Biochemical Correlates of Estivation Tolerance in the Mountainsnail Oreohelix (Pulmonata: Oreohelicidae)

13 Pages·1993·5.6 MB·English

by  B B Rees

Reference: Biol Bull 184: 2.10-242. (April, 1993)
Biochemical Correlates of Estivation Tolerance in the
Mountainsnail Oreohelix (Pulmonata: Oreohelicidae)
BERNARD B. REES1 AND STEVEN C. HAND
Department ofEnvironmental, Population ami Organismic Biology, University ofColorado.
Boulder. Colorado 80309-0334
Abstract. Biochemical changesoccurringover 7 months Introduction
of estivation were studied in two species of land snail,
Oreohelix strigosa (Gould) and O. subrudis (Reeve), to Thesuccessofgastropod mollusks in terrestrial habitats
determine whetherdifferential mortality during estivation has been due to various structural, physiological, and be-
is related to different energetic strategies. Laboratory- havioral specializations (Riddle, 1983). One specialization
maintained snails, which were fed adlibitum prior to es- that is well developed among the pulmonate land snails
tivation, were compared with snails collected from the is the capacity to enter the dormant state of estivation
field and induced to estivate without augmenting their during periods ofhot and dry environmental conditions.
energy reserves. In all groups, polysaccharide was catab- Byenteringestivation, snailsareabletoendurepotentially
olized early in estivation, and protein was the primary desiccating climatic conditions until the return of more
metabolicsubstrateafterpolysaccharide reserveswerede- favorable conditions. Some species are capable of esti-
pleted. Lipid was catabolized at a low rate throughout vating for remarkable periods oftime, ranging up to sev-
estivation. Rates of catabolism were largely statistically eral years in duration (Stearns. 1877; Machin, 1967).
equivalent between species. Urea and purine bases ac- There are limits to the duration ofestivation that can
cumulated during estivation as a result ofprotein catab- be tolerated, though, and mortality eventually increases
olism, with the former being quantitatively more impor- as estivation is prolonged. Because there is no intake of
tant. In both laboratory-maintained and field-collected foodstuffs during estivation, the period ofestivation that
snails, the rate of urea accumulation was greater in O. can be survived may be limited by the exhaustion ofen-
subrudis. resulting in higher tissue urea contents in this dogenousenergy reserves(Pomeroy, 1969; Schmidt-Niel-
species at the end ofthe 7-month experiment. The tissue sen el til.. 1971 ). Metabolic rate reduction, which would
concentrations of urea at 7 months ranged from about serve to prolong the energy stores ofthe animal, occurs
150 to 300 mA/and were positively correlated (r = 0.99, during estivation, and desert-dwelling species display
P = 0.006) with mortality in these snails. Methylamine lower rates than species from more mesic environments
compounds, a class ofcompounds that can offset disrup- (Schmidt-Nielsen el a/.. 1971; Herreid, 1977; Rees and
tive effects ofelevated urea, were measured in one group Hand. 1990). These observations have been taken assup-
ofO. strigosa at 7 months ofestivation and found to be portingthe ideathat energyreservesarelimiting. Butsince
low relative to urea levels. We suggest, therefore, that in the rates of metabolism and evaporative water loss are
the absence of elevated levels of counteracting com- highly correlated in land snails (Barnhart, 1986), the re-
pounds, ureamayreachtoxiclevelsand may beone factor duction of metabolic rate may reflect an adaptation to
limitingtheduration ofestivation that issurvived by these conserve water rather than energy. A comparison ofsur-
land snails. vivorship in snails with differing levels ofenergy reserves
priortoestivation would moreclearlyaddressthequestion
ofenergy limitation.
Received 2June 1992;accepted 9 December 1992. The duration ofestivation may also be limited by the
1 Presentaddress: HopkinsMarineStation, DepartmentofBiological accumulation ofnoxious end-products ofprotein catab-
Sciences, Stanford University. PacificGrove. CA 93950. olism. Depending upon the species and activity pattern.
230
BIOCHEMICAL CHANGES IN ESTIVATION 231
land snails can dispose ofnitrogen derived from protein Mitchell Creekdrainage werecollected alongtheeastbank
m
catabolism in the form of uric acid and other purines, of the creek, approximately 100 downstream of the
ureaorgaseousammonia (Bishop el <//., 1983). In species Mitchell Creek Fish Hatchery (3942', 10722'W; 1850
that produce urea, this compound can reach very high m). near Glenwood Springs, Colorado. Along the East
levels in the tissues during estivation: levels of260 jimol Rifle Creek, snails were collected from areas located ap-
g ' wet mass (ca. 300 mM) have been measured in the proximately 1 km upstream and 3 km downstream ofthe
tissues of Biilinnihts dealbatits (Home, 1971), and 440 Rifle Falls Fish Hatchery (3942', 10742'W; 2100 m).
mAI in the blood of Strophocheilus oblongns (Tramell Theupstream sitewasabout25 m westofthecreekamong
and Campbell, 1972). At these levels, urea can have sig- rock slide rubble in Rifle Gorge, and thedownstream site
nificant deleterious effects on the function ofseveral bio- was adjacent to the creek at the Rifle Falls campground.
logical processes (Yancey el til.. 1982: Yancey, 1985; The three collection locales will be referred to as the
Yancey and Berg, 1990). In other organisms displaying Mitchell Creek, Rifle Gorge, and Rifle Falls sites. The
elevatedtissuecontentsofurea, methylaminecompounds, Mitchell Creek site has previously been referred to as the
which can offset the disruptive effects of urea, are com- Glenwood Springs collection site (Rees, 1988).
monly accumulated. It is not known whether methyl- The climatic conditions prevailing during the summer
amines accumulate during estivation in snails with high months in the Mitchell Creek and East RifleCreek drain-
urea. If not, then urea could reach toxic levels and be a ages are shown in Table I. Further information on the
factor limiting the duration ofestivation. conditionsat the two Rifle sites wasobtained with a hand-
In the present study, we have investigated the extent held temperature-humidity sensoron several daysduring
to which the exhaustion of energy reserves and the ac- thesummersof 1990and 1991. Measurementswere made
cumulation of nitrogenous compounds correlate with 2-5 cm above the ground between 06:00 and 08:00, and
mortalitydifferencesobservedduringlaboratoryestivation again between 13:00 and 16:00 h. On average, the early
in two species ofthe mountainsnail Orcohclix. We mea- morning humidity was 4% higher, and the mid-day hu-
sured the biochemical composition ofO slrigosa and O. midity was 5% higher, at the Rifle Falls than at the Rifle
xubrudis over a 7-month period oflaboratory estivation. Gorgesite. Taken together,thesedataillustratethat mois-
From thesedatawe haveestimated rates ofcatabolism of ture availability at the three collection sites decreases in
protein, polysaccharide, and lipid. We compared snails the order Mitchell Creek > Rifle Falls > Rifle Gorge.
that had been fedadlibitumpriortoestivation withsnails
that had been collected from the field and induced to Animals andspecies identification
estivatewithout feedingtoascertain theeffectsofelevated
energy stores. We also measured the accumulation ofni- Snails were collected in June and August of 1987 and
trogenous end-products ofprotein catabolism. Estivating in November of 1989. They were either sacrificed im-
snails were found to accumulate large quantities ofurea, mediately for determination ofthe biochemical compo-
and we measured the tissue content ofmethylamines to sition ofanimalsin thefield, orbroughtintothelaboratory
addressthe possible counteraction ofurea effectsby these and used for estivation studies (see below). The average
compounds. shell-free tissue mass of snails prior to estivation in the
Finally, tolerance todesiccation under laboratory con- laboratory was 0.453 0.014 g (SEM, n = 63) for O.
ditions has been correlated with the distribution ofa va- strigosa and 0.394 0.013 g for O. siibntdis (n = 41).
rietyofland snail speciesin nature, with the more tolerant Both speciesarehermaphroditicand bearliveyoung. Only
species occurring in drier habitats (Machin, 1967; Cam- individuals without developing young in their oviducts
eron, 1970; Arad el a/., 1989). The genus Orcohclix is were used in this study.
widely distributed in western North America, ranging After the snails had been sacrificed for biochemical
from mesic riparian areasto semi-arid habitats(Bequaert analyses(seebelow), thespecieswasdeterminedbystarch
and Miller, 1973; Rees, 1988). In the present study, we gel electrophoresis of proteins (Rees, 1988). During the
have characterized the climatic conditions prevailing at present study, additional, faster-migratingalleleswerere-
three collection sites in western Colorado, and we have solved in O. strigosa at the phosphoglucomutase and
evaluated the distribution ofO. strigosa and O. xubrudix phosphoglucose isomerase loci. This finding does not
at these sites in light oftheir differing capacities for pro- compromise the utility ofthis technique in speciesdeter-
longed laboratory estivation. mination, however, as the occurrence ofthe slow alleles
attheseloci remainsdiagnostic ofO. siibntdis. Individuals
Materials and Methods
that were not electrophoretically genotyped (snails col-
Collection sites lected in June 1987 and those which died during the es-
Oreohelixspp. werecollected inwesternColoradoalong tivation series) were separated into species by their shell
Mitchell and East Rifle Creeks. The snails from the morphology (Rees, 1988).
B. B. REES AND S. C. HAND
Table I
Climalu-iniul/lu>n<i iliinni; the \iininier<>/ IWtlin llicMitchellCreek andKust Ri/lcCreekdrainages
Site
BIOCHEMICAL CHANGES IN ESTIVATION 233
70C for purine analysis. The remainder of the per- were omitted. Polysaccharide content was expressed as
chloric acid extract was centrifuged at 10,000 X g for 15 0.9 x glucose mass.
min. The pellets were washed once with 0.7-0.8 ml of 1 For urea analysis, samples were thawed and clarified
Nperchloricacid andcentrifuged asabove. Theperchloric by centrifugation at 10,000 X g for 10 min. Urea was
acidinsoluble material wassavedforDNA measurement. measured colorimetrically asammonia aftertreatment of
Perchloric acid supernatants for each individual were the samples with urease (Sigma Diagnostic Kit No. 640).
pooled, neutralized with 5 Af K2CO3, and centrifuged at Blankswithout ureaseweresubtracted from each sample.
10,000 x g for 10 min to remove perchlorate salts. Two Purine baseswere analyzed with high performance liq-
hundred to400n\ ofthe neutralized extractwascombined uidchromatographyessentiallyasdescribed bySimmonds
with two volumes of95% ethanol and stored at -70C and Harkness (1981). A LDC/Milton Roy HPLC system
for polysaccharide assays, and the remainder was saved was employed in conjunction with a Waters /uBondapak
at 70C for urea measurements. C-18 column (30 cm X 3.9 mm i.d.). The lithium car-
bonate solutions were thawed, diluted, neutralized, and
Biochemical analyses filtered through Gelman SuporO.45 ^m membrane filters.
Twenty n\ were injected onto the column, and purines
Protein was measured by the method of Lowry el al. wereeluted isocratically with abufferof4mA/potassium
(1951),as modified by Peterson(1977), with bovineserum phosphate (pH 3.6) containing 1% (v/v) methanol. Ab-
albumin asthestandard. Forcalculations ofnitrogen bal- sorbancewasmonitored at 265 nm, and uricacid,guanine
ance, it was necessary to determine the mass ofnitrogen and xanthinewerequantified by integration ofpeak area.
in snail protein. The protein in a perchloric acid homog- Total lipidwasdetermined afterextraction ofthetissues
enate was recovered by centrifugation after the nucleic in chloroform:methanol (Folch el al.. 1957; Ways and
acids had been digested by heating(see below). Lipid was Hanahan, 1964). For each snail, lyophilized tissues were
removed by washing the PCA-insoluble material with homogenized in 4 ml chloroform:methanol (2:1) with a
methanol. The amount ofnitrogen in the PCA-insoluble Virtis micro-ultrashear apparatus for 1 min and filtered
fraction was determined by a micro-Kjeldahl procedure through a fritted disc funnel. The residue was rehomog-
that includes direct nesslerization ofammonia following enized in 4mlchlorofornrmethanol andfiltered. Theres-
digestion of the proteins (Koch and McMeekin, 1924). idue was finally washed with another 2 ml ofchloroform:
The Nessler reagent was obtained from Sigma Chemical methanol and the filtrates combined. The filtered chlo-
Company. The amount ofnitrogen in protein determined roform:methanol homogenate was mixed with 0.25 vol-
in this manner was not different in the two species and ume 0.88%- (w/v) KC1 in water, and after separation, the
was found to account for 16.8 0.9% (S.D., n = 4) of aqueousphasewasaspirated. Theremainingorganicphase
the protein mass measured by the Lowry assay. was mixed with 0.25 volume methanol:water (1:1), and
DNA
was determined by the diphenylamine assay of theaqueous phase wasaspirated afterseparation. The or-
Burton (1956) with modifications suggested byGiles and ganic phase was then decanted into a pre-weighed alu-
Myers (1965). Briefly, perchloric acid insoluble material minum planchet and evaporated to dryness under a
wassuspended in 1.0 ml 1.5 A'perchloricacid and heated stream ofnitrogen. Thedried lipid was held over Drierite
70C
at for 20 min. Following centrifugation at 10,000 a further 24 h and weighed to the nearest 0.1 mg.
X gfor 20 min, an aliquot (50-100 ^1) ofthesupernatant In one group of estivating snails, methylamine com-
was brought to 2.5 ml with 1.5 N perchloric acid and pounds were measured by reineckate precipitation pro-
combined with 1.5 ml 4% (w/v) diphenylamine made in tocol modified from Kermack et al. (1955). Lyophilized
glacial acetic acid and 0.1 ml 0.16 mg ml ' acetaldehyde tissues from a whole snail were homogenized in 30 vol-
made in water. The color was allowed to develop for 20 umes of40%> ethanol and centrifuged at 20,000 X g for
h in the dark at room temperature. To correct for non- 15 min. The pellet was washed with another 30 volumes
specificcolordevelopment, anabsorbancedifference (A600 of 40% ethanol, and the combined supernatants were
- A700) was determined for each sample. Calf thymus boiled for 10 min to precipitate proteins. The ethanolic
DNA was the standard. extract was centrifuged at 10.000 X g for 20 min, lyoph-
Polysaccharide (glycogen plus galatogen), which pre- ilized, and redissolved in 1.0 ml 0.1 TV HC1. Saturated
cipitated in the ethanolic extract, was collected by cen- ammonium reineckate, prepared in waterand titrated to
trifugation at 10,000 X g for 20 min, washed once with pH 1 with 5.0 N HC1, was added to the each sample in
1.0 ml 95% ethanol and centrifuged again. The pellets the ratio 3:1 (reineckate:sample). Reineckate salts were
4C
were air-dried and redissolved in 1.0 ml waterby heating allowed to precipitate at overnight and were collected
at 70C. Polysaccharide was measured by the anthrone by filtration on polycarbonate membrane filters(Nucleo-
method described by Jermyn (1975), except that the ad- pore, 0.2 ^m). After washing the precipitate three times
ditions of hydrochloric and formic acid to the samples with 3 ml diethyl ether, the precipitate and membrane
234 B B. REES AND S. C. HAND
Table II
i^ilinn atlul'tiivlnrynuiinuiincilOrcohelix
Compound
BIOCHEMICAL CHANGES IN ESTIVATION 235
bases totaled to 64-79 ^mol g~' dry tissue, similar to the Snails of either species collected late in the summer
tissuecontents ofother non-estivating snails(Jezewska el demonstrated much more variable urea contents than
al.. 1963: Home, 1971). On a molar basis, uric acid ac- snails in the laboratory-maintained or early summer
counted for about 70% ofthe total purine, with guanine groups (Fig. ID). Among the laboratory-maintained
and xanthine accounting for approximately 20 and 10% snails, only 17% had urea contents greater than 1 ^mol
ofthe total purine, respectively, in both O. strigosa and g ' dry mass, and among the snails collected early in the
O. subrudis. Hypoxanthine was not found in the tissues summer, this percentage was 22%. In these groups, the
ofthesesnails. Taken together, thesecompoundsaccount highest urea content measured was 11.7 /urno! g ' dry
for more than 80% ofthe dry mass ofthese snails. The mass. Among the snails collected later in the summer,
unaccounted fraction is presumed to be other low mo- urea was higher than 1 /^mol g ' dry mass in 33% ofthe
lecularweightorganiccompounds(e.g.. aminoacids)and snails, and the highest value was 93.0 /xmol g"1 dry mass.
inorganic ash. Since urea accumulates duringestivation (see below), the
occurrence ofelevated urea in snails collected late in the
Biochemicalcomposition offield-collectedOreohelix summer suggests that many of these animals had been
estivating in the field.
Compared with the values obtained for laboratory-
maintained snails, both O. strigosa and O. subrudis dis- Mortality during estivation
played lower polysaccharide levels in the field-collected Both species ofOreohelixexperienced mortality during
groups (Fig. 1A). Protein constituted a correspondingly the later months ofestivation. In the group ofsnails that
larger portion ofthe dry mass in both species (Fig. IB), had been maintained in thelaboratory priortoestivation.
and lipid wassomewhat higher in O. strigosacollected in 1 ofthe remaining 13 O. strigosa had died at 7 months,
the late summer (Fig. 1C). These differences in biochem- whereas9 of30 O. subrudishaddied. Forsnailsthatwere
ical composition reflect the effects ofad libitum feeding brought in from the field, the mortality at 7 months in
in the laboratory-maintained group and suggest that snails both species was higher: 10 of 24 O. strigosa had died,
feed less regularly or on food ofdiffering qualities in the whereas 28 of34 O. subrudis had died. Among the field-
field. Ofthesnailscollected in thelatesummer, O. strigosa collected snails, the proportion ofdead O. subrudis at 7
displayed significantly higherlevelsofpolysaccharide than months was significantly greater than the proportion in
O. subrudis. Differences in polysaccharide content may O. strigosa (G-test, P < 0.05). These results demonstrate
influence the capacity ofthese snails for long-term esti- that O. strigosa tolerates extended periods ofestivation
vation (see Discussion). in the laboratory better than O. subrudis.
120
A- (27) (|221'1
ti O
g E200 E 80
o JL
51 -a 60
' en 40
25
S 20
500 E
? 15
0.250
o*
Figure 1. Biochemicalcompositionoflaboratory-maintainedand field-collectedO. strigosa(openbars)
and O .subrudis (solid bars). A. Polysacchande content. B. Protein content. C. Lipid content. D. Urea
content. Error bars indicate one standard error ofthe mean. Asterisks indicate that the content ofthis
constituentissignificantlydifferentfromthatmeasuredinlaboratory-maintainedsnailsofthesamespecies,
and thecrossesindicate that species meansaresignificantlydifferent forthatsamplinginterval.
236 B. B. REES AND S. C. HAND
Analysis oj changes during estivation with large polysaccharide stores, and in these snails, ca-
tabolism ofthissubstratecontinued forthe first 4 months
We were interested in whether the two species have ofestivation (Fig. 2A). During the first month ofestiva-
different rates of substrate depletion or end-product ac- tion, the rate ofpolysacchande depletion was significantly
cumulation during c>; vation. Because variation in the fasterin O. suhntdis(Table III). Between 1 and4months,
size ofindividuals among the sampling intervals and be- carbohydrate catabolism continued at moderate rates that
tween species would tend to obscure these rates, we have were similar in thetwo species. After4 months, the poly-
normalized the tissue mass, watercontent,andthecontent sacchande content ofthe snails was much reduced and
ofbiochemical constituentsto an average snail size based itsrateofutilization wascorrespondingly low. Inthe field-
upon shell diameter (see Materials and Methods). Note collected snails, the polysaccharide stores were smaller,
that, since dry mass, water content, and biochemical and consequentlytheyweredepleted earlier(Fig. 2B). Al-
composition can be determined only once for any indi- though the initial rates ofutilization were similar in the
vidual, the ratesofchangedescribed belowreflectaverage two species, carbohydrate lasted longer in O. strigosa.
ratesoflossoraccumulation amonggroupsofindividuals which had begun estivation with larger stores. As in the
rather than rates ofchange within individual snails. Fur- estivation series begun with laboratory-maintained snails,
thermore, shell diameterswerenot measured on the field- rates of polysaccharide utilization were much reduced
collected snails sacrificed prior to estivation (day 0). and during the later phases of estivation and statistically
consequently the data for this group begin at 10 days of equivalent between species.
estivation. Upondepletion ofthepolysaccharidestores, netprotein
catabolism occurred (Fig. 3). In the laboratory-maintained
Loss oftissue mass and water during estivation snails, the onset ofnet protein depletion occurred atabout
Freshtissue mass, drytissue mass, and waterdecreased 2 months ofestivation (Fig. 3A). Before this time, no net
significantlyinboth speciesoWreo/ie/ixduringestivation.
When tissue mass and watercontent data were corrected
forsize differences among individuals, rates ofloss in the Zb
two species were not significantly different. The loss of
tissue was characterized by parallel decreases in both dry
tissue mass and tissue water. These losses were biphasic,
occurring more quickly at the onset ofestivation as the
snailsenteredestivation, andthen reachingasteadyslower
rate after the initial drop. By 7 months ofestivation, the
tissue mass and water content ofsnails were reduced by
approximately 35% in all groups.
The loss oftissue water from estivating Oreolielixwas
not reflected in a decrease in the percent tissue water be-
cause the dry mass decreased proportionately. The per-
centage oftissuewaterremained between 78 and 81% tor
both species in both experimental series. In fact, among
the laboratory-maintained snails, there was a slight but
statistically significant increase in thepercenttissuewater
overthe 7 months ofestivation despite the overall loss of
water. Thusa constant percentage tissue watercannot be
interpreted as indicating no loss of water, as has been
assumed previously for other species ofestivating snails
(Schmidt-Nielsen et at.. 1971).
Catabolism ofenergy reserves during estivation
Polysaccharide, protein, and lipid were all catabolized
during estivation, but the substrates that were utilized
changed as estivation proceeded (Figs. 2-4, Table III).
Polysaccharide was the primary metabolic fuel for the
initial months ofestivation (Fig. 2). Snails that had been
maintained in the laboratory began the estivation period
BIOCHEMICAL CHANGES IN ESTIVATION 237
Table III
<>/polysaccharide, protein, andlipidcatabolism amiureaamipurineaccumulation inOreohehx v/>/> duringestivation
Compound Experiment Interval O suhnulis
Polysaccharide
238 B B. REES AND S. C. HAND
60 theircapacity for long-term estivation. Differences in the
patternsofbiochemical changes mayaccount, in part, for
50 (26,21)(12.12) the observed difference in mortality. Below, we evaluate
the relationships between mortality and both the exhaus-
.0..4) tion ofenergy storesand theaccumulation ofnitrogenous
We
end-products ofprotein catabolism. also discuss the
ct: distributionsoftheseOreohelixspeciesinthe field in light
Q. o.20 oftheir different survivorship during desiccation stress.
10
Mortality anil exhaustion ofenergystores
Ifthe duration ofestivation is limited by the depletion
60 ofenergy storagecompoundsduringestivation, then snails
B. with larger stores prior to estivation would be predicted
50 (111.1(11)4.9) (13.10) to survive estivation proportionately longer. Wewere able
to elevate the level ofpolysaccharide, the primary meta-
(4.9) (12.6) bolic substrate during early estivation, by feeding snails
O o ad libitum in the laboratory prior to estivation. Subse-
rr quently, when these snails were allowed to estivate, poly-
saccharide storeslasted longer, and mortality in both spe-
cies was lower than when snails collected from the field
10
01234567
DURATION OF ESTIVATION (mo)
(17.12)
Figure 3. Protein content during estivation in O sirigosa (O) and
O xnhnidix (). All valueshavebeen adjustedtoasnail ofaveragesize
based upon shell diameter. A. Laboratory-maintained snails. B. Field- o 6
collectedsnails.Samplesizesaregiven inparentheseswiththevaluefor CO
O.strigosaappearingfirst. Barsindicateonestandarderrorofthemean. en
Q
Over this period, the amount ofammonia produced by CL
8 snails ofeither species was below the limit ofdetection
(0.02
Levels ofurea-counteracting solutes
Methylamine compoundswere measured in onegroup
offield-collected O. .strigosa after 7 months ofestivation
and found to be 2.68 0.27 ^mol snair' (n = 5). HPLC 6
analyses ofselected extracts ofboth species have shown en
en
thatbetaineisthepredominant methylaminecompound,
and that polyhydric alcohols, another class ofprotective
compounds, do not significantly accumulate in snail tis- 9 3
Q.
sues during estivation (data not shown).
0123456
Discussion
In the present study, we undertook an analysis ofthe DURATION OF ESTIVATION (mo)
biochemical changesthat occurin Oreohelixstrigosaand
O. .siibniclisduring a period oflaboratory estivation. The Figure4. Lipid contentduringestivation in O. slrigosa(O)and O
xiihritdix (). All values have been adjusted to a snail ofaverage size
temporal nature ofsubstrate utilization and nitrogenous based upon shell diameter. A. Laboratory-maintained snails. B. Field-
end-productaccumulation weredescribed forthefirsttime collectedsnails. Samplesizesaregiven inparentheseswiththevaluefor
in congeneric species ofland snails that are dissimilar in O.strigosaappearingfirst. Barsindicateonestandarderrorofthemean.
BIOCHEMICAL CHANGES IN ESTIVATION 239
estivated without prior laboratory feeding. In addition, 70
among the field-collected snails. O. strigosa began with
A.
higher polysaccharide levels than O. suhrudis, and the
former displayed only halfthe mortality by 7 months of "650
estivation. Withdata from fourgroupsofsnails(2 species ^40
X 2experimental series),wetestedthecorrelation between o
pre-estivation polysaccharide stores and percent mortality I30
at 7 months ofestivation. Since snails with higher poly-
saccharide storeswere predicted to surviveestivation bet- < 20 (10.13)
Ld
ter (i.e., show lower mortality), the test was one-tailed.
9510
The negative correlation between pre-estivation polysac-
charide stores and mortality was statistically significant
(r = -0.91, P = 0.045). The observation that polysac- /u
charide storeswereexhausted several months priortothe
onset ofmortality, however, suggests that mortality is not
due to the depletion of this substrate in sensu stricto.
Rather, the correlation between polysaccharide stores and
mortality likely reflects other biochemical changes that
are initiated upon the depletion ofthe polysaccharide re-
serves (see below).
Mortalityand the accumulation ofnitrogenous
end-products
Upon the exhaustion of polysaccharide, protein was
catabolized, and both O. strigosa and O. suhrudis were
found toaccumulate ureaasthe majorproductofprotein
metabolism. Based upon rates ofprotein catabolism and
end-product accumulation during the estivation interval
of net protein depletion (2-7 months for laboratory-
maintainedsnailsand 10days-7 monthsforfield-collected
snails), urea accumulation in the tissues accounted for
approximately 50% ofthe nitrogen derived from protein
catabolism, whereastheaccumulation ofpurinesonlyac-
counted forabout 10%oftheprotein nitrogen. Ammonia
production was below measurable levels, corresponding
to less than 1% ofthe calculated nitrogen liberated from
protein catabolism. A portion ofthe unaccounted fraction
ofnitrogen was probably lost during sample preparation
(blotting ofhemolymph can account for the loss ofup to
25% ofthe urea nitrogen), and nitrogen may have accu-
mulated in compounds not measured in this study (e.g.,
amino acids; c.f., Wieser and Schuster, 1975). Further
studies of nitrogenous compounds in hemolymph ofes-
tivating snails may elucidate the nature of the missing
nitrogen fraction.
In both experimental series, the rate oftissue urea ac-
cumulation was found to be faster in O. suhrudisthan in
O. strigosa. resulting in higher ureacontents in the former
species. Because ureacaneasilycrossmostcell membranes
(Forster and Goldstein, 1976), the urea measured in ex-
tracts ofwhole snails is likely to be uniformly distributed
throughout the tissues ofthe snails. This assumption was
supported by measuringurea in hemolymph, foot muscle