121
(two diffusion intensities (forward angle and
sideward light scatter) and three fl uorescence
intensities (red, orange, and yellow fl uorescences))
are recorded for each single particle (cell). Cells
sharing similar optical properties are therefore
grouped together when plotted in 2D projections
of the numerous variables collected by the fl ow
cytometer (2 light scatter and three fl uorescences,
with for each one the area under the curve, the
peak and length of the curve, etc.). In addition, an
“image-in-fl ow” device mounted in the Cytosense
takes pictures of cells of interest after their passage
through the 488 nm laser beam.
In parallel to fl ow cytometry analyses, hydrological variables were also measured by automated
sensors (ISUS for nitrate concentration and
Hydrolab probe for temperature, salinity, turbidity,
chlorophyll a content, pH) in the water pumped
from the sampling point (43°28′10.57 N, 5°10′9.91
E, 2.5 m depth) to the fi eld laboratory. Water was
carried out to the laboratory through a 250 m hose
(50 mm inner diameter) at a fl ow rate of 30 dm
3
/
min during 17 min before each analysis (in order
to completely fl ush the entire hose). Each hour,
temperature, salinity, turbidity, pH, nitrate, and
chlorophyll a concentrations were measured by
the various sensors installed in an 80 dm
3 tank
receiving the pumped water. A dedicated volume
of 1 dm
3
, set up between the pipe and the 80 dm
3
tank, was used for the fl ow cytometry analyses
performed by the Cytosense. An in situ HOBO
®
Pendant
® Temperature/Light Data Logger has been
installed next to the pipe inlet in order to measure
the incident light intensity at the very sampling
depth. A strainer brass was set in situ at the end of
the hose to prevent biofouling and the passage of
larger particles (several millimeters long) which
may clog the hose and/or the instruments.
4
Results
4.1
Phytoplankton Clusters
Defi ned by Flow Cytometry
During the October 2011 monitoring, up to 12
separate clusters of photoautotrophic cells
(arbitrary labeled C1 to C12) have been resolved
thanks to their optical properties recorded by the
scanning fl ow cytometer (Fig. 3 ). Clusters showed
a proportional relation between light scatter
(related to cell size) and red fl uorescence (related
to chlorophyll a pigment content) intensities.
The length of the cells has been determined both
from the pictures taken by the image-in-fl ow and
from calibration microspheres (Polysciences) of various sizes (from 1 to 20 µm in diameter). It covered a
large range of sizes, from 0.9 ± 0.1 µm (for C12) to
56.4 ± 12.2 µm (for C1) (Table 1 ). The largest cells
pictured by the image-in-fl ow camera correspond to
clusters C1 to C4 and belong to the microphytoplankton class. As revealed by the pictures, they were
monospecifi c and composed of Akashiwo sanguinea (Hirasaka) for C1, Prorocentrum micans
(Ehrenberg) for C2, Scrippsiella sp. (Balech) for
C3, and Gymnodinium sp. (Stein) for C4.
4.2
Dynamics of Akashiwo
sanguinea
and of the Environmental
Variables During
the Sampling Period
The chlorophyll a concentration of the overall
phytoplanktonic community strongly varied in
the Berre lagoon during the sampling period,
with maxima correlated to high nitrate concentrations ( n = 215, correlation coeffi cient 0.53,
p < 0.001). Proliferation of microphytoplanktonic
cells, including A. sanguinea , has led to several
peaks of chlorophyll a concentration which
reached up to 21.81 µg/dm
3 on October 5 at
05:30 pm. Such high biomass have not been
detected by the monthly monitoring carried out
in the Berre lagoon; actually, in October the
lowest concentration has been measured (Fig. 4 ).
During the sampling period, one major forcing
that occurred in the lagoon was a turbulent
mixing induced by a strong mistral event (wind
from 330° to 360° from October 6 to 10) (Fig. 5 ).
The mean temperature of the water dropped
down by 5.2 °C (from 23.2 ± 0.2 °C to 18.0 ± 0.3 °C
between October 6 and 7). Nitrates and micro- and
nanophytoplankton concentrations decreased
and Akashiwo sanguinea abundance dropped
Monitoring of a Potential Harmful Algal Species in the Berre Lagoon by Automated…
(two diffusion intensities (forward angle and
sideward light scatter) and three fl uorescence
intensities (red, orange, and yellow fl uorescences))
are recorded for each single particle (cell). Cells
sharing similar optical properties are therefore
grouped together when plotted in 2D projections
of the numerous variables collected by the fl ow
cytometer (2 light scatter and three fl uorescences,
with for each one the area under the curve, the
peak and length of the curve, etc.). In addition, an
“image-in-fl ow” device mounted in the Cytosense
takes pictures of cells of interest after their passage
through the 488 nm laser beam.
In parallel to fl ow cytometry analyses, hydrological variables were also measured by automated
sensors (ISUS for nitrate concentration and
Hydrolab probe for temperature, salinity, turbidity,
chlorophyll a content, pH) in the water pumped
from the sampling point (43°28′10.57 N, 5°10′9.91
E, 2.5 m depth) to the fi eld laboratory. Water was
carried out to the laboratory through a 250 m hose
(50 mm inner diameter) at a fl ow rate of 30 dm
3
/
min during 17 min before each analysis (in order
to completely fl ush the entire hose). Each hour,
temperature, salinity, turbidity, pH, nitrate, and
chlorophyll a concentrations were measured by
the various sensors installed in an 80 dm
3 tank
receiving the pumped water. A dedicated volume
of 1 dm
3
, set up between the pipe and the 80 dm
3
tank, was used for the fl ow cytometry analyses
performed by the Cytosense. An in situ HOBO
®
Pendant
® Temperature/Light Data Logger has been
installed next to the pipe inlet in order to measure
the incident light intensity at the very sampling
depth. A strainer brass was set in situ at the end of
the hose to prevent biofouling and the passage of
larger particles (several millimeters long) which
may clog the hose and/or the instruments.
4
Results
4.1
Phytoplankton Clusters
Defi ned by Flow Cytometry
During the October 2011 monitoring, up to 12
separate clusters of photoautotrophic cells
(arbitrary labeled C1 to C12) have been resolved
thanks to their optical properties recorded by the
scanning fl ow cytometer (Fig. 3 ). Clusters showed
a proportional relation between light scatter
(related to cell size) and red fl uorescence (related
to chlorophyll a pigment content) intensities.
The length of the cells has been determined both
from the pictures taken by the image-in-fl ow and
from calibration microspheres (Polysciences) of various sizes (from 1 to 20 µm in diameter). It covered a
large range of sizes, from 0.9 ± 0.1 µm (for C12) to
56.4 ± 12.2 µm (for C1) (Table 1 ). The largest cells
pictured by the image-in-fl ow camera correspond to
clusters C1 to C4 and belong to the microphytoplankton class. As revealed by the pictures, they were
monospecifi c and composed of Akashiwo sanguinea (Hirasaka) for C1, Prorocentrum micans
(Ehrenberg) for C2, Scrippsiella sp. (Balech) for
C3, and Gymnodinium sp. (Stein) for C4.
4.2
Dynamics of Akashiwo
sanguinea
and of the Environmental
Variables During
the Sampling Period
The chlorophyll a concentration of the overall
phytoplanktonic community strongly varied in
the Berre lagoon during the sampling period,
with maxima correlated to high nitrate concentrations ( n = 215, correlation coeffi cient 0.53,
p < 0.001). Proliferation of microphytoplanktonic
cells, including A. sanguinea , has led to several
peaks of chlorophyll a concentration which
reached up to 21.81 µg/dm
3 on October 5 at
05:30 pm. Such high biomass have not been
detected by the monthly monitoring carried out
in the Berre lagoon; actually, in October the
lowest concentration has been measured (Fig. 4 ).
During the sampling period, one major forcing
that occurred in the lagoon was a turbulent
mixing induced by a strong mistral event (wind
from 330° to 360° from October 6 to 10) (Fig. 5 ).
The mean temperature of the water dropped
down by 5.2 °C (from 23.2 ± 0.2 °C to 18.0 ± 0.3 °C
between October 6 and 7). Nitrates and micro- and
nanophytoplankton concentrations decreased
and Akashiwo sanguinea abundance dropped
Monitoring of a Potential Harmful Algal Species in the Berre Lagoon by Automated…