(Homarus Gammarus) Exposed to Lower Ph at Two Different Temperatures

Home , Homarus gammarus

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C and 1217 µatm Climate ◦ Sciences Sciences Dynamics Chemistry C. Long-term expo- of the Past Solid Earth ◦ Techniques Geoscientific Methods and and Physics and Atmospheric Atmospheric Atmospheric Data Systems Geoscientific erent Earth System Earth System Measurement Instrumentation Hydrology and Ocean Science Annales Biogeosciences ff The Cryosphere Natural Hazards Natural Hazards and Earth System and Earth Model Development Geophysicae ect the ability to find food, sexual ff ects of temperature and projected ff treatment, 31.5 % of the larvae were 2 CO p , respectively, and 20 % in juveniles exposed were deformed, compared with 0 % in larvae 2 7580 Open Access Open Access Open Access Open Access Open Access Open Access Open Access Open Access Open Access Open Access Open Access Open 7579 Access Open Access 2

Homarus gammarus CO CO erences in total length and dry weight at Stage 1 at p , at temperatures 10 and 18 p ff 2 C the development from Stage 1 to 4 lasted from 14 ◦ Climate CO Sciences Sciences C and in high Dynamics p ◦ Chemistry of the Past Solid Earth Techniques Geoscientific Methods and and Physics and Advances in Atmospheric Atmospheric Atmospheric ) is among those species at risk. A project was initiated in Data Systems Geoscientific Geosciences Earth System Earth System Measurement Instrumentation Hydrology and C. At 18 Ocean Science Biogeosciences ◦ The Cryosphere Natural Hazards Natural Hazards EGU Journal Logos (RGB) EGU Journal Logos and Earth System and Earth Model Development erences in carapace length at the various larval stages between the dif- . Some of the deformities will possibly a ff 2 CO p levels of ambient water (water intake at 90 m depth, tentatively of 380 µatm were also larger in size and heavier by dry weight compared with 727 µatm. ), 727 and 1217 µatm Homarus gammarus 2 2 2 C, Stage 2 at both temperatures, producing larvae slightly larger in size and lighter ◦ CO CO CO This discussion paper is/has been under review for the journal Biogeosciences (BG). Please refer to the corresponding final paper in BG if available. Deformities were however observed inof both the larvae larvae and juveniles. exposed Atraised to in 10 elevated pH above 8.0. Atdeformed. 18 Occurrence of deformitiesjuveniles after raised 5 in months ambient of andto exposure low high was 33 andpartner 44 % (walking in legs, claw(tail-fan and damages). antenna), respiration (carapace), and ability to swim ferent treatments, but there were di 10 by dry weight in thep exposed treatments. Stage 3 larvaeUnfortunate raised circumstances in 18 precluded a full comparison across stages and treatment. increases in ocean acidificationp on the lifep cycle of lobster.sure Larvae lasted were until exposed 5 to to months ambient of water age. at Thereafter 14 to the 16 surviving days, as juveniles predictedresulted were under in transferred normal only two pH larvaesignificant values. Growth di reaching was Stage very 4 slow in at the 10 ambient treatment. There were no Trends of increasingence temperatures benthic marine andster resources, ocean ( especially acidification calcifying organisms.2011 are aiming The expected to European investigate to lob- long-term synergistic influ- e Abstract Biogeosciences Discuss., 10, 7579–7615, 2013 www.biogeosciences-discuss.net/10/7579/2013/ doi:10.5194/bgd-10-7579-2013 © Author(s) 2013. CC Attribution 3.0 License. exposed to lower pH attemperatures two di A.-L. Agnalt, E. S. Grefsrud, E. Farestveit,Institute M. of Larsen, Marine and Research, F. P.O. Keulder Box 1870 Nordnes, 5817Received: Bergen, 31 Norway March 2013 – Accepted: 9Correspondence April to: 2013 A.-L. – Agnalt Published: ([email protected]) 2 MayPublished 2013 by Copernicus Publications on behalf of the European Geosciences Union. Deformities in larvae and juvenile European lobster ( 5 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 3 of ect 2 ff CO p ected by ff , focusing on H. gammarus 60 mm CL (Fac- > induced acidosis in C by 2100. The ab- ◦ 2 ). CO H. gammarus 2 − 3 juveniles increased calcifica- ) in , if thermal tolerance limits are 2 2800 µatm) for 60 days but at a . 2 CO p 1200 µatm (Arnold et al., 2009). Calci- CO consists of 4 larval stages, a juvenile p 2 CO p Hyas araneus is currently around 380 ppmv, but is predicted 7582 7581 levels ( 2 ) to carbonate (CO ects of OA ( − 3 2 Homarus ff absorption varying greatly with latitude and tem- 2 Homarus americanus CO uptake is higher and pH lower (7.84) in the eastern Homarus gammarus p 2 into the marine environment leads to an increase in total 2 50 mm carapace length CL) and adult of ∼ Morocco to the colder areas near the Arctic Circle i.e. Tysfjord ff N) (Agnalt et al., 2009). This distribution covers a large latitudinal 100 m to 50 m by 2100 at high latitudes (Fabry et al., 2008). The ◦ ects of acidification may vary greatly for subpopulations across the by the ocean and ocean acidification (OA) occurs mostly in the upper is found along the continental shelf in the northeast Atlantic, extending ∼ ff 2 as carbon emissions from burning fossil fuels keep increasing (IPCC, 2007; 2 ects of both warming and OA, especially calcifying marine organisms. One such ff and converting bicarbonate (HCO Only one study has investigated e H. gammarus + H to harden the shell whichstress on is the energetically animal. costly Low and pHbe resulting therefore devastating from puts to OA great increases moulting physiological physiologicalatures, juveniles stress predicted and already may to under co-occur metabolic withon stress. increased juvenile Warmer OA, lobsters, temper- may as haveexceeded seen its in (Walther added the metabolic et crab stress al.,tion 2010). by 600 %very under high very cost high toto survival (Ries be et initiated al., by 2009). actively The increasing increase pH in calcification at are the thought calcifying centres, therefore reducing 1200 µatm may have beenbly due to acid-base energy regulation being (Arnold channelledthe et towards process al., growth of and 2009). moulting, possi- GrowthMoulting occurring in is more lobsters highly frequently takes temperature in placeperature dependent, juveniles through changes making compared juveniles with (Waddy highly adults. et vulnerable al., to 1995). tem- Moulting also involves depositing of CaCO northern Europe for several centuriesin (Agnalt, Norway 2008). In were the depleted 1960s,covery below lobster has sustainable populations levels been and slow.size due Any further to additional may low push factors recruitment this that species the reduce to re- recruitment the brink andlarvae of population at extinction in the these predicted areas. futurefication scenario (Ca of and Mg) wason significantly growth reduced observed. in The Stages decrease 3 in and calcification 4, observed with no in direct Stage e 4 at a stage, a sub-adult stage ( tor, 1995). Larvae aresettle pelagic during larval in stage thejuveniles 4. three less Little than first is 40 stages known mmis (stage CL about esteemed in the 1–3), as the benthic a after wild stages valuable which (Linnane of marine et they resource, al., and 2001). has The supported European lobster the coastal fishery in distribution range. The life cycle of perature, the e saturated, from Arctic region is alreadyMarine warming organisms up in and colder the regionsthe warming are e is therefore at predicted a tospecies greater increase is risk the further. of European being lobster a from the warm waters o and Nordfolda (68 and temperature range. With CO Regional monitoring of DIC atof Station 2140 M (Skjelvan in et the al.,with eastern 2008), which Norwegian recent is Sea, modeling above shows the a thatNorwegian global value Sea average CO of which 2026. has Thislevels agrees cold will waters most of likely decrease Atlantic the origin depth (Olsen at et which al., calcium carbonate 2006). becomes Low under- pH sorption of atmospheric CO dissolved inorganic carbon (DIC), which changesresulting the in chemistry a and decreased acid-base seawater balance age pH sea (Dickson et water al., pH 2007).al., is Currently 2008). about the It 8.05 global is units aver- predictedthe and to same is drop time, ocean by associated temperature 0.3 withsorption is to a of predicted 0.4 DIC to CO pH increase of units by100 2026 by 2–4 m, 2100 (Fabry and (Feely et varies et with al., 2009). latitude At and temperature (Orr et al., 2005; Fabry et al., 2008). The world’s atmosphere is increasinglytion of becoming CO more saturatedCaldeira with et the al., concentra- 2005). Atmosphericto CO increase to 780 ppmv and 1200 ppmv by 2100 and 2200 respectively. Increased ab- 1 Introduction 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 2 N, 0 52 ◦ solution ) and pH 2 Ω treatment). Cl aiming to be g 2 0.2 ppt. Tem- 12 µatm, and 2 (and other lob- ± ± CO 2 AGA 25 kg gas CO p ) were collected in p × T C 8.06 in ambient treat- = ects of temperature and ff H. gammarus 7.62 in high . The flow rates were regulated 2 = treatment (Table 1), combined for erent stages of the life cycle. The 2 ff CO p and studies concerning other crustacean 7584 7583 were then calculated. Hausser electrodes, using the National Bureau of . treatment and pH T) + C was also included, but due to a limited number of C, to simulate a lower limit and an optimum threshold 2 ◦ treatment the actual values were 727 ◦ 2 H. gammarus CO p CO of approximately 750 µatm and with high p ) and total dissolved inorganic carbon ( T was bubbled into two enclosures, using 2 2 134 µatm in the high A C and 18 2 ◦ ± H. gammarus 0.53 m. Six of these units were used for the ambient, medium CO erent impacts occurring in di p × of 380 µatm occurring in the natural oceanic environment, and treat- ff C the pH in the larval experiments was on average 8.01 from 28 ◦ 2 C, until 25 October 2012 of which they were about 1 year old. The treatment run at two temperatures. For the larval rearing, two 40 L ◦ 2 0.87 CO ects of warming and OA on the life cycle of p × ff 7.79 in medium CO p = ¨ ortner et al., 2004; Spicer et al., 2007). Bicarbonate production is energetically E) over a period of five months, lasting from 28 September 2011 to 22 March 0 35 ◦ Unfortunately, the water quality in ambient water, i.e. intake at 90 m depth, did not stay stable. At 10 ing Orbisint CPS11D from Endress Standards (NBS) scale. Tonected calibrate to Refrigerated the Heating pH, Circulatorsors a Julabo TD301A F12 spectophometry from combined SAIV Hitachi with A/Sin temperature U-2900 was sen- µatm, used. con- total Samples alkalinity for ( analysis350 mL of amber seawater content glass CO bottleswas with added minimal to headspace. 300 preserve µL theon of the sample. satured total Aragonite H hydrogen and ion calcite scale saturation (pH state ( by controllers to be ablement, to pH reach the desired seawaterWater pH from (pH the enclosurestanks. were Temperature, pH supplied and oxygen to were andtank monitored continuously circulated by in through each probes experimental the connected experimental to a computer. The pH was measured every minute us- larvae this had to bebe excluded. The at current experiments were run atments ambient with water, medium believed to 1200 µatm, to simulate low andspectively. medium In OA the scenarios medium predicted forcorrespondingly 2100 and 1217 2200, re- both temperatures. CO bottles, providing a mixture of ambient air and pure CO The salinity during theperature course was of run at the 10 experimentfor was homarid on lobsters, respectively average (Wickins 33.7 andtially Lee, a 2002; Kristiansen middle et temperature al., of 2004). 14 Ini- 2.1 Water quality and high incubators (Hughes kreisel) werejuveniles were placed kept individually in in trays, each5 placed months in unit of the experimental (Fig. age, units the 1a). (Fig.conditions surviving 1b). For at From juveniles on-growing, 14 were the continuedlarvae monitored and but juveniles were under all ambient kept in 8 to 10 hours light and 16 to 18 h dark. Experiments combining OA with temperature05 were conducted at IMR-Matre2012. (60 Raw water was pumpedstation, from thus 90 m representing depth, ambient watertank water. intake Each of located experimental close 0.87 unit to consisted the of field a 400 L synergistic e ster species) are unknowninitiated and in urgently 2011 needs aiming to toprojected be investigate increases long-term studied. in ocean synergistic A acidification e project onand the was juvenile early phase therefore life of cycle of lobster, i.e. the larval 2 Material and methods 1978; P costly and may beclimate reduced change over indicate the thatgions. long warming The term one and study if OA on energyspecies OA will in reserves suggest be are di most low. pronounced Trends in in colder re- crustaceans is usually compensated for by increasing bicarbonate production (Truchot, 5 5 25 15 20 10 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | , 2 2 CO CO p p sp. Lar- , as pre- H. amer- Artemia -control system was (Fig. 3). In these ex- 2 C, to induce hatching 2 ◦ population in Øygarden C in individual 75 L tanks Leucothrix mucor ◦ CO C, and recorded to closest ◦ p the pH in the larval phase was ◦ 7 µatm ± treatment. Despite this, the term ambi- H. gammarus 2 CO p 7586 7585 had two replicates. Every third day the incubators were 2 spring from female American lobster and male European ff 0.02). In other words, pH in ambient water was low and did not ± C. pH was stable in the experiments run at medium and high ◦ E) during the commercial fishing season September to October 2011, 0 50 ◦ 28 cm) in a CT room with the lighting set to a 12 h dark: 12 h light cycle. erence in age were not mixed due to increased risk of cannibalism. Maximum × ff N, 4 ), especially between stage 2 and 3 and between stage 3 and 4 (Templeman, 0 52 35 × ◦ gram did not go as scheduled. The freezer containing many of the later larval stages posed for a minimumwere of three recorded days using before arecorded sampling. dissecting after All 3 measurements microscope. days of Dry ofmicrogram CL weights drying (µg) and of in using TL Termaks individual Mettler drylook larvae Toredo oven for scale were at intermediate (AG204 60 stages, Delta asicanus Range). this Care has was been taken observed1936; to in Wells American and lobster Spraque,observed 1976; in Charmantier hybrids and i.e.lobster Aiken, (A. o 1987). L. This Agnalt, personal has communication, also 2010). Unfortunately, been the sampling pro- Hatchery, Padstow UK, personal communication4), 2009). according The to larvae Sars wereopment staged (1875) stage (1 (1 and to to Herricktreatment 4) (1909). were from A collected each for total incubator measurementtip of (parallels) of of in 10 CL, rostrum each larvae total to temperature length at the and (TL; each end measured devel- of from the telson) and dry weight. The stage 1 larvae had been ex- incubators (plankton Kreisler; Hughes et11 L al., 1974). per The incubators minutevae were sea supplied hatching with over water. a Thelarger period di larvae of were 3 days feddensity were daily for mixed each with in incubator thement frozen was i.e. same temperature set incubator. and to Larvae CO treated 1000 with with larvae, Chloramid-T or to 25 controlviously growth larvae experienced of per in the litre. other bacterium Each lobster rearing treat- systems (D. Boothroyd, National Lobster Each of the20 individual mm tanks water with hosehatched leading the larvae. to ovigerous Larvae separate normally females containers hatchthe had during equipped outflow late an with night/dawn containers overflow a and each through were filter collected morning, a to from counted retain the and transferred to the 40 L upstream 2.3 Larval rearing and sampling operative from early Octoberearly 2011, September and the the temperaturethat hatching was commenced was 28 slowly postponed September increasedhatched accordingly. 2011. to during In Of 18 the a experimental total period.110, 113 The of and sizes 14 135 of mm females, therior carapace only ovigerious rim length 4 females (CL; of had were measured the 91, eggs as eye15 the socket that 000 to distance larvae the from in the posterior total. poste- edge of the carapace). The females hatched Ovigerous females were(60 collected from the and transported to IMR-Matre.(52 They were acclimatized at 6 Lobsters were fed frozen shrimps and fish twice a week. The CO also lower than expected,periments corresponding there to was no 706 truewas control at either, since the the same water levelent quality as has in in the been the ambient kept medium water juveniles throughout from the the paper. exposed InpH treatments the varied grown around experiments in 7.95 monitoring ambient to the 7.96 water surviving (data until not 8 shown). 2.2 months of age, Brood stock of the larval periodrelatively stable (Fig. (7.80 2). Inaccount ambient as water a control at inlarval 18 period any at of 10 the larvalwith experiments, only except a in few the outliers early (Fig. phase 2). of In the the juvenile experiments, pH in ambient water was September until 11 November then dropped to 7.92 and even down to 7.84 by the end 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 2 CO 0.05, 1 mm p × p > 6.123, re- erent = ff C and 224 at C, TL and dry ◦ 0.01, ANOVA, erences in TL, F ◦ ff ered significantly p > ff treatments at Stage 1 ) produced by Nofima ) produced by Nofima treatment with medium 2 2 4.6308 and CO C, development from Stage ◦ p CO = C, and after 5 weeks none of ◦ treatment, as predicted under p F 2 treatment with ambient ( 2 CO 13.44 for dry weight ANOVA), also 5.27 respectively ANOVA), and high p 0.05, = = CO F p F p < 7588 7587 6.35 and = erences in TL between erences in CL in larvae in the same development ff F ff erence between lobsters undergoing the di and temperature treatments (all gave ff 2 C from 22 March to 25 October 2012. The purpose was 11.38 for TL and ◦ = 0.05, CO F p ). On 22 March, TL and CL were recorded for each of the sur- ). At the end of the experiment, CL and TL were recorded for p < treatment. Water flow was set to 18 L per minute. The lobster levels, growth was very slow at 10 http://www.norwegian-lobster-farm.com/no http://www.norwegian-lobster-farm.com/no 2 2 C( ◦ 0.05, erent ff CO CO treatments at these stages ( p p p < 2 CO 53 cm). Each unit was given water quality according to ambient, medium ered significantly at stage 3 comparing high p ff × or high treatments produced slightly larger larvae. Dry weight also di 87 2 2 C giving on average 20 larvae for each treatment. × ◦ There were no significant di CO CO http://www.nofima.no/ http://www.nofima.no/ ANOVA). p between spectively, ANOVA). Larvae raised inand ambient heavier water at stage were 2 lighterweight compared in di with weight the at treatedand stage larvae ambient (Fig. 1 ( 4). Atproducing 18 slightly larger and heavier larvae.or There dry were no weight significant comparing di stage 3 from medium stages. stage between di Fig. 4). There were significantand Stage di 2 at 10 3 Results 3.1 Growth Irrespective of the larvae moulted into Stageent 4. treatment, Eventually, but only died two within larvae the1 reached following to two Stage 4 days. 4, At lasted in 18 normal from ambi- pH 14 to values. 16 Of days the independent 450 of larvae investigated, none were found in intermediate To determine any significant di and temperature treatments, two-way analysis of variance (ANOVA) was used. 2.6 Statistics From five months, the juvenilesthe were raw all water kept intake, into at ambient verify 14 water if i.e. the thequality water deformities of quality higher were pH, in retained asjuveniles or describen were in lost fed 2.1 through special pH moulting varied commerciallygian between produced when Lobster 7.95 pellets kept and Farm (5 in 7.96. mm), The ( ( patented water lobster by Norwe- each surviving juvenile. gian Lobster Farm ( ( viving juveniles. 2.5 Continued monitoring of surviving juveniles in ambient water until 1 yr old While still pelagic, stageconsisting 4 of larvae were single-cell collectedsisted compartments, one of made by 30 one ofholes) and to black to transferred ensure PVC 40 to water plastic. flow. trays individual(87 Three Each to compartments four tray trays with were con- p placed perforated together bottoms in 400 (1juveniles litres mm were units fed special commercially produced pellets (2 mm), patented by Norwe- resulted in a muchtotal lower 409 sampling larvae number have for size18 each recordings, larval 185 stage for than the experiments first run planned. at In 10 2.4 Long-term exposure of juveniles; five months broke at a point, and the samples decayed and could not be processed further. This 5 5 20 15 10 20 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | . 2 CO C and p ◦ erence /twisted ff ff treatment treatment C, 12 % of 2 ◦ 2 CO CO p p . At high exposure, erences in CL as a 2 ff treatment 70 % of the 2 CO p CO C only 20 % were deformed p ◦ , 71 % of the deformed juveniles ected part was curled carapace, (Fig. 8). In ambient and medium 2 were misshaped, all with a curled ff 16), accounting for the di 2 2 = CO could be conducted. After 5 months p CO CO N 2 p p CO p 7590 7589 treatments respectively, were deformed com- 18.4 ANOVA, Fig. 5), but the sample size was 2 = /twisted walking leg as illustrated in Fig. 10. Of ff treatment ( F CO 2 p C (Fig. 7), two Stage 2 larvae had developed defor- erences between ambient and high C, 19 % of the larvae were deformed. Curled cara- ◦ ff ◦ CO C successfully moulted into Stage 4, this was the only 0.05, y carapace (39 %). When the juveniles from ambient ◦ p of around 1217 µatm. At 10 ff 2 p < CO p erence in the shape of a body part or organ compared to the y carapace. Of these, 24 % had also developed deformed claws. ff treatment had deformed claw(s) about 54 % had also developed erent abnormalities (Fig. 10). At high ff treatment. After five months of exposure, 44 % of the juveniles at ) and medium ff 2 2 22 % of the larvae were misshaped. Half of the deformed larvae had 2 y carapace with sti 2, 1.38 ANOVA). treatment, at 10 ff were deformed, compared with 20 % in high treatment ( CO CO CO ered multiple deformities. The most a = 2 2 2 p p ff p . As much as 31 % of the larvae were deformed when raised in 18 CO 2 F p CO CO CO p p p CO p were misshaped, with curled carapace as the only deformity. At 18 treatment, deformed claws were most often found (56 %), followed by sti 2 2 0.109, /twisted walking legs. In comparison, at high = ff At one year age 76 of the 148 juveniles had survived. Mortality was equally high Of the 148 juveniles that survived after five months, 40 were classified as morpho- In the ambient treatment at 10 As only larvae raised at 18 CO CO p in the same medium perate, most likely through moulting. However, ofas the normal 108 juveniles at there five were months classified of age, 15 had seven months later4 developed deformities. Discussion Deformities were observedhigher both in theexposed larval for high and level juvenile phase when exposed to Overall, 28 % of the juveniles(28 %) were deformed. were The most pu commonthe occurring 40 deformities deformed juvenilesmonths. found Of 22 these, March six 2012, wereone only still additional 12 deformed deformity). (four had of In survived the other another juveniles words, seven even 50 developed % at of least the survivors had managed to recu- and medium sti had developed a pu In overall, about 50 %had of two the or juveniles three from di juveniles the had ambient multiple deformities. and medium for those juveniles that had been exposed until 5 months in ambient or in high logically deformed (see Table(i.e. 2). medium In overall, 33pared with and 21 44 % % inp of juveniles the exposed juveniles to inwalking high ambient legs (39 %) and pu much as 31.5 % ofcarapace. the larvae exposed to high 9 November and sampledmedium 22 November, i.e.pace at represented the 45 % period of when thea deformations, pH bent followed by dropped. rostrum damages In to (22p %). the the tail Concurrently, (33 approximately %) and 20 %the larvae of raised the in larvae the(33.3 raised ambient %), treatment in curled were high carapace deformed (33.3at (Fig. %) medium 7), or with damages a bent toa rostrum bent the rostrum, tail 38 fan % with (33.3 %). a Concurrently, curled carapace and 12 % had damages to the tail fan. As No larvae su occurring in 59 % oftrum was the found deformed in larvae 27 %, when and combining damages all to treatments. themities tail A (tail-fan fan bent damages in ros- and 14 % bent of rostrum). all However, the these deformed larvae larva. had been hatched ( 3.2 Deformities Deformities i.e. aaverage di or normal shape wasble found 3). in The both the morphologicalpace larvae abnormalities (Fig. (Table 2) 6), in and damages the the to larvae juveniles the were (Ta- tail fan classified or as that curled the rostrum cara- was bent or curved (Table 2). temperature were long-term exposureof to exposure 148 juveniles hadresult survived. of There were significanthowever di low at thesince medium there were no significant di 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ect ff ydd et C may ect lar- ◦ ff ff H. amer- could potentially of 1200 µatm. Mg- Crassostrea gigas 2 2 CO2 levels of around CO p CO p p should therefore not a 2 and Ries et al. (2009) with CO and Pacific Oyster p ) essential for the calcification process. The 7592 7591 + larvae with concentrations varying from 0.01 2 3 C treatment could also be a result of increased ◦ H. gammarus , CO C that had a true control in place. No larvae raised Mytilus edulis ◦ − 3 C was surprising since we expected a greater e . ◦ 2 levels. Growth above normal may result in thinning of the and not something that normally occurs in this species? C is below the lower optimal temperature limit (metabolic CO 2 ◦ 2 p H. gammarus CO CO p p C. One reason for the higher prevalence of deformities at 18 ◦ was at the lowest 1.12 in this study, but above the threshold limit of C, but does not explain why the degree of deformation is similar be- , and found a significant reduction at elevated ◦ ect on e.g. formation of the new exoskeleton. The higher % of defor- 2 ff − C would pose greater metabolic stress on the larvae than at the optimal ◦ Aragonite is more soluble than pure calcite or aragonite (Andersson et al., 2008). Even Ω . This could however be due to less experience with larvae and juveniles of C. Assuming that 10 3 ◦ Our study shows that deformities occur in lobster larvae from Deformities have not been reported in the two ocean acidification studies conducted A major concern with the present study is the lack of control. It was only in the first (Gazeau et al., 2007; Melzner et al., 2011). A thinner shell in lobster larvae may not mities observed in larvaeat at 10 18 range), 10 temperature of 18 be explained by thebonate water ions chemistry (total parameters such alkalinity,increased HCO as deformities under found saturation in of thegrowth the due 18 to car- elevated shell as reported in blue mussels to 0.02 µg mm CaCO though 1.0 thus carbonatestill ions have were an not e under saturated, elevated erected within a few hours.even The in trait the “bent juvenile rostrum” stages, and was found again across only all in larval exposed727 µatm individuals. stages, at 10 tween 727 and 1217 µatm.decapod Whiteley larvae (2011) is stated unmineralisedval that and conditions. the elevated exoskeleton However, oftions Arnold planktonic of et the al. carapace (2009) of analysed the magnesium concentra- by one experienced person onlysified (main blindly author), and i.e. asoriginally without well, assigned the knowing as individuals the “seems werenot to treatment. clas- seen be Traits this in as before”, arostrum but the moulting in later curled process, newly-hatched this but larvae carapace showed a is to was bit bent be odd when related braking since to out I exposed of have larvae the only. egg The shell, but will be might miss these traits. In this study, all measurements and classifications were made phase were found in the carapace, tail fan or in the rostrum. Inexperienced personnel 2013; Aspaas, 2012; Trengereid, 2012). Theareas hatching at experiments Kvitsøy were made andpair in Øygarden. open of We the have only periopods,or in or two a claws, moults. few could None occasionsexperiments of be found with these misshaped, elevated that misshapes but the looked this first like trait the was claw deformities loston found after lobster, in one Arnold the et al.icanus (2009) with Homarus sp. under normal conditions. In the present study, deformities in the larval due to high exposure The authors of this paper havejuvenile since European the early lobster 1990’s for hatched various and1999, studies produced 2001, larvae as Agnalt and e.g 2008), fitness stockrying and enhancement genetic capacity (Agnalt studies (Agnalt, et (Jørstad 2013) al., et and al., conditioning 2001, 2005), juveniles car- for release purposes (Agnalt, Linnane et al., 2000;et Jørstad al., et 2004; Agnalt, al., 2008;2009). 2001, Arnold 2005; et Wickins al., and 2009, Lee, Ries 2002; et al., Kristiansen 2009,part Schmalenbach of et the al., larvalin phase sea of water the with 10 juvenile pH phase above of 8.0 the developed experimentbother deformities. was trying On without to a publish the this true other work, control. hand, and In the still, other why entire words, are why we even so sure that the deformities are ster larvae and juvenilesAmerican have lobster, not although previously larvaeperimental been and studies, reported, juveniles including ocean neither haveal., acidification been in 1975; and produced European Capuzzo stock and for or enhancement Lancaster, severalnister, 1979; (Gru Latrouite ex- 1994; and Uglem Lorec, et 1991; al., Addison and 1995; Ban- Agnalt et al., 1999, 2001; Nicosia and Lavalli, 1999; 70 % of the deformed juveniles had developed multiple deformities. Deformities in lob- 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | + 2 3 to CO − 3 joints in the C may also ◦ ff ), lack of spines y or swollen thus ff , therefore resulting in the 3 walking legs observed during ff Strombus gigas in the shell results in the incomplete 3 C may have caused severe metabolic . ◦ 3 ecting the carapace (pu walking legs, twisted claws, sti antenna. The latter is of vital importance in ff ff ff 7594 7593 sp. consists of chitin, protein, calcium, magnesium, ) and queen conch ( ected the depositing of CaCO ect various cuticle layers and various parts in the moulting ff ff Homarus can a 2 CO Pecten maximus p ˚ asand et al., 1998; Tsukamoto et al., 1999; and references therein). In in the shell. So that too high deposits, thought to be a compensation 3 erent layers (Al-Sawalmih et al., 2008; Kunkel et al., 2012). The epicuticle ff C were most likely due to the low pH values prevalent in the ambient water in ◦ levels. This may have a 2 ected had also developed multiple deformities i.e. more severe damages. Deformities due to OA has been documented on the embryonic stages for a few Returning juveniles to “control” water at 14 The high % of deformities observed in juvenile lobsters in the ambient treatment In the juveniles, the deformities were a ff greater scallop ( but the walking legsswollen would carapace would eventually become be normal replaced after by several moultings. normal walkingspecies, legs, often and even resultingKawaguchi in et al., low 2010;(Kurihara, or Byrne, 2008; even 2011), Byrne no butever, also hatching et hatchery-induced in changes success al., the haveet 2011; (Parker larval been al., stages et and described 1998; in Sv for al., references ashellfish, a therein). 2009; most few number changes species In of documented species are aquaculture morphological (Olla how- as e.g. lower shell strength in thus resulting in metabolichave exhaustion varied and dramatically, therefore with death. water TheCO taken pH directly at from 14 90high m % without of regulating deformities pHdeformities observed or as in the e.g. surviving damages juveniles. to We the found that tail some fan of could the not be repaired through moulting, average global pH ofilar 8.05 to (Fabry that et al., inmay explain 2008). the the In slightly 727 µatm lower fact, %occurred ambient treatment, of in pH though deformities juveniles in values a this exposed werea treatment. bit to sim- That higher 1217 fewer µatm deformities in was some unexpected, but instances, the which onesstress that which were led toresources thermal high shock. The enough juveniles to may regulate not be their able metabolism to (below maintain their energy metabolic scope), at 18 Masfjorden. Compared to pH(pH 7.98, in Andersen ambient et watermunication, al., 2013) 2013) at located and IMR’s in Parisvatn outer researchfjorden (pH coastal seems stations 8.11, areas to in A. Austevoll have the L. quite same a Agnalt, region, large personal the natural com- water variation in and Mas- may drop to levels well below uncalcified and rapidly needswords, to elevated harden by depositioncycle. Thus, of the calcium deformities carbonate.of may In have Mg-CaCO other been causedfor by low irregularities haemolymph in the pHour depositing (acidosis), study. Whereas resulted too in littleformation the depositing of of sti CaCO certain structuresenergy in resources the (to exoskeleton. maintainand This therefore homeostasis) the could depositing required be of for enough related converting CaCO to HCO depleted consists of a thin calcite-containingamorphous layer while calcium the carbonate rest isof that fully calcium. is mineralized, The mostly highly with endocuticleprocess soluble (Greenaway, consists and 1985; of Dillaman acts et crystallinereabsorption as al., calcite. of 2005; a Moulting calcium Politi transient et occurs is al., source from a 2010), the complicated and old for exoskeleton instance, before it is shed. The new is Growth in crustaceans requires replacement ofexoskeleton, the in old a exoskeleton by process a called newthree moulting. and larger The layers lobster epi-, exoskeleton can2007; exo- be Romano and et divided al., into endocuticle 2007; Al-Sawalmih2009). et (e.g. The al., exoskeleton Sachs 2008; of Sachs et et al.,phosphate al., 2008; and Fabritius a 2006; et al., few Boßelmann otherin compounds et the (Ba, al., Mn, di Sr),is although particularly variable in rich composition in calcium, magnesium and phosphate. The outermost exocuticle where water flow continuously makes a pressure on the exoskeleton. often leaving part of the gillsabdomen, exposed), tail sti fan damagescommunication and in even lobster, sti among others finding a partner (Johnson and Atema, 2005). be strong enough to keep its shape and especially when covering organs like the gills 5 5 25 15 20 10 25 20 10 15 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ) Sci- in the ect the ff ) stock at ort needs to be ff ) in Norway; Com- Palaemon Homarus gammarus from hatching to one year erentiation in lobster, shell ect of this has not yet been ff ff Homarus gammarus Homarus gammarus southwestern Norway (Kvitsøy), in: Stock ff H. gammarus on erentiation in the claws in lobster Homarus ff 2 7596 7595 CO at the Institute of Marine Research – Matre Research p ff ´ eguer et al., 2008, 2010). The deformities were reported ) or lack of di ˚ asand, T., Blackwell Publishing Ltd., Oxford, 415–426, 2001. ˚ aen, E., and Ydstebø, L.: Stock enhancement of European lobster ); a large-scale experiment o ect of elevated ff We thank the sta ˚ asand, T.: Enhancing the European lobster ( has not yet been investigated. This study shows deformities in the larval Trochus niloticus 2 , University of Bergen, Norway, 140 pp., 2008. ˚ asand, T., Blackwell Science Ltd., Oxford, 401–419, 1999. ˚ asand, T., Kors CO ects the ecosystem and life processes in the fjords. p ff ect adult individual mortality and egg production. It is not sure the causes for these ff Homarus gammarus characteristics of the world’s northernmostin stock Tysfjord and of Nordfolda, European northern lobster Norway, ( New Zeal. J. Mar. Fresh., 43, 47–57, 2009. entiarium E., Sv ( enhancement and sea ranching,and Fishing Sv News Books, edited by: Howell, B., Moksness,O.I., E., and Sv Kvitsøy Islands; Perspectives ofsea rebuilding ranching Norwegian developments, stocks, pitfallsBlankenship, in: H. and L., Stock opportunities, and enhancement edited Sv and by: Leber, K. M., Kitada, S., a review, Crustaceana, 67, 131–155, 1994. parisons of reproduction, growth and movement between wild and cultured lobster, Dr. The variation in pH measured in the ambient water used in the present study shows Agnalt, A-L., Farestveit, E., Gundersen, K., Jørstad, K. E., and Kristiansen, T. S.: Population Agnalt, A.-L., Jørstad, K. E., Kristiansen, T. S., Nøstvold, E., Farestveit, E., Næss, H., Paulsen, Agnalt, A.-L., van der Meeren, G. I., Jørstad, K. E., Næss, H., Farestveit, E., Nøstvold, Agnalt, A.-L.: Stock enhancement of European lobster ( Acknowledgements. Station for helpful assistance duringtute of the Marine project Research period. through This the study project was number supported 13193-01, by Ocean the Acidification Insti- – Lobster. References Addison, J. T. and Bannister, R. C. A.: Re-stocking and enhancement of clawed lobster stocks: (i.e. no exchange oftopography water and for hydrology a of periodinto the the of system, fjord. days, the When weeks changeput or water in into months) from water monitoring quality depending outside fluctuation may onthis the be in a the fjord dramatic. water More is quality e flushed throughout the water column and how it fluctuates between seasons and years is scarce. Deep water in fjords may be old ambient water on the Oregonsummer coast was of variable, 2009. pH varied Knowledge from about 7.6 the to 8.2 pH in in the early the Norwegian fjord systems and how there is a negative e of age. Working with early25 yr, stages no of deformities this described species into on the confirm present a study yearly these has basis findings everwithin during been a the the observed. temperature experiment last However, range 20– has that is to within be the repeated species optimalthat with range. the a water true qualitydepths control may and of change dramatically 90 m. within Also a Barton short period et of al. time, (2012) even showed at that the carbon chemistry in the 5 Conclusions and future work Although the present studyperiod, the lack high a ratio true of control deformed throughout lobster most larvae and of juveniles the strongly experimental indicates that Gironde estuary in France (B to a deformities, stress or environmentallower contamination. However, a possibleand response juvenile phase to of Europeanability lobster. 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Analysis of nutrition gave estimates of nitrate − T ) exposed to elevated C Seawater parameters measured and calculated during the OA experiment. Classification of deformities found in larval stages 1 to 4 in European lobster ( CategoryCurled carapace Organ Carapace The carapace was curled at the Description edge, often forming Tail-fan damages UropodBent rostrum Damages to parts of the tail fan, or Rostrum even lacking one The rostrum was bent, as if not yet straighten out Table 2. gammarus alkalinity, (12.3 µmol kg Table 1. tions. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

Homarus piece, ﬀ

cult to 2 2 CO CO p ﬃ p Ambient High - - Tank 11 Tank Tank 10 Tank Tank 12 Tank Medium Medium C ° C - ° ° 18 18 18 b) Juvenileb) phase larval (two incubators as parallels in

” when touching. Di ﬀ (a) 15b 14b 13b Incubator Incubator Incubator y/swollen, or up folded on one side often ﬀ 2 2 7606 7605 CO 15a 13a 14a p CO p 13 Ambient High High - - Medium Medium Incubator Tank Tank 15 Tank C Incubator Incubator Tank 14 Tank - levels.

C 18

18 2 18 CO sometimes “frozen” in an arbitrary/twisted position. observe when animal was out of water. p juvenile phase with trays with individual compartments (several 2b (b) 3b 4b Incubator

Incubator Incubator ected 2 ﬀ 2 2a 3a CO 4a

a 2nd antenna Segments of the antenna were fused, as if the joints were p CO p Ambient - High High Incubator Incubator Tank 2 Tank - Incubator Tank 3 Tank Medium Tank 4 Tank C -

C exposed to elevated

C Larval phase Larval 10

10 Classiﬁcation of deformities found in juvenile stages of European lobster (

a) 1. Figure Overview of the experimental setup during the

y Carapace Carapace pu /twisted 2nd–5th The joints were fused together as if the joints were over ff ff ff 769 770 771 772 773 774 775 776 Pu Category Organ Description carapaceSti walking legs pereopodMiss-shapedclaw 1st pereopod/ calsified. The entire pereopod Various shapes legBent of was the like one cheliped, sti but leavingrostrum most some often parts twisted, of the gills exposed. Tail-fan chelipeddamages RostrumTwisted Uropod deviating fromabdomen normal. The rostrumSti was bent, Abdomen as if notantenna yet Damages erected to after parts hatching. of the tail fan, Abnormal or shape even of lacking one the or abdomen as if both some of of the the tail segments fans. were once broken and then grown back in a wrong shape. over calcified. Felt “sti Fig. 1. trays in each tank unit). each tank unit) and in the gammarus) Table 3. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

◦ C in 2011. ◦ 18 (b) Ambient MediumpCO2 pCO2 High

C and ◦ 10 b) 18°C b) (a) 8,1 8,0 7,9 7,8 7,7 7,6 7608 7607 C were monitored, as non reached stage 4 in 10 ◦ Ambient MediumpCO2 pCO2 High in the juvenile phase until 22 March 2012 in the experiments run at in the larval phase run at NBS NBS

8,1 8,0 7,9 7,8 7,7 7,6 pH

a) 10°C a)

3 Figure

7,7 7,6 8,1 8,0 7,9 7,8 Variation in pH Variation in pH

re 2. re

pH C. Note that only juveniles in 18 ◦

Figu

33 treatment and C from newly ◦

2 32 CO

14 14 14 4 4 4 raised in 18 13 13 13 medium 3 3 3 treatments. Ambient is raw wa- 12 12 12 (b) 2

2 2 2 Larval stage Larval 11 11 11 18°C CO p Ambient MediumpCO2 pCO2High 10 10 10 1 1 1

9 9 9 6 4 2 0 8 6 8 4 0 16 14 12 10 16 12

8 8 8

ambient water, 2 survived 2 days 2 survived 2 7610 7609 Homarus gammarus 4 4 4

Carapace length (mm) length Carapace 7 7 7 (a) 3 3

3 6 6 6

C for the various ◦ 5 5 5 10°C 2 2 2 Larval stage Larval 4 4 4 1 1 1 and 18

◦ 5 0 5 0 5 0 . 10 30 25 20 15 10 30 25 20 15 10 30 25 20 15

6 4 2 0 8 4 0 8 6

16 12

16 14 12 10

Number of juveniles of Number length (mm) length Carapace µg) ( wieght Dry Total lengh (mm) lengh Total

Figure 5. Figure for 10

Figure 4 Figure

787 788 786 789 790 791 792 treatment. Size recordings were made 22 March 2012. 2 CO p Carapace length(mm) of juvenile Carapace length (mm), total length (mm) and dry weight (µg) at each larval stage of high Fig. 5. hatched larvae until 5(c) months of age in ter intake at 90decayed. m depth. Note that lacking bars are due to a freezer broke and the samples Homaru gammarus Fig. 4. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | than 8.1.

34 NBS 7612 7611 with deformities in the larval phase. larvae raised in environment with lower pH Homarus gammarus Homarus gammarus 0

. 20 10 40 30

7. % deformed %

6 Figure

Figure Figure

Percentage of A Stage 3

806 807 808 809 810 795 796 797 798 799 800 801 802 803 804 805 793 794 Fig. 7. Fig. 6. The carapace is curled, leavingas “curls” a on deformity. the side (indicated with the arrow). This is classiﬁed

76 Total 148 C/Ambient pCO2 C/Medium pCO2 C/High 22March2012 25October 2012 4 deformities 4 ° ° ° 18 18 18 47 C/High C/High ° pCO2 85 18 3 deformites 3 4 7614 7613 pCO2 18 C/Medium C/Medium ° 18 for ﬁve months. 2 deformities 2 2 CO 25 p 45 C/Ambient ° 18 1 deformity 1

0 . 20 10 80 70 60 50 40 30

60 50 40 30 20 10 70

% Occurence % % deformed %

Figure 8. Figure

Figure 9 Figure The percentage of juveniles with deformities. Light grey bar represents juveniles surviv- The percentage of lobster juveniles with single or multiple occurrence of deformity when

Fig. 9. exposed to elevated levels of Fig. 8. ing 5 months of exposurewater and only. Total darker number grey of bar juveniles in the each juveniles category after is another given 7 above months each in column. ambient 820 818 819

Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | lower than 8.1 since NBS /twisted walking legs. ﬀ 7615 y carapace and sti ﬀ juvenile, one year of age, exposed to pH

10 Figure

Homarus gammarus 840 832 833 834 835 836 837 838 839 822 823 824 825 826 827 828 829 830 831 821 birth displaying two deformities, pu Fig. 10.