Diversity and community structure of eukaryotic microalgae in surface sediments in the central Bohai Sea,China, based on a metabarcoding approach

Sediment samples were collected at 17 stations in the central Bohai Sea, China, and the diversity and community structure of eukaryotic microalgae were assessed by metabarcoding the V4 region of 18S rDNA. A total of 930 operational taxonomic units (OTUs) were detected for microeukaryotes, including 98 algal OTUs. The algal communities comprised 42 genera belonging to 19 classes of six phyla, and they were dominated by chrysophytes and dinoflagellates. Dinoflagellates were also the most diverse microalgal group. The nano-sized dinoflagellates Biecheleria halophila and Azadinium trinitatum occurred abundantly in the study area; however, they have not been reported previously, as they may be overlooked or misidentified in light microscopy. Many pico-sized chlorophytes were detected in the sediment samples. Sixteen of the detected OTUs were assigned to potentially harmful and/or bloom-forming microalgae, suggesting some potential risks of harmful algal blooms in the central Bohai Sea. The capacity of metabarcoding to detect morphologically cryptic and small species makes this method a sufficiently sensitive means of detection for assessing eukaryotic microalgae in sediments.

     

Complementary DNA sequencing (cDNA): an effective approach for assessing the diversity and distribution of marine benthic ciliates along hydrographic gradients

The Yellow Sea Cold Water Mass(YSCWM) is a distinct hydrographic phenomenon of the Yellow Sea,and the distribution pattern of meio-and macrobentho s diffe rs inside and outside of the YSCWM.However,such a pattern has never been observed in the microbenthic ciliate communities.Therefore,we hypothesized that benthic ciliates followed a similar distribution pattern as meio-and macrobentho s,but this pattern has not been uncovered by morphological methods.We evaluated the diversity and distribution of benthic ciliates at five stations along hydrographic gradients across the YSCWM and adj acent shallow water by using morphology and DNA and complementary DNA(cDNA) high-throughput sequencing of the V4 region of 18 S rRNA gene.Results showed that the diversity of benthic ciliate s detected by DNA(303 OTU s),and the cDNA(611 OTUs) sequencing was much higher than that detected by the morphological method(79 species).Morphological method detected roughly different ciliate communities inside and outside of the YSCWM,but without statistical significance.No clear pattern was obtained by DNA sequencing.In contrast,cDNA sequencing revealed a distinct distribution pattern of benthic ciliate communities like meioand macrobenthos,which coincided well with the results of the environmental parameter analysis.More than half of the total sequences detected by DNA sequencing belonged to planktonic ciliates,most(if not all) of which were recovered from historic DNA originating through the sedimentation of pelagic forms because none of them were observed morphologically.The irrelevant historic DNA greatly influenced the recovery of rare species and thus limited the understanding of the benthic ciliate diversity and distribution.Our research indicates that the methods used have significant effects on the investigation of benthic ciliate communities and highlights that cDNA sequencing has great advantages in estimating the diversity and distribution of benthic ciliates,as well as the potential for benthic environmental assessments.     

NGS-metabarcoding revealing novel foraminiferal diversity in the Western Pacific Magellan Seamount sediments

Seamount is a unique deep-sea ecosystem widely distributed in the world.Its biodiversity is vibrant due to its specific geographical and hydrological conditions.However,the diversity and features of foraminifera in such an environment have rarely been studied.We extracted environmental DNA(eDNA) in sedime nts and amplified the partial small subunit ribo somal DNA(SSU rDNA) of fo raminife ra to understand the foraminiferal diversity from four sites in Magellan Seamount(Western Pacific Ocean).Partial S SU rDNA sequencing was conducted and 912 979 foraminiferal reads were obtained and gathered into 266 operational taxonomic units(OTUs).In the available dataset,a high proportion of rare OTUs and low identity OTUs in each studied sample showed that the Magellan Seamount foraminiferal community might have a high genetic novelty.The relative abundance of foraminifera varied between replicates probably due to the existence of bias in amplification process and patchiness of the deep-sea floor.It showed that the Magellan Seamount has a relatively high benthic foraminiferal diversity characterized mainly by monothalamiids(76.37% of total reads) in association with rotaliids(19.03 % of total reads),including planktic foraminiferal sequences(38.58% of rotaliids;7.36% of total reads).The remaining reads were assigned to miliolids(0.83 % of total reads) and textulariids(0.66% of total reads),and 3.11% of total reads are unassigned to a specific family.The co mparative analysis with foraminiferal assemblage s from coastal and deep-sea environme nts indicated that seamounts could aggregate species from a nearby deep-sea.     

Screening for lipophilic marine toxins and their potential producers in coastal waters of Weihai in autumn, 2020

Lipophilic marine toxins (LMTs) produced by some microalgae in the sea could accumulate in shellfish and pose potential threats to the health of seafood consumers. Phytoplankton and shellfish samples were collected from coastal waters of Weihai in Shandong Peninsula, China in autumn, 2020, and screened for lipophilic marine toxins and their potential producers using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and high throughput sequencing of partial DNA (V4 region of the 18S rRNA gene) extracted from phytoplankton. Pectenotoxin-2 (PTX2), trace amounts of azaspiracid (AZA1 or AZA40), and 13-desmethyl spirolide C (13-DesMe-C) were detected in phytoplankton samples, while PTX2 and gymnodimine (GYM) were detected in shellfish samples. The toxin content in shellfish samples was much lower than the regulatory limit or values reported previously. Results suggest that lipophilic marine toxins should have low risk in coastal waters of Weihai in autumn. Based on the data of high throughput sequencing, the OTUs were assigned to 5 identified species of Alexandrium, including A. ostenfeldii capable of producing 13-DesMe-C and GYM. Two OTUs were found closely related to the toxic species in genus Dinophysis, but it is impossible to assign them to any identified species due to the low resolving power of the V4 region for Dinophysis. The OTUs could not be assigned to any identified species in the genus Azadinium, suggesting the existence of unidentified species in this region.

     

Comparison of fungal community composition within different intestinal segments of tilapia and bighead carp

Although intestinal fungi play important roles in host health and disease,the composition and diversity of fungal communities remain poorly reported in fish.In this study,fungi in the fore-,mid-,and hindintestine of tilapia(Oreochromis mossambicus) and bighead carp(Aristichthys nobilis) from Hongchaojiang Reservoir in Guangxi,China were investigated by ITS sequencing.Based on this,we obtained 1 763 478 high-quality tags,which clustered into 1 089 operational taxonomic units(OTUs).In total,404 OTUs were annotated,of which 310,68,and 26 belonged to Ascomycota,Basidiomycota,and other,respectively.Results show significant differences in the community composition of intestinal fungi between tilapia and bighead carp but not within their different intestinal segments.Furthermore,154 of the 404 annotated OTUs were considered reliable and were classified into three trophic modes and nine guilds.The three trophic modes consisted of 108 OTUs of saprotrophic fungi,41 OTUs of pathotrophic fungi,and five OTUs of symbiotrophic fungi.The top three most abundant OTUs overall(i.e.,Otu000002,Scopulariopsis acremonium; Otu000018,Alternaria palandui; Otu000034,Aureobasidium pullulans) showed lower abundance in the hind-intestinal segments of bighead carp,suggesting uneven distribution of these fungi in this species.In addition,saprotrophic and pathotrophic fungi were markedly decreased in the hindintestine.It is indicated that the fungal community was not only related to host species specificity but also to the respective physiological functions of different intestinal segments.These findings provide valuable information on the composition,structure,and potential function of the intestinal fungi community associated with different intestinal segments in tilapia and bighead carp under natural conditions,thus highlighting the importance of fungi as an integral part of the inte stinal microbiota in maintaining host health.     

Enhancing the culturability of bacteria from the gastrointestinal tract of farmed adult turbot Scophthalmus maximus

Eighteen agar media were tested for the culture of gut-associated bacteria from farmed adult turbot(Scophthalmus maximus),including 16 agar media with or without 1% gastrointestinal(GI)supernatant,or with 2% or 4% GI supernatant.A total of 1 711 colonies were analyzed and 24 operational taxonomic units(OTUs)were identifi ed.The greatest bacterial diversity was isolated on Zobell 2216E/Zobell 2216E+ agar media,whereas MRS/MRS+ agar media produced a low diversity of colonies.Agar media with GI supernatant(1%,2%,or 4%)showed increased diversity and yielded different profi les of OTUs from the corresponding original media,suggesting that GI supernatant provides substances that enhance the culture effi ciency of bacteria from the turbot GI tract.The large majority of the colonies(82%)were γ-Proteobacteria,whereas 15.6% and 2.4% of colonies were Firmicutes and Actinobacteria,respectively.At the genus level,49.4% of all colonies were assigned to Vibrio.Other potential pathogens,including Pseudomonas,Photobacterium,and Enterobacter,and potential probiotics,including Bacillus,Paenibacillus,and Pseudomonas,were also isolated on agar media.Most cultured bacteria belonged to species that were fi rst described in the turbot GI tract.The impact of these species on turbot physiology and health should be investigated further.     

Integrating a DNA barcoding project with an ecological survey: a case study on temperate intertidal polychaete communities in Qingdao, China

In this study, we integrated a DNA barcoding project with an ecological survey on intertidal polychaete communities and investigated the utility of CO1 gene sequence as a DNA barcode for the classification of the intertidal polychaetes. Using 16S rDNA as a complementary marker and combining morphological and ecological characterization, some of dominant and common polychaete species from Chinese coasts were assessed for their taxonomic status. We obtained 22 haplotype gene sequences of 13 taxa, including 10 CO1 sequences and 12 16S rDNA sequences. Based on intra- and inter-specific distances, we built phylogenetic trees using the neighbor-joining method. Our study suggested that the mitochondrial CO1 gene was a valid DNA barcoding marker for species identification in polychaetes, but other genes, such as 16S rDNA, could be used as a complementary genetic marker. For more accurate species identification and effective testing of species hypothesis, DNA barcoding should be incorporated with morphological, ecological, biogeographical, and phylogenetic information. The application of DNA barcoding and molecular identification in the ecological survey on the intertidal polychaete communities demonstrated the feasibility of integrating DNA taxonomy and ecology.     

Marine ciliate community in relation to eutrophication of coastal waters in the Yellow Sea

We assessed the potential of marine ciliate community as an indicator to coastal water quality using water samples collected from four stations in the Yellow Sea in the summer 2000. The four stations were characterized by different levels of pollution. The ciliate communities consisted primarily of tintinnids and aloricate ciliates that were <30 μm. A total of 78 species were classified: 55 species at Station 2, 51 species each at Stations 1 and 4, and 47 species at Station 3. The mean number of species at each site was 29.2±2.0 (Station 1), 28.5±2.9 (Station 2), 27.8±1.7 (Station 3), and 24.5±2.3 (Station 4). The abundance was highly variable: 19 331±11 187 ind./L at Station 1, 7 960±5 639 ind./L at Station 2, 29 015±12 999 ind./L at Station 3, and 8 190±4 658 ind./L at Station 4. Our results suggest that neither the simple chemical analysis (e.g. chemical oxygen demand, dissolved inorganic nitrogen, and phosphate) nor the eutrophication/pollution index adequately described the water quality at the four stations. The same was true of the number of species and their abundance, both of which had no correlation with the chemical indices. In contrast, Margalef’s diversity index values (3.12 at Station 2, 2.89 at Station 1, and 2.64 at Stations 3 and 4) generally discriminated the water quality status of the four stations. The difference in water quality among the stations was strongly supported by the pattern of species richness (i.e. the total number of species) of ciliates at each station. Our evaluation was consistent with the results of long-term water quality monitoring at the four stations. With increasing eutrophication, we observed also a compositional and functional shift in the ciliate assemblages from algivorous oligotrich/choreotrich to nonselective-omnivorous gymnostomatids to bacterivorous-detrivorous scuticociliatids. Thus, ciliates may be used to indicate the coastal water quality status of a given site.     

Applications of three DNA barcodes in assorting intertidal red macroalgal flora in Qingdao, China

This study is part of the endeavor to construct a comprehensive DNA barcoding database for common seaweeds in China. Identifications of red seaweeds, which have simple morphology and anatomy, are sometimes difficult solely depending on morphological characteristics. In recent years, DNA barcode technique has become a more and more effective tool to help solve some of the taxonomic difficulties. Some DNA markers such as COI (cytochrome oxidase subunitⅠ) are proposed as standardized DNA barcodes for all seaweed species. In this study, COI, UPA (universal plastid amplicon, domain V of 23S rRNA), and ITS (nuclear internal transcribed spacer) were employed to analyze common species of intertidal red seaweeds in Qingdao (119.3 -121 E, 35.35 -37.09 N). The applicability of using one or a few combined barcodes to identify red seaweed species was tested. The results indicated that COI is a sensitive marker at species level. However, not all the tested species gave PCR amplification products due to lack of the universal primers. The second barcode UPA had effective universal primers but needed to be tested for the effectiveness of resolving closely related species. More than one ITS sequence types were found in some species in this investigation, which might lead to confusion in further analysis. Therefore ITS sequence is not recommended as a universal barcode for seaweeds identification.     

Microbial diversity in the sediments of the southern Mariana Trench

Microbial diversity in the abyssal sediments beneath the seafloor of 30, 94, and 151 cm near the southern end of the Mariana Trench was analyzed in the Illumina HiSeq 2500 platform. Results show that the microbial populations were dominated by bacteria but merely no archaea were identified at the three depths. In the bacterial community,Proteobacteria and Firmicutes dominated the total taxon tags, followed by Bacteroidetes, Actinobacteria, Planctomycetes, Cyanobacteria, and Chloroflexi, which together account for over 99% of the total population. Similar to that in the seawater in the trench, the operational taxonomic units(OTUs) belonging to Gammaproteobacteria from the sediment samples showed high abundance.However, common bacterial OTUs in the water of the trench including Nitrospirae and Marinimicrobia were hardly found in the sediments from the southern Mariana Trench or the hadal region. Therefore, this study documented for the first time the compositions of microbial diversity in the trench sediments, revealed the difference in microbial diversity in water and sediment of the trench and will enrich the knowledge on the microbial diversity in the abyssal areas.     

Zooplankton community analysis in the Changjiang River estuary by single-gene-targeted metagenomics

DNA barcoding provides accurate identification of zooplankton species through all life stages. Single-gene-targeted metagenomic analysis based on DNA barcode databases can facilitate longterm monitoring of zooplankton communities. With the help of the available zooplankton databases, the zooplankton community of the Changjiang (Yangtze) River estuary was studied using a single-gene-targeted metagenomic method to estimate the species richness of this community. A total of 856 mitochondrial cytochrome oxidase subunit 1 (cox1) gene sequences were determined. The environmental barcodes were clustered into 70 molecular operational taxonomic units (MOTUs). Forty-two MOTUs matched barcoded marine organisms with more than 90% similarity and were assigned to either the species (similarity>96%) or genus level (similarity<96%). Sibling species could also be distinguished. Many species that were overlooked by morphological methods were identified by molecular methods, especially gelatinous zooplankton and merozooplankton that were likely sampled at different life history phases. Zooplankton community structures differed significantly among all of the samples. The MOTU spatial distributions were influenced by the ecological habits of the corresponding species. In conclusion, single-gene-targeted metagenomic analysis is a useful tool for zooplankton studies, with which specimens from all life history stages can be identified quickly and effectively with a comprehensive database.     

Polyphasic examination on Merismopedia tenuissima CHAB 7021 from Ganjiang River,China revealed the polyphyly of the genus Merismopedia(Cyanobacteria)

Species in the cyanobacterial genus Merismopedia are present in freshwaters at different trophic levels, with some species even as the components of cyanobacterial blooms. However, species diversity in this genus was not fully verified by molecular investigation and polyphasic taxonomic studies. In this study, Merismopedia-like strain tenuissima CHAB 7021 was isolated from Ganjiang River in Jiangxi Province, China, and polyphasic characterization of this strain was performed by morphological observation, ultrastructural examination, chemical detection of pigments and phylogenetic analysis based on 16 S rRNA gene sequences. Morphological identification of the strain was supported by the ultrastructural features, as the tiny species Merismopedia tenuissima Lemmermann. The phylogeny based on 16 S rRNA gene sequences revealed at least three clades formed by the strains of Merismopedia. The three M. tenuissima strains including M. tenuissima CHAB 7021 was gathered in clade III with distant relationship to the clade I formed by the six Merismopedia strains including the type species M. punctata, and such a genetic distance may propose Merismopedia tenuissima to separate from Merismopedia genus. However intermixture relationship in between strains of M. punctate and M. glauca in the phylogenetic tree still complicated the taxonomic status in the genus Merismopedia. The process for taxonomic revision in the Merismopedia genus still await for examination and further information on more strains of type species M. punctata.     

侧孢短芽孢杆菌对铜绿微囊藻藻际微生物群落的影响

【目的】探究铜绿微囊藻(Microcystis aeruginosa)藻际微生物群落组成以及与溶藻细菌侧孢短芽孢杆菌(Brevibacillus laterosporus)Bl-zj共培养对藻际微生物群落的影响。【方法】将侧孢短芽孢杆菌Bl-zj与铜绿微囊藻共培养4 d后(BL组),收集样品,以单独培养的铜绿微囊藻(BG组)为对照,提取样品总基因组DNA后进行16S rDNA扩增、构建文库、测序及生物信息学分析,比较BL组与BG组间藻际微生物群落的差异。【结果】测序平均获得高质量序列85390条,946个OUT(Operational Taxonomic Unit),BL组特有OTU 339个。物种组成分析显示,BG组微生物群落主要以微囊藻属(Microcystis)为主,其余相对丰度由高到低分别为假单胞菌属(Pseudomonas)、中慢生根瘤菌属(Mesorhizobium)、短芽孢杆菌属(Brevibacillus)和红杆菌属(Rhodobacter)。BL组微生物群落主要以短芽孢杆菌属(Brevibacillus)为主。BL组的微生物多样性相较于BG组显著下降(P<0.05)。经预测,BG组微生物功能集中在能量代谢与外源生物降解与代谢,BL组微生物功能集中在碳水化合物代谢和氨基酸代谢功能。【结论】侧孢短芽孢杆菌Bl-zj与铜绿微囊藻共培养,打破了铜绿微囊藻藻际微生物群落的平衡,与Bl-zj的溶藻进程有关。     

Dynamic genetic features of eukaryotic plankton diversity in the Nakdong River estuary of Korea

Estuaries are environments where freshwater and seawater mix and they display various salinity profiles.The construction of river barrages and dams has rapidly changed these environments and has had a wide range of impacts on plankton communities.To understand the dynamics of such communities,researchers need accurate and rapid techniques for detecting plankton species.We evaluated the diversity of eukaryotic plankton over a salinity gradient by applying a metagenomics tool at the Nakdong River estuary in Korea.Environmental samples were collected on three dates during summer and autumn of 2011 at the Eulsukdo Bridge at the mouth of that river.Amplifying the 18 S rDNA allowed us to analyze 456 clones and 122 phylotypes.Metagenomic sequences revealed various taxonomic groups and cryptic genetic variations at the intra-and inter-specific levels.By analyzing the same station at each sampling date,we observed that the phylotypes presented a salinity-related pattern of diversity in assemblages.The variety of species within freshwater samples reflected the rapid environmental changes caused by freshwater inputs.Dinophyceae phylotypes accounted for the highest proportion of overall diversity in the seawater samples.Euryhaline diatoms and dinoflagellates were observed in the freshwater,brackish and seawater samples.The biological data for species composition demonstrate the transitional state between freshwater and seawater.Therefore,this metagenomics information can serve as a biological indicator for tracking changes in aquatic environments.     

First record of Pantropical spotted dolphins Stenella attenuata in the Yellow Sea,China

On October 1, 2009, sixteen dolphins were obtained from fishermen by incidental catching in the Yellow Sea, China. As the dolphins' skin color was ambiguous, morphological parameters were measured, and mitochondrial DNA cytochrome b(Cyt b) gene sequence was studied to identify the species. Morphological characteristics were consistent with Pantropical spotted dolphins, S tenella attenuata. Furthermore, a partial mitochondrial DNA cytochrome b(Cyt b) gene sequence as long as 328-bp was studied by extracting genomic DNA from the skins, and six haplotypes were detected in the sixteen dolphins. By comparing homologous sequences available in Gen Bank(www.ncbi.nlm.nih.gov), all the six haplotypes had maximal genetic similarity with Pantropical spotted dolphin. Eight species of cetacean(whales and dolphins) are now recognised in the Yellow Sea. To the best of our knowledge, this is the first record of Pantropical spotted dolphins from this region. Despite this species being listed as a Grade II National Key Protected Animal since 1988, little is known of its biology in Chinese waters. We recommend remedial research be undertaken to ensure appropriate management.     

The loss of genetic diversity during captive breeding of the endangered sculpin, Trachidermus fasciatus, based on ISSR markers: implications for its conservation

Inter-simple sequence repeat (ISSR) markers were used to determine the genetic variation and genetic differentiation of cultured and wild populations of Trachidermus fasciatus, an endangered catadromous fish species in China. Six selected primers were used to amplify DNA samples from 85 individuals, and 353 loci were detected. Relatively low genetic diversity was detected in the cultured population (the percentage of polymorphic loci PPL = 73.80%, Nei’s gene diversity h = 0.178 2, Shannon information index ...     

Temporal and spatial variation of fish assemblages in Dianshan Lake,Shanghai, China

Using multi-mesh gillnets and trawls, the fish communities in Dianshan Lake at 6 stations from Oct. 2009 to Jul. 2010 were investigated seasonally to reveal the biodiversity and its spatial and temporal distribution patterns. The long-term changes in their structural characteristics were then analyzed to identify the main infl uencing factors and several measures for lake restoration were put forward. Thirty six species, belonging to 9 family and 30 genera, were collected, amongst which, the order Cypriniformes accounted for 61.1% of the total species number. In terms of importance value, Cypriniformes was the predominant group, Coilia nasus the dominant species, while Cyprinus carpio and Rhinogobius giurinus were the subdominant taxa. The community types did not differ among stations, but between seasons. There were no significant differences between seasons and among stations in species diversity, but richness differed both spatially and seasonally. Along with the process of eutrophication and the drastic reduction of the area colonized by macrophytes from 1959 to 2009–2010, the fish diversity declined markedly, and species numbers of herbivores and piscivores declined proportionately more than those of invertivores, omnivores, and planktivores. The decline of potamophilus and river-lake migratory fish was more marked than those of sedentary, river-sea migratory, and estuarine fishes. Eutrophication concomitant with sharp reduction of macrophyte area and overfishing may be the main reasons for the decline in fish diversity in Dianshan Lake.     

3种江珧同工酶遗传标记

通过对江珧科的栉江珧、旗江珧及二色裂江珧三物种的 5种组织 (消化盲囊、肾组织、外套膜、后闭壳肌和鳃组织 )进行垂直板状聚丙烯酰胺凝胶电泳分离技术 ,研究江珧科的 SOD、EST同工酶酶谱 ,结果表明 ,不同种间的个体其酶谱表型有稳定的差异 ,同属的种间差异小于不同属的种间差异 ,即酶谱的差异程度与形态分类学中的亲缘关系相关。可利用其酶谱表型具有种特异性作为一种蛋白分子标记 ,应用于江珧物种尤其是经济品种的鉴定上