Locomotory, ventilatory and metabolic responses of the subterranean Stenasellus virei (Crustacea, Isopoda) to severe hypoxia and subsequent recovery

0 Acadbmie des sciences / Elsevier. Paris Biologie et pathologie animales / Animal biology and pathology

Locomotory, ventilatory and metabolic responses of the subterranean Stenasellus virei (Crustacea, Isopoda) to severe hypoxia and subsequent recovery Reponses locomotrices, ventilatoires et m&aboliques du Crustac6 isopode souterrain en hypoxie s&&e et en &up&-a tion FRCD~RIC HERVANT’*, JACQUES MATHIEU*, GIUSEPPE MESSANA~ ’ Hydrobiologie et kcorologie souterraines (Esa CNRS 5023), universitk Claude-Bernard-Lyon-I, 2 Centro di Studio per la Faunistica ed Ecologia Tropicali del CNR, 50125 Florence, Itab

G9G22 I/ilkurbanne cedex, France;

Les activiks locomotrices

et ventilatoires ainsi que l’kolution du mttabolisme intermkdiaire et en hypoxie s&&e (Po2 < O,O3kPa) et lors d’une phase de rCcuptration post-hypoxique chez le crustace isopode aquatique hypogt Stenasellus virei. Les diffkrents buts de ce travail ont ktk i) de determiner pourquoi les esptces souterraines prbentent des temps de survie en hypoxie plus ClevCs que la plupart des crustaces superficiels, ii) d’inttgrer ces rbultats k ceux obtenus prCcCdemment sur quatre autres crusta& 6pigCs et hypog&, iii) de comparer les rkponses B I’hypoxie s&&e de crusta& hypogks isopodes et amphipodes et iv) de mewe en tvidence certaines des adaptations permettant aux organismes souterrains d’assurer leur survie. En hypoxie s&&e, S. virei prksente un mkcabolisme anatrobie principalement bask sur la fermentation simultanke du glycoghne et du glutamate, caract&isC par une accumulation de lactate et d’alanine et par une diminution des teneurs en ATP et en phosphagkne. Le lactate produit est de plus largement excrttk dans le milieu extkrieur, phtnomkne inhabituel chez les crustacts. Le temps de survie Clew? de 5’. virei en hypoxie s&&e esc en partie expliquk par une kduction de la depense 6nergCtique like g la locomotion et B la ventilation, associke ?I d’importances &serves en glycogtne et en phosphagPne. ComparC g d’autres crustads &pig&s, lors d’une phase de rtcupkration l’isopode S virei posstde une capacitt glycontogknique plus importante, associke g une resynthkse plus rapide de I’ATP et des reserves en phosphagkne. Les rksultats pr&entCs dans ce travail mettent en evidence des rCponses similaires g celles prtktdemment obserkes sur deux autres crustaces amphipodes hypogks, except6 que cet isopode ne synthkcise pas de succinate en anakrobiose.

Cnergtkique

ont Ctt ttudites

Mats cl& : isopodes, crustac&, hypogks, ventilation, locomotion, m&ubolisme,

ration

Note pr&ende pat Yvon Le Maho Note remise le 28 octobre 1996, accept& aprks rkision *Correspondence

le 27 janvier 1997

and reprints

C. R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 1997. 320, 139-148

hypoxie. r&up&

F. Hervant et al.

AESSTRACT The locomotory and ventihtory activities and the intermediary and energy metabolism mod.$cations of the hypogean aquatic isopod crustacean Stenasellus virei were investigated in severe hypoxia (Paz < 0.03 kPa) and subsequent recovey. The aims of this study were i) to determine why the subterranean species displayed a greater tolerance of hypoxia than numerous other epigean crustacean, ii) to confirm previous results obtained with four hypogean and epigean crustaceans, iii) to compare the responsesto severe hypoxia in hypogean amphipod and isopods, and iv) to better understand the ecologicalproblems of the bypogean organisms survival in subterranean habitats. S. virei responded to experimental long-term, severe hypoxia with classical anaerobic metabolism mainly characterized by a decrease in adenosine tripbospbate (ATP) andpbospbagen, utilization ofglycogen andglutamate, and accumulation of lactate and alunine. Lactate was also hrgely excreted by this organism, which is unusual for crustaceans in general. Compared to most other epigean crustaceans, the isopod S. virei showed high amounts ofstoredglycogen and argininepbospbate. These d.iferences inglycogen andpbospbagen stores, and the ability to reduce energetic expenditures linked to locomotion and ventilation, extended the survival of S. virei under experimental anaerobiosis. During recovery, the isopod S. virei showed a higher capacityfor glyconeogenesis from Lactateand a fzster and total replenishment ofATP and arginine phosphate 1eve.h than epigean crustaceans. Data concerning responses to hypoxia and subsequent recovery in S. virei are similar to those previously obtained with two other bypogean ampbipods, except that this isopod did not synthesize succinate in anaerobiosis. Key words: isopod, crustacean,

hypogean,

ventilation,

locomotion,

metabolism,

hypoxia, reco-

very

VERSION

ABRGGGE

Des travaux r¢s ont montre que certaines eaux souterraines, karstiques ou interstitielles, presentent de facon transitoire des pressions partielles en oxygene tres faibles. De ce fait, les organismes qui les peuplent subissent des alternances de conditions hypoxiques - parfois longues et severes - et normoxiques. Face a ce type de milieu extreme, les organismes aquatiques doivent obligatoirement mettre en jeu des reponses et/au adaptations comportementales, physiologiques et metaboliques pour assurer leur survie. Des rtsultats obtenus precedemment ont montre que certains crustaces amphipodes aquatiques souterrains presentent des durtes moyennes de survie en hypoxie severe tres ClevCes, comparees a celles de la plupart des crustads tpiges. Cette difference semble principalement due a une reduction de la dtpense tnergetique lice a la locomotion et a la ventilation chez les hypoges, reduction associee B un metabolisme anaerobie base sur la fermentation simultanie du glycogene et des acides amines, energetiquement plus (( effrcace Nque la classique glycolyse anatrobie. De plus, ces crustaces amphipodes hypoges presentent d’importantes reserves en glycogene et en phosphagene leur permettant d’alimenter longtemps et effrcacement leur metabolisme en hypoxie. Lors dune reoxygenation, ils montrent de plus une tres importante capacite de resynthese du glycogtne de reserve (voie de la glyconeogen&se) a partir du lactate accumult? en hypoxie. Le present travail complete ces etudes des reponses locomotrices, ventilatoires et metaboliques entreprises sur des crustaCCSamphipodes et isopodes places en hypoxie severe puis en recuperation post-hypoxique. Ses principaux buts ont et6 i) de determiner pourquoi les esptces souterraines prtsentent des temps de survie en hypoxie plus &eves que la plupart des crustaces superficiels, ii) d’integrer ces rtsultats a ceux obte-

nus prectdemment sur quatre autres crustads (deux epiges et deux hypoges), iii) de comparer les reponses a l’hypoxie severe de crustaces isopodes et amphipodes et iv) de mettre en evidence quelques-unes des adaptations permettant aux organismes souterrains d’assurer leur survie. Pour cela, les activitCs locomotrices et ventilatoires ainsi que l’kolution du mttabolisme intermediaire et Cnergetique ont CtC ttudiees en hypoxie severe (PO, c 0,03 kPa) et lors d’une phase de recuperation post-hypoxique chez le crustad isopode hypoge Stenasehs virei, vivant en milieu aquatique interstitiel. Cette espece, trts rtpandue, represente un ecotype de ce type de milieu souterrain en France. Les animaux ont et6 maintenus en Clevage et experiment& dam une enceinte climatisee a 11 “C. 11sont ttC nourris regulitrement de faqon a realiser les experimentations sur des individus homogenes quant aux reserves Cnergetiques. Les individus ttmoins ont ttC experiment& dans des conditions normoxiques. L’ttude de la survie de 5’. virei a et6 r&&see en plaqant les individus dans des flacons remplis d’eau rendue hypoxique (sans variation de pH et de PCO,) par bullage d’un melange d’azote et de CO2 (contenant moins de 1~10~~ % de 0,). La survie des individus dans cette eau pratiquement anoxique (PO, < 0,03 kP a ) a et6 observee en lumitre rouge de faible tnergie (n’entrainant pas de reponses mttabolique, locomotrite ou ventilatoire). La mesure individuelle des activites locomotrice (nombre de d’ep 1acements/min) et ventilatoire (nombre de battements des pleopodes/min) en hypoxie skere a et6 egalement faite en lumitre rouge, avec un protocole experimental identique a celui de l’etude de la duree de survie. A l’issue de ces observaC. R. Acad. Sci. Paris, Sciences de lo vie / Life Sciences

1997. 320, 139-148

Hypoxia and recoven/ in hypogean crustacean tions,

l’eau d&oxyg&nCe a && remplacee

par de I’eau nor-

moxique pour permettre la mesure de ces deux activites d’une phase de r&up&ration posthypoxique.

lors

L’etude des modifications du metabolisme intermediaire et dnergkique, en considerant les reserves metaboliques (glucose, glycogene, acides aminks), I’etat tnergitique (ATP, arginine phosphate), I’excrCtion azotee (NH*‘), les produits terminaux du metabolisme anatrobie accumulCs et/au excr& tes (lactate, alanine, succinate, propionate) ainsi que le malate et le glyc&ol, a &C rialis&e B I’obscuritt sur des animaux plac&s en hypoxie s&&e (PO, < 0,03 kPa) pendant 2, 5, I5 ou 24 h, puis lors d’une phase de &oxygCnation constcutive g un stress hypoxique de 24 h. En hypoxie s&&e, S. virei presente une duree l&ale pour 50 % de la population (DL50) de 6 1.7 h B 11 “C, beaucoup plus elevee que chez la plupart des crustaces &pig& dijg &udiCs. En hypoxie et en comparaison de la plupart des crustaces epig&, S. virei montre une faible hyperactivite (absence de comportement de fuite), trb atypique chez les crusta&, associCe B une diminution rapide de l’activite ventilatoire. 11en r&he une tconomie d’energie en anaerobiose qui permet vraisemblablement une augmentation du temps de survie. En hypoxie s&&e S. virei prCsente aussi un metabolisme anaCrobie principalement base sur la fermentation simultante du glycogene et du glutamate, caractCrise par une accumulation de lactate (9 1 % de la synthese totale en produits terminaux) et d’alanine (9 %), et par une diminution des teneurs en ATP et en phosphagene. A la diffkrence des deux amphipodes hypoges pr&demment &tudi&, cet isopode ne montre pas de synthese de succinate en anaCrobiose. Le lactate produit est, de plus, largement excrete (i 24 %) dans le milieu exttrieur, ph&nom&ne inhabituel chez les crustac&.

Introduction Aquatic subterranean biotopes, including porous, fissured and karstic aquifers, are characterized by two main properties. Firstly, these environments are relatively stable with respect to abiotic factors such as darkness, high moisture, temperature and water chemistry, as well as with regard to biotic factors such as predator and food limitations. Secondly, numerous aquatic subterranean organisms have to cope, more or less frequently and severely, with hypoxia or near anoxia, with sometimes a very rapid switch from high to low Po2 [l-lo]. In temporarily anaerobic interstitial or karstic habitats, the survival of aquatic animals requires specific biochemical, behavioural, and/or physiological adaptations [l 11. We know very little about the adaptations that contribute to extend the survival of hypogean organisms in their alternately hypoxic and normoxic habitats. C. R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 1997. 320. 139-148

Le temps de survie elevC prCsentt par S. virei en hypoxie shere est done en partie explique par une rCduction de la depense energetique Me ?J une locomotion et B une ventilation reduites, associee g d’importantes rtserves en glycogene et en phosphagkne lui permettant d’alimenter plus longtemps son metabolisme anaerobic et de maintenir constante pendant au moins 15 h sa concentration tissulaire en ATP. Lors d’une phase de rt!cuptration, S. virei presente une importante hyperventilation, caracteristique d’un organisme cherchant B combler la dette en oxyghne acquise lors du stress hypoxique, ainsi qu’un metabolisme pr&Rrentiellement a&obie. ComparC aux crusta& epig&, I’isopode S. virei montre aussi une forte aptitude g utiliser le lactate accumult en hypoxie s&&e pour resynthitiser du glycogkne (voie de la glycondogen&se). De plus, ses contenus en ATP et en arginine phosphate sont restaur& beaucoup plus rapidement, ce qui, associC g un taux tleve de resynthttse des reserves en glycogene, represente du point de vue &ologique, un important avantage fonctionnel. En effet, lors d’une deuxieme ptriode d’hypoxie, les organismes hypoges ayant rapidement restaurC leurs reserves mCtaboliques seront 2 meme d’assurer h nouveau leur survie. Chez I’isopode S. virei, I’absence d’augmentation de l’excr& tion azotee lors de la phase de rtcuperation semble montrer que la protPolyse n’intervient pas dans la fourniture &erg&ique. Le glycogkne &ant activement resynthktisi et la concentration en glycerol augmentant de faGon importante apres un stress hypoxique, c’est I’oxydation des reserves lipidiques qui parait assurer la majeure partie du remboursement de la dette en oxyghne et de la fourniture d’ATP pendant cette ptriode. En r&urn& les rPsultats prtsentes dans cet article mettent en tvidence cha l’isopode S. virei des rCponses etlou adaptations locomotrices, ventilatoires et metaboliques similaires ?J celles pr&edemment observees sur deux autres crusta& amphipodes hypoges, except6 que celui-ci ne synthetise pas de succinate en anakrobiose.

In the last 20 years, survival, behaviour, physiology and metabolism of aquatic epigean crustaceans (especially marine decapods) in anaerobiosis have been the subject of a few investigations [I 21. It was found that the majority of epigean crustaceans show little tolerance to severe hypoxia or anoxia. They are generally very mobile [12] and, therefore, can easily move to sites of higher PO, (behavioural compensation). Consequently, it is probably not necessary for them to become adapted to long-term environmental hypoxia. On the contrary, some aquatic hypogean populations living permanently in groundwaters generally cannot escape anaerobic conditions and cannot select oxygenated waters. Therefore, it is necessary for them to become adapted to long-term environmental hypoxia. Previous investigations have shown that two widespread species of hypogean aquatic amphipod crustaceans, Niphargus rhenorhodanensis and N. virei, which

141

F. Hervant et al.

have to cope with hypoxic conditions for several months per year during the hydrological cycle, displayed very high survival times under severe hypoxia [13, 141. When compared to epigean crustaceans, the most significant adaptive characteristics of these species appear to be i) the ability to decrease their metabolic rate during hypoxia by reducing levels of activity, ventilation and glycolysis, ii) their low respiration rates and iii) their high amounts of stored glycogen and phosphagen arginine phosphate. In addition, both Niphargus species displayed an important glyconeogenic ability during posthypoxic recovery [13-l 61. These results partly explained the high survival times found for these organisms.

(PO2 = 0.0 f 0.03 kPa) was checked using an electrode (TriOx EO 200) coupled to an O2 meter (WTW Oxi 2000). The flasks containing individuals were then sealed and mortality was noted over time. The results of this survival experiment were taken into account when planning the duration of hypoxic exposure in the experiments outlined later. To investigate changes in key metabolites in S. virei during severe hypoxia, batches of five individuals were maintained under hypoxic conditions as described earlier and removed at intervals of 2, 5, 15 and 24 h. To investigate recovery from severe hypoxia, batches of five individuals were maintained under hypoxia conditions, as described earlier, for either 24 h, before the deoxygenated water was replaced with oxygenated water. Animals were then removed at intervals of 2, 5 and 24 h after oxygenation. Once removed, batches of five individuals were immediately frozen in liquid nitrogen, before being lyophilized (VIRTIS lyophilisator, Trivac D4B). The concentration of ammonia (NH,+ + NH,) in the water contained

Therefore, experiments were conducted on the hypogean isopod Stenasellus virei (an ecotype of the French interstitial biotope) under experimental severe hypoxia (Paz < 0,03 kPa) and subsequent recovery, with the following objectives: i) to determine the metabolic changes, the use and resynthesis of energy reserves, and the locomotory and ventilatory adaptations to hypoxia and hence the ecological importance of these processes, ii) to compare the responses to severe hypoxia in hypogean amphipods and isopods, and iii) to extend the study of locomotory, ventilatory, and metabolic responses of the hypogean aquatic amphipods, N. rhenorhodanensis and N. virei, to severe hypoxia and subsequent recovery [13-l 61.

within the incubation flask was determined as described later. The pH of the water was measured using a pH electrode and meter (TACUSSEL lssis 20000) accurate to + 0.01 py units and the CO, content determined using the Gran titration method [17]. The remaining water was then lyophilized and the concentrations of lactate and succinate determined as outlined later.

Materials and methods

In order to measure ammonia excretion in normoxia, experiments were carried out with animals treated as described earlier, but after 24 h under normoxic conditions.

Animals Stenasellus virei (hypogean isopods, fresh weight = 10.8 f 0.4 mg) were collected in groundwater hydraulically connected with the River Lot, using special pumps lowered into piezometers in an interstitial system at Cantepau (AEP d’Albi), France. Animals were maintained in recirculating aquaria containing groundwater (pumped from the underground water table of the University of Lyon-l) and were fed with minced meat every 2 weeks. The tanks which held S. virei contained clay and stones removed from the collection site. Aquaria were kept in the dark in a controlled temperature facility (T = 11 “C) and animals (males only) were removed for experimentation as required. Experimental protocol In order to examine

the effect of severe hypoxia on survival of S. virei, the following experiment was carried out. Individuals were introduced into glass incubation flasks (vol. = 25 mL ). The air in each flask was then displaced using special pure nitrogen gas (containing 0.1 ppm 02) before being filled with hypoxic water (Paz < 0.03 kPa). Hypoxic water was generated by bubbling nitrogen with CO, added (500 ppm) to prevent alkalosis during equilibration, through water contained in a 500 mL glass flask for 1 h. The oxygen tension of the water after this time

142

For measuring locomotory activity (number of animal displacements/min, defined by the number of swimming periods between rest periods/min) and ventilatory activity (number of pleopod beats/min), as described in Hervant et al. 1151, animals were placed individually in severe hypoxia and subsequent recovery in 25 mL incubation flasks as described earlier. Sample preparation The lyophilized tissues of the batches of five individuals and the dry powder resulting from lyophilization of the 25 mL incubation water of each flask were prepared for enzymatic assay according to Hervant et al. [15]. Metabolite assays The following metabolites were determined by standard enzymatic methods [18]: alanine, ammonia (NH,+ + NH,), arginine and arginine phosphate, aspartate, ATP, glucose, glutamate, glycerol, glycogen, lactate, propionate and succinate. All assays were performed in a Uvikon 940 recording spectrophotometer (Kontron) at 25 “C, except for ATP assays, performed in a LKB 210 luminometer. Enzymes, coenzymes and substrates used for enzymatic assays were purchased from Bee hringer (Mannheim, Germany) and Sigma Co (Saint Louis, MO, USA). C. R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 1997.320. 139-148

Hypoxia and recovery in hypogean crustacean Units and statistics

hypoxia. During recovery, the activity until control value was attained at 24 h.

Values are presented as means rt SE. Student’s t-test was used for the statistical comparison of experimental and control values.

recommenced,

Ventilatory activity in severe hypoxia and subsequent recovery S. virei presented a hyperventilation (+25%) at the beginning of the hypoxic stress (figure 2), then ventilation decreased and reached 72% of the initial value after 24 h of severe hypoxia. The duration of hyperventilation was 2 h (8% of the hypoxic total stress duration). During recovery, a hyperventilation rapidly appeared for at least 13 h, then decreased until the control value was attained at 18 h.

Results pH and CO* concentrations changes in the incubation water The pH of the control incubation water used in all treatments was 7.14 * 0.5 and no significant pH difference (P > 0.05) was detected in hypoxic water and in reoxygenated water. The CO, concentration was 557 f 42 pmol/ L in normoxic incubation water and no significant changes (P > 0.05) were measured in hypoxic water and in reoxygenated water.

Effects of severe hypoxia and subsequent recovery on metabolite concentrations The propionate, succinate and aspartate concentrations in whole body and incubation water were too low to be detected by the enzymatic assay used. No significant changes in whole body ATP concentration were observed up to 15 h of severe hypoxia (P > 0.05) (figure 3a). Then ATP concentration decreased to 76% of its initial value after 24 h. During reoxygenation, ATP concentration was re-established after 24 h in S. virei.

Severe hypoxia survival time The LT,, (lethal time for 50% of the population) for S virei, as estimated by the Trimmed Spearman-Karber method [19], at 11 “C was 61.7 h (57.3-64.5 h; 95% confidence). Mortality was 0% prior to 26 h and 100% at 102 h.

Arginine phosphate concentration decreased to 52% of its initial value after 24 h severe hypoxia (figure 3b). Arginine phosphate concentration returned to control value after 24 h recovery. Arginine concentration changed in a pattern reciprocal to that of arginine phosphate concentration up to 15 h severe hypoxia, then decreased. After

Locomotory activity in severe hypoxia and subsequent recovery S. virei presented no hyperactivity at the beginning of the experiments but rather displayed an immediate decrease in activity (figure 1). Activity ceased after 4 h of severe

:

Hypoxia

Recovery

*

. 20

10

2,

. Ib

;O Tie

figure

1. Locomotory

activity

(number

of periods of locomotion/min)

in severe hypoxia and subsequent

recovery

of

24 (h)

Stenasellusvirei at

in darkness. Values are means f SE, for n = 15 individuals;

* indicates

C. R. Acad. Sci. Paris,Sciences de la vie / Life Sciences 1997.320, 139-148

a value that was significantly

different

from normoxic

control

(P < 0.051.

11 “C,

F. Hervant et al.

Hypoxia

. :0

Figure 2. Ventihtory in darkness. Valuesare

means f

activity

(number

of pleopods

SE, for n = 15 individuals;

beats/min)

* indicates

in severe hypoxia

a value that was significant/y

;o 24 Time (h)

and subsequent

different

recovery

from normoxic

of Stenasellus virei at 11 “C,

control

(P < 0.05).

24 h severe hypoxia, only 7 pmol/g dry weight (DW) arginine were produced while 15.3 pmol/g DW arginine phosphate were utilized, which corresponds to a consumption of 8.3 pmol arginine/g DW (figure 3b). During recovery, arginine concentration changed in a pattern reciprocal to that of arginine phosphate and returned to control value after 24 h recovery.

During recovery, whole body lactate concentration decreased rapidly, reaching control value after 24 h (P > 0.05). Over the same period, lactate concentration in the incubation water remained constant (P > 0.05) (figure 3f), showing that neither excretion nor consumption had occurred. Total lactate consumption during recovery was 106.3 pmol/g DW/24 h.

Glycogen content decreased and reached 65% of its initial concentration after 24 h of severe hypoxia (figure 3c), which corresponded to a utilization of 107.3 pmol glycosylic unit/g DW. During recovery, glycogen content increased and reached 83% of the initial value after 24 h, which represented a resynthesis of 56.7 umol glycosylic unit/g DW.

Whole body alanine concentration markedly increased by 162% in 24 h severe hypoxia, which corresponds to a production of 19.6 pmol alanine/g DW/24 h (figure 3g). During recovery, alanine concentration decreased rapidly (17.8 pmol/g DW were metabolized), reaching control values after 24 h (P> 0.05). No alanine was detected in the incubation medium.

Glucose concentration (figure 3d) increased, reaching 123% of the control value after 5 h hypoxia, then decreased to control value after 15 h. There were no significant differences in glucose concentrations during posthypoxic recovery.

There were no significant differences in whole body glycerol concentrations during severe hypoxia in S. virei (P > 0.05) (figure 3h). During recovery, glycerol concentration markedly increased by 73% in 24 h, which corresponds to a production of 4.1 pmol glycerol/g DW/24 h.

Glutamate concentration decreased and reached 51% of the control value after 24 h of severe hypoxia, which corresponds to a consumption of 5.9 umol/g DW (figure 3e). After 24 h recovery, it increased up to 113% of the initial value, corresponding to a resynthesis of 7.5 umol/g DW.

Under normoxic conditions, S. virei excreted ammonia (NH4+ + NH3) at an average rate of 2.01 + 0.2 umol/g DW/h (calculated from the cumulative ammonia excretion during an incubation period of 24 h). During 24 h hypoxia the average ammonia excretion was 33% of its initial level, at 0.66 f 0.1 pmol/g DW/h (calculated from cumulative ammonia release over the whole hypoxic period). During recovery, excretion of ammonia increased until it reached 79% of the control value, at 1.59 f 0.1 umol/g DW/h (calculated from cumulative ammonia release over the whole recovery period).

During 24 h severe hypoxia, whole body lactate concentration increased from 10.6 k 1.2 to 118.1 + 6.5 umol/ g DW (figure 30. During this time, S. virei released 81.5 + 7.3 umol lactate/g DW into the incubation water. Therefore, the total production after 24 h was 189 umol/g DW.

144

C.

R. Acad. Sci. Paris,Sciences de la vie / Life Sciences 1997.320,139-148

Hypoxia and recovery in hypogeon crustacean

Hypoxia

Recovery

Recovery

20

24’0

10

T

Recovery

24 ~Ime (h)

20

nme(h)

nme(h)

Figure 3. Changes in the levels of metaboiites in severe hypoxia and subsequent recovery of Stenasellus virei at f I “C, in darkness. Values are means rt SE, for n = 7-8 batchesof five individuals; * indicates a value that was significantly different from normoxic control (P < 0.05). dw = dry weight.

C. R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 1997.320, 139-146

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F. Hervant et al.

Discussion

a large proportion severe hypoxia.

Survival times under severe hypoxia

As in other amphipods [14, 151, the origin of the NH2 groups of alanine is partly unclear in the case of the isopod S. virei, because only 69% could be accounted for by the fermentation of glutamate 1281. The most convincing hypothesis might be a glutamate utilization linked to the transamination of arginine or other amino acids (certainly originating from a proteolysis: for S. virei, the excretion of ammonia was not suppressed during hypoxia, indicating that protein degradation played a role in anaerobic energy production). In contrast with both hypogean amphipods N. virei and N. rhenorhodanensis, 36% of the NH, groups of alanine could be accounted for by the fermentation of arginine (originating from the utilization of arginine phosphate) in the isopod S. virei (figure 3).

Crustaceans show very little tolerance to anoxia or severe hypoxia, with LT50 values generally lower than 24 h at 1O-l 5°C [12]. The present study shows that the subterranean isopod S. virei, like the hypogean amphipods Niphargus rhenorhodanensis and N. virei 114, 151, is better adapted to environmental anaerobiosis than the widely spread epigean isopod Asellus aquaticus [141 and than most of the crustaceans previously studied [12, 151. Locomotory and ventilatory and recovery

responses in severe hypoxia

Compared to epigean species such as Asellus aquaticus or Gammarus fossarum [14, 151, the hypogean isopod S. virei reduced its energetic expenditure linked to locomotion and ventilation in severe hypoxia, as shown by the absence of hyperactivity and the weaker hyperventilation at the beginning of the hypoxic stress, and also the very fast onset of immobility (i.e., the absence of an escape behaviour). Both hypogean amphipods, Niphargus rhenorhodanensis and N. virei, showed the same kind of behavioural adaptation 113, 141. During posthypoxic recovery, the large hyperventilation noted in S. virei may indicate that oxygen consumption markedly increased: anaerobic energy production was quickly shifted to aerobic and S. virei tried to repay the oxygen debt contracted during the lack of oxygen [14, 16, 20, 211. Metabolic

responses under severe hypoxia

Like the hypogean amphipods N. vireiand N. rhenorhodanensis [14, 151, the hypogean isopod S. virei maintained a high ATP concentration in tissues during the first 15 h of hypoxic stress. In contrast, there was a drastic decrease throughout the stress in the epigean isopod A. aquaticus [14]. These results could be explained by the higher amounts of stored arginine phosphate in S. virei than in A. aquaticus and numerous other epigean crustaceans 1141. Like the amphipods N. virei [141 and N. rhenorhodanensis [15], and like numerous bivalves [22-241, S. virei preferentially used stored phosphagen in order to maintain a high ATP concentration in tissues during the first hours of severe hypoxia. In S. virei under severe hypoxia, lactate was the major anaerobic end product (91% of the metabolic response), while alanine was the minor end product (9%). These results suggested that in anaerobiosis this species, like other crustaceans [15] and among them isopods [25, 261, used the simultaneous fermentation of glycogen and amino acids. Contrary to both hypogean amphipods N. virei and N. rhenorhodanensis, the isopod S. virei did not synthesize succinate in anaerobiosis. Like numerous other invertebrates, but very rarely crustaceans [12, 14, 15, 21, 25, 271, S. virei excreted in the incubation water

146

(24%) of the lactate synthesized

during

Not surprisingly, this study showed that glycogen was the most important and significant substrate of anaerobic metabolism for this species, while glutamate was the minor substrate. In S. virei, anaerobiosis resulted in a marked increase in the rate of glycogen use, in order to compensate for the much lower efficiency of ATP production by fermentation than by respiration. In the isopod S. virei, like in other subterranean amphipod crustaceans [14, 151, body glucose concentration was high during severe hypoxia. This high glucose level suggested that intertissue transport of substrates (glucose and amino acids) via the hemolymph may play a major role in anaerobic energy production. In the first hours of hypoxia, the high rate of glycogen utilization (high substrate demands of fermentation) is reflected in a transitory increase of glucose concentration. Santos and Colares [29], Hervant et al. 114, 151 and Zou et al. [30] documented a similar pattern of change in the hemolymph or body glucose concentration. Santos and Colares I.291 found that changes of hemolymph glucose concentration were controlled by an eyestalk hormone known as the crustacean hyperglycemic hormone (CHH). Whether there was a CHH involvement in the body glucose concentration change in subterranean crustaceans under hypoxia needs to be investigated. S. virei, like both species of Niphargus, presented higher amounts of stored arginine phosphate and glycogen than epigean crustaceans, even the most hypoxia tolerant ones [12, 14, 15, 221, making the fuelling of environmental anaerobiosis possible for a longer time. These results, associated with a high ability to accumulate end products and to decrease energetic expenditures in severe hypoxia (with in particular slower and shorter hyperactivity and hyperventilation than epigean crustaceans), might partly explain the high resistance to the lack of oxygen found in subterranean organisms in general. Metabolic

responses during recovery

No further accumulation of any anaerobic end product was detected in the tissue of S. vireior in the water during its recovery, which confirms an exclusively aerobic recoC. R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 1997. 320, 139-148

Hypoxia and recovery in hypogean crustacean very process. In the case of S. virei, posthypoxic recovery was characterized by a total restoration of energy reserves, except for stored glycogen, and a total removal of the accumulated end products. Glycogen reserve restoration was greater in hypogean species than in epigean ones (53% restoration in S. virei, 49% in N. virei and 47% in N. rhenorhodanensis versus 8% in C. fossarum and 19% in A. aquaticus, in 24 h recovery). A high glycogen resynthesis capacity is ecologically very advantageous for subterranean organisms, especially for fuelling a new hypoxic period. Metabolized end products accounted for 91% of the glycogen resynthesized after 24 h recovery in S. virei. This isopod seems to have used preferably the glyconeogenesis pathway from lactate, whose existence has been demonstrated in several epigean crustaceans [31-341 and in the hypogean amphipod Niphargus virei [16]. On the contrary, oxidation and excretion were major mechanisms in the disposal of end products in epigean crustathat hypoxia-tolerant ceans [14, 151. It appeared hypogean crustaceans are better adapted for the metabolization of accumulated anaerobic end products than epigean species.

Like in other epigean and hypogean species 114, 151, body glucose level was maintained strictly constant in S. virei. It probably indicated a normal glycolytic recovery rate. For the hypogean isopod S. virei, as for the hypogean amphipod N. virei and the epigean isopod A. aquaticus [141, posthypoxic recovery resulted in an increase in ammonia excretion when compared to hypoxia, but the excretion rates of ammonia after 24 h were lower than during normoxic experiments. Increased rates of ammonia excretion during posthypoxic recovery were probably due to the clearance of accumulated ammonia and/or alanine. There was no evidence of protein utilization for energy metabolism during recovery, since no significant increase in NH,+/NH, production was observed in S. virei during posthypoxic recovery. Since glycogen stores were actively resynthesized and since glycerol concentration was markedly increased during the same period, it can be hypothesized that the main substrate for the oxidation and the payment of the 0, debt [14, and unpublished data] in this species were the triglycerids stores.

Acknowledgements: This research

was supported by the French Ministry of Education, France, grant No 93-l 154, and by funds from the University Claude-Bernard-Lyon-l and the National Centre of French Scientific Research (CNRS). The authors thank C. Bou for his valuable assistance in collecting individuals of Stenasellus virei.

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