Grantee Research Project Results
2008 Progress Report: Quantifying Grazing on Harmful Algae with a Novel, qPCR-based Technique
EPA Grant Number: R833222Title: Quantifying Grazing on Harmful Algae with a Novel, qPCR-based Technique
Investigators: Juhl, Andrew , Dyhrman, Sonya
Institution: Lamont Doherty Earth Observatory of Columbia University , Woods Hole Oceanographic Institution
EPA Project Officer: Packard, Benjamin H
Project Period: March 15, 2007 through March 14, 2010
Project Period Covered by this Report: March 15, 2008 through March 14,2009
Project Amount: $409,856
RFA: Ecology and Oceanography of Harmful Algal Blooms (2006) RFA Text | Recipients Lists
Research Category: Aquatic Ecosystems , Water
Objective:
The overall objective of this project is to develop a quantitative PCR-based method to assay
zooplankton grazing rates on harmful algae. The specific objectives are to: 1) optimize the qPCR assay for quantitative detection of ingested Alexandrium, 2) calibrate and test the qPCR-based measure of grazing rate in laboratory experiments, and 3) apply the qPCR-based grazing technique to quantify grazing by copepods on an in-situ Alexandrium bloom.
Progress Summary:
Considerable progress was been made during the 3rd year of this project and we are grateful for the EPA support. With support from the EPA grant, we are pleased to report the progress made to date, focusing on year 3.
Obj. 1) Optimize the PCR-based assay for quantitative detection of ingested Alexandrium:
This objective was completed in year one of this project and the details were presented in an earlier report. To date, our results suggest that feeding strategy and digestion play a significant role in the recovery of detectable prey DNA.
The qPCR method optimized and used here makes use of a well-tested primer set specific for an rDNA gene present in Alexandrium from the fundyense/tamarense/catenella species complex. However, one issue that has not been resolved is the degree of variability of this target gene among different Alexandrium strains, and even within a single strain over time. Knowledge of the amount of target gene per cell is essential for the correct determination of the cell number using qPCR. We are currently analyzing experiments to address this question. Specifically, Alexandrium fundyense cells were grown in f/2 medium at 15°C in cool white fluorescent light (~200 μmol quanta m-2 s-1) on a 14 h light:10 h dark cycle under nutrient replete, -N (20 μM) and –P (2 μM) conditions. To assess changes in rRNA gene copy number at various points during the light:dark cycle, Alexandrium cells were removed from the nutrient replete culture and individual cells (~50) were transferred to Buffer ATL for subsequent DNA extraction. To assess difference in rRNA copy number brought on by nutrient limitation/growth phase, Alexandrium cells were removed from the –N and –P cultures and individual cells (~50) were transferred to Buffer ATL for DNA extraction. Growth in the cultures was determined via cell counts of acid-Lugol’s preserved samples. DNA extractions on these samples is pending.
Obj. 2) Calibrate and test the qPCR-based measure of grazing rate in laboratory experiments:
The key elements of this objective were completed in years 1 and 2 of the project, and the details were presented in an earlier report. The work has been presented at numerous national and international meetings. A manuscript is in prep.
We have continued work to satisfy this objective in year 3 by validating this approach for use with bivalve grazers. Blue mussels, Mytilus edulis, were collected from Eel Pond, a small embayment in Woods Hole, MA. Mussels were cleaned and placed in filtered seawater. To assess the efficacy of PCR to distinguish between bivalves fed Alexandrium from those not fed Alexandrium, 5 mussels of roughly the same size were transferred to each of two different treatments (a and b) in 100-mL tri-corner beakers: a) 100 mL filtered (0.2 μm) seawater amended with 100 Alexandrium cells mL-1 and b) 100 mL filtered seawater. The bivalves were incubated for 24 h at room temperature. After 24 h, the bivalves were washed with filtered seawater. The bivalves were immediately transferred into liquid nitrogen. Once frozen, the bivalves were removed to a petri dish and the gill and digestive gland were dissected out. These tissues were placed into separate screw-cap tubes containing 0.4 mL Buffer ATL. Dissecting tools were cleaned between mussel individuals.
In the mussels that were allowed to graze on Alexandrium, an Alexandrium DNA signal was detected by PCR in triplicate samples of both gill and digestive gland tissues. In contrast, an Alexandrium DNA signal was not detectable in the dissected tissues (gill and digestive gland) from the mussels that did not feed on Alexandrium (Figure 1). This is a very promising result, as it suggests utility of this approach for other predator:prey combinations.
Obj. 3) Apply the qPCR-based grazing technique to quantify grazing by copepods on an in-situ Alexandrium bloom:
We collaborated with Dr. Don Anderson and Bruce Keafer (both at WHOI) for collection of copepod samples from the Gulf of Maine during Alexandrium bloom surveys. This specific objective was completed in years 1 and 2 of the project, and the results were presented in earlier reports. Data obtained from completing this objective has also been presented at several national and international meetings. A manuscript describing the results has been submitted for publication in the Journal of Plankton Research, and is currently in review.
Research related to this objective is ongoing in year 3, as we are continuing to collaborate with Emil Vahtera, a Post Doctoral Fellow in Don Anderson’s lab, on an ongoing in-situ grazing project. The project is focused on investigating toxic Alexandrium blooms in Nauset Marsh on Cape Cod, MA. This area is supported largely by shellfishing, but over recent years resource has had nearly annual PSP events. PSP is a recurrent problem in areas “downstream” of the marsh, and thus export of toxic Alexandrium cells from the system is a concern as well. One of the objectives of this work is to map the distribution and abundance of A. fundyense resting cysts within Nauset Marsh to determine the potential for local initiation of blooms. To do this, samples were collected in Spring 2010 with an emergence trap. The volume of water collected was sieved and washed with filtered seawater. The sample was backwashed into a 50 mL tube to collect zooplankton (if present) and stored on ice until returning to the lab. The sample was filtered onto a 5 μm Durapore filter approximately 1-2 h after collection and then stored in liquid nitrogen. Alexandrium cells were collected in a similar fashion, but the water sample was sieved through a 20 μm mesh after passing through the 80 μm mesh. Early counts suggested a loss of cells after emergence from the sediment. Given the large number of zooplankton that were observed in the collected samples, the suspicion was that the zooplankton had grazed the emerging cysts/cells. Zooplankton grazing on these cells could significantly affect estimates of cell emergence, and increase the detection limit for the approach.
To test whether zooplankton at the water-sediment interface had grazed emerging cells, we used the PCR assay optimized for this study to detect ingested Alexandrium. To begin DNA extractions, individual zooplankton (~20) were hand picked from the Durapore filter on a cold (< 0˚ C) dissecting microscope stage, inspected to ensure that no Alexandrium cells were stuck to appendages, and transferred to a screw-cap tube containing 0.4 mL Buffer ATL. Since these were field samples, there were, at times, different species of copepods. Only copepods of the same species and with visibly pigmented guts were collected. Copepods in buffer were stored at -20ºC until proceeding with subsequent DNA extraction steps.
Zooplankton samples were extracted using the DNeasy Blood and Tissue kit with a minor modification in the lysis procedure. Lysis was performed by adding ~250 μL zirconium/silica beads (0.5 mm diameter) to the thawed volume of Buffer ATL and vortexing (250 rpm) for 1 min. The resulting cell lysate was digested with proteinase K for 3.5 h at 55ºC with rotation in a hybridization oven. The resulting lysate was transferred to a clean tube and processed as per the remaining steps of the DNeasy protocol. The eluted DNA was stored at -20°C for later PCR analysis.
Ingested Alexandrium DNA was detected using PCR. PCR primers were designed to amplify a 183-bp sequence of the ribosomal large subunit (LSU) gene. The forward primer (5’-GCAAGTGCAACACTCCCACCAAGCAA-3’) was designed from an alignment of publicly available Alexandrium strain sequences. The reverse primer (5’-GCAAGTGCAACACTCCCACCAAGCAA-3’) was modified from a previously designed oligonucleotide (NA1), specific for toxic North American ribotypes of the Alexandrium fundyense/tamarense/catenella species complex. PCR amplification was performed using 5 μL gDNA in a final reaction mixture (25 μL) containing 10X PCR buffer, 0.25 mM deoxynucleoside triphosphates, 1.5 mM MgCl2, 150 nM of each forward and reverse primer, and 1 unit of Taq DNA polymerase. Reactions were cycled with an iCycler using a temperature profile of 98°C for 5 min, 60°C for 1 min (1X), 96°C for 30 s, 63°C for 30 s, and 72°C for 30 s (45X), and a final extension of 72°C for 7 min (1X). PCR products were resolved on a 2% agarose gel, stained with ethidium bromide, and imaged with a Gel Logic 440 imaging system.
Nine separate zooplankton field samples collected over a wide temporal range were investigated. The dominant copepod observed in all samples was a benthic harpacticoid. Work is ongoing to identify the genus and species of this copepod. With our PCR assay, no Alexandrium DNA was detected in any of these 9 field samples. This suggests that zooplankton grazers were not responsible for the loss of Alexandrium cells observed in the emergence samples. We are confident that PCR inhibition is not a problem, as a spike of the field samples with Alexandrium DNA was resolved with our assay. Work is ongoing, however, to investigate whether the lack of signal may be a result of DNA degradation and to look at grazing of Alexandrium cells by these benthic copepods within a laboratory setting to confirm ingestion. To our knowledge, little work has been done to investigate grazing on Alexandrium by benthic copepods.
Education/Outreach activities:
Outreach activities in the first few years of the project were focused on the Artistic Oceanographer Program (AOP). With the publication in year 2 of this work in Science and Children, teachers have contacted Dyhrman from SC, MA, CA, KY and WA about implementing the program in their school districts. After several years of the AOP, it remains a powerful instructional tool to introduce students to marine phytoplankton through the merging of art and science.
Future Activities:
We are currently operating in a no cost extension. We do not anticipate any major changes from the three outlined objectives. A strong focus will be the preparation of two additional manuscripts.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 7 publications | 3 publications in selected types | All 3 journal articles |
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Type | Citation | ||
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Haley ST, Dyhrman ST. The Artistic Oceanographer Program: encouraging ocean science literacy through multidisciplinary learning. Science and Children 2009;46(8):31-35. |
R833222 (2007) R833222 (2008) R833222 (Final) |
Exit Exit |
Supplemental Keywords:
RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Oceanography, algal blooms, Ecological Risk Assessment, marine ecosystem, bloom dynamics, HAB ecology, water qualityProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.