r Printed in Great Britain

J. Exp. Bil. (1972), 56, 49-55 49 *~"ith 2 text-figures r Printed in Great Britain WATER CONTENT AND WATER ACTIVITY IN THE CUTICLE OF TERRESTRIAL ISOPODS BY OSSI V. LINDQVIST,* INGA SALMINEN AND PAUL W. WINSTON Zphysilgical Labratry, Department f Zlgy, University f Turku, Finland, and Department f Bilgy, University f Clrad, Bulder, Clrad {Received 3 May 1971) INTRODUCTION The terrestrial ispds make an interesting parallel t insects in their adaptatins t life n land (cf. Edney, 1968). In general, ispds have a prer resistance t desiccatin, but their behaviural reactins t external humidity cntribute strngly t their success in terrestrial cnditins, as reviewed by Edney (1968) and Lindqvist (1968). A thrugh study f the permeability characteristics f the ispd cuticle is that by Bursell (1955), wh dealt with Oniscus asellus. Since the effect f envirnmental humidities n terrestrial ispds is mre prnunced than n mst insects, we culd expect that the water cntent and water activity f their cuticle als wuld reflect this difference. The insects Lcusta and Periplaneta, bth f which are relatively hardy in dry cnditins, have a reduced water activity in the cuticle as cmpared t that f their bld (Winstn, 1967; Winstn & Beament, 1969); different envirnmental humidities d nt affect the cuticular water cntent and nly slightly the cuticular water activity in these insects. The present paper aims at assessing the free water cntent and the smtic characteristics f the cuticle f several species f terrestrial ispds, in the hpe that it may thrw sme light n the mde and the site f water regulatin in the cuticle. MATERIAL AND METHODS Prcelli scaber Latr. and Cylisticus cnvexus De Geer were cllected frm the suth-western cast f Finland and Armadillidium vulgare Latr. and Oniscus asellus L. frm near Visby, Sweden. In the labratry they were reared in small plastic cntainers prvided with paper twelling which was mistened at times t keep the relative humidity in the cntainers in excess f 90%. The rm temperature was thermstatically cntrlled at abut 24 C and the experiments were cnducted at the same temperature. The animals were fed n fresh carrt, but befre each experiment they were starved fr 24 h. Only adult intermult animals f bth sexes were used; n difference was bserved between the sexes in the cuticular prperties studied. As the water cntents f the cuticles frm different tergites f the same animal differed slightly but irregularly, the samples were taken frm the third tergite nly, and in the fllwing way. The animal was decapitated and the third thracic shield Present address: Department f Bilgy, University f Daytn, Daytn, Ohi 45409. 4 E x B 56

50 0. V. LlNDQVIST AND OTHERS was excised as rapidly as pssible. The cuticle was wiped with a piece f filter pape' t remve any remnants f tissue, and the lateral edges f the tergite were trimmed t a rectangle weighing 1-1*5 m - This was wrapped in aluminium fil and weighed n a Cahn Electrbalance t the nearest *i mg. The aluminium fils with the cuticle inside were dried in vacua ver CaCl 2 at rm temperature fr 24 h and reweighed t btain the free water cntent. The dissectin f the animals was cnducted in a chamber having a relative humidity f abut 98 %; the balance was situated in the same chamber. The chamber was prvided with a glass windw and tw hles thrugh which the peratr culd push his hands t wrk inside. The smtic equilibrium pint f the cuticles was determined by wrapping ther samples in cups f aluminium fil and weighing them; after this the cups were pened and were left hanging in small glass jars cntaining different NaCl slutins t cntrl humidity. Humidities were calculated accrding t the tables f Washburn (1928). Then the jars were put int large well-insulated bxes t minimize and smth ut any changes in temperature. Any errrs resulting frm pssible precipitatin were crrected by the use f empty cntrl cups. After 3 days the cups were weighed again and dried fr 24 h t btain the free water cntent. Befre the cuticles were excised fr equilibratin, the animals were dried ver silica gel fr 30 min t remve the free water in the cuticle and t reduce variability. The experiments reprted here were cnducted during the perid frm December t March. Bld smtic pressures were determined by the methd f Grss as described in a previus paper (Lindqvist, 1970). RESULTS Bth Armadillidium vulgare and Prcelli scaber are relatively hardy in terms f water lss under terrestrial cnditins, and fr this reasn their cuticular water cntent was measured first. In bth species the free water cntent f the cuticle remained rather stable during desiccatin f live animals fr varius times (Fig. 1). The mean water cntent was 54-0 ±078% (N = 70) fr P. scaber and 52*7 ± i*n% (N = 28) fr A. vulgare. The difference between the species is nt significant. There was n trend as regards the desiccatin time, nr did the bdy weight affect the cuticular water cntent. The mean weight f P. scaber was 51-1 mg (range 34-1-77-6 mg) and that f A. vulgare 70-0 mg (range 40-3-109*7 mg). In thse animals which were dehydrated fr up t 7 h the cuticular water cntent was nt significantly changed, even when the water lss frm the bdy had amunted t abut 25 % f the initial bdy weight. The animals which died, hwever, after having lst 30 % r mre f bdy weight had cuticular water cntents distinctly lwer than thse shwn abve; values frm abut 40% t as lw as 25% were btained depending upn the length f time the animals had been dead. This difference between living and dead animals wuld indicate that in dead animals water was being lst frm the cuticle faster than it culd enter frm the bld, and that an active mechanism therefre maintains the high water cntent f the cuticle in living animals. In these tests P. scaber shwed a slight tendency fr lwer water cntents in the cuticle after sme desiccatin, as cmpared t nn-desiccated animals. This was tested again in a further experiment at anther time; there was a significant drp frm

Water cntent and activity in ispd cuticle 60 CJ50 I 40 c "30 0) - eft 0 - -. «.r " * t i V^ J^ D ^20 10-1 1 1 30 60 90 120 150 180 210 Time (min) Fig. 1. The water cntent f the cuticle f A. vulgare (pen circles) and f P. scaber (clsed circles) after desiccatin f intact animals fr varius times ver silica gel. The unbrken line is the regressin curve fr A. vulgare, the brken ne fr P. scaber. +80 r +70 + 60 _ +50.2 +40 1 +30 +20 00 c + 10 OC 5-10 -20-30 100 200 mm/l NaCI 300 400 100 200 mm/l NaCI 300 400 Fig. 2. The change in weight in percentage f the excised cuticle after equilibratin at varius humidities prvided by different mlal cncentratins f NaCI. A, P. scaber; B, A. vulgare; C, O. asellus; D, C. cnvexus. Vertical bars indicate the standard errrs f means f the number f animals shwn by the adjacent figures. 4-2

52 O. V. LlNDQVIST AND OTHERS 56-8 ± i-66% t 50-4+ I-6I% after 30 min. dehydratin (N = 7 in bth cases). Irl A. vulgare there was n change in the cuticular water cntent after this shrt desiccatin perid. Fr measurements f the water activity f the excised cuticle, tw less hardy species, Oniscus asellus and Cylisticus cnvexus, were included. Bth have been fund t have cnsiderably higher rates f lss than either P. scaber r A. vulgare (cf. Warburg, 1965, and unpublished data). The water activity f cuticle in living, nrmal animals was in equilibrium with 200-270 mm/1 NaCl (Fig. 2, Table 1). This is the cncentratin f salt ver which the excised cuticle neither gained nr lst weight, i.e. it had the same vapur pressure as the slutin. (The values are estimatins f the pint at which a plt crsses the line fr n gain in weight, rather than direct measurements.) In cntrast, the freezing-pint determinatins shwed the bld pressures t be higher, averaging between 290 and 330 mm-nacl (equiv.) per litre fr the fur species (Table 1). (The values fr bld smtic pressure f P. scaber and O. asellus are apprximatins frm an earlier study (Lindqvist, 1970), while measurements f A. vulgare and C. cnvexus are 331-2 and 319-6 mm/1 NaCl respectively (N = 8 in bth cases) under similar cnditins.) Thus the water activity f the cuticle is higher than that f the bld. Armadillidium vulgare Prcelli scaber Oniscus asellus Cylisticus cnvexus Table 1. The smtic relatinship between excised cuticle and bld in fur species f terrestrial ispds (The equilibrium pints are estimatins f the pints at which plt (Fig. 2) crssed the line fr n weight gain r lss.) Equilibrium Osmtic Difference in pint f the pressure f smtic pressure excised cuticle the bld (equivalent t between cuticle and bld Water cntent f the excised cuticle (mm/1 NaCl) mm/1 NaCl) in atm. after equilibratin 270 213 202 260 c 33 c. 33 c. 290 c. 320 -i-5-28 21 -i-5 50-8 ±0-85% (iv = 36) 59-2 ± 1 s % (AT = 36) 6i±3-29% (N = 496 ±1-25% (iv = The free water cntents f cuticles determined after equilibratin (Table 1) were apprximately 59% in P. scaber, 51 % in A. vulgare, 61 % in O. asellus, and 50% in C. cnvexus. These values did nt differ significantly frm thse btained at the same time frm nn-desiccated (and nn-equilibrated) animals. In all fur species, hwever, there is a statistically significant (P < 0-05) negative crrelatin between the water cntent and the smtic equilibrium pint f the cuticle (r = -0-979; Table 1). It can be seen that the higher the water cntent f the cuticle, the higher the water activity. The measurements f cuticular water described here were cnducted frm December 1969 till late March 1970. There appeared t be a definite pattern in the water cntents; the values btained in December were all higher (especially in nn-desiccated animals) than thse btained in the spring fr bth P. scaber and A. vulgare. The envirnmental cnditins were kept as stable as pssible during this perid and they prbably did nt cntribute t this gradual change. It is cnceivable that the cuticular water is cntrlled peridically, linked with seasns r the breeding cycle. Gupta (1963) reprted 10) 36)

Water cntent and activity in ispd cuticle 53 that bth P. scaber and O. asellus shw an annual rhythm in the intensity f their humidity preference in a humidity chamber; whether this behaviural preference shws any relatin t the cntrl f cuticular water remains t be seen. DISCUSSION The transpiratin rates f terrestrial ispds shw a characteristic curve in dry air: at first the rate f water lss is high and nly after sme time des it reach a steady level (Edney, 1951; Bursell, 1955; Lindqvist, 1968). In Armadillidium vulgar e, fr instance, the initial rate 6/tg/mm 2 /h was abut six times higher than the plateau level (abut i/tg/mm 2 /h) (Lindqvist, 1968), which was reached nly after 120-150 min desiccatin. This is reflected in n way in the cuticular water cntent, indicating a strng degree f regulatin f water levels in the face f high evapratin rates. Prcelli scaber shwed nearly as gd regulatin, but there was always a difference f 1-5% in cuticular water cntent between desiccated and nn-desiccated specimens. The water cntent and the water activity f the cuticle are nt necessarily cmparable (Winstn & Beament, 1969), but in these ispds they appear t be s. It is apparent in Table 1 that when the water cntent is high the water activity is als high (the equilibrium pint is lw), and vice versa. Thus, such a relatinship wuld be expected t hld in ther situatins and, in general, when we speak f changes in ne, it will mean changes in the same directin in the ther. The fact that the free water cntent f the cuticle remained almst unchanged during desiccatin mst prbably means that there are sme mechanisms, wrking in the living animal nly, that drive water int the cuticle at rates which make up the evaprative lsses. This is the mre remarkable as the animal may lse 25 % f its bdy weight withut a significant drp in the free water cntent f the cuticle. As the bdy water cntent f nrmal P. scaber is between 65 and 72% (unpublished data), such lsses culd mean an increase f up t 45 % in smtic pressure f bdy fluids, prvided that water levels were reduced by the same amunt in all cmpartments. T explain the high sustained water activity in the cuticle, there may exist at least the fllwing three alternatives. First, there may be special mechanisms in the epidermis r in the cuticle itself which tend t maintain the stability f the cuticular water activity (thugh nt in smtic equilibrium with the haemlymph) in spite f smtic changes. Winstn & Beament (1969) shwed this pattern in Periplaneta americana and Lcusta migratria. Secnd, the bld smtic pressure may nt appreciably change with desiccatin, but the bld vlume wuld instead decrease with cncmitant withdrawl f slutes frm the haemlymph. Hence, the bld bathing the cuticle wuld be smtically stable, reducing the energy needed t maintain the differential between the haemlymph and the cuticle. Quite recently Hrwitz (1970) reprts that during desiccatin f P. scaber its bld smtic values remained relatively cnstant fr sme perid f time; this result definitely refers t this kind f regulatin. Amng the insects sme cases are knwn where the bld smtic pressure is regulated against the effects f hydratin and dehydratin (Edney, 1966; Djajakusumah & Miles, 1966; Wall, 1970), and the best example f such regulatin knwn is in Leucphaea maderae where the water cntent f the cuticle (Winstn & Hffmeier, 1968) and the bld smtic pressure

54 O. V. LlNDQVIST AND OTHERS (Laird, 1970) are unaffected by lng perids f desiccatin and starvatin. The ptamnid crab Sudannautes africanus shws increased cncentratins f several plasma ins except sdium at mild desiccatin (Lutz, 1969). It may be f sme interest t nte here that the fresh-water ispd Asellus aquaticus has abut 20-30% f its ttal bdy sdium utside the haemlymph and mst f it is cncentrated in the Zenker's rgan (Lckwd, 1959). The rle f this sdium is nt knwn, but it might be used in regulating the bld sdium levels. A third alternative may be that the bld smtic pressures change smewhat accrding t hydratin and dehydratin, but all tissues are regulated smtically and kept stable and the cuticle is just anther tissue. Hwever, this is nt very prbable, as Hrwitz (1970) fund that in P. scaber muscles at least lse water int the haemlymph during desiccatin and sme water may als be drawn in frm the gut. Wrk has been started t investigate this prblem mre clsely. Further evidence fr an active cuticular regulatry principle is the drp in water cntent at death. This wuld indicate that a barrier, pssibly at the base f the cuticle, breaks dwn when the animal dies, allwing the rest f the cuticle t equilibrate with the bld. Mre water wuld be drawn ut f the cuticle if the bld smtic pressure als rises at this time. Thus these animals smehw pssess a mechanism akin t the 'water pump' f sme insects (Winstn, 1967; Winstn & Beament, 1969) which maintains cuticular water cntent and activity in the face f changing rates f water lss. The adaptive advantage t keeping the water activity abve that f bld even during desiccatin is nt clear. The few insects in which a 'pump' is knwn (Winstn, 1967; Winstn & Beament, 1969) shw just the ppsite effect; water activity is kept lwer in the cuticle than in the bld, a cnditin which wuld seem t aid in the reductin f transpiratin. In the terrestrial ispds the relatively pr waterprfing f the cuticle allws rapid water lss and, apparently, water must be 'pumped' in t maintain the level in the cuticle. If it were nt, the cuticle wuld becme dry and brittle, an bvius disadvantage, and the many sense rgans f the cuticle pssibly culd nt functin prperly in such a milieu. Als, a high cuticular water cntent may be advantageus in view f the fact that the terrestrial ispds seem t excrete mst f their nitrgen as ammnia in gaseus frm thrugh bdy surfaces (Wieser & Schweizer, 1970). SUMMARY 1. The water cntent f the cuticle f bth desiccated and nn-desiccated terrestrial ispds Prcelli scaber and Armadillidium vulgare was measured. The animals were desiccated fr varius times (up t 3 h) ver silica gel and the mean water cntent f the cuticle was 54-0+ 0-78% fr P. scaber and 52-7+ i-n% fr A. vulgare. There was n trend as regards the desiccatin time, nr did the bdy weight affect the water cntent. 2. The water cntent f the cuticle remained virtually unchanged as lng as the animal was alive in the desiccatr. It drpped significantly after the animal had died after having lst sme 30 % f its bdy weight. 3. The cuticular water cntent f nn-desiccated P. scaber tended t be slightly higher than that f desiccated nes. In A. vulgare n significant difference was bserved between nn-desiccated and desiccated specimens.

Water cntent and activity in ispd cuticle 55 4. The water activity f the excised cuticle f the abve tw species and f Oniscus asellus and Cylisticus cnvexus was abve that f the haemlymph and therefre nt in smtic equilibrium with it. The smtic equilibrium pints were belw the smtic pressures f the bld; the difference amunted frm 1*5 t 2-8 atm. in different species. 5. The difference in water activity between bld and cuticle, the maintenance f water cntent with desiccatin, and the drp in water level at death, all indicate the presence f an active mechanism regulating the cuticular water in terrestrial ispds. This study was aided by grants frm the Natinal Research Cuncil fr Sciences f Finland t ne f us (O.V.L.). REFERENCES BURSELL, E. (1955). The transpiratin f terrestrial ispds. jf. exp. Bil. 32, 238-55. DJAJAKUSUMAH, T. & MILES, P. W. (1966). Changes in the relative amunts f sluble prtein and amin acid in the haemlymph f the lcust, Chrticetes terminifera Walker (Orthptera: Acrididae), in relatin t dehydratin and subsequent hydratin. Austr.J. Bil. Sci. 19, 1081-94. EDNEY, E. B. (1951). The evapratin f water frm wdlice and the milliped Glmeris. J. exp. Bil. 28, 91-115. EDNEY, E. B. (1966). Absrptin f water vapur frm unsaturated air by Arenivaga sp. (Plyphagidae, Dictyptera). Cmp. Bichem. Physil. 19, 387-408. EDNEY, E. B. (1968). Transitin frm water t land in ispd crustaceans. Am. Zl. 8, 309-26. GUPTA, M. (1963). Seasnal variatin in the humidity reactin f wdlice, Oniscus asellus L. and Prcelli scaber Latr. (Crustacea: Ispda). Prc. natn Inst. Sci. India 29, 203-6. HOROWITZ, M. (1970). The water balance f the terrestrial ispd Prcelli scaber. Ent. exp. & appl. 13, 173-8. LAIRD, T. B. (1970). Humidity effects n hemlymph in Leucphaea maderae F. Am. Zl. 10, 233. LINDQVIST, O. V. (1968). Water regulatin in terrestrial ispds, with cmments n their behavir in a stimulus gradient. Ann. zl. Fenn. 5, 279-311. LINDQVIST, O. V. (1970). The bld smtic pressure f the terrestrial ispds Prcelli scaber Latr. and Oniscus asellus L., with reference t the effect f temperature and bdy size. Cmp. Bichem. Physil. 37, 503-10. LOCKWOOD, A. P. M. (1959). The extra-haemlymph sdium f Asellus aquaticus (L.). J. exp. Bil. 36, 562-5. LUTZ, P. L. (1969). Salt and water balance in the West African fresh-water/land crab Sudannautes africanus africanus and the effects f desiccatin. Cmp. Bichem. Physil. 30, 469-80. WALL, B. J. (1970). Effects f dehydratin and rehydratin n Periplaneta americana. J. Insect Physil. 16, 1027-42. WARBURG, M. R. (1965). Water relatin and internal bdy temperature f ispds frm mesic and xeric habitats. Physil. Zdl. 38, 99-109. WASHBURN, E. W. (1928). Internatinal Critical Tables, vl. 3. New Yrk: McGraw-Hill. WIESER, W. & SCHWEIZER, G. (1970). A re-examinatin f the excretin f nitrgen by terrestrial ispds. J. exp. Bil. 52, 267-74. WINSTON, P. W. (1967). Cuticular water pump in insects. Nature, Lnd. 214, 383-4. WINSTON, P. W. & BEAMENT, J. W. L. (1969). An active reductin f water level in insect cuticle.^, exp. Bil. 50, 541-6. WINSTON, P. W. & HOFFMEIER, P. (1968). Influence f ambient humidity n water cntent f cckrach cuticle. Prc. Int. Unin Physil. Sci., Washingtn 7, 471.