Hemolymph osmoregulation in several myriapods and arachnids

Camp Biochem. Physiol. Vol. SOA,No. 3, pp 313-323, 1985

0300-9629/85$3.00+ 0.00 Pergamon Press Ltd

Printed in Great Britain

HEMOLYMPH OSMOREGULATION IN SEVERAL MYRIAPODS AND ARACHNIDS WAYNE A. RIDDLE Department of Biological Sciences, Illinois State University, Normal, IL 61761, USA. Telephone: (309) 4382661 (Received 22 May 1984) Abstract-l.

Osmotic

responses

to dehydration

and rehydration

were examined

in a millipede,

Cylindroiulus londinensis (Leach), a centipede, Lithobius forficatus L., a harvestman, Leiobumon vittatum (Say) and a spider, Argiope trifasciata (Forskbl).

2. Osmoregulatory responses to dehydration, assessed using a graphical method, differed considerably between species. Male millipedes regulated hemolymph osmolality better than females. 3. Size and temperature influenced water content and hemolymph osmolality in millipedes. 4. Rectal fluid became hyperosmotic to the hemolymph in dehydrated harvestmen.

INTRODUCTION

A substantial and growing body of literature is becoming available on the osmoregulation of terrestrial arthropods, primarily insects: Reviews of the water relations of insects by Maddrell (1980), Stobbart and Shaw (1974) and Willmer (1982) and as well as those concerning land arthropods in general by Berridge (1970) and Edney (1977) include discussions of osmoregulation. Mantel (1979) has reviewed the literature on osmoregulation in land invertebrates other than insects. However, considering the literature dealing with myriapods and arachnids, it becomes evident that our understanding of osmotic and ionic regulation in these groups of arthropods is quite limited. Among the myriapods, osmoregulation has been examined in the woodland millipede, Pachydesmus crassicutis and in the desert millipede, Orthoporus ornatus. In the former species, Woodring (1974) found that millipedes, even when slowly dehydrated, regulated hemolymph osmolality less efficiently than did locusts and cockroaches. The extent of regulation of hemolymph osmolality in this millipede, following rehydration, was influenced by the rate of prior dehydration. In the desert millipede, strong regulation of hemolymph osmolality was noted following moderate dehydration in females but not in males (Riddle et al., 1976). Further dehydration resulted in substantial increases in hemolymph osmolality in both sexes. Excellent regulation however was evident following rehydration. Seasonal variations in hemolymph osmolality have also been noted in this species (Crawford, 1978; Pugach and Crawford, 1978). Variations in hemolymph osmolality were also noted in 0. ornatus and in Archispirostreptus syriacus in response to starvation and dehydration exposure (Crawford and Warburg, 1982). Information on the osmotic regulation of centipedes is limited to a single study of the weight changes of littoral and terrestrial geophilomorph centipedes in various dilutions of seawater (Binyon and Lewis, 1963). Aspects of the hemolymph chemistry of myriapods have been examined (Sundara Ragulu, 1974; Bedford and Leader, c BP *0,3*-n

313

1975; Gowri and Sundara Rag&, 1976; Pugach and Crawford, 1978). In arachnids, exclusive of the acarines, osmoregulation has only been investigated well in scorpions. Comparatively poor hemolymph osmoregulation was evident during dehydration in species studied by Riddle et al. (1976) and by Warburg et al. (1980). Interestingly, Robertson et al. (1982) found much better osmotic and ionic regulation in a xericadapted scorpion than in a more mesic-adapted species. For spiders, Edney (1977) cites the work of Ueda (1974) on the orb-weaving spider Nephila clavata, which suggests that rectal resorption of water is significant in regulating hemolymph osmolality. In contrast to the limited information on osmoregulation of spiders, their hemolymph chemistry has received greater interest (see Cohen, 1980; Punzo, 1982, 1983; Schartau and Leidescher, 1983 and references therein). The present paper examines osmotic responses to dehydration and rehydration in two myriapods (a millipede and centipede) and in two arachnids (a harvestman and spider). In addition to assessing and comparing the osmoregulatory abilities of these arthropods, it examines the influence of size and temperature on hemolymph osmolality in millipedes and the possible role of rectal resorption of water in osmoregulation in harvestmen and spiders. Finally, it utilizes two analytical approaches which may be useful in uniformly comparing the osmoregulatory capacities of other groups of terrestrial arthropods. MATERIALS AND METHODS

Collection and maintenance of animals

Male and female Cylindroiulus londinensis (Leach) were collected from leaf litter at the author’s residence in May and June 1981 and 1982. Animals were taken to the laboratory on moist soil and litter the same day. After weighing and hemolymph sampling, specimens were killed by freezing, sexed and dried to constant weight at 70°C. Millipedes to be used in all other experiments were placed on moist paper in covered plastic containers for approx. 48 hr. This post-collection laboratory treatment (designated “pretreatment”) tended to minimize variations between

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WAYNE

individuals in water content and hemolymph osmolality, by allowing animals to defecate and drink. Mean water content and hemolymph osmolality values of these pretreatment groups were considered to reflect a standardized initial condition of hydration and hemolymph concentration prior to dehydration and rehydration. Lithobius forficatus (Linnt) centipedes were collected from the same habitat as millipedes from August to October 1981, and from May to September 1982. “Pretreatment” exposure was the same as for millipedes except that animals remained separated. Light-coloured specimens or those that had a purplish-gray coloration, indicating a recent moult, were not used. With the exception of very small animals, oocytes were found in the ovary of all females following dissection. Only females which were obviously gravid, as indicated by a large mass of oocytes visible through the integument, were not used in experiments. Adult male harvestmen, Leiobunum vittatum (Say) were collected in a roadside grassland area near Normal, Illinois in July and August 1982. Animals were either examined immediately (“field” groups) or placed on moist paper in large plastic containers for approx. 48 hr (“pretreatment” group). Animals used in dehydration experiments were similarly exposed prior to desiccation. Adult female Argiope trifasciata (Forskil) were collected from roadside grassland habitats near Normal, Illinois from August to October 1982. Spiders not immediately sampled were placed in small plastic screen cyhnders, lightly sprayed with tap water in a partially covered container. This treatment gave an RH of 8&95% during pretreatment, thus avoiding the saturated humidity conditions that were poorly survived by spiders. Specimens were killed by freezing and the abdomen of each was bisected to roughly determine reproductive state. In order to minimize possible variations in water content and osmolality, due to differences in reproductive state, only non-gravid animals were considered. Specimens with egg mass one-sixth, or more, of the cross-sectional area of the abdomen were considered gravid. Sampling

of hemolymph

and

rectal@id

Hemolymph samples were drawn into l-p1 pipettes, sealed at both ends with Vaseline, and frozen at -20°C for later osmometry. In millipedes hemolymph was taken from a dorsal puncture made in the anterior one-third of the animal. Centipedes were lightly anesthesized with CO,, secured to a smooth surface with tape and sampled for hemolymph after they had regained activity. In harvestmen, hemolymph was removed from a cut leg or pedipalp. Rectal fluid was secured by removing fluid exuded from the anus following a gentle massage of the abdomen. Pipettes were sealed with Vaseline and at one end with a dense pipettesealing clay. Samples were centrifuged at 1000 rpm for 10 min. Hemolymph and rectal fluid was taken from spiders in a similar manner. Unlike the slightly opaque rectal fluid of harvestmen, spider fluid was dark and contained considerable suspended material. Rectal fluid samples could seldom be drawn from spiders which had experienced even moderate dehydration. Centrifugation rendered only about hah” of the spider samples clear-enough for melting points to be determined. The “rectal fluid” sampled could be described as “hindgut” or “posterior intestine” fluid in harvestmen and would also likely be composed of the contents of the stercoral pocket in spiders. Dehydration

and rehydration

exposure

Water loss rates varied considerably among the arthropods examined in the present study. The millipedes and centipedes occupy essentially a hygric soil habitat and avoid desiccating conditions by moving more deeply into litter and soil. For these animals, laboratory desiccation was accomplished by exposing animals to moving air at a high humidity. Circulating air was bubbled through a saturated solution of potassium dichromate (K,CrO,) which increased

A. RIDDLE the relative humidity from room conditions (about 6O”/,) to about 95% over approx. 2 hr. During the initial low humidity conditions, centipedes were extremely active and experienced comparatively high water loss rates. Harvestmen and spiders were collected at or above the soil surface in vegetation and as such are normally exposed to temperature and humidity conditions more closely approaching those of ambient air. Harvestmen were typically taken within tall grasses and forbs while spiders were collected from webs more fully exposed to sun and wind. Harvestmen were exposed in screen top jars and spiders within small screen cylinders to circulating air dried to O-5% RH with silica gel. Following dehydration, animals were allowed to rehydrate for lo-12 hr by providing wet paper (millipedes, centipedes, harvestmen) or by water droplets on screen cylinders (spiders). Dehydrated animals of all species began drinking immediately after water was made available. Millipedes also rehydrated through anal water uptake. Relative humidity was measured using cobalt thiocyanate humidity papers (Solomon, 1957). The water content of dehydrated animals prior to rehydration was estimated in the following way: the water content of animals prior to dehydration was assumed to be reflected by that of animals in an approprtate pretreatment group. Using this water content, expressed as a percentage of live weight, the initial water mass of animals prior to dehydration was estimated. Non-fecal weight changes during dehydration were assumed to be due entirely to water loss. This weight loss was subtracted from, and then divided by, the estimated initial water mass. The resulting value, representing the percentage loss of initial water content, was multiplied by the pretreatment water content in mg H,O/mg dry weight. The resulting value was subtracted from the pretreatment water content to arrive at a water content estimated to be present prior to rehydration. Intuence

of stze and temperature

on hemolymph

osmolality

A large collection of millipedes made on 11 May 1982 was exposed to pretreatment conditions and divided into two groups by sex. To investigate the influence of size on osmolality, each group was subdivided into groups of small, medium and large mdividuals which were kept for 4-5 weeks on moist soil and litter in darkness at room temperature (23 + 2°C). To examine the effect of temperature, additional groups of medium-sized animals of each sex were similarly maintained at 5 f 1°C m a refrigerator, or at 30 + 1°C over a covered water bath. Medium-sized animals used in size-osmolality experiments also constituted a room temperature (23°C) group. Osmometry Hemolymph and rectal fluid osmolality were determined using a melting-point osmometer adapted from that described by Ramsey and Brown (1955) and used in a previous study (Riddle et al., 1976). Using this device, melting points could be determined to kO.O5”C, giving osmolality values which varied correspondingly in increments of 26 mosm/kg. Assessment

of osmoregulatory

capacity

Previous studies of osmoregulation in arthropods have used a variety of parameters to estimate the severity of dehydration stress experimentally applied to animals (Edney, 1977). In those studies which have not used changes in hemolymph volume to reflect the extent of dehydration, percentage whole body water loss has been commonly used. Percentage whole body water loss, while suitable in studies comparing the osmotic responses of related taxa, may be less useful in comparative studies in which species vary widely in water content, as they do in this study. It follows that arthropods with low water contents would experience more severe dehydration following a given percentage weight loss than those of a higher water content.

Osmoregulation

in myriapods

In addition to percentage weight loss, changes in whole animal water content have been used to measure the extent of dehydration. Bursell (1970), using data from Edney (1966) graphically depicted observed changes in hemolymph osmolality with changes in percentage water content. He compared the trend evident in the distribution of points with that of a curve describing the relationship between water content and osmolality predicted in the absence of osmoregulation. The present study utilizes a similar technique but describes the observed relationship between hemolymph osmolality and whole animal water content, expressed as mg H,O/mg dry weight (DW) using linear regression. Osmotic responses predicted in the absence of regulation are depicted by constructing a line from a point describing the water content/osmolality status of normally hydrated animals (pretreatment groups) to a water content/osmolality coordinate expected following a given reduction in water content due to dehydration. For example, pretreatment animals with a water content of 3.0mg H,O/mg DW (75%) and a mean hemolymph osmolality of 400 mosm, which lost 50% of total water to a water content of 1.5 mg H,O/mg DW (60x), would increase hemolymph osmolality to 800mosm in the absence of osmoregulation. In order to assess the osmoregulatory capacities of each species, the observed osmotic response, reflected by the slope (b) of a linear regression equation in the form Y = a + bX (Y = hemolymph osmolality, a = intercept, and X = water content) is compared with a predicted (no regulation) slope value. An index of the osmoregulatory capacity is provided by the ratio of the observed slope/predicted slope. For a species displaying good osmoregulatory ability, the slope ratio value should be small; for a poorer regulator, a higher value would be expected. This analytical scheme has the advantage that it allows comparisons to be made of the osmoregulatory capacities of species which differ substantially in water content and in dehydration tolerance. The practical advantage of utilizing regression analysis is compromised however by the inability of linear regression to accurately depict relationships that may be essentially curvilinear. In addition to the analytical scheme described above, assessment of osmoregulatory capacity will utilize a method originating in volume regulation studies (see Machin, 1975), which has been applied to a study of whole animal and cellular osmoregulation in larvae of the desert tenebrionid beetle, Onymacris (Machin, 1981). In this procedure, water content (in mgH,O/mg DW) appears on the vertical axis and hemolymph osmolality (expressed as I/osm/kg) on the horizontal axis. This method gives a regression line, fitted to observed water content/osmolality correlates, that is positive in slope. Greater slope values, and consequently higher negative intercept (a) values, indicated better osmoregulation. The intersection of a vertical line extending from a hemolymph osmolality value (X) of normally hydrated animals, with the regression line, provides a corresponding estimate of the water content (Y) for animals at that hemolymph osmolality. A line drawn from this intersection to the origin represents changes in water content predicted in the absence of osmoregulation. Differences between the slopes of the observed regression line and that of the predicted (passive) osmotic response to changes in water content, can be used to assess osmoregulatory capacity. The present paper modifies this technique to utilize the value of the observed slope/predicted slope as a second index of osmoregulatory capacity. In this method larger values of these indices were considered to reflect better osmoregulatory capacities. Linear regression equations were examined statistically by analysis of variance to determine if slopes differed significantly from zero (Sokal and Rohlf, 1969). The slopes of two regression lines were compared using the method described by Sokal and Rohlf (1969, p. 455). Mean values for hemolymph osmolality and water content were

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and arachnids

05

cl6

07

08

09

Water Content

10

(mg HIO/mg

11

12

13

D.W.)

Fig. 1. Influence of dehydration and rehydration on the water content and hemolymph osmolality of male C. londinensis. The solid line is a regression line fitted to the points. The regression equation is: Y = 344.73 - 107.32X, r = -0.227, F = 2.9 (P > 0.05), N = 55. Water content and hemolymph osmolality values are presented for field collected (m), pretreatment (a) and rehydration (0) groups. The dashed line depicts a predicted osmotic response of pretreatment animals to dehydration in the absence of osmoregulation. Vertical and horizontal bars are 95% confidence limits. Limits of the mean osmolality value for field animals is less than symbol size. presented Student’s

+95x t-test.

confidence

limits (CL) and compared

using

RESULTS

Millipedes

The relationships between water content and hemolymph osmolality in male and female C. londinensis are presented in Figs 1 and 2 respectively. Water content values (mg H,O/mg DW) in females for two collections (*95x confidence limits) (N) were 1.038 kO.027 (20) on 30 May 1981 and 1.020 + 0.026 (20) on 22 June 1981. Corresponding hemolymph osmolality values were 235.8 + 11.6 (20) and 230.6 + 7.3 mosm (20). No significant differences were found in water content or osmolality between the collections. A single group of males taken 22 June 1981 gave values of 1.015 f 0.036 mg H,O/mg DW (20) and 225.4 + 7.3 mosm (20). These values did not statistically differ from those found in either collection of females. Figures 1 and 2 indicate that osmolality values in field animals of both sexes (22

05

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Water Content

09

10

(mg H,O/mg

11

D W

12

)

13

Fig. 2. The influence of dehydration and rehydration on the water content and hemolymph osmolality of female C. londinensis. Regression equation for the solid line is: Y = 581.31 -351.72X, r = -0.817, F = 132.5 (P < O.OOl), N = 68. Other symbols are as in Fig. 1.

WAYNE A. RIDDLE

316

June) were slightly greater than the corresponding pretreatment values. In female C. londinensis, field and pretreatment osmolality values differed significantly (P < 0.001) despite the statistical similarity in water content. In males, field and pretreatment water content and osmolality did not differ significantly. Neither did water content nor osmolality differ statistically between pretreatment males and females. The influence of dehydration on hemolymph osmolality depicted by the slopes of regression lines in Figs 1 and 2 indicate substantially better osmoregulation in males than in females. Observed/ predicted slope ratios were 0.262 (- 107.32/-409.88) for males and 0.908 (-351.72/-387.50) for females. Regression line slopes of males and females differed significantly (F = 14.1, P < 0.001). Among the groups of desiccation-protected animals used in size-osmolality experiments (discussed below), significant regression relationships between water content (X) and osmolality (Y) were found. These regression equations were: Y = 275.05 - 46.308 (N = 59, P < 0.05) for males and 307.94 - 87.65X (N = 60, P < 0.01) for females. Although the slopes of these lines did not differ from each other, they did differ significantly from zero, and were greater in females than in males. In addition, these slopes were considerably lower in both sexes than those presented in Figs 1 and 2. In males the slope of -46.30 did not differ from that of - 107.32 (Fig. l), while that of females (- 87.65) differed (P < 0.001) from that of - 351.72 (Fig. 2). It should be emphasized that these animals were provided moist leaf litter and soil prior to sampling and as much resembled field animals. That significant effects of water content on osmolality are evident in animals provided food and moisture suggests that individual variations in water content, although not attributable to dessication exposure, do nonetheless influence hemolymph osmolality. Millipedes dehydrated for 48 hr prior to rehydration experienced a weight loss of 18.1% (males, N = 17; 0.38%/hr) and 17.4% (females, N = 19; 0.36%/hr). This dehydration resulted in a reduction in water content (mg H,O/mg DW) from pretreatment levels of 1.049 + 0.035 (males, N = 20) and 1.058 + 0.057 (females, N = 13) to 0.663 and 0.699 (39.7 and 33.9% loss of original water, respectively). In 17 males and 11 females, presumed near death by

water contents were 0.596 and dehydration, 0.601 mg H,O/mg DW respectively. A comparison of the latter values with those estimated prior to rehydration indicates that pre-rehydration water loss probably approached lethal levels. Rehydration reduced hemolymph osmolality from regression predicted values of 281 (males) and 370 mosm (females), to 191.7 + 10.2 (N = 19) and 176.7 + 6.4 mosm (N = 19) respectively. Rehydration water content and osmolality values of males differed significantly (P < 0.001) from pretreatment values. Among females, significant differences were noted only in hemolymph osmolality (P < 0.001). Following rehydration, males and females increased live weights, 1.9 and 2.4% respectively, over predesiccation weights. The influence of live weight on water content and hemolymph osmolality in C. londinensis is presented in Table 1. In males, despite significant differences in water content associated with live weight, hemolymph osmolality was not affected. Significant positive regression relationships (P < 0.05 and P < 0.01) were found between both live weight and dry weight (X) and water content (Y). No significant relationship was found between either live weight or dry weight and osmolality in males. Females had mean osmolality values which were higher in smaller animals despite statistically similar water contents between size groups (Table 1). Among females, no significant regression relationship was found between either live weight or dry weight and water content. In contrast to males, a significant inverse regression relationship existed between both live weight and dry weight and hemolymph osmolality (both P < 0.05). The influence of temperature on the water content and hemolymph osmolality of C. londinensis is presented in Table 2. The general trend evident therein is an association of greater hemolymph osmolality with exposure to lower temperatures. In the 5”C- and 24”C-acclimation groups the higher osmolality of the 5°C group appears largely attributable to temperature because water content values between acclimation groups did not differ significantly. When results for 30”C-acclimated animals are considered, variations in hemolymph osmolality cannot be clearly ascribed to temperature because of the association of significantly higher water contents with acclimation to higher temperatures. Considering the

Table 1. Influence of live weight and water content on hemolymph osmolality in C. londznenszs* Group (N) (Lwe weight range in mg)

water content (mg H,O/mg DW) (&95X CL) (P)

Hem&mph osmolality (mosm) (595% CL) (P)

Small males (30) (3 1.I&64.0)

0 992 rf: 0.049

228.0 + 7.5

<0.05

NS

Large males (29) (64.1-99.1)

1.051 + 0.032

227.6 + 6.8

Small females (36) (26 5-l 10 3)

1.013 f. 0.042

231.6 5 5.6

NS


Large females (36) (110.4-167.0)

1.014 + 0.029

213.6 + 6.6

*StatIstical comparisons of water content and osmoiality between sexes in similar size groups indicated differences only in the hemolymph osmolality of large males and females (P i 0.01).

Osmoregulation in myriapods and arachnids

317

Table. 2. Influence of temperature on water content and hem&mph in C. londinensis Water content (mg H,O/mg Dw) (f95% CL) (P)

Acclimation group (A? Males* 5°C (30) 24°C (25) 30°C (25) Females*

1.009NS * 0.035 1.055 + 0.038 <0.05 1.102 + 0.028

osmolality

Hemolymph osmolality (mosm) (595% CL) (P)

1 10.001 1

10.001 260.1 +_8.4 228.5 + 1.1 10.05 218.1 k 6.4

1


1

10.01 243.9 + 7.2 1 0.951NS + 0.029 232.9 + 4.7
inverse relationship between water content and osmolality among animals not exposed to dehydration, it can be conservatively concluded that temperature, possibly by exerting an effect on whole animal water content, influences hemolymph osmolality. Centipedes

Figures 3 and 4 respectively depict relationships between water content and osmolality of male and female L. forjicatus. In males no significant differences were noted between field, pretreatment and rehydration groups in water content, although significant differences existed in osmolality values between field animals and both the rehydration and pretreatment groups (P < 0.001 and P < 0.01 respectively). Among females, significant differences (P < 0.05) were found between the mean water contents of rehydration and pretreatment groups. Water content and hemolymph osmolality of the rehydration and pretreatment groups also differed respectively from that of the field group at P < 0.05 and P < 0.001 (water content) and at P < 0.001 and P < 0.01 (osmolality). The slopes of regression lines depicting the influence of dehydration on hemolymph osmolality did not differ significantly between males and females. Observed/predicted slope ratios for

I 15

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Water Content

I 25

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(mg H20/mg

I 35

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D.W.)

males and females were: 0.773 (- 178.91/-231.52) and 0.626 (- 138.65/-221.36), respectively. Water loss rates of centipedes at 1.83%/hr (N = 39) were the highest of all the arthropods examined. Strong post-rehydration regulation of water content and osmolality in centipedes is evident in Figs 3 and 4, despite extensive prior dehydration. Percentage weight loss of original water content for males and females before rehydration were 40.5 and 41.9x, respectively, giving estimated pre-rehydration water contents of 1.93 and 1.87 mg H,O/mg DW. That prior dehydration was severe was confirmed by the observation that within these groups estimated water contents of 9 males and 6 females showing highly impaired locomotion were 1.87 and 1.77 mg H,O/mg DW, respectively. Unlike millipedes, which exceeded pre-desiccation live weights upon rehydration, males regained only 97.2% and females only 97.6% of their original live weight. Also unlike millipedes, field-collected centipedes had water contents and hemolymph osmolality values that were lower, and in most cases significantly lower, than values in the pretreatment groups. The increase in water content over that of field animals during pretreatment exposure may be attributable to both water uptake and the clearance of gut dry matter by freshly collected animals. Considering the strong influence of

I

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25

30

Water Content

(mg H20/mg

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D.W.)

Fig. 4. The influence of dehydration and rehydration on the water content and hemolymph osmolality of female L. for$catus. Regression equation for the solid line is: Y = 960.08 - 178.91X, r = -0.822, F = 137.9 (P < O.OOl), Y = 858.77 - 138.65X, r = -0.788, F = 65.6 (P < O.OOl), N = 68. Other symbols are as in Fig. 1. N = 42. Other symbols are as in Fig. 1.

Fig. 3. The influence of dehydration water content and hemolymph forjicutus. Regression equation

and rehydration on the osmolality of male L. for the solid line is:

318

WAYNE

A.

ibXlLE

. ...

.A

“i*

mom.200 --“I

’ 300

400

Rectal

Fluid

500

’ 600

Osmolallty

1 700

i 800

(mOsm)

35

Fie. 5. The influence of dehydration and rehvdration on thi water content and heiolymph osmola&y of male L. vitsatum. Regression equation for the sotid line is: Y=827.44-175.13X, r= -0.780, F=53.9 (P
The influence of water content of hemolympk osmolality in male L. v~ttat~ is presented in Fig. 5. In addition to the results presented there, water content and osmolality were examined from three field collections of 20 animals each during August 1982. The first collection (3 August) was made on a particularly hot and dry afternoon, and a second early the following morning, The third collection was made on the morning of 7 August, following a rainshower the previous evening. Mean water contents (mg H,O/mg DW) and hemolymph osmolality (mosm) +95x confidence limits were: 2.261 + 0,078, 403.2 & 6.4 (3 August); 2,464 + 0.018, 401.8 _t 7.4 (4 August); and 2.460 & 0.105, 404.4 + 7.9 (7 August). Water cotitent of animals in the first collection (afternoon) differed significantly (P < 0.01) from that of the later collections (morning and after rain) and from the pretreatment mean (Fig. 5). Hemolymph osmolallty was statistically similar among the field and pretreatment groups. These field collections indicate that, despite appreciable natural changes in water content, hemolymph osmolality appears to be well regulaxed. The pattern of similarity between the field and pretreatment water content and osmolality of harvestmen differs slightly from that in C. t~~~~~~~~ but contrasts markedly from that evident in L. forjkatus. An observed/predicted slope ratio of 0.528 ( - 175,13/ - 33 1.73) was determined for L. vittatum, Unlike the osmotic responses to dehydration displayed by millipedes and centipedes, the general distribution of points in Fig. 5 appears more curvilinear. Hemol~~h osmolality appeared to be less influenced by water contents greater than approx 2.0 mg H,O/mg DW than below that level. This trend of better osmoregulation at higher water contents

Fig. 6. Rdationship between rectal fluid and hemolymph osmoIality in dehydrated and rehydrated L. vit~txalum (larger figure and insert respectively). Numbers associated with points indicate the number of identical values. Dashed lines depict isoosmotic relationships between variables. The equation for dehydrated animals is: regression Y = 121.44 + 0&33X, r = +0.861, F = 259.9 (P < O.OOl), N = 93. The regression equation for rehydrated animals (insert> is: Y=283.46+0.211X, r = +0.411, F=3.5 {P > 0.05), N = 19. resulted largely from values for lightly dehydrated animals having high pre-desiccation water contents. Estimated mean water content of harvestmen prior to rehydration was I .XI mg H@fmg DW. Considering that the mean content of a separate group of 17 dehydrated animals, showing obvious impairments was 1.58 mg HzOjmg DW, prein locomotion, rehydration desiccation was quite severe. Mean water loss rate for this group was 0.83%/hr. Upon rehydration, water content in L. vittatum increased to 3.092 + 0.091 mg H@/mg I?W. This value, and the associated osmolality (360.3 -i_ 1I .7 mosm), differed significantly (P < 0.001) from corresponding pretreatment values. The substantial increase in water content and decrease in osmolality with rehydration in L. vittatum contrasts with the strong postrehydration regulation of L. for$kattu and moderate re~la~o~ of C. ~~~~~~~~sj~. Live weights after rehydration in harvestmen exceeded predesiccation weights by 4.92x, an increase greater than that in millipedes and in centipedes. The influence of dehydration and rehydration on the osmotic gradient between hemolymph and rectal fluid in L. vittatum is presented in Fig. 6. In the larger figure, a comparison of the slope of the solid line with the dashed “‘isoosmotic” line reveals a trend of slightly greater osmolality in rectal fluid than in hemolymph in the same specimen, Contributing considerably to the slope of the regression line are hemolymph/rectal fluid correlates for more severely dehydrated animals, although an appreciable gradient is evident in lightly dehydrated animak as well. The relationship between hemolymph and rectal fluid osmolality among rehydrated animals (open circles, insert) shows no evidence of the production of a hypoosmotic rectal fluid. Considering that hemolymph and rectal fluid were sampled some lo-12 hr following rehydration, a fairly rapid production and subsequent removal of a h~oosmotic rectal fluid several hours following rehydration would not have been noted. That two specimens had rectal fluid hypoosmotic to the hemolymph by more than

Osmoregulation in myriapods and arachnids

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15

35

waterC’:“te”t ~mg21-S12O/mg D.vYl Fig. 7. Influence of water content on rectal fluid and hemolymph osmolality m L. vzttatum. Regression equation for the solid line (rectal fluid) is: Y = 807.27- 156.322 I = -0.627, F = 58.9 (P < O.OOl), N = 93. Regression equation for the dashed line (hemolymph, points not plotted) is: Y = 731.18- 133.392 Y= -0.675. F= 76.1 (P < O.OOl),N = 93. lOOmosm, does suggest the possibility of the production and retention of a dilute rectal fluid. The relationship between rectal fluid osmolality and whole animal water content in L. vittatum is depicted in Fig. 7. Considering the fairly close association between rectal and hemolymph osmolatity, and also the relationship between water content and hemolymph osmolality (Fig. 5), increasing rectal fluid osmolality with decreasing water content is to be expected. A slightly higher osmolality of rectal fluid than hemolymph in dehydrated animals is apparent in the slope and position of the solid and dashed lines in Fig. 7. Although osmotic gradients between rectal fluid and hemolymph greater than 100 mosm exist among dehydrated animals (Fig. 6) differences in the position of the two lines in Fig. 7 predicts smaller osmotic gradients among individuals in the entire sample. In Fig. 7, as in Fig. 5 for hemolymph, the distribution of points suggests better regulation of rectal fluid osmolality at water contents above about 2,0mgH,O/mgDW. Below that water content hemolymph osmolality (Fig. 5) and particularly rectal fluid osmolatity (Fig. 7) appear to increase more sharply. Spiders

The influence of dehydration and rehydration on the water content and hemolymph osmolality in A. trzyasciata is presented in Fig. 8. Significant differences were noted in water content (mg H,O/mg DW) between field collected animals (2.62 f 0.12) and both the pretreatment (2.87 f 0.16) and rehydration group (3.25 f 0.17) at P < 0.05 and P < 0.001, respectively. Hemolymph osmolality (mosm) of field ‘animals of 403.1 t_ 9.5 differed significantly (P < 0.001) from that of rehydrated animals (371.4 f 12.6), but was statistically similar to that in pretreatment animals (400.1 + 9.4). Estimated pre-rehydration water content for spiders was 2.3 1 mg H,O/mg DW representing a moderate dehydration compared with that of the other species. Despite this comRaratively mild prior dehydration, water contents and hemolymph osmolality values of the rehydration group differed significantly from those of pretreatment animals (P < 0.01 and P < 0.001, respectively).

I

,

15

35

water‘,mte”t (ms’SH,O/mg D ;, Fig. 8. The influence of dehydration and rehydration on the water content and hemolymph osmolality of female A. trifasciata. Regression equation for the solid line is: Y = 616.02-44.91X, I = -0.370, F = 21.6 (P < O.OOl), N = 138. Other symbols are as in Fig. 1. slope ratio for A. triwas the lowest of all the arthropods examined. Unlike the other species however, the regression line describing the influence of water content on osmolality came far from intersecting the pretreatment water content/osmolality mean. Also unexpected was that this regression line predicted hemolymph osmolality values which were in the order of 100 mosm higher than field, pretreatment and rehydration means. Considering that animals in all three groups above were exposed to fairly high humidities prior to sampling, it appears possible that some effect of exposure to dry air, apart from its influence on decreasing water content, was responsible for increasing hemolymph osmolality. Attempts to secure suitable rectal fluid samples from even moderately dehydrated A. trifnsciata were not successful. Consequently, the role of the hindgut and/or the stercoral pocket in water and solute absorption during dehydration could not be assessed. Rectal fluid and corresponding hemolymph samples were however removed from groups of spiders taken immediately from the field (N = 26) and following pretreatment exposure (N = 23). The water content of field and pretreatment groups of 2.620 and 2.912 mg H,O/mg DW, respectively were similar to corresponding means in Fig. 8. No significant differences were found between the osmolality of rectal fluid (433.9 mosm) and hemolymph (418.9 mosm) in field animals, or in pretreatment animals (411.3 and 402.3 mosm, respectively). Comparisons made between means of pooled samples (N = 49) also showed no significant differences. These results indicated that in the well-hydrated spiders sampled, rectal fluid and hemolymph were essentially isoosmotic. The observed/predicted

fasciata of 0.161 (-44.91/-278.92)

Alternate assessments of osmoregulatory capacity

Table 3 presents relationships between water content and hemolymph osmolality for the arthropods examined in the present study and for larvae of the desert tenebrionid beetle Onymacris, using a technique modified from that of Machin (1981). Beetle larvae used in that study included dehydrated animals as well as those rehydrated through vapor water uptake. The observed/predicted slope ratios based on a pooled sample of dehydrated, rehydrated

320

WAYNE

A.

RIDDLE

Table 3. Regression reiationships between hemol~ph osmolahty and water content for varmns arthropods techmque modified from that of Machin (1981)

species

C. londinensis C. londmensis L f~r~ca~~s L .f@iC~atws L. ulttatwn A. trijkciata Onymacris sp.

N/sex*

94M 1OOF 104M 66F 178M 188F 24-

a

f0251 +0.430 +0.175 t 0.065 -0.215 +0.652 -1.80

Hemolymph osmolality (l/omUkg)

W

+0.161 +0.120 +I.140 -+1.218 + 1.057 +0.909 +2 12

4.651 4.X78 2.664 2.671 2.469 2.499 2.560

Water1 content Predicted (mgimg DW) -_______ stop+ 1.000 0.215 1.015 0.208 3.212 1.206 3.318 1 242 2.395 0.970 2.924 1.170 3.627 1.417

using a

Observed/ predicted slope§ 0 149 0 571 0.945 0.98 1 1.090 0.771 1.496

*Groups mclude ali dehydrated, rehydrated and pretreatment ammals. PRegression equations are in the form: Y = a + bX. Y = water content (mg/mg DW) and X = osmolahty (l/osm/kg). $Normal water content predtcted by regression for ammals of normal (pretreatment) osmolality indicated in column 5. ~Predicted slopes determined by dividing water content (cohnnn 6) by osmoiahty (column 5). Obse~~/predicted values determined by dividing the slope (6) by the predicted slope value.

and pretreatment arthropods used in the present study gave a single overall assessment of the osmoregulatory capacity in each species. An examination of the right column of Table 3 shows an osmoregulation ability of ~nyma&ris, reflected by the high slope ratio of 1.496, to be superior to that of all the arthropods examined in the present study. These slope ratios show roughly comparable osmoregulation abilities for centipedes and harvestmen. Increasingly poorer regulation is evident in spiders, male millipedes and female millipedes. With the exception of harvestmen, values of the intercept (a) are positive in the animals used in the present study. That positive intercepts are found suggests that animals have overall osmoregulatory responses that are poorer than that represented by the predicted slope which characterizes a passive “no regulation” response. Consequently, observed/predicted slope ratio values in those animals are less than 1. Table 4 presents regression information for arthropods exposed to dehydration in the present study and compares slope ratios with those determined using the original analytical technique. As in Table 3, centipedes and harvestmen appear the best regulators during dehydration with poorer regulation evident in millipedes and spiders. A comparison of observed/ predicted slope ratios determined using the technique modified from that of Machin and those found using the original technique (in parentheses) indicate much different relative osmoregulatory capacities among the arthropods examined. The comparatively high values indicate good regulation in centipedes and harvestmen ~0.84~1*036), whereas values of 0.528 and 0.773 denote rather intermediate osmoregulatory capacities using the original technique. Differences in apparent osmoregulatory capacity using the two techniques are particularly evident for millipedes and

spiders. Male millipedes which show the poorest regulation (0.4Og), appear good regulators (0.262) using the original technique. The apparent osmoregulatory capacity of spiders also differs considerably with the analytical technique employed. DISCUSSION

Osmotic responses to dehydration in mil~pedes depicted in Figs 1 and 2 clearly show substantially better regulation in males than in females. These unexplained differences contrast with those of Riddle et al. (1976) for the desert millipede Urthoporus ornatus, which indicated poorer regulation in males than in females following moderate dehydration. The virtual absence of defecation during dehydration in both male and female C. Zondinensis precludes the excretory loss of osmolytes unless small volumes of undetected fluid material were voided. Regulation of osmolality during dehydration, at least in males, may be due to movement of water from the tissues or gut to the hemolymph. If the Malpighian tubules of C. ~o~di~ns~s produce an isoosmotic urine as doe% the pill millipede, Glomeris marginatu (Farquaison, 1974), absorption of water in the hindgut or rectum during dehydration would contribute to the regulation of hemolymph osmoIatity. That resorption of water from hindgut or rectal contents is influenced by dehydration has been shown in Pachydesmaus cr~si~ut~s and ~rthopor~s texicolens (Stewart and Woodring, 1973). In a study by Woodring (1974) on the hygric Pachydesm~s cr~s~~utis, slowly dehydrated specimens (18% weight loss over 60 hr or 0.3%/hr) increased hemolymph osmolality from 165 to 182 mosm. The predicted final osmolality in the absence of regulation, based on changes in hemo-

Table 4. Regresston relationship between hemolymph osmoiality and water content for dehydrated arthropods technique mod&d from that of Machin (19811

Specres C i~ndi~ensis C. londinensis L. forficatus L foFgkztus L. ulitat~ A. trifasciata

N/sex * SSM 68F 68M 42P 134M 138F

P

b

Hemolymph osmolality W=/W

1-0.543 i-o.349 +0.138 -0.120 Jr0 343 -+0.995

i-0.080 +0.13s f1.159 +I 307 -to.164 +a.737

4.651 4.878 2.664 2.671 2.469 2.499

Water content (w/w DW)

Predicted slope

0.913 1.021 3.224 3.372 2.230 2.838

0.196 0.209 1.210 1262 0.903 1.136

“Column designations are as in Table 3 except that only dehydrated animals were considered. iValues m parentheses are observed/predicted slope ratios detenmned usmg the original technique

using a

Observed/ predicted dOpet

0 408(0.262) 0.660(0.908) 0 958(0.773) 1 036(0.626) O.g46(0.528) 0.64?(0.161)

Osmoregulation in myriapods and arachnids lymph volume, was 224 mosm, thus indicating appreciable regulation. Using the initial water content of these animals prior to dehydration (73.5% or 2.78 mgH,O/mg DW) and an initial osmolality of 165 mosm, a reduction of 50% of original water content (to 1.39 mg H,O/mg DW) would be expected to double osmolali~ to 330mosm in the absence A predicted change of of osmoregulation. 165 mosm for a change in water content of 1.39 mg HzO/mg DW gives a slope of - 119 mosm per unit change in water content. The change in osmolality observed in P. &~~~~~~~~~ following 18% water loss (to 2.10mg H,O/mg DW) was only 17 mosm (182-l 65 mosm) or 25 mosm/unit change in water content. An estimate of osmoregulatory capacity obtained by dividing the observed slope by the predicted slope gives a value of 0.21 (- 25/ - 119) for P. crassicutis. This value is comparable to that of the slope ratio for male C. Zondinensis(0.262) but differs from that of females (0.908). Although the rate of dehydration of P. cr~s~cutis (0.3%~hr) was comparable to that of C. londinensis $0.36-0.38x), the severity of dehydration in the latter species was greater due to the lower initial water content of about 1.Omg H,O/mg DW. Considering this, and also the finding by Woodring (1974) that more rapidly dehydrated millipedes showed poorer osmotic regulation, a slower rate of dehydration of C. londinensis may have resulted in better regulation than that observed. Upon rehydration, C. lond~nensis increases water content and reduces hemolymph osmolality to levels above and below, respectively, those characteristic of pretreatment animals. The osmotic dilution evident may be explained in part by the greater live weights of males and females following rehydration than prior to dehydration. Moreover, post rehydration defecation may have had the effect of increasing water content values by removing gut dry matter, and of reducing osmolality through the possible excretory loss of osmolytes. The significant rehydration depression of hemolymph osmolality in C. londinensis differs from the strong post-rehydration regulation apparent in slowly dehydrated P. crassicutis ~oodring, 1974) and 0. o~natus (Riddle et al., 1976). In P. crassicutis, animals rehydrated on moist leaf litter returned only to their original predesiccation weight. Live weight and dry weight were found to have a significant positive association with water content in male C. Zondinensis. This trend differs from that found by Riddle et al. (1976) for scorpions and that found by Baker (1980) in Ommatoiudusmoreletti. In the latter study both male and female millipedes of greater dry mass had lower percentage water contents. No relationships between live weight and water content was noted in three iulid millipedes examined by Barlow (1957). The Iack of sexual differences in water content in C. lond~~e~s~salso differs from a consistent pattern of greater percentage water contents among female 0. moreletti (Baker, 1980). The influence of size and temperature on hemolymph osmolality in C. ~ondi~e~sis(Tables I and 2) was comparable to that noted by Lindqvist (1970) for the terrestrial isopod Porcellio scaber. The inverse size-osmolality relationship found in C. londinensis females was similar to the trend found in P. scabev.

321

The physiological significance of these sizeosmolahty relationships, or of the positive relations found in other arthropods (Riddle eb al., 1976) remains unclear. Temperature appeared to have a rather consistent influence in increasing water contents, and likely thereby in decreasing hemolymph osmolality in C. ~ondinensis,particul~ly in males. In P. scaber, Lindqvist found that a regression estimate of hemolymph osmolality of medium size (60 mg) animals acclimated at 3°C exceeded that found at 30°C by 28 mM NaCl(52 mosm). The latter value is comparable to the 42 mosm difference found between 5- and 30”C-acclimated male C. londinensis (Table 2). Osmotic responses to dehydration and rehydration in L. forficatus (Figs 3 and 4) contrast with those of millipedes in two ways. First, no large differences in regulation ability were noted between male and female centipedes. Second, centipedes show particularly strong post-rehydration regulation of hemolymph osmolality. Part of this regulation may be att~bu~ble to the control of uptake by drinking. Also significant in the control of osmolality on rehydration, but possibly limiting regulation ability during dehydration, is the functioning of the excretory system in this species. Wenning (1978) found that in starved and in well fed L. for~~atus, primary urine sampled at the ampullae of the malpighian tubules was only slightly hyperosmotic to the hemolymph, but became hypoosmotic in the hindgut, p~tic~arly in starved specimens. The production of a dilute final urine clearly would minimize the loss of hemolymph osmolytes, and contribute to regulation of hemolymph osmolality upon rehydration. Consistent with the limited hemolyrnph osmoregulation observed in L. ~~~~at~ during dehydra~on is Wenning’s finding that this species cannot produce a hyperosmotic final urine. Coxal glands located near the base of the hind legs of L. ~~~cu~~~ may be important in osmoregulation. Rosenburg and Seifert (1977) have shown that these glands in three species of geophilomorph centipedes have typical transport epithelia. They have suggested that these glands have a water uptake and water removal function in dry and moist habitats respectively. Lewis (1981) cites work indicating that lithobiid species from hygric habitats have more numerous and larger coxal pores. Three patterns of osmotic response to dehydration and rehydration are evident in harvestmen. First, there is an indication of comparatively good osmoregulation following moderate dehydration. This regulation at higher water content ranges is consistent with the pattern evident in freshly collected animals apparently exposed to moderate dehydration under natural conditions. Second, comparatively poor regulation is evident at lower water contents. Third, there is evidence that harvestmen are able to increase water content subs~ntially during rehydration without a pronounced dilution of the hemolymph. This regulation is not primarily based on controlling the extent of water uptake, as it may be in centipedes, but rather seems associated with the exchange of water and osmolytes between the hemolymph and with tissues or with gut water compartments. Clearly worthy of investigation is the possible role of the comparatively spacious gut cavity and

WAYNEA. RIDDLE

322

associated caecae (Kaestner, 1968) in water and solute exchange. In addition to the pattern of excellent osmoregulation in A. trz~asc~ata evident in Fig. 8, is an unexplained pattern of substantially higher hemolymph osmolality values among spiders even briefly exposed to desiccation. Work by Ueda (1974) as cited by Edney (1977) on tequila clavata has suggested a role of the hindgut in osmotic regulation in spiders, In that species hemolymph osmolality varied between 340 to 435 mM NaCl equivalents (626800 mosm) at relative humidities from 75 to 10’4. Corresponding rectal fluid osmolality values from 330 to 680 mM NaCl (607-1253mosm) were noted. Although the hemol~ph osmolality values in that species were much higher than found in the present study (approx. 400-600 mosm), the extent of variation (approx. 170mosm) was comparable. That h~molymph and rectal fluid were essentially isoosmotic in well hydrated A. trifasciata is consistent with Ueda’s findings. Additional work on the ultrastructure and transport function of the hindgut or stercoral sac is clearly needed. Also of interest would be work examining possible changes in the relative concentrations of hemol~ph carbohydrates and free amino acids with dehydration and rehydration exposure. High hemolymph carbohydrate concentrations which could contribute substantially to total osmolality have been noted in adult female A. trifaciata (Cohen, 1980). Moreover, proline has been found the predominant free amino acid in spider hemolymph (Punzo, 1982, 1983; Schartau and Leidescher, 1983) and has been firmly implicated in hemolymph osmoregulation in insects (Woodring and Blakeney, 1980). The present study has utilized an essentially descriptive approach in examining osmotic regulation in groups of land arthropods that have received comparatively little experimental attention. Deserving of greater emphasis in the future is work directed at the mechanisms of water and solute exchange occurring between internal water compartments in these groups of arthropods. Such an emphasis would seem particularly appropriate in that it should enable productive comparisons to be made with the better understood excretory and osmoregulatory systems of insects. Acknowledgements-The author greatly appreciates the of ~y~~n~oiu~~ londinensis by Richard Hoffman and of _ Leiobunum vittatum by James

identi~~ation

Cokendolpher. helpful.

Conversations with David Mead were very REFERENCES

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