A physiological study of the development of the egg of Fasciola hepatica L., the common liver fluke

Comp. Biochem. Physiol.,

1967, I,roL 21, pp. 307 to 320. Pergamon Press Ltd. Printed in Great Britain

A PHYSIOLOGICAL STUDY OF THE DEVELOPMENT OF THE EGG OF F A S C I O L A H E P A T I C A L., THE COMMON LIVER FLUKE* R. A. W I L S O N t Imperial College Field Station, Sunninghill, Ascot, Berkshire, England (Received

21 N o v e m b e r 1966)

Abstract--1. Forty per cent of the egg contents of Fasciola are utilized as food during development. 2. In the developing egg the QO~ increases from less than 1 to 2"5. 3. The respiratory quotient (RQ) increases from 0"7 at day 1 to 0"94 at day 3, and to 0-62 at day 9. 4. The results are discussed in terms of changes of chemical composition of the egg during development. INTRODUCTION IN COMMON with other trematodes the liver fluke has an ectolecithal egg, and the food reserves necessary for development are contained in discrete vitelline cells, about thirty in number, which surround the ovum. T h e developmental physiology of this type of egg has attracted little attention. Ranzoli (1956) has described the development of the ovum and vitelline cells using histochemical techniques, and concluded that carbohydrate was the main food reserve. Horstmann (1962) studied oxygen consumption and glycogen utilization of the developing egg, and Bogolomova (1957) described the histochemistry of the miracidium. T h e investigations reported in this paper fall into four sections 1. T h e chemical constitution of the vitelline cells determined by histochemical means. 2. T h e growth of the embryo and concomitant changes in the dry weight of eggs. 3. Respiration of developing eggs and miracidia. 4. Changes in the chemical constituents of the egg during development. * Part of a thesis approved by the University of London for the award of the Ph.D. degree. t Present address: Department of Biology, University of York, Heslington, York, England. 307

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Size variations of individual Fasciola eggs, as indicated by absorption measurements, may be as high as + 40 per cent. Large samples of eggs were therefore used in all experiments. A more serious source of variations was indicated by Horstmann (1962), in that some eggs always failed to develop and this renders comparison between experiments difficult. In order to minimize this variation, the percentage development of each sample of eggs was determined and experimental results were corrected to 100 per cent development for comparative purposes. T h e effect of these and other variations make it unlikely that an overall accuracy of more than +_5 per cent was attained in any one experiment. I n some cases, the variation was probably as high as + 10 per cent and interesting metabolic changes might therefore have gone undetected. Eggs used in experiments were incubated at 25°C in 1-cm depth of distilled water, in conical flasks. At this temperature development to maturity takes 11 days. Age of embryo was therefore a useful criterion of development.

1. Histochemical techniques In order to obtain preparations of vitelline cells suitable for histochemical studies, undeveloped eggs were fixed in Baker's formaldehyde fixative for several days. A small n u m b e r of eggs was transferred to an albumen-smeared slide and excess fluid was removed. T h e y were then squashed gently under a coverslip and the vitelline cells were freed. Cells were made to adhere to the slide by running a drop of formalin fixative under the coverslip and leaving the slide for 5 min. Nucleic acids were detected using the methyl green-pyronin Y technique and ribonuclease extraction (Kurnick, 1955). T h e distribution of protein was investigated using the mercuric chloride-bromophenol blue test (Mazia et al., 1953). Carbohydrates were located principally using the periodic acid-Schiff (PAS) reaction (McManus, 1946), also Best's carmine method (Best, 1906) for glycogen, and alcian blue and toluidine blue techniques (Pearse, 1960) for acid mucopolysaccharides. T h e distribution of lipids was studied using Sudan Black B as a general stain, also the Nile Blue method (Cain, 1947) for neutral and acidic lipid and the Nile Blue method (Menschik, 1953) for phospholipids. The results of staining for lipids were assessed using temporary preparations.

2. Growth and weight determinations A simple technique was used to estimate growth of embryos. The lengths and breadths of a random sample of embryos were measured at daily intervals during development. The embryo has for most of its development an ovoid shape most nearly described as a prolate spheroid, and volumes were therefore calculated by applying the formula V = 4/3 zraM, where a and b are the major and minor semi-axes respectively. Dry and fresh weight changes of undeveloped eggs were measured using a Cahn microbalance. Weighing boats consisted of 0-5-cm circles of 400-mesh phosphor-bronze gauze. Using a Pasteur pipette, the suspended sample of eggs was transferred to a weighed grid resting on absorbent paper. T h e water ran through the grid and eggs were strained out. Excess water was removed by placing the grid on clean dry blotting paper. The grid plus eggs was then rapidly weighed (fresh weight), dried overnight at 105°C, cooled in a desiccator and reweighed to find the dry weight. At daily intervals, samples of eggs incubated at 25°C were removed to the refrigerator at 4°C to arrest development. Washing and weighing of eggs was then carried out under uniform conditions at the end of the experiment and daily changes in weight during development were determined.

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DEVELOPMENT OF EGG OF F A S C I O L A H E P A T 1 C A

T h e dry weight of egg shells was determined by collecting samples of hatched eggs, washing and treating as above (the absence of the operculum was ignored). T h e dry weight of miracidia was more difficult to determine since they easily passed through the mesh of grids during manipulation. This could be overcome by killing miracidia immediately prior to transfer onto the grid, but a technique for concentrating miracidia without damaging them is required.

3. Respiration A capillary respirometer designed by Dr. F. Call of Imperial College, and based on the method of Tobias & Gerard (1941), was used to study respiration. T h e technique and apparatus are described here in full as their use has not hitherto been published. T h e apparatus is diagrammatically illustrated in Fig. 1.

J

Respira vessel

Fine bore

capillary

Rotator copil I01 holder

,Coarse capil lary

I

5cm

J

FIc. 1. Capillary respiration apparatus. T h e biological material was placed in 100-/zl flasks with ground-glass necks. A section of coarse capillary (1.0 m m bore x 1 cm long), fused to a length of precision-bore capillary (0"1 m m bore x 15 cm long) was inserted into the neck of each flask. Assembled flasks and capillaries were inserted into a rotatable frame which was then enclosed in a large glass chamber fitted with a tap. T h e glass chamber was mounted in a constant-temperature water-bath (Techne Tempunit, with an accuracy of + 0"05°C).

Experimental procedure (i) Respiration vessels and capillaries were cleaned by washing in ether, acetone, water, acid cleaning mixture (overnight), water, acetone and ether.

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(ii) Washed eggs were transferred to respiration vessels and excess water removed to leave the eggs suspended in 10--15/zl of water. (iii) T h e ground section of the capillary was smeared with silicone high-vacuum grease, and a piece of W h a t m a n No. 50 filter paper inserted into the coarse section. In rubes in which oxygen uptake was to be measured, 1/~l of 20~o potassium hydroxide was applied to the paper. T h e capillary was then inserted into the respiration vessel and the joint sealed. (iv) Indicator fluid (2, 7-dimethyl octane) was introduced into the capillary. This was effected by heating the respiration vessel between the fingers for 20 sec and applying a drop of indicator fluid to the open end of the tube. As the vessel cooled, the indicator fluid was drawn into the capillary to form a drop 2-5 m m long. (v) T h e tubes were assembled in the frame, which was then placed in the large vessel, and the joint sealed with stopcock grease. T h e vessel was then equilibrated for 1 hr at 25°C, with the tap open to let air escape. T h e tap was then closed and the first reading taken after 5 rain. T h e movement of the indicator droplet in the capillary was measured using a Vernier microscope reading to 0'02 ram, from a reference mark scored on each capillary. T h e rate of respiration was measured at daily intervals during development. Half the vessels (three sample and two thermobarometer) were used to measure total oxygen consumption. T h e other half (three sample and two thermobarometer) were used to measure changes in total gas volume (O~ taken up - C O 2 given off). Readings were taken at 15-rain intervals for 3 hr. T h e apparatus was then dismantled, the total number of eggs in each vessel (around 1000) counted and percentage development determined. Results were corrected for fluctuations in the thermobarometer tubes, and distance moved by the indicator droplet was then used to calculate the volume change in microlitres of gas. Respiratory quotient (RQ) and QO~ were then calculated. It is necessary in measuring changes in total gas volume to correct for carbon dioxide dissolved in the medium. A factor K was therefore devised ~vf K= 1+~-, where a is the absorption coefficient (0-76 at 25°C for CO~), vf is the volume of fluid present (/~1) and V is the volume of the gas space ( = volume of chamber minus vl). Putting typical values, 0-76 × 15 K=I-~ 1-1. 115 Calculated values of carbon-dioxide evolution were therefore multiplied by K to give the true reading. In order to determine the rate of respiration of miracidia, fully developed but unhatched eggs were transferred to vessels and excess water removed. T h e eggs were allowed to hatch and rate of respiration was determined. T h e miracidia were then killed and counted. As rate of respiration of miracidia was ten times that of unhatched eggs, the presence of a few unhatched eggs was ignored.

4. The chemistry of development Protein. T h e nitrogen content of undeveloped and developing eggs and of shells was determined by a method based on Levy (1936), King (1951) and Ballentine (1957), using glycine as a standard. Soluble protein was determined by the method of Lowry et al. (1951). Samples of 5000 eggs were homogenized, the homogenate and washings transferred to a graduated tube and made up to 5 ml. The tube was then centrifuged at 4000 rev/min for 5-10 m i n and 1-ml aliquots were assayed for protein content. A freeze-dried protein extract was also electrophoresed to test its homogeneity, using a Shandon horizontal apparatus and cellulose acetate paper. Barbitone buffer (0"05 M, pH 8"6) was used and protein was visualized with mercuric chloride-bromophenol blue stain.

DEVELOPMENT OF EGG OF F A S C I O L A H E P A T I C A

311

Carbohydrate. Total carbohydrate was determined using the anthrone method of Kahan (1953). Carbohydrates were extracted from eggs by homogenizing in 5% trichloroacetic acid, followed by centrifugation to remove the precipitate; 1-ml aliquots of supernatant were then assayed.

Lipid. Histochemical techniques indicated that lipid present was predominantly neutral, i.e. triglycerides. The colorimetric method of Rapport & Alonzo (1955) was therefore used to assay this. Lipids were extracted by homogenizing with alcohol-ether (3 : 1) mixture. The homogenate was incubated at 60°C for 1 hr, centrifuged and the supernatant removed. The residue was extracted with more alcohol-ether, and finally twice with ether alone. The four supernatants were combined and evaporated to dryness. The residue was dissolved in 5 ml of absolute alcohol and the lipid content of 1-ml aliquots determined. A standard of glycerol tripalmitate (100 pg/ml) was used. Nucleic acid. Nucleic acid was extracted from homogenates using 5% perchloric acid. The absorption spectrum of the extract was determined using a Beckman DB spectrophotometer, and from this the nucleic acid concentration was calculated. RESULTS

1. Histochemical techniques Unfixed vitelline cells have an orderly structure of aggregations of granules and vacuoles (Fig. 2a) which is readily destroyed by fixatives. This may account for the lack of structure shown by vitelline cells in situ in sections of vitellaria. Each vitelline cell contained a prominent DNA-positive nucleus, but little detectable RNA in the cytoplasm. Conversely, the ovum, and later the embryo, contained large amounts of RNA uniformly distributed in the cytoplasm. This was particularly well marked during the early part of development but the cytoplasm of the germinal cells remained rich in RNA throughout development. The vitelline cells contained dense aggregations of protein (Fig. 2b), much of it in small granules. This protein was rapidly utilized during development. Large amounts of carbohydrate were also present in the cytoplasm of vitelline cells (Fig. 2c) and in the developing embryo. Best's carmine test indicated that most of this was glycogen (Fig. 2d). Staining with Sudan Black B indicated that little lipid was present in undeveloped eggs (Fig. 2e) but that the amount detectable on the second and successive days was greatly increased. Obviously, a synthesis of lipids was occurring in the early stages of development. By the sixth day, lipids predominated in the vacuolated cytoplasm of the vitelline cells. The granules reported by many workers as surrounding the papilla from the eighth to tenth day of development are in fact lipid. Results of other lipid tests indicated that the lipid was predominantly neutral, i.e. triglycerides. 2. Growth and weight determinations The summarized daily measurements of embryo size are illustrated graphically in Fig. 3a. From these results the change in volume of the developing embryo was calculated (Fig. 3b). Increase in both length and breadth of the embryo occur until the sixth day of development, at which point the embryo has an ovoid shape. This period

312

R.A. WILSON

~

b

FIC. 2. Histochemistryof vitelline cells. (a) Unfixed, unstained vitelllne cell. (b) Bromophenol blue method for protein. (c) Periodic acid-Schiff technique for carbohydrate. (d) Best's carmine method for glycogen. (e) Sudan BlackB method for lipids.

120 I00

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80

.2

60

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40

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20

I

I

l

I

I

t

I

I

I

I

2

4

6

8

I0

2

4

6

8

I0

Time,

doys

Time,

dQys

FIQ. 3. Growth of embryo. (a) Change in dimensions. (b) Logarithmic plot of volume.

DEVELOPMENT OF EGG OF F A S C I O L Y l H E P A T I C A

313

(day 0-6) is one of rapid cell division. Thereafter, cell differentiation occurs and this is reflected in an elongation of the embryo. The ratio of length to breadth increases from 1.25 : 1 on the sixth day to 2.5 : 1 on the tenth day. The plot of volume indicates that up to the seventh day of development "growth" occurs at a uniform rate. Beyond this point increase in volume is replaced by cell differentiation. The daily increment in growth up to the seventh day is 0.8, i.e. the embryo almost doubles its size each day. The fresh weight per sample of 1000 undeveloped eggs was determined as 747/~g, and the dry weight of 1000 as 181.5/~g. The change in total dry weight/ 1000 eggs measured daily during development is given in Table 1. The mean total change recorded was from 181.5/~g/1000 eggs to 142-4/~g/1000 eggs representing a decrease in dry weight of 22 per cent. In further experiments, changes of 175-128/~g/1000 eggs and 165-116/~g/1000 eggs were recorded. No significant change in fresh weight of eggs during development could be detected. T A B L E 1 - - C H A N G E S IN DRY W E I G H T OF DEVELOPING EGGS

Day

No. of samples

0 1 2 3 4 5 6 7 8 9 10

3 3 3 3 2 2 2 3 2 2 3

Mean dry weight (/zg/1000eggs + S.E.) 181.5 + 7.5 174-0 + 1.7 165.8 + 1.4 176.3 + 4-8 161-6 + 16.1 158.0+ 1"5 154.3 + 4"0 149-9 + 3"4 150-0+ 2.3 141.3 _+ 0"8 142.4 + 5"2

The dry weight of shells was determined as 39.1/~g/1000 shells (mean of seven experiments). The shell therefore accounts for 20 per cent of the dry weight of the egg. By subtracting this figure from the total dry weight, the true decrease in egg constituents can be assessed (approximately 40 per cent of egg contents are utilized as food during development.) The results of dry weight determinations on miracidia varied from 39.2 to 73.0tzg/1000 with a mean of 61.0 (five experiments). The wide variation is probably a reflection on the inaccuracy of the method employed.

3. Respiration Sample data from a respiration experiment are summarized in Fig. 4 (day 0 of development). Inspection of the graph shows that uptake and evolution of gases remained constant under experimental conditions for at least 2.5 hr. The 2-hr figure was therefore used to calculate hourly rates of respiration. The figures for

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daily rate of gas exchange and the calculated respiratory quotients (RQs) are summarized in Table 2. Oxygen consumption is maintained at a steady level during the phase of embryonic cell division, but from the sixth day onwards there is a rapid rate of oxygen uptake corresponding to the cell differentiation phase. Finally, as development nears completion, there is a slight fall in the rate of oxygen consumption.

0,6 1155 ]

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TABLE 2--RESPIRATION OF DEVELOPING EGGS

Oxygen uptake/lO00 eggs Day 0 1

2 3 4 5 6 7 8

9 10

No. of samples

Oxygen uptake - C02 evolved/ 1000 eggs

Mean (/~l/hr 4- S.E.)

No. of samples

Mean (/~l/hr_+S.E.)

RQ

0-098 4-0"010 0-119 + 0"007 0"123 + 0"007 0-111 4-0.004 0"118 4-0'017 0"138 4-0"017 0.090 _ 0"004 0.143 4-0"003 0.183 + 0"010 0"218 ± 0"001 0"192 ± 0-017

3 3 3 2 3 3 2 3 3 3 3

0"035 + 0"002 0'067 _ 0"011 0"035 +_0"008 0"044 + 0"003 0"017 4-0.004 0-034__.0.009 0"018 _ 0.003 0"056 __0'004 0"067 4-0"016 0'097 _+0"006 0"061 ± 0"012

0"7 0"48 0"72 0"66 0'94 0'83 0"88 0"64 0"69 0"62 0"75

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D E V E L O P M E N T OF E G G O F F A S C I O L A H E P A T I C A

In order to allow comparison with other work, particularly that of Horstmann (1962), the QO~ was calculated. This is defined as (/~1 of oxygen taken up/mg dry weight of tissue)/hr. The dry weight figures recorded earlier were used, corrected to 100 per cent development. Figures for oxygen consumption also corrected to 100 per cent development are given in Fig. 5.

2.0

t/$

I-O

T 2

I 4 Time,

I 6

r 8

i0

days

FIG. 5. Q02 of developing eggs. Needham (1931) gives a useful check on the results of dry weight experiments. The total carbon dioxide evolved during development can be calculated from respiration figures. One microlitre of carbon dioxide evolved -'-2/~g of carbon dioxide and is equivalent to the combustion of 0.76/~g of lipid or 1"4/~g of carbohydrate. The estimated total carbon dioxide evolved during development (corrected to 100 per cent development) was 78.85/~g equivalent to 30/~g of lipid or 55/~g of carbohydrate/1000 eggs. T h e actual loss in dry weight/1000 eggs (corrected to 100 per cent development) was 55/~g so that figures are in close agreement. T h e results of experiments on the respiration of miracidia depart markedly from those of Horstmann. The QO~ of the miracidia appeared to be related to the length of incubation of eggs after full development and prior to hatching. Thus, the (202 of miracidia hatched from eggs incubated for 15 days was 18.3; for 21 days, 9.8; and for 23 days, 7.25. The decrease in activity during otherwise uniform conditions must be attributed to the utilization of food reserves during the post-developmental phase. T h e RQ of the miracidium was calculated as 0.97 indicating carbohydrate as the principal energy source. Horstmann observed that darkness had a depressing effect on the QO2 of miraeidia and that the QO2 declined with time. No such effect was observed here.

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R . A . WILSON

The rate of oxygen consumption remained constant for 2-3 hr, after which it increased steadily. Miracidia removed from respiration vessels were cytolysed and moribund. The cytolysis presumably results from an uncoupling of oxidative phosphorylation, with diminution of ATP production followed by loss of membrane integrity and oxidation of structural components in an attempt to satisfy energy requirements.

4. The chemistry of development Protein. The changes in total nitrogen content of eggs during development are given in Table 3. The results were statistically significant at the 1 per cent level. A repeat experiment, however, gave mean figures of 8.0/~g N/500 eggs at the beginning of development declining to 7.2/zg/500 eggs at the end. These results, corrected to 100 per cent development, indicate a loss of 1.6-3-0 tzg N/1000 eggs during development. In terms of protein this would amount to 20-37/~g/1000 eggs, the total protein content at the beginning of development being 80-100 t~g. TABLE 3--CHANGES IN NITROGEN CONTENT OF DEVELOPING EGGS

Day

M e a n decrease O~g N / 5 0 0 eggs ± S.E.)

No. of samples

0 2 5 8 10

6.4±0.5 6.2±0.4 5"4±0'3 5-0±0.6 5.1±0.2

5 5 5 5 5

The nitrogen content of shells was determined as 2.8/~g/500, a figure equivalent to 37.5/~g of protein and in excellent agreement with the determination of shell dry weight. The nitrogen content of miracidia was determined as 1.7/~g/500 (mean of five determinations). This would be equivalent to 21/~g of protein/1000, a low figure when compared with a dry weight of about 60/~g/1000. The soluble-protein content of the undeveloped egg was around 20/~g/1000 and the amount present decreased during development to 10-15/~g/1000 in fully developed eggs. This decrease in soluble protein could indicate either combustion as a food source or combination into structural components. The protein extract appeared very labile and difficult to freeze-dry. When electrophoresed at pH 8.6 for 1 hr, the extract separated into two components, a major and a minor, which both migrated towards the cathode. Carbohydrate. The total carbohydrate content of undeveloped eggs ranged from 38 to 56/~g/1000 (mean, 47/~g). The amount detected at the end of development ranged from 8 to 38/~g/1000 eggs (mean 17.7/~g corrected to 100 per cent development). In other words, 30/~g of carbohydrate/1000 eggs are used up as a food source during development. Carbohydrate thus accounts for 26 per cent of

DEVELOPMENT OF EGG OF F A S C I O L A H E P A T1CA

317

total dry weight at the beginning of development and 14 per cent at the end. (This is in good agreement with Horstmann's figures of 32 and 15 per cent respectively.) Lipid. The mean lipid content of undeveloped eggs was 16/~g/1000 and of developed eggs, 22/~g/1000. Superficially, the role of lipid would not appear to be great. However, it was noted in histochemical observations that the amount of lipid present increased in the early stages of development. Accordingly, lipid content of eggs as determined on the second day of development was found to be 40/~g/1000 eggs, and this confirmed the rapid synthesis of lipid during the early stages of development. Nucleic adds. The method used was not sufficiently sensitive to give precise information on changes in nucleic acid content. Undeveloped eggs were recorded as containing 1.1/~g/1000 and developed eggs 1.9/~g. This is equivalent to a nucleic acid content of 0.8 per cent at the beginning of development and 3.1 per cent at the end. Key

Undeveloped egg

i~1 J" Protein

• []

Nucleic aeiel Rest

Fully developed egg t

50/~g

FIG. 6. Summary of changes in chemical composition/1000 eggs during

development. The data on chemical composition of eggs are summarized in Fig. 6. The mean loss in dry weight of 55/~g/1000 eggs can be accounted for mainly by a reduction in the amount of carbohydrate and protein present. These appear to be the main energy source in a ratio of 3 : 2. This ratio can be used to convert the values calculated for total carbon dioxide evolved into material combusted. The 21.3/~g of carbon partitioned between carbohydrate and protein gives an equivalent of 54.2/~g of solid/1000 eggs used as food during development. DISCUSSION

No other study of an ectolecithal egg appears to have been recorded in the literature, apart from Horstmann's account of respiration, and it is therefore difficult to draw comparisons. The chemical composition of the eggs is unlike any other recorded. Water content (80 per cent) seems a little high when compared with other invertebrates such as molluscs, 50-75 per cent (Needham, 1931) or Ascaris, 70 per cent (Fairbairn, 1957). The ready plasmolysis of the fluke egg in air accounts for the great difficulty in measuring wet weight accurately. The contribution of shell material to total dry weight (20 per cent) is considerable and appears to be predominantly protein. Most of the remaining 80 per cent of egg solids can be accounted for by 27 per cent protein, 26 per cent carbohydrate

318

R.A. WILSON

and 10 per cent lipid. Carbohydrate on account of its low energy-to-volume ratio generally plays little role in embryonic development and molluscs alone make use of it, usually storing it as galactogen. The development of the embryo, both histologically and biochemically, can be divided into two phases. The first, a period of rapid cell division, is followed from day 6 onwards by a phase of cell differentiation and an increase in embryonic length. The process of development is obligatorily aerobic and 38 per cent of the egg contents are used as an energy source. The figures for respiration recorded here are two to three times greater than those of Horstmann, but QOz curves show a similar configuration. One puzzling feature of Horstmann's work is his use of buffered 0-9~/o saline as a culture medium, since fresh water is the natural environment of the egg. It is possible that the saline had a depressing effect on respiration. The maximum QO2 observed during development was 2.5. This is very similar to the figure of 2.7 at 30°C for Ascaris eggs, recorded by Fairbairn (1957). The calculated values for RQ reflect metabolic changes occurring within the egg and types of substrate being utilized as an energy source. On the basis of RQ values it is possible to make a tentative division of developmental metabolism into three stages: (a) an event occurring at the onset of development which markedly depresses RQ; (b) a period from day 2 to day 6 with a mean RQ of 0.81 indicating a mixed substrate with carbohydrate as a major energy source; (c) a period at the end of development (days 7-10) with a mean RQ of 0.68 indicating a lipid substrate. The environment of an egg is said by Needham to be the determining factor in the type of food reserve stored and utilized. In general, in aquatic eggs, more protein (62 per cent of total) is utilized than in terrestrial eggs (6-8 per cent of total). Conversely, in terrestrial eggs more lipid (81 per cent of total) is utilized than in aquatic eggs (32 per cent of total). The pattern in Fasciola fits neither of these situations. Overall changes in constituents during development show that carbohydrate and protein are the main food stores. The added complication of the conversion of one or both of these into lipid at the beginning of development and consequent utilization of lipid in the later phases of development cannot be easily explained. The situation is doubtless related to the unique structure and origins of the ectolecithal egg. If these eggs are, as Hyman (1951) suggests, modified ova, then this would explain the high carbohydrate content and lipid synthesis could be regarded as late maturation of the modified ova. On the other hand, the synthesis of lipid could be connected with some, as yet, unsuspected event in development. Very little is known of the dynamics of the embryo-vitelline cell system. Early workers (e.g. Schubmann, 1905 ; Ortmann, 1908) considered that the vitelline cells simply degenerated into a mass of yolk which was subsequently used by the developing embryo. This is certainly not the case and available evidence points to an active and ordered metabolism of the food stores by the vitelline cells themselves. Degeneration appears to occur in the very last stages when nuclei and mitochondria disappear and such an activity could well be controlled by lysosomes, although none have been detected. The utilization of the food reserves must, however, be "organized" by the embryo. The fact that at the end of development

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two to five vitelline cells still remain, implies that, as d e v e l o p m e n t is completed, the strength of the organizing force diminishes and the e m b r y o becomes quiescent; a theory s u p p o r t e d by the terminal decline in respiration rate.

SUMMARY I. The histochemistry of the vitelline cells in the egg of Fasciola was described. Large quantities of carbohydrate and protein, but little lipid, were present. 2. The growth rate of the embryo was measured and development divided into two phases: the first of rapid cell division, the second of cell differentiation. 3. Changes in dry weight of the egg during development were determined. 4. The QO~ and RQ of developing eggs were measured at daily intervals. 5. Changes in protein, carbohydrate, lipid and nucleic acid content of the eggs during development were also determined. 6. The development of the liver fluke egg was discussed with reference to the findings.

Acknowledgements--I wouldliketo thank ProfessorO. W. Richards in whosedepartment this workwas carried out, also Dr. F. Call and ProfessorB. G. Peters for their many helpful criticisms. REFERENCES BALLENTINE R. (1957) Determination of total nitrogen and ammonia. In Methods in Enzymology. Vol. 3. Academic Press, New York. BEST F. (1906) Quoted in PEARSEA. G. E. (1960). BOGOMOLOVA N. A. (1957) Cytochemical investigations on the miracidium of Fasciola hepatica. (In Russian.) Dokl. Akad. Nauk S S S R 117, 313-315. CAiN A. J. (1947) Quoted in PEARSEA. G. E. (1960). FAIRBAIRND. (1957) The biochemistry of Ascaris. Expl Parasitol. 6, 491-554. HOI~TMANN H. J. (1962) Sauerstoffverbrauch und Glycogengehalt der Eier yon Fasciola hepatica w~ihrend der Entwicldung der Miracidien. Z. ParasitKde 21, 437-445. HYMANL. (1951 ) The Invertebrates. Vol. 2 (Platyhelminthes and Rhyncocoela). McGraw-Hill, New York. KAHAN J. (1953) A rapid photometric method for the determination of glycogen. Archs Biochem. Biophys. 47, 408418. KING E. J. (1951) Microanalysis in Medical Biochemistry. Second edn. Grune & Stratton, New York. KORNICK N. B. (1955) Quoted in PEARSEA. G. E. (1960). LEVY M. (1936) Studies on enzymatic histochemistry--XVII. A micro-Kjeldahl estimation. C. r. Tray. Lab. Carlsberg 21, 101-110. LowRY O. H., ROSEBROImHN. J., FARRA. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MCMANtlS J. F. A. (1946) Histological demonstration of mucin after periodic acid. Nature, Lond. 158, 202. MAZtA D., BREWERA. P. & ALFm~TM. (1953) The cytochemical staining and measurement of protein with mercuric bromophenol blue. Biol. Bull., Woods Hole 104, 57-67. MENSCmK Z. (1953) Quoted in PEARSEA. G. E. (1960). NEEImAM J. (1931) Chemical Embryology. University Press, Cambridge. ORTMANN W. (1908) Zur Embryonalentwicklung der Leberegels (Fasciola hepatica). Zool. fib. 26, 255-292.

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