Effects of acidification on carbon and nitrogen stable isotopes of benthic macrofauna from a tropical coral reef

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2008; 22: 2955–2960

) DOI: 10.1002/rcm.3694
Published online in Wiley InterScience (www.interscience.wiley.com
Effects of acidification on carbon and nitrogen

stable isotopes of benthic macrofauna from

a tropical coral reef

Joanna Kolasinski1*, Karyne Rogers2 and Patrick Frouin1

1Laboratoire d’Ecologie Marine, Universite de La Reunion, 15 avenue Rene Cassin, BP7151, 97715 Saint-Denis messag cedex 9, La Reunion,

France2National Isotope Centre, GNS Science, 30 Gracefield Road, PO Box 31 312, Lower Hutt, New Zealand

Received 5 May 2008; Revised 23 July 2008; Accepted 24 July 2008

*CorrespoUniversitSaint-DenE-mail: joContract/

Stable isotope analyses are widely used to determine trophic levels in ecological studies. We have

investigated the effects of carbonate removal via acidification on the stable carbon and nitrogen

isotopic composition of 33 species of tropical benthic macrofauna, and we report guidelines for

standardizing this procedure for higher taxa in tropical coral reef ecosystems. Many tropical benthic

invertebrates are small in size, and therefore body tissue isolation (separating organic carbon from

inorganic structures) is difficult and time-consuming. Literature reviews of invertebrate studies show

a lack of consistent procedures and guidelines for preparation techniques, especially for carbonate

removal via acidification of whole individuals. We find that acidification decreases the d13C values of

samples containing carbonate, with shifts ranging from 0.21 to 3.20%, which can be related to CaCO3

content (assessed by a carbonate proxy), justifying acid pre-treatment. Carbonate-containing taxa

benefiting from acidification included Amphinomida, Terebellida (Annelida), Anomura, Brachyura,

Caridea, Amphipoda, Tanaidacea (Arthropoda) and Edwardsiida (Cnidaria). The d13C shifts of

samples containing no carbonate varied up to 0.02W 0.20%. As this induced d13C shift was lower

than the range of an average trophic level shift (0.5 to 1%), we conclude that acid pre-treatment is

unnecessary. Carbonate-free taxa consisted of Eunicida, Phyllodocida (Annelida) and Mollusca. We

note minimal impact of acidification on d15N values except for Brachyura, which showed a shift of

0.83W 0.46%, which is still lower than a single trophic level shift (2.9–3.8%). We conclude that for

trophic level studies, both the d13C and the d15N of carbonate-rich macrofauna can be determined

from the same acidified sample. Copyright # 2008 John Wiley & Sons, Ltd.

Coral reefs are tropical biotopes of great ecological value,

well known for their remarkable biodiversity,1 trophic

complexity,2 and increasingly for their high vulnerability

in coping with anthropogenic stresses. The decline of these

ecosystems is caused primarily by pollution and nutrient

enrichment (eutrophication) from surface runoff or ground-

water.3 Benthic macrofaunal communities (sediment-dwelling

organisms of between 1 and 20mm) rapidly respond to

various human-induced disturbances in a predictable

manner.4 There is growing interest in using benthic

communities as a quality indicator tool for marine ecosys-

tems,5 especially through their trophic structure.6 However,

the food web structures of many coral reef communities are

still poorly known,7 restricting their use as reliable

indicators. Traditional approaches to food web analyses

usually focus on gut content analysis, together with field and

laboratory observations. These approaches are difficult and

ndence to: J. Kolasinski, Laboratoire d’Ecologie Marine,e de La Reunion, 15 avenue Rene Cassin, BP7151, 97715is messag cedex 9, La Reunion, [email protected] sponsor: French Conseil Regional de La Reunion.

time-consuming due to the typically small size of organisms

and low population density in coral reef habitats. Rapid, low-

cost analyses such as the use of stable isotopes are

progressively being developed to study macrofaunal com-

munities in coral reef ecosystems.8,9

More recently, stable isotope analysis has been established

as an alternative approach for investigating relative trophic

positions of organisms.10,11 The d13C values provide

information about the source of carbon entering a food

chain12 whereas the d15N value is used as an indicator of the

relative trophic level.13,14 Isotope ratios of both elements

usually show a constant change along different trophic levels

with a mean trophic split of consumer tissue relative to their

food source of around 2.9 to 3.8% for d15N values and 0.5 to

1% for d13C in marine systems.10,11,15,16 Compared with

temperate systems, few stable isotope studies have been

conducted in coral reef systems, with most dealing with

Scleractinian corals17,18 and primary producers such as

macroalgae,19,20 seagrasses,21 and cyanobacteria.22 Isotope

studies of sediment fauna from coral reefs6,18,23,24 are scarce,

and preparation techniques of tissue samples are often

unclear and differ between studies. Furthermore, some

authors who investigate the carbon stable isotope compo-

Copyright # 2008 John Wiley & Sons, Ltd.

2956 J. Kolasinski, K. Rogers and P. Frouin

sition of whole individuals do not take into consideration the

presence of calcareous structures inside their tissues.

Calcium carbonate (CaCO3) is the principal source of

inorganic carbon in coral reefs25 and one of the major mineral

components of the structure of an organism26 (i.e. exoskeleton

or internal structure). CaCO3 is significantly more enriched in13C than organic carbon27 due to differences between organic

matter synthesis and calcium carbonate precipitation.

Removal of the animal’s internal (i.e. spicules, radula) and

external (i.e. operculum, claw) carbonate structures is thus

required prior to analysis to ensure that only organic carbon is

quantified.28 In most cases, manual extraction of an organ-

ism’s calcareous component is impossible due to its small size

or time constraints.

Sample acidification with diluted hydrochloric acid

(1–2M) has been widely used and is the standard treatment

method for marine sample demineralization.27,29,30 How-

ever, some authors have shown that acidification could lead

to significant decreases in d15N values.27,31 It is assumed that

all nitrogen in animal tissue is organically bound, and is not

removed during the acidification process. A simple verifica-

tion procedure is usedwhich analyzes both the acidified sub-

sample and a portion of the untreated sample to ensure that

the d15N signature is unchanged.

This study provides new data on the effects of acidification

(using HCl) on the isotopic signature of common benthic fauna

found in tropical coral reefs. We report guidelines for

standardizing this procedure in tropical regions. We focus

on higher coral reef macrofauna taxa, which include Annelida

(polychaetes), Arthropoda (crustaceans), Cnidaria (anemones)

and Mollusca (bivalves and gastropods). A key aspect of this

study is the very large number of tropical taxa processed,which

gives a good assessment of the acidification efficiency on each

taxum. For each taxum we determined whether the acidifica-

tion improves the accuracy of the organic d13C values, and the

effects of acidification on nitrogen isotopic composition. This

work contributes to refining methods for food web investi-

gations through stable isotope analysis in tropical ecosystems.

Table 1. List of species and higher taxa submitted for isotopic an

Phylum Class OrderHigher taxa (Infra Ordeor Super Family)

Annelida Polychaeta Palpata AmphinomidaEunicida

PhyllodocidaTerebellida

Arthropoda Malacostraca Decapoda AnomuraBrachyuraCaridea

Peracarida AmphipodaTanaidacea

Cnidaria Anthozoa Actiniaria EdwardsiidaMollusca Bivalvia Veneroida Tellinacea

Gastropoda Cephalaspidea ActeonoideaHaminoeidea

Sorbeoconcha Littorinimorpha

Neogastropoda

N is the mean number of individuals pooled per sample (mean� SD).


EXPERIMENTAL

SamplesStable isotope analyses were performed on faunal taxa

resident in the coral sediments of La Saline fringing reef,

located on the west coast of Reunion Island (218S, 558E), inthe south-western Indian Ocean. Macrofauna were sampled

using an air lift system from the top 15 cm of sediment

(maximum depth reached by macrofauna) to collect a large

variety of species. Nine replicates of 0.2m2 of sediment were

sieved on 1mm mesh to isolate organisms. Due to low

biomass of macrofauna specimens, large numbers of

replicates had to be collected to obtain enough material

for reliable detection. Samples were collected during March

2007 within a short time interval (1 week) to minimize

temporal changes in isotopic compositions.

Sample preparation and analysisMacrofauna species were dissected and cleaned using Milli-

Q deionized water under a dissecting microscope (Leica MZ-

16; Leica Microsystems GmbH, Weltzwar, Germany)

adapted with a cold light source. Dissecting tools were

acid-washed, rinsed with distilled water and roasted at

4508C for 4 h. Laboratory gloves were worn at all times

during macrofauna preparation to minimize carbon and

nitrogen contamination. Entire Annelida, Arthropoda and

Cnidaria individuals were rinsed three times, and detritus

and gut contents were manually removed with fine forceps.

Mollusca species were prepared by peeling back the

calcareous external skeleton (shell) and the operculum,

andmuscle tissue was rinsed three times in deionized water.

All invertebrates were identified to the lowest possible

taxonomic level. Whole individuals from each selected

species were pooled to obtain enough material for reliable

analysis (Table 1). All samples were kept frozen at �208Cuntil they were freeze-dried and ground to a fine homo-

geneous powder.

alyses

rSpecies N

Eurythoe complanata, Eurythoe sp1, Linopherus sp1 2.0� 0.9Arabella iricolor caerulea, Arabella sp1,Onuphis holobranchiata, Eunice cincta

4.7� 2.6

Nematonereis unicornis 23.0� 13Cirratulus africanus, Cirratulus sp1 8.6� 3.8Calcinus latens 3.0� 1.2Portunus longispinosus, Callapa hepatica 4.1� 1.6Alpheidae sp1, Alpheidae sp2 4.7� 2.1Amphitoe alluaudi, Gammaropsis latipalma 26.2� 15.1Tanaidacea sp1, Leptochelia sp1 38.5� 13.2Edwardsiidae sp1, Edwardsiidae sp2 33.6� 13.8Tellina rhomboides, Macoma sp1 2.0� 0.4Bullina vitrea 3.5� 1.7Haminoea cymbalum 1.5� 0.3Natica areolata, Polinices melanostoma,Strombus mutabilis, Cymatium encausticum

2.4� 0.4

Conus coronatus, Cronia sp1,Nassarius albescens gemmuliferus, Hebra horrida

2.5� 0.5


DOI: 10.1002/rcm

Effects of acidification on benthic macrofauna 2957

Samples of macrofauna were divided into two sub-samples,

the first sub-sample was analyzed without any pre-treatment

to serve as a control, while the second was treated with 5mL

1M HCl30 until no further CO2 degassing was observed29

(18h max.). Acidified samples were neutralized with deio-

nized water,31,32 centrifuged three times (3600 rpm for 5min)

and the supernatant discarded before the sample was

freeze-dried and homogenized again. We chose this method

above other methods (e.g. dropwise addition of concentrated

or dilute HCl and evaporation30,33) to ensure the preservation

and longevity of the chemicals inside the combustion tube in

the elemental analyzer during the analytical phase.

Powdered samples were weighed into 6� 4mm tin

capsules. Carbon and nitrogen content and isotopic compo-

sition were analyzed in duplicate at the Stable Isotope

Laboratory, GNS Science, New Zealand, using a Europa Geo

20/20 isotope ratio mass spectrometer (PDZ Europa Ltd.,

Crewe, UK), interfaced to an ANCA-SL elemental analyzer

(PDZ Europa Ltd.) in continuous flow mode (EA-IRMS).

Carbon dioxide and nitrogen gases were separated on a GC

column at 608C, and analyzed for isotopic abundance. The13C/12C and 15N/14N compositions are expressed in

conventional delta notation in per mil (%) relative to the

levels of 13C in Vienna Pee Dee Belemnite (VPDB) and 15N in

atmospheric air. Repeated measurement of an internal

standard exhibited a precision of �0.1% for d13C and

�0.3% for d15N.

Statistical analysis was undertaken with Statistica 7.0

software (StatSoft, Inc., Tulsa, OK, USA). Acidification shifts

were expressed as a mean� standard deviation (SD). Data

were tested for normality by the Shapiro-Wilk’s test, and the

Levene’s test was used for homogeneity. Differences

amongst untreated and acidified samples were examined

with the paired Student’s t-test. The non-parametric

Wilcoxon test was used when the data did not fulfil

requirements. A probability level of less than 0.05 was

considered significant for comparison tests. As the CaCO3

Figure 1. Effect of acidification on d13C (a) and d15N (b) values

content is estimated by carbonate proxy. Plots represent higher tax

4, Terebellida; 5, Anomura; 6, Brachyura; 7, Caridea; 8, Amph

Acteonoidea; 13, Haminoeidea; 14, Littorinimorpha; 15, Neogastro

r2¼ 0.806, N¼ 15, p< 0.001.


content of each sample was not quantified, a carbonate proxy

was used to analyze the effect of CaCO3 on the d13C and

d15N values of untreated and acidified samples.30 Carbonate

proxy¼ ([C:N]untreated/[C:N]acidified) – 1 where [C:N]untreatedis the C:N ratio of an untreated sample and [C:N]acidified is the

C:N ratio of an acidified sample. This proxy is linearly related

to CaCO3 content given that the tissue C:N ratio is

independent of sample CaCO3.

RESULTS AND DISCUSSION

With a total of 131 samples processed, this work is one of the

largest experimental studies of acid pre-treatment effects on

d13C and d15N signatures of marine invertebrates. Our

research was carried out on 33 species belonging to four

major phyla found in sediments: Annelida, Arthropoda,

Cnidaria and Mollusca. We evaluated 15 higher taxa levels,

namely Infra Order or Super Family (Table 1), to produce a

broad tool that can be used in other tropical ecosystems. This

was possible as no significant differences were found in the

isotopic shifts after acidification at species level (one-way

analysis of variance (ANOVA), p> 0.05 in each case). The

species are listed in Table 1. The inter-species variability for

isotopic shift is shown by SD bars in Fig. 1. The precision of

acidified material was generally poorer (d13C: 0.21%;

d15N: 0.17%) than that of the untreated samples (d13C:

0.14%; d15N: 0.11%); however, the acidifiedmaterial was still

considered to be within the analytical error boundaries for

trophic studies.

Tissue acidification resulted in d13C shifts which were

positively related to sample CaCO3 content (linear

regression: r2¼ 0.806, N¼ 15, p< 0.001; Fig. 1(a)). Acidifica-

tion affected the d13C values of samples containing carbonate

(defined by carbonate proxy >0.03),30 with shifts ranging

from 0.21 to 3.20% (Fig. 1(a)), as inorganic carbon is more

enriched in 13C than the organic carbon.27 The magnitude of

the d13C shift depended on the carbonate content of the

and relationship with the CaCO3 content. Sample CaCO3

a mean�SD. 1, Amphinomida; 2, Eunicida; 3, Phyllodocida;

ipoda; 9, Tanaidacea; 10, Edwardsiida; 11, Tellinacea; 12,

poda. Significant linear regression y¼�0.215þ 10.677� x,


DOI: 10.1002/rcm


animal, where a larger d13C shift suggests that acidification of

carbonate-rich samples effects a loss of inorganic carbon,

rather than a loss of acid-soluble organic carbon.

A negative carbonate proxy value was found for Eunicida

polychaetes (Fig. 1) where acidification of these samples

decreased the nitrogen content proportionally more than the

carbon content of tissue. However, Jacob et al.30 suggested

there was no need to correct carbonate proxy values for this

type of effect, as it is assumed to be independent of sample

CaCO3 content. Acidification of many samples containing no

carbonate (carbonate proxy�0.03)30 often induced a negative

effect with more positive d13C values (Fig. 1(a)). The range of

d13C shifts for carbonate-free samples was from 0.02 to

�0.37% and corresponded to values previously reported

concerning the acidification of some marine invert-

ebrates.30,33,34 These negative shifts could, however, be

caused by the removal of acid-soluble 13C-rich compounds in

biological tissues such as proteins, amino acids and

polysaccharides.35 Another hypothesis points to the loss of

hydrolyzed organic matter dissolved in the deionized water

discarded during the rinsing step.31 The mechanisms

controlling this undesirable change remain unclear and

should be further investigated.

Significant differences were found between d13C untreated

and d13C acidified samples of all taxa which contain

carbonate (i.e. carbonate proxy values >0.03, except the

Haminoeidea, Mollusca group) (Table 2). Two polychaete

taxa, Amphinomida and Terebellida, had mean d13C shifts of

0.59� 0.41% and 0.64� 0.32%, respectively. Arthropoda

(Anomura, Brachyura, Caridea, Amphipoda, Tanaidacea)

ranged from 0.88� 0.39% to 3.20� 0.39%, while Cnidaria

Edwardsiida had mean d13C shifts of up to 0.82� 0.26%(Table 2). Removal of the organism’s inorganic carbonate

structures via acidification revealed that the process

significantly decreases the d13C signature. Moreover, the

induced d13C shift from acidification of these organisms was

equal to or higher than the range of 0.5–1% typically

identified for a single trophic level shift. Hence acidification

of the carbonate-containing taxa appears essential to

Table 2. Carbonate proxy values and d13C and d15N isotopic shifts a

outputs

Higher taxa (Infra Orderor Super Family) N

Carbonate proxy(mean� SD)

d13C(mea

Amphinomida 21 0.047� 0.049 0.59Eunicida 5 �0.02� 0.026 �0.07Phyllodocida 5 0.00� 0.004 �0.37Terebellida 6 0.055� 0.044 0.63Anomura 10 0.106� 0.048 1.55Brachyura 10 0.230� 0.035 3.20Caridea 5 0.066� 0.021 0.88Amphipoda 5 0.068� 0.023 1.39Tanaidacea 5 0.219� 0.042 1.76Edwardsiida 24 0.180� 0.039 0.82Tellinacea 6 0.020� 0.030 �0.25Acteonoidea 7 0.017� 0.020 �0.16Haminoeidea 5 0.042� 0.027 0.21Littorinimorpha 6 0.030� 0.027 �0.17Neogastropoda 11 0.004� 0.024 0.02

N¼number of samples.�p< 0.05; ��p< 0.01; ��p< 0.001; ns: non-significant.


producing unbiased values for ecological interpretation

and comparison of food sources.

However, Haminoeidea is an exception (carbonate proxy:

0.042) that shows that some samples with carbonate proxy

<0.05 CaCO3 do not induce bias on the d13C measurement.

We therefore suggest acidification of taxa with a carbonate

proxy �0.05. This threshold is lower than those defined by

Mateo et al.36 with a carbonate proxy >0.09 or Jacob et al.30

with a carbonate proxy >0.08. For carbonate proxies which

lie between 0.03 and 0.05 we recommend testing the

efficiency of the acidification step for all taxa.

The significance of d13C shifts from the acidification of

Amphinomida and Terebellida polychaetes has not pre-

viously been reported. We suggest that common preparation

techniques such as rinsing specimens with deionized water

after dissection would not be effective, as particulate

carbonate material can often be trapped in numerous large

chetae (found in Amphinomida) or dense tentacles which

are able to retain particles (found in Terebellida). This

highlights the importance of taking into account not only the

chemical composition of the organism, but also its physical

environment when devising standard preparation pro-

cedures, particularly in coral reefs where carbonates are

dominant. We have not found any published isotopic studies

of Cnidaria, Anomura, Amphipoda and Tanaidacea to

compare with this study. Brachyura showed a high

d13C shift (3.20� 0.39%) after acidification, which was in

accordance with results by Carabel et al.,29 who found a

significant decrease up to 2.4% for two similar Atlantic taxa

(Polybius henslowii, Necora puber, Portunidae). Bosley and

Wainright37 did not find any significant effects of acidifica-

tion treatment for a temperate Caridea species (Crangon

septemspinosa). This conflicts with results found in this study

which had d13C shifts of 0.88� 0.40 (Table 2). The variability

found in carbon shifts may be due to discrepancies in

methods (e.g. sampling effort, acid form) or geographical

changes in water chemistry such as temperature, pH or ion

chemistry38,39 that can influence the composition of the

structure of an organism and hence its response to the

fter acidification, with Wilcoxon and Student matched-pair test

shiftn� SD)

d15N shift(mean� SD)

d13C shifttest outputs

d15N shifttest outputs

� 0.41 0.39� 0.36 Z¼ 3.484�� ns� 0.14 0.12� 0.31 ns ns� 0.07 0.21� 0.21 ns ns� 0.32 0.11� 0.21 Z¼ 2.023� ns� 0.37 0.02� 0.14 Z¼ 2.803�� ns� 0.39 0.83� 0.46 t¼ 4.205�� Z¼ 2.034�

� 0.40 0.29� 0.39 Z¼ 2.068� ns� 0.22 0.09� 0.24 Z¼ 2.201� ns� 0.41 0.06� 0.19 Z¼ 2.521� ns� 0.26 �0.20� 0.30 Z¼ 5.077�� ns� 0.28 �0.16� 0.35 ns ns� 0.37 �0.16� 0.26 ns ns� 0.11 �0.01� 0.29 ns ns� 0.20 �0.51� 0.20 ns Z¼ 2.201�

� 0.20 �0.20� 0.22 ns ns


DOI: 10.1002/rcm

Effects of acidification on benthic macrofauna 2959

acidification treatment. The latter hypothesis highlights the

importance of conducting preliminary assessments of

sample preparation techniques in each typical oceanographic

zone.

No significant d13C shift after acidification was observed

for any of the carbonate-free samples (Table 2): Mollusca

(mean d13C shift ranged from�0.25� 0.28% to 0.02� 0.20%),

and the polychaetes Eunicida (�0.07� 0.14%) and Phyllo-

docida (�0.37� 0.07%). These results suggest that any small

amounts of carbonate preserved internally within Mollusca,

Eunicida and Phyllodocida tissue (e.g. jaws) do not affect the

overall isotopic signature. Moreover, the induced d13C shifts

were still below the average trophic level shift for carbon

(0.5–1%) and we conclude that acid pre-treatment of these

groups is unnecessary and should be avoided. These results

agree with literature data of Mollusca d13C shifts;33 however,

there is no comparable literature data for polychaete taxa.

The effects of sample acidification on d15N values are

presented in Table 2 and Fig. 1(b). We observe that the

acidification process leads to changes in the d15N values of

some species. There was no statistically significant corre-

lation between tissue d15N acidification shift and CaCO3

content (linear regression: r2¼ 0.315, N¼ 15, p¼ 0.252). Loss

of 15N has previously been reported during acidifica-

tion33,34,40 and is attributed to small losses of acid-soluble

proteins and amino acids via the volatilization of oxidized

nitrogenous organic compounds.41,42 It has been found that

the deionized water rinsing step can also induce a loss of

nitrogen compounds.29 Acidification induced both positive

and negative changes in d15N values, which were lower than

the precision range of the EA-IRMS setup (0.3%) for 12

higher taxa out of 15 (d15N shifts from�0.20% to 0.29%). The

three remaining taxa that show d15N shifts exceeding the

precision range after acidification are Littorinimorpha,

Amphinomida and Brachyura (Table 2). Although signifi-

cant d15N shifts were found in Littorinimorpha after

acidification (�0.51� 0.20%), it is not considered an issue

as acidification of this taxa is not required. Whereas

Brachyura and Amphinomida required acid pre-treatment

before analysis, only Brachyura showed a significant

d15N shift of 0.83� 0.46% after acidification. From the 33

species studied, we suggest that acidification of carbonate-

containing taxa in this study will not lead to an under-

estimation of trophic level if we assume a 2.9–3.8% shift in

d15N values between consumer and prey.15,43 Under these

conditions, dual carbon and nitrogen isotope values

(d13C and d15N) can be determined from the same acidified

sample without compromising trophic levels. When more

precise d15N values are required,44,45 the d15N value should

be measured from a sub-sample of the untreated sample.

CONCLUSIONS

Acidification of whole specimens appears to be a necessary

process for accurate assessment of d13C values in many

higher tropical macrofauna taxa such as Amphinomida and

Terebellida (Annelida), Anomura, Brachyura, Caridea,

Amphipoda, Tanaidacea (Arthropoda) and Edwardsiida

(Cnidaria). The need for acidification is related to the CaCO3


content in body tissue. Samples with a carbonate proxy�0.05

must be acidifed, while acidification efficiency should be

tested for taxa with carbonate proxies which lie between 0.03

and 0.05. Acid pre-treatment should be avoided for tropical

taxa which have no carbonate content (carbonate proxy

<0.03) (e.g.Mollusca, Eunicida, Phyllodocida) to prevent any

loss of soluble N and C compounds which could affect their

bulk isotopic signature. When acidifying the taxa reported in

this study for food web analysis via EA-IRMS, quantification

of the d13C and d15N values can be performed simultaneously

on the same acidified sample, as the d15N shift effects are

minimal and do not interfere with trophic level interpret-

ation. This study provides guidelines for processing tropical

macrofauna samples for ecological analyses, and it could be

extended by further investigations in other higher tropical

taxa.

AcknowledgementsThe authors would like to thank J. K.-L. Leung for laboratory

assistance. DrM. Potier is thanked for helpful suggestions on

a draft of themanuscript. This researchwas supported by the

French Conseil Regional de La Reunion. J.K. also benefited

from support of the Conseil Regional de La Reunion and the

European Social Fund through a PhD grant.

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