Electronic Supplementary Materials
Table S1: Life history parameters for anemonefish in the Amphiprion
akallopisos/perideraion/sandaracinos species complex used to estimate ages of our
sampled skunk anemonefish
TL at sexual maturity (mm)
A. akallopisos
721, 45-77 (males)2, 78-105 (females)2
A. perideraion
47 (males)5, 54 (females)5, 541
A. sandaracinos
511
Age at sexual maturity (yr)
Max TL (mm)
Age at TL (yr)
1053, 1104
1.75-1.83 (both sexes)5
1003, 1006
10.77
1406
1
(Allsop and West 2003); 2 Aldabra islands in the Indian Ocean (Fricke 1979); 3East Indies:
(Allen and Erdmann 2012); 4(Fautin and Allen 1992); 5Eniwetok Atoll: (Allen 1975); 6IndoPacific & Caribbean including the Red Sea: (Lieske and Myers 1994); 7Calculated from growth
data for a dominant pair (Allen 1975).
We wished to age the A. akallopisos sampled and measured, but age at length has not
been determined for A. akallopisos, only for closely related anemonefish species, such as
the pink skunk anemonefish, A. perideraion (Timm et al. 2008). Sizes at sexual maturity
and maximum length are comparable for these two species (Table S1), thus we assumed
that life history parameters of A. perideraion would be very similar to A. akallopisos.
Using a complete growth record of a dominant pair from recruitment to a TL of 54 and
70 mm and the mean growth rates of 0.4, 0.7 and 0.3 mm per month for the three largest
size classes of A. perideraion (50.0-59.9; 60.0-69.9; 70.0-79.9mm) (Allen 1975), we
estimated that a maximum length of 100mm corresponds to an age of approximately
10.7-13.3 years old (Table S1). We do not know whether the individual A. akallopisos we
sampled had obtained their maximum size, therefore we propose a conservative estimate
of maximum age at eight years for the largest A. akallopisos that we sampled (112 mm).
We therefore conservatively examined thermal stressor events over the last eight years
from 2003-2010.
References
Allen GR (1975) The Anemonefishes: their classification and biology. Second edition.
Tropical fish hobbyist publications, Hong Kong
Allen GR, Erdmann MV (2012) Reef fishes of the East Indies. University of Hawai'i
Press, Perth, Australia
Allsop DJ, West SA (2003) Constant relative age and size at sex change for sequentially
hermaphroditic fish. J Evol Biol 16:921-929
Fautin D, Allen G (1992) Field guide to anemonefishes and their host sea anemones,
Francis Street, Perth: Western Australia Museum
Fricke HW (1979) Mating system, resource defence and sex change in the anemonefish
Amphiprion akallopisos. Zeitschrift für Tierpsychologie 50:313-326
Lieske E, Myers R (1994) Collins Pockets Guide. Coral reef fishes. Indo-Pacific &
Caribbean including the Red Sea. Harper Collins Publishers
Timm J, Figiel M, Kochzius M (2008) Contrasting patterns in species boundaries and
evolution of anemonefishes (Amphiprioninae, Pomacentridae) in the centre of
marine biodiversity. Mol Phylogenet Evol 49:268-276
Table S2: Individual size of each Amphiprion akallopisos sampled, their estimated hatch
date after estimating their age from growth rates for A. perideraion (Allen 1975), the
summer during which the thermal event occurred at each site. When the thermal event
preceded hatch date, the year during which the greatest maximum summer sea surface
temperature and when the individuals were alive is given.
Site
Individual sizes (mm)
Estimated hatch date
Year of
New thermal
(of smallest if >1)
thermal event
event year
IG1
84, 88, 100
March 2002
2010
IG2
48, 62, 87, 89, 107, 112
July 2009
2010
JDN1
50, 62, 70, 78, 79, 83
June 2009
2010
JDN2
55, 84
June 2008
2009
JDN3
57, 75, 80, 93
January 2008
2010
JDN4
65
September 2006
2005
2010
JDN4
66
August 2006
2005
2010
JDN4
70
January 2006
2005
2010
JDN4
86, 98
February 2000
2005
EUR1
68, 82, 86, 90, 103
January 2006
2010
EUR2
73
March 2005
2004
2009
EUR2
76
June 2004
2004
2009
In order to verify that each of our individuals experienced the thermal stressors at each
site, we estimated their ages based on growth rates for A. perideraion (Allen 1975) and
their estimated hatch date is given in Table S2. If they hatched after the thermal event
between 2003-2010 for each site (shown in Table S2), the year during which the
maximum summer SST occurred that they would have experienced was used (shown in
Table S2). The new maximum summer SST was then used in the final models.
References
Allen GR (1975) The Anemonefishes: their classification and biology. Second edition.
Tropical fish hobbyist publications, Hong Kong
Table S3: Density of anemonefish predators, anemonefish egg predators and anemone
predators present in the Îles Eparses that may cause a stress response in anemonefish,
Amphiprion akallopisos. A list was drawn up from Fricke (1975) and Moyer (1980) and
other species within the families were included based on their known diet. We visually
censused three 50x5m belt-transects (250-m2) distributed on either side of three replicate
20-m Line Intersect Transects to record fish species density (juveniles and
adults)(Chabanet et al. 2002).
EUR1
EUR2
Balistapus undulatus
0.7
0.7
Caranx ignobilis & C. melampygus
7.3
0.7
IG1
IG2
JDN1
JDN2
JDN3
JDN4
Balistidae
0.3
Carangidae
6.3
23.3
1.0
1.0
Elagatis bipinnulata
0.7
1.0
Chaetodontidae
Chaetodon auriga & C. lunula
4.0
1.3
4.0
1.3
0.7
3.0
5.3
Fistulariidae
2.0
1.0
Fistularia commersoni
0.3
1.0
Labridae
Bodianus axillaris
1.0
0.3
1.0
0.3
Cheilinus trilobatus
0.3
0.3
Epibulus insidiator
0.7
0.7
0.3
7.0
0.3
0.3
Thalassoma ballieui, T. hardwicke
& T. lunare
3.7
4.3
1.3
3.3
Lethrinidae
Gnathodentex aureolineatus
Lethrinus harak
10.7
2.3
0.3
Lutjanidae
Aphareus furcatus & A. rutilans
1.0
0.3
Aprion virescens
1.0
0.3
0.3
Lutjanus bohar, L. fulvus, L.
gibbus, kasmira, L. monostigma &
L. rivulatus
4.7
6.7
2.0
4.3
2.7
Macolor niger
0.7
0.3
0.7
Parupeneus cyclostomus
0.3
1.0
0.7
14.0
3.3
Mullidae
0.7
Pomacentridae
Amblyglyphidodon leucogaster
Dascyllus trimaculatus
0.3
0.7
10.0
6.7
lacrymatus
3.0
4.0
2.0
Aethaloperca
1.0
1.3
1.0
Cephalopholis
9.3
7.7
11.0
Epinephelus
4.0
1.3
5.3
Plectroglyphidodon dickii & P.
6.3
5.3
8.7
1.7
0.3
0.7
0.3
0.3
1.7
3.7
3.7
5.0
1.0
0.3
Serranidae
Plectropomus
Variola
Total density per site
0.3
2.3
0.7
1.3
42.0
29.7
1.7
56.3
39.7
1.0
19.3
28.0
0.7
33.0
References
Chabanet P, Bigot L, Naim O, Garnier R, Moyne-Picard M (2002) Coral reef monitoring
at Reunion Island (Western Indian Ocean). Proc 9th Int Coral Reef Symp 2:873878
Fricke HW (1975) Selektives feinderkennen bei dem anemonenfisch Amphiprion
bicinctus (Rüppell). J Exp Mar Biol Ecol 19:1-7
Moyer JT (1980) Influence of temperate waters on the behavior of the tropical
anemonefish Amphiprion clarkii at Miyake-Jima, Japan. Bull Mar Sci 30:261-277
18.3
APPENDIX 1: Validation of cortisol kit for Amphiprion akallopisos
Blood samples were taken from thirty-six skunk anemonefish, Amphiprion akallopisos,
laterally from the caudal vein using heparinised (Sigma H-0878) plastic 1 ml syringes
fitted with a 30 gauge needle and kept on ice until processing (Godwin and Thomas
1993; Mills et al. 2010). Half of the specimens (15 in total) were immediately released
back on their host anemone, whereas the other half (21 in total) were taken aboard the
Marion Dufresne and kept in aquaria. Individual blood samples were transferred to 75 µl
haematocrit capillaries and centrifuged (Beckman Coulter TJ-25 Centrifuge) at 10,000g
for 5 minutes. The supernatant, a yellow plasma layer, was collected without disturbing
the white buffy layer or the blood cells. Individual plasma samples, as well as a pool of
approximately twenty samples, were stored at -80 ºC for one month, transported back to
France and kept at -80 °C for three months when the hormone measurements were made.
Morphological sex identification
The 21 specimens in aquaria were anesthetized, frozen at -80 °C for one month,
transported back to France and kept at -80 °C until dissection after a further month.
Gonads were removed and sex was determined morphometrically by two observers blind
to their group size and structure.
Hormone measurements
Plasma cortisol was measured using an EIA kit (Cortisol EIA Kit, No. 500360, Cayman
Chemicals, SPI BIO, France) as described in Mills et al. (2010). 50 µl of the 8 standards
or 50 µl of the blood plasma samples were added with 50 µl of cortisol-
acetylcholinesterase (AChE) conjugate and 50 µl of cortisol-specific rabbit antiserum to a
96-well plate. During 18 hours at 4°C, cortisol AChE and sample or standard cortisol
competed for a limited number of cortisol-specific rabbit antiserum binding sites whose
complex attached to the mouse monoclonal anti-rabbit IgG antibody previously attached
to the well. The plate was washed five times to remove any unbound reagents and 200 µl
of Ellman’s reagent, that contains the substrate to AChE, was added to the wells. The
plate was placed on an orbital shaker in the dark for 80 mins. The intensity of the yellow
colour was measured spectrophotometrically (Beckman Coulter AD 340
Spectrophotometer) at 405nm and is proportional to the amount of cortisol AChE bound
to the well, which is inversely proportional to the amount of free cortisol present.
Sample cortisol concentrations were determined by interpolation from the
standards calibration curve using a common functional model for calibration curves. The
data were plotted as % maximum bound (% B/Bo) versus log concentration using a logitlog curve fit as recommended for this kits (www.caymanchem.com/analysis/eia).
Validation comprised: 1) parallel displacement of serially diluted plasma to the
standard curve; 2) precision from intra- and inter-assay variabilities.
1) Parallelism was evaluated by measuring cortisol concentrations in pooled
plasma samples, serially diluted in EIA buffer provided with the kits. The dilution ratios
were: 1:19, 1:34, 1:60, 1:108, 1:182, 1:323, 1:561, and 1:989. The maximum bound (%
B/Bo) for each sample dilution and for the standards were plotted against their relative
log dilution and the shapes of the resulting curves were compared. These curves must be
parallel to support the assumption that the antibody-binding characteristics of standard
and sample are similar enough to allow the determination of antibody levels in the diluted
plasma sample. An ANCOVA was carried out to determine the homogeneity of slopes
between the sample dilutions and those of the kit’s standards. In addition, regression
analysis of the diluted sample was used to determine the dilution factor that corresponds
to 50 % of antibody bound.
2) Precision was assessed by examining intra- and inter-assay variability of
samples with different hormones levels. Intra-assay variability was determined by
evaluating 49 plasma samples in duplicate within the same run of the assay. Inter-assay
variability was determined by evaluating nine samples in quadruplicate across two runs
of the assay. Variabilities or coefficients of variation (CV) of repeated measures of
samples were assessed. CV was calculated according to the formula: CV = (SD X -1) x
100. A kit was considered to have good precision if the CV was < 20% as per the
guidelines in Plikaytis et al. (1994) and Sukovaty et al. (2006).
RESULTS
Pooled plasma of field skunk anemonefish, A. akallopisos was screened for parallelism
with six dilutions of the cortisol kit’s standards and were found to run parallel to the
cortisol standard curve (Table AI; Fig. A1). Regression analysis enabled the appropriate
dilution factor for 50 % of antibody bound for the skunk anemonefish to be determined
with the cortisol kit (Table AI). A. akallopisos also showed high accuracy and precision
with the cortisol kit determined from intra- and inter-assay variabilities respectively;
13.3% (n = 49) and 16.9% (n = 7).
Cortisol concentrations of field skunk anemonefish, A. akallopisos, based on the
mean (± SE) of the pooled plasma from 36 individuals was 54.14 ± 4.26 ngml-1. This
mean is comparable to cortisol levels previously recorded for the fire clownfish A.
melanopus (Bleeker, 1852) (mean plasma cortisol values: males = 17 ± 4 ngml-1 and
females = 16 ± 3 ngml-1; (Godwin and Thomas 1993; Buston et al. 2004) and for
aquarium and field A. percula and A. chrysopterus: 18.98 ± 1.59 ngml-1; and 21.53 ± 2.46
ngml-1 respectively (Mills et al. 2010).
In conclusion, cortisol EIA assay kit was tested for use on A. akallopisos and the
dose-response curve was parallel to the cortisol EIA assay kit standards (Fig. A1; Table
AI) and high precision was obtained from intra-and inter-assay variabilities (< 20%) with
A. akallopisos. Consequently, this kit can be confidently used for measuring cortisol in A.
akallopisos.
Table AI. ANCOVA on homogeneity of slopes for sample dilution versus standard
dilution curves for cortisol kits in Amphiprion akallopisos. The dilution factor
(dilution) for 50 % of antibody bound determined from regression analyses is also
given.
Assay
Species
df
Mean square
F
p
Dilution factor
Cortisol
A. akallopsisos
1,27
0.003
0.340
0.565
1:335 (0.003)
df, degrees of freedom; F, test statistic: p, probability.
Figure A1. Dose-response curve for cortisol obtained using 6 kit standards and pooled
plasma from Amphiprion akallopisos (kit standards: y = -40.50 x - 66.81, R2 = 0.97, N =
12, p < 0.001; samples: y = -39.21 x - 49.03, R2 = 0.98, N = 16, p < 0.001).
Dashed line and arrow represents 50 % bound (see Table I for corresponding dilution
factors).
= Pooled sample plasma;
= Cortisol kit standards.
100
% B/Bo
80
60
40
20
0
-5.0
-4.0
-3.0
-2.0
-1.0
0.0
Relative log dilutions (%)
References
Buston PM, Munday PL, Warner RR (2004) Sex change and relative body size in animals.
Nature 428
Godwin JR, Thomas P (1993) Sex change and steroid profiles in the protandrous
anemonefish Amphiprion melanopus (Pomacentridae, Teleostei). General and
Comparative Endocrinology 91:144-157
Mills SC, Mourier J, Galzin R (2010) Plasma cortisol and 11-ketotestosterone enzyme
immunoassay (EIA) kit validation for three fish species: the orange clownfish
Amphiprion percula, the orangefin anemonefish Amphiprion chrysopterus and the
blacktip reef shark Carcharhinus melanopterus. Journal of Fish Biology 77:769777
Plikaytis BD, Holder PF, Pais LB, Maslanka SE, Gheesling LL, Carlone GM (1994)
Determination of parallelism and nonparallelism in bioassay dilution curves.
Journal of Clinical Microbiology 32:2441-2447
Sukovaty RL, Lee JW, Fox J, Toney K, Papac DI, Grover TA, Wells DS (2006)
Quantification of recombinant human parathyroid hormone (rhPTH(1-84)) in
human plasma by immunoassay: Commercial kit evaluation and validation to
support pharmacokinetic studies. Journal of Pharmaceutical and Biomedical
Analysis 42:261-271