Simultaneous Detection of Viability and Concentration of Microalgae Cells Based on Chlorophyll Fluorescence and Bright Field Dual Imaging.
ABSTRACT: Ship ballast water contains high concentration of plankton, bacteria, and other microorganisms. If the huge amount of ballast water is discharged without being inactivated, it will definitely spell disaster to the marine environment. Microalgae is the most common species exiting in ballast water, so the detection of the concentration and viability of microalgae is a very important issue. The traditional methods of detecting microalgae in ballast water were costly and need the help of bulky equipment. Herein, a novel method based on microalgae cell intracellular chlorophyll fluorescence (CF) imaging combines with cell bright field (BF) microscopy was proposed. The geometric features of microalgae cells were obtained by BF image, and the cell viability was obtained by CF image. The two images were fused through the classic image registration algorithm to achieve simultaneous detection of the viability and concentration of microalgae cells. Furthermore, a low-cost, miniaturized CF/BF microscopy imaging prototype system based on the above principles was designed. In order to verify the effectiveness of the proposed method, four typical microalgae in ballast water (Platymonas, Pyramimonas sp., Chrysophyta, and Prorocentrum lima) were selected as the samples. The experimental results show that the self-developed prototype can quickly and accurately determine the concentration and the viability of microalgae cells in ship ballast water based on the dual images of BF and CF, and the detection accuracy is equivalent to that of commercial microscope. It was the first time to simultaneously detect the viability and concentration of microalgae cells in ship ballast water using the method that combining the fluorescence and bright field images; moreover, a miniaturized microscopic imaging prototype was developed. Those findings expected to contribute to the microalgae detection and ship ballast water management.
Project description:Biological invasions from ballast water are a severe environmental threat and exceedingly costly to society. We identify global hot spots of invasion based on worldwide patterns of ship traffic. We then estimate the rate of port-to-port invasion using gravity models for spatial interactions, and we identify bottlenecks to the regional exchange of species using the Ford-Fulkerson algorithm for network flows. Finally, using stochastic simulations of different strategies for controlling ballast-water introductions, we find that reducing the per-ship-visit chance of causing invasion is more effective in reducing the rate of biotic homogenization than eliminating key ports that are the epicentres for global spread.
Project description:The introduction and establishment of nonindigenous species (NIS) through global ship movements poses a significant threat to marine ecosystems and economies. While ballast-vectored invasions have been partly addressed by some national policies and an international agreement regulating the concentrations of organisms in ballast water, biofouling-vectored invasions remain largely unaddressed. Development of additional efficient and cost-effective ship-borne NIS policies requires an accurate estimation of NIS spread risk from both ballast water and biofouling. We demonstrate that the first-order Markovian assumption limits accurate modeling of NIS spread risks through the global shipping network. In contrast, we show that higher-order patterns provide more accurate NIS spread risk estimates by revealing indirect pathways of NIS transfer using Species Flow Higher-Order Networks (SF-HON). Using the largest available datasets of non-indigenous species for Europe and the United States, we then compare SF-HON model predictions against those from networks that consider only first-order connections and those that consider all possible indirect connections without consideration of their significance. We show that not only SF-HONs yield more accurate NIS spread risk predictions, but there are important differences in NIS spread via the ballast and biofouling vectors. Our work provides information that policymakers can use to develop more efficient and targeted prevention strategies for ship-borne NIS spread management, especially as management of biofouling is of increasing concern.
Project description:The impact of NaOH as a ballast water treatment (BWT) on microbial community diversity was assessed using the 16S rRNA gene based Ion Torrent sequencing with its new 400 base chemistry. Ballast water samples from a Great Lakes ship were collected from the intake and discharge of both control and NaOH (pH 12) treated tanks and were analyzed in duplicates. One set of duplicates was treated with the membrane-impermeable DNA cross-linking reagent propidium mono-azide (PMA) prior to PCR amplification to differentiate between live and dead microorganisms. Ion Torrent sequencing generated nearly 580,000 reads for 31 bar-coded samples and revealed alterations of the microbial community structure in ballast water that had been treated with NaOH. Rarefaction analysis of the Ion Torrent sequencing data showed that BWT using NaOH significantly decreased microbial community diversity relative to control discharge (p<0.001). UniFrac distance based principal coordinate analysis (PCoA) plots and UPGMA tree analysis revealed that NaOH-treated ballast water microbial communities differed from both intake communities and control discharge communities. After NaOH treatment, bacteria from the genus Alishewanella became dominant in the NaOH-treated samples, accounting for <0.5% of the total reads in intake samples but more than 50% of the reads in the treated discharge samples. The only apparent difference in microbial community structure between PMA-processed and non-PMA samples occurred in intake water samples, which exhibited a significantly higher amount of PMA-sensitive cyanobacteria/chloroplast 16S rRNA than their corresponding non-PMA total DNA samples. The community assembly obtained using Ion Torrent sequencing was comparable to that obtained from a subset of samples that were also subjected to 454 pyrosequencing. This study showed the efficacy of alkali ballast water treatment in reducing ballast water microbial diversity and demonstrated the application of new Ion Torrent sequencing techniques to microbial community studies.
Project description:Green autofluorescence (GAF) has been described in the short flagellum of golden and brown algae, the stigma of Euglenophyceae, and cytoplasm of different life stages of dinoflagellates and is considered by some researchers a valuable taxonomic feature for dinoflagellates. In addition, green fluorescence staining has been widely proposed or adopted to measure cell viability (or physiological state) in areas such as apoptosis of phytoplankton, pollutant stresses on algae, metabolic activity of algae, and testing treatment technologies for ships' ballast water. This paper reports our epifluorescence microscopic observations and quantitative spectrometric measurements of GAF in a broad phylogenetic range of microalgae. Our results demonstrate GAF is a common feature of dinoflagellates, diatoms, green algae, cyanobacteria, and raphidophytes, occurs in the cytoplasm and particularly in eyespots, accumulation bodies, spines, and aerotopes, and is caused by molecules other than chlorophyll. GAF intensity increased with time after cell death or fixation and with excitation by blue or UV light and was affected by pH. GAF of microalgae may be only of limited value in taxonomy. It can be strong enough to interfere with the results of green fluorescence staining, particularly when stained samples are observed microscopically. GAF is useful, however, for microscopic study of algal morphology, especially to visualize cellular components such as eyespots, nucleus, aerotopes, spines, and chloroplasts. Furthermore, GAF can be used to visualize and enumerate dinoflagellate cysts in marine and estuarine sediments in the context of anticipating and monitoring harmful algal blooms and in tracking potentially harmful dinoflagellates transported in ships' ballast tanks.
Project description:<h4>Background</h4>Ascidians play a key role in the ecology and biodiversity of marine ecosystem. Ascidians can be transported in ship ballast water and while attached to ship and boat hulls. Heavy traffic by domestic and international ships as well as cargo vessels between the major and minor ports warrants continuous monitoring for new introductions of ascidians. The Mandapam coast is situated in the Gulf of Mannar, India, a marine hot spot area in the Indian Ocean which provides an environment suitable for the settlement of ascidians.<h4>New information</h4>A total of 30 species of ascidians were reported from Mandapam coastal waters, of which 26 species were new to the study area and five species: Ecteinascidia turbinata, Eudistoma carnosum, Trididemnum caelatum, T. vermiforme and Didemnum spadix, were new to India.
Project description:In this article, we determined the concentration levels of metals including Zn, Al, Sr, and Co and metalloid of As of ballast water in commercial ships entering Bushehr port, along the Persian Gulf. Ballast water samples were taken from commercial ships entering Bushehr port from 34 ports around the world during 15 February and 25 August 2016. The concentration levels of metals and metalloid were determined by using a graphite furnace absorption spectrometer (AAS).
Project description:Over the past several decades, much attention has been focused on the dispersal of aquatic nonindigenous species via ballast tanks of shipping vessels worldwide. The recently reclassified dinoflagellate <i>Pseudocochlodinium profundisulcus</i> (previously identified as <i>Cochlodinium</i> sp., <i>Cochlodinium geminatum</i>, or <i>Polykrikos geminatus</i>) was not reported in China until 2006. However, algal blooming events caused by this organism have been reported almost every year since then in the Pearl River Estuary and its adjacent areas in China. Whether <i>P. profundisulcus</i> is an indigenous or an invasive species has thus become an ecological question of great scientific and practical significance. In this study, we collected the sediments from ballast tanks of ships arriving in the ports of China and North America and characterized dinoflagellate resting cysts via a combined approach. We germinated two dark brownish cysts from the tank of an international ship (Vessel A) arriving at the Jiangyin Port (China) into vegetative cells and identified them as <i>P. profundisulcus</i> by light and scanning electron microscopy and phylogenetic analyses for partial LSU rDNA sequences. We also identified <i>P. profundisulcus</i> cyst from the ballast tank sediment of a ship (Vessel B) arriving in the port of North America via single-cyst PCR and cloning sequencing, which indicated that this species could be transported as resting cyst via ship. Since phylogenetic analyses based on partial LSU rDNA sequences could not differentiate all sequences among our cysts from those deposited in the NCBI database into sub-groups, all populations from China, Australia, Japan, and the original sources from which the cysts in the two vessels arrived in China and North America were carried over appeared to share a very recent common ancestor, and the species may have experienced a worldwide expansion recently. These results indicate that <i>P. profundisulcus</i> cysts may have been extensively transferred to many regions of the world via ships' ballast tank sediments. While our work provides an exemplary case for both the feasibility and complexity (in tracking the source) of the bio-invasion risk via the transport of live resting cysts by ship's ballast tanks, it also points out an orientation for future investigation.
Project description:Indicator organisms and antibiotic resistance were used as a proxy to measure microbial water quality of ballast tanks of ships, and surface waters in a tropical harbor. The survival of marine bacteria in ballast tanks appeared to diminish over longer water retention time, with a reduction of cell viability observed after a week based on heterotrophic plate counts. Pyrosequencing of 16S rRNA genes showed distinct differences in microbial composition of ballast and harbor waters. The harbor waters had a higher abundance of operational taxonomic units (OTUs) assigned to Cyanobacteria (Synechococcus spp.) and ?-proteobacteria (SAR11 members), while marine hydrocarbon degraders such as ?-proteobacteria (Ocenspirillaes spp., Thiotrchales spp.) and Bacteroidetes (Flavobacteriales spp.) dominated the ballast water samples. Screening of indicator organisms found Escherichia coli (E. coli), Enterococcus and Pseudomonas aeruginosa (P. aeruginosa) in two or more of the ballast and harbor water samples tested. Vibrio spp. and Salmonella spp. were detected exclusively in harbor water samples. Using quantitative PCR (qPCR), we screened for 13 antibiotic resistant gene (ARG) targets and found higher abundances of sul1 (4.13-3.44 x 102 copies/mL), dfrA (0.77-1.80 x10 copies/mL) and cfr (2.00-5.21 copies/mL) genes compared to the other ARG targets selected for this survey. These genes encode for resistance to sulfonamides, trimethoprim and chloramphenicol-florfenicol antibiotics, which are also known to persist in sediments of aquaculture farms and coastal environments. Among the ARGs screened, we found significant correlations (P<0.05) between ereA, ermG, cfr and tetO genes to one or more of the indicator organisms detected in this study, which may suggest that these members contribute to the environmental resistome. This study provides a baseline water quality survey, quantitatively assessing indicators of antibiotic resistance, potentially pathogenic organisms and a broad-brush description of difference in microbial composition and diversity between open oceans and tropical coastal environments through the use of next generation sequencing technology.