scholarly journals A Novel Immunofluorescence Flow Cytometry Technique Detects the Expansion of Brown Tides Caused by Aureoumbra lagunensis to the Caribbean Sea

2014 ◽  
Vol 80 (16) ◽  
pp. 4947-4957 ◽  
Author(s):  
F. Koch ◽  
Y. Kang ◽  
T. A. Villareal ◽  
D. M. Anderson ◽  
C. J. Gobler
Keyword(s):  
Flow Cytometry ◽  
Brown Tide ◽  
Economic Damage ◽  
Content Type ◽  
Relative Standard ◽  
Wide Range ◽  
Brown Tides ◽  
Indian River ◽  
Brown Tide Bloom ◽  
Aureoumbra Lagunensis

ABSTRACTDuring the past 3 decades, brown tides caused by the pelagophytesAureococcus anophagefferensandAureoumbra lagunensishave caused ecological and economic damage to coastal ecosystems across the globe. While blooms ofA. lagunensishad previously been confined to Texas, in 2012, an expansive brown tide occurred on Florida's East Coast, causing widespread disruption within the Indian River and Mosquito Lagoons and generating renewed interest in this organism. A major impediment to detailed investigations ofA. lagunensisin an ecosystem setting has been the absence of a rapid and reliable method for cell quantification. The combination of their small size (3 to 5 μm) and nondescript extracellular features makes identification and enumeration of these cells with conventional methods a challenge. Here we report the development of an immunological-based flow cytometry method that uses a fluorescently labeled antibody developed againstA. lagunensis. This method is species specific, sensitive (detection limit of 1.5 × 103cells ml−1), precise (1% relative standard deviation of replicated samples), and accurate (108% ± 8% recovery of spiked samples) over a wide range of cell concentrations. Furthermore, this method effectively quantifiesA. lagunensisin both glutaraldehyde- and formalin-preserved samples, yields a high throughput of samples (∼35 samples h−1), and is cost-effective, making it an ideal tool for managers and scientists. This method successfully documented the recurrence of a brown tide bloom in Florida in 2013. Bloom densities were highest in June (>2.0 × 106cells ml−1) and spanned >60 km from the Ponce de Leon inlet in the northern Mosquito Lagoon south to Titusville in the Indian River Lagoon. Low levels ofA. lagunensiscells were found >250 km south of this region. This method also quickly and accurately identifiedA. lagunensisas the causative agent of a 2013 brown tide bloom in Guantanamo Bay, Cuba, and thus should prove useful for both quantifying the dynamics of ongoing blooms ofA. lagunensisas well as documenting new outbreaks of this harmful alga.

scholarly journals Bivalve Feeding on the Brown Tide Aureoumbra lagunensis in a Shallow Coastal Environment

2021 ◽  
Vol 8 ◽  
Author(s):  
Eve Galimany ◽  
Jessica Lunt ◽  
Christopher J. Freeman ◽  
I. Segura-García ◽  
M. Mossop ◽  
...  
Keyword(s):  
Clearance Rate ◽  
Indian River Lagoon ◽  
Brown Tide ◽  
Bivalve Species ◽  
Clearance Rates ◽  
Interspecific Differences ◽  
Brown Tides ◽  
Indian River ◽  
Aureoumbra Lagunensis ◽  
Fish And Shellfish

Brown tides formed by Aureoumbra lagunensis decrease light penetration in the water column and are often followed by hypoxic events that result in the loss of fish and shellfish. To understand the ability of bivalve filter feeders to control and prevent A. lagunensis blooms, we exposed eastern oysters (Crassostrea virginica), hooked mussels (Ischadium recurvum), and hard clams (Mercenaria mercenaria) to a naturally co-occurring brown tide in the Indian River Lagoon (IRL), Florida, United States. Bivalves were exposed in the laboratory to multiple concentrations (104 to 106 cells mL–1) of isotopically labeled (13C and 15N) A. lagunensis cells. The standard clearance rate (herein clearance rate) of each bivalve species was calculated using flow cytometry to quantify A. lagunensis cell removal. The highest clearance rates were at 104 cells mL–1, but values varied across bivalve species (2.16 ± 0.30, 3.03 ± 0.58, and 0.41 ± 0.12 L h–1 for C. virginica, I. recurvum, and M. mercenaria, respectively). Although clearance rates decreased with increasing bloom concentrations, bivalves were still consuming algal cells at all concentrations and were retaining and assimilating more cells at the highest concentrations, as revealed by δ13C and δ15N values. We highlight interspecific differences among bivalve species in the removal of A. lagunensis, supporting the importance of healthy and diverse filter feeding communities in estuaries, especially as threats of brown tides and other HABs are increasing in the Anthropocene.

scholarly journals Characterization of the Proteomic Profiles of the Brown Tide Alga Aureoumbra lagunensis under Phosphate- and Nitrogen-Limiting Conditions and of Its Phosphate Limitation-Specific Protein with Alkaline Phosphatase Activity

2012 ◽  
Vol 78 (6) ◽  
pp. 2025-2033 ◽  
Author(s):  
Ming-Ming Sun ◽  
Jin Sun ◽  
Jian-Wen Qiu ◽  
Hongmei Jing ◽  
Hongbin Liu
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Specific Protein ◽  
Brown Tide ◽  
Mass Spectrometry Analysis ◽  
Phosphate Limitation ◽  
Content Type ◽  
P Limitation ◽  
Aureoumbra Lagunensis

ABSTRACTThe persistent bloom of the brown tide algaAureoumbra lagunensishas been reported in coastal embayments along southern Texas, but the molecular mechanisms that sustain such algal bloom are unknown. We compared the proteome and physiological parameters ofA. lagunensisgrown in phosphate (P)-depleted, P- and nitrogen (N)-depleted, and nutrient-replete cultures. For the proteomic analysis, samples from three conditions were subjected to two-dimensional electrophoresis and tandem mass spectrometry analysis. Because of the paucity of genomic resources in this species, ade novocross-species protein search was used to identify the differentially expressed proteins, which revealed their involvement in several key biological processes, such as chlorophyll synthesis, antioxidative protection, and protein degradation, suggesting thatA. lagunensismay adopt intracellular nutrient compensation, extracellular organic nutrient regeneration, and damage protection to thrive in P-depleted environments. A highly abundant P limitation-specific protein, tentatively identified as a putative alkaline phosphatase, was further characterized by enzyme activity assay on nondenaturing gel and confocal microscopy, which confirmed that this protein has alkaline phosphatase activity, is a cytoplasmic protein, and is closely associated with the cell membrane. The abundance, location, and functional expression of this alkaline phosphatase all indicate the importance of organic P utilization forA. lagunensisunder P limitation and the possible role of this alkaline phosphatase in regenerating phosphate from extra- or intracellular organic phosphorus.

scholarly journals Field‐Validated Detection of Aureoumbra lagunensis Brown Tide Blooms in the Indian River Lagoon, Florida, Using Sentinel‐3A OLCI and Ground‐Based Hyperspectral Spectroradiometers

GeoHealth ◽  
2020 ◽  
Vol 4 (6) ◽  
Author(s):  
Taylor J. Judice ◽  
Edith A. Widder ◽  
Warren H. Falls ◽  
Dulcinea M. Avouris ◽  
Dominic J. Cristiano ◽  
...  
Keyword(s):  
Indian River Lagoon ◽  
Brown Tide ◽  
Indian River ◽  
Aureoumbra Lagunensis

scholarly journals Immunofluorescence Flow Cytometry Technique for Enumeration of the Brown-Tide Alga, Aureococcus anophagefferens

2008 ◽  
Vol 74 (22) ◽  
pp. 6931-6940 ◽  
Author(s):  
Beth A. Stauffer ◽  
Rebecca A. Schaffner ◽  
Catherine Wazniak ◽  
David A. Caron
Keyword(s):  
Flow Cytometry ◽  
Fluorescein Isothiocyanate ◽  
Cross Reactivity ◽  
Brown Tide ◽  
Enzyme Linked Immunosorbent Assay ◽  
Aureococcus Anophagefferens ◽  
Elisa Method ◽  
Intact Cells ◽  
Microscopy Technique ◽  
Wide Range

ABSTRACT A new immunologically based flow cytometry (IFCM) technique was developed to enumerate Aureococcus anophagefferens, a small pelagophyte alga that is the cause of “brown tides” in bays and estuaries of the mid-Atlantic states along the U.S. coast. The method utilizes a monoclonal antibody conjugated to fluorescein isothiocyanate (FITC-MAb) to label the surface of A. anophagefferens cells which are then detected and enumerated by using a flow cytometer. Optimal conditions for FITC-MAb staining, including solution composition, incubation times, and FITC-MAb concentrations, were determined. The FITC-MAb method was tested for cross-reactivity with nontarget, similarly sized, photoautotrophic protists, and the method was compared to an enzyme-linked immunosorbent assay (ELISA) using the same MAb. Comparisons of the IFCM technique to traditional microscopy enumeration of cultures and spiked environmental samples showed consistent agreement over several orders of magnitude (r 2 > 0.99). Comparisons of the IFCM and ELISA techniques for enumerating cells from a predation experiment showed a substantial overestimation (up to 10 times higher) of the ELISA in the presence of consumers of A. anophagefferens, presumably due to egested cell fragments that retained antigenicity, using the ELISA method, but were not characterized as whole algal cells by the IFCM method. Application of the IFCM method to environmental “brown-tide” samples taken from the coastal bays of Maryland demonstrated its efficacy in resolving A. anophagefferens abundance levels throughout the course of a bloom and over a large range of abundance values. IFCM counts of the brown-tide alga from natural samples were consistently lower than those obtained using the ELISA method and were equivalent to those of the polyclonal immunofluorescence microscopy technique, since both methods discriminate intact cells. Overall, the IFCM approach was an accurate and relatively simple technique for the rapid enumeration of A. anophagefferens in natural samples over a wide range of abundance values (103 to 106 cells ml−1).

scholarly journals Metatranscriptome Library Preparation Influences Analyses of Viral Community Activity During a Brown Tide Bloom

2021 ◽  
Vol 12 ◽  
Author(s):  
Eric R. Gann ◽  
Yoonja Kang ◽  
Sonya T. Dyhrman ◽  
Christopher J. Gobler ◽  
Steven W. Wilhelm
Keyword(s):  
New York ◽  
Community Dynamics ◽  
Brown Tide ◽  
Aureococcus Anophagefferens ◽  
Brown Tides ◽  
Viral Communities ◽  
Brown Tide Bloom ◽  
Selection For ◽  
Virus Community

There is growing interest in the use of metatranscriptomics to study virus community dynamics. We used RNA samples collected from harmful brown tides caused by the eukaryotic alga Aureococcus anophagefferens within New York (United States) estuaries and in the process observed how preprocessing of libraries by either selection for polyadenylation or reduction in ribosomal RNA (rRNA) influenced virus community analyses. As expected, more reads mapped to the A. anophagefferens genome in polyadenylation-selected libraries compared to the rRNA-reduced libraries, with reads mapped in each sample correlating to one another regardless of preprocessing of libraries. Yet, this trend was not seen for reads mapping to the Aureococcus anophagefferens Virus (AaV), where significantly more reads (approximately two orders of magnitude) were mapped to the AaV genome in the rRNA-reduced libraries. In the rRNA-reduced libraries, there was a strong and significant correlation between reads mappings to AaV and A. anophagefferens. Overall, polyadenylation-selected libraries produced fewer viral contigs, fewer reads mapped to viral contigs, and different proportions across viral realms and families, compared to their rRNA-reduced pairs. This study provides evidence that libraries generated by rRNA reduction and not selected for polyadenylation are more appropriate for quantitative characterization of viral communities in aquatic ecosystems by metatranscriptomics.

Advances in sample digestion using microwave-ultraviolet radiations: phosphorus and sulfur determination in animal feed

2020 ◽  
Vol 16 ◽  
Author(s):  
Diogo L. R. Novo ◽  
Priscila T. Scaglioni ◽  
Rodrigo M. Pereira ◽  
Filipe S. Rondan ◽  
Gilberto S. Coelho Junior ◽  
...  
Keyword(s):  
Inductively Coupled Plasma ◽  
Animal Feed ◽  
Optical Emission ◽  
Emission Spectrometry ◽  
Official Method ◽  
Dissolved Carbon ◽  
Inductively Coupled ◽  
Relative Standard ◽  
Wide Range ◽  
Sulfur Determination

Background: Conventional analytical methods for phosphorus and sulfur determination in several matrices present normally analytical challenges regarding inaccuracy, detectability and waste generation. Objective: The main objective is proposing a green and feasible analytical method for phosphorus and sulfur determination in animal feed. Methods: Synergic effect between microwave and ultraviolet radiations during sample preparation was evaluated for the first time for the animal feed digestion associated with further phosphorus and sulfur determination by ion chromatography with conductivity detection. Dissolved carbon and residual acidity in final digests were used for the proposed method assessment. Phosphorus and sulfur values were compared with those obtained using conventional microwave-assisted wet digestion in closed vessels associated with inductively coupled plasma optical emission spectrometry and with those obtained using Association of Official Analytical Chemists International official method. Recovery tests and certified reference material analysis were performed. Animal feeds were analyzed using the proposed method. Results: Sample masses of 500 mg were efficiently digested using only 2 mol L -1 HNO3. The results obtained by the proposed method was not differing significantly (p > 0.05) from those obtained by the conventional and official methods. Suitable recoveries (from 94 to 99%), agreement with certified values (101 and 104%) and relative standard deviations (< 8%) were achieved. Phosphorus and sulfur content in commercial products varied in a wide range (P: 5,873 to 28,387 mg kg-1 and S: 2,165 to 4,501 mg kg-1 ). Conclusion: The proposed method is a green, safe, accurate, precise and sensitive alternative for animal feed quality control.

scholarly journals Transfer of Plasmid DNA to Clinical Coagulase-Negative Staphylococcal Pathogens by Using a Unique Bacteriophage

2015 ◽  
Vol 81 (7) ◽  
pp. 2481-2488 ◽  
Author(s):  
Volker Winstel ◽  
Petra Kühner ◽  
Bernhard Krismer ◽  
Andreas Peschel ◽  
Holger Rohde
Keyword(s):  
Plasmid Dna ◽  
Genetic Manipulation ◽  
Current Knowledge ◽  
Virulence Determinants ◽  
Coagulase Negative Staphylococci ◽  
Reliable Technique ◽  
Content Type ◽  
Research Activities ◽  
Wide Range ◽  
Genetic Barriers

ABSTRACTGenetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a uniqueStaphylococcus aureusstrain via a specificS. aureusbacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinicalStaphylococcus epidermidisisolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

scholarly journals Use of Proteins Identified through a Functional Genomic Screen To Develop a Protein Subunit Vaccine That Provides Significant Protection against Virulent Streptococcus suis in Pigs

2017 ◽  
Vol 86 (3) ◽  
Author(s):  
Susan L. Brockmeier ◽  
Crystal L. Loving ◽  
Tracy L. Nicholson ◽  
Jinhong Wang ◽  
Sarah E. Peters ◽  
...  
Keyword(s):  
Immune Response ◽  
Vaccine Trial ◽  
Streptococcus Suis ◽  
Protein Subunit ◽  
Content Type ◽  
Degree Of Protection ◽  
Wide Range ◽  
Associated Proteins ◽  
Clinical Protection ◽  
Cellular Immune

ABSTRACT Streptococcus suis is a bacterium that is commonly carried in the respiratory tract and that is also one of the most important invasive pathogens of swine, commonly causing meningitis, arthritis, and septicemia. Due to the existence of many serotypes and a wide range of immune evasion capabilities, efficacious vaccines are not readily available. The selection of S. suis protein candidates for inclusion in a vaccine was accomplished by identifying fitness genes through a functional genomics screen and selecting conserved predicted surface-associated proteins. Five candidate proteins were selected for evaluation in a vaccine trial and administered both intranasally and intramuscularly with one of two different adjuvant formulations. Clinical protection was evaluated by subsequent intranasal challenge with virulent S. suis . While subunit vaccination with the S. suis proteins induced IgG antibodies to each individual protein and a cellular immune response to the pool of proteins and provided substantial protection from challenge with virulent S. suis , the immune response elicited and the degree of protection were dependent on the parenteral adjuvant given. Subunit vaccination induced IgG reactive against different S. suis serotypes, indicating a potential for cross protection.

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