Hemolymph osmoregulation and urea retention in the woodland snail, Anguispira alternata (say) (endodontidae)

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HEMOLYMPH OSMOREGULATION AND UREA RETENTION IN THE WOODLAND SNAIL, ANGUfSPfRA ALTERNA7’A (SAY) (ENDODONTIDAE) WAYNEA. RIDDLE Department of Biological Sciences, Illinois State University, Normal, IL 61761 U.S.A. (Received 28 October 1980) Abstract--1. Mantle water storage in Anguispira alrernara snails depressed elevations in hemolymph osmolality during dehydration. 2. Osmolality of non-urea solutes was not regulated during dehydration. 3. During rehydration, urea that accumulated during dormancy was rapidly excreted, while that retained helped minimize changes in total hemolymph osmolality. 4. Seasonal variations in water content, osmolality and hemolymph urea concentration were detected in field animals.

batus, has shown that protein is catabolized during dormancy and that whole body urea and uric acid content increases. In that species urea biosynthesis during dormancy was considered adaptive in alleviating ammonia toxicity (Horne, 1973b). The slug Limux flaws has also been found to synthesize urea during periods of activity and dormancy (Horne, 1977). In the South American land snail Strop~oc~e~~us ob~ongus hemolymph urea concentrations were found to be low during activity (De Jorge et al., 1965; Horne, 1977) but rose during dormancy to concentrations as high as 44OmM, greatly exceeding levels found in the tissues of some individuals (Trammel & Campbell, 1972). In contrast, the helicid snails Helix aspersa and Otala lactea synthesize urea, but degrade it by urease to ammonia and carbon dioxide, thereby facilitating calcium carbonate deposition in the shell (Speeg & Campbell, 1968). Ammonia volatilization and urease activity have been identified recently in a variety of terrestrial snails and similarly considered significant in shell formation (Loest, 1979a,b). The present study examined changes in hemolymph osmolality in the woodland snail, Anguispira alternata during dehydration and rehydration. In order to assess the extent of hemolymph osmoregulation on this species, observed changes in hemolymph osmolality resulting from reductions in water content were compared with expected changes predicted in the absence of osmoregulation. The effects on hemolymph osmolality of urea accumulation during dormancy and of urea excretion following rehydration were also examined. Seasonal changes in water content, hemolymph osmolality and urea concentration experienced by animals in their habitat were also examined.

INTRODUCTION ft is evident

from literature recently reviewed by Machin (1975) that land snails and slugs experience large variations in body water content under natural conditions, Dehydration and rehydration result in changes in hemolymph volume, tissue hydration and in the concentration of inorganic solutes (Burton, 1964, 1966). Machin’s review documents evidence showing an association between reduced water content during dormancy and increased hemolymph osmolality. Evidence of this association supports, but does not ex~rimentally confirm, an emerging generalization than land snails and slugs simply tolerate changes in hemolymph osmolality with changes in body water content. In a study which directly addressed this problem, Little (1968) found appreciable regulation of hemolymph osmolality during dehydration in air in the amphibious ampullariid snail, Pomucea lineata. In that paper it was speculated that during dehydration hemolymph osmolytes may have been removed from solution and combined to form larger molecules. Regulation of total hemolymph protein concentration during dehydration was clearly evident. It is generally assumed that mollusc cells are in osmotic equiIibrium with surrounding extracellular fluid (Schoffeniels & Gilles, 1970) and that in the open circulatory system of snails, no physical separation exists between interstitial fluid and hemolymph (Machin, 1975). While these conditions indicate that appreciable osmotic gradients between the hemolymph and cells are unlikely, they do not preclude the possibility that hemolymph osmolytes could be sequestered in regions such as the gut, or otherwise removed from solution, thereby minimizing increases in hemolymph osmolality during dehydration. The potential problem of limiting the elevation of hemolymph osmolality during dormancy in snails and slugs is accentuated in those species that synthesize and retain urea during dormancy. Work by Horne (1971, 1973a) on the semi-desert snail, Bulimulus deal<.B.P. 69 3r -_I

MATERIALSAND METHODS Animuls Snails were collected from a single population in a mesic deciduous woodland 1.5km north of Normal, Illinois. Animals used in dehydration and rehydration experiments were kept in a screen-top container and rinsed with cool tap water once daily for 3 days, allowing them to void gut

493

494

WAYNEA. RIDDLE

contents and accumulated urine This hydration “pretreatment” served to reduce variations between snails in gut dry matter, water content, hemolymph urea concentration and hemolymph osmolality. In all but one experiment, most of the water stored in the mantle cavity (pallial water) was removed by blotting after forcing animals to retract into shells. Osmometry

nnd determination

of water content

A single 10 ~1 sample of hemolymph was removed from each snail from a puncture made on the abumbilical surface of the shell in a region about l-1/4 whorls from the aperture. Hemolymph osmolality was determined using a Wescor Vapor Pressure Osmometer. Following hemolymph sampling, snails were killed by freezing. Soft tissues were removed and dried with the shell to constant weight at 60°C. Water mass was determined by subtracting shellfree dry weight from shell-free live weight. Water mass divided by shell-free dry weight provided an estimate of whole animal water content, and was expressed as mg H,O/mg shell-free dry weight. Urea

Hemolymph urea concentrations were determined from a separate 1~1 sample of hemolymph taken prior to osmometry. This sample was applied to a filter paper disk, dried at room temperature and stored in a freezer. Hemolymph urea concentration was determined by the Berthelot reaction modified from that described by Chaney & Marbach (1962). Individual disks were added to 6ml test tubes which contained 100 ~1 of glass distilled water and 100 ~1 of urease buffer reagent. Samples were agitated and incubated at 37°C for 10min in a water bath. On returning tubes to room temperature, 200 ~1 of phenol nitroprusside solution, 200~1 of alkaline hypochlorite solution and l.Oml of distilled water were immediately added. Tubes were briefly agitated and allowed to develop color for 30min at room temperature. Absorbance was determined at 570 nm with a Spectronic 20 spectrophotometer using a pair of matched quartz cuvettes. A standard urea concentration: absorbance curve prepared with 1~1 samples of known urea concentration was used to determine hemolymph urea concentrations. Since aqueous solutions of urea less than 0.33 M exert osmolalities in osm kg- 1 equivalent to their molality, urea concentrations were expressed as either mM or mOsm. Urease and reagent solutions were secured from Sigma Chemical Co. Estimates were made of the rate of removal of urea by excretion in snails rehydrated following prolonged dormancy. Snails dormant at room conditions for 115 days were placed in individual plastic petri dishes. The shell and aperture region of each snail was rinsed and cleaned with cotton swabs to remove adhered urea over a 30-min period as snails aroused. Animals were thoroughly blotted, placed in a clean dish containing approximately 2 ml of distilled water and allowed to further rehydrate and urinate for I hr. Shells and apertures were cleaned again with cotton swabs and each snail transferred to a clean dish. Petri dishes occupied by snails and swabs used in cleaning were dried at 60°C and stored in a freezer. Plates containing dried mucous and urine were washed in 10ml of distilled water. This water and cotton swabs were added to a test tube and briefly agitated using a vortex mixer. From this 10 ml sample, 100 ~1 was removed and added to 100 ~1 of urease buffer solution. Urea was determined colorimetritally as above. Rates of urea excretion were expressed as pm01 g - ’ shell-free dry weight hr- ’ during each collection period. Known amounts of urea were. applied to petri dishes, dried and similarly diluted in constructing a standard curve. This method was considered adequate in determining if urea was excreted and if so, when in time most excretion occurred.

Dehydration

und rehydrution

experiments

The first experiment examining the influence of dehydration on hemolymph osmolality utilized animals collected in July 1978. Following hydration pretreatment and removal of pallial water, 74 snails were desiccated in circulating air passed over Drierite desiccant at room temperature for 15-18 days. The role of mantle (pallial) water in influencing osmotic response during dehydration was examined in a group of 59 snails allowed to retain mantle water after initial hydration pretreatment. Following desiccation at room humidity for 7 days, hemolymph was sampled in dormant animals (N = 30) and also for those active for 30min to 1 hr following arousal by brief exposure to 5°C (N = 29). With the development of the urea detection technique, three additional experiments were conducted. In the first, 66 snails were exposed to dehydration under room conditions for I7 days. From among these animals (N = 20). total osmolality and urea concentrations were determined. Remaining snails were placed in individual petri dishes with 2-3 ml of tap water and allowed to rehydrate. Hemolymph osmolality and urea concentration were determined in snails removed over a 23 hr period. A second experiment examined hemolymph osmolality and urea concentrations during prolonged dormancy, permitting an assessment to be made of the extent to which osmolality of non-urea hemolymph solutes was regulated. A single group of animals was removed and examined after 7, 16, 22, 31, 46, 55 and 80 days of dormancy at room conditions, These parameters were also measured in a group of snails which had remained dormant at 5 +, 1‘C for a period of 15 months. The third experiment esttmated the rate of excretion of urea following rehydration of 16 snails dormant at room conditions for I15 days. Hemolymph osmolality and urea concentrations for a group of 10 snails similarly desiccated but not rehydrated was also examined. Srasonul collections offield animuls Hemolymph osmolality. urea concentration and water content were determined following collection in groups of 2c-25 animals during the year. To insure animals were available in winter, a group of snails collected 23 November 1979 was divided into smaller groups and placed in clean containers. These containers remained outdoors under temperature conditions considered similar to those of natural hibernacula. Animals in winter dormancy retained distinctive epiphragms from 23 November through a sample made 20 March 1980. Animals sampled on 9 April had terminated winter dormancy and were active in containers. This group was exposed to unnatural post-arousal conditions in being denied access to food and having only droplets of condensed water with which to rehydrate. Hemolymph samples were taken from this group, but a remaining group was placed in a depression with leaf litter and allowed to feed and hydrate normally for 5 days. Subsequent spring and summer collections were made directly from the habitat. Statisticul

analysis

Mean values of hemolymph osmolality. urea concentration, water content and the concentration of non-urea solutes were expressed *950/;, confidence intervals. Statistical comparisons were made using f-tests. Other data was analyzed using linear regression. Analysis of variance was performed on linear regression equations and resulting F-values used to determine if slopes differed significantly from zero. RESULTS AND DISCUSSION Relationships between water content and total osmolality following dehydration are depicted in

Osmoregulation

in a land snail

495

Fig. 1. Relationships between whole body water content and hemolymph osmolahty in A. afternura. Uppermost regression line depicts relationship for snails dehydrated 15-18 days in circulating dry air (Y = 424.748 -34.7369 r = -0.6190, F = 44.73, P < 0.04X). Lower solid and dashed regression lines respectively indicate relationships for dormant (a) and aroused (Cl)snails allowed to retain mantle water during dehydration at room conditions for 7 days. Regression equations are: Y = 184.404 - 7.607X. r = -0.3182. F = 3.27, P > 0.05 and Y = 199,637 - 11.828X, r = -0.4383, F = 6.90, P <: 0.05) respectively.

Fig. 1. Considerable variation in final water content is evident m both treatment groups due to individual differences in water loss rates and initial water content. More important are the lower slopes of regression lines describing osmotic response in snails allowed to retain palhat water prior to dehydration. The influence of pallial water storage rather than the osmotic effects of urea accumulated over an 8-11 day longer period of dormancy most likely accounts for the diminished osmotic response. Blinn (1964) found that in Mesodon thyroideus and AIIgona profindu palha1 water accounted for body weight variations of up to approximately 20%. He further indicated that variations in pallial water in A. alrernata were essentially similar to those in the species above, but presented no data. Particularly relevant to the present study was his observation that in M. thyroideus withdrawal and epiphragm formation occurred only after palliai water had disappeared or was substantially diminished. In 37 A. alternata aliowed to retain mantle water following hydration pretreatment, water contents varied from 5.87 to 7.85 (mean = 6.86 + 0.18 mg H20/mg D.W.), and differed significantly (P < 0.05) from those of animals in which pallial water was removed (6.57 + 0.03 mg HzO/mg D.W., N = 16). Whole animal live weights of a group of 50 dormant snails allowed to retain mantle water were 7.04 f 1.25% greater than in the same animals following removal of water. These observations indicated that A. alternata retains variable but appreciable quantities of pallial water on the assumption of dormancy. Variations in water content associated with differences in the volume of palhal water stored had no significant effect on hemolymph osmolality. This was confirmed in the group of 37 snails above, for which a regression equation relating osmolality (Y) to water content (X) (Y = 123.745 + 0.975X) lacked a significant regression coefficient (F = O.tMO; F = 0.057). Results of the present paper confirms the “extrasomatic” nature of pallial water suggested by Blinn (1964), and indicate that utilization of palhal water during

desiccation exposure diminishes elevations in hemolymph osmolality. In order to assess the extent to which the osmolahty of osmolytes other than urea is controlled, the relationship depicted by the solid line in Fig. 2 was compared to that expected in the absence of any osmotic regulation. In a group of 16 snails experiencing hydration pretreatment and removal of palhal water, mean water content was 6.57 f 0.03 mg H,O/mg D.W.; total osmolality, 139.88 +_6.19 mOsm and urea concentration 10.75 f 2.83 mM. By subtracting the contribution of urea from total osmolality a value of 129.13 mOsm was determined, represented at the extreme right of the lower dashed line in Fig. 2. In the absence of any regulation following a loss of 500/, of original water (reduction in water content to 3.29 mg H20/mg D.W.), an approximate doubling of osmolality of non-urea osmolytes would be predicted. It is clear that the observed osmotic response corresponds closely to the predicted response in the absence of regulation. These results indicate that A. a~~ernuta is incapabfe of regulating hemolymph osmolality during dehydration. Concentrations of total hemolymph solutes other than urea and presumably protein simply change in concentration in proportion to changes in water content. A comparison of the positions and slopes of the uppermost lines in Figs 1 and 2 show that a greater totaf osmolality is predicted at virtually all water contents in Fig. 2. These higher osmolalities are most likely due to the effects of longer duration of dormancy in most of the animals sampled and the effect of the higher (room) humidity to which they were exposed. Higher humidity during dormancy has been associated with higher rates of metabolism and presumabty protein catabolism in some species of snails (Riddle, 1975, 1977; Herreid, 1977) and with higher levels of accumulated urea (Horne, 1973a). In a group of snails kept at 5°C for 15 months, mean water content was extremely low’ (3.12 f 0.22 mg HzO/mg D.W.). Corresponding total

496

WAYNEA. RIDDLE

L. 3.0

Water Gtent

(mg &mg

6.0 D.W.)

7.0

Fig. 2. Relationship between water content and hemolymph osmolality in A. ulternatu following prolonged dormancy. Upper dashed regression line depicts relationship for total osmolality (individual points not plotted); Y = 592.920 - 62.211X, r = -0.6225, F = 37.33, P < 0.001. Lower solid line indicates corresponding relationship for total non-urea solutes (Y = 358.950 - 33.776X. r = -0.7150, F = 61.73, P c 0.001). Lower dashed line predicts osmotic response of normally hydrated animals undergoing dehydration to SO”/,of original water content.

hemolymph osmolality and urea concentrations were 328.10 + 9.89 mOsm and 53.35 f 13.12 mM respectively. Total osmolality in these specimens was considerably below that predicted at their mean water content by regression in Fig. 2 (399 mOsm) due to the comparatively low concentrations of urea. However, observed osmolality attributable to non-urea solutes (274 mOsm) was comparable with a predicted value of 254mOsm. This last observation indicates that while temperature and humidity conditions may affect the rate of urea accumulation, they appear to have little influence on the relationship between water content and the concentration of non-urea osmolytes. Figure 3 indicates a reduction in the concentration of non-urea osmolytes over several hours with water

2501

F O 200 s

I

uptake during rehydration. No comparable trend is discernible in the reduction of urea concentration over the same period, although urea concentrations are generally below the mean level of dehydrated ,animals. The relative stability of the concentration of non-urea solutes from hr 6 to hr 23 when snails were active and excreting urea suggests that the loss of osmolytes in mucous production (Burton, 1965) and in urine formation had little influence on hemolymph osmolality. Results presented in Table 1 indicate that urea is rapidly excreted in severely dehydrated snails following rehydration. This finding is consistent with the rapid clearance of urea found in other terrestrial snails (Martin, et al., 1965; De Jorge, et al., 1969). Of Table 1. Mean rates of urea excretion in olternuta during rehydration

.

A.

A

Total Osmolality (0) l

Collection interval (hr)

Activity condition*

Mean rate ("moles gD.W.-1 h -')

.

Fig. 3. Changes in total hemolymph osmolality. urea osmolality and osmolality of non-urea solutes over time following rehydration. Arrows identify mean osmolality values *9S% confidence intervals for 20 snails desiccated for 17 days but not rehydrated. Curves are eye-fitted lines.

4 o-1

A

99.7 2 4&s+

1-2

A

167.9 + 69.0

2-5

A

90.7 + 27.5

5-7

A

80.1 + 30.9

7-10

A

53.3 + 13.6

10-20

w

21.4 +

4.4

20-31

0

20.9 t

9.0

31-40

D

10.4 +

4.9

40-48

D

14.0 +

4.4

* A = Snails fully or partly extended during collection period; W = some snails withdrawn at beginning of collection period: D = all snails withdrawn at beginning of collection period, subsequently aroused. t +95x confidence intervals; N = 16.

Osmoregulation in a land snail

497

Month

Fig. 4. Seasonal changes in hemolymph osmolality (0) water content (0) and hemolymph urea concentration (A) in freshly-collected snails. Arrows identify dates of missing urea samples. Vertical limits are 957; confidence intervals: N = 16-25 per mean.

the total amount of urea excreted by A. altemata over 85% was removed during the first 24 hr. This rate of removal is comparable to the reduction in whole body urea content of dormant Bulimulus dealbatus following rehydration on moist paper towels (Horne, 1971). In that species, approximately 920/, of the total reduction in whole body urea content (from about 7.5 to 1.0 mg/g animal) occurred during the first day of rehydration. Urea remained detectable at about 0.5 mg/g animal in B. dealbatus after 8 days of rehydration. In urea collection experiments, repeated application of water to the animals placed them under unusually severe conditions of potential osmotic dilution of body fluids. Mean water content, hemolymph osmolality and urea concentration of a group of 10 animals similarly dormant for 115 days but not rehydrated was 4.16 + 0.71 mg H,O/mg D.W., 506.00 Ic_ 34.28 mOsmo1 and 174.99 f 19.22 mM, respectively. Rehydration of severely dehydrated animals resulted in excessive hydration as confirmed by the unusually high final water contents (8.69 + 0.60 mg H20/mg D.W.) and the turgid appearance of foot and mantle tissue. Despite differences in the extent of previous dehydration and rehydration, total osmolality in groups of rehydrated animals did not differ significantly. For animals exposed to the standard hydration pretreatment total hemolymph osmolality (139.88 mOsm) was similar to that in 14 moderately dehydrated animals (131.36 + sampled after 19-23 hr rehydration 7.71 mOsm Fig. 3) and after rehydration of severely dehydrated animals (132.81 f 7.85 mOsm). When the contribution of urea to total osmolality is considered in these groups (10.8, 19.0 and 27.5 mOsm respectively) it becomes apparent that total hemolymph osmolality is effectively stabilized by the osmotic contribution of urea. Substantial seasonal changes in hemolymph osmolality, urea concentration and water content of field animals are evident in Fig. 4. Under the dry con48 hr, approximately

ditions of late August through late September, hemolymph osmolality increased as water content fell. Although a moist period preceded the November sampling, water contents remained low, as expected in snails entering winter dormancy. Hemolymph osmolality increased between the late September and November sampling to a greater extent than predicted, based on changes in water content. This change in osmolality was not accompanied by a change in hemolymph urea concentration, however, which remained similar from September through December. The simplest explanation for the stability of urea concentrations prior to the assumption of winter dormancy is that snails did periodically arouse and excrete urea. The comparatively low urea concentration found in January is unusual since arousal and urination during winter would not be expected. Removal of nitrogenous waste by ammonia volatilization during dormancy is known to occur in certain helicid snails (Speeg & Campbell, 1968) and is common in active A. altemata (Loest, 1979a). In dormant A. altemata however, significant ureolytic activity would not appear likely, considering the low urease activity reported in dormant snails which retain urea (Horne, 1973b). Hemolymph osmolality rose in overwintering animals examined from 23 November to 20 March, although water contents did not differ significantly. Increases in osmolality during overwintering however were only partly attributable to urea. This interpretation can be supported by applying the regression equation relating water content to the osmolality of non-urea solutes in Fig. 2 to data on overwintering animals. Since snails placed in artificial hibernacula were derived from a single collection made on 23 November (non-urea osmolality = 173 mOsm, water content = 4.88 mg H*O/mg D.W.), subsequent osmolality values can validly be compared with animals sampled on that date. Snails sampled 9 December, 14 January, 21 February, 8 March and 20 March had observed osmolality levels which differed from

WAYNE A. RIDDLE

498

expected levels by -6, 4, 27, 14 and 10 mOsm respectively. These differences between observed and predicted osmolalities suggest the appearance and disappearance of additional osmolytes during the overwintering period. Resumption of activity in early April was associated with increased body water content and lower nemolymph osmolality. Results from animals sampled on 9 April indicate the influence of the hydration of non-feeding animals with condensation water in containers. The comparatively high urea levels found at this sample are consistent with laboratory results associating urea retention with rehydration in the absence of feeding. Resumption of normal feeding activity was clearly associated with a reduction in hemolymph urea in the mid-April sample. In animals taken from their habitat under moist conditions in late April and June, no significant differences in hemolymph osmolality or hemolymph urea levels were found, despite significant differences (P < 0.001) in water content.

oblongus musculus (Becquaert)

Camp.

Biochem.

Physiol.

14, 3542.

DE JORGEF. B., PETERSEN J. A. & DITADI A. S. F. (1969) Variation in nitrogenous compounds in the urine of Strophocheilus (Pulmonata, Mollusca) with different diets. Experentia 25, 614615. HERREIDC. (1977) Metabolism of land snails (Or& lacteu) during dormancy, arousal and activity. Camp. Biochem. Physiol. XiA, 21 l-215. HORNEF. R. (1971) Accumulation of urea by a pulmonate snail during estivation. Camp. Biochem. Physiol. 38A. 565-570. HORNEF. R. (1973a) The utilization of foodstuffs and urea production by a land snail during estivation. Biol. Bull. mar. biol. Lab., Woods Hole 144, 321-330. HORNE F. R. (1973b) Urea metabolism in an estivating terrestrial snail Bulimulus dealbatus. Am. J. Physiol. 224, 781-787. HORNEF. R. (1977) Regulation of urea biosynthesis in the slug, Limax~auus Linne. Camp. Biochem. Physiol. 56B. 63-69. LITTLEC. (1968) Aestivation and ionic regulation in two species of Pomacea (Gastropoda, Prosobranchia). J. e.xp. Biol. 48, 569-585.

Acknowledgements-The author thanks Robert Preston for use of his laboratory, Becky Pavledes for her expert technical assistance and Marie Knight for typing the manuscript. Comments on the manuscript made by Francis Horne and John Machin were greatly appreciated.

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