ECOHAB: Van Dolah_F- Karenia brevis Cell Cycle Analysis for Determination of In Situ Growth Rates (NODC Accession 0000538)

The reported growth rates of Karenia brevis vary from 0.2 to
0.5 divisions per day, both in laboratory and field
populations observed. This growth rate alone is not
sufficiently high to account for its dominance in the water
column. However, careful studies have not been carried out
to determine if the documented slow growth rate of K. brevis
persists throughout bloom development, or if different rates
of growth might characterize bloom initiation, growth,
maintenance, and termination phases. For example, it is
conceivable that an "explosive" growth stage occurs early
during bloom initiation/growth stages that serves to boost
the population size, which has not been previously
documented. Like most autotrophic dinoflagellates studied,
cell division in K. brevis blooms is phased to the diel
cycle. Diel phasing of cell division imposes a maximum
potential growth rate of 1 division per day in
dinoflagellates. The occurrence of an "explosive growth
stage" would require the release of K. brevis cells from
mechanisms which regulate this circadian rhythm.
To address this question, diel phasing of the cell cycle of
K. brevis was first documented in laboratory isolates of K.
brevis. In situ diel studies were then carried out on
naturally occurring blooms of K. brevis during the R/V
Pelican cruise (1996) and during ECOHAB process cruises
carried out during years 1-5 of ECOHAB Florida (1997-2001).
Cell cycle distribution of K. brevis cells and correlation
of cell cycle events with vertical migration was determined
by sampling at multiple depths (minimum of surface and
bottom, depending on depth). For shipboard field studies,
whole water samples of K. brevis (1-2 L) were collected
every 2 or 3 h throughout a diel cycle, fixed in
glutaraldehyde and stored in the dark. Further processing of
samples was carried out in the laboratory following
completion of the cruise. The flow cytometry method method
of Van Dolah and Leighfield was used (1999. J. Phycology
35:1404-1411). Glutaraldehyde-fixed whole water samples were
filtered through a 100 micrometer nitex screen to remove large
phytoplankton/zooplankton/debris, then filtered by gravity
flow through a 10 micrometer nylon screen to collect K. brevis
cells. Cells were collected from the screen by rinsing with
2% glutaraldehyde into a 50 ml polypropylene centrifuge
tube, then centrifuged at 1000 x g. The cell pellet was then
treated with 2 ml of 20 degrees C methanol overnight to
remove pigments. Methanol extracted cells were collected by
centrifugation (500xg for 3 min) and resuspended in
phosphate buffered saline + 0.5% Tween 20 containing 10 micrograms.
mL-1 propidium iodide (PI, Sigma, St. Louis, MO) and 10
mg/ml Rnase (Sigma, St. Louis, MO). DNA analysis was carried
out on an Epics MXL4 flow cytometer (Coulter, Miami, FL)
using a 5 W argon laser with a 488 nm excitation wavelength
and 635 nm emission wavelength. Cell aggregates were
eliminated by gating all histograms within the linear range
using a peak-area cytogram for PI fluorescence. Cell cycle
distribution was analyzed using Multicycle software (Phoenix
Flow Systems, San Diego, CA).

Purpose:
The research conducted in this portion of the Florida ECOHAB
Program will provide insight into which the role of
endogenous cellular rhythms in regulating the accumulation
growth phase of K. brevis blooms decline by conducting cell
cycle studies. This work entails both both laboratory and
field experiments. Laboratory studies will be carried out to
identify cell cycle regulatory mechanisms in K. brevis,
using western blotting to identify cyclins and cyclin
dependent kinases in K. brevis, and to determine their
relative levels of expression in actively growing versus
stationary phase K. brevis populations. In addition we will
characterize their expression during the course of the
circadian day, as it correlates with cell cycle phase. In
situ labeling of K. brevis cells with fluorescently labeled
antibodies to cyclins will assist in discerning whether
cyclin expression might be suitable for use as a biomarker
of actively dividing blooms in the field. Field studies will
use flow cytometry to determine in situ growth rates of
blooms of K. brevis during the yearly ECOHAB process cruises
in order to establish a multiyear record of growth rates in
early and late bloom stages.