Introduction:

Since the start of the industrial revolution, atmospheric levels of carbon dioxide (CO2)
have been rising at a far greater rate than previously experienced in the Earths history
!his is primarily a result of burning fossil fuels !he oceans are a natural carbon sin" and
have so far absorbed approximately half of all anthropogenically produced CO2
(Siegenthaler # Sarmiento $%%&) 'hen CO2 enters the ocean it reacts (ith sea(ater and
alters the chemical properties of the sea itself ()eebe #'olf*+ladro( 2,,$) -mong
other things this process produces hydrogen ions thus increasing the acidity reflected in a
lo(ering of the value of p. Sea(ater p. currently ranges bet(een /0 and 02 and is
already on average ,$ p. unit lo(er than it (as prior to the industrial revolution
(Caldeira # 'ic"ett 2,,&) 1redictions, based on realistic scenarios for future CO2
emissions, suggest that ocean p. (ill decrease by a further ,&2,3 by 2$,, (Caldeira #
'ic"ett 2,,&), a phenomenon termed ocean acidification 'hile the magnitude of impact
(ill vary (ith depth (Caldeira #'ic"ett 2,,&), latitude (Orr et al 2,,4) and habitat, the
effects of ocean acidification on sea(ater chemistry (ill affect all marine organisms -n
organism can be affected by ocean acidification in t(o (ays5 firstly through reduced p.
and secondly through increased CO2 (hypercapnia) .ere (e use the term ocean
acidification to include both
6ifferent species and groups of marine animals vary in their ability to cope (ith, and
compensate for, hypercapnia and lo(ered p. (eg 1o7rtner et al 2,,3,2,,4) (ith
implications for marine trophic interactions Species (ith calcium carbonate s"eletons,
such as molluscs, crustaceans and echinoderms, are particularly susceptible to ocean
acidification -s p. decreases, so too does carbonate availability that has led some
authors to conclude that ocean acidification (ill result in reduced rates of calcification
(eg +attuso et al $%%%) and shell dissolution (eg 8eely et al 2,,3) for all calcified
organisms, as (ell as metabolic depression resulting in reduced gro(th (eg 9ichaelidis
et al2,,4) Echinoderm s"eletons are composed of magnesium calcite that is particularly
susceptible to dissolution as ocean p. decreases (Shirayama # !hornton 2,,4)
'or" on the effect of acidification on echinoderms is currently restricted to investigations
of survival, gro(th and extracellular acid2base balance in a limited number of groups,
mainly echinoids (eg Shirayama #!hornton 2,,45 9iles et al 2,,/) One of the "ey
characteristics of many echinoderm species is their ability to regenerate, (hich involves
alterations in calcification rates (:o(mer # ;eegan $%0&5 :annisteret al 2,,4)
.o(ever, (e "no( nothing of the effect of CO2*induced acidification on such
regeneration Conse<uently, (e have investigated the effect of CO2*induced acidification
on the ability of a calcifying organism (the ophiuroid brittlestar -mphiura filiformis) to
regenerate calcium carbonate structures (arms) =n addition, (e have examined the
potential energetic costs associated (ith regeneration in terms of metabolism and
reproduction -mphiura filiformis is a "ey species in many seafloor communities >sing
sediment*filled cores supplied (ith filtered sea(ater, brittlestars (ere exposed for 3,
days to varying degrees of acidification5 nominally a control of p. 0,, the (orst case
scenario for the end of the century of p. //, a 2&,, scenario of p. /& and finally p.
?0
Hypothesis:

Material & Methods:
(a) Experimental set-up
!he experiment (as carried out in a mesocosm facility ('iddicombe # @eedham
2,,/) -mphiura filiformis collected from 1lymouth Sound, >;, (ere maintained in
sediment cores (five individuals per core) supplied (ith filtered sea(ater of the allocated
p. (p. modified using CO2) Each p. treatment (0,, //, /& and ?0) had four cores
(2, individuals per p.) .alf the individuals in each treatment had one arm removed and
the remaining had t(o arms removed !otal carbon dioxide content (!CO2), p.@=S!,
salinity and temperature of the sea(ater (ere measured for each treatment three times a
(ee" (figure 3)
(b) Oxygen uptake
Closed*bottle respirometry techni<ue adapted from 1omory # Aa(renc($%%%)
6issolved O2 measured using an automated titration system (ith photometric endpoint
(c) Arm regeneration
Begenerated arm discernable from original arm by lighter colour 9easured to ,,4 mm
(ith vernier calipers
(d) Measurement of calcium content
-rms (ere digested in nitric acid and the total calcium content (as determined using
atomic absorption spectrophotometer (Carian Spectr-- 4,) as in Spicer # Eri"sson
(2,,&)
(e) Arm structure and measurement of egg size
Central discs (ere embedded in methacrylate (Ae(is # :o(en $%04), sectioned (ith a
glass "nife and stained (Aees methylene blueDbasic fuchin) Egg feret diameter (as
measured by image analysis soft(are (=9-+E*1BO 1A>S v 34 9edia Cybernetics)
using a digital image (E3, mag Beichert 1olyvar microscope)
(f ) Statistical analyses
-ll statistical analyses (ere run using 9=@=!-: v $3 !he t(o*(ay analysis of variance
(-@OC-) (as used to test for effects of p. treatment or number of arms regenerating on
O2 upta"e, calcium content, arm regeneration rate, egg siFe and arm structure -
;olmogorov2Smirnov test (as used to test for normality
Results:
3 E!!E"# O! O"EA$ A"%&%!%"A#%O$ O$
"A'"%!%"A#%O$ %$ A !%'%!O(M%S
One of the most surprising results is that there (as no decrease in the total amount of
calcium carbonate in individuals exposed to acidified (ater =ndeed, individuals from
lo(ered p. treatments had a greater percentage of calcium in their regenerated arms than
individuals from control treatments, indicating a greater amount of calcium carbonate
(t(o*(ay -@OC- using log transformed data, table $c) Established arms had a
significantly lo(er percentage of calcium carbonate content than regenerated arms (figure
$c)5 ho(ever, number of arms removed had no effect !he interaction bet(een p. and
arm type (established or regenerated) (as significant due to the different amount of
calcium found in each of the arm types5 the regenerated arms having significantly greater
calcium levels than established ones (figure $c) !his (as due to the more developed
s"eletal structure seen in the established arms !hroughout the exposure, and therefore
during the period of regeneration, the brittlestars (ere maintained in sediment cores (also
collected from 1lymouth Sound) in order to simulate natural conditions for the species
!he inorganic carbon levels (G) for this sediment are 22,/ (+,$/?5 S 'iddicombe
2,,4, 2,,/, unpublished data) .o(ever, it is unli"ely that thesediment is being used as a
carbon source for the calcification process5 a previous study found no change in the
carbon (!=C) content of the same type of sediment (fine muddy) containing species
including - filiformis after a 2,*(ee" exposure to p.s of /&, ?4 and 4? ('iddicombe
et al submitted)
!he sediment p. profiles from another acidification study using sediment cores (S
'iddicombe 2,,4, 2,,/, unpublished data) sho( that the p. of the sediment is lo(er
than that of the overlying (ater even under normocapnic conditions5 the p. is /?3 at a
depth of 4 cm, the depth at (hich - filiformis is typically found .o(ever, after a four*
(ee" exposure to mild hypercapnic conditions (overlying (ater p. //) the sediment p.
at 4 cm deep (as still /?3, (hile more severe hypercapnia (p. /& and ?4) only reduced
sediment p. at 4 cm depth by ,$? and ,22 p. units, respectively =n a study by
'iddicombe et al (in preparation), cores of both muddy and sandy sediment (ere
exposed to acidified sea(ater (p. /0, /3 and ?0) for ?, days -fter this time oxygen
profiles (ere measured through the sediment =t (as demonstrated that sea(ater
acidification had no significant impact on the sediment oxygen profiles in either the sand
or the mud, indicating no increase in sediment anoxia p. imaging of @ereis succinea
burro(s sho(ed that the pore(ater p. (as dependent on the burro( profile, animal siFe
and rate of irrigation, (ith high pore(ater p. associated (ith periods of irrigation ()hu
et al 2,,?) -mphiura filiformis continually ventilate their burro(s by arm undulation5
therefore, the p. of their burro( pore(ater is expected to be related to surface (ater p.
rather than the surrounding sediment -s such, the burro(ing lifestyle of this study
species is not counteracting or altering the experimental p. conditions created for the
purposes of this study and the results sho(n are as a result of altering sea(ater p.
!o disentangle the direct chemical effect of p. on the calcium carbonate (ithin -
filiformis arms from the active biological processes used by the species to maintain
calcium carbonate structures, a separate /*day exposure at all four p. treatments (as
carried out on Hdead arms !he arms (ere removed from the animal, froFen for a period
in excess of / days to ;0,8C to "ill, and then brought bac" to sea(ater temperature =n
this experiment, the dead arms (ere placed in small pots (ith no sediment and supplied
continuously (ith sea(ater of appropriate p. >nder these conditions calcium levels
decreased (ith p. (figure 2) -s these arms (ere detached from the individual and
therefore could not replenish the calcium carbonate s"eleton, the decrease in calcium
indicates that this structure is susceptible to dissolution at lo(ered p. !herefore, in live
(attached) arms, an increased rate of calcification is re<uired merely to maintain calcium
carbonate structures in their original condition =n regenerated arms, calcium levels (ere
greater in those organisms exposed to acidified sea(ater than in those held in untreated
sea(ater (figure $c) !his (as true for all three levels of acidified sea(ater !he data
from the detached (dead) arms (figure 2) sho(ed that lo(ered p. caused dissolution of
arm calcium carbonate !herefore, (here these three lo(ered p. treatments appear to
have had a similar response, there (as actually an increasing rate of calcification (ith
lo(ered p. Calcium carbonate in established arms (as also affected by lo(ered p. -t
p. ?0, calcium levels increased and at p. // and p. /&, calcium levels (ere e<ual to
the control indicating that - filiformis actively replaced calcium carbonate lost by
dissolution
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A !%'%!O(M%S ME#A*O'%SM
Bates of oxygen (O2) upta"e (as a measure of metabolic rate), or 9O2, (ere
significantly greater at reduced p.s(//, /& and ?0) than in controls (p. 05 figure $a)
.o(ever, 9O2 (as not significantly different bet(een the three lo(ered p. treatments
(figure $a) =ncreased rates of physiological processes that re<uire energy are paralleled
by an increase in metabolism5 this relationship is seen (ith gro(th and metabolism here
in our results
+ E!!E"# O! O"EA$ A"%&%!%"A#%O$ O$
A !%'%!O(M%S ,(O-#. A$& (E,(O-#.
Sea(ater acidification stimulated arm regeneration -fter the 3,*day exposure, the
length of the regenerated arm (as greater in acidified treatments than in the controls
(t(o*(ay -@OC-, table $b5 figure $b) !his increased rate of gro(th coincided (ith
increased metabolism Begeneration (as not affected by the number of arms removed,
nor (as there a significant difference in any of the physiological parameters
measured as a result of having t(o arms regenerating instead of one !he ability to
regenerate lost arms faster meant a reduction in the length of time animal function (eg
burro( ventilation and feeding) (as compromised by reduced arm length
/ #.E *%O'O,%"A' "OS# O! O"EA$
A"%&%!%"A#%O$
!he internal structure of - filiformis arms (as affected by p. (figure &)5 muscle (astage
occurred at lo(ered p. Aongitudinal sections of the arm sho(ed distinct loss of
musclemass as p.decreased =n each arm segment there are four sections separated by the
calciumcarbonate s"eleton =n the control individuals, these sections (ere filled (ith
muscle -s p. decreased, large empty spaces (ere clearly visible (figure &) Candia
Carnevali et al (2,,$) found that muscle de*differentiation occurred in regenerating arms
as a result of 1C:s, (hich appeared visually similar to muscle loss.o(ever, the de*
differentiatedmuscle sho(ed a change in structure not seen in our study5 the arm muscle
from lo(ered p. samples has the same visual structure as the controls, (ith Iust less
present =n addition, our results sho(ed muscle loss in established arms as (ell as
regenerating, (hereas Candia Carnevali et al (2,,$) found de*differentiation in the
regeneration process !he absence of muscle as a result of lo(ered p. is not de*
differentiation or an inability to synthesiFemuscle tissue under hypercapnia, but rather
muscle loss =n conclusion, arms can be regenerated under hypercapnic conditions but
they are unli"ely to function as (ell as arms regenerated under normal conditions
-mphiura filiformis uses its arms to collect food particles and irrigate its burro(
!herefore,muscle loss can be expected to result in a loss of armmovement (hich in turn
(ill affect both feeding and respiration and ultimately survival !his species is also
predated on by the commercial flatfish dab, Aimanda limanda (:o(mer # ;eegan $%0&),
(hich crop the arms extended into the (ater column =f the muscle mass in these arms is
significantly reduced, so too is the nutritional value, indicating the effects of ocean
acidification could be transferred bet(een trophic levels
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A !%'%!O(M%S (E1(O&2"#%O$ A$& MO(#A'%#3
Egg siFe (feret diameter) and structure (ere not affected by sea(ater acidification (t(o*
(ay -@OC-, table $d5 figure $d ) .o(ever, the timing of this study (6ecember2
January) falls in a latent period of egg gro(th5 development of eggs laid do(n the
previous autumn typically begins in 9arch (:o(mer $%02) !herefore, (hile no
degeneration of eggs (as found in this study, egg development may still be affected by
hypercapnia - further experiment encompassing the egg gro(th phase is re<uired to
assess the impact of ocean acidification on egg development - study by Ao(e et al (in
preparation) has found that the process of vitellogenesis in the surface d(elling ophiuroid
Ophiura ophiura (as disrupted by lo(ered p., highlighting the potential for disruption in
the gro(th phase Spermatogonia (ere not investigated in the current study as all
individuals sampled (ere female 'hile the sex ratio of - filiformis is thought to be $ K
$, patchiness in the distribution of sexes has been documented (:o(mer $%02), (hich
may explain the reason for the absence of males from the samples fixed for gonadal
assessment
'hile some ophiuroids reallocate energy from gonadal to somatic gro(th, and a
decrease of egg siFe is seen (hen arm regeneration is underta"en, this (as not seen in -
filiformis (figure $d ) Ocean acidification has the potential to also affect reproductive
success indirectly5 as a broadcast spa(ner, - filiformis must come to the sediment
surface to spa(n !his behaviour re<uires the arms to move the individual through the
sediment and should arm muscle (astage reduce motility then individuals may release
gametes (ithin their burro(s and far fe(er gametes (ould enter the (ater column5
significantly reducing reproductive success
!he duration of this experiment (3, days) (as chosen to investigate long*term
physiological responses to hypercapnia Shirayama # !hornton (2,,4) have elegantly
demonstrated (ith echinoids that mortality as a result of a ,,4 p. decrease (4?, ppm)
may only occur after several months =nterestingly, even at high levels of hypercapnia
(the ?0 p. treatment crosses the threshold into acidic (ater, ie p.!/,) investigated
here, no mortality (as observed =n light of the results regarding the trade*off bet(een
calcification and muscle mass it is probable that mortality at lo( p. (ill occur as an
indirect result of lo(ered p., and this may ta"e longer than the experimental duration
-ny loss, or impairment, of an important ecosystem engineer ( Jones et al $%%3) (ill
profoundly affect the biotic and abiotic environments (here they occur5 therefore, the
potential for loss of this species (ould alter ecosystems on a large geographical scale
Disscusion:
-ll previous ocean acidification studies on benthic marine invertebrates have reported
reduced calcification rates (+aFeau et al 2,,/) and hypometabolism (9ichaelidis et al
2,,/) as common outcomes .ere (e have sho(n the opposite5 that in some species at
least, ocean acidification can increase both the rate of calcification and metabolism
!hese results change the face of predictions for future marine assemblages (ith respect to
ocean acidification 'hereas it (as previously assumed that all calcifiers (ould be
unable to construct shells or s"eletons, and inevitably succumb to dissolution as
carbonate became undersaturated, (e no( "no( that this is not the case for every
species .o(ever, by investigating the functional conse<uences of hypercapnia and
lo(ered p. at an organism level rather than focusing on a single process, (e have also
detected a cost to these increased activities -rm muscle mass decreased (ith p., ie as
calcification and metabolism increase !here (as a trade*off bet(een maintaining
s"eletal integrity and arm function p. decreased the arm muscle mass by causing the
brittlestar to use the muscle as an energy source -s muscle loss (as seen in established
as (ell as regenerated arms, it is clearly not Iust a failure to synthesiFe muscle tissue
under hypercapnic conditions 8or this particular ophiuroid species, the loss of muscle
mass experienced at lo( p. has implications for survival and ecosystem function5 arm
movement is necessary for feeding (Aoo et al $%%?), burro( aeration ('oodley $%/4)
and predator avoidance (OBeilly et al 2,,?) =n areas (here this animal is present,
burro( creation and irrigation by - filiformis is responsible for up to 0,G of all
bioturbation (Copel et al 2,,&)5 therefore, the effects of ocean acidification (ill also alter
the surrounding environment Besults of a previous study indicate that this trade*off of
increased calcification against reduced muscle mass is occurring in other species5
Shirayama # !hornton (2,,4) found that the decrease in test thic"ness did not account
for total mass loss of the echinoderms .emicentrotus pulcherrimus and Echinometra
mathaei exposed to hypercapnic conditions !hese species may also be decreasing muscle
mass as a cost of increasing calcification and metabolism .ere (e sho( that -
filiformis, and almost certainly other species, (ill attempt to cope (ith changes in
sea(ater acid2base balance >nfortunately, it appears that the physiological responses to
combat the effects of ocean acidification may themselves reduce survival and fitness as
much as acidification itself
Reference: