Cuticle water activity and water content of beetles and scorpions from xeric and mesic habitats

CUTICLE WATER ACTIVITY AND WATER CONTENT OF BEETLES AND SCORPIONS FROM XERIC AND MESIC HABITATS WAYNEA. RIDDLE Department of Biological Sciences, Illinois State University, Normal, IL 61761, U.S.A. (Receiwd

I1 June 1980)

Abstract-l. Cuticle water activity (a,,,) and water content were determined for beetles and scorpions collected from relatively xeric and mesic habitats. 2. The hemo~ymph-cuticle a, gradient was higher in the species collected from a xeric habitat. 3. Cuticle water contents were signifi~antiy lower in scorpions and beetles taken from a xeric habitat.

Transpiration through the integument of terrestrial insects and arachnids involves progressive movement of water molecules from the hemolymph through epidermal cells, endocuticle and epicuticle. The importance of the lipoidal epicuticle as a major barrier to water movement has been firmly established (Neville, 1975; Edney, 1977). In contrast to the interest in the epicuticle, the role of epidermal cells in influencing the permeability of the entire integument has received little experimental attention. In a simple model describing the movement of water through the integument, Berridge (1970) made a significant contribution to our understanding of cuticle water relations by identifying epidermal cells as possible barriers to transpiratory water movement. Water movement between the organism and its external environment, and between internal water compartments such as hemolymph and the integument is best discussed in terms of gradients of water activity. The useful concept of water activity has been fully discussed by Wharton & Devine (1968) and by Edney (1977). Using a method of exposing pieces of excised cuticle over the humidities provided by NaCl solutions of different molalities, Winston & Beament (1969) found that the water in living cuticles of locusts and cockroaches is not in osmotic equilibrium with the hemolymph. In terms of water activity (a,) values, this meant that cuticle a, was less than hemolymph a,. These investigators proposed that the lowering of cuticle water activity below that of the hemolymph was due to some form of active regulation, and that the living epidermal cell was the site of this regulation Berridge (1970) asserted that an active mechanism need not be envisaged to explain hemolymphcuticle (I,, gradients across the epidermal cell layer. Alternatively, he proposed that these gradients could be established if the apical epidermal cell membrane were a passive diffusion barrier which restricted water movement out of the cuticle. The mechanisms of water movement through the arthropod integument presented by Berridge and discussed by Edney (1977) identify the apical epidermal

cell membrane and epicuticle as major potential resistance barriers. According to one mechanism. if the main site of resistance to water movement is provided by the epicuticle, rather than the apical membrane, then the hemolymphhcuticle a, gradient would tend to be small. Alternatively, if the total resistance is more evenly shared between the apical membrane and epicuticle, a, gradients between hemolymph and cuticle may exist, and tend to increase in magnitude with greater apical membrane resistance. Toolson & Hadley (1977) found that surface densities of lipids and hydrocarbons in scorpion cuticle were inversely related to cuticular permeability. They noted that in a more xeric-adapted species of scorpion both surface densities of hydrocarbons and the proportion of long-chained branched hydrocarbons were higher. Significant seasonal changes in cuticular permeability in the scorpion Cmrruroidrs s~ui~r~~rl1fu.s have also been associated with qualitative changes in epicuticular lipid composition (Toolson & Hadley, 1979). These data clearly indicate that at least part of the low cuticular permeabilities characteristic of desert arthropods is attributable to the increased resistance to water movement that cuticuiar lipids and hydrocarbons provide. It can be reasoned that if a major adaptation in the cuticle of desert arthropods has been an increased integumentary resistance conferred by lipids, rather than increased resistance of the apical membrane, then certain predictions can be made concerning the hemolymph-cuticle a, gradients which exist in desert arthropods. Berridges’s model predicts that if the major proportion of the total integumentary resistance is attributable to epicuticular resistance, as it may generally be in desert arthropods. then the comparatively low apical membrane resistance should be manifested in a small hemolymphcuticle a, gradient. Alternatively, if the low integumentary permeability of xeric-adapted arthropods is due to both greater resistance in the epicuticle and in the apical cell membrane, then greater hemolymphcuticle a, gradients should exist. Experiments described in the present paper investigate the role of the epidermis as a ~rmeability barrier, considering the ahernative hypotheses outlined above. These experiments examine and compare the 231

_7 3 -7

WAYSIA.

RIIX,I

I

cuticle water iictibit? and cuticle water contents of scorpions and beetles found in relatively seric and mesic habitats. tlATERIAl
AND

METHODS

Adult liimale scropionr. Pu~u~-oc~to~rr.~utrr/tu~r.sis (Stahnke) (Vaejovidac) and adult female beetles. Elrodes hispilrrhris Say (Tenehrmnidac) were collected in desert grassland areas near Albuquerque. New Mexico in April and May 1977. Scorpions were kept ~~vernight in the laboratory rn darkness at Z.? & I C and at about \)o”~,relative humidity (RH). The lollowing d:tr\? scorpions were placed at room humidity (3s40”,, RH) for 2-4 hr before cuticle samples were taken. Beetles were collected during the morning and kept at room conditions l’or Z-4 hr just before cuticle samples were removed. Two groups of 20 beetles were established to examine the effect of desiccation on cuticle water activity and water content One group was desiccated (mean = 3.?“,, non-fecal weight loss) by exposing it for 10 days to dry moving air in B device described previously (Riddle or pi.. 1976). A second group of beetles was protected from desiccation by exposing them for I I days to high humidity conditiorls without free water or food. Desiccated be&es were treated like freshly collected animals prior to cuticle sampling. Non-desiccated beetles were kept for 2 4 hr at 9?-Y”,, RH to minimize desiccation stress prior lo cuticle sampling. All experiments utilizing P. u~rrhr,nsi.\ and E. hi,spi/trhri\ were conducted at the [Jniversity of New Mexico. Adult female scorpions, C‘eutruroidrs tittatus (Say) (Buthidae) were collected from open forest glade areas near St Louis, Missouri in October. 1978. Crntruroides scuIptwutus scorpions collected near Phoenix, Arizona were shipped by air to Illinois and maintained in the laboratory in the same manner as C. ritrottrs. Beetles. Alohatrs penn.~~,~~u~i~Q(DeGeer) (Tenebrlonidae) which closely resemble E. ~i.~piru~r~s morphologically. were collected from under the bark of fallen logs in a pme-oak woodland near Gainesville. Florida. Scorpions and beetles were transferred by car to Illinois State (Jnirerslty where experiments were conducted. Specimens of all species collected in 1978 were kept for up to 6 weeks in plastic shoeboxes at room temperature (IO-23 C) and humidity (5@70”,, RH) under controlled light conditions determined by laboratory use. Scorpions were provided free water and fed immature cockroaches. Beetles were given free water and fed Iaboratory rat food itd fib. Animals were denied food and water for 4--j hr before cuticle samples were taken

Hemolymph samples from P. utulr~trsi.s and E. hispilrhri~ were taken and osmolality determined using procedures described in a previous paper (Riddle ct ~1.. 1976). Hemolymph osmolalrty of freshly collected scorpions was estimated in a separate group of 28 animals taken at the same time as those used for cuticle experiments. Hemolymph samples from 6’. ili.\~,i/ahris were taken immediately prior to the removal of cuticle samples and were frozen for later osmometry. Hemolymph osmolafity of C rittatus, C. sczdpTUMIUS and A. f’ctrirs~iror?icft were determined using a Wescar Vapor Pressure Osmometer

Following hemolymph sampling, specimens were taken from room temperature and humidity into a controlled temperature,‘humlditv chamber where cuticle samples were prepared. Chamber donditions were 26 i I -C/93:95”;, RH (University of New Mexico) and 29 + I C/90. 97”,, RH (Illinois S&te linlversity). Scorpions w&-e cut tran&rsely along the posterior margin of the prosoma then along the

After weighing. each sample wah placed on a piece 01 screening ober a NaCI holutlon of known molaht\ 111 ,I 250 ml jar. Jars containing individual cuticle iample\ LICK kept In Insulated boxes fool- 20 2-l hi. After cxpo\urt~ each cuticle sample was removed and quickly uergheti under hrph humtdit> condltionr (00 92 or ‘) 95”,, RHI. :1II CIIOcle samples \\cre then drtrd at 60 C’ under vacuum ln or&r that estimates of cuticle \\ater cc)ntcnt ccluld he made. Cuticle water content was e~pre5~ed 25 ‘1 pcrccntagc i,l original cuticle weight and also a\ mg H,O per mp Jr! cuticle wrtght. Water activity of cuticle sampies was calculated h! trslng the molality of NaCl at which samples neither galned nor lost water following equilihratlon. In order to catimatc thic “equilibrium” concentration. mean v+rlght change of cuticle samples (ordmate) was plotted against the I\iaCl mol;llit! over uhlch they were exposed (abscl\sa). The cyuihbrlum concentration was Indicated by the tntersectlon 01‘ :t lmc connecting points of minlmal sample weight gain and los\ during equilibration with :I line cztcnding horizontally from a point on the >-as1\ Indicating no weight change. StatistIcal comparisons \ an;iiysts of variance.

Rt3C

LTS

Weight changes of beetle cuticle samples after cquilibration over NaCl solutions are presented in Fig. I. These results indicated that cuticle samples of E. I~r.spilubris were in osmotic equilibrium with a solutton of about 565 mM NaCI (I 039 mOsm). The osmotic concentration of the bemol~m~h t cYY’,, confidence intervals) was 536.X ) 18.5mOsm (191 mM Natl) (r~ = 66). Weight gains of cuticle samples for desiccated and non-desiccated beetles over 4OOmM NaC’I did not differ significantly (Fig. I I. However. weight changes over 400 mM NaCl of field-collected and desiccated beetles did differ (P < 0.05). Cuticle weight changes in 4. prrlrl.s!,/t,ctrtic,tr Indicated that the cuticle was in equilibrium with a solution of approx 265mM NaCl (489 mOsm). This equilibrium concentration exceeded the hemoiymph osmolality of 349.3 & Il.9 mOsm (189 mM) by some l4~mOsm (76 mM NaCI). Clearl\. the cutickhemolymph osmotic gradient (about 470 mOsm) in E. iiispilahris was substantially greater than that existing in A. parm,hxuzicu (140 mOsm). Cuticle water contents of beetles arc presented in Table 1. These results indicated that desiccation lowered cuticular water content in E. hi.spikzhri.s below that of non-desiccated specimens but not below that of the field controls. Significantly higher cuticular

Cuticle +5

233

water relations

r -+-

r

hispllobrls

-=-

5

pennsylvanica

+4 +3 t

300 of

NaCI

600

500

400 Molollty

solution

(mM)

I. Percentage

weight changes of excised cuticle pieces from Ekodes hispilabris and Alobnres pennsylbeetles after equilibration over NaCl solutions. The open square @) indicates mean weight changes obtained over 400mM NaCl of cuticle samples from desiccated E. hispilnbris, while the open triangle (A) is from non-desiccated E. hispilabris. Open circle (0) and solid circle (0) with horizontal bars indicate mean hemolymph osmolality (as mM NaCI) of E. hispilabris and A. pennsyloanica respectively. Vertical and horizontal bars are 95% confidence intervals. Numbers associated with means are sample sizes. Fig.

uanica

water contents were found in A. pennsyloanica than in E. hispilabris. Weight changes for scorpion cuticle samples following equilibration are indicated in Fig. 2. In P. utnhensis the cuticles were in equilibrium with a solution of approx 510 mM NaCl (938 mOsm). Hemolymph osmolality (+9S% confidence intervals) was 584.7 k 8.3 mOsm (317 mM NaCl) (n = 28). These results indicated that a cuticle-hemolymph osmotic gradient of about 350mOsm existed in P. urahensis. Hemolymph osmolality of C. oirrarus was 572.1 f 6.2 mOsm (310 mM NaCl) (n = 44) only slightly lower than that of P. utahensis, and did not differ significantly from that of C. sculprurarus (576.4 k 6.7 mOsm, II = 23). Cuticle samples of C. uitrarus were in equilibrium with a concentration (about 295mM NaCl or 543 mOsm) which was slightly lower than the hemolymph osmolality. These results indicated that in living C. uirrnrus the cuticle was essentially in osmotic equilibrium with the hemolymph.

Table

I. Cuticle water content

of Eleodes

During equilibration of C. sculprurarus cuticle samples over NaCl solutions, chamber temperature changed and condensation occurred. These cuticle samples, while not suitable for a, estimates, were used for determinations of water content. Using osmolality values corresponding to NaCl molalities with which cuticle samples were in equilibrium, cuticle water activity (a,) values were calculated. Those values and estimates of a, gradients between hemolymph and cuticle for arthropods in the present and in previous studies are presented in Table 2. Cuticle water contents of C. uirrarus were 34.97 k 1.25% (n = 28) and 0.5413 k 0.0295 mg H,O/mg D.W. (n = 28). Both measurements of cuticle hydration were higher (P < 0.001) in C. cirrarus than in P. urahensis (29.09 k 0.83%, 0.41 12 _t 0.0162, mg H,O/mg D.W.. II = 79). Cuticle water contents for C. 0.4416 mg sculpturarus were 30.42 + 1.70% and H,O/mg D.W. (~7= 23). Water contents of C. sculprurarus and P. urahensis samples did not differ signifi-

hispilabris

Water Species and Treatment Eleodes

and AIobares content

%

pemsylranica

beetles

f SE (II)* mg H,O/mg

dry wt

hispilabris

field desiccated non-desiccated .4loharrs

*Means

pemsylvanica

IO.15 f O.l25(66)abc 9.83 f 0.229(17)ab 10.69 + 0.236(18)c 12.75

followed

f

0.396(3l)d

0.1 126 + O.OOt4(66) abc 0.!066 i O.O019(17)ab 0.1184 +O.O028(18)c 0.1461 f 00054(3l)d

by at least a single common letter do not dilfer srgnrtic,~;rtly. are significantly different. Probability levels of P < 0.05 and P < 0.01 were associated with y0 and mg H,O/mg dry wt comparisons respectively between groups of E. hispilahris. Levels for inter-species comparisons were all P < 0.001.

Means with no letters in common

WAYNE A. RIDDLE

234

-+--

.

P -c

utahems wttatus

400 of NOW solution

300 Molality

500 (mM)

600

Fig. 2. Percentage weight changes of excised cuticle pieces from Parurocronus uruhensis and Corrruroides t!ittarus scorpions after equilibration over NaCl solutions. Open circle (0) and solid circle (0) with horizontal bars represent mean hemolymph osmolality (as mM NaCI) of P. rrquilonrtlis and f. rirrcrrus respectively. Vertical and horizontal bars are 95”a confidence intervals. Numbers associated with means are sample sizes.

cantly. Water contents tarus

samples

of C.

sc~~pturat~s

did differ significantly

and C.

vit-

(P < 0.001) in

comparisons using both expressions of water content. DISCUSSION

Lindqvist et al. (1972) found that cuticle G in several species of land isopods was slightly greater than that of the hemolymph (Table 2). They proposed that water was actively supplied to the cuticle to keep pace with high cuticular transpiration, thereby preventing drying of the cuticle. It is clear that the epidermis does not provide an effective resistance barrier to water movement in the isopods studied. In contrast, Table 2 indicates

that. with the exception

of C. cit-

tutus, insects and arachnids have hemolymph~uticle a, gradients which reflect appreciable epidermal cell resistance. Most significantly, the hemolymph-cuticle rr, gradients presented in Table 2 show that the xericadapted arthropods examined in the present study had a,,, gradients that were greater than those found in mesic species. If Locusta migratoria can be considered to occupy a xeric habitat and Periplanetu americana a mesic habitat, as indicated by Edney (1977), then the greater hemolymph-cuticle 4, gradient found in t. migrutoria (Table 2) would be expected, based on the results in the present study. The differences in G gradients found in cockroaches and locusts should be interpreted with caution because these species are not as closely related as are

Table 2. Cuticle and hemolymph water activity in groups of terrestrial arthropods Water activity (4,)* Group/Species

Hemolymph

Cuticle

Difference

0.9892 0.9892 0.9905 0.9895

0.99 I I 0.9930 0.9933 0.99 14

-0.0019 - 0.0038 - 0.0028 -0.0019

0.9942 0.9942 0.9938 0.9904

0.9863t 0.9837f 0.9913 0.9816

0.0079 0.0105 0.0025 0.0088

0.9898 0.9896

0.99 I3 0.9834

Reference

ISOPODA Armudillidium wigare Porcellio se&r Oniscus asellus Cyfisticus c5mt~.ws

Lindqvist Lindqvist Lindqvist Lindqvist

et al. (1972) cr al. (1972) cr rrl. ( 1972) rr al. I 1972)

INSECTA Pl,ripluf~etu u~ner~cu}zu Locustu migruraria .4lohutrs pennsyluanica EIrodes hispilahris

Winston & Beament (1969) Winston & Beament (1969) Present study Present study

SCORPIONIDA Centruroides Pururoctonus

oittatus utahensis

-0.0015

0.0062

Present study Present study

* Cuticle and hemolymph a,, values were calculated using the expression:

aw =%I5106

55.5106 mol ke-’ mol kg-’ + osmol kg-’

(Wharton & Devine, 1968). Hemolymph osmolality and cuticle equifibrium concentrations were converted from values presented by authors in mM NaCt to osmol kg-’ and substituted in the second term of the denominator in the expression above. t Values based on graphic presentation of equilibrium molality of cuticle

samples.

235

Cuticle water relations those used for comparisons in the present study. It is

possible that they differ in cuticle structure to the extent that the relatively small differences noted in the a, gradients could be due to factors other than differences in the resistance provided by the epidermis. Cuticle water contents were significantly lower in xeric-adapted scorpions and beetles than in the mesic species. This finding further suggests that the epidermis is more significant in restricting water movement into the cuticte in the xeric-adapted species. Restriction of water movement by the epidefmis into the cuticle would tend to reduce cuticle water content and would be manifested in hemolymph-cuticle aru gradients. While the presence of a,,, gradients indicates that an epidermal permeability barrier exists, reducing cuticle a, below that of the hemolymph, to the extents observed, could in itself have little influence of the vapor pressure gradient and consequently on the rate of diffusion of water across the integument. This contention can be demonstrated by using formulae presented by Edney (1977) which describe the relationship between vapor pressure and rd. For Locusta m~grator~~the reduction found in the cuticle a, below that of the hemoiymph (Table 2) represents a decrease in the vapor pressure of the cuticle relative to that of the hemolymph of only 0.33 mmHg at 30°C (0.0105 x 3 1.824 mmHg). Such a reduction could only minimally decrease the substantial vapor pressure gradient which would normally exist between the cuticle and subsaturated air. More important than 4y gradients is the potential influence of reduced cuticle water content on the permeability of the entire integument. That the epidermis in a living arthropod can influence cuticular water content has been shown by Winston & Beament (1969) in ~er~~~a?~etaAmericana and ~c~sta migratoria. It was found that cockroach and locust cuticles would contain 20 and 60”/, more water respectively if the hemolymph and cuticle were in osmotic equilibrium. If cuticle hydration influences the overall resistance of the integument to water movement, a possibility that has been discussed by Edney (1977), then a reduction in cuticle water content could result in reduced transpiratory water loss. Unfortunately, no clear relationship has been established between cuticle water content and cuticular permeability. Edney (1977) indicates that diffusion formulae predict that the rate of diffusion of water through the integument should partly depend on the absolute water content of the cuticle. Evidence suggesting a lack of correlation between cuticle hydration and ~rmeability has come from work with ticks by Hafez ef al. (1970). In that study it was found that whole body water loss, rather than a reduction in cuticle hydration, was associated with lower cuticle permeability. Results of the present study indicate that prior desiccation did increase water uptake in excised cuticle pieces in E. hispilabris (Fig. 2). While these results suggest a reduced water content in cuticle samples taken from desiccated beetles, significant differences in cuticle water content were not found between field collected and desiccated beetles (Table 1). In view of the possibility that the high water contents of nondesiccated beetles may have been due to the unusually

high relative humidity (93-95x) to which these animals were exposed immediately prior to sampling, water contents of these animals probably cannot be validly compared to those of the desiccated and field groups. The simplest tentative conclusion that can be drawn from the results presented is that the higher hemolymph~uticle 4~ gradients and lower cuticular water contents in the xeric-adapted species indicate that the epidermis plays a more important role in restricting water movement in these species. Results of the present study, while they do support the interpretation above, are too limited to rigorously substantiate any broad generalizations. Additional work on a variety of arthropods is needed to assess the contribution of the epidermis to total cuticular permeability. Also needed is work examining the tentative association made herein between the physiological significance of the epidermis and adaptation to a xeric habitat. ~c~~~~~~~ge~~llfs-I wish to thank C. S. Crawford. E. 8. Edney and E. C. Toolson, for useful discussions and for reviewing previous drafts of the manuscript. I also thank Robert Woodruff, Division of Plant Industry, University of Florida for identification of AI&ares pefl~s~/~a~icu. I appreciate the help of J. F. Anderson. University of Florida, and of E. L. Mockford of my department in collecting specimens. This research was supported in part by an ISU Faculty Research Grant.

REFERENCES BERRIDGEM. J. (1970) Osmoregulation in terrestrial arthropods. In Chemical Zoology, Vol. 5. Part A (Arthropoda) (Edited by FLORKIN M & SCHEER B. T.), pp. 287-319. Academic Press, New York. EDNEY E. B. (1977) ~ooph~sio/o~~ and ~co~o~}l. Vol. 9. Water Balance in Land Arthropods. Springer-Verlag, New York. HAFEZM., EL-ZIADYS. & HEFNAWYT. (1970) Biochemical and physiological studies of certain ticks (Ixodoidea). Cuticular permeability of Nvulomma (H.) dromedarii Koch (Ixodidae) and &thodoros (0.) saaiylyi (Audouin) (Argasidae). J. Parosirol. 56, 154-I 68. LINDQVIST0. V., SALMINEN1. & WINSTONP. W. (1972) Water content and water activity in the cuticle of terrestrial isopods. J. exp. Biol. 56, 49-55. NEV~LLE A. C. (1975) Zoophysiology and Ecology. Vol. 415, Biology of the Arthropod cuticle. Springer-Verlag. New York. RIDDLEW. A.. CRAWFORDC. S. & ZEITOONE A. M. (1976) Patterns of hemolymph osmoregulation in three desert arthropods. J. camp. Phpsiol. 1 It. 295-305. TOOLSONE. C. & HADLEYN. F. (1977) Cuticular permeability and epicuticular lipid composition in two Arizona vejovid scorpions. Physiol. Zoo/. 50. 323-330. T~~LXIN E. C. & HADLEYN. F. (1979) Seasonal effects on cuticular permeability and epicuticular lipid composition in Centruroides sculptuwtus Ewing 1928 (Scorpiones: Buthidae). J. camp. Physiol. 129. 319-325. WHARTONG. W. & DEVINET. L. (1968) Exchange of water between a mite Laelaps echidnina, and the surrounding air under equilibrium conditions. J. insrcf Physiol. 14, 1303-1318. WINSTON

P. W. & BEAMENTJ. W. L. (1969) An active reduction of water level in insect cuticle. J. r.xp. Biol. 50, 54 l-546.