Introduction
Emiliania huxleyi (Lohmann) Hay and Mohler is a cosmopolitan
, genetically diverse
,
morphologically variable marine
photosynthetic and calcifying unicellular haptophyte
algae species and the most abundant of the coccolithophores. It produces
calcite (CaCO3) plates called coccoliths which cover the cell. As a
photosynthetic organism, E. huxleyi shifts the seawater carbonate
system towards [CO32-], but as a calcifier it shifts the seawater
carbonate system towards [CO2]. Therefore, part of the interest in
E. huxleyi derives from its role in the global carbon cycle.
In particular, extensive blooms might impact
air–sea gas exchange . Climate-change-induced surface water stratification was shown to trigger E. huxleyi blooms .
The ratio of particulate inorganic carbon (PIC) and particulate organic
carbon (POC) influences surface water–atmosphere gas exchange as well as the
composition of matter exported from surface waters to the deep ocean
. The response of PIC and POC production and
their ratio in the prolific species E. huxleyi to temperature is a
necessary first step towards an understanding of its possible impact on
global biogeochemical cycles.
The relationship of PIC production/PIC : POC and temperature in E. huxleyi is not clear. found that PIC production was
higher at lower temperatures in a strain grown at 13 and 18 ∘C,
while found the opposite in another strain grown at 10,
15 and 20 ∘C. found higher PIC : POC ratios at
lower temperatures for a strain of E. huxleyi and
found a similar relationship for a strain of the species
Coccolithus pelagicus. , however, found a different
relationship for the PIC : POC ratio in another strain of E. huxleyi, which is not supported by the experiment of on
the same strain. did not find differences in the PIC : POC
ratio in another strain grown at 20 and 24 ∘C. These discrepancies
between studies might stem from different experimental setups and a lack of
knowledge of the optimum growth temperature or indeed strain-specific
differences . Therefore, it is necessary to test more than
one strain for its temperature response under otherwise identical conditions.
This we have done in the present study.
Apart from biogeochemical considerations, global warming might also be of
interest in terms of the ecological success of coccolithophores, because
different groups of organisms might be differently affected by warming and
therefore ecological succession patterns, grazing pressure etc. might
change. The latter was proposed to depend on coccolith morphology more than
it does on PIC production . The effect of temperature on
coccolith morphogenesis is evident in field observations
and is best assessed with respect to the
optimum growth temperature in laboratory experiments. While the effect of
supra-optimal temperature is unequivocally detrimental
, it is not clear whether there is an effect of
sub-optimal temperature at all . A
temperature increase in the sub-optimal range is probably what most
coccolithophore clones will experience in the course of global warming
this study, and therefore
this temperature range is particularly interesting. In the present study we
focus on coccolith morphology under sub-optimal temperature, doubling the
amount of data currently available, and thereby clarifying whether
sub-optimal temperatures can cause malformations. We selected three strains
of E. huxleyi from a single area, the Japanese coast in the North
Pacific Ocean, in order to assess the plasticity within strains originating
from a particular environmental setting.
Materials and methods
Pre-culture and batch culture experiments
Clonal cultures of Emiliania huxleyi were obtained from the Roscoff
Culture Collection. We selected three strains of E. huxleyi, two
from the Japanese coast in the North Pacific Ocean (RCC1710 – synonym of NG1
and RCC1252 – synonym of AC678 and MT0610E) and a third strain from the
same region but of unknown exact origin and strain name, named here IAN01.
Strain RCC1710 was collected off Nagasaki at Tsushima Strait (Japan) and
RCC1252 at Tsugaru Strait (Japan); both places are strongly influenced by
the Tsushima warm current. Additional information about the strain RCC1252
can be found at http://roscoff-culture-collection.org/.
The culture media was sterile-filtered North Sea water (filtered through
0.2 µm pore size sterile Sartobran 300 filter cartridges,
Sartorius, Germany) supplemented with nutrients (nitrate and phosphate),
metals and vitamins according to . Cell densities were
determined using a Multisizer 3 Coulter Counter (Beckman Coulter for particle
characterization). To prevent significant changes in seawater carbonate
chemistry, maximum cell densities were limited to ≈1×105cellsmL-1 e.g.. We used a 16/8
light/dark cycle, and an irradiance of ≈300µmolphotonss-1m-2. The three strains were grown for at least 20
generations.
The dilute batch culture experiments were conducted in triplicate, for the
strains RCC1710 and RCC1252 at 10, 15, 20 and 25 ∘C of temperature,
and for IAN01 at 15, 20 and 25 ∘C. The strains were grown in 2 L of sea water within
transparent sterilized 2.3 L glass bottles. Cell
density at inoculation was 500 to 1000 cellsmL-1, and at harvest it was a maximum of 1×105cellsmL-1. Harvesting was done 9 h after the onset of the
light period.
Growth rate was calculated from exponential regression according to
µ=(lnc1-lnc0)Δt-1,
where c1 and c0 are the final cell concentration and the initial
cell concentration, respectively, and Δt is the duration of
incubation in days. Averages of triplicates and SD were used in tables and
figures (Table and Fig. a).
Growth rate and cellular PIC, POC, and TPN content and production
of the three strains of E. huxleyi at different temperatures.
Standard deviation of the triplicates in parentheses. Measured growth rates
for extra temperatures from the pre-experiments are included, but PIC, POC
and TPN were not measured for these temperatures.
Strain
T
Growth rate
PIC
POC
TPN
PPIC
PPOC
PTPN
(∘C)
(μ)
(pgcell-1)
(pgcell-1)
(pgcell-1)
(pgcell-1d-1)
(pgcell-1d-1)
(pgcell-1d-1)
RCC1710
6.5
0.19
RCC1710
10
0.26 (0.00)
15.31 (0.15)
8.91 (0.29)
1.54 (0.07)
3.98 (0.03)
2.32 (0.08)
0.40 (0.01)
RCC1710
15
0.75 (0.01)
14.07 (0.40)
9.90 (0.11)
1.47 (0.01)
10.55 (0.41)
7.42 (0.16)
1.10 (0.01)
RCC1710
20
1.15 (0.02)
11.47 (0.09)
12.05 (0.79)
1.71 (0.06)
13.16 (0.15)
13.82 (0.63)
1.98 (0.04)
RCC1710
25
1.24 (0.01)
10.80 (0.24)
9.30 (0.80)
1.38 (0.04)
13.34 (0.33)
11.48 (0.99)
1.70 (0.06)
RCC1710
27.5
1.04
RCC1710
30
0.23
RCC1252
6.5
0.18
RCC1252
10
0.26 (0.04)
8.29 (0.49)
6.35 (0.11)
1.16 (0.03)
2.15 (0.39)
1.64 (0.23)
0.30 (0.04)
RCC1252
15
0.73 (0.00)
9.92 (0.32)
8.64 (0.29)
1.34 (0.03)
7.22 (0.23)
6.29 (0.22)
0.97 (0.02)
RCC1252
20
1.15 (0.14)
9.89 (0.28)
8.75 (0.71)
1.35 (0.07)
12.01 (0.74)
9.99 (1.13)
1.56 (0.26)
RCC1252
25
1.22 (0.02)
12.20 (0.21)
10.19 (0.75)
1.41 (0.02)
14.84 (0.38)
12.39 (0.86)
1.72 (0.02)
RCC1252
27.5
1.02
RCC1252
30
0.00
IAN01
6.5
0.12
IAN01
15
0.81 (0.01)
10.18 (0.30)
9.89 (0.43)
1.47 (0.08)
8.20 (0.19)
7.97 (0.30)
1.18 (0.06)
IAN01
20
1.17 (0.00)
8.12 (0.21)
8.95 (0.43)
1.75 (0.09)
9.46 (0.25)
10.43 (0.51)
2.04 (0.11)
IAN01
25
1.32 (0.03)
11.21 (0.36)
9.95 (0.11)
1.46 (0.01)
14.84 (0.49)
13.17 (0.22)
1.94 (0.03)
IAN01
27.5
1.01
IAN01
30
-0.11
Results at different temperatures. Growth rate (a) (extra
temperatures from pre-experiments are included and shown as empty symbols);
cellular PIC and its concomitant calcite (b), POC (e) and
TPN (h) content; PIC (c), POC (e) and TPN
(i) production (linear trend lines and r squared values are shown);
and PIC : POC ratio (d) and POC : TPN ratio (g). Standard
deviations of the triplicate experiment results are shown. Three different
strains of E. huxleyi were used.
Carbonate chemistry
The seawater carbonate system was monitored because temperature and
coccolithophore production alter the system. We employed the dilute batch
method to minimize production effects.
During the harvesting, samples for total alkalinity (TA) measurements were
sterile-filtered (0.2 µm pore size) and stored for less than 2
months prior to measurement in 25 mL borosilicate flasks at 4 ∘C.
TA was calculated from linear Gran plots after
potentiometric titration (in duplicate) .
Samples for dissolved inorganic carbon (DIC) were sterile-filtered
(0.2 µm pore size) with gentle pressure using cellulose-acetate
syringe filters and stored bubble-free for less than 2 months prior to
measurement at 4 ∘C in 5 mL borosilicate flasks. DIC was measured,
in triplicate, using a Shimadzu TOC 5050A.
The carbonate system was calculated from temperature, salinity
(32 ‰), TA and DIC, using the program CO2SYS ,
applying the equilibrium constants from , refitted by
. For an overview of carbonate chemistry final conditions
in all treatments, see Table .
The carbonate system final values. Standard deviation of the
triplicates in parentheses.
Strain
T
TA
DIC
pH
pCO2
HCO3-
CO32-
Omega
(∘C)
(µmolkg-1)
(µmolkg-1)
(total scale)
(µatm)
(µmolkg-1)
(µmolkg-1)
calcite
RCC1710
10
2138 (23)
2012 (3)
7.95 (0.07)
482 (74)
1893 (14)
98 (15)
2.38 (0.36)
RCC1710
15
2167 (14)
2023 (12)
7.92 (0.01)
530 (13)
1893 (11)
111 (3)
2.69 (0.07)
RCC1710
20
2291 (25)
2110 (4)
7.92 (0.06)
571 (84)
1953 (19)
139 (18)
3.39 (0.45)
RCC1710
25
2306 (24)
2123 (7)
7.86 (0.03)
688 (55)
1961 (4)
142 (11)
3.51 (0.28)
RCC1252
10
2249 (8)
2095 (12)
8.02 (0.03)
427 (30)
1959 (16)
117 (6)
2.84 (0.15)
RCC1252
15
2219 (57)
2065 (6)
7.94 (0.12)
533 (136)
1925 (21)
119 (32)
2.90 (0.78)
RCC1252
20
2212 (20)
2043 (15)
7.91 (0.01)
571 (10)
1896 (11)
129 (4)
3.15 (0.09)
RCC1252
25
2229 (8)
2052 (10)
7.85 (0.04)
670 (64)
1896 (19)
137 (11)
3.37 (0.26)
IAN01
15
2206 (9)
2064 (16)
7.92 (0.02)
551 (33)
1932 (19)
111 (4)
2.70 (0.11)
IAN01
20
2249 (28)
2106 (6)
7.84 (0.05)
698 (86)
1969 (5)
115 (14)
2.80 (0.34)
IAN01
25
2243 (2)
2066 (4)
7.85 (0.01)
677 (13)
1910 (5)
137 (2)
3.37 (0.05)
Particulate organic and inorganic carbon, particulate nitrogen and calcite
Duplicate samples for the determination of total particulate carbon (TPC) and
total particulate nitrogen (TPN) were filtered onto pre-combusted
(500 ∘C; 12 h) 0.6 µm nominal pore size glass fibre
filters (Whatman GF/F), placed in pre-combusted Petri dishes
(500 ∘C; 12 h), oven-dried (60 ∘C 24 h) and stored at
-20 ∘C. Before analysis, TPC and TPN samples were dried for 24 h
in a drying cabinet at 60 ∘C prior to measurement. All samples were
then measured on a Euro EA analyser (Euro Vector).
Particulate inorganic carbon (PIC) was calculated measuring calcium content
of samples with 3.6×106 E. huxleyi cells filtered onto
47 mm polycarbonate (PC) filters (0.8 µm pore size). PC filters
were immersed overnight in an acid solution of 1% HNO3 to
dissolve calcite. Calcium was determined by analysing an aliquot of the
samples using an inductively coupled plasma mass spectrometer (ICP-MS,
Agilent model 7500ce). Cellular PIC was calculated from the molecular mass of
calcite, using the following equations:
PICcell-1=PICsc⋅Vs,wherePICs=[Ca2+]s⋅12.010740.078,
where PICcell-1 is the cellular PIC (in pg),
PICs is the PIC sampled contained in the filter (in pg), c
is the cell concentration (in cellsL-1), Vs is the
volume sampled (in L), [Ca2+]s is the calcium content
in the sample (in pg), 12.0107 corresponds to the relative atomic mass
of carbon, and 40.078 corresponds to the relative atomic mass of calcium.
Particulate organic carbon (POC) was calculated as the difference between TPC
and PIC. PIC, POC and TPN production (PPIC, PPOC,
PTPN) were estimated as the product of cellular PIC, POC or TPN,
and growth rate. Calcite (CaCO3) per cell (concomitant of PIC) can
also be estimated, substituting in Eq. () the calcium carbonate
molecular mass (100.0869) in place of the relative atomic mass of carbon. The
ratio between PIC and POC (PIC : POC) and the ratio between POC and TPN
(POC : TPN) were also calculated.
Coccolith morphology – by scanning electron microscopy
Thirty millilitres of culture was filtered onto polycarbonate filters
(0.8 µm pore size) and dried at 60 ∘C for 24 h. A
small portion (∼0.7 cm2) of each filter was mounted on an
aluminium stub and coated with gold (EMITECH K550X sputter coater). Images
were captured along random transects using a ZEISS-EVO MA10 scanning electron
microscope (SEM).
Emiliania huxleyi SEM images were used to measure and categorize 300
coccoliths per sample e.g.; the coccoliths were on
coccospheres. The tube width (width of the tube elements cycle) of each
coccolith (Fig. c) was the average of the tube width
measured on the two semi-minor axes (along the coccolith width) on the distal
view of the coccolith. Tube width measurements were manually taken using the
program Gimp-2.8. Examples of the tube width variations in the three
different strains are shown in Fig. . The 300 coccoliths
were classified as normal, malformed or incomplete
e.g., as described in Table , with
examples in Figs. and .
Examples of tube width variations observed in E. huxleyi
RCC1710 (a–c), RCC1252 (d–f), and IAN01 (g–i)
coccoliths. Tube width (c) was measured along the two semi-minor axes (along
the coccolith width) of each coccolith and averaged. Scale bar equal to
1 µm.
Examples of malformed coccoliths found in E. huxleyi
RCC1710 (a), RCC1252 (b), and IAN01 (c). Scale bar
equal to 1 µm.
Examples of incomplete coccoliths of E. huxleyi RCC1710
(a), RCC1252 (b), and IAN01 (c). Scale bar equal
to 1 µm.
Morphological categorization of coccoliths (from SEM images) of
E. huxleyi used in this study.
Morphological category
Description
Normal
Regular coccolith in shape, with well-formed distal shield elements aligned forming a symmetric rim. Considered normal when zero or only two malformations were present.
Malformed
Irregular coccolith in shape or size of individual elements and a general reduction in the degree of radial symmetry shown; teratologicalmalformation (Young and Westbroek, 1991). Considered malformed when three or more malformations were present in the coccolith.
Incomplete
Coccolith with variations in its degree of completion according to its normal growing order, with no malformations. Primary calcification variation (Young, 1994).
Coccolith length and mass – by polarized light microscopy
Between 10 and 30 mL of culture was filtered with ∼ 200 mbar
onto cellulose nitrate filters (0.2 µm pore size) and dried
at 60 ∘C for 24 h. A radial piece of filter was embedded and made
transparent in immersion oil on microscope slides
e.g..
Images were taken at a magnification of 1000× with a Leica DM6000B
cross-polarized light microscope (LM) equipped with a SPOT Insight camera
e.g.. Between 50 and 200 image frames from each
sample were taken along radial transects and analysed using SYRACO software
. A minimum of 300 coccolith images were
automatically identified by the software and measured in pixels. The software
also automatically measures the grey level for each pixel by a birefringence
method based on the coccolith brightness when viewed in cross-polarized light
. Coccolith length and mass were subsequently calculated
from the pixels and from the measured grey level, respectively, following
and . Therefore, coccolith length
was converted from pixels to micrometres, where 832 pixels correspond to
125 µm, and coccolith mass was converted from grey level units to
picograms, where 2275.14 grey level units were equivalent to 1 pg of
calcite.
Statistics
For the three E. huxleyi strains together, ANOVA (two-factor with
replication) was used to test whether a response variable (i.e. growth rate,
element variables, morphological variables and mass) presented significant
(p<0.05) differences between the temperature treatments, to test whether the
effect was strain-independent or strain-specific (p<0.05), and to test
whether there were significant differences in the interaction between treatment and
strain (p<0.05) and therefore whether the different strains respond similarly or
not regardless of whether they were presenting differences between them. If the
temperature effect was strain-specific, further ANOVA was used for pairs of
strains.
If a response variable presented significant differences between the
temperature treatments, and the variable also presented a significant
strain-independent response to temperature, or at least the same response on
two of the strains, the variable for the similar strains was analysed with
simple and multiple linear regressions, including CO2 partial
pressure (pCO2), CO32- concentration and pH, in order to
find the useful coefficients (t statistics, p<0.05) of the
significant equation (F test, p<0.05) that would estimate the
assessed variable value, e.g. the single or combined variables significantly
estimating growth rate.
Results
Population growth
The three strains of E. huxleyi presented a stable growth rate (per
day) that changed with temperature (Fig. a,
Table ), with significant differences between the temperature
treatments (F=244.11, p=0.000). The strains RCC1710 and RCC1252
presented similar growth rates, not statistically different from one another
(F=0.372, p=0.550). From 15 to 25 ∘C, the IAN01 growth rate
was significantly different from the other two E. huxleyi strains
(F=4.53, p=0.025), but there was no significant difference in the
interaction between treatment and strain (F=0.71, p=0.597), so the
three strains behaved significantly similarly. The optimum temperature for the
three strains was 25 ∘C. When RCC1710 and RCC1252 were analysed
together, changes in growth rate only depended significantly on temperature
(linear regression: R2=0.91, F=229.58, p=0.000); the carbonate
system variables (Table ) did not much increase the
coefficient of determination (maximum to an R2=0.92) and none of them
were significantly useful in predicting growth rate when used together with
temperature (t statistics: p>0.05). According to
Eq. (), on the three strains, a minimum of one duplication per
day was obtained from 15 to 27.5 ∘C.
Element measurements, ratios and production
Cellular PIC (and its concomitant calcite), POC and TPN
(pgcell-1) did not show a consistent trend related to
temperature when comparing the three strains of E. huxleyi
(Fig. b, e, h; Table ). When cellular PIC and TPN
response to temperature (from 15 to 25 ∘C) were statistically
analysed (ANOVA), significant differences were found between treatments (F=113.42, p=0.000 and F=36.52, p=0.000, respectively), but these were
not strain-independent (F=182.86, p=0.000 and F=33.32, p=0.000, respectively). Cellular POC, conversely, did not show significant
differences between strains (F=1.71, p=0.209), nor did between the temperature treatments (F=0.09, p=0.908). There was no consistent explanatory variable for cellular PIC, POC,
and TPN when analysing the three strains independently.
In the three strains, production of PIC (and its concomitant calcite), POC
and TPN (pgcell-1day-1) showed a positive relationship with
temperature (Fig. c, f, i; Table ). Highest PIC and
POC production was in general reached at 25 ∘C, except for RCC1710, which reached it at 20 ∘C. From the statistical analysis, PIC and
POC production response to temperature, when comparing the three strains of
E. huxleyi together, was significantly different between the
temperature treatments (F=8.36, p=0.003) and the response was
strain-independent (F=0.89, p=0.428). Highest TPN production was in
general reached at 20 ∘C, except for RCC1252, which reached it at
25 ∘C. The latest was supported statistically, as TPN production
response, with significant differences between temperature treatments (F=499.96, p=0.000), was strain-specific (F=65.92, p=0.000) when
comparing the three strains of E. huxleyi together, and yet still
the strains RCC1710 and IAN01 presented a similar interaction between
treatment and strain (F=3.52, p=0.062); thus, the two strains had a
similar behaviour in the TPN production response despite the different values
between the strains (F=19.02, p=0.000).
Changes in PIC production on the three strains of E. huxleyi mostly
depended on temperature (linear regression: R2=0.89, F=217.36, p=0.000); pCO2 with [CO32-], when used together
with temperature, only slightly increased the coefficient of determination
(R2=0.93). Changes in POC production on the three strains of E. huxleyi only depended significantly on temperature (linear regression: R2=0.85, F=157.71, p=0.000).
The PIC : POC ratio decreased from 10 to 20 ∘C in the three
strains of E. huxleyi (Fig. d). POC was higher than PIC
only in the strains RCC1710 and IAN01 at 20 ∘C. From the statistical
analyses, the only significant similitude obtained was in the interaction
between treatment and strain for RCC1252 and IAN01 (F=2.12, p=0.163),
which means that the PIC : POC ratio behaves similarly towards temperature
in these two strains.
The POC : TPN ratio (Fig. h) relationship with temperature was
strain-specific (F=9.59, p=0.001). The differences between the
temperature treatments were significant (F=16.95, p=0.000). There
were no significant differences between the strains RCC1710 and RCC1252 (F=2.71, p=0.119), in which the lowest POC : TPN ratio was found at
10 ∘C; however, there were significant differences in the interaction
between treatment and strain (F=3.52, p=0.039), as observed in the
different temperatures at which maximum POC : TPN ratios were found for each
strain (20 and 25 ∘C, respectively). The strain IAN01 showed a much
different relationship with temperature, with a minimum POC : TPN ratio
found at 20 ∘C.
Coccolith morphology and mass
Although there was great variation between replicates, mean tube width of
coccoliths (Fig. a,
Table ) presented a positive trend with
temperature, independent of the strain of E. huxleyi (F=1.73,
p=0.204). Changes in tube width on the three strains of E. huxleyi only depended on temperature (linear regression: R2=0.47, F=28.09, p=0.000); pCO2 and [CO32-] did not much
increase the coefficient of determination (R2=0.51) and none of
them were significantly useful in predicting tube width when used together
with temperature (t statistics: p>0.05).
Coccoliths morphology and mass. Standard deviation of the
triplicates is shown in parentheses.
Strain
T
Tube width
Coccolith length
Coccolith mass
Malformed
Incomplete
(∘C)
(µm)
(µm)
(pg)
(%)
(%)
RCC1710
10
0.20 (0.02)
2.03 (0.06)
0.99 (0.11)
33.18 (2.02)
2.39 (0.75)
RCC1710
15
0.22 (0.03)
2.12 (0.03)
1.63 (0.25)
29.19 (4.50)
2.38 (2.36)
RCC1710
20
0.26 (0.02)
2.05 (0.04)
1.75 (0.09)
33.66 (5.85)
8.60 (4.51)
RCC1710
25
0.28 (0.02)
2.16 (0.05)
2.48 (0.16)
37.75 (7.90)
20.10 (5.24)
RCC1252
10
0.21 (0.04)
2.06 (0.00)
1.61 (0.00)
56.39 (3.54)
1.22 (0.51)
RCC1252
15
0.26 (0.05)
2.15 (0.09)
1.97 (0.07)
7.65 (5.29)
1.28 (1.25)
RCC1252
20
0.28 (0.04)
2.27 (0.03)
2.49 (0.30)
10.09 (3.21)
7.09 (5.01)
RCC1252
25
0.27 (0.02)
2.30 (0.03)
3.00 (0.18)
9.09 (3.67)
5.08 (4.85)
IAN01
15
0.22 (0.03)
2.15 (0.06)
2.02 (0.19)
52.13 (8.41)
2.58 (0.66)
IAN01
20
0.25 (0.03)
2.24 (0.00)
2.63 (0.00)
47.09 (2.92)
3.05 (1.78)
IAN01
25
0.27 (0.02)
2.26 (0.02)
2.66 (0.27)
41.18 (4.01)
8.95 (3.01)
Changes in coccolith morphometry (a, b) and mass
(c), at different temperatures. Standard deviations of the
triplicate experiment results are shown. Three different strains of
E. huxleyi were used.
Coccolith length (Fig. b,
Table ) showed a positive trend with
temperature, especially on strains RCC1252 and IAN01. The positive trend in
strain RCC1710 was not so clear; however, minimum length was also found at
10 ∘C and maximum length also at 25 ∘C. Strains RCC1252 and
IAN01 were analysed together in a multiple linear regression analysis, as
they did not present significant differences between them (F=2.12, p=0.171); temperature gave the highest coefficient of determination (R2=0.62, F=24.03, p=0.000) and was the only useful coefficient in
estimating coccolith length when making any combination with
pCO2, [CO32-] or pH. The strain RCC1710 was analysed
independently of the other two strains: temperature presented a low and non-significant coefficient of determination (R2=0.28, F=3.55, p=0.092); instead, pH presented the highest coefficient of determination (R2=0.65, F=16.87, p=0.002).
The positive relationship of the mean tube width with temperature reflects
the increased coccolith calcite quota at higher temperature. Coccolith mass
and coccolith size are positively correlated. Why coccolith mass or size
should increase with temperature cannot be decisively answered based on our
data.
Regardless of the strain, coccolith calcite mass
(Fig. c,
Table ) showed a positive trend with
temperature; significant differences were found between treatments (F=35.59, p=0.000) and no significant differences were found in the
interaction between treatment and strain (F=2.53, p=0.08). The
strains RCC1252 and IAN01 were analysed together as they did not show
significant differences between them (F=0.65, p=0.425). Temperature
presented the highest coefficient of determination for RCC1252 and IAN01
(R2=0.75, F=45.93, p=0.000) and also for RCC1710 (R2=0.87,
F=58.58, p=0.000), and adding other coefficients was not
significantly useful in estimating coccolith mass. On average, coccolith mass
increased with temperature ∼ 2.2× from 10 to 25 ∘C,
∼ 1.5× from 15 to 25 ∘C, and ∼ 1.2× from 20 to
25 ∘C; on average, coccolith mass increased 1.28× (or
0.45 pg) each 5 ∘C.
The percentage of malformed coccoliths per sample (Fig. a,
Table ) did not show a consistent trend with
temperature when comparing the three strains of E. huxleyi (F=113.21, p=0.000). Only one strain (RCC1252) presented significant
differences between the temperature treatments, with higher percentage at the
lowest experimented temperature.
Percentage of malformed (a) and incomplete (b)
coccoliths in three E. huxleyi strains grown at different
temperatures. Standard deviations of the triplicate experiment results are
shown.
Only in strain RCC1710, the percentage of incomplete coccoliths presented a
significant increase with temperature (Fig. b,
Table ). Higher percentages of incomplete
coccoliths in strain RCC1710 were found at 25 ∘C. ANOVA results
showed that, between the three strains, there were no significant differences
between only the strains RCC1252 and IAN01 (F=0.06, p=0.810) and
their interaction between treatment and strain (F=2.33, p=0.139),
though in this case (analysed from 15 to 25 ∘C) there were also no
significant differences between the temperature treatments (F=3.78, p=0.053). Significant strain-independent and strain-specific responses of E. huxleyi to temperature, found in the three strains of this study, are summarized in Table 5.
Discussion
Growth rate, elemental production and incomplete coccoliths
All three E. huxleyi strains investigated here displayed similar
growth rate versus temperature relationships, with an optimum at
20–25 ∘C (Fig. a). This is a typical range for many
E. huxleyi strains
e.g..
We expect that strains isolated, for example, in the Arctic will have a lower
temperature optimum, though. Also not untypical, elemental production (PIC,
POC, TPN) increased with temperature over the sub-optimum to optimum
temperature range Fig. ;. It is
intuitive that, approaching optimum, higher temperature increases elemental
production, because biochemical rates are temperature-dependent. It is also
intuitive that the percentage of incomplete coccoliths should increase with
higher PPIC, as indeed observed in RCC1710
(Fig. b). The idea underlying this intuition is that less
time is taken to produce one coccolith and that the production process is
stopped before the coccolith is fully formed. A comparison of RCC1710 and
RCC1252 shows how wrong this idea is (Table ). The
percentage of incomplete coccoliths increases in the former only. While it is
true that coccolith production time in RCC1710 decreases from 31 min at
10 ∘C to 22 min at 25 ∘C, this decrease is even more
pronounced in RCC1252 (from 88 to 23 min). Hence, RCC1252 should show a
steeper increase in incompleteness than RCC1710. This is not the case. Please
note that the increase in incompleteness in RCC1252
(Fig. b) is not significant, because the increase is well
below 10% and the error bars overlap see alsofor a
discussion of this criterion. Another piece of evidence which does not fit
the “premature release of coccoliths because of time shortage” idea is that
both RCC1710 and RCC1252 manage to produce heavier coccoliths in a shorter
time at higher temperature (Tables and
). We do not know why the stop signal for coccolith
growth is affected by temperature in RCC1710. Nothing is known about the
biochemical underpinning of that stop signal, so it is unfortunately
impossible to speculate about the mechanism of a temperature effect. It was,
however, argued that the processes involved in the stop signal are different
from those producing teratological malformations
. This is supported by our data,
because there is no correlation between incompleteness and malformations
(Fig. ). We will discuss malformations in Sect. .
Significant strain-independent and strain-specific responses of
E. huxleyi to temperature found in the three strains of this
study.
Strain-independent responses
Strain-specific responses
– Growth rate optimum temperature was 25 ∘C. – Highest PIC, POC, and TPN production values were found at 20 or 25 ∘C. – The PIC : POC ratio decreased from 10 to 20 ∘C. – Tube width increased with temperature, from ∼ 0.20 µmat 10 ∘C to ∼ 0.27 µm at 25 ∘C. – Maximum coccolith length was found at 25 ∘C. – Coccolith mass increased with temperature (∼ 2.2× from 10 to 25 ∘C, ∼ 1.5× from 15 to 25 ∘C, and ∼ 1.2× from 20 to 25 ∘C; on average, 0.45 pg each 5 ∘C).
– Cellular PIC, POC and TPN (pg per cell). – POC : TPN ratio. However, in the two strains tested at 10 ∘C (RCC1710 and RCC1252), the POC : TPN ratio was lowest at 10 ∘C. – Percentage of malformed coccoliths per sample. – Percentages of incomplete coccoliths. – Coccolith length, although in strains RCC1252 and IAN01 was positively correlated with temperature.
Coccolith production time. Standard deviation of the triplicates is
shown in parentheses. Lith: coccolith; d: day; h: hour; min: minutes.
Strain
T (∘C)
pgPIClith-1
lithcell-1
lithcell-1d-1
lithcell-1h-1
minlith-1
pgPICh-1
RCC1710
10
0.12 (0.01)
121 (2)
31 (0)
2.0 (0.0)
31 (0)
0.25 (0.00)
RCC1710
15
0.20 (0.03)
74 (14)
55 (10)
3.4 (0.6)
18 (3)
0.66 (0.03)
RCC1710
20
0.21 (0.01)
53 (0)
61 (1)
3.8 (0.1)
16 (0)
0.82 (0.01)
RCC1710
25
0.30 (0.02)
36 (2)
45 (2)
2.8 (0.1)
22 (1)
0.83 (0.03)
RCC1252
10
0.19 (0.00)
43 (2)
11 (2)
0.7 (0.1)
88 (18)
0.13 (0.02)
RCC1252
15
0.24 (0.01)
42 (1)
31 (1)
1.9 (0.1)
31 (1)
0.45 (0.01)
RCC1252
20
0.30 (0.04)
35 (6)
42 (4)
2.6 (0.2)
23 (2)
0.75 (0.05)
RCC1252
25
0.36 (0.02)
34 (3)
41 (3)
2.6 (0.2)
23 (2)
0.93 (0.02)
IAN01
15
0.24 (0.02)
42 (3)
34 (2)
2.1 (0.2)
28 (2)
0.51 (0.01)
IAN01
20
0.32 (0.00)
26 (1)
30 (1)
1.9 (0.0)
32 (1)
0.59 (0.02)
IAN01
25
0.32 (0.03)
35 (5)
47 (6)
2.9 (0.4)
21 (3)
0.93 (0.03)
Interestingly coccolith mass is positively correlated with temperature (and
PPIC) in all strains tested here. The positive correlation of
coccolith mass and PPIC was also observed by in
a carbonate chemistry manipulation experiment and is the basis of using
coccolith mass as a proxy for PPIC . This is
an interesting option, because in field samples coccolith mass might be a
promising indicator of PPIC. There are only few proxies available
to reconstruct past coccolithophore PPIC, the traditional one
being the calcite Sr / Ca ratio, established at the turn of the millennium
. Analysing Sr / Ca, however, requires either a sizable sample
or comparatively sophisticated secondary ion mass spectrometry (SIMS)
measurements . Recently, coccosphere diameter
and coccolith quota were introduced as growth rate proxies .
However, complete coccospheres are the exception rather than the rule in
sediment samples, so it is important to have a proxy based on individual
coccoliths. Hence, coccolith mass and size (which are correlated;
Fig. ,
Table ) are an option which it is worthwhile
exploring in the future.
Emiliania huxleyi PIC : POC response
As detailed in the introduction there is considerable variability in the
PIC : POC response of E. huxleyi to temperature changes. This
variability cannot be traced back to strain-specific features, but it might
partly reflect the fact that different temperature ranges were investigated,
mostly without the knowledge of the optimum temperature. Other
experimental conditions, such as light intensity and nutrient concentrations,
varied and might have also played a role . In this study we ran
three strains under identical conditions and, for the first time, are
presented with a coherent picture. All three strains display a bell-shaped
curve with lowest PIC : POC close to the optimum growth temperature
(Fig. d). Although our data on the right-hand side of the
PIC : POC minimum are not conclusive for RCC1252, the bell-shaped curve is
discernible in the latter strain. This finding seems to fit data on other
E. huxleyi strains and on C. pelagicus . This comparison is, however, not
straightforward since two of the studies
employed two temperatures, one of the studies employed three temperatures
, only without determining the optimum temperature in all
three studies. Be that as it may, based on our data, we might conclude that
E. huxleyi tends to show the lowest PIC : POC close to its optimum
growth temperature. In the context of global warming, that would mean that, in
the future, E. huxleyi and possibly coccolithophore PIC : POC will
tend to decrease because most strains live at sub-optimal temperatures in the
field . This trend might be
pronounced because global warming is accompanied by lower surface water
nutrient levels and ocean acidification . All
these changes apparently cause a decrease in E. huxleyi's
PIC : POC our data;. A marked
decline in coccolithophore PIC : POC will have implications for long-term
carbon burial and might even affect surface water carbonate chemistry on
short timescales, i.e. 1 year .
Coccolith malformations
The coccolith shaping machinery is, besides the ion transport machinery, an
essential part of coccolith formation
for an overview see. The latter commences with heterogeneous nucleation on an
organic template, the so-called base plate. The nucleation determines crystal
axis orientation. Crystal growth proceeds in principle inorganically, with
the notable exception that crystal shape is strongly modified by means of a
dynamic mould, which essentially consists in the coccolith vesicle shaped by
cytoskeleton elements and polysaccharides inside the coccolith vesicle.
Malformations can be due to an abnormal base plate which would affect crystal
axis orientation, aberrations in the composition or structure of the
polysaccharides, and disturbance of cytoskeleton functionality. The last of
these would most likely also cause a decline in growth rate, which is why this
mechanism was disregarded in the case of carbonate-chemistry-induced
malformations . By the same reasoning, temperature-induced
malformations might be due to cytoskeleton disturbance, because temperature
does also alter growth rate (Fig. a). However, it is not
straightforward to see why lower than optimum temperature should disturb
cytoskeleton functionality see also. At any rate,
coccolith malformations are most likely detrimental to fitness, because
malformed coccoliths result in fragile coccospheres, which are regarded as
instrumental in coccolithophore fitness
. One of the many
hypotheses concerning function of calcification is that the coccosphere
confers mechanical protection . After more than a
century of research, it still remains the most plausible hypothesis.
Coccolith malformations, i.e. disturbances of the coccolith shaping
machinery, occur in both field and culture samples, but usually more so in
the latter . The causes of malformations are
only partly known. In cultured samples, artificial conditions (not present in
the field) such as cell densities of 106cellsmL-1, cells
sitting on the bottom of the culture flask, stagnant water, and confinement
in a culture flask play a role in inducing the surplus of malformations
compared to field samples . However, in the
field malformations do occur, and sometimes in considerable percentages
. The environmental conditions leading to
elevated levels of malformations have long since been disputed. Besides
nutrient limitation , temperature and carbonate chemistry
are conspicuous candidates. Although the range of temperatures used here
exceeds 2100 projections , we used it not only on
physiological grounds but also for ecological reasons. Over the course of
the year, coccolithophores in the North Pacific experience the whole range
of temperatures used here (http://disc.sci.gsfc.nasa.gov/giovanni/,
maps in the Supplement). In a seminal experimental study it was
shown that moving away from the optimal growth temperature increases
malformations in E. huxleyi . This result was
confirmed for higher than optimum temperature in another strain
but could not be confirmed for sub-optimal temperature in
two strains . The sub-optimal temperature range
is of particular interest because most clones live at sub-optimal
temperatures in the field. Here we investigated sub-optimum to optimum
temperatures in three further strains. While RCC1710 showed no change in the
percentage of malformations and IAN01 featured a shallow gradual increase
from 25 to 15 ∘C, RCC1252 was insensitive over the latter range but
displayed a steep increase in malformations at 10 ∘C
(Fig. ). Based on our own and the literature data, we
conclude that the sub-optimal temperature effect on morphogenesis is
strain-specific. The fact that the base level of malformations in cultured
coccolithophores differs between species and strains (and also varies with
time) has been recognized for many years and is now well documented
e.g.. Also, the response of the
morphogenetic machinery to environmental factors is strain-specific
. We currently do not have enough accessory information to
formulate a hypothesis why exactly one strain differs from another. The fact
that they do indeed differ, however, probably reflects the high genetic
diversity in E. huxleyi.
Can we see a pattern in this strain specificity? It is intriguing that
E. huxleyi clones fall into two distinct groups characterized by
their temperature preference: the warm-water and the cool-water group
. Of the strains analysed for morphology, the following
belong to the warm-water group: BT-6 , RCC1710, RCC1252,
and possibly RCC1238 . The latter was unfortunately not
included in the study by . Since these strains display
different responses to temperature, their being part of the warm-water group
does unfortunately not help finding common features of sensitive strains.
However, only a few strains have been studied so far, and it might be worthwhile
testing a statistical number from the warm-water and the cool-water group.