Optimized Separation of Estuarine Plankton to Determine Associations with Vibrio SpeciesE. Shultz1,2, E. Deyett1,2, M. Hartwick1,2, E. Urquhart1,2, and S. Jones1,3

IntroductionVibrio related illness is a complex public health problem that is intrinsically linked

to ecosystem conditions that support the proliferation of pathogenic strains. Since

1996, the rate of reported Vibrio-related food borne illness in the United States has

increased by 115%. Most transmission occurs indirectly through contaminated

seafood consumption, especially raw oysters. Nutrient-rich microenvironments

associated with plankton selectively enrich Vibrio species to higher densities than

the surrounding water column, and Vibrio parahaemolyticus is highly associated

with zooplankton blooms. To study the interactions between Vibrio species and

plankton in estuaries, a series of experiments were designed to adapt a fresh water

plankton separator created by Nancy Leland under the direction of Dr. Jim Haney

of UNH, for use in an estuarine ecosystem. The plankton separation device utilizes

phototactic behavior to separate phytoplankton from zooplankton with minimal

intermixed contamination to allow quantification of Vibrio concentrations in

separated plankton samples. Optimization of this method required repeated study

over discreet time periods to determine the purity of plankton separation and the

effectiveness of this method.

Figure 1. Protocol for sampling for plankton time trial separation

Results and DiscussionA key outcome of this initial study is that the separation time with the least cross-

species contamination occurs between 30-40 minutes. On the 07/08/2014

sampling date, there was an initial quality separation at 10 minutes, followed by

negative phototaxis of the zooplankton from the zooplankton fraction up into the

phytoplankton fraction, but quality separation was achieved between 30-40

minutes. On the 07/28/2014 sampling date, standard deviations and variability in

data were high, but provided valuable data. There was no separation from 0-20

minutes, zooplankton and phytoplankton were moving back and forth between

fractions.

The current results provide important starting points for continuing to optimize

both the methods and the separation time needed to efficiently separate

phytoplankton species from zooplankton species in estuarine ecosystems. The

upcoming sampling season will benefit from this study because it will help

produce valuable data for understanding the association of plankton species with

Vibrio species in order to determine its potential use in the monitoring of Vibrio

species. The methods outlined in this current study could be utilized in current

empirical modeling methods in coastal regions enabling prediction and prevention

of disease outbreaks from contaminated shellfish. This is a mathematical

representation that attempts to model how plankton species behave in the natural

world, and further how they can be used as a proxy for Vibrio species

concentrations in coastal waters and shellfish found in those waters. This model

takes into consideration environmental factors, nutrient availability and uptake,

community composition, and human influence. Future research will include an in-

depth look at seasonal shifts, same-day tide comparisons, and taxonomic shifts in

plankton composition in order to determine how those relate to Vibrio species

populations and rate of human exposure.

Objectives1. Develop and standardize methods for using a fresh water plankton separator in

estuarine environments.

2. Determine the optimum plankton separation time in estuarine environments.

3. Determine efficacy of separated plankton species to inform molecular and

remote sensing applications.

MethodsChlorophyll a and phycocyanin concentrations were determined by fluorometry

and evaluated at varying time periods on two different sample dates to identify

optimal separation time, where there is the least cross-contamination between

phytoplankton and zooplankton. This optimum time corresponds to when there is

the lowest chlorophyll a concentration in the zooplankton fraction and the highest

chlorophyll a concentration in the phytoplankton fraction. Separated samples were

massed to determine zooplankton mass and thus the time required for the most

zooplankton to move out of the phytoplankton fraction into the zooplankton

fraction via phototaxis. Community analysis was used to determine the

concentration and make-up of zooplankton species in the zooplankton fractions.

Community analysis was accomplished by using an Amscope compound

microscope, counting a minimum of 200 individuals per subsample volume,

taxonomically sorting each individual into its genus/species, and then adjusting the

count data to reflect the total sample volume.

Figure 2: Map of Adams Point and Jackson Estuarine Laboratory in Durham, NH.

Phytoplankton

Zooplankton

Shellfish

Vibrio species

Vibriosis

Water

Sediment

Why does this Research Matter?

1 Northeast Center for Vibrio Disease and Ecology, University of New Hampshire, Durham, NH

2 Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH

3 Department of Natural Resources and the Environment, University of New Hampshire, Durham, NH

Figure 3: Separation efficiency graphs where phytoplankton concentrations are represented by Chloropyll a and Phycocyanin, and zooplankton concentrations are represented by

community analysis counts sorted into Acartia hudsonia, Pseudocalanus, Copepodites, and Nauplii. Quality separation is achieved between 30-40 minutes; no quality separation is

seen from 0-20 minutes.

AcknowledgementsA special thank you to Amanda Murby,

Nancy Leland, Stephanie Rodriguez,

Jackie Lemaire, and Jim Haney PhD

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Figure 1: Ecology of Vibrio species.

Figure 4: Collection, processing, and separation methodology.