Grantee Research Project Results
Final 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 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 was to develop a quantitative PCR-based method to assay zooplankton predation rates on harmful algae. The specific objectives were to: 1) optimize the qPCR assay for quantitative detection of ingested Alexandrium, 2) calibrate and test the qPCR-based measure of predation rate in laboratory experiments, and 3) apply the qPCR-based technique to quantify predation by copepods on an in-situ Alexandrium bloom.
Summary/Accomplishments (Outputs/Outcomes):
With EPA support, we are pleased to report the cumulative progress made and summary of findings in relation to our proposed objectives.
Obj. 1) Optimize the PCR-based assay for quantitative detection of ingested Alexandrium:
To find the best DNA extraction procedure, we compared three different, commonly-used, commercial DNA extraction kits for their ability to quantitatively recover high-quality DNA from Alexandrium cells. We followed the manufacturer’s instructions for each kit and also tested the effectiveness of modified extraction protocols. Using the ratio of light absorbance at 260 and 280 nm to assess the purity of nucleic acids and the extraction efficiency (based on ~ 200 pg DNA/cell) a modified procedure, based on the DNeasy Kit, provided both the highest quantitative DNA recovery (> 90% of Alexandrium DNA) and also the highest DNA purity of all procedures tested. We also tested a number of modifications to a standard PCR reaction, including modifying the initial amount of template, the PCR cycle number, and the annealing temperature before settling on a set of optimized conditions that provided the best signal to noise for detecting our target Alexandrium sequence. Standard curves prepared using hand-picked Alexandrium cells provided high r2 (> 0.99) and also showed high qPCR efficiency (E = 1.95). It should be noted that although we could demonstrate that the DNA of our experimental predators did not inhibit PCR reactions needed to amplify and quantify Alexandrium DNA, including predator DNA with our standards did alter the standard curves. Inclusion of predator DNA in the standards did not change the standard curve slope, but did shift the y-intercept. This is an important methodological point that is particularly critical for quantification of low cell numbers.
Additional work tested amplification and signal recovery from Alexandrium cells ingested by a broader range of zooplankton predators than originally proposed. We can now demonstrate recovery of Alexandrium DNA after feeding experiments from predators as diverse as copepods, snail veliger larvae, blue mussels and ciliates. Predators were incubated with and without Alexandrium prey. Amplifiable DNA was recovered by PCR from all of the different zooplankton predators tested that had fed on Alexandrium. No Alexandrium DNA was recovered from predators that did not feed on the dinoflagellate. This work demonstrated the potentially broad applicability of the concept underlying the project, although our work also brought to light important limitations that have to be considered (see below).
A final step in the optimization of the PCR-based assay was to resolve the degree of variability of the target gene (LSU rRNA) among different Alexandrium strains, and 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. To address this, Alexandrium fundyense cells were grown under 5 conditions: A) nutrient replete (L1 media), B) low nitrogen (20 µM total N), C) low phosphorus (2 µM), D) nitrogen re-fed (N-amended after nitrogen depletion), and E) phosphorus re-fed (P-amended after P depletion). To assess differences in gene copy number brought on by nutrient limitation/growth phase, individual Alexandrium cells (~50 per treatment) were transferred one by one to Buffer ATL for DNA extraction and quantification of the rRNA gene using qPCR. There was no difference in the gene copy number between the different nutrient conditions (ANOVA, p = 0.1165). At present, few data are available in the literature concerning the number of rRNA gene copies per cell in dinoflagellates, yet variability in copy number could affect the level of accuracy of the quantitative real-time PCR assays. The above results related to Obj. 1 were presented at several scientific meetings and will be further disseminated through a manuscript that is currently in prep and nearing submission to the peer-reviewed journal, Limnology and Oceanography: Methods.
Obj. 2) Calibrate and test the qPCR-based measure of predation rate in laboratory experiments:
To test the effectiveness of qPCR as a tool to measure zooplankton predation rates on harmful algae, laboratory experiments were initially conducted using the copepod grazer, Acartia hudsonica, fed a sole diet of Alexandrium (GTCA01). Ingestion rates were calculated by measuring cell loss over time using microscopic cell counts, and were determined to be comparable to other studies. For qPCR analysis of ingestion rate, A. hudsonica individuals were hand picked, cleaned of any Alexandrium cells that were stuck to appendages, and these individuals were processed for DNA extraction. qPCR amplification of standards and samples were performed in triplicate in a real-time detection system. Cell numbers of total ingested Alexandrium cells for each copepod sample were determined through a comparison to the standard curve. The ingestion rates determined by qPCR of gut content was much lower (~0.01 cells copepod-1) than the rates calculated by cell counts. Moreover, this rate did not increase with prey concentration. It is worth emphasizing that there was a recoverable Alexandrium signal in the fed copepods, but the signal was too low to be quantitatively related to ingestion rate. This led us to assess potential grazer differences. We tested another zooplankton predator, Nassarius veliger larvae, with a similar experimental set-up. Ingestion rates were calculated as above and were similar to other studies. Veligers were collected for DNA extraction and qPCR analysis using the same method as for the copepods. Approximately 20%-40% of the ingested Alexandrium DNA was recovered from veliger gut contents as measured by qPCR. This is in sharp contrast to the copepods, in which less than 0.1% of ingested DNA was detected by qPCR. Moreover, unlike the copepods, Alexandrium DNA in veliger gut contents did increase with increasing prey concentration and was quantitatively related to ingestion rate (r2 = 0.69). It is hypothesized that the difference in recoverable Alexandrium DNA between the two predator types was a result of their different feeding strategies. Veliger larvae ingest prey cells whole, and digestion is internal. However, copepods tend to macerate prey, and prey DNA may be lost through sloppy feeding or more rapid digestion. To test this hypothesis, we ran grazing experiments with a tintinnid ciliate, Favella sp., which also directly engulf whole dinoflagellate cells. These experiments yielded results similar to the veligers, with a quantitative relationship between gut content and ingestion rate. These experiments suggest that feeding strategy and digestion play a significant role in the recovery of detectable prey DNA. Results related to Obj. 2 were presented at several scientific meetings and will be further disseminated through a manuscript that is currently in prep and nearing submission to the peer-reviewed journal, Limnology and Oceanography: Methods.
Obj. 3) Apply the qPCR-based grazing technique to quantify grazing by copepods on an in-situ Alexandrium bloom:
We collaborated with the Anderson lab (WHOI) for collection of copepod samples from the Gulf of Maine during Alexandrium bloom surveys. Of the samples collected, the dominant copepod was Calanus finmarchicus. Alexandrium counts corresponding to the copepod samples ranged from undetectable to <1000 cells per L. Qualitative PCR was used to detect recent ingestion of Alexandrium cells by the copepods and results indicated that C. finmarchicus will ingest Alexandrium cells whenever they are present, apparently even when Alexandrium concentrations are as low as ~ 10 cells L-1. Such low concentrations could represent the earliest stages of incipient blooms. These results demonstrate that Calanus graze Alexandrium cells even when the Alexandrium are at extremely low concentration, although the predation rates are likely very low. Nevertheless, using a simple model of Alexandrium growth, we demonstrated that even very low predation rates could contribute to significant inhibition of net Alexandrium population growth during the early stages of bloom formation. The general concept, that low predation rates can have a large impact on small prey populations, is potentially important for modeling other types of prey populations. Models of phytoplankton blooms generally recognize phytoplankton mortality as a key parameter that is nevertheless commonly oversimplified. Specifically, models that parameterize predation loss as proportional to population size will not show the sensitivity to predation at low population size demonstrated to occur here. This work may therefore be used to improve how predation losses for HABs and other phytoplankton populations are represented in predictive models. Results related to this objective were presented at an international HAB meeting and were recently published in the Journal of Plankton Research.
We continued to collaborate with the Anderson lab on an ongoing in-situ grazing project focused on investigating toxic Alexandrium blooms in Nauset Marsh on Cape Cod, MA. This area is economically supported largely by shellfishing, but over recent years the resource has had nearly annual PSP events. PSP is a recurrent problem in areas “down-current” from 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. Based on early work, it was suspected that emerging cells/cysts had been lost to zooplankton grazing. Using our PCR assay, we tested whether zooplankton at the water-sediment interface had grazed emerging cells. Zooplankton samples were extracted using the DNeasy kit and the potential for ingested Alexandrium DNA in the copepods was assessed using PCR. Our PCR assay did not detect Alexandrium DNA in any field samples. This suggests that zooplankton grazers were not responsible for the loss of Alexandrium cells observed in the emergence samples. To our knowledge, this is the first work to investigate grazing on Alexandrium by benthic copepods. It also shows the applicability and ease of application of the PCR approach for investigating grazing by a wide range of predators in different habitats.
The project also supported completion of related work studying the uptake, retention, and depuration of PSP toxins from Alexandrium by Acartia copepods. This work was conducted in the lab of Prof. Hans Dam of the Univ. of Connecticut. Our project supported final analyses and write up of the work. Two Acartia populations, one that co-occurs with Alexandrium in the Gulf of Maine, and one that does not (New Jersey), were compared to understand the physiological mechanism underlying resistance to PSP toxins in the Maine population. The Acartia population from Maine retains less of the PSP toxins from ingested Alexandrium prey than the naive population from New Jersey. This result helps explain how copepods are able to continue grazing on Alexandrium populations despite the PSP toxins the Alexandrium produce. It also is valuable in terms of predicting toxin transfer up the food chain from Alexandrium to copepods to higher trophic level organisms. Results of this work were recently published in the peer-reviewed journal, Marine Ecology Progress Series.
In summary, zooplankton predation on harmful algae is a critical but poorly constrained factor in HAB development and termination. Our qPCR-based method yielded different results depending on the predator:prey combination investigated. It was hypothesized that the difference in recoverable Alexandrium DNA between the different predator types was a result of their different feeding strategies (e.g., maceration, engulfment). These results suggest that feeding strategy and digestion play a significant role in the recovery of detectable prey DNA. Therefore, the utility of the qPCR-based approach may depend on the feeding strategy of the predator being investigated. The method and approach developed here could be tailored for use in other systems, with the appropriate understanding of its limitations.
Despite the limitations of the quantitative PCR approach, using qualitative PCR to detect the presence/absence of Alexandrium DNA in potential predators proved to be very successful. The method was tested in a broad range of predators in the lab and applied to field populations of copepods. Particularly useful was our ability to detect ingestion of prey even at exceedingly low prey concentrations. This would be extremely challenging without the sensitivity provided by the PCR-based method. At low prey concentrations, ingestion rates on Alexandrium may have been as low as 1 cell copepod-1day-1. Nevertheless, simulations of Alexandrium population growth suggest that a few predators L-1 have the potential to curb the early development of a slow-growing bloom, even if ingestion rates are extremely low. Low predation rates can still have a large impact when prey populations are small. These results may lead to improved representation of phytoplankton mortality in predictive models of bloom formation, thereby aiding the forecasting and mitigation of Alexandrium blooms by coastal managers.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 7 publications | 3 publications in selected types | All 3 journal articles |
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Dam HG, Haley ST. Comparative dynamics of paralytic shellfish toxins (PST) in a tolerant and susceptible population of the copepod Acartia hudsonica. Harmful Algae 2011;10(3):245-253. |
R833222 (Final) |
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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) |
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Haley ST, Juhl AR, Keafer BA, Anderson DM, Dyhrman ST. Detecting copepod grazing on low-concentration populations of Alexandrium fundyense using PCR identification of ingested prey. Journal of Plankton Research 2011;33(6):927-936. |
R833222 (Final) |
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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.