Retention of a transgenerational marker (137Barium)in tissues of adult female anemonefish and assessmentof physiological stress

Alexandra-Sophie Roy & Ashley J. Frisch &

Craig Syms & Simon R. Thorrold &

Geoffrey P. Jones

Received: 10 October 2011 /Accepted: 20 April 2012 /Published online: 22 May 2012

Abstract Recently it was shown that female fishinjected with enriched stable isotopes maternally trans-mit a chemical signature to larval otoliths. Validation ofthis larval marking technique requires laboratory experi-ments to determine appropriate injection concentrationsand to assess any negative effects on larval and adultcondition. This study investigated the temporal profileof 137barium assimilation and retention in tissues ofadult female anemonefish Amphiprion melanopus(Pomacentridae) following intraperitoneal injectionwith either 2 or 4 μg 137Ba g−1 bodymass. Mean bariumisotope ratios (138Ba:137Ba) in the two groups of treatedfish were not significantly different from each other, butwere significantly different from those in the control groupup to 56 days post-injection. This pattern of 137Ba

retention was consistent across gonad, muscle, liver andbone tissues. Mean plasma cortisol concentration (an in-dicator of non-specific physiological stress) was not sig-nificantly different among groups and was considered tobe representative of unstressed fish. Together, these resultsindicate that (1) A. melanopus suffer minimal physiolog-ical stress and cope well after treatment with 137Ba,(2) 137Ba is retained in female A. melanopus for a pro-longed period (at least 56 days), such that multipleclutches of offspring are likely to be marked with anisotopic signature, and (3) a lower dosage of 2 μg137Ba g−1 appears sufficient for transgenerational mark-ing. It is concluded that 137Ba is suitable for use as atransgenerational marker and is a powerful tool to re-solve long-standing enigmas such as larval dispersaldistances and the fishery benefits of marine reserves.

Keywords Amphiprion melanopus . Coral reeffish . Larval tagging . Batch marking . Otolithmicrochemistry . Plasma cortisol

Introduction

Due to widespread declines in the condition of coralreefs and the fisheries they support (Bellwood et al.2004; Newton et al. 2007; Knowlton and Jackson2008), there is burgeoning interest in the developmentof networks of marine reserves for protecting biodiver-sity and for sustaining fisheries in areas adjacent tomarine reserves (Gell and Roberts 2003; Almany et al.

Environ Biol Fish (2013) 96:459–466DOI 10.1007/s10641-012-0029-y

A.-S. Roy :A. J. Frisch :G. P. JonesARC Centre of Excellence for Coral Reef Studies,and School of Marine and Tropical Biology,James Cook University,Townsville, QLD 4811, Australia

C. SymsInstitute of Marine and Antarctic Science,Hobart, TAS 7001, Australia

S. R. ThorroldBiology Department,Woods Hole Oceanographic Institution,Woods Hole, MA 02543, USA

A.-S. Roy (*)GEOMAR - Helmholz-Zentrum für Ozeanforschung Kiel,Kiel, Germanye-mail: sroy@geomar.de

# Springer Science+Business Media B.V. 2012

2009; McCook et al. 2010). However, our understand-ing of how reserves function has been hindered by lackof knowledge regarding the fate of larvae produced byadult fish within reserves and their relative contributionto fisheries in outlying areas (Sale et al. 2005). Resolu-tion of these long-standing enigmas is therefore a prior-ity issue for natural resource managers.

Most species of coral reef fishes have broad geo-graphic ranges that encompass numerous, seeminglyisolated populations that may be separated by tens tohundreds of kilometres. Although adult reef fish arerelatively sedentary, it has traditionally been assumedthat there is a high level of connectivity among thesepopulations via the larval stage, which inhabits theplanktonic environment for 10–50 days depending onspecies (Leis and McCormick 2002; Mora and Sale2002; Jones et al. 2009). To understand connectivity ofreef fish populations and to develop effective networksof marine reserves, it is first necessary to quantify larvaldispersal distances and rates of localised self-recruitment(Almany et al. 2009; Botsford et al. 2009). A range ofapproaches, including otolith microchemistry (Thorroldet al. 2007), population genetics (Hellberg et al. 2002)and biophysical models (Cowen et al. 2006) have pro-vided new insights into population connectivity, but allof these require validation with direct estimates of larvaldispersal and self-recruitment. Ultimately, direct estima-tion of these parameters necessitates use of mark-recapture methods.

The larvae of coral reef fishes are characterised bysmall size, pelagic habit and highmortality. A successfulmark-recapture program therefore requires ability tomass mark (or batch mark) larvae. This was firstachieved by Jones et al. (1999), who mass marked fishlarvae in situ by temporarily immersing nests ofspawned embryos in tetracycline – a fluorescent com-pound that subsequently forms an identifiable mark inotoliths of juvenile fish. However, the utility of thismethod is limited because most reef fishes are pelagicspawners and methods to manipulate spawned embryosin situ are lacking. To overcome this problem, Thorroldet al. (2006) mass marked fish larvae via maternaltransmission of a chemical marker from gravid femalesto their offspring. The transgenerational marker was arare, stable isotope of barium (137Ba) that was adminis-tered to female fish via abdominal injection. The markeroperates by altering barium isotope ratios in otoliths ofdeveloping embryos, which are subsequently spawnedby treated fish. Once deposited in the core of the

embryonic otoliths, the isotopic signature remains pres-ent throughout the life of the fish. Measurement ofbarium isotope ratios via laser ablation Inductively Cou-pled Plasma Mass Spectrometry (ICP-MS; Arai andHirata 2006) can then be used to identify marked andunmarked offspring after hatching and dispersal(Thorrold et al. 2006). In 2007, Almany et al. success-fully applied the technique to estimate rates of selfrecruitment of both a benthic and a pelagic spawningreef fish. More recently, Williamson et al. (2009) foundthat stable isotope (138Ba) treatment of a commerciallyimportant reef fish did not pose a significant healthrisk to humans who may consume treated fish. Inaddition, research has commenced to assess the utilityof 137Ba as a transgenerational marker for freshwaterfishes (Munro et al. 2009).

Although transgenerational marking provides apowerful new tool for use in empirical investigationsof larval dispersal and population connectivity, thetechnique is yet to be optimized for maximum effi-ciency. The following questions, for example, remainunanswered: What is the optimal dosage of 137Ba formaternal fish? What is the temporal profile of 137Baassimilation in maternal fish and is 137Ba retained forsufficient time to mark multiple clutches of offspring?Is 137Ba retained in different maternal body tissues andin what relative proportions? Another outstanding is-sue is the potential effects of 137Ba on reproductivebehaviour, egg production and offspring quality. Pre-vious studies have shown that physiological stress,caused by capture, handling or chemical treatment,can impair reproductive output (Campbell et al.1992; Schreck 2010). This effect is mediated by thesteroid hormone cortisol, which is released after acti-vation of the hypothalamic-pituitary-interrenal endo-crine axis (Barton 2002; Pankhurst 2011). Elevatedplasma cortisol concentration is therefore regarded asa non-specific indicator of physiological stress and anearly warning sign of future reproductive impairment(Frisch and Anderson 2000; Schreck 2010).

This study aims to advance the development of stableisotope treatment of maternal fish as a method for trans-generational marking of fish larvae. The specific objec-tives of the study were to (1) define the temporal profileof 137Ba assimilation and retention in representativetissues (gonad, muscle, liver and bone) of adult femaleanemonefish Amphiprion melanopus (B. Specie’s au-thority), (2) compare the efficacy of two different dos-ages (2 and 4 μg 137Ba g−1 body mass) for treating

460 Environ Biol Fish (2013) 96:459–466

maternal fish, and (3) assess the degree of physiologicalstress in A. melanopus caused by maternal treatmentwith 137Ba. Plasma cortisol concentration was used anan indicator of chronic physiological stress because cir-culating cortisol levels remain elevated for days to weeksin fish exposed to chronic stressors such as heavy metals(Schreck and Lorz 1978) and crowding (Pickering andPottinger 1989; Frisch and Anderson 2005).

Materials and methods

Study species

Amphiprion melanopus (Pomacentridae) is a protan-drous hermaphroditic fish that lives mutualisticallywith sea anemones on Indo-Pacific coral reefs (Fautinand Allen 1992). It is a benthic spawner that typicallylives in groups of one dominant reproductive pair anda few non-breeding adults and juveniles (Fautin andAllen 1992; Wilkerson 2003). Amphiprion melanopuswas chosen for use in this study because it is highlyamenable to laboratory experiments and because allpost-settlement stages display a very high level of site-fidelity in the wild. These characteristics render A.melanopus a prime candidate for use in future mark-recapture studies that employ transgenerational mark-ing techniques. In addition, baseline (unstressed) plas-ma cortisol levels in A. melanopus have beenpreviously established (Godwin and Thomas 1993),thereby allowing comparative assessment of physio-logical stress levels in the fish used here.

Maintenance of experimental fish

Sixty-one adult A. melanopus (size range 69–116 mm total length) were collected from theGreat Barrier Reef and maintained in separate100 l plastic aquaria at the Marine and Aquacul-ture Research Facilities Unit at James Cook Uni-versity, Townsville, Australia. All aquaria weremaintained on a single recirculating system withconstant water flow, aeration, controlled tempera-ture (29–30°C), salinity (30–32 ppt) and pH (8·0).Dissolved ammonia (NH3), nitrite (NO2) and ni-trate (NO3) concentrations within the system allremained below 0·01 mg l−1 throughout the study.Experimental fish were fed ad libitum daily withFish Dinner Marine Green (Fish Fuel Co.Tm,

Thebarton, Australia) and fortnightly with a freshmix of minced fish and prawn. All experimentalfish were subject to an acclimation period of atleast 90 days prior to commencement of experi-mental trials.

Gender identification and experimental treatment

To identify fish gender, the morphology of genitalpapilla was examined using a stereo microscope. Eachindividual was lightly anaesthetized in a solution ofclove oil, ethanol and salt water at a ratio of 1:5:10.Characteristically, male A. melanopus have a smallpinnacle at the urogenital opening and mature femaleshave an ovipositor that can be observedwhen pressure isapplied to each side of the urogenital orifice (Wilkerson2003). Once the gender of each fish was identified,females (n036) were weighed on a digital balance(±0·01 g) and randomly assigned to one of two treatmentgroups or one control group. The treatment consisted ofan intraperitoneal injection of either 2 or 4 μg 137Ba g−1

body mass. These dosages were thought to approx-imate the minimum dosages required to create anunequivocal isotope signature in the otoliths ofoffspring, and were based on the preliminary workof Thorrold et al. (2006). The enriched stableisotope (137Ba) was obtained as a solution of bar-ium chloride from Oak Ridge National Laboratory(Oak Ridge, Tennessee, U.S.A.) and was deliveredto the peritoneal cavity of experimental fish usinga sterile hypodermic needle (27·5 gauge) attachedto a 1 ml syringe. Injection volumes ranged from0·01 to 0·13 ml per fish, depending on fish massand treatment group. The control group did notreceive an injection because (1) captive A. melanopushave a barium isotope ratio that is indistinguishablefrom natural ratios (Thorrold et al. 2006) and (2) theinjection process may evoke a physiological stress re-sponse (i.e. the aim was to ensure plasma cortisol levelsin the control group were representative of unstressedfish). After treatment, fish were revived in a bucket ofclean seawater and then returned to their aquaria.

The availability of fish was limited by logisticalconstraints, so sample sizes were stratified on the basisof expected variability in barium isotope ratios (seeThorrold et al. 2006). Four fish from each treatmentgroup and two fish from the control group were sam-pled at 2, 7, 21 and 56 days post-injection (n.b. thistrial period was designed to encompass up to four

Environ Biol Fish (2013) 96:459–466 461

potential reproductive episodes; Wilkerson 2003). Dueto accidental mortality (e.g. entrapment of fish in therecirculation system), the sample size of some treat-ment groups was reduced to three. Despite this, smallsample sizes were considered adequate for this studybecause barium isotope ratios in control fish are verystable, and because barium isotope treatment generatesa very distinct response that is statistically easy todetect (Thorrold et al. 2006; Munro et al. 2009; Wil-liamson et al. 2009). On each sampling day, the rele-vant fish were rapidly captured with a dip-net andimmediately anaesthetised in a solution of clove oil(details as above). Fish were secured in a foam cradleand blood (mean volume00·5 ml) was extracted fromthe caudal artery using a 27·5 gauge hypodermic nee-dle on a 1 ml syringe that contained traces of theanticoagulant fluoride heparin (Sigma-Aldrich, CastleHill, Australia). Blood samples were immediatelytransferred to a 2 ml plastic vial and centrifugedfor 5 min at 3,000 rpm to separate plasma, whichwas stored at −20°C until further analysis (Frischet al. 2007a). Each fish was then euthanized anddissected to extract gonad, muscle and liver tissue,as well as three vertebrae from the mid-section(cleaned of soft tissue with a scalpel). Sampleswere then freeze-dried for 50 h and subsequentlystored in a sealed container with silica gel untilfurther analysis.

Analysis of isotope ratios and cortisol concentrations

Dried tissue samples were weighed and then digestedfor 2 h in a solution containing 2·5 ml of 70 % Supra-Pure nitric acid (HNO3), 1 ml of hydrofluoric acid(HF) and 1 ml of perchloric acid (HClO4) (Sigma-Aldrich, Castle Hill, Australia). Samples were heatedin a microwave oven (high power for 2 min), dried inair, and then dissolved in 100 ml ultrapure water. Theconcentrations of 137Ba and 138Ba were measuredusing a Varian 820-MS inductively coupled plasmamass spectrometer (ICP-MS) and subsequently con-verted to ratios (138Ba:137Ba) as per Thorrold et al.(2006). The ICP-MS machine was externally calibrat-ed using a series of barium standard solutions and anindependent standard solution was used to check thevalidity of the calibrations. All samples were continu-ously passed through the ICP-MS machine to ensure ahigh degree of precision.

Cortisol concentration was measured by radioim-munoassay (RIA) following extraction from plasmawith ethyl acetate using the protocol described byFrisch et al. (2007b). Extraction efficiency (94 %)was determined by recovery of 3H-labelled steroidfrom triplicates of a plasma pool, and cortisol assayvalues were adjusted accordingly. Assay specificitywas verified by confirming parallelism in the bindingcurves of serially diluted plasma extracts and cortisolstandards. The minimum detectable plasma cortisolconcentration, estimated as two standard deviationsof the standard curve at zero cortisol concentration,was 0·08 ng ml−1.

Statistical analysis

Two-factor analysis of variance (ANOVA) was used totest for significant differences among mean isotoperatios (138Ba:137Ba) in gonad, muscle, liver and bonetissues (treatment and time were regarded as fixedfactors) followed by post hoc comparisons of groupmeans using Tukey’s HSD test (Zar 1999). Assump-tions of ANOVAwere verified a priori using Levene’stest and normal probability plots (Zar 1999). Hetero-scedastic data were transformed (log10 [x+1]) and thenanalysed as above. All analyses were performed usingSTATISTICA software (StatSoft Inc., Tulsa, U.S.A.)and a significant difference was considered to exist ifp<0·05. All data presented in the text and figures arethe arithmetic mean±one standard error (SE) of un-transformed data.

Results

Temporal profile of barium assimilation and retention

Barium isotope (137Ba) was quickly assimilated intotissues of treated A. melanopus, as indicated by alteredisotopic ratios. Within 2 days of administering either 2or 4μg 137Ba g−1 bodymass, mean ratios of 138Ba:137Bain gonad, muscle, liver and bone tissues of treatedfish were significantly reduced relative to controlfish (Fig. 1 and Table 1). Furthermore, mean ratiosof 138Ba:137Ba in treated fish (2 and 4 μg 137Ba g−1)remained significantly different from control fishfor the duration of the experiment, indicating thatthe isotopic marker would be available for trans-generational transfer to developing embryos for at

462 Environ Biol Fish (2013) 96:459–466

least 56 days. Typically, mean ratios of 138Ba:137Ba ingonad, muscle, liver and bone tissues of treated fish(2 and 4 μg 137Ba g−1) were 2⋅4–12⋅5 fold lower thanthose in control fish at each sampling time. Mean ratiosof 138Ba:137Ba in gonad, muscle, liver and bone tissuesof control fish were remarkably constant and in therange of 5⋅93–6⋅31 (Fig. 1).

Dosage (2 or 4 μg 137Ba g−1) had no apparenteffect on the temporal profile of 137Ba assimilationor retention, since no significant differences couldbe detected among mean ratios of 138Ba:137Ba ingonad, muscle, liver or bone tissues at any timeduring the experiment (Tukey’s HSD test; Fig. 1and Table 1). Furthermore, there was no significantinteraction between treatment (dosage) and time(Table 1). Surprisingly, the pattern of 137Ba assim-ilation and retention was similar among all fourtissues examined, with two possible exceptions.Firstly, mean ratios of 138Ba:137Ba in muscle andliver tissues (Fig. 1b and c, respectively) showedsome evidence of partial recovery (i.e. partial

clearance of 137Ba) after 56 days. Secondly, indi-vidual variation in isotope ratio tended to be great-er in bone tissue (Fig. 1d) than in muscle or livertissues (Fig. 1b and c, respectively).

Fig. 1 Temporal profile ofbarium isotope retention ina gonads, b muscle, c liverand bone d of femaleAmphiprion melanopus aftertreatment with 2 μg 137Bag−1 body mass (long dashedline) or 4 μg 137Ba g−1 bodymass (short dashed line).Untreated fish (solid line)were used as controls. Val-ues represent mean ± stan-dard error. Group means(within dosage) were com-pared using Tukey’s HSDtest; means with the samealphabetic letter are not sig-nificantly different (appliesto the 2 μg 137Ba g−1

treatment)

Table 1 Effect of treatment (2 or 4 μg 137Ba g−1 body mass)and time (2, 7, 21 or 56 days after injection) on mean ratios of138Ba:137Ba in tissues of female Amphiprion melanopus. Nu-merical figures are F values from ANOVA

Treatment Time Treatment × Timed.f.02, 24 d.f.03, 24 d.f.06, 24

Gonad 3⋅787* 2⋅314NS 0⋅468 NS

Liver 4⋅121* 6⋅290** 0⋅848 NS

Muscle 6⋅870** 7⋅292** 0⋅725 NS

Bone 4⋅546* 3⋅050 NS 0⋅701 NS

NS, non-significant (p>0⋅05)*, p<0⋅05**, p<0⋅01

Environ Biol Fish (2013) 96:459–466 463

Physiological stress

There were no significant differences in mean plasmacortisol concentrations between control or treated fish(2 or 4 μg 137Ba g−1) after 2, 7, 21 or 56 days post-injection (Fig. 2 and Table 2). Furthermore, there wasno significant effect of time and no significant inter-action between treatment (dosage) and time (Table 2).The highest observed mean cortisol concentrationswere 3⋅89±0⋅38 ng ml−1 for control fish (56 days),6⋅59±2⋅79 ng ml−1 for fish treated with 2 μg 137Ba g−1

(21 days), and 6⋅61±3⋅47 ng ml for fish treated with4 μg 137Ba g−1 (21 days).

Discussion

Decreased mean ratios of 138Ba:137Ba in gonad, mus-cle, liver and bone tissues of treated fish (2 or 4 μg137Ba g−1) after 2 days indicate that injected 137Ba wasquickly assimilated into a range of maternal tissues.This is consistent with the results of Williamson et al.(2009), who found that enriched barium isotope(138Ba) was rapidly incorporated into tissues of adultfemale coral trout (Plectropomus leopardus). Al-though barium is primarily incorporated into calcifiedstructures such as bones or otoliths (Dietz et al. 1992;Foster et al. 1998), it is apparent from the presentstudy that a portion of the administered 137Ba remainsfree in the soft tissues (gonad, muscle, liver) andprobably also in blood. Retention of the enrichedisotope in soft tissues such as gonad is a critical

requirement for maternal inheritance of the isotopicmarker into developing embryos (Thorrold et al.2006).

After 56 days, there were indications that 137Ba wasbeing slowly eliminated from muscle and liver tissues,but not from gonad or bone tissues. Retention of 137Bain the gonad for at least 56 days after treatment sug-gests that multiple clutches of offspring may bemarked via a single injection of 137Ba (see alsoThorrold et al. 2006). Assuming that adult females ofA. melanopus produce one clutch of 400 eggs every14 days (Wilkerson 2003; authors’ pers. obs.), thenmaternal treatment with a single injection of 137Ba willpotentially produce 1,600 marked larvae over 56 days.This prolonged retention of 137Ba in maternal tissuesis ideal if the aim is to mass mark larvae throughout aspawning season.

Mean ratios of 138Ba:137Ba in gonad, muscle,liver and bone tissues of control fish were remark-ably constant and not dissimilar to the natural ratioof 138Ba:137Ba (i.e. 6·385; Thorrold et al. 2006).Thus, the isotopic signature in treated fish wasunequivocal, regardless of dosage (2 or 4 μg137Ba g−1) and time since injection (2–56 days).There was, however, a high degree of variation inratios of 138Ba:137Ba among individual fish. Thisvariation may be the result of differences in theefficiency of injection, since leaching or excretionof the injectant via the dermal puncture wound orthe intestinal tract (respectively) may have oc-curred in some fish.

Despite the use of two different dosages (2 or 4 μg137Ba g−1), there were no significant differences inmean ratios of 138Ba:137Ba amongst treated fish, re-gardless of time since injection. Furthermore, thehigher dosage (4 μg 137Ba g−1) did not appear toincrease physiological stress, since there was no sig-nificant difference in mean plasma cortisol concentra-tions among the two groups of treated fish. Based on

Fig. 2 Mean (± SE) plasma cortisol concentration in femaleAmphiprion melanopus after treatment with 2 μg 137Ba g−1

body mass (long dashed line) or 4 μg 137Ba g−1 body mass(short dashed line). Untreated fish (solid line) were used ascontrols

Table 2 Effect of treatment (2 or 4 μg 137Ba g−1 body mass)and time (2, 7, 21 or 56 days after injection) on mean cortisolconcentrations in plasma of female Amphiprion melanopus.Numerical figures are F values from ANOVA

Treatment Time Treatment×Timed.f.02, 24 d.f.03, 24 d.f.06, 24

Plasma cortisol 1⋅124NS 0⋅345NS 1⋅130NS

NS, non-significant (p>0⋅05)

464 Environ Biol Fish (2013) 96:459–466

these results, a dosage of 2 μg 137Ba g−1 appearssufficient for transgenerational marking of fish larvae,although the higher dosage of 4 μg 137Ba g−1 may bedesirable in certain circumstances given that no addi-tional physiological stress was observed.

The utility of enriched barium isotopes fortransgenerational marking of fish larvae dependson whether the marker can be administered tomaternal fish without adversely affecting reproduc-tive processes. It is well established that physio-logical stress impairs reproductive output andquality of larvae, and that this phenomenon ismediated by the action of cortisol (Gagliano andMcCormick 2009; Schreck 2010; Pankhurst 2011).Importantly, this study demonstrates that 137Ba canbe delivered to, and retained for at least 56 daysby, adult female A. melanopus without significant-ly increasing the levels of cortisol that circulatearound the body. Importantly, mean plasma cortisolconcentrations in control and treated fish (2 or4 μg 137Ba g−1) were normal for rest ingaquarium-acclimated individuals of reef fishes gen-erally (Begg and Pankhurst 2004; Frisch andAnderson 2005; Frisch et al. 2007b) and of A.melanopus specifically (Godwin and Thomas1993). Together, these results suggest that individ-uals of A. melanopus suffer minimal physiologicalstress and appear to cope well after treatment withdosages up to 4 μg 137Ba g−1. It is thereforeanticipated that maternal treatment with 137Ba isunlikely to bias reproductive output or larval qual-ity via stress-mediated maternal effects.

In conclusion, treatment of A. melanopus withenriched barium isotope, delivered at an appropri-ate dosage for maternal transmission of an isoto-pic marker, appears to be an ideal method formass-marking larvae in the field. Hence, transge-nerational marking is forecast to facilitate resolu-tion of long-standing enigmas such as larvaldispersal distances and the fishery benefits of ma-rine reserves.

Acknowledgments Funding for this work was provided bythe Australian Research Council Centre of Excellence for CoralReef Studies. The authors are grateful for logistical supportprovided by S. Weaver, G. Duffin and J. Morrison at the Marineand Aquaculture Research Facilities Unit, Yi Hu at the Ad-vanced Analytical Centre, and N. Pankhurst at the Fish Endo-crinology Laboratory, James Cook University. Technicalassistance from D. Lemke and K. Markey was also greatly

appreciated. This research was undertaken with permission fromthe James Cook University Animal Ethics Review Committee(approval no. A1134).

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