indicate that A. erythraea has diet-specific feeding performance in different processes, and a quantitative assessment of copepod feeding is needed to accurately determine its functional role in the energy and matter uptake from marine food webs.

Genetic basis of embryo and juvenile physiological responses to salinity changes in freshwater pipefish (Hippichthys heptagonus)

DEAR EDITOR,Syngnathidae species commonly inhabit ocean environments.However,some have adapted to live exclusively in freshwater over long-term adaptive evolution but continue to retain physiological adaptations to saltwater environments.The genetic basis underlying the adaptive strategies and molecular regulation of freshwater syngnathids to freshwater and saltwater remains unclear.Here,we investigated the molecular characteristics and core gene expression in freshwater belly pipefish(Hippichthys heptagonus)embryos and juveniles through salinity stress experiments and transcriptome analysis.Results showed that embryonic exposure to salinity at a concentration of 30‰down-regulated cell cycle-associated genes vital to embryonic development.Retinol metabolism,neuroactive receptor interaction,and peroxisome proliferator-activated receptor signaling pathways were significantly enriched in up-regulated genes in the embryos.Notably,there was no significant change in the expression of ion transport and energy metabolism genes.Conversely,juvenile exposure to 30‰salinity up-regulated ion transport-related genes and significantly enriched immune-related signaling pathways,including lysosome,phagosome,autophagy,and mitophagy signaling pathways.Carbohydrate metabolism genes were also up-regulated,whereas oxidative phosphorylation genes were significantly down-regulated.These results suggest that brood pouch protection during the embryonic stage and salinity adaptation plasticity in juveniles may be strategic adaptations in freshwater pipefish.

Effects of DNA extraction and universal primers on 16S rRNA gene-based DGGE analysis of a bacterial community from fish farming water

Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.

RNAi knockdown of PIK3CA preferentially inhibits invasion of mutant PIK3CA cells

AIM: To explore the effects of siRNA silencing of PIK3CA on proliferation, migration and invasion of gastric cancer cells and to investigate the underlying mechanisms. METHODS: The mutation of PIK3CA in exons 9 and 20 of gastric cancer cell lines HGC-27, SGC-7901, BGC-823, MGC-803 and MKN-45 was screened by polymerase chain reaction (PCR) followed by sequencing. BGC-823 cells harboring no mutations in either of the exons, and HGC-27 cells containing PIK3CA mutations were employed in the current study. siRNA targeting PIK3CA was chemically synthesized and was transfect- ed into these two cell lines in vitro . mRNA and protein expression of PIK3CA were detected by real-time PCR and Western blotting, respectively. We also measured phosphorylation of a serine/threonine protein kinase (Akt) using Western blotting. The proliferation, migration and invasion of these cells were examined separately by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT), wound healing and Transwell chambers assay. RESULTS: The siRNA directed against PIK3CA effectively led to inhibition of both endogenous mRNA and protein expression of PIK3CA, and thus significantly down-regulated phosphorylation of Akt (P < 0.05). Furthermore, simultaneous silencing of PIK3CA resulted in an obvious reduction in tumor cell proliferation activity, migration and invasion potential (P < 0.01). Intriguing, mutant HGC-27 cells exhibited stronger invasion ability than that shown by wild-type BGC-823 cells. Knockdown of PIK3CA in mutant HGC-27 cells contributed to a reduction in cell invasion to a greater extent than in non-mutant BGC-823 cells. CONCLUSION: siRNA mediated targeting of PIK3CA may specifically knockdown the expression of PIK3CA in gastric cancer cells, providing a potential implication for therapy of gastric cancer.

Cloning and expression analysis of a ubiquitin gene (Ub_(L40)) in the haemocytes of Crassostrea hongkongensis under bacterial challenge

Ubiquitin, a highly conserved stress-related protein, is assigned multiple functions, such as DNA processing, protein degradation, and ribosome synthesis. The Crassostrea hongkongensis ubiquitin gene (designated ChUbL40) was cloned by a combination of suppressive subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The full-length cDNA of ChUbL40 is 496 bp in length, consisting of a 5' untranslated region (UTR) of 34 bp, a 3'-UTR of 75 bp and an open reading frame of 387 bp encoding a ubiquitin fusion protein of 128 amino acids. Analysis of the amino acid sequence of ChUbL40 reveals that UbL40 is highly conservative during evolution. The expression patterns of ChUbL40 gene in various tissues were examined by real-time PCR. The expression level of ChUbL40 in haemocytes is down-regulated at 4 h and gradually returned to its original level from 6 h to 24 h after Vibrio alginolyticus challenge. Our results suggest that ChUbL40 is ubiquitously expressed and plays an important role in immune defense against bacterial challenge.

The effects of four transplantation methods on five coral species at the Sanya Bay

Coral transplantation is considered as one of the major tools to increase coral abundance for degraded coral reefs.To investigate the effects of various methods and coral species in transplantation, coral fragments（n=902） of five coral species were transplanted by four methods at Luhuitou, the Sanya Bay, Hainan Province, China, where the reef has been over-exploited and is still threatened by human activities and natural disasters. Ten months after the transplant, the average survivorship of the transplanted corals was 45.5%. Methodologies had different effects on the transplanted corals, but none of them was efficacious for all coral species. Methodology could not change the decreasing trend for Montipora foliosa and Acropora hyacinthus, although it did slow down their decline. All transplants of A. hyacinthus and M. foliosa had high mortalities and significant decrease on survival area, while Porites andrewsi and Galaxea fascicularis had lower mortalities and partial mortalities. Only one method had significant effect on increasing survival area of G. fascicularis, same as P. andrewsi. Out of the five transplanted coral species, Pocillopora damicornis was the only species that had living tissue area increase in all applied methods, while the others had decreased live tissue area in one or more methods. The results of this study suggested that performing coral transplantation in a highly threatened area was not efficient unless the threats were diminished or erased. Moreover, proper species selection for coral transplantation is crucial, especially in a disturbed environment. Methodology, although having limited effects on improving results of coral transplantation, cannot compensate the maladjustment of vulnerable species to the stresses on the Luhuitou Reef. Coral transplantation on Luhuitou Reef should not be performed unless the stresses are under controlled,and corals with good tolerance to the environment should be considered first.

Long duplication of 18S ribosomal DNA in Cynoglossus lineolatus(Pleuronectiformes： Cynoglossidae)： novel molecular evidencefor unequal crossing over model

Although 18 S r DNA sequence is extremely conservative, the polymorphism still has been found in few species.In the present study, three types(Type A, B and C) of 18 S r DNA sequence coexisted in Cynoglossus lineolatus genome, suggesting a non-concerted evolution process, rather than a strictly concerted evolution fashion.Based on the differences of sequence variation, GC content, secondary structure and minimum free energy,Types A and B were speculated as the potential pseudogenes. Additionally, a fascinating finding was a 189-bp duplication of 18 S r DNA in Type A sequence. To our knowledge, this is the first report on such a long duplication in teleostean ribosomal DNA. Compared with several theories accounting for the formation of tandem repeats, the unequal crossing over model was thought to be the most likely mechanism to generate the189-bp duplication of 18 S r DNA. These results not only provide a novel molecular evidence for the unequal crossing over model, but also benefit for the further study on 18 S r DNA in fishes.

Histological and Histochemical Analyses of the Cuttlebone Sac of the Golden Cuttlefish Sepia esculenta

The secretion function of mantle is closely related to shell formation in some bivalves and gastropods. Up to now, few researches have been reported for cuttlebone formation in the class Cephalopoda. In this study, the structure and secretion function of cuttlebone sac of the golden cuttlefish Sepia esculenta was analyzed using the histological and histochemical methods. The results showed that high and columnar cells located in sac epithelium, and flat cells existed near the base membrane. A lot of fibroblasts were found in the lateral mantle collective tissue. Some mucus, mucopolysaccharide and alkaline phosphatase (ALP) were found in the sac. The ultrastructural characteristics of Quasi-connective-tissue-calcium cells (QCTCC) were observed using a transmission electron microscope (TEM). The relationship between cuttlebone sac secretion function and shell formation was discussed.

Environmental Drivers of Temporal and Spatial Fluctuations of Mesozooplankton Community in Daya Bay, Northern South China Sea

The response of zooplankton to the ecological environment in Daya Bay is unclear under the influence of both climate changes and anthropogenic activities on a seasonal to inter-annual scale.Based on monthly surveys and historical data,we found the zooplankton community had changed temporally and spatially.A total of 134 species was recorded during the study,and copepods dominated numerically in terms of diversity and abundance.Both copepods and cladocerans were the main contributors to zooplankton abundance.The community structure of zooplankton was temporally classified into the warm and cold groups,and spatially into the three groups located in the marine cage-culture area(MCCA),the outflow of nuclear power plants(ONPP)and unpolluted waters(UPW).The zooplankton was characterized by low biomass(dry weight),high diversity and abundance in the warm period in contrast to that in the cold period.Compared with the other two groups,the MCCA group of zooplankton showed high abundance,low diversity and biomass.Variations in dominant species were closely related to temperature,salinity and chlorophyll a concentration.Species diversity and dry weight decreased in comparison with 30 years ago,while zooplankton abundance increased.The seasonal variation in zooplankton was affected mainly by temperature that was controlled by monsoon,while the spatial difference in the community structure was probably due to eutrophication in the MCCA and thermal water discharge from ONPP.The zooplankton community is undergoing great changes with the tendency of miniaturization and gelatinization in recent 30 years in Daya Bay.

Effect of Shrimp(Litopenaeus vannamei) Farming Waste on the Growth, Digestion, Ammonium-Nitrogen Excretion of Sea Cucumber(Stichopus monotuberculatus)

In this study, specific growth rate(SGR), ingestion rate(IR), food conversion ratio(FCR), apparent digestion ratio(ADR) and ammonium-nitrogen excretion were determined for sea cucumber(Stichopus monotuberculatus) reared in plastic containers(70 L; 4 containers each diet treatment). Sea cucumbers were fed with five diets containing different amounts of farming waste from shrimp(Litopenaeus vannamei)(100%, 75%, 50%, 25% and 0) and a formulated compound(20% sea mud and 80% powdered algae). Sea cucumbers grew faster when they were fed with diet D(25% shrimp waste and 75% formulated compound) than those fed with other diets. Although IR value of sea cucumber fed with diet A(shrimp waste) was higher than those fed with other diets, both the lowest SGR and the highest FCR occurred in this diet group. The highest and the lowest ADR occurred in diet E(formulated compound) and diet A group, respectively, and the same to ammonium-nitrogen excretion. The contents of crude protein, crude lipid and total organic matter(TOM) in feces decreased in comparison with corresponding diets. In the feces from different diet treatments, the contents of crude protein and TOM increased gradually as the contents of crude protein and TOM in diets increased, while crude lipid content decreased gradually as the crude lipid content in diets increased.

Temperature-Dependent Fatty Acid Composition Change of Phospholipid in Steelhead Trout (Oncorhynchus mykiss) Tissues

In this study, the changes of the fatty acid composition of phospholipid in different tissues(muscle, heart, brain and spleen) of steelhead trout(Oncorhynchus mykiss) were analyzed when the water temperature decreased gradually from 16 to 12℃℃, 8℃, 6℃, 4℃, 2 and 1℃℃. Three fish individuals each tank(average weight 70.32 g ± 9.12 g) were collected and used to analysis at each designed temperatures. At normal temperature(16℃), the fatty acid composition of phospholipid of muscle and heart was similar each other. The highest concentration of saturate fatty acids(SFA) was found in the phospholipid of spleen. The brain phospholipid contained higher oleic acid(18:1 n9) than the phospholipid of other tissues at 16℃. When the environmental temperature decreased, the concentration of unsaturated fatty acids of phospholipids in all tissues increased, and accordingly the ratio pf the unsaturated to saturated fatty acids(U/S) and unsaturation index(UI) increased, indicating that steelhead trout can compensate temperature-dependent changes in membrane fluidity by remodeling the fatty acid composition of phospholipids. The changes in the fatty acid composition of phospholipid were tissue-specific. At the early stages of the experiment(16 to 8℃℃), the fatty acid composition of phospholipid changed remarkably in muscle, heart, and spleen. When temperature decreased to less than 8℃, an obvious response of phospholipid fatty acid was observed in all tissues. The change of phospholipid composition of steelhead trout tissues may be affected by both cold stress and starvation when the temperature decreased to 2℃, and the change of phospholipid composition of muscle was very obvious.

Distribution of reactive aluminum under the influence of mesoscale eddies in the western South China Sea

To understand the distribution of aluminum （A1） under the influence of mesocale eddies in the western South China Sea （SCS）, sea level anomaly, geostrophic current, environmental parameters and reactive A1 were investigated in the western SCS in August 2013. The highest reactive A1 concentration （（180±64） nmol/L） was observed in the surface waters, indicating a substantial atmospheric input. Vertically, the reactive A1 decreased from the surface high concentration to the subsurface minima at the depth of chlorophyll a （Chl a） maxima and then increased again with depth at most of the stations. The average concentration of reactive A1 in the upper 100 m water column was significantly lower in the cyclonic eddy （（137±6） nmol/L） as compared with that in the non- eddy waters （（180±21） nmol/L）. By contrast, the average concentrations of Chl a and silicate in the upper 100 m water column were higher in the cyclonic eddy and lower in the anticyclonic eddy. There was a significant negative correlation between the average concentrations of reactive Al and Chl a in the upper 100 m water column. The vertical distribution of reactive AI and the negative correlation between reactive A1 and Chl a both suggest that the reactive A1 in the upper water column was significantly influenced by biological removal processes. Our results indicate that mesoscale eddies could regulate the distribution of reactive A1 by influencing the primary production and phytoplankton community structure in the western SCS.

Application of the first internal transcribed spacer(ITS-1)of ribosomal DNA as a molecular marker to population analysis in farrer's scallop Chlamys farreri

Sequence variation of the first internal transcribed spacer of ribosomal DNA （ ITS - 1 ） was examined and its application to the study of genetic variation was explored in four populations of farter＇ s scallop Chlamys farreri. ITS - 1 fragments, with a length of about 300 bp,of 78 individuals collected from Dalian, Qingdao, Yantai in China and Korea respectively were amplified via PCR, cloned and sequenced. Intra-genomic variation was examined by sequencing several clones of single individuals. Alignment and polymorphism analysis detected 44 haplotypes and 50 polymorphic sites which consist of 30 substitutions and 20 indels, indicating a high level of polymorphisms. Sequence analysis also showed a very low level of intra-individual variation. All these features validated the feasibility of application of ITS - 1 fragment to population analysis. Polymorphism analysis showed that the Korea sample has the richest genetic variation, followed by Yantai and Qingdao samples. AMOVA （analysis of molecular variance） showed that the majority （96.26%） of genetic variation was distributed within populations and 3.74% resulted from among populations, but with P 〈 0.05 （ = 0.042）, indicating that the populations in this study have significant divergence. This output was basically concordant with the result arising from RAPD data and different from that from mitochondrial 16S rDNA sequence data. Discussion on this inconsistency was made accordingly.

Zooplankton in the Huangyan Atoll,South China Sea:A comparison of community structure between the lagoon and seaward reef slope

Relatively few studies have investigated the community structure of zooplankton in the atolls of the central South China Sea. Zooplankton were sampled at nine stations within the lagoon and four facing the seaward reef-slope in the Huangyan Atoll during May 2015. Lagoonal samples contained 48 zooplankton taxa( H' =2.25±0.95; J =0.70±0.23) with planktonic larvae, copepods, and euphausids contributing 95.63% of total abundance. Seaward oceanic samples contained 114 zooplankton(H ' =5.27±0.27; J =0.90±0.03) with copepods, siphonophores, chaetognaths, appendicularians, and planktonic larvae accounting for 93.66% of abundance. Lagoonal zooplankton abundance was lower than that on the seaward reef-slope. Zooplankton community dif fered significantly between the lagoon and reef-slope groups(R =0.708, P =0.001). The former was dominated by Macruran larvae, Brachyuran larvae, S tylocheiron carinatum, and Acartia spp., while the latter was predominately represented by O ikopleura longicauda, Chelophyes contorta and C opilia mirabilis. Chlorophyll a concentration was significantly higher in the lagoon than the reef slope regions( P <0.01), which may be due to the nutrient input from fishing vessels near the lagoon mouth. Zooplankton abundance was negatively correlated with chlorophyll a concentration(P <0.01). A clear dif ference was observed statistically in the species composition and abundance of zooplankton between the lagoon and the seaward slope at Huangyan Atoll, which suggests that it may be resulted from the influence by natural and anthropogenic interactions.

Roles of interleukins in antibacterial immune defense of the brood pouch in the lined seahorse Hippocampus erectus

Seahorse embryos are brooded in the enclosed nutrient-rich environment of the male brood pouch,which may be prone to bacterial infection.The immune responses of interleukin(IL)genes in the brood pouch have not been well studied.We identifi ed 13 interleukins in the lined seahorse Hippocampus erectus.Tissue-specifi c expression analysis revealed increased mRNA expression levels of il-1β,il-18,and il-8 in the brood pouch.When challenged with lipopolysaccharide or Vibrio parahaemolyticus,il-1βand il-18 were active as part of the acute and chronic infl ammatory responses,respectively.Importantly,il-8 may be involved in powerful antibacterial immune responses and may be induced by il-1βand il-18 via a process involving the nuclear factor-κB signaling pathway.These results suggest that il-1β,il-18,and il-8 may play key roles in the antibacterial immune defense of the brood pouch in male seahorses.

Global-Scale Diversity and Distribution Characteristics of Reef-Associated Symbiodiniaceae via the Cluster-Based Parsimony of Internal Transcribed Spacer 2 Sequences

Photosynthetic species belonging to Symbiodiniaceae play important ecological roles in coral reef ecosystems. Nine phylogenetically supported Symbiodiniaceae clades(each containing multiple genetically distinct molecular types) have been detected in reef-associated animals and in seawater or sediments near coral reef areas. Although major advances in research on Symbiodiniaceae diversity have been made, information on the global-scale diversity and distribution characteristics of Symbiodiniaceae clades/types is still limited or must be expanded because most studies on Symbiodiniaceae have mainly focused on a limited number of hosts associated with Symbiodiniaceae or coral reef areas. Here, the diversity and distribution characteristics of Symbiodiniaceae data derived from reef-associated animals and seawater or sediments near coral reef areas were analyzed using 3899 qualified Symbiodiniaceae internal transcribed spacer 2(ITS2) sequences retrieved from the GenBank database in May 2017. These sequences were annotated as Symbiodiniaceae ITS2 types via sequence alignment against database of Symbiodiniaceae ITS2 types. Results showed that photosynthetic Symbiodiniaceae ITS2 sequences could be annotated as 119 Symbiodiniaceae ITS2 types within nine clades(A–I), and Symbiodiniaceae ITS2 types mainly inhabited coral reefs between 30?N and 30?S. Symbiodiniaceae ITS2 types could be associated with multiple host taxa. Interestingly, these results implied that the distribution of Symbiodiniaceae ITS2 types was distinct in the Atlantic Ocean, the Red Sea, the Mediterranean Sea, and the Indo-Pacific Ocean. Although future field research should be performed to verify our results, this study provided a broad perspective of the diversity of Symbiodiniaceae clades/types on a global scale.

Fast acclimation of phytoplankton assemblies to acute salinity stress in the Jiulong River Estuary

Mixing of freshwater and seawater creates the well-known salinity gradients along the estuaries. In order to investigate how phytoplankton respond to the acute salinity changes, we exposed natural phytoplankton assemblies from the Jiulong River Estuary to differential saline field water while continuously monitoring their photosynthetic performances under both indoor-and outdoor-growth conditions. When the natural cell assemblies from salinity 30 field water were exposed to series low saline field water(salinity 25, 17, 13 and 7.5), the effective Photosystem II quantum yield(ΔF/Fm′) decreased sharply, e.g., to one-fifth of its initials after 5 min exposure to salinity 7.5 field water, and then increased fast during the following 40 min and almost completely recovered after 320 min. During such an exposure process, non-photochemical quenching(NPQ) sharply increased from 0 to 0.85 within 5 min, and then decreased to nearly 0 within the following 70 min. When these cells re-acclimated to salinity 7.5 field water were exposed to series high saline field water(salinity 13, 17, 25 and 30), a similar response pattern was observed, with the decreased ΔF/Fm′ accompanied with increased NPQ, and followed by the recovery-induced increase in ΔF/Fm′ and decrease in NPQ. A similar response pattern as ΔF/Fm′to the acute osmotic stress was also observed in the photosynthetic carbon fixation capacity according to radiocarbon(14C) incorporation. Our results indicate that estuarine phytoplankton assemblies could rapidly recover from the acute osmotic stress, implying a potential cause for their frequent blooms in coastal-estuarine waters where despite drastically varying salinity, available nutrients are abundant due to the land-derived runoffs or mixing-caused relaxations from sediments.