Re-identifying Grateloupia yangjiangensis(Rhodophyta, Halymeniaceae) based on morphological observations, life history and rbcL sequence analyses

On the basis of morphological observations, life history and molecular phylogeny, Grateloupia yangjiangensis, which is similar to G. filicina, G. orientalis, G. catenata, and G. ramosissima in appearance, was re-examined. The results are as follows：（1） the auxiliary-cell ampullae of G. yangjiangensis were of Grateloupia type, thalli was fleshy and gelatinous in texture, and the erect axes were compressed; the cortex was 0.25–0.30 mm thick, consisting of five to seven outer layers, and there were five inner layers of triangular or stellate cells;（2） there was no filamentous stage in the development of the carpospores;（3） the ribulose-1,5-bisphosphate carboxylase/oxygenase gene（rbcL） sequence of four G. yangjiangensis examined showed that there was no intergeneric divergence among them, and for the phylogenetic tree, four sequences of G. yangjiangensis formed a single monophyletic subclade within the large Grateloupia clade of Halymeniaceae. In conclusion, G. yangjiangensis was a single species within the genus Grateloupia. This research provided criterion for identification and cultivation of G. yangjiangensis.

Diversity and seasonal variation of marine phytoplankton in Jiaozhou Bay, China revealed by morphological observation and metabarcoding

Phytoplankton are central components of marine environments,and are major players in the production and respiration budgeting.However,their diversity and distribution patterns are still poorly understood due largely to their small sizes and inconspicuous morphology that have been determined via the application of traditional morphology methods over the past two decades.To better understand the composition and diversity of phytoplankton in Jiaozhou Bay,China,seasonal sampling was carried out in 2019 and samples were analyzed with morphological observations and high-throughput sequencing,from which obvious seasonal variations in phytoplankton composition and proportional abundances were uncovered.Metabarcoding revealed far more diversity and species richness of phytoplankton than morphological observations,especially with respect to dinofl agellates.Diatoms were the most dominant phytoplankton group throughout the year,of which Thalassionema and Skeletonema were co-dominant in the bay.Parasitic dinofl agellates(e.g.Amoebophrya),which is often overlooked in the morphological observations,were in dominance and high diversity in the metabarcoding dataset,thus more attention should be paid to exploring the potential role of parasitic dinofl agellates.Temperature,chlorophyll a,and nutrient levels were the main infl uential factors on the distribution of phytoplankton.This study provided a comprehensive morphological and molecular description of phytoplankton and clearly demonstrated the importance of molecular technology in exploring phytoplankton communities.More-widespread use of molecular technology will facilitate deeper understanding of the ecological importance of the diff erent species.

Makinoella parva,a new species of the genus Makinoella(Oocystaceae,Trebouxiophyceae,Chlorophyta)

Makinoella Okada is a genus of coccoid green algae belonging to the family Oocystaceae with typical morphological characteristics.Reports to date included only one type species.Since the genus is rarely reported,there is a lack of taxonomic research.In this study,17 strains of this genus were collected from several different places in China,and their taxonomic studies were conducted based on morphological and molecular phylogenetic analysis.The phylogenetic results based on the analysis of ITS,chloroplast genes rbc L and tuf A showed that the genus was divided into two branches.One of the branches comprised newly collected algae.Compensatory base changes(CBCs)and hemi-compensatory base changes(hemi-CBCs)within the secondary structure of the ITS-2 confirmed the branches as two species.Compared with the type species of Makinoella tosaensis,the cell size of this new branch was only about 50%,and included 2-4 colonial cells.Therefore,based on the smaller cell size,simpler colony composition,independent phylogenetic position and CBCs and hemi-CBCs,we suggest that the new clade should be designated as a new taxon of Makinoella,namely Makinoella parva.Among the four molecular markers using in this study,the rbc L and newly introduced ITS are recommended for species separation,which can help further studies to revise the species and generic concepts of the family.

Primary culture and identification about brain microvascular endothelial cells of rabbits

Objective:To establish a simple and efficient culture method of primary rabbit brain microvascular endothelial cells,provide important carriers and tool cells for the research of related cerebrovascular diseases.Methods:The cerebral cortexes of rabbits were collected aseptic and inoculated after cutting,passing through cell sieve,bovine serum albumin density gradient centrifugation,typeⅡcollagenase digestion,finally inoculated and cultured.The cultured cells were identified by cell morphological observation and angiogenesis experiment.Results:Under the inverted microscope,the cells were short fusiform or polygonal,and grew in clusters and adhere to the wall.After the cells were densely fused,they would be in a typical monolayer flat,“pebbled"mosaic arrangement.Tube formation test had the ability to form tubes structure.Conclusion:This method can successfully separate and cultivate primary rabbit brain microvascular endothelial cells.

Dynamical changing patterns of histological structure and ultrastructure of liver graft undergoing warm ischemia injury from non-heart-beating donor in rats

AIM： To investigate the histological and ultra-structural characteristics of liver graft during different of warm ischemia time （WIT） in rats and to predict the maximum limitation of liver graft to warm ischemia. METHODS： The rats were randomized into 7 groups undergoing warm ischemia injury for 0, 10, 15, 20, 30, 45 and 60 min, respectively. All specimens having undergone warm ischemia injury were investigated dynamically by light and electron microscopy, and histochemistry staining. After orthotopic liver transplantation （OLT）, the recovery of morphology of liver grafts after 6, 24 and 48 h was observed. RESULTS： The donor liver from non-heart-beating donors （NHBD） underwent ischemia injury both in the warm ischemia period and in the reperfusion period. Morphological changes were positively related to warm ischemia injury in a time-dependent manner during the reperfusion period. The results demonstrated that different degrees of histocyte degeneration were observed when WIT was within 30 min, and became more severe with the prolongation of WIT, no obvious hepatocyte necrosis was noted in any specimen. In the group undergoing warm ischemia injury for 45 min, small focal necrosis occurred in the central area of hepatic Iobule first. In the group undergoing warm ischemia injury for 60 rain, patchy or diffused necrosis was observed and the area was gradually extended, while hepatic sinusoid endothelial cells were obviously swollen. Hepatic sinusoid was obstructed and microcirculation was in disorder.CONCLUSION： The rat liver graft undergoing warm ischemia injury is in the reversible stage when the WIT is within 30 min. The 45 min WIT may be a critical point of rat liver graft to endure warm ischemia injury. When the WIT is over 60 min, the damage is irreversible.

Phenotypic plasticity and taxonomy of Cladophora gracilis(Griffiths)Kützing(Cladophorales,Chlorophyta)in the western Yellow Sea with the implication of its DNA barcode

Due to extensive morphological plasticity,the taxonomy of Cladophora species remains unclear.As one of the widely distributed species,C.gracilis was reported to hold many varieties,which make it difficult to identify the species.This study explored the morphology diversity of nine C.gracilis samples collected along the coast of western Yellow Sea.Some samples showed extremely varied characteristics,by which one cannot classify them correctly.Hence,18S rDNA and nuclear ribosomal DNA internal transcribed spacer regions(ITS)sequences were employed to delimit species.For 18S rDNA,sequence similarity ranged from 99.6%to 100%.For ITS region,the similarity ranged from 98.7%to 100%.Molecular data strongly suggested that the morphologically heterogeneous samples were actually the same species.Characteristics comparison of the samples revealed that the taxonomy criteria including branching pattern and density,thallus color,height and texture varied widely,influenced by environmental conditions and age of alga.Besides,cell dimensions,as the relatively stable criterion,also exhibited intraspecific variance.Successful application of 18S rDNA and ITS sequences indicated that molecular method can be a powerful assistant as DNA barcodes to traditional morphology taxonomy.