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.     

Sequence analysis and quantitative detection of Norwalk-like viruses in cultured oysters of China

We isolated 4 Norwalk-like viruses (NLVs) contaminated oysters from 33 Chinese oysters collected from local commer-cial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase (RdRp) region of NLVs genomes with RT-PCR, the open reading frame 1 (ORF1) of the RdRp was sequenced and subjected to multiple-sequence alignment. The re-suits showed that NLVs in the four isolates belong to genogroup Ⅱ. The sequence comparison showed that the similarity between four Chinese oyster isolates were higher than 99.0%, which indicated that NLVs prevalent in close areas have high homogeneity in genome sequences. In addition, the most conserved sequences between diverse NLVs were used to design primers and TaqMan probes, then the real-time quantitative PCR assay was performed. According to the standard curve of GII NLVs, the original amounts (copies) of NLVs in positive patient's fecal isolate, positive Japanese oyster isolate, and the Chinese oyster isolate were 8.9×108, 1.25×108 and 4.7×101 respectively. The detecting limit of NLVs was 1×101 copies. This study will be helpful for routine diagnosis of NLVs pathogens in foods and thus for avoiding food poisoning in the future.     

Development of a Label-Free Colorimetric Aptasensor with Rationally Utilized Aptamer for Rapid Detection of Okadaic Acid

Okadaic acid(OA)is a typical marine toxin with strong toxicity causing diarrheic shellfish poisoning(DSP).Aptamers show great advantages in toxin detection and attract increasing attentions in the field of food analysis.In this study,a label-free col-orimetric aptasensor was constructed for visual and rapid detection of OA in shellfish.To exploit the binding capability of the anti-OA aptamer,the inherent molecular recognition mechanism of aptamer and OA was studied,based on molecular docking,fluorescent assay,and biolayer interferometry.Consistent results showed that the stem-loop near the 3’terminal of the aptamer exhibit dominate binding capacity.Based on the revealed recognition information,the aptamer was thus rationally utilized and combined with AuNPs and cationic polymer polydiallyl dimethyl ammonium chloride(PDDA)for the development of the label-free colorimetric aptasensor,in which the 3’terminal was thoroughly exposed to OA.The aptasensor provided robust performance with a linear detection range of 100-1200 nmol L-1,a limit of detection of 41.30 nmol L-1,recovery rates of 91.6%-106.2%,as well as a high selectivity towards OA in shellfish samples.The whole detection process can be completed within 1 h.To our best knowledge,this is the first time that the anti-OA aptamer was thoroughly studied,and a label-free colorimetric aptasensor was rationally designed in this way.This study not only provides a rapid detection method for highly sensitive and specific detection of OA,but also serves as a reference for the design of efficient aptasensors in the future.     

Development and validation of 89 novel expressed sequence tag-derived microsatellite markers in blood clam, <Emphasis Type="Italic">Tegillarca granosa</Emphasis>

Blood clam, Tegillarca granosa, is an important shellfish in Chinese mariculture industry. Investigative research in this species, such as genetic linkage mapping, requires a large panel of molecular markers. In present study, a total of 89 polymorphic microsatellite markers were developed in T. granosa using the sequence database of Life Sciences Technology 454 next generation sequencing technology. All 89 loci were characterized in 20 individual clams from a natural population inhabiting Yueqing Gulf, Zhejiang Province, China. The number of alleles per polymorphic locus varied between 2 and 15, while the observed heterozygosity, expected heterozygosity and polymorphic information content varied between 0.000 and 1.000, 0.102 and 0.921, and 0.048 and 0.886, respectively. Of the 89 loci identified, 32 loci deviated significantly from Hardy-Weinberg equilibrium following Bonferroni correction. Thirty nine markers, which were shown to be polymorphic in a full-sibling family, were tested in Mendelian segregations. As expected, 32 loci were co-dominantly segregated in a Mendelian fashion. These novel developed microsatellite markers represent useful research tools for investigation of population genetic structure and genetic diversity in this species.     

A new identification method for five species of oysters in genus Crassostrea from China based on high-resolution melting analysis

The high phenotypic plasticity in the shell of oysters presents a challenge during taxonomic and phylogenetie studies of these economically important bivalves. However, because DNA can exhibit marked differences among morphologically similar species, DNA barcoding offers a potential means for oyster identification. We analyzed the complete sequences of the cytochrome oxidase subunit I （COI） of five common Crassostrea species in China （including Hong Kong oyster C. hongkongensis, Jinjiang oyster C. ariakensis, Portuguese oyster C. angulata, Kumamoto oyster C. sikamea, and Pacific oyster C. gigas） and screened for distinct fragments. Using these distinct fragments on a high-resolution melting analysis platform, we developed an identification method that does not rely on species-specific PCR or fragment length polymorphism and is efficient, reliable, and easy to visualize. Using a single pair of primers （Oyster- COI-1）, we were able to successfully distinguish among the five oyster species. This new method provides a simple and powerful tool for the identification of oyster species.     

方斑东风螺两种养殖模式的比较

在室内水泥池,利用沙层自净养殖模式和直接铺沙养殖模式对不同规格的方斑东风螺(Babylonia areolataLink)进行了高密度养殖的研究。结果表明,沙层自净养殖模式养殖小螺、中螺、大螺组日均增重分别为0.031、0.088、0.098 g/d,沙层NH4+-N最高含量w分别为1.3、2.1、3.1 mg/L,H2S最高含量w分别为0.03、0.07、0.14mg/L,各规格组东风螺保持正常生长和活动,成活率92.9%以上;直接铺沙养殖模式养殖小螺、中螺、大螺日均增重分别为:0.023、0.051、0.068 g/d,成活率分别为95.2%、86.7%、84.9%,沙层NH4+-N最高含量w达到13.7mg/L,H2S最高含量w达到0.47 mg/L,沙层底质恶化,东风螺活动异常、不摄食。可见,沙层自净养殖模式对方斑东风螺的生长、成活率、沙层水质控制效果显著,在一定程度上克服了直接铺沙养殖底质恶化问题。     

Optimization of the purification methods for recovery of recombinant growth hormone from Paralichthys olivaceus

This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone (r-fGH) was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield (2.605 mgL-1) than that from soluble protein (1.964 mgL-1). Of the tested recovery methods, addition of renaturing buffer (pH 8.5) into denatured inclusion body yielded the best recovery rate (17.9%). This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture.     

Effect of substrate component on the growth and survival of juvenile sunray surf clam (<Emphasis Type="Italic">Mactra chinensis</Emphasis> Philippi)

Substrate is a critical environmental factor affecting the activity of bivalves. To examine the effect of the substrate component on the growth and survival of juvenile sunray surf clam(Mactra chinensis Philippi), a series of short-term experiments were conducted using a variety of substrates with different ratios of sand to mud. The experimental group cultured without substrate showed poor survival, with all juveniles died after day 20. The juveniles cultured in mud without sand showed a lower survival rate(25.54% ± 0.40% on day 45) than those in other groups. The juveniles cultured in sand without mud, or the mixtures of sand and mud with a ratio of 1:1 and 2:1, respectively, exhibited modest survival at day 45. Maximal weight gain rate(WGR), shell length growth rate(LGR), and specific growth rate(SGR) were observed when the sand concentration was 61.97%, 77.69%, and 64.64%, respectively. As the fast growth and high survival were observed when the sand to mud ratio was 1:1(50% sand) and 2:1(67% sand), a sand concentration of more than 50% is optimal. The optimal concentration of sand in the substrate for rearing juvenile sunray surf clams was 67% which resulted in the fastest growth and highest survival. These results can be used to developing a nursery/farming technique of improving the yield of sunray surf clams.     

企鹅珍珠贝附壳珍珠的培育

以7种聚乙烯塑料核型为珠核,正常壳形与二壳隆起的畸形企鹅珍珠贝为手术贝,培育附壳珍珠。养殖6个月,结果表明:壳形正常的育珠贝成活率为73.3%～93.8%,成珠率55.0%～82.0%;畸形贝成活率为60%,成珠率53.3%。统计分析表明:珠核高度是影响育珠贝成活率和成珠率的主要因素,珠核越高,育珠贝成活率和成珠率越低。畸形贝与正常壳形贝成活率和成珠率存在极显著差异(P<0.01),畸形贝不宜用作手术贝。     

Warming hiatus and evergreen conifers in Altay-Sayan Region,Siberia

"Warming hiatus" occurred in the AltaySayan Mountain Region, Siberia in c. 1997–2014. We analyzed evergreen conifer(EGC) stands area(satellite data) and trees(Siberian pine, Pinus sibirica Du Tour, Siberian fir, Abies sibirica Ledeb.) growth increment(dendrochronology data) response to climate variables before and during the hiatus. During the hiatus, EGC area increased in the highlands(1000 m)(+30%), whereas at low and middle elevations(1000 m. a.s.l.) the EGC area decreased(-7%). The EGC area increase was observed on the rain-ward northwest slopes mainly. In highlands, EGC area increase mainly correlated with summer air temperature, whereas at low and middle elevations EGC area decrease correlated with drought index SPEI and vapor pressure deficit(VPD). EGC mortality(fir and Siberian pine) in lowland was caused by the synergy of water stress(inciting factor) and barkbeetle attacks(contributing factor). Tree growth increment(GI) dynamics differs with respect to elevation. At high elevation(1700 m) GI permanently increased since warming onset, whereas at the middle(900 m) and low elevations(450 m) GI increased until c. 1983 yr. with followed depression. That GI "breakpoint" occurred about a decade before hiatus onset. In spite of growth depression, during hiatus GI was higher than that in pre-warming period. At high elevation, GI positively responded to elevated June temperatures and negatively to moisture increase(precipitation, root zone moisture content, VPD, and SPEI). At low elevation GI negatively responded to June temperatures and positively to moisture increase. For both, low and high elevation, these patterns persisted throughout the study period(1967–2014). On the contrary, at middle elevations GI dependence on climate variables switch after breakpoint year(1983). Before breakpoint, June air temperature(positive correlation) and moisture(negative correlations) controlled GI. Further temperature increase leads GI depression and switched correlation signs to opposite(from positive to negative with temperature, and from negative to positive with moisture variables).     

Coral reef recovery status in south Andaman Islands after the bleaching event 2010

The Andaman and Nicobar Islands are one of the Union Territories of India, located in the eastern part of the Bay of Bengal. In 2010 summer, the increment in sea surface water temperature (up to 34℃) resulted in the bleaching of about 74% to 77% of corals in the South Andaman. During this event, coral species such as Acropora cerealis, A. humilis, Montipora sp., Favia pallida, Diploastrea sp., Goniopora sp. Fungia concinna, Gardineroseries sp., Porites sp., Favites abdita and Lobophyllia robusta were severely affected. This study is to assess the recovery status of the reef ecosystem by estimating the percentage of Live Coral cover, Bleached coral cover, Dead coral with algae, Rubble, Sandy flat, Algal assemblage and other associated organisms. The sedimentation rate (mg cm-2 d-1) and coral coverage (%) were assessed during this study period. The average sedimentation rate was ranged between 0.27 and 0.89 mg cm-2 d-1 . The observed post bleaching recovery of coral cover was 21.1% at Port Blair Bay and 13.29% at Havelock Island. The mortality rate of coral cover due to this bleaching was estimated as 2.05% at Port Blair Bay and 9.82% at Havelock Island. Once the sea water temperature resumed back to the normal condition, most of the corals were found recovered.     

引进印尼种大珠母贝繁育F1母贝植核育珠研究

以印度尼西亚引进大珠母贝亲本繁育的子代F1群体进行植核育珠,育珠贝数量8086只,在海区休养20d后,统计育珠母贝的成活率与留核率,比较3月与4月植核手术育珠贝的育珠性状差异。结果表明：休养期结束后,育珠贝的成活率与留核率分别为97.57％和90.69％;育珠期3个月时（6月17日）育珠贝的成活率为81.22％;4月植核育珠母贝的留核率与成活率明显高于在3月的植核育珠母贝,但两者之间差异不具统计学意义（P〉0.05）。     

钦州湾牡蛎线粒体16S rRNA和COⅠ基因片段的序列变异分析

利用线粒体16S rRNA基因和线粒体细胞色素氧化酶亚基Ⅰ(cytochrome oxidaseⅠ,COⅠ)基因片段初步研究钦州湾牡蛎(Ostrea)的物种组成。以特异引物进行PCR扩增,对扩增产物进行纯化、测序,分析表明,16S rRNA基因部分长度为413bp,COⅠ基因部分长度为535bp,2种牡蛎序列的碱基组成均显示出较高的A+T比例:16S rRNA基因59.8%;COⅠ基因60.5%。对位排序比较表明,16S rRNA片段种内个体间变异较小,存在7个变异位点,4种单倍型,其中包括5个转换位点突变,2个颠换位点突变;COⅠ片段有30个碱基存在变异,7种单倍型,其中包括16个转换位点突变,14个颠换位点突变。运用MEGA4软件计算出不同个体间的遗传距离,并构建了NJ和UPGMA系统树。香港牡蛎(Crassostrea hongkongensis)16S rRNA和COⅠ序列与白肉牡蛎的遗传距离均为0.000,有明巨牡蛎(Crassostrea ariakensis)16S rRNA和COⅠ序列和红肉牡蛎(red meat ostrea)的遗传距离分别为0.000和0.011,白肉牡蛎16S rRNA和COⅠ序列和红肉牡蛎之间的遗传距离分别为0.035和0.146。结果表明,钦州湾牡蛎分为2个不同的种,线粒体16S rRNA和COⅠ基因在种间存在明显的多态性,证实了16S rRNA和COⅠ基因序列适用于牡蛎的系统学分析。     

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.

     

钦州湾牡蛎线粒体16SrRNA和COI基因片段的序列变异分析

利用线粒体16SrRNA基因和线粒体细胞色素氧化酶亚基Ⅰ（cvtocllrome oxidase Ⅰ,COI）基因片段初步研究钦州湾牡蛎（Ostxea）的物种组成。以特异引物进行PCR扩增,对扩增产物进行纯化、测序,分析表明,16SrRNA基因部分长度为413bp,COI基因部分长度为535bp,2种牡蛎序列的碱基组成均显示出较高的A＋T比例：16SrRNA基因598％;COI基因605％。对位排序比较表明,16SrRNA片段种内个体间变异较小,存在7个变异位点,4种单倍型,其中包括5个转换位点突变,2个颠换位点突变;COI片段有30个碱基存在变异,7种单倍型,其中包括16个转换位点突变,14个颠换位点突变。运用MEGA4软件计算出不同个体间的遗传距离,并构建了NJ和UPGMA系统树。香港牡蛎（Crassostrea hongkongensis）16SrRNA和COI序列与白肉牡蛎的遗传距离均为0000,有明巨牡蛎（Crassostrea ariakensis）16SrRNA和COI序列和红肉牡蛎（redmeatostxea）的遗传距离分别为0000和0011,白肉牡蛎16SrRNA和COI序列和红肉牡蛎之间的遗传距离分别为0035和0146。结果表明,钦州湾牡蛎分为2个不同的种,线粒体16SrRNA和COI基因在种间存在明显的多态性,证实了16SrRNA和COI基因序列适用于牡蛎的系统学分析。     

Effect of cadmium on the defense response of Pacific oyster Crassostrea gigas to Listonella anguillarum challenge

Heavy metal pollution can affect the immune capability of organisms.We evaluated the effect of cadmium(Cd) on the defense responses of the Pacific oyster Crassostrea gigas to Listonella anguillarum challenge.The activities of several important defensive enzymes,including superoxide dismutase(SOD),glutathione peroxidase(GPx),acid phosphatase(ACP),Na+,K+-ATPase in gills and hepatopancreas,and phenoloxidase-like(POL) enzyme in hemolymph were assayed.In addition,the expression levels of several genes,including heat shock protein 90(HSP90),metallothionein(MT),and bactericidal/permeability increasing(BPI) protein were quantified by fluorescent quantitative PCR.The enzyme activities of SOD,ACP,POL,and GPx in hepatopancreas,and the expression of HSP90 were down-regulated,whereas GPx activity in the gill,Na+,K+-ATPase activities in both tissues,and MT expression was increased in Cdexposed oysters post L.anguillarum challenge.However,BPI expression was not significantly altered by co-stress of L.anguillarum infection and cadmium exposure.Our results suggest that cadmium exposure alters the oysters’ immune responses and energy metabolism following vibrio infection.     

Study on elemental fingerprint of traditional marine Chinese medicine oysters from Jiaozhou Bay, China

In order to investigate the relationship between the trace elements and the characteristics of the oysters, we analyzed the trace elements present in the germplasm of oysters from different producing areas in the Jiaozhou Bay. The element fingerprints were established to reflect the elemental characteristics of the oysters. Concentration patterns of the elements were deciphered by principle component analysis (PCA) and hierarchical cluster analysis (HCA). The six regions were discriminated with accuracy using HCA and PCA based on the concentration of 16 trace elements. The elements were viewed as characteristic elements of the oysters and the fingerprints of these elements could be used to distinguish the quality of the oysters.     

Development and Validation of 89 Novel Expressed Sequence Tag-Derived Microsatellite Markers in Blood Clam,Tegillarca granosa

Blood clam, Tegillarca granosa, is an important shellfish in Chinese mariculture industry. Investigative research in this species, such as genetic linkage mapping, requires a large panel of molecular markers. In present study, a total of 89 polymorphic microsatellite markers were developed in T. granosa using the sequence database of Life Sciences Technology 454 next generation sequencing technology. All 89 loci were characterized in 20 individual clams from a natural population inhabiting Yueqing Gulf, Zhejiang Province, China. The number of alleles per polymorphic locus varied between 2 and 15, while the observed heterozygosity, expected heterozygosity and polymorphic information content varied between 0.000 and 1.000, 0.102 and 0.921, and 0.048 and 0.886, respectively. Of the 89 loci identified, 32 loci deviated significantly from Hardy-Weinberg equilibrium following Bonferroni correction. Thirty nine markers, which were shown to be polymorphic in a full-sibling family, were tested in Mendelian segregations. As expected, 32 loci were co-dominantly segregated in a Mendelian fashion. These novel developed microsatellite markers represent useful research tools for investigation of population genetic structure and genetic diversity in this species.     

Multiple excitation wavelength fluorescence emission spectra technique for discrimination of phytoplankton

In vivo fluorescence methods are efficient tools for studying the distribution of phytoplankton in nature.Different algae species usually have different pigments with different ratios,which results in different fluorescence emission spectra.Based on multiple excitation wavelength fluorescence emission spectra,a discrimination technique is established in this study.The discrimination method,established by multivariate linear regression and weighted least-squares,was used to differentiate the samples cultured in the laboratory and collected from Jiaozhou Bay near Qingdao at the division level.The correctly discriminated samples were ≥ 86% for single algae samples,≥ 88% for simulatively mixed ones,≥ 91% for physically mixed ones and 100% for samples collected from Jiaozhou Bay.The result in this research is more definite for the physically mixed samples in the laboratory.The method described here can be employed to monitor the phytoplankton population in the marine environment.