A study of carbohydrate metabolism in Planorbis exustus

The processes of embryogenesis, namely differentiation and growth, and also the maintenance of the organism, require energy. Needham (1950) claimed that during development carbohydrate, protein and fat metabolism came into operation in that order. This sequence may differ in some species (Løvtrup, 1959a, b). The importance of -SH groups in the morphogenesis of Planorbis exustus was earlier studied using chloroacetophenone (CAP), an -SH inhibitor, which caused arrest of development, delay in cleavage, exogastrulation etc. when cleavagestage embryos were treated (Mulherkar & Sherbet, 1963). Since -SH reactants are also inhibitors of carbohydrate metabolism, we felt it desirable to find out if carbohydrate metabolism preponderates during the cleavage period and thereby decide whether the action of CAP is to inhibit this metabolism or to interfere with the -SH groups taking part in the formation of the mitotic spindle, regarding which Rapkine put forward the theory of reversible denaturation. We have, therefore, undertaken a study of the metabolism of P. exustus using sodium azide and iodoacetamide (IAA). There is reason to believe that the development of this mollusc has different kinds of metabolism dominant in its different phases of development.

The egg masses of P. exustus were detached from the undersurface of Nymphaea leaves and were treated with sodium azide at concentrations of 0·001,0·002 and 0-003 M (Series A, B and C) for 1 hr. or indefinitely. In some experiments the egg masses were returned to normal pond water to remove the block to development. lodoacetamide, in concentrations of 0 ·001,0 · 0005,0·00025 and 0 · 0001 M, was the other inhibitor employed (Series D). Proper controls were maintained in all the experiments. The method of treatment was the same as that followed by Mulherkar & Sherbet (1963). The control and experimental egg masses were allowed to grow in standard glass Petri dishes (50 mm. diameter). Not more than ten embryos were placed in each Petri dish containing 10 ml. of the control or experimental solution. The development of the embryos was carefully recorded until they succumbed to the treatment or hatched. The text-figures were drawn after narcotizing the animals with menthol. The animals were fixed in Bouin’s fluid, serially sectioned at 10 μ and stained by iron-haematoxylin.

In these experiments 1033 embryos were treated with azide and IAA with 613 embryos serving as controls. The results of the experiments with azide (continuous treatment) are given in Table 1.

A few words of explanation of the data given are required.

The mortality of experimental embryos given in column 6 refers only to deaths occurring before any specific abnormal development or malformation was noticed. This does not include the total mortality observed by the end of the experiments. For example, in some experiments involving cleavage-stage and gastrulating embryos, gastrulation stopped and the embryos became hydropic, all ultimately dying without further differentiation, but this has not been considered as 100 per cent, mortality.

Under this head we have included embryos grown beyond 72 hr. which have enlarged and become transparent due to accumulation of fluid. Such accumulation of fluid took place in the region posterior to the cephalic ganglia, in the cephalic region, in the foot or all over the organism.

This abnormality occurred on treating gastrulating embryos with azide or when those already in azide reached the gastrula stage. Normally, in such cases, gastrulation stops halfway, so that the archenteric cavity does not fill the blastocoel and the embryos become enlarged and transparent with a mass of cells in the centre. Text-fig. 1A represents a section of hydropic embryo produced by treatment of 3rd-cleavage embryos with 0·001 M azide. Text-fig. IB is a section of a normal embryo (approximately 48 hr. old) and is intended for comparison with Text-fig. 1A. It should be noted that in the normal embryo the differentiated albumen cells completely fill the interior of the embryo, but that in the hydropic embryo though these cells are differentiated they do not fill up its interior. We would like to mention here that the albumen cells of hydropic embryos differ in structure from those of the normal embryos in that the large albumen vacuoles are present at the pole of the cell which is away from the archenteron in the normal embryo whereas in the hydropic embryo these albumen vacuoles are situated at the archenteron end of the cells.

This included disproportionate shells or those which were very small and remained as a cap on the visceral hump. Embryos which lacked shells altogether have also been included under this head.

A 15 min. treatment of some of the cleavage stages (up to approximately 6-hr.-old embryos) did not produce any effect on the development and the experimental embryos hatched as normally as did the controls. The cleavage stages were therefore given 1 hr. or continuous treatment in view of the observation of Hall & Moog (1947) that the developmental block produced by azide is reversible. It was observed that at whatever stage the treatment commenced the eggs cleaved normally until they reached the gastrula stage. The embryos did not develop further, however, they became hydropic and finally degenerated when allowed to remain in azide in this arrested state of development. It is interesting to note that there was no delay in cleavage and at each step the cleavage divisions in the experimental embryos coincided with those in the controls. This means that the embryos continued their development without inhibition for 18–24 hr. despite the presence of azide in the surrounding medium.

Continuous treatment of 24-hr.-old embryos caused them not to develop at all, to become hydropic and finally to degenerate. Embryos 48-hr. old showed some development, unlike the 24-hr.-old ones. But it was retarded from 50-108 hr. as compared to that of control embryos. Hydropia was of usual occurrence, and whenever it occurred there was no further development. If development did take place, retardation, Vesiculation of embryos and shell abnormality occurred (Text-fig. 2A).

A 1-hr. treatment of this stage did not seem to produce any effect. Continuous treatment caused retardation of development of 120 to 168 hr. compared with the control embryos, and also Vesiculation and shell abnormalities. In some development was first retarded and then ceased.

These showed Vesiculation in the head region, the foot or the visceral hump, and shell abnormalities (Text-fig. 2B & C), retardation or a complete block in development.

A curious observation in the azide experiments involving 60- and 72-hr.-old embryos needs to be mentioned here. Though many developed normally and only showed retardation in development, such embryos like those which showed shell abnormalities or Vesiculation, failed to hatch from their capsules. However, if they were freed from their capsules they moved about like any normal embryo. In one batch the experimental embryos were under observation for 20 days after the corresponding control embryos had hatched out. These embryos were in azide for 77 hr. only. What prevents apparently normal embryos, let alone abnormal ones, from hatching is a question which we are unable to answer.

The results of the experiments in this series were similar to those of Series A. The cleavage-stage embryos developed normally till gastrulation began and then ceased to develop. When gastrulating embryos were treated the process of gastrulation stopped almost immediately. Hydropia developed as usual, but in many cases the embryos degenerated before hydropia became visible. Sixty-hour-old embryos showed delay in development which ultimately came to a standstill. The 72-hr.-old embryos when placed in azide ceased to develop any further.

The results of these experiments differed from those of the two earlier series. It was observed that the experimental embryos showed delay in cleavage ranging . from 10 to 35 min. The development then proceeded apparently normally up to gastrulation and then stopped and the embryos became hydropic. The 24-hr.-old embryos stopped gastrulating and became hydropic. In the 48-hr.-old ones, also, the development was arrested but the embryos degenerated before hydropia became visible. The older stages (60 and 72 hr.-old) underwent a complete block of development.

The IAA experiments were restricted to a few embryos (135 with 87 controls) since they were required to serve a limited purpose. With high concentration of IAA (0·001M) cleavage-stage embryos showed arrest of development, slight enlargement and rounding off of the blastomeres. Older embryos also died as a result of the treatment. The lower concentration (0·0005 M) again had similar effect on cleavage-stage embryos but older ones showed delay in development and, in a low percentage, Vesiculation or developed normally. Even with the lesser concentration of 0·00025 M, cleavage-stage embryos showed the same abnormalities but older ones developed normally. The lowest concentration tried (0·0001M) did not affect the cleavage-stage embryos. Unlike CAP, IAA did not produce exogastrulation, which may be because the concentration was not suitable; for example, a concentration between 0·00025 and 0·0001 M was not tried. A detailed study was not undertaken because we merely wanted to see if any similarity existed between the effect of CAP and IAA. In fact, arrest of or delay in development, rounding off of the blastomeres and their enlargement, are produced by both the substances.

The interference in the development of P. exustus by sodium azide resulted in retardation in development and certain shell abnormalities. Hall & Moog (1948) studied the growth rate in some amphibian embryos in the presence of azide at different concentrations and showed that the rate of growth gradually declined to zero; they believed that this was a result of a progressive decrease in certain factors affected by azide. We presume this is the case in the present experimental embryos, too, in which we have observed everything from slight retardation in development to its complete cessation.

Raven (1958) refers to the work of Mancuso who found that azide treatment of early cleavage embryos of Physa produced exogastrulae and of Attardo who observed similar exogastrulation in Bithynia. However, we did not find any effect on cleavage or exogastrulation in embryos of Planorbis which stopped development during the process of gastrulation.

Brachet (1950) stated that azide is an inhibitor of cytochrome oxidase. Cytochemical study (see Raven, 1958) showed that shell glands of Aplysia, Bithynia or shells of Physa and Limnaea gave positive cytochrome oxidase reactions. In fact, treatment with azide or potassium cyanide causes various shell abnormalities (Raven, 1958). These observations might well explain the irregularities in the shell formation noticed in our experiments.

Accumulation of fluid causing hydropia and Vesiculation was of very common occurrence in the azide treated embryos. The abnormal water distribution caused in CAP-treated P. exustus has earlier been thought to be due to an alteration in the -SH and -SS-balance. Lithium, also, was found to cause a disturbance of the water balance. Raven (1952) suggests that this might be due to a general effect on the protoplasmic colloids or a specific inhibition of the larval kidney. Geilenkirchen & Nijenhuis (1959) believe that ‘impairment of morphogenetic potencies’ of the endoderm inevitably entails hydropia. According to Hess (see Geilenkirchen & Nijenhuis, 1959) if there is no normal intimate contact between the endoderm and the ectoderm, Vesiculation and hydropia occur. This is quite understandable because we find that accumulation of fluid begins only after gastrulation has been blocked. As already stated in later stages also accumulation of fluid took place resulting in Vesiculation of embryos. A large number of such abnormal embryos were produced by azide; a few cases were also found among the IAA-treated embryos. It would thus appear that this abnormal water distribution is an unspecific reaction.

Sodium azide is said to inhibit certain processes of synthesis and assimilations (Clifton, 1946; Spiegelman, 1946). It is found to produce effects similar to those produced by dinitrophenol which is said to effect an uncoupling of the oxidations and harnessing by phosphorylation the energy thus liberated (Loomis & Lipmann, 1948). Hall & Moog (1948), who studied its effect on certain amphibian species, were of the opinion that their findings could be explained on the basis that azide prevents phosphorylations which supply energy for the developmental processes and probably that the succinate oxidizing system is affected.

We are, therefore, inclined to suggest that the pre-gastrulation and postgastrulation phases have different energy producing systems. In the former a metabolism which involves protein or fat and not a carbohydrate probably dominates. Thus the succession of energy sources seems to be different for the species studied. This seems reasonable when we consider the work of Buglia, reported by Raven (1958), who found that the respiratory quotient (R.Q.) during the cleavage of Aplysia was 0·8–0·85. Mayerhof (see Raven, 1958) stated that fat is the principal fuel. However, Baldwin (1935) found an R.Q. of 1·05 and suggested that carbohydrate metabolism is dominant throughout the development of Limnaea stagnalis. Another group of animals which apparently does not conform to the normal sequence of energy succession is the amphibia.

We shall now discuss the possible interference by IAA in the pre-gastrulation stage of development. It would be recalled at the outset that Mulherkar & Sherbet (1963) found that integrity of the -SH groups was of great importance in the morphogenesis of P. exustus. They also found that the phase of development from the 2nd cleavage to the 4th cleavage seemed to be CAP-susceptible and the period between the 3rd cleavage and the 3rd cleavage-flattened stages seemed to be critical from the point of view of production of exogastrulae. The CAP-susceptible phase, in general, coincided with the process of ooplasmic segregation taking place in the cleaving eggs and the critical phase in particular coincided with the establishment of the -SH rich ectoplasm. The rounding off of the blastomeres and their enlargement observed in the IAA-treated embryos could also be explained on the basis that the -SH groups present in the cortical region of the eggs are acted upon by the chemical.

Let us now consider the question of arrest of development caused by IAA. Two explanations are possible. Rapkine & Brachet (1951) have observed that IAA rapidly blocks development of certain amphibian embryos primarily by its action on the spindle. Mazia (1954,1955,1958) has shown that oxidation of the -SH groups into -SS-causes the folding of the fibrous proteins into globular proteins. CAP or IAA might then act in this way by inhibiting the formation of the spindle. An alternative explanation is that these chemicals interfere with th’e energy requirements of cleavage. The energy source (‘ energy reservoir ’) might be a phosphorylated compound. A further connection between carbohydrate metabolism and cleavage has also been indicated. Enzymes involved in both anaerobic and aerobic carbohydrate metabolism contain -SH groups in their active centres. The -SH reactants affect the integrity of these active centres and inhibit the metabolism. When the energy source falls below a certain level cleavage stops. Earlier in this paper it was suggested that during cleavage some metabolism other than that involving carbohydrates might be dominant and probably, on the whole, the carbohydrate metabolism is eclipsed by the dominant metabolic system. It is conceivable that the energy requirements of cleavage might form a small fraction of the total energy requirements of the cleaving egg. If it were so, only high concentrations of azide might be expected to affect cleavage. These expectations, in fact, are fulfilled. The Series C experiments with 0·003M azide have indicated that cleavage-stage embryos are affected to some extent by azide and gastrulation movements are inhibited. Very high concentrations were not tried because azide might then act by sheer toxicity rather than by interfering with carbohydrate metabolism.

The vulnerability of gastrulation to azide has been striking. This clearly indicates that the substrate oxidized to meet the energy requirements of these morphogenetic activities is a carbohydrate. It is known, for example, that ‘a high carbohydrate metabolism is required for the morphogenetic movements of gastrulation’(Brachet, 1960).

In the post-gastrulation phase, azide seems to dissociate growth metabolism from the metabolism of maintenance. The 48- and 60-hr.-old embryos stopped development or showed delay in development, and the 72-hr .-old embryos also behaved in the same manner. In Series B and C experiments, a complete cessation of development was observed in all 48-hr.-old, most of the 60-hr.-old and all the 72-hr.-old embryos. Thus carbohydrates seem to be essential for growth metabolism.

The main conclusions could be summarized as follows : In the development of P. exustus, a metabolism involving other substrates than a carbohydrate is dominant in the pre-gastrulation phase. The energy requirements of cleavage, which conceivably form a small part of the total requirements of the embryo, are met by carbohydrate metabolism. The importance of carbohydrate metabolism for morphogenetic activities of gastrulation is evidenced by the vulnerability of these stages to azide. The carbohydrate metabolism is also important for the processes of differentiation and growth.

1. On a étudié le métabolisme de Planorbis exustus à l’aide de nitrure de sodium et d’iodacétamide (IAA) à différentes concentrations.

2. Les embryons en cours de segmentation traités au nitrure (0,001, 0,002 et 0,003 M) se sont développés normalement jusqu’à ce qu’ils atteignent la gastrulation, stade où le développement s’est arrêté. Les gastrulas ont cessé toute activité morphogénétique. Ceci a été généralement suivi d’hydropisie. Il n’y a pas eu d’exogastrulation. Les embryons de 48, 60 et 72 heures ont montré un retard de développement, une vésiculisation et des anomalies coquillières, ou ont subi un blocage du développement.

3. Le traitement à l’IAA a provequé un arrêt du développement, un arrondissement et une légère dilation des blastomères.

4. Le développement de cette espèce de Mollusques semble être divisible en deux phases, prégastrulation et post-gastrulation (y compris la gastrulation). Dans la phase de prégastrulation domine un métabolisme impliquant d’autres substrats qu’un hydrocarbone. Les besoins en énergie de la segmentation, qui constituent sans doute une petite fraction des besoins totaux d l’embryon, sont couverts par le métabolisme des hydrocarbones. L’importance de ces derniers pour les activités morphogénétiques est mise en évidence par la vulnérabilité de ces stades au nitrure de sodium. Le métabolisme hydrocarboné est également important pour les processus de différenciation et de croissance.

We are deeply indebted to Professor C. H. Waddington, F.R.S., for reading the paper and making suggestions for its improvement. We thank Professor D. R. Newth for helping us to prepare it and Dr Leela Mulherkar for providing facilities of work in the Department of Zoology, University of Poona. One of us (G.V.S.) is grateful to the Government of India for the award of a National Research Fellowship and the other (M. S. L.) wishes to express her gratitude to the University Grants Commission of India for a Junior Research Fellowship.