AQUATIC PATHOGENS IN MARINE SNOW

Got Snow? Tracking Marine Pathogens in Aquatic Ecosystems

M. Maille Lyons1*, J. Evan Ward1, Steven B. Roberts2, Roxanna Smolowitz3, Joseph Vallino3, Bassem Allam4
1
University of Connecticut, Groton, CT; 2 University of Washington, Seattle, WA; 3 Marine Biological Laboratory, Wood Hole, MA; 4 Stony Brook University, NY

Marine aggregates (i.e., marine snow, organic aggregates, organic detritus, flocs) are ubiquitous in aquatic ecosystems. The photomicrographs presented above demonstrate the diversity of marine
aggregate shape, size, porosity, and biological composition. Some are primarily unrecognizable detritus, but others have structures that elude to their mechanism of formation. All photos at 100x.

Abstract Data and Results

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Marine aggregates (i.e., marine snow, flocs, detritus) were evaluated for their role in the Examples of the type of data we have collected
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ecology of aquatic pathogens using underwater video surveys coupled with direct to evaluate the association between marine
n 120
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aggregates and aquatic pathogens. o e
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collection of aggregates in modified settling cones. Six locations, in New York, Macroaggregates (l )
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Macroaggregates
i 100 n 600
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Locations F (MA) and D (CT), clam habitats )t
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Microaggregates (ml -1) i 60
Microaggregates (
 500
a 80 h
Connecticut, and Massachusetts, were surveyed over 8 months to explore differences in MA
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CONCENTRATION: The concentration of i

the characteristics of aggregates found in habitats populated by clams (Mercenaria
ln a 300
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microaggregates (<500 µm) was always greater v
c 20
200

mercenaria) and oysters (Crassostrea virginica). Microaggregate (< 500 µm) than the concentration of macroaggregates.
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concentrations were always greater than macroaggregate (> 500 µm) concentrations, but n 120
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700
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PATCHINESS: Patchiness (variance to mean o Macroaggregates (l -1) e 

peak concentrations of macroaggregates and microaggregates, mean size of particles,
Macroaggregates
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Microaggregates (ml -1)
ratio for 3 videos) was greater for microaggregates t
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Microaggregates (
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and volume fraction of aggregated material varied among the six shallow-water habitats.
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and varied throughout the study. CT r
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i
Concentrations (colony forming units per ml) of total heterotrophic bacteria (THB) and total
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SIZE: The mean size of aggregates was always c 20
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mesophilic pathogenic bacteria (MPB) from samples of aggregates were significantly less than 500 µm.
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different among the four locations bordering Long Island Sound (LIS). The highest
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VOLUME FRACTION: On the next set of graphs, Concentration

concentrations and enrichment factors in aggregates were observed in August for THB volume fraction is a combination of mean
Patchiness Size
and June for MPB. Significant correlations were detected for salinity and the concentration and size.
concentrations and enrichment factors of THB in aggregates, and the concentrations and
percentages of MPB in seawater samples. Significant correlations were also detected for
temperature and the concentrations of MPB in aggregates and the enrichment factors for )
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ENRICHMENT FACTORS PERCENT PATHOGENIC BACTERIA
p
THB and MPB (marginal significance). Bacterial species identified in association with p
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aggregates included Vibrio cholerea, V. parahaemolyticus, V. vulnificus, V. alginolyticus, n

Oyster Clam t
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% Oyster Clam
Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, and Mycobacteria sp.
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Habitat Habitat
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These results have important implications for the way in which aquatic pathogens are c
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Aggregates
Seawater
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Seawater
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collected and quantified for risk-based surveillance in shallow-water ecosystems. f

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Study Locations
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i APR MAY JUN JUL AUG SEP OCT NOV APR MAY JUN JUL AUG SEP OCT NOV
t 200 500 200 500 e 50 50
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m 80 200 80 200 r
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Northern Quahog or Hard Clam l 40 100 40 100
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APR MAY JUN JUL AUG SEP OCT NOV
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APR MAY JUN JUL AUG SEP OCT NOV APR MAY JUN JUL AUG SEP OCT NOV

Suspension-feeding bivalves would have maximum exposure to The percentages of pathogenic bacteria in samples of marine
B A. NY oyster: Mattituck Inlet, NY aquatic pathogens (via marine aggregates) when a high volume aggregates (solid bars) were frequently higher than in samples
A B. NY clam: Hashamomuck Pond, NY fraction of aggregated material (black bars) overlaps with a high of seawater (striped bars) suggesting the importance of
C. CT oyster: Mystic River, CT enrichment factor of pathogenic bacteria (red bars). An example of monitoring marine aggregates in aquatic ecosystems. The
D. CT clam: Stonington Harbor, CT such exposure occurs at the CT oyster habitat in June, suggesting presence of pathogen-laden aggregates in environments prone
NY E. MA oyster: Martha’s Vineyard, MA the importance of tracking pathogens in aggregates during this part to disease outbreaks would suggest a means for the spread
F. MA clam: Barnstable Harbor, MA Eastern Oyster
1 in = 19.2 miles of the year. Whether or not this exposure translates into an and survival of pathogens, and would provide a target for
Crassostrea virginica
outbreak of disease will be the next step to evaluate. environmental surveillance of those pathogens.

Methods Conclusions
Quantifying Collection of Microbiological
Marine Aggregates Marine Aggregates Analysis Marine aggregates are ubiquitous so their distribution will overlap with a variety of hosts and
Marine Agar ChromAgar Vibrio
pathogens. This research highlights the importance of evaluating marine aggregates as potential
plates plates reservoirs and vectors for aquatic pathogens.

Imhoff
Settling Cone
Marine Marine
Using microbiological techniques, the bacterial species identified in association with marine
aggregate
sample
aggregate
sample
aggregates included Vibrio cholerea, V. parahaemolyticus, V. vulnificus, V. alginolyticus,
Aeromonas hydrophila, Pseudomonas aeruginosa. Also identified, but not included in total counts
because of their scarcity, were Shigella sonnei, Stenotrophomonas maltophila, Photobacterium
damsela, and Burkholderia cepacia. Virulence was not assessed.
Seawater Seawater
sample sample

15 ml Samples of marine aggregates and Data from all six location is available:
Falcon Tube seawater were plated (in triplicate) on two
types of agar: Marine Agar (for culturable Lyons, M. M., Y. Lau, W. E. Carden, J. E. Ward, S. B. Roberts, R. Smolowitz, J. Vallino, B. Allam.
heterotrophic bacteria) and ChromAgar 2007. Characteristics of marine aggregates in shallow-water ecosystems: Implications for
Vibrio (for culturable pathogenic bacteria).
Marine aggregates were quantified Marine aggregates were collected in disease ecology. EcoHealth 4(4): 406-420
using an underwater camera and settling cones with Falcon tubes Enrichment factors for both heterotrophic
image analysis as described in attached to the bottom as described in bacteria and pathogenic bacteria were

Acknowledgements
(Lyons et al., 2007) (Lyons et al., 2005) calculated as the concentration (CFUs/ml)
of bacteria in aggregates divided by the
Many flocs concentration of bacteria in seawater
(YELLOW ARROWS).
The authors thank Y. Lau, W. Carden, C. Romano, R. Bogin, M. Perrigault, and S. Dahl for laboratory assistance with processing samples and T.
The percentage of pathogenic bacteria
Marcotti, K. Uhlinger, B. Holohan, D. Kach, D. Frank, J. Thiel, and P. Bagnell for assistance with field collections. We also thank B. MacDonald and A.
Few flocs for both marine aggregates and seawater Bennett for recommendations on image processing and T. Getchis (CT Seagrant) for assistance with outreach. This research was funded by an NSF
was calculated as the concentration of Graduate Research Fellowship to M. Lyons and NSF-NIH Ecology of Infectious Disease Grants to J. E. Ward (0429004), R. Smolowitz and J. Vallino
2 examples of digital still Species composition of bacteria on
bacteria on ChromAgar Vibrio plates (0429018), and B. Allam (0429051) along with a NOAA Grant to B. Allam (R/FBF-17 award NA16RG1645) and an NRAC Grant to R. Smolowitz.
frames grabbed from analog ChromAgar Vibrio plates was determined
divided by the concentration of bacteria on
videos. Each square is 1 cm2 biochemically using API 20E (Biomerieux)
the Marine Agar plates (BLUE ARROWS). *Corresponding author: M. Lyons at [email protected]