Nitrogen excretion and changes in blood components during emersion of the subtidal spider crab Maia...

Comparative Biochemistry and Physiology Part A 127 (2000) 259–271

Nitrogen excretion and changes in blood components duringemersion of the subtidal spider crab Maia squinado (L.)

Fabrice Durand *, Nicolas Devillers, Francois H. Lallier, Michele RegnaultEquipe Ecophysiologie, Obser6atoire Oceanologique de Roscoff (CNRS, UPMC, INSU), Station Biologique, BP 74,

F-29682 Roscoff Cedex, France

Received 7 February 2000; received in revised form 13 June 2000; accepted 27 July 2000

Abstract

Survival ability of Maia squinado to emersion and subsequent reimmersion was determined in winter and summerconditions. Male spider crabs were less tolerant of emersion than females. Emersion (up to 24 h in summer and to 48h in winter) induced a marked reduction of nitrogen excretion, especially ammonia excretion. Increase in blood ammoniacontent was rapid and very high in summer (1750 mmol l−1), but non-lethal levels. Estimation of the body ammoniaoverload showed that only 30% of unexcreted ammonia accumulated in blood. The ammonia release at reimmersionindicated that ammonia also accumulated in other body compartments. Increase in blood urate content, which indirectlyreduces ammonia production, was similar at both seasons. Emersed M. squinado was rapidly resorting to anaerobicmetabolism, especially in summer when its blood haemocyanin content is low. A strong hyperglycemia was developed inthe first 12 h of emersion at both seasons. Mortality occurring beyond 24 h of reimmersion, when the body ammoniaoverload is cancelled and the recovery of most of blood components is achieved, remains unexplained. © 2000 ElsevierScience Inc. All rights reserved.

Keywords: Ammonia; Emersion/reimmersion; Nitrogen excretion; Urate; Glucose; Lactate; Maia squinado

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1. Introduction

The spider crab Maia squinado is an infralit-toral species common in the Channel and alongthe east Atlantic coast from Ireland to Spain. Itexhibits some specific biological characteristics,i.e. a definitive pause in moulting after the puber-tal moult (Teissier, 1935), large autumnal migra-tions from shallow waters towards deep waters,which are determined by its reproductive cycle(De Kergariou, 1984; Latrouite and Le Foll,1989), successive spawnings (up to four) in sum-mer without further mating (Gonzales-Gurriaran

et al., 1998) and a very low blood haemocyanincontent (Zuckerkandl, 1960). In spite of thesepeculiar properties, the physiological andmetabolic rates of this species, except for oxygenconsumption (Aldrich, 1975), have scarcely beeninvestigated. It is all the more surprising when oneconsiders the economical interest in this speciesand problems inherent in its commercialization,especially, transport in aerial conditions. Forother commercial crustacean species, Nephropsnor6egicus (Spicer et al., 1990; Schmitt andUglow, 1997), Homarus gammarus (Whiteley andTaylor, 1990, 1992) and Jasus edwardsii (Morrisand Oliver, 1999), the physiological response tosuch conditions has nevertheless been studied.Therefore, this prompted us to analyse this prob-

* Corresponding author. Tel.: +33-2-98292312.E-mail address: [email protected] (F. Durand).

1095-6433/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved.PII: S1095-6433(00)00253-1

F. Durand et al. / Comparati6e Biochemistry and Physiology, Part A 127 (2000) 259–271260

lem in M. squinado in order to determine itsability to withstand short-term and long-termemersions.

Emersion is mainly known for impedingbranchial exchanges in marine crustaceans withobvious consequence on respiration. Limitation ofenvironmental water to the small branchial watervolume and its possible reduction, leading to gillcollapse and reduction of the exchange surface, isthought to be the main cause of emersion-inducedphysiological troubles. These are generally morepronounced in infralittoral species (Taylor andWhiteley, 1989; Spicer et al., 1990; Whiteley andTaylor, 1992; Schmitt and Uglow, 1997; Morrisand Oliver, 1999), which rarely experience airexposure, except when imposed, than in intertidaland supralittoral species (Truchot, 1975; DeFurand Mc Mahon, 1984; Varley and Greenaway,1992; Morris et al., 1996). Reduction of both O2

supply and CO2 elimination induces internal hy-poxia and respiratory acidosis. As emersion isprolonged, a shift to anaerobic metabolism andthe subsequent development of lactic acidosis dis-turb the extra- and intra-cellular acid–base bal-ance (for review, see Burnett (1988), DeFur (1988)and Wheatly and Henry (1992)). Impediment ofgaseous and ionic exchanges between hemolymphand branchial water through branchial epitheliumis also affecting ammonia excretion. Ammonia(NH3+NH4

+) is the main nitrogenous waste ex-creted by marine crustaceans and its excretion viathe gills is realised by passive diffusion and activeNa+/NH4

+ exchange (Regnault, 1987). Therefore,emersion is also resulting in a body ammoniaoverload, which may be critical if nitrogenmetabolism is not regulated.

Few studies have been devoted to the effects ofair exposure on ammonia excretion and, conse-quently, on nitrogen metabolism of marine crabs.A marked increase in blood ammonia content wasobserved in Panulirus argus (Vermeer, 1987), Can-cer pagurus (Regnault, 1992), N. nor6egicus(Schmitt and Uglow, 1997), Necora puber(Durand and Regnault, 1998) and Penaeus japoni-cus (Chen and Chen, 1998), as a result of severalhours of air exposure. Reduction of nitrogen ex-cretion during prolonged emersion and compen-sating ammonia release following re-immersionwere studied in only three marine species, such asC. pagurus (Regnault, 1994), N. puber and Carci-nus maenas (Durand and Regnault, 1998). Thepost-reimmersion excretion was also investigated

in N. nor6egicus (Schmitt and Uglow, 1997) andan amphibious crab Potamonautes warreni (Mor-ris and Van Aardt, 1998). Recently, emersion-in-duced changes in tissue amino acid content of twomarine crabs were described for the first time(Durand et al., 1999).

In the present study, M. squinado was submit-ted to various air-exposure periods in winter andsummer conditions. Its survival ability and theemersion-induced changes in its nitrogen excre-tion are presented. Some blood components (am-monia, urate, lactate and glucose), theconcentration of which providing a rapid insightinto the physiological state of crabs, were regu-larly measured throughout both emersion andsubsequent re-immersion periods.

2. Material and methods

2.1. Animals

The spider crabs were collected at the north ofBatz Island (N-Brittany) by local fishermen inearly December. At this time of the year, availablemales were large (\2 kg) and, consequently, onlyfemales, not ovigerous at this period, were stud-ied. The mean body weight of these winter fe-males was 588910 g (n=48). Summer crabswere collected from mid-June to mid-July. Allfemales were ovigerous and these, as well as themales, were studied; mean body weight was 557910 g (n=24) and 590911 g (n=34), respectively.They were kept in indoor concrete tanks (450 l) inrunning seawater (flow rate 15 l min−1, opensystem), under natural photoperiod but soft lightfor two weeks at least, being fed three times aweek with fish meat (Merlangus merlangus). Allexperiments were run in this room (R.H. around90%) when seawater and air were at ambienttemperature, i.e. 1091°C in winter and 2091°Cin summer.

2.2. Experimental emersion/reimmersion

The day before the experiment, 10–14 crabswere transferred into individual buckets in run-ning seawater. At this time, their blood was sam-pled (1.5 ml) and their body weight determined.Crabs quickly compensated for the reduced bloodvolume by drinking, provided that the blood with-drawn represented less than 2.6% of body mass

F. Durand et al. / Comparati6e Biochemistry and Physiology, Part A 127 (2000) 259–271 261

(Greco et al., 1986). Thus, blood sampling(B1% of body mass) was considered not to af-fect survival of the spider crabs. They were leftunfed for 24 h, then transferred into dry PVCcontainers. In order to measure ammonia excre-tion during emersion, 280 ml of seawaterproviding a 2 mm layer on the container bottomwere given to each crab. For emersions lasting18 h or less, blood was sampled only at the endof the emersion. For prolonged emersion (up to48 h), blood was also sampled at mid-time. Aspreviously observed for C. pagurus (Regnault,1992), this double blood sampling did not affectsurvival of M. squinado. Reimmersion was donein a new series of PVC containers previouslyfilled with 20 l of air-saturated seawater; thiswater (continuously and gently aerated) was reg-ularly sampled for determination of the excre-tion rate during the 6 h following reimmersion.Afterwards, crabs were set back into their initialbuckets in running seawater for further 42 h,since total mortality (TM) was recorded at thistime. Some crabs were also bled after 6 and 24h of reimmersion for studying the recovery ofthe blood parameters.

2.3. Measurement of metabolic rates

Ammonia excretion of immersed crabs wasdetermined as above, but over an 8-h period,seawater being sampled every 2 h. The ammoniaamount released by each crab within the first 2h being affected by the handling stress (Reg-nault, 1986; Hunter and Uglow, 1993) was notused for calculation of the mean excretion rate.Urea and amine excretion rates were determinedover a 12-h period for immersed crabs and overthe whole emersion period for emersed crabs.Oxygen consumption was determined over suc-cessive periods of 2 h. The O2 content of seawa-ter was recorded every 20 min using apolarographic electrode and a WTW oxymeter,after crab introduction into the 20 l of O2 satu-rated seawater. Recording of the water O2 con-tent was stopped as soon as the O2 saturationlevel was down to 75–80% and water was fullyresaturated with air stones. A new recording pe-riod started when all containers again presentedan O2 saturated water. This simple method givesreliable values in winter conditions.

2.4. Blood and seawater analysis

Prebranchial haemolymph (1.5 ml), collectedfrom the venous sinus at the base of apereiopod using a sterilised syringe and a 20 Gneedle, was rapidly centrifuged at 8000×g and4°C. The supernatant was divided into four sub-samples. The first one was immediately analysedfor its ammonia (NH3+NH4

+) content using aflow injection per gas diffusion technique(Clinch et al., 1988), as usually (Regnault, 1992).The second subsample was rapidly deproteinisedwith Ba(OH)2 and ZnSO4 for enzymatic deter-mination of glucose content (Sigma kit c510).The third subsample was deproteinised with 10%TCA for enzymatic determination of its lactatecontent (Sigma kit c826, modified according toGraham et al. (1983)). The last subsample wasanalysed for its urate content using a Sigma kit(c685). All analyses were performed in thehours following blood sampling, supernatantsbeing kept at 4°C. Haemocyanin content ofwhole haemolymph was determined fromchanges in absorbance at 335 nm after additionof Na2SO3 (Lallier, 1988) using the millimolarextinction coefficient (E1cm=2.33 cm mmol−1)of Nickerson and Van Holde (1971).

Ammonia content of seawater samples (2×5ml) was determined according to Solorzano(1969), sodium dichloroisocyanurate being sub-stituted for hypochlorite, using for blank, dilu-tions and the (NH4)2SO4 standard solutions, theseawater used daily for filling the experimentalcontainers. Urea content of seawater was deter-mined by the monoxime method (Newell et al.,1967) and amine content by the fluorescaminemethod (buffer pH]9, North, 1975). Accordingto North (1975), compounds that give a maxi-mum fluorescence when the reaction is per-formed at pH\9 represent amino acids andamides.

2.5. Expression of results and statistics

All results are expressed as mean value9S.E.M, n being indicated in the results. The sig-nificance of differences between means wasassessed for PB0.05 with Student’s t-test, usingeither a paired (intra-group comparisons) or un-paired (inter-group comparisons) as needed.


3. Results

3.1. Physiological characteristics of crabsimmersed in normoxic seawater

The relative importance of ammonia, urea andamines in total nitrogen excreted by M. squinadowas first determined. Winter crabs (680926 g)unfed for 24 h excreted 696956 mg ammonia-Nkg−1 h−1 (n=10), 51.495.2 mg urea-N and17.292.7 mg amine-N kg−1 h−1. Thus, ammoniawas making up 91% of excreted nitrogen whileurea and amines accounted for 6.7 and 2.2% ofexcreted nitrogen, respectively.

The ammonia excretion rate of this species wasdetermined in winter conditions (non-ovigerousfemales, at 10°C) and in summer conditions(ovigerous females and males, at 20°C). In allcases, the mean rate was measured on 24 h unfedand unstressed individual crabs in normoxic wa-ter; it represented the routine excretion rate.

In winter conditions, the mean ammonia excre-tion rate was 45.795.06 mmol kg−1 h−1 (n=28).It increased almost 4-fold in response to handlingstress (transfer from buckets to experimental con-tainers), but this stress effect disappeared withinless than 2 h. Following a month of starvation,the mean ammonia excretion rate was reduced to14.493.6 mmol kg−1 h−1 (n=10), as was itsrelative increase in response to handling stress(Table 1). In these conditions, the mean oxygenconsumption rate of regularly fed M. squinadowas 24.6591.03 mg O2 kg−1 h−1 and an atomicO:N ratio of 30 was obtained. A month of starva-tion also reduced O2 consumption rate (14.194

mg O2 kg−1 h−1), but to a lesser extent thanammonia excretion, and a mean atomic O:N ratioof 60 (individual values ranging from 20 to 95)was obtained.

In summer conditions, the mean ammonia ex-cretion rate of males was 64.07913.42 mmolkg−1 h−1 (n=18). A higher rate (100.8920.0mmol kg−1 h−1) was observed in ovigerous fe-males, but it has to be kept in mind that bothexcreted ammonia amounts and body weight wereobviously affected by the egg mass and its associ-ated microorganisms. In response to the abovehandling stress, ammonia excretion rate was in-creased by 50% in females, but not in males.

Blood components were measured on crabs atrest and 24 h before emersion experiments; thesevalues (Table 1) were considered as representativeof the pre-emersion level of each component.

For lactate and glucose, no significant differ-ence was observed between females and males insummer or between winter females and summerfemales. However, in winter females, these bloodcomponents were affected by one month of star-vation when blood lactate (PB0.05) decreasedand blood glucose increased (PB0.001), signifi-cantly. Blood ammonia contents was significantly(PB0.01) lower in winter females than in summerfemales and males, whereas no significant differ-ence was observed regarding sex in summer. Inwinter, prolonged starvation did not significantlyaffect blood ammonia level.

Following one month of starvation in winterfemales, both increase in blood glucose and un-changed blood ammonia level were in agreementwith the starvation effects on ammonia excretion

Table 1Physiological parameters of M. squinado immersed in normoxic sea watera

Winter conditions (à) Summer conditions (à and ß, fed)

Fed Starved Females Males

n=28Ammonia excretion (mmol kg−1 h−1) n=10 n=10 n=18100.8920.014.493.6 64.1913.445.795.1Routine rate

170926 44.899.7Handling stress 158.0945.2 65.8915.7Blood components n=45 n=15 n=30 n=32Haemocyanin (g l−1) 43.391.6 42.292.2 21.191.9 24.792.4

53.793.2 58.0396.23Ammonia (mmol l−1) 69.493.9 68.595.350.093.2Urate (mmol l−1) 89.994.8 97.2910.3 61.392.8

0.16490.013 0.15990.026 0.17390.017Lactate (mmol l−1) 0.11990.0160.20290.0160.20990.018 0.17590.0170.86790.026Glucose (mmol l−1)

a In winter all crabs were non-ovigerous females and some were starved for one month; all others crabs were regularly fed except forthe 24 h-period before blood sampling and rate measurements.; mean values9S.E.M.


Table 2Mortality of M. squinado as a function of the length of the emersion period, the season and the physiological state of crabsa

12 h (%)Emersion periods 18 h (%) 24 h (%) 36 h (%) 48 h (%)

Winter conditionsNon-o6igerous femalesEM 0 4 11 42

0TM 7 28 61

Summer conditionsO6igerous femalesEM 0 16 n.d.

9 25TMMalesEM 7 n.d. 100

50TM

a Percentages are calculated for 25–30 animals. EM, mortality at the end of the emersion; TM, total mortality following 48 h ofre-immersion.

rate and the atomic O:N ratio. Blood urate con-tent was significantly (PB0.001) higher in winterfemales than in summer crabs, and although meanblood urate levels were of the same order insummer crabs, a significant (PB0.01) differencecould be observed between summer males andfemales. However, in winter females, blood uratelevels were not significantly affected by starvation.The blood hemocyanin content (which indicatesthe oxygen carrying capacity of haemolymph) wassignificantly higher in winter crabs compared withsummer crabs; besides, the non-utilisation of thisreserve protein after one month of starvationcorroborated the above data showing that starvedfemales were resorting to carbohydrates for en-ergy requirements in these conditions.

3.2. Emersion effects

At the end of a prolonged emersion (up to 48h), the mortality rate was recorded (EM), butfurther mortality occurred after reimmersion. Thispost-reimmersion mortality was observed only be-tween the 24 and the 48th h of reimmersion in M.squinado. Therefore, two mortality rates are given(Table 2), i.e. EM, mortality subsequent to emer-sion by itself and TM, total mortality recordedafter 48 h of reimmersion.

According to a TM not exceeding 10%, thefemales of M. squinado tolerated 24 and 12 h ofemersion in winter and summer conditions, re-spectively. For the males, the LT50 was alreadyreached after 12 h of emersion. Females, oviger-ous or not, appeared to be more resistant than

males to prolonged emersion and the seawatervolume stored in the egg incubation chambermight possibly explain this difference. The volumeof this external seawater reserve (measured beforeemersion) was 3492 ml (n=21) in non-ovigerousfemales of 602918 g mean body weight (BW); nowater remained in the incubating chamber at theend of a 24- or 36-h emersion. After 24 h ofemersion, the measured global water loss (6% ofBW) was not significantly different (P\0.85)from the water loss at the expense of this waterreserve, suggesting that this water reserve wasused for reducing either branchial water loss orbody dehydration. However, after 36 h of emer-sion, global water loss was increased by 2% andthis loss was significantly higher (PB0.001) thanthe loss of water from the incubation chamberreserve.

At the end of a 36-h emersion in winter condi-tions, M. squinado had excreted 8219252 mgammonia-N kg−1, 170925 mg urea-N and 7509183 mg amine-N. Total nitrogen excreted duringemersion (1740 mg N kg−1) represented only 7%of total nitrogen excreted by immersed crabs dur-ing 36 h. Hourly excretion rates of emersed crabsestimated from these values show that ammoniaexcretion was reduced to 3.5% of its pre-emersionvalue, urea excretion was reduced by 90% andamine excretion was maintained at its pre-emer-sion level. Thus, the marked reduction of nitrogenexcretion was mainly due to ammonia excretionhindrance. Furthermore, the relative importanceof each nitrogenous waste in total nitrogen ex-creted was modified (Table 3), nitrogen as ammo-


Table 3Effect of emersion on nitrogen excretion of M squinado in winter conditionsa

Excreted nitrogen (kg−1 h−1)

Immersed crabs Air exposed crabs

mmol mg N Total N (%) mmol mg N Total N (%)

45.795.1 640Ammonia 91 1.690.5 22.8 471.68 47.1Urea 6.7 0.1790.02 4.8 9.81.13 15.8 2.2Amines 1.4990.36 20.8 43

703Total 48.4

a The rates of ammonia, urea and amine excretion were recorded in crabs immersed at 10°C in normoxic water and in crabs exposedto air for 36 h at the same temperature; mean value9S.E.M. (n=10).

nia and amines being excreted in similar amountsduring the present emersion period.

In summer, ammonia excretion of ovigerousfemales being biased by the egg mass and itsepibionts, only males were used for measurementof nitrogen excretion during emersion and subse-quent reimmersion. In these conditions, malecrabs had excreted 90.62913.58 mmol ammoniakg−1 at the end of a 12 h-emersion when im-mersed crabs excreted 770 mmol ammonia kg−1.At the end of a 24 h-emersion, 151.098.3 mmolammonia kg−1 were excreted versus 1538 mmolkg−1 by immersed crabs. In both cases, ammoniaexcretion was reduced to 10% of its pre-emersionlevel. Similarly, urea excreted by emersed crabs(5.890.3 and 7.590.8 mmol urea kg−1) indi-cated a 90% reduction of urea excretion duringemersion. Amine excretion was not measured atthis time.

Ammonia content of pre-branchialhaemolymph was markedly affected by emersion;its increase was strongly influenced by seasonalconditions (initial rate of ammonia excretion andtemperature, Fig. 1A). In winter conditions, bloodammonia content did not change during the first 6h of emersion, and a significant increase (PB0.001) in blood ammonia was observed after 12 hof emersion. Then, ammonia concentration in-creased at a relatively steady rate (22 mmol l−1),reaching around 1000 mmol l−1 after 48 h ofemersion. In summer conditions, blood ammoniaconcentration increased more rapidly, a signifi-cant higher (PB0.001) ammonia level being ob-served as early as 3 h of emersion (210 mmol l−1),and this blood ammonia level would correspondto that observed after 15 h of emersion in winter.The increase rate of blood ammonia concentra-

tion was around 6-fold as high as in winter. Veryhigh, but not letal, concentrations (1746965.0mmol l−1, n=25) were observed in males as wellas ovigerous females after 12 h of emersion.

Fig. 1. Changes in ammonia (A) and urate (B) contents ofprebranchial hemolymph of M. squinado during experimentalemersions in winter conditions (1091°C) and summer condi-tions (2091°C); mean value9S.E.M.; Pre-emersion values,n=see Table 1; emersion, 105n50.30. Significant changesduring emersion according to the pre-emersion value are indi-cated (*).


Fig. 2. Changes in lactate (A) and glucose (B) contents ofhemolymph of M. squinado during emersions in winter andsummer conditions; mean value9S.E.M.; Pre-emersion val-ues, n=see Table 1; emersion, 105n50.30. Significantchanges during emersion according to the pre-emersion valueare indicated (*).

emersion in winter, but was not letal, as crabswere surviving beyond 48 h of reimmersion.

In response to emersion, M. squinado exhibitedhyperglycaemia. Increase in blood glucose contentdid not depend either on seasonal conditions, orsex or length of the emersion period (Fig. 2B). Inall cases, hyperglycaemia was fully developedwithin the first 12 h of emersion and, at this time,similar blood glucose contents (2.0990.18 mmoll−1, n=18 in winter and 1.7290.22 mmol l−1,n=25 in summer) were noted. According to thewinter experiments, the emersion-induced hyper-glycaemia was maintained throughout the emer-sion period. Much higher individual values ofblood glucose concentration (up to 4 mmol l−1),recorded in summer crabs which survived after 48h of reimmersion, showed that the above value (2mmol l−1) concentration was not a thresholdvalue.

In order to detect the critical concentration ofthese blood components, these were also mea-sured in crabs found dead at the end of emersion.In summer male crabs, which did not survive a24-h emersion, blood ammonia content was34509280 mmol l−1 (n=14), urate content was189.9919.5 mmol l−1, lactate content was 30.591.3 mmol l−1 and glucose content was 3.2490.41mmol l−1.

3.3. E6ents following re-immersion

Ammonia excretion following reimmersion wasstudied in winter female crabs emersed for 24 hand in summer male crabs emersed for 12 h. Assoon as M. squinado were reimmersed in nor-moxic water, they released a large ammoniaamount in few minutes.

After a 24-h emersion at 10°C, 462 mg NH4+-N

per crab (i.e. 0.78 mg NH4+-N kg−1) were re-

leased within the first 5 min following reimmer-sion and this would indicate a 14-fold increase inwinter ammonia excretion rate (Fig. 3A). Follow-ing a 12-h emersion at 20°C, a much greateramount of ammonia (9.95 mg NH4

+-N kg−1) wasreleased at this time and this corresponded to a100-fold increase in the summer ammonia excre-tion rate (Fig. 3B). Such an increase in anymetabolic rate is not possible and this initialammonia release could only be justified by anammonia output from an external body compart-ment. Furthermore, the 10-fold increased excre-tion rate observed in the next hour in summer

Blood urate content also increased during pro-longed emersion but, in contrast to blood ammo-nia, changes in blood urate content were poorlyaffected by seasonal conditions (Fig. 1B). Signifi-cant increase in blood urate was observed after 24h of emersion in winter and after a 6 h emersionin summer, but a similar increase rate (around 7mmol l−1 h−1) was observed at both seasons andfor males and females.

Any increase in blood lactate content indicatesthat some body compartments are hypoxic oranoxic. A partial shift to anaerobic metabolismwas observed early in the emersion period (Fig.2A); then, anoxia was progressively developing asemersion was prolonged. Changes in this bloodcomponent appeared to be related to environmen-tal temperature with a faster accumulation insummer. A mean blood lactate content of 28.292.0 mmol l−1 (n=15) was reached after 48 h of


conditions indicated that some ammonia originat-ing from this compartment was still superimposedon the body ammonia naturally excreted throughgills (Fig. 3B).

In the first hour following this initial ammoniaoutput in winter and in the second hour in sum-mer, ammonia excretion rate was 4.5- and 4.2-foldas high as the corresponding pre-emersion rate,respectively. These values represented, at thistime, a physiological increase in the excretionrates. In every case, ammonia excretion raterapidly returned to its pre-emersion level aftereither 5 h (39.9695.50 mmol kg−1 h−1) or 3 h(79.24923.96 mmol kg−1 h−1) of reimmersion.

Recovery of blood components following thereimmersion of non-ovigerous females emersed

Fig. 4. Changes in the urate (A), lactate (B) and glucose (C)contents of hemolymph of M. squinado following reimmersionafter a 36-h emersion in winter conditions (females), an 18-hemersion (females) and 12-h emersion (males) in summerconditions. For each blood component, its level before emer-sion (pre-E) and at the end of the emersion period (E) areindicated; mean value9S.E.M., n=12. Significant differencesbetween mean values are indicated by * according to thepre-emersion value, and by c according to the value observedat the end of the prolonged emersion.

Fig. 3. Ammonia excretion rate of M. squinado before emer-sion (pre-E), during emersion (Em) and after reimmersion. (A)Non-ovigerous females emersed for 24 h in winter conditions.(B) Males emersed for 12 h in summer conditions. The largeammonia amount released within the first minutes of reimmer-sion is expressed as mmol kg−1 h−1 for comparison; meanvalue9S.E.M., n=10. Significant changes during emersionaccording to the pre-emersion value are indicated (*).

for 36 h in winter conditions, ovigerous femalesemersed for 18 h and males emersed for 12 h insummer conditions is presented in Fig. 4.Haemolymph of crabs was sampled after 6 (R6)and 24 h (R24) of reimmersion (females only).Blood ammonia content rapidly decreased, ac-cording to the recorded ammonia excretion rate,and its recovery was already achieved at R6.Thus, only recovery of blood urate, lactate andglucose contents is shown.


A regular decrease in blood urate level occurredat reimmersion in both emersion conditions (Fig.4A); the decrease rate (between 4 and 5 mmol l−1

h−1) was slightly lower than the increase rateobserved during emersion. However, at R6, de-crease in blood urate of winter and summer fe-males was not significant, whereas a significantdecrease of blood urate was observed for summermales at this time. After 24 h of reimmersion,blood urate concentrations (102.9919.7 and79.6910.6 mmol l−1, n=10) had returned totheir corresponding pre-emersion values (86.394.9 and 61.392.8 mmol l−1), and the recovery ofblood urate was fully achieved at this time.

In summer crabs, blood lactate content de-creased rapidly and decreased significantly afterthe first 6 h of reimmersion, whereas in winterfemale, blood lactate concentration remained con-stant during this period. After 24 h of reimmer-sion, blood lactate levels were still significantlyhigher than pre-emersion levels, although, it wasstrongly lowered especially in summer females. Inwinter females, although, same level was reachedat the end of emersion, only 60% of the recoverywas realised and crabs were still enduring a highblood lactate content (8.3690.97 mmol l−1, n=10).

Decrease in blood glucose content was morerapid in summer crabs than in winter crabs, butafter 24 h of reimmersion, the recovery of theblood glucose was achieved in both summer andwinter females.

3.4. Estimation of the ammonia o6erload andproduction in emersed M. squinado

Estimation of the blood ammonia overload andthe ammonia production of crabs emersed for 24h at 10°C were attempted. A new batch of non-ovigerous females (n=12; mean weight, 660 g)was submitted to various experiments for (1) de-termination of their nitrogen excretion, first inseawater and then during a 24-h emersion; (2)determination of their blood ammonia and uratecontents in these two conditions; (3) measurementof the ammonia amounts excreted in the 6 hfollowing their reimmersion and recording ofsimultaneous changes in blood components. Allparameters were calculated for a 1-kg crab with a300 ml blood volume. Besides, breakdown of onemole of urate producing 4 mol of ammonia, urateaccumulated in blood was considered as an am-

monia reserve and expressed as ammoniaequivalents.

Increase in blood ammonia concentration (+180 mmol l−1) indicated that 54 mmol accumu-lated in this body compartment during emersion.Also, increase in blood urate concentration (+53mmol l−1) corresponded to the retention of 64mmol of ammonia. The blood ammonia overloadresulting from these emersion conditions was 118mmol for a 1-kg crab. On account of this overloadand the nitrogen amounts excreted as ammoniaand amines during emersion (80 mmol), ammoniaproduction of an emersed crab would be around200 mmol, when that of a similar immersed crabwas 1080 mmol.

After 6 h of reimmersion, the whole bloodammonia overload was cancelled and 14% of theurate overload was metabolised; this was leadingto the release of 63 mmol of ammonia. Meanwhile,477 mmol of ammonia were released by a reim-mersed crab while a non-emersed crab excreted270 mmol Blood clearing from ammonia and uratewas making up 30% of the 207 mmol excreted inexcess, suggesting an important ammonia storagein some other body compartments. On account ofthis excretion in excess together with the remain-ing blood urate (55 mmol of ammonia) and thenitrogen actually excreted during emersion, am-monia production during emersion would bearound 340 mmol, i.e. 30% of that of non-emersedcrabs. The same calculation was made for malecrabs exposed to air for 12 h in summer condi-tions. Emersion resulted in a much more impor-tant blood ammonia overload; 670 mmoles for a1-kg crab (ammonia; 540 mmol and urate ex-pressed as ammonia equivalent; 130 mmol). In thefirst 6 h following reimmersion, the ammoniarelease of emersed crabs was 2210 versus 492mmol kg−1 for non-emersed crabs. Blood clearingfrom ammonia and 18.5% of its urate (total, 564mmol) represented 33% of the ammonia excretedin excess of the non-emersed crabs. At both sea-sons, only one third of the ammonia producedand non-excreted during emersion accumulated inblood.

Crab muscle, which makes around 25% of bodyweight, is known to have an ammonia contentone order of magnitude higher than blood; fur-thermore, ammonia storage in this compartmentduring prolonged emersion appeared to be a char-acteristic of subtidal crab species (Durand, 1999).Therefore, the present blood ammonia overload


partly illustrated the body ammonia overload.Consequently, ammonia production during emer-sion was very likely more important than the oneestimated above.

4. Discussion

Branchial exchanges of M. squinado arestrongly impeded when this species is deprived ofenvironmental water. This was corroborated bythe strong reduction in ammonia excretion aspreviously observed in other air-exposed marinecrabs (Regnault, 1994; Durand and Regnault,1998). Urea excretion was also reduced whereasexcretion of amine was unaffected by emersion, itsrelative importance in nitrogen excretion ofemersed crabs being emphasised. Ammonia excre-tion nevertheless prevailed and, as in all studiedspecies (for review, see Durand, 1999), emersiondid not induce any major changes in the nitrogencatabolism pathways of M. squinado. However,total ammonia excreted within either a 36-h emer-sion in winter or a 12-h emersion in summer,corresponded to maintenance of the pre-emersionrate for less than 1.5 h. Thus, the relative reduc-tion of ammonia excretion would be similar atboth seasons, but the rate of the decrease inammonia excretion was different and likely de-pendent on ambient temperature. In C. maenas(Durand and Regnault, 1998), the ammonia ex-creted within the first 12 h of emersion in summerconditions would also correspond to main-tainance of the pre-emersion excretion rate forless than 1.5 h. Thus, estimation of the strongreduction of the ammonia excretion that actuallyoccurs in marine crabs when air exposed, has tobe modulated according to the length of the emer-sion period.

Changes observed in ammonia content ofhemolymph within the first hours of emersioncorroborated the effect of ambient temperature onthe excretory response. The lake of increase inblood ammonia content during the first 6 h ofemersion then its slow increase in winter M.squinado showed that reduction of ammonia ex-cretion occurred lately and progressively. In con-trast, the very early and important changes inblood ammonia of summer animals showed arapid impediment of ammonia excretion. Seawa-ter availability for emersed crabs confines itself tothe small volume of branchial water that would be

rapidly saturated of ammonia. According toDurand (1999),am monia oversaturation ofbranchial water would be responsible for ammo-nia excretion hindrance. Consequently, increase inblood ammonia would be enhanced by a reversalof the diffusion gradient and/or reversal of activeNa+/NH4

− exchanges. Although the emersion-in-duced increase in blood ammonia is common tothe most studied crustacean species (Regnault,1992; Schmitt and Uglow, 1997; Chen and Chen,1998; Durand and Regnault, 1998), blood ammo-nia levels as high as those observed in M.squinado appeared to be very unusual. No appar-ent regulation of blood ammonia content wasdisclosed in any of these subtidal crustacean spe-cies whereas such regulation was observed in theintertidal C. maenas (Durand and Regnault,1998). Besides, our estimation of the body ammo-nia overload in air exposed M. squinado indicatedthat a large part of ammonia accumulated inbody compartments others than blood, muscletissue being a possible site as shown in the subti-dal crab N. puber (Durand and Regnault, 1998).

As soon as M. squinado was reimmersed, itreleased a large amount of ammonia as did othercrab species (Regnault, 1994; Durand and Reg-nault, 1998; Morris and Van Aardt, 1998). In M.squinado, the importance of this ammonia burstappeared to be related to both pre-emersion rateand body ammonia overload rather than to lengthof the emersion period. As previously discussed(Regnault, 1994; Durand and Regnault, 1998),this ammonia burst originated from the clearancefrom stored ammonia of a body compartmentdirectly communicating with the environmentalmedium. The glycosaminoglycans (GAG) of thegills, the content of which increased in C. maenasduring prolonged emersions (Regnault andDurand, 1998) were proposed to be implied in thisammonia storage. The increased number of an-ionic sites of these anionic polymers able to retaincations during emersion and to release them at theonset of reimmersion, owing to the GAG proper-ties and changes in the electrolyte concentrationof the external medium at this time, might explainpart of the ammonia burst not justified by anammonia excretion rate at it is highest metaboliclevel.

Reduction of both oxygen consumption rateand blood PO2 in air exposed subtidal Crustacean(Taylor and Whiteley, 1989; Spicer et al., 1990;Morris and Oliver, 1999) leads to internal hy-


poxia. Consequently, some oxygen-dependent en-zymes could be inhibited, as observed for uricasewhich insures a low blood urate content throughurate breakdown (Dykens, 1991). Thus, increasein blood urate content is considered as an indiceof a developing internal hypoxia in air exposedcrabs (Regnault, 1992; Durand and Regnault,1998; Morris and Oliver, 1999) rather than apossible shift to uricogenesis. Urate accumulationis certainly beneficial since it avoids a furtherincrease in blood ammonia, and may increasehemocyanin oxygen affinity as shown in someother species (Lallier, 1993). In air exposed M.squinado, blood urate content increased at a simi-lar rate in both seasons, but changes occurredearlier in summer crabs than in winter ones. Curi-ously, increase in blood urate was observed whensignificant lactate amounts were already present inblood. This suggested that some body compart-ments were rapidly oxygen depleted during emer-sion, while others were still supplied with oxygen,even at a low rate. Also, urate production, whichis mainly due to xanthine and hypoxanthine oxi-dative reduction via either xanthine oxidase(XOD) or xanthine dehydrogenase (XDH), mightbe reduced during the first hours of emersionwhen oxygen availability is diminished. SinceXOD requires molecular oxygen as cofactor in theurate production reaction, this would indicatethat XOD is the prevailing form of the xanthineoxidoreductase in M. squinado.

M. squinado had a higher blood hemocyanincontent in winter than in summer and this can berelated to changes in blood lactate content duringemersion. This together with the ambient temper-ature could explain the observed rate of lactateaccumulation in blood, which was almost half asgreat in winter as in summer. A similar effect oftemperature on blood lactate was observed in airexposed P. japonicus (Chen and Chen, 1998) butits blood lactate level never exceeded 12 mmoll−1, whereas much higher levels (up to 28 mmoll−1) still compatible with survival were observedin M. squinado.

Increase in blood glucose content in response tovarious stress is usually observed in Crustacea.The emersion-induced hyperglycemia is alwaysdeveloping within the first hours of emersion andits highest level (between 1.5 and 2.0 mmol l−1) isreached in less than 12 h of emersion. Increases inblood glucose appeared to be dependent on theambient temperature (Spicer et al., 1990) but not

on the ambient humidity level (Schmitt andUglow, 1997). Also, hyperglycemia could be tran-sient (Johnson and Uglow, 1985; Spicer et al.,1990) or maintained throughout the whole emer-sion period (Morris and Oliver, 1999). Changes inblood glucose content of M. squinado presentedthe above general pattern: increase during the first3 h and a maximal value (around 2 mmol l−1)reached after 8–12 h of emersion; however, thispattern was not affected by seasonal conditionsand was similar for the all considered crabs. Thewinter females (Fig. 2B) and the recovery experi-ments showed that hyperglycemia was maintainedduring the whole emersion period.

Overall, the ability of M. squinado to withstandprolonged emersion appears to be limited. Femalecrabs were able to survive 48 h of emersion at10°C and possibly 24 h at 20°C. In view ofmortality rates and levels of the present bloodparameters reached after either a 36-h emersion at10°C or an 18-h emersion at 20°C, no differencewas observed between ovigerous and non-oviger-ous females. In summer conditions, male crabswere much less resistant than female crabs. Thefemales were observed to have a water reserve intheir egg incubation chamber and this water, be-ing used during emersion, very likely contributedto the reduction of body water loss.

The mortality recorded between 24 and 48 h ofreimmersion was rather unexpected. Recovery ofblood ammonia, urate and glucose was achievedafter 24 h of reimmersion. This mortality couldnot be attributed to blood lactacte levels sincepostponed mortality occurred even if blood lac-tate recovery has been achieved (i.e. males insummer conditions). This postponed mortalitysuggested that air exposed M. squinado did sufferdefinitive damages, these being more pronouncedas emersion was extended. Since ammonia excre-tion was again active and internal hypoxia hadceased, what vital function or metabolic pathwayhas been impaired by prolonged air exposure? Inwhat extent this damage may be compensatedcalls for further investigations. Finally, the physi-ological response of M. squinado to prolongedemersion appears to be similar, as far as nitrogencatabolism is concerned, to that observed in N.puber (Durand and Regnault, 1998). It was re-cently demonstrated that regulation of the bodyammonia overload induced by air exposure in-volved an active amino acid synthesis and that thesubtidal N. puber could not resort to this detoxify-


ing process (Durand et al., 1999). This appearsalso to be lacking in the subtidal M. squinado.

Acknowledgements

This study was supported by IFREMER (grantc 98-5-57 4408 DRV).

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