Visualization of Literature Information on Postmortem Interval Estimation Indexed by CNKI Database from 1990 to 2020.

OBJECTIVES: To explore the development process of the postmortem interval (PMI) research in China from January 1990 to August 2020, research hotspots in different periods, authors and cooperation between institutions, and to provide a reference for the better development of PMI inference research through the visualization of the literature information of the PMI estimation research indexed in China National Knowledge Infrastructure (CNKI). METHODS: The information visualization analysis software CiteSpace 5.7.R1 was used to carry out big data analysis on hotspots, high-frequency keywords, authors, institutions and other information in the research literature on PMI inference from January 1990 to August 2020 indexed in CNKI. RESULTS: The peak time of publication of PMI was from 2006 to 2010 with 114 articles. In keyword co-occurrence network, the effective hot words were forensic entomology, DNA content analysis and some emerging words such as artificial intelligence and big data. In the cooperation network of institutions, the high-frequency institutions were mainly the scientific research institutions. The author cooperation network showed a trend of co-aggregation and multi-cooperation. CONCLUSIONS: With the development of science and technology, the research on PMI estimation based on traditional methods is mature and novel strategies are emerging. Big data and artificial intelligence combined with forensic science provide new research directions on PMI estimation.

Metatranscriptome analysis reveals environmental and diel regulation of a Heterosigma akashiwo (raphidophyceae) bloom.

Despite numerous laboratory studies on physiologies of harmful algal bloom (HAB) species, physiologies of these algae during a natural bloom are understudied. Here, we investigated a bloom of the raphidophyte Heterosigma akashiwo in the East China Sea in 2014 using metabarcode (18S rDNA) and metatranscriptome sequencing. Based on 18S rDNA analyses, the phytoplankton community shifted from high diversity in the pre-bloom stage to H. akashiwo predominance during the bloom. A sharp decrease in ambient dissolved inorganic phosphate and strong up-regulation of phosphate and dissolved organic phosphorus (DOP) uptake genes, including the rarely documented (ppGpp)ase, in H. akashiwo from pre-bloom to bloom was indicative of rapid phosphorus uptake and efficient utilization of DOP that might be a driver of the H. akashiwo bloom. Furthermore, observed up-regulated expression of mixotrophy-related genes suggests potential contribution of mixotrophy to the bloom. Accelerating photosynthetic carbon fixation was also implied by the up-regulation of carbonic anhydrase genes during the bloom. Notably, we also observed a strong morning-to-afternoon shift in the expression of many genes. Our findings provide insights into metabolic processes likely important for H. akashiwo bloom formation, and suggest the need to consider timing of sampling in field studies on this alga.

Transcriptomic and physiological analyses of the dinoflagellate Karenia mikimotoi reveal non-alkaline phosphatase-based molecular machinery of ATP utilisation.

The ability to utilize dissolved organic phosphorus (DOP) is important for phytoplankton to survive the scarcity of dissolved inorganic phosphorus (DIP), and alkaline phosphatase (AP) has been the major research focus as a facilitating mechanism. Here, we employed a unique molecular ecological approach and conducted a broader search for underpinning molecular mechanisms of adenosine triphosphate (ATP) utilisation. Cultures of the dinoflagellate Karenia mikimotoi were set up in L1 medium (+P), DIP-depleted L1 medium (-P) and ATP-replacing-DIP medium (ATP). Differential gene expression was profiled for ATP and +P cultures using suppression subtractive hybridisation (SSH) followed by 454 pyrosequencing, and RT-qPCR methods. We found that ATP supported a similar growth rate and cell yield as L1 medium and observed DIP release from ATP into the medium, suggesting that K. mikimotoi cells were expressing extracellular hydrolases to hydrolyse ATP. However, our SSH, qPCR and enzymatic activity assays indicated that 5'-nucleotidase (5NT), rather than AP, was responsible for ATP hydrolysis. Further gene expression analyses uncovered that intercellular purine metabolism was significantly changed following the utilisation of ATP. Our findings reveal a multi-faceted machinery regulating ATP utilisation and P metabolism in K. mikimotoi, and underscore AP activity is not the exclusive indicator of DOP utilisation.

High throughput sequencing of 18S rRNA and its gene to characterize a Prorocentrum shikokuense (Dinophyceae) bloom.

Comparing phytoplankton non-bloom and bloom communities using rRNA and its coding gene can help understand the shift of dominant species and its driving processes (e.g., intrinsic growth or grazing). Here we conducted high-throughput sequencing of 18S rRNA and its coding gene for studying non-bloom and bloom plankton communities in East China Sea. The non-bloom community was dominated by diatoms whereas during the bloom it was dominated by the dinoflagellate Prorocentrum shikokuense (formerly P. donghaiense). P. shikokuense rRNA abundance and rRNA:rRNA gene ratio both increased markedly in the bloom, indicating that the bloom arose from active growth. In contrast, some non-bloom species showed low DNA abundances during the bloom albeit high rRNA:rRNA gene ratios, suggesting that cell loss processes such as grazing might have prevented these species from blooming or that these species might be at an early stage of bloom development. Furthermore, Pearson's correlation analysis showed that dinoflagellate abundance was positively correlated with temperature and negatively related to dissolved inorganic phosphate (DIP) concentrations, suggesting warm and DIP-poor environment as a niche space for P. shikokuense. Our results demonstrate the usefulness of combined analysis of rRNA and its gene in characterizing phytoplankton bloom development to shed light on the complex phytoplankton dynamics and regulating mechanisms in the course of bloom development.

Metatranscriptomic Signatures Associated With Phytoplankton Regime Shift From Diatom Dominance to a Dinoflagellate Bloom.

Diatoms and dinoflagellates dominate coastal marine phytoplankton communities as major players of marine biogeochemical cycles and their seasonal succession often leads to harmful algal blooms (HABs). What regulates their respective dominances and the development of the HABs remains elusive. Here we conducted time-sequential metatranscriptomic profiling on a natural assemblage that evolved from diatom dominance to a dinoflagellate bloom to interrogate the underlying major metabolic and ecological drivers. Data reveals similarity between diatoms and dinoflagellates in exhibiting high capacities of energy production, nutrient acquisition, and stress protection in their respective dominance stages. The diatom-to-dinoflagellate succession coincided with an increase in turbidity and sharp declines in silicate and phosphate availability, concomitant with the transcriptomic shift from expression of silicate uptake and urea utilization genes in diatoms to that of genes for light harvesting, diversified phosphorus acquisition and autophagy-based internal nutrient recycling in dinoflagellates. Furthermore, the diatom-dominant community featured strong potential to carbohydrate metabolism and a strikingly high expression of trypsin potentially promoting frustule building. In contrast, the dinoflagellate bloom featured elevated expression of xanthorhodopsin, and antimicrobial defensin genes, indicating potential importance of energy harnessing and microbial defense in bloom development. This study sheds light on mechanisms potentially governing diatom- and dinoflagellate-dominance and regulating bloom development in the natural environment and raises new questions to be addressed in future studies.

Glyphosate Shapes a Dinoflagellate-Associated Bacterial Community While Supporting Algal Growth as Sole Phosphorus Source.

Glyphosate is a widely used herbicide that can potentially be a phosphorus (P) source for phytoplankton and microbes when discharged into the coastal ocean. In contrast to bacteria, few eukaryotic phytoplankton species appear capable of directly utilizing glyphosate. In this study, we observed, after a long delay (>60 days), Prorocentrum donghaiense, a dinoflagellate known to cause major harmful algal blooms in the East China Sea, could grow in a medium with glyphosate as the sole P source; suggesting that P. donghaiense growth was through bacterial mediation. To understand how the bacteria community might respond to glyphosate, we analyzed the 16S rRNA genes of the microbial community present in P. donghaiense cultures when grown under lower (36 µM) and higher (360 µM) glyphosate concentrations. Based on both Sanger and Illumina high throughput sequencing, we obtained more than 55,323 good-quality sequences, which were classified into six phyla. As the concentration of glyphosate rose, our results showed a significant increase in the phyla Proteobacteria and Firmicutes and a decrease in the phylum Bacteroidetes. Further qPCR (Quantitative PCR) analysis showed higher abundances of two specific phylotypes in the higher-glyphosate P. donghaiense cultures when compared to the lower-glyphosate and no-glyphosate cultures. Correspondingly, qPCR displayed the same trend for the abundance of a gammaproteobacterial type of phnJ, a gene encoding Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P lyase, which is responsible for phosphonate degradation. In addition, Tax4Fun analysis based on our 16S rRNA gene sequences results in higher predicted abundances of phosphonate metabolizing genes in glyphosate-treated cultures. This study demonstrates that glyphosate could selectively promote the growth of particular groups of bacteria within an algal culture and in glyphosate enriched coastal waters, this interaction may potentially further facilitate the growth of alga.

Screening for Suitable Reference Genes for Quantitative Real-Time PCR in Heterosigma akashiwo (Raphidophyceae).

The raphidophyte Heterosigma akashiwo is a globally distributed harmful alga that has been associated with fish kills in coastal waters. To understand the mechanisms of H. akashiwo bloom formation, gene expression analysis is often required. To accurately characterize the expression levels of a gene of interest, proper reference genes are essential. In this study, we assessed ten of the previously reported algal candidate genes (rpL17-2, rpL23, cox2, cal, tua, tub, ef1, 18S, gapdh, and mdh) for their suitability as reference genes in this species. We used qRT-PCR to quantify the expression levels of these genes in H. akashiwo grown under different temperatures, light intensities, nutrient concentrations, and time points over a diel cycle. The expression stability of these genes was evaluated using geNorm and NormFinder algorithms. Although none of these genes exhibited invariable expression levels, cal, tub, rpL17-2 and rpL23 expression levels were the most stable across the different conditions tested. For further validation, these selected genes were used to normalize the expression levels of ribulose-1, 5-bisphosphate carboxylase/oxygenase large unite (HrbcL) over a diel cycle. Results showed that the expression of HrbcL normalized against each of these reference genes was the highest at midday and lowest at midnight, similar to the diel patterns typically documented for this gene in algae. While the validated reference genes will be useful for future gene expression studies on H. akashiwo, we expect that the procedure used in this study may be helpful to future efforts to screen reference genes for other algae.

The Symbiodinium kawagutii genome illuminates dinoflagellate gene expression and coral symbiosis.

Dinoflagellates are important components of marine ecosystems and essential coral symbionts, yet little is known about their genomes. We report here on the analysis of a high-quality assembly from the 1180-megabase genome of Symbiodinium kawagutii. We annotated protein-coding genes and identified Symbiodinium-specific gene families. No whole-genome duplication was observed, but instead we found active (retro)transposition and gene family expansion, especially in processes important for successful symbiosis with corals. We also documented genes potentially governing sexual reproduction and cyst formation, novel promoter elements, and a microRNA system potentially regulating gene expression in both symbiont and coral. We found biochemical complementarity between genomes of S. kawagutii and the anthozoan Acropora, indicative of host-symbiont coevolution, providing a resource for studying the molecular basis and evolution of coral symbiosis.