Metabolic rate depression in the ampullariid snail Pomacea urceus (Müller) during aestivation and anaerobiosis

Camp. Biochem. Physiol. Vol. 102A, No. 4, pp. 675-678, 1992 Printed in Great Britain

0

0300-9629/92 S5.00 + 0.00 1992 Pergamon Press Ltd

METABOLIC RATE DEPRESSION IN THE AMPULLARIID SNAIL P~~~C~~ URCEUS (MILLER) DURING AESTIVATION AND ANAEROBIOSIS M. A.

THOMAS

and J. B. R. ACARD

Department of Zoology, The University of the West Indies, St Augustine, Trinidad, West indies (Received 13 January 1992) AbstracC---1.Using oxygen consumption and glycogen utilization rates as indices of metabolic rate, facultative metabolic rate depression was shown to accompany aestivation and anaerobiosis in Pomacea urceus. 2. Anaerobiosis resulted in lactate accumulation; juveniles and adults both died at the same body tissue lactate concentration. 3. Juveniles depressed metabolic rate by greater orders of magnitude and survived longer in anoxia, per unit mass, than adults. 4. Larger adults survived longer periods in anoxia than smaller ones.

INTRODUCTlON

neotropical, amphibious snail, Po(Miiller), aestivates in periods of drought and survives by burying itself in the ground and depressing rates of oxygen consumption and utilization of glycogen reserves (Burky et al., 1972; Cedeiio-Leitn, 1984). Adult females oviposit at the beginning of the dry season, before the onset of aestivation. Eggs hatch soon after laying and juveniles aestivate beneath the females until the start of the rainy season (Lum-Kong and Kenny, 1989). Burky (1974) pointed out that breeding at the beginning of the dry season assures the hatched juveniles an opportunity to exploit fully the favorable conditions for growth which exist in the rainy season. Also, if breeding was postponed until the beginning of the rainy season, many adults would not reproduce since there is a high adult mortality near the end of the dry season. Significantly, broods survive beneath dead females. Aestivating through the dry season, however, makes above normal metabolic demands on both adults and juveniles. Regulated glycogen utilization is vital to the survival of aestivating animals. Such animals do not feed but rely on glycogen reserves for their energy needs. In addition, after rain when the soil is wet, the exchange of gases with the air is reduced and ground oxygen is rapidly depleted. In such instances, aestivating individuals may experience anaerobic conditions. The stress accompanying these metabolic demands seem to be greater for juveniles than for adults. In juveniles, unlike adults, aestivation is not preceded by a period of active feeding and accumulation of reserves as it begins immediately upon hatching from the eggs. Also, only 19% of an ad&t female’s nonrespired assimilation of the rainy season preceding breeding goes into reproduction (Burky, 1974) and this is shared between up to 93 eggs (Lum-Kong and Kenny, 1989). Juveniles, thus, appear to have more restricted energy stores for use while aestivating than The dioecious,

macea

urceus

675

adults. Further, while adults, by virtue of their size, can access air from above the ground with their siphons and escape their burrows rapidly when soil conditions are anaerobic, juveniles cannot. In fact, for juveniles to escape the burrows with any speed, the adult females above need to remove themselves first. Periods spent in anoxic conditions are, therefore, frequently longer for juveniles than for adults. Metabolic rate depression serves to maximize the survival time of individuals in unfavorable conditions and usually accompanies successful aestivation and anaerobiosis (Storey and Storey, 1990). As the stresses of these metabolic states are apparently greater for juveniles than for adults, juveniles should possess an enhanced ability to depress metabolic rate. This investigation tests this hypothesis using oxygen consumption and glycogen utilization as indices of metabolic rate. MATERIALS

AND METHODS

In the laboratory, active snails were kept in fresh water and fed ad lib. on leaves of the dasheen plant (C~~ocff~~). A batch of randomly selected snails was induced to aestivate by being placed in tanks containing mud which was allowed to dry. The temperature in the laboratory was 30 & 3°C. Oxygen consumption The oxygen consumptions of aestivating and active snails were measured at 30 + 3°C using Scholander-type constant volume respirometers (Scholander, IPSO). Following this, body wet mass, exclusive of shell and operculum, and body dry mass after desiccation at 60°C for 48 hr, were determined for each snail. Ability to sun&e anoxia Adult and juvenile snails, which bad been aestivating for 1 month were used. The snails were weighed and divided into two groups. One group was placed in 100% nitrogen while the other was placed in air. Absence of oxygen was verified with alkaline pyrogallic acid. Survival time was recorded for each snail. The instant of death was taken as the time of release of a great amotmt of mucus for ad&s, and the time at which the heart, visibIe through, the shell,

616

M. A. THOMAS and J. B. R. AGARD Table I. Oxygen consumption

for active and aestivating

snails at 30 + 3°C

Juveniles Active ml 0, ind/hr

Aestivatine

0.38 * 0.05 (13) 0.30 rl_0.03 (13) 1.43io.15 (13)

ml 0,/g wet weight/hr ml 0,/g dry weight/hr

Adults Active

0.06 f 0.01 (9) 0.04 f 0.01 (9) 0.14 * 0.03 (9)

4.27 * 0.19 (10) 0.05 + 0.01 (10) 0.30 * 0.01 (IO)

Aestivatine 1.94+0.12 (11) 0.02 * 0.01 (11) 0.09 * 0.01 (11)

Mean k SE (sample size).

was no longer beating for juveniles.

Dead adult individuals

were sexed.

glucose was determined enzymatically with glucose and peroxidase (Bergmeyer and Bemt, 1963).

Body lactic acid

RESULTS

Aestivating snails were weighed and placed in jars containing either 100% nitrogen or air. When a snail in an anaerobic jar was observed to be dead, it’s tissues were removed and frozen. The same was done for snails in air after 28 days. The tissues were then homogenized and lactate contents of the homogenates were estimated quantitatively by the calorimetric method of Barker (1957). Lactate concentrations were expressed in pmoles lactate/g dry weight using lactate values obtained and a correlation (P < 0.05) found between whole mass and dry mass of tissues for aestivating snails. Glycogen

utilization

For juveniles, aestivating individuals from a single cohort were divided into groups of about 10. The glycogen contents of tissues of group one and group two snails were determined immediately upon the introduction of group three into 100% nitrogen and group four into air. After periods of 4 and 24 days, respectively, the glycogen contents of tissues of group three and group four snails were also determined.

For adults, individuals aestivating for 3 months were used. Adults were divided into two groups, one of which was placed in 100% nitrogen while the other was placed in air. Pieces of foot and hepatopancreas were dissected out from the snails of both groups immediately before, and 2 days after, introduction into the test atmospheres. Samples were taken under sterile conditions, through holes made in the shell of each snail. To determine glycogen contents of the given tissues, the samples were weighed and the glycogen they contained was isolated using the method of Hassid and Abraham (1957). Following acid hydrolysis of the glycogen (I .2 M HCI).

'-1

1

0

-d

2

1

Oxygen

consumption

Table I compares respiratory rates during activity and aestivation. The results show that, irrespective of units of measurement, during aestivation active oxygen consumption rates decreased (P < 0.05) by about 610 times in juveniles but only by 2-3 times in adults. Resistance of anoxia

All individuals of the group kept in air were still alive after 2 months. The times survived by juveniles in an anoxic atmosphere ranged from 2.1 to 8.4 days with the mean time survived per individual being 5.3 f 0.5 days. Adults survived from 1.5 to 10.1 days in 100% nitrogen with the mean time survived per individual being 5.6 k 0.6 days. There was a linear relationship (r = 0.464; onesided P < 0.05) between whole mass and time survived in 100% nitrogen for pooled adults (Fig. 1). This linear relationship is defined by the equation: r, = 0.0232 Mb + 1.3887 where T, is the survival time, in days, of an individual in anoxia, and M, is the whole mass, in grams, of the individual. No significant (P -C0.05) correlations were found between T, and M,, when adult males and females were considered independently of each other. There was no significant difference (P < 0.05) between T, values for adults and juveniles. However, if juvenile survival in anoxia was also defined by the equation found for adults, the expected juvenile survival time (I .41 + 0.01 days) is significantly less (P < 0.05) than the observed survival time. Body lactate content

Table 2 shows lactate concentrations in the body tissues of snails aestivating in air and killed by anoxia. The results suggest that lactate did not acTable 2. Lactate (qmles/gdry weight) contents of aestivating snails alive in air and dead after exposure to anoxia Individual

100

150

1. Duration

250

200

WHOLE

Fig.

oxidase

MASS

300

Adults

@)

of adult survival in anoxia. represent males.

Juveniles

Solid circles

Alive in air

Dead in 100% nitrogen

n.d. (12) n.d. (9)

62.37 f 5.86 (12) 48.95 f 5.61 (11)

Mean f SE (sample size). n.d.: Not detected; detec-

tion limit l.l9~moles/g.

Metabolic rate depression in the ampullariid snail Table 3. Glycogen (mg glycogen/g wet weight) contents of aestivating juveniles in aerobic and anaerobic atmospheres Atmosphere Time in atmosphere (days) Initial glycogen content Final glycogen

content

Air

100% Nitrogen

24 17.82 k 0.51 (15) 13.50 + 0.62 (15)

4 15.42 f 0.15 (15) 15.32 f 0.17 (15)

Mean k SE (sample size).

cumulate in detectable amounts in snails aestivating under normoxic conditions. Accumulation did occur, however, in the bodies of snails exposed to 100% nitrogen. Further, the mean lethal lactate concentration for juveniles was not significantly different (P < 0.05) from that for adults. This implied that lactate was equally toxic to both juvenile and adult snails. The mean lethal concentration of lactate (in pmoles/g dry weight) was found to be 55.95 f 4.22 when juvenile and adult results were pooled. utilization rates

Glycogen

Glycogen contents of aestivating juveniles and the times spent in aerobic and anaerobic atmospheres are shown in Table 3. The mean glycogen content, after 24 days in air, was found to be significantly less (P < 0.05) than the mean initial glycogen content. The aerobic rate of glycogen utilization suggested by these values is 0.18 mg glycogen/g wet weight/day. In anoxic conditions, the mean initial glycogen content was not significantly different (P < 0.05) from the mean glycogen content after 4 days. These results indicate that, in juveniles, anaerobic glycogen utilization is both very low and lower than aerobic glycogen utilization. For adults, glycogen utilization rates found for individuals kept in air and nitrogen are shown in Table 4. For both foot and hepatopancreas, the mean aerobic rate of glycogen utilization was not significantly different (P < 0.05) from the mean anaerobic rate. Hence, while glycogen utilization rate in juveniles decreased in the transition from aerobiosis to anaerobiosis, it remained about the same in adults. DISCUSSION

Pomacea urceus snails are limited in their ability to elude environmental extremes and so have adapted metabolically to endure environmental stresses. Thus, drought induces aestivation and low oxygen conditions initiate anaerobiosis. Both aestivation and anaerobiosis are accompanied by reversible entry into a hypometabolic state where energy expenditures are less than the corresponding amounts in active snails. Thus, the rate of oxygen consumption in aestivating individuals is significantly less than that in active ones Table 4. Glycogen utilization rates (fig glycogen/g wet/weight day) found for foot and hepatopancreas aerobic and anaerobic atmospheres Tissue used Aerobic Anaerobic

Foot

rate

0.78 + 0.26 (13) I .34 + 0.34 (13)

rate

Mean f SE (sample

size).

tissue used/g of adults in

Hepatopancreas 16.56 f 5.24 (13) 7.99 + 2.98 (13)

617

(Table 1). Also, a reverse Pasteur effect (Hochachka, 1987) is seen, as glycogen utilization rate decreases or remains the same when juveniles and adults, respectively, experience anoxic conditions. Juveniles have a greater ability to depress metabolic rate than adults. In the transition from activity to aestivation, juveniles reduce their mass specific rate of oxygen consumption by about 610 times, while adults reduce theirs by only 2-3 times. Further, in anoxia, juveniles reduce glycogen consumption rates to values significantly less than those for aerobic aestivation. In adults, anoxic rates of glycogen consumption are not significantly different from normoxie aestivating rates. The main benefit of facultative metabolic rate depression is to maximize the survival time of individuals when conditions are unfavorable (Storey and Storey, 1990). Juveniles have more restricted energy stores for use during aestivation and are subject to longer periods in anoxia than adults. The ability of juveniles to depress metabolic rates by greater orders of magnitude than adults, is thus consistent with theoretical expectations as the stresses experienced in, and the duration of, unfavorable conditions are greater for juvenile snails. The enhanced ability of juveniles to depress metabolic rate enables them to slow down the utilization of their restricted glycogen reserves while aestivating. Additionally, this enhanced ability permits juveniles to have a survival time in anoxia 34 times greater than would be expected if juveniles and adults showed the same relationship between survival time and body mass. Anaerobiosis leads to the accumulation of lactate, which is toxic as an end product of glycogen fermentation (Hochachka and Somero, 1984). Death in juveniles and adults occurs at about the same lactate level, the mean value of which was found to be 55.95 k 4.22 pmoles/g dry weight when juvenile and adult results were pooled. The enhanced ability of juveniles to depress metabolic rate means that rates of lactate accumulation in anoxia are, likewise, reduced by greater degrees in juveniles than in adults. In anaerobic conditions, juveniles thus take longer than adults to accumulate lactate to the lethal level. The more extended survival time in 100% nitrogen by the larger of the adults (Fig. 1) need not be explained in terms of metabolic rate depression. Generally, in the mollusca, metabolic rate decreases as size increases (Ghiretti, 1966). This appears also to be the case for Pomacea urceus (data not shown here). Consequently, larger snails have lower metabolic rates and take longer for lactate to accumulate to the lethal level than smaller snails. Adult females are, in general, larger than adult males (Lum-Kong, 1986) and generally have lower metabolic rates. This is especially advantageous since, with the onset of rains, many burrowed females have their shell apertures blocked by juveniles. This blockage prevents the females from extending their bodies and escaping the anaerobic soil rapidly. No such blockage is found in males. The molecular mechanisms involved in metabolic rate depression involve specific controls on key regulatory enzymes to reorganize metabolism (Brooks and Storey, 1990). Additionally, mechanisms of metabolic rate depression prominent in regulation of

678

M. A. THOMAS and J. B. R. AGARD

glycolysis during anaerobic metabolism also serve in glycolytic rate control in aerobic aestivation. While this study did not investigate the actual mechanisms involved in depression of metabolic rate in Pomacea urceus, those listed by Storey (1988) and Storey and Storey (1990) are universal and are assumed to be present. The mechanisms which lead to metabolic rate depression have been shown to reduce metabolic rates by orders of magnitude in the same animal. In Otala Zactea (Mtiller), the changes that result in metabolic rate depression in mantle tissue of an individual, are more pronounced in anoxia than in aestivation (Whitman and Storey, 1990). This study shows that metabolic rate is depressed in Pomacea urceus by different orders of magnitude as well. However, the degree by which metabolic rate is depressed is not different for an individual, of a given state of maturity, as it undergoes different transitions. Instead, metabolic rate is depressed by greater degrees, in a given transition, when an individual is a juvenile than when it is an adult. SUMMARY

In Pomacea urceus (Miiller), aestivation and anaerobiosis are both accompanied by facultative metabolic rate depression which maximizes survival times in unfavorable environmental conditions. Juveniles have more restricted glycogen stores for use while aestivating and experience longer periods in anoxia than adults. To cope with these greater stresses, juveniles depress metabolic rate by greater orders of magnitude than adults. This enables juveniles to survive greater periods of aestivation on their restricted reserves and, in anoxia, juveniles take longer periods than expected to accumulate lactate to the lethal level. Larger adults have lower metabolic rates and take longer periods to accumulate lactate to lethal levels than smaller ones. Acknowledgemenu-We wish to thank Mr N. Thomas for snail collection, Prof. E. J. Duncan for sterile facilities, Dr J. B. Davidson for enzymes and MS R. Singh and MS S. Mohammed for their support during the perigd of research. We also wish to thank Dr I. Omah-Maharaj and Prof. J. S. Kenny for their encouragement and critical review of the manuscript.

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