Dataset: Chemical Defenses-5: Park and Dam 2021

Name: Cell-growth gene expression reveals a direct fitness cost of grazer-induced toxin production in red tide dinoflagellate prey
Published: 2021-06-16
Keywords: oceans

Cite This Dataset

DOI:10.26008/1912/bco-dmo.853900.1

Spatial Extent: N:41.320717 E:-72.06196 S:41.320717 W:-72.06196

Temporal Extent: 2011-09 - 2018-05

Project:

Chemical Defenses in a Toxic Dinoflagellate: Mechanisms and Constraints (Chemical Defenses)

Principal Investigator:

Hans G. Dam (University of Connecticut, UConn)

Contact:

Hans G. Dam (University of Connecticut, UConn)

Gihong Park (University of Connecticut, UConn)

BCO-DMO Data Manager:

Shannon Rauch (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version:

Version Date:

2021-06-16

Restricted:

Validated:

Yes

Current State:

Final no updates expected

Cell-growth gene expression reveals a direct fitness cost of grazer-induced toxin production in red tide dinoflagellate prey

Abstract:

Data include Paralytic Shellfish Toxins (STX and its derivatives) of the marine dinoflagellate Alexandrium catenella, cell growth rates, and relative gene expression (RGE) level of the toxin gene compared to the reference gene as values of the independent and dependent variables. Independent variable: exposure time (per day) of cells either without grazers (control) or with grazers (treatment) Dependent variables: cell density (cells per liter), cell toxin content (mol per cell), cell growth rate (per day), and RGE (unitless) Data were published in: Park, G., & Dam, H. G. (2021). Cell-growth gene expression reveals a direct fitness cost of grazer-induced toxin production in red tide dinoflagellate prey. Proceedings of the Royal Society B, 288(1944), 20202480. https://doi.org/10.1098/rspb.2020.2480

Expand/Collapse All

Archival Copy

Version Date	Archive	Persistent Identifier (PID)	Date PID Issued
2021-06-16	Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)	10.26008/1912/bco-dmo.853900.1	2021-06-17

Methods & Sampling

Refer to the Methods section of Park & Dam (2021).

Sample collection and culture:
The toxigenic dinoflagellate, Alexandrium catenella (strain BF-5, isolated from the Bay of Fundy, Canada) was grown in F/2 medium without silicate. Cultures were maintained in the exponential growth phase and all experiments were conducted in an environmental chamber kept at 18°C and illuminated with fluorescent lighting (~100 µM m⁻² s⁻¹) set to a 12 h:12 h light:dark photoperiod. The calanoid copepod Acartia hudsonica, historically co-occurring with toxic A. catenella, was collected from Casco Bay, Maine, U.S.A. (43°39′N, 74°47′W), a location in which blooms of A. catenella are common. Triplicate copepod cultures were maintained with a mixed diet of Rhodomonas sp., Tetraselmis sp., and Thalassiosira weissflogii.

Grazer-induced toxin production assay:
Alexandrium catenella cells were placed in 500 ml bottles (300 cells ml⁻¹) either without copepods (controls: constitutive toxin production) or with 20 adult female A. hudsonica (treatments) for a period of 96 h. Experiments were done in quadruplicate sets for both control and treatment, and carried out at 18 °C in a walk-in environmental chamber as described above. For the time series analysis, A. catenella cells were harvested at the conclusion of each exposure time (0, 4, 8, 24, 48, 72, and 96 h) after which cells were gently separated by wet-sieving onto a 63 μm mesh to remove copepods, nauplii, eggs, and fecal pellets. Two aliquots from each bottle were filtered onto 5 μm pore size polycarbonate membranes; one for toxin analysis and another for RNA extraction.

Toxin and gene expression analysis:
PST concentrations were determined by reverse-phase ion-pairing high performance liquid chromatography (HPLC) using the post-column oxidative fluorescence method. Gene expression analyses were conducted using reverse transcription quantitative PCR (RT-qPCR). From previous studies several pairs of primers were designed and tested for specificity and PCR efficiency by RT-qPCR. All quantitative PCRs (qPCRs) were performed on a StepOnePlus™ real-time PCR system (Applied Biosystems) and run in 10 μL reactions with Fast SYBR® Green Master Mix (Applied Biosystems).

Data Processing Description

Data Processing:
A Kruskal-Wallis one-way ANOVA on ranks was used and all pairwise comparisons among controls/treatments were assessed with the Student-Newman-Keul’s (SNK) post-hoc procedure. The effects of day, presence of grazer, and their interaction on cell concentration, toxin content, toxin profile, growth rate, and relative gene expression (four independent replicates per test and two samples for PST analysis) were tested by two-way ANOVA. Time-dependent changes in these variables were also tested separately by regression analysis for the control and treatments. Ingestion rates were tested with a Mann-Whitney U Test to compare the Frost equation to our modified equation. All statistical analyses were performed using SigmaPlot version 11.0 and SPSS version 26 software.

Data Files

File
park_dam_2021.csv (Comma Separated Values (.csv), 4.73 KB) MD5:268dae7956bb150a223dd17a6d71bc06 Primary data file for dataset ID 853900

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-1130284

Deployments

None available yet

Instruments

High-Performance Liquid Chromatograph

Supplied Name: HPLC system (Waters, Milford, MA)

Supplied Description:

High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase). Scanning Fluorescence Detector (Waters 474, Waters, Milford, MA) was used for in-vitro diagnostic testing to analyze compounds of STX and its derivatives.

Instrument Type

Generic Name: High-Performance Liquid Chromatograph

Acronym: HPLC

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB11/

Generic Description:

A High-performance liquid chromatograph (HPLC) is a type of liquid chromatography used to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of the mobile phase, a pump, an injector, a separation column, and a detector. Compounds are separated by high pressure pumping of the sample mixture onto a column packed with microspheres coated with the stationary phase. The different components in the mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase.

Homogenizer

Supplied Name: Fastprep-24 Tissue and Cell Homogenizer (MP Biomedicals, Solon, OH)

Supplied Description:

The FastPrep of benchtop instrument utilizes bead-beating technology to lyse, homogenize and grind routine and difficult samples in 40 seconds or less. A completely self-contained system, FastPrep instrument eliminates the risk of cross-contamination and time-consuming clean up. The use of interchangeable adapters offers exceptional versatility in sample size and temperature with ambient, cryogenic and all-metal options.

Instrument Type

Generic Name: Homogenizer

Generic Description:

A homogenizer is a piece of laboratory equipment used for the homogenization of various types of material, such as tissue, plant, food, soil, and many others.

Inverted Microscope

Supplied Name: Olympus IX70 inverted system microscope

Supplied Description:

The IX70 inverted tissue culture microscope is a research-level instrument capable of imaging specimens in a variety of illumination modes including brightfield, darkfield, phase contrast, Hoffman modulation contrast, fluorescence, and differential interference contrast.

Instrument Type

Generic Name: Inverted Microscope

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB05/

Generic Description:

An inverted microscope is a microscope with its light source and condenser on the top, above the stage pointing down, while the objectives and turret are below the stage pointing up. It was invented in 1850 by J. Lawrence Smith, a faculty member of Tulane University (then named the Medical College of Louisiana).

Inverted microscopes are useful for observing living cells or organisms at the bottom of a large container (e.g. a tissue culture flask) under more natural conditions than on a glass slide, as is the case with a conventional microscope. Inverted microscopes are also used in micromanipulation applications where space above the specimen is required for manipulator mechanisms and the microtools they hold, and in metallurgical applications where polished samples can be placed on top of the stage and viewed from underneath using reflecting objectives.

The stage on an inverted microscope is usually fixed, and focus is adjusted by moving the objective lens along a vertical axis to bring it closer to or further from the specimen. The focus mechanism typically has a dual concentric knob for coarse and fine adjustment. Depending on the size of the microscope, four to six objective lenses of different magnifications may be fitted to a rotating turret known as a nosepiece. These microscopes may also be fitted with accessories for fitting still and video cameras, fluorescence illumination, confocal scanning and many other applications.

qPCR Thermal Cycler

Supplied Name: StepOnePlus™ real-time PCR system (Applied BiosystemsTM)

Supplied Description:

The system can be setup in a variety of configurations and comes ready to use, out of the box, with intuitive data analysis and instrument control software. Utilizing robust LED based 4-color optical recording, the StepOnePlus™ Real-Time PCR System is designed to deliver precise, quantitative Real-Time PCR results for a variety of genomic research applications.

Instrument Type

Generic Name: qPCR Thermal Cycler

Acronym: qPCR

Generic Description:

An instrument for quantitative polymerase chain reaction (qPCR), also known as real-time polymerase chain reaction (Real-Time PCR).

Parameters

Date and time formats are described in python strftime() syntax.

Supplied Name	Supplied description	Supplied Units	Standard Name
Name	Alexandrium catenella cells, either without grazers (control) or with grazers (treatment)	unitless	sample
Day	Experimental time for a period of 4 days	days	time_elapsed
Hour	Harvested time at the conclusion of each exposure hr (0, 4, 8, 24, 48, 72, and 96 hr)	hours	time_elapsed
Cell_Number	Cell concentration	cells per milliliter (cells ml-1)	cell_concentration
Growth_Rate	Net growth rate	per day (d-1)	growth
Toxin_Content	Total cellular toxin content	femtomoles per cell (fmol cell-1)	no_bcodmo_term
utox	Toxin production rate	per day (d-1)	no_bcodmo_term
Rtox	Net toxin production rate	femtomoles per cell per day (fmol cell-1 d-1)	no_bcodmo_term
GTX3	gonyautoxin 3	femtomoles per cell (fmol cell-1)	no_bcodmo_term
GTX4	gonyautoxin 4	femtomoles per cell (fmol cell-1)	no_bcodmo_term
NEO	neosaxitoxin	femtomoles per cell (fmol cell-1)	no_bcodmo_term
STX	saxitoxin	femtomoles per cell (fmol cell-1)	no_bcodmo_term
C2	C-2 toxin	femtomoles per cell (fmol cell-1)	no_bcodmo_term
RGE_stxA4	Relative gene expression level of the stxA4 gene compared to the reference gene (lbpn)	unitless	no_bcodmo_term
RGE_stxG	Relative gene expression level of the stxG gene compared to the reference gene (lbpn)	unitless	no_bcodmo_term
RGE_cyc	Relative gene expression level of the cyc gene compared to the reference gene (lbpn)	unitless	no_bcodmo_term
g	Grazing rate	per day (d-1)	no_bcodmo_term
g_modified	Modified grazing rate (this study)	per day (d-1)	no_bcodmo_term
Cm	Average cell concentration	cells per milliliter (cells ml-1)	no_bcodmo_term
Cm_modified	Modified average cell concentration (this study)	cells per milliliter (cells ml-1)	no_bcodmo_term
I	Ingestion rate	cells per copepod per day (cells cop-1 d-1)	no_bcodmo_term
I_modified	Modified ingestion rate (this study)	cells per copepod per day (cells cop-1 d-1)	no_bcodmo_term

Status message

Database

Contribute Data

Dataset: Chemical Defenses-5: Park and Dam 2021