Appendix 1.
Freeze-dried algal samples (50-150 mg) were homogenized by grinding with a mortar and
pestle, weighed and transferred into 1.5 ml Eppendorf tubes. DCM (500 μl) as added and the
samples were sonicated for 30 minutes. Samples were then centrifuged at 10,000 x g for 10
minutes and approximately 400 μl of supernatant was transferred into another, clean 1.5 ml
Eppendorf tube. This process was repeated a total of three times to yiel a final extract volume
of approximately 1200 μl, which was evaporated overnight in a fume cupboard. The dried
extract was resuspended in 1 ml HPLC-grade ethyl-acetate (EtOAc) and stored in the dark at
-20 ⁰C until quantification.
GC-MS was performed on diluted samples containing internal standard F8 using an Agilent
6890N gas chromatograph (GC) and a polyimide-coated fused silica capillary column (ZB1MS, 30m length, 0.25 mm ID). All injections (1 µl) were performed in the splitless mode
with an inlet pressure of 11.4 psi. The injection port was held at 250˚C and the GC-MS
interface at 300˚C. The GC was held at 100˚C for 3 min, ramped at 10˚C/min to 200˚C and
then ramped at 20˚C/min to 280˚C, with a final run time of 17 minutes. The carrier gas was
helium. Mass spectrometry was performed on an Agilent Technologies 5973 inert mass
spectrometer. Ions characteristic of the internal standard and the analytes were monitored in
the selected ion monitoring (SIM) mode. The GC-MS system was controlled by Agilent
Chemstation software (D.01.00 Build 75 2003). The ratio of peak areas (analyte/internal
standard) in the sample was calculated and converted to concentration by reference to a
standard curve. Concentrations of the four main furanone compounds (furanones1-4) in each
sample were calculated as μg/mg freeze-dried material.
Appendix 2.
Polymerase chain reaction (PCR) volumes were 25 μl, including 12.5 μl Econo Taq 2 X
mastermix (Lucigen), 0.25 μl of 10 μmol forward and reverse primers (27F, labelled with 6FAM fluorophore :5'-AGAGTTTGATC(AC)TGGCTCAG-3' and 519R: 5’GWATTACCGCGGCKGCTG-3’), template DNA and molecular water. ‘Touchdown’
(variable annealing temperatures) PCR was conducted in a thermocycler, where the samples
were initially denatured at 94 °C for 3 minutes, followed by 27 cycles of denaturation at 94
°C for 30 seconds, primer annealing at 55 °C for 45 seconds, followed by enzyme extension
for 2 minutes at 72 °C, another 30 seconds at 94 °C, 45 seconds at 54 °C, another 2 minutes
at 72°C, 30 seconds at 94°C, 45 seconds at 53 °C, 2 minutes at 72 °C, 30 seconds at 94 °C, 45
seconds at 52 °C and another 2 minutes at 72 °C. This was followed by a final extension at 72
°C for 5 minutes.
PCR products were checked on 1% agarose gels stained with gel red (BIOTIN), purified
using a Zymo DNA Clean and Concentrator Kit 5 (Zymo Research) and quantified using a
nano-drop ND1000 (Thermo Scientific). Approximately100 ng of purified PCR product was
digested in 25 µl reactions containing 5 units of the restriction enzyme RsaI (NE Biolabs)
with 14 μl 1 X NE buffer. Digestion reactions were incubated at 37 ⁰C for four hours.
Digestion products were de-salted and re-purified using the Zymo DNA Clean and
Concentrator Kit 5 (Zymo Research), as high salt concentrations can interfere with
electrokinetic injection into capillaries during TRFLP. Finally, 2 μl of each desalted, purified,
digestion product (with an approximate DNA concentration of 10-20 ng/μl) was submitted to
the Ramaciotti Centre at UNSW for fragment analysis using an AB3730 capillary sequencer
(Applied Biosystems) in genetyping mode. Electropherograms were visualised and data were
extracted using ‘Peak Scanner v 1.0’ (Applied Biosystems).
Appendix 3.
Five-factor ANOVA comparing the prevalence of bleaching in D. pulchra populations from
Bare Island and Long Bay across three sampling years in deep and shallow water.
Source of variation
Sampling year “Y”
Season “S”
Sampling time(Season) “T(S)”
Location “L”
Depth “D”
YxS
YxL
YxD
SxL
SxD
LxD
Y x T(S)
T(S) x L
T(S) x D
YxSxL
YxSxD
YxLxD
SxLxD
Y x T(S) X L
Y x T(S) X D
T(S) x L x D
YxSxLxD
Y x T(S) x L x D
Error
df
2
1
1
1
8
2
2
2
1
1
1
12
8
8
2
2
2
1
9
12
8
2
9
471
MS
1.146
2.393
0.127
0.009
0.233
0.056
0.091
0.103
0.008
1.787
0.340
0.241
0.174
0.192
0.044
0.321
0.107
0.488
0.009
0.147
0.334
0.147
0.243
0.080
F-ratio
14.247
29.769
2.089
0.108
2.894
0.697
1.129
1.275
0.098
22.214
4.227
3.957
2.864
3.166
0.549
3.987
1.332
6.071
1.574
2.415
5.500
1.823
3.997
p
0.000*
0.000*
0.036*
0.742
0.089
0.499
0.324
0.280
0.755
0.000*
0.040*
0.001*
0.006*
0.001*
0.578
0.019*
0.265
0.014*
0.112
0.004
0.001
0.163
0.001
Appendix 4.
b
a
a
c
Concentrations of ‘total’ furanones (means +SE) from healthy algae collected during summer
and winter at deep and shallow populations (data from locations pooled as there was no effect
of location) with n=5-8. Shared letters indicate statistical similarity according to Tukey’s
multiple pairwise comparisons tests.
Appendix 5.
Table 2.9. ANOVA comparing the proportional areas of furanone (+/-) thallus bleaching
exposed to high and low temperatures and the presence or absence of microbes. As there was
no effect of individual incubators (which were nested within each temperature) the data from
each incubator within each temperature were pooled for simplicity. ‘*’ indicates significant
effect of factor on the extent of thallus bleaching.
Source of variation
Temperature “T”
Furanones “F”
Antibiotics “A”
TxF
TxA
FxA
TxFxA
Error
df
1
1
1
1
1
1
1
35
MS
0.336
1.497
2.391
0.004
0.030
0.274
0.000
0.050
F-ratio
6.70
29.80
47.60
0.08
0.60
5.46
0.01
p
0.014*
0.000*
0.000*
0.776
0.443
0.025*
0.915