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The mode of hatching of the egg of Fasciola hepatica II. Colloidal nature of the viscous cushion
EXPERIMENTAL
6, 131-142 (1957)
PARASITOLOGY
The Mode of Hatching of the Egg of Fusciolu hepatica II. Colloidal Nature of the Viscous Cushion. William
B. Rowan
I,2
Department of Veterinary Parasitology, Oklahoma Agricultural and Mechanical College, Stillwater, Oklahoma (Submitted
for publication,
17 July
1956)
In a recently published paper concerned with the mode of hatching of the egg of Fasciola hepatica it was reported that light stimulates the miracidium to release an enzyme, prior to hatching, which digests a proteinaceous substance that binds the operculum of the egg to t,he shell allowing the egg to open (Rowan, 1956). The report referred to a viscous, granular cushion of material at the opercular end of the egg which lies like a concave-convex lens against the inner surface of the vitelline membrane. This viscous cushion has attracted the attention of numerous writers in their observations on the hatching of trematode eggs. Barlow (1925) and Bennett (193G) both observed the material in the eggs of Fasciolopsis buski and G’ot:ylophoron cotylophorum, respectively. They referred to it as the “mucoid plug” and stated that it forms a hwrier to the escape of the miracidium. According to Barlow the miracidium uses an “erosive” and dissolves partially the viscous cushion to escape. Bennett, on the other hand, believed that the miracidium removes mechanically the viscous cushion to escape by brushing it repeatedly with it,s anterior cilia. Mattes (1926) and Roman (1956), working with the egg of F. hepatica, reported entirely different findings from those of Barlon and Bennett. They found that the cushion expands just before the release of the operculum, and fills nearly one-half of the cavity of t*he egg. Mattes suggested that expansion of the cushion increases the internal pressure of the egg sufficiently to force the operculum from the shell, 1 Public Health Service Postdoctorate Research Fellow of tute for Allergy and Infectious Disease, National Institutes * Present address: Puerto Rico Field Station, P. 0. Box Disease Center, Public Health Service, U. S. Department of and Welfare, San ,Jllan, Puerto Rico. 131
the National Inst,iof Health. 52, Communicable Health, Eclucat.ion,
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WILLIAM
B.
ROWAN
but Rowan demonstrated that expansion of the cushion does not, in itself, cause the egg to open. In his experiments several conditions caused expansion of the cushion without release of the operculum. Heating the eggs to 70°C or submerging them in dilute solutions of silver nitrate, mercuric chloride, formalin and many other killing agents had this effect. The cause of expansion of the cushion was not determined at that time. The viscous cushion also has been described as playing a role in hatching after the release of the operculum. Onorato and Stunkard (1931), working with the operculate egg of Spirorchis sp., observed that the material of the viscous cushion flows out of the egg before the escape of t,he miracidium and forms a flexible envelope in the aqueous medium outside the egg. They noted that at first the wall of this envelope merely gives under the probing art,ion of the emerging miracidium in the envelope, but, with repeated application of pressure by the miracidium “the elast,icity of the membrane is gradually reduced until it ruptures and the larva is able to escape.” They found that “if miracidia are liberated into water as soon as they emerge from the shell they usually disintegrate,” and they believed it, probable that during the time the larvae are in the envelope “they become adjusted to life in water.” Rowan (1956) has observed also the formation of a flexible envelope from the material of the viscous cushion of the F. hepatica egg when it hatches either in tap water, dilute NaCl or certain other dilute salt solutions. He found, however, that when an egg hatches in Pyrex-redistilled water the cushion material simply disperses as it flows from the egg. An understanding of the volume and viscosity changes of the cushion material is essential for a full appreciation of the mode of hatching of the trematode egg, and forms the subject of this paper. The source of materials and the general methods used are the same as those used previously. New methods relating specifically t,o individual experiments will be described more fully in the text. THE
hVELOPE
It has just been stated that when an egg of F. hepatica is put into Pyrex-redistilled water and allowed to hatch the material of the viscous cushion flows out of the egg and disperses in the water. On the other hand, if an egg is put into either tap water, dilute NaCl, or other salt solut8ions the material of the viscous cushion does not disperse as it flows from the egg, but forms a flexible envelope with a capacity of approximately one-half that of the egg. The fully formed envelope is thick walled,
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FIG. 1. Hatched egg of F. hepatica with the “envelope” outlined in ink. The shell, thick wall of the envelope, which tapers to t,he point, of att,aehment to the .E is indicated by parallel lines.
the thickness tapering to an opening at the point of attachment to the shell (Figs. 1, 2). The wall of the envelope has the consistency of 1% agar or stiff gelatin. It is resilient, flexible and homogeneous through out. The wall can be torn with a dissecting needle without causing the col-
FIG. 2. Hatched egg of F. hepatica showing the envelope as it actually appears.
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lapse or contraction of the enclosed cavity. The miracidium partly enters t,he envelope as it emerges from the shell. It gently probes the inner wall in an hnsuccessful effort to escape, and then usually turns back on itself and breaks out near the point where the envelope contacts the shell. If the envelope is undisturbed after hatching has occurred, it may remain int,act for several hours. Since the envelope forms when an egg hatches in dilute salt solutions but does not form when an egg hatches in Pyrex-redistilled water, experiments were undertaken to observe the effect of alternately exposing the envelope first to dilute salt solution and then to Pyrex-redistilled water. These experiments were done in the following manner. Fifty or more fully developed eggs were drawn with a medicine dropper from a darkened culture bottle and put on a glass slide in a drop of 0.15 M phosphate buffer solution (pH 7.2) between two parallel ridges of Vaseline. A cover slip was placed over the drop and pressed down to form a seal with the Vaseline. Thus the cover slip, slide and Vaseline ridges enclosed a shallow channel running parallel with the length of the slide and measuring lo- to 15-mm wide by 0.5- to l-mm deep. Fluids could be made to flow slowly and continuously through this channel by adding them dropwise at the right of the channel and removing the overflow with absorbent filter paper at the left. The eggs usually remained fixed in place beneath the cover and were bathed by the fluids which flowed past. After this preparation each slide was placed beneath a fluorescent light to stimulate hatching of the eggs. The eggs began to hatch usually within a few minutes and as each egg opened the material of the viscous cushion flowed out into the buffer solution to form a typical envelope. After a few minutes the slide was placed on the microscope stage and a single hatched egg with its attached envelope was brought into focus at a magnificat,ion of 100X. Then a drop of Pyrex-redistilled water (pH 7.2) was put on the slide at the right of the channel and this water was caused to flow through the channel by placing absorbent filter paper at the left. Within seconds after the addition of the pure water the wall of the envelope in the field of vision began to expand and as it expanded it lost its refractive properties and became almost invisible. Expansion of the envelope wall continued until it was two or three times its original size. The volume of the cavity enclosed by the wall of the envelope changed only slightly. After exposing the envelope momentarily to a stream of the Pyrex-redistilled water, a drop of the 0.15 M buffer solution was added to the right of the channel and a fresh piece of filter paper
HATCHING OF FASCIOLA HEPATICb
EGG. II
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was placed at the left. The buffer solution replaced the pure water in the vicinity of the egg and as it did so the wall of the envelope contracted again to its original size. The experiment just described was repeated many times and with several variations, In several instances nigrosin was used in the buffer solution to surface stain t,he envelope wall and render it] visible during the expansion in pure water. Another variation tried involved freeing the envelope from the egg before subjecting it t,o the solutions. The envelope was caused t,o break away from the egg by tapping t,he ~OWI slip directly above the egg with a dissecting needle. The freed envelope, torn open at t’he side, was t,hen exposed alternately to water and buffer solution. All of these experiments produced similar results. Pyrex-redistilled water caused the wall of the envelope to expand and phosphate buffer solution caused it to contract,. However, in every case it was essential that the buffer solution be replaced by Pyrex-redistilled water soon after expansion of t,he envelope wall, for when pure water was allowed to remain in contact with the envelope for longer t,han a fen minutes the wall of the envelope dispersed and failed to return t)o t,hc contracted condition following the addition of buffer solution. The results of these experiments suggest that’ the wall of the envelope has the properties of a colloidal system that contracts to form a gel on contact, with either the ions of t,ap water, phosphat,e buffer solut’ion, or dilute NaCl solution, and expands to form a sol when these ions are removed or greatly diluted by the addition of pure water. 11 THEORETIC!.~L EXPLAXATIOX FOR Ex~a~sro~ OF THE VISCOUS CUSHIOK DURIXG HATCHING
The observations suggest a plausible explanation for the phenomenon of expansion of the viscous cushion during normal hatching, and for expansion of the viscous cushion when t,he unhatched egg is treated with heat or exposed to various toxic solutions. If the material of the cushion has the properties of a colloidal system after hatching, it probably exists also as a colloidal system while it is within t*he egg. It follows that if the concentration of ions wit,hin the egg were to be lowered markedly in some manner, such as through the osmosis of salts or other materials from the egg to the surrounding medium, the viscous cushion within the egg would be expected to change from a gel to a sol, and the change would he accompanied by expansion. The writ’er believes that expansion of the cushion is accompanied by au exosmosis of salt’s or ot,her ma-
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terials. Hereafter “salts” will refer to those undetermined materials. It is suggested that the exosmosis of “salts” occurs as a result of naturally or artificially induced damage to the vitelline membrane. Exposure of eggs to heat or to toxic chemicals could readily damage the selective permeability of the vitelline membrane and initiate an exosmosis of “salts.” This assumption is supported by work reviewed by Needham (1931, p. 798) indicating that when the trout egg is injured or poisoned the permeability of its membranes increases and salts diffuse out of the egg in measurable amounts. During normal hatching, damage to the vitelline membrane of the egg with subsequent exosmosis of “salts” and expansion of the viscous cushion, could result from either of two causes or from a combination of them. In an earlier paper (1956) the writer demonstrated that the miracidium releases a proteolytic enzyme prior to hatching and that this enzyme destroys an opercular cement. Possibly this enzyme also damages or destroys the vitelline membrane. In this connection it can be said that fishery workers according to Needham (1931, p. 1598) are familiar with the fact that “the membranes of the trout egg, while at first thick and parchment-like, become at hatching thin and brittle.” It is stated that the hatching enzyme released by the trout embryo is powerful enough to kill the embryo and there is “literally a race as to whether hatching or death will take place first.” Many embryos are “so weakened or necrosed at hatching that they cannot long survive it . . . and such necrosed embryos have the tips of their tails and fins eaten away.” A second factor may account for the proposed naturally-induced damage to the vitelline membrane with an accompanying exosmosis of “salts.” The enzymatic release of the operculum is undoubtedly a gradual process. Leakage probably occurs about the digesting opercular border as the operculum gradually splits away from the remainder of the shell. Since both the shell and the vitelline membrane serve normally as differentially permeable barriers to the passage of salts, leakage would upset the particular osmotic balance between the vitelline membrane and the shell. Such a change could produce damage to the vitelline membrane and cause an exosmosis of “salts” from the egg. In his statements concerning the egg of C. cotylophorum, Bennett (1936) notes that rough handling of an egg in one case loosened its operculum and allowed the entrance of water, and that this change resulted in immediate expansion of the viscous cushion. Similar rough handling was found occasionally to cause expansion of the viscous cushion of the unhatched F. hepatica egg.
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THE EXOSMOSIS OF “SALTS”
In support of the hypothesis that has been presented a series of 20 experiments were done over a period of several weeks which demonstrate that there is a decrease in the vapor pressure of wat,er surrounding unhatched eggs of F. hepatica following the expansion of t,he viscous cushion. By virtue of the conditions of each experiment,, it is presumed that the decrease in vapor pressure is the result, of t,he osmosis of “salts” from the eggs into the surrounding water. In each experiment over 100 fully developed, but unhat’ched eggs of F. hepatica were removed from a darkened culture bottle and plunged into Pyrex-redistilled water at a t,emperature of 45°C. They were held at that temperature for 30 minutes. This exposure to heat killed the miracidia within t)he eggs but. did not, cause either expansion of the viscous cushion, release of t,he operculum, or any other visible change in the eggs. Exposure to 45°C apparently inactivates at least temporarily any hatching enzyme present in the eggs, since those which had undergone this exposure frequent,ly remained unchanged for periods of as long as 72 hours. After the exposure to heat, these fully developed, unhatched eggs containing dead larvae were transferred immediately to a Syraruse watch glass containing Pyrex-redistilled water and washed thoroughly with several changes. Any damaged, abnormal or undeveloped eggs were removed and the remaining ones were transferred to another Syracuse watch glass of Pyrex-redistilled water. These eggs were caused to collect, on the bottom of the watch glass towards the center by gently swirling the dish. One end of a fine capillary tube (600 microns outside diameter, 400 microns inside diameter) was int’roduced into the watch glass, and using the method of manipulation of the capillary tube dewrihed prcviously by the writ.er (19.56) three droplets of water were drawn into the tube. The droplets in the tube measured approximately 900 microns in length and were separated from each other by short air spaces (GO0microns in length). Following the introduction of t,he three water droplets, approximately one-half of the eggs on the bot,tom of the watch glass were drawn into the capillary tube as a part of a fourth droplet of water. The egg-containing droplet was adjusted to a length of approximately 900 microns, the adjustment being accomplished without removing t,he capillary tube from beneath the surface of the water. A fourth air space was interposed and three additional water droplets separated by air spaces were drawn into the t,ube. Aft.er t’he seven separate droplets of wat.er were centered in t’he tube both ends were sealed by fire. During
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the sealing procedure the droplets were protected from the heat by covering the section with the fingers. A second tube was filled and sealed in exactly the same manner using the remaining eggs in the watch glass. The first tube was immersed in a water bath, temperature 65”C, for 10 minutes, and after removal was fixed to a microscope slide. The second tube was not exposed to the hot water, but was fixed beside the first one on the slide. Microscopic examination revealed that the hot water had caused the expansion of the viscous cushion in each of the eggs in the egg-containing droplet of the heat treated tube. The eggs in the eggcontaining droplet of the untreated tube remained unchanged. As previously stated, 20 experiments were done and in each two capillary tubes prepared as described were used. Each tube contained seven droplets of Pyrex-redistilled water of nearly equal length separated by air spaces and the central droplet in each tube contained 25 to 75 eggs, The two tubes in each experiment differed in that one had been exposed to a temperature of 65°C for 10 minutes while the other had not. The eggs in the experimental tube contained fully developed, dead larvae and the viscous cushion of each egg was expanded. The eggs in the control tube also contained fully developed, dead larvae and the viscous cushion of each egg was unexpanded-unchanged in volume from the normal. The temperature of the room was recorded and the length of each of the droplets in each tube was measured microscopically, using an ocular micrometer at a magnification of 52.5X. After the measurements had been taken the glass slide bearing the two tubes was placed in an incubator and maintained at a temperature of 25 to 27°C for 24 hours. The slide was removed from the incubator, to a room temperature adjusted to the temperature of the previous day, and the droplets in each tube again were measured. Then both tubes were opened with a glass file and their contents expelled into separate water glasses containing Pyrex-redistilled water. A record was made of both the number of eggs in each tube and the number of these in each tube which contained a viscous cushion in the expanded condition. The results of the 20 experiments are recorded in Table I. It is evident, from the data in Table I that in each experiment the egg-containing droplet of the tube subjected to heat increased measurably in length, while the length of the egg-containing droplet in the control tube changed only slightly or not at all. Likewise, the water droplets on either side of the egg-containing droplet, decreased in length in each experimental
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TABLE I Changes in Droplet Length in Capillary Tubes Used as a Measure of Vapor Pressure Changes Within the Droplets Experimental
-. T otal number of eggs -
Experiment
,
25 37 28 35 42 65 53 58 39 75 46 84 68 44 54 57 52 61 53 67 52
1
2 3 4 5 6 7 8 9 10 11
12 13 14 15 16 17 18 19 20 Average -
--
25 36 28 35 42 65 53 58 39 75 46 84 68 44 54 56 52 61 53 67 52
Control tube
Percent change in length of the three center droplets
I! 5
tube
4 5% rd’c1 3
5 5% cd-c 3
-12.0 -10.2 -8.6 0 -7.6 -6.8 -10.8 -7.6 -3.5 -4.8 -11.7 -3.5 -7.1 -3.1 -5.7 -5.8 -1.4 -6.1 -4.0 -10.7 -6.5
rota1 num,er of eggs
f21.4 +18.9 +8.2 +16.4 +16.8 +12.7 f7.5 +10.0
+8.8 $5.9 +6.4 +4.6 +10.5 +10.0 +10.0
$8.8 +4.8 +9.4 +8.1 $8.9 -i-10.4
-11.3 -10.0
-7.5 -20.0 -8.7 -11.4 -11.5 -11.3 -6.2 -6.4 -4.9 -4.5 -4.4 -6.1 -3.4 -4.2 -1.6 -5.8 -2.5 -3.2 -7.2
-
- T 5 4 8 B 31 30 29 48 34 45 48 57 64 43 68 46 55 28 70 39 53 55 53 69 48
-
- ~.%Z ;gp !ge 3 0 0 0 3 0 2 3 4 0 0 2 2 3 3 0 5 2 2 3 2
1Percent change in length of the three center droplets
-1.6 0 0 -3.1 -1.9 0 -5.7 -4.8 0 0 0 0 0 0 0 0 f2.4 0 0 0 -0.7 -
-
$1.9 i-1.7 -0.9 f5.4 +2.7 0 +1.4 +4.9 0 f2.9 0 +1.3 0 +1.6 f1.6 0 0 0 $0.9 0 $1.3
-1.9
-4.4 i-2.1 -5.7 -2.9 -0.8 0 0 -4.9 -4.6 0 0 -1.9
-2.2 0
-
-1.0 0 0 0 0 -1.4
tube, but their lengths changed very little or not at all in the control tubes. The procedure described above is a modification of the capillary tube method of Halket (1913) for determining the osmotic pressure of unknown solutions. It has been used recently by Hayes (1953) to find the osmotic pressure of the blood of the mosquito, Aedes aegypti. The method incorporates a very simple and useful physical principle. If two solutions of different salt concentration are separated from each other by an air space, but are hermetically sealed in a tube, water molecules will diffuse through the air from the solution of lower concentration
140
WILLIAM
B. ROWAN
which, however, has a higher vapor pressure, to the solution of higher concentration (lower vapor pressure). Applied to the experiment just described it means that in each case the egg-containing droplet of the experimental tube must have had, during the incubation period, a higher “salt” concentration (lower vapor pressure) than the bracketing but airseparated droplets of Pyrex-redistilled water. The ‘$alt” concentration of the egg-containing droplet of each control tube, on the other hand, must have been essentially the same as that of the bracketing droplets of Pyrex-redistilled water. Since the experimental and control tubes in each experiment were prepared in the same manner the egg-containing droplets in both tubes had the same “salt” concentration (vapor pressure) at the time they were sealed. The change in concentration in the egg-containing droplet of the experimental tubes can readily be accounted for by assuming an osmosis of “salts” from the experimental eggs into the egg-containing droplet. The fact that the experimental eggs differ visibly from the control eggs only in having an expanded viscous cushion leads one to believe that the exosmosis of “salts” is associated with and perhaps accounts for the expansion of the cushion. THE CHEMICAL
NATURE
OF THE VISCOUS CUSHION
Results of attempts to determine the chemical nature of the viscous cushion were inconclusive. The colloidal nature of the cushion material suggested tests for protein. In fact Onorato and Stunkard (1931) stated that the cushion material from hatched Spirorchis eggs gives a protein reaction. The envelope formed from the cushion material of hatched 8’. hepatica eggs failed to give the characteristic reaction for protein when heated in 2% ninhydrin reagent. On the other hand, the potassium ferrocyanide reaction for protein of Hartig-Zacharias (Gomori, 1952) was positive. Furthermore, the envelope was rapidly destroyed by artificial digestive fluid (1 g dry pepsin, 1 ml HCl, 100 ml water) and by a solution of trypsin (10 mg per ml in 0.15 M phosphate buffer solution, pH &LO), but the envelope was not destroyed by either hyaluronidase from bovine testes (10 mg per ml in 0.15 M phosphate buffer solution, pH 6.4), by malt diastase at the same concentration (pH (i.9), or by control solutions of any of these four enzymes. These results would seem to indicate that the cushion material is at least in part composed of protein. The negative ninhydrin reaction, however, casts some doubt on this conclusion.
HATCHING OF FASCIOLA HEPATICA EGG. II
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DISCUSSION
The viscous cushion within the egg of F. hepatica is described as a colloid which changes from a gel to a sol with changes in the ion constitution of the egg contents. The writer believes that any expansion of the viscous cushion within the egg results from damage to the vitelline membrane-damage which permits an osmosis of salts or other materials from the egg into the surrounding medium. If this explanation is correct then expansion of the viscous cushion plays no active role in egg hatching. The phenomenon simple accompanies hatching as a natural result of chemical and physical changes that are in process within the egg at this time. It will be recalled that Onorato and Stunkard (1931) were the first to mention the formation of a flexible envelope from the material of the viscous cushion of a trematode egg as the material flows out during normal hatching. The envelope traps the emerging miracidium and delays its escape, and they suggest that in the case of Spirorchis sp. the envelope gives t,hc miracidium a necessary period in which to adjust gradually to the new environment outside the egg. The writer does not believe that the miracidium of F. hepatica requires an adjustment period after release of the operculum. He has watched repeatedly perfectly normal miracidia emerge in a matter of seconds from eggs hatching in Pyrex-redistilled water, and swim energetically away to live for several hours, and yet it will be recalled that the envelope does not form when an egg hatches in Pyrex-redistilled water. The developing miracidium within the trematode egg is surrounded by two delicate, differentially permeable membranes and is therefore vulnerable to many physical and chemical factors in the medium surrounding these membranes. It should be productive to determine the effect of various penetrable materials on the development of the miracidium and on its subsequent ability to infect an intermediate host. The possibility of using a safe, inexpensive prophylactic drug against the miracidium should be explored. SUMMARY
1. A viscous and granular cushion of material at the opercular end of the fully developed egg of Fasciola hepatica expands within the egg just prior to the release of the operculum during normal hatching. This expansion is shown to play a passive role in the hatching phenomenon.
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2. The cushion resemblesa colloid and consists at least in part of protein. It changes from a contracted gel to an expanded sol with changes in the ion constitution of the fluid bathing it. 3. Evidence is presented to show that expansion of the cushion within the unhatched egg is accompanied by osmosis of salts or other materials from the egg. This exosmosis is believed to result under natural conditions from damage to or destruction of the vitelline membrane by the direct or indirect action of the hatching enzyme. 4. Expansion of the cushion within the egg can be artificially induced by exposing the egg to heat or to toxic solutions. This change provides a valuable visual clue to the penetrability to toxic materials and may play a useful role in ovicide research. ACKNOWLEDGMENT The writer is grateful to Dr. Wendell H. Krull for his helpful criticism of the manuscript.
(Oklahoma A and M College)
HXPERENCES BARLOW, C. H. 1925. The life cycle of the human intestinal fluke Fasciolopsis buski (Lankester). Am. J. Hyg. Monogr. Ser. 4, July, 98 pp. BENNETT, H. J. 1936. The life history of Cotylophoron cotylophorum, a trematode from ruminants. Illinois Biol. Monogr. 14, 1-119. GOMORI, G. 1952. Microscopic Histochemistry. The Univ. of Chicago Press,
Chicago, 273 pp. HALKET, A. D. 1913. On various methods for determining osmotic pressures. New Phytologist 12, 164-176. HAYES, R. 0. 1953. Determination of a physiological saline solution for Aedes aegypti (L.). J. Econ. Entomol. 46, 624-627. MATTES, 0. 1926. Zur Biologie der Larvenentwichlung von Fasciola hepatica,
besonders tiber den Einfluss der Wasserstoffionenkonsentration auf das Ausschltipfen der Miracidien. Zool. Anz. 69, 133-156. NEEDHAM, J. 1931. Chemical Embryology. Cambridge Univ. Press. Cambridge, 2921 pp. ONORATO, A. R., AND STUNKARD, H. W. 1931. The effect of certain environmental factors on the development and hatching of the eggs of blood flukes. Biol. Bull. 61. 129132. ROWAN, W. B. 1956. The mode of hatching of the egg of Fasciola hepatica. Exptl. Parasitol. 6, 118-137.
6, 131-142 (1957)
PARASITOLOGY
The Mode of Hatching of the Egg of Fusciolu hepatica II. Colloidal Nature of the Viscous Cushion. William
B. Rowan
I,2
Department of Veterinary Parasitology, Oklahoma Agricultural and Mechanical College, Stillwater, Oklahoma (Submitted
for publication,
17 July
1956)
In a recently published paper concerned with the mode of hatching of the egg of Fasciola hepatica it was reported that light stimulates the miracidium to release an enzyme, prior to hatching, which digests a proteinaceous substance that binds the operculum of the egg to t,he shell allowing the egg to open (Rowan, 1956). The report referred to a viscous, granular cushion of material at the opercular end of the egg which lies like a concave-convex lens against the inner surface of the vitelline membrane. This viscous cushion has attracted the attention of numerous writers in their observations on the hatching of trematode eggs. Barlow (1925) and Bennett (193G) both observed the material in the eggs of Fasciolopsis buski and G’ot:ylophoron cotylophorum, respectively. They referred to it as the “mucoid plug” and stated that it forms a hwrier to the escape of the miracidium. According to Barlow the miracidium uses an “erosive” and dissolves partially the viscous cushion to escape. Bennett, on the other hand, believed that the miracidium removes mechanically the viscous cushion to escape by brushing it repeatedly with it,s anterior cilia. Mattes (1926) and Roman (1956), working with the egg of F. hepatica, reported entirely different findings from those of Barlon and Bennett. They found that the cushion expands just before the release of the operculum, and fills nearly one-half of the cavity of t*he egg. Mattes suggested that expansion of the cushion increases the internal pressure of the egg sufficiently to force the operculum from the shell, 1 Public Health Service Postdoctorate Research Fellow of tute for Allergy and Infectious Disease, National Institutes * Present address: Puerto Rico Field Station, P. 0. Box Disease Center, Public Health Service, U. S. Department of and Welfare, San ,Jllan, Puerto Rico. 131
the National Inst,iof Health. 52, Communicable Health, Eclucat.ion,
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B.
ROWAN
but Rowan demonstrated that expansion of the cushion does not, in itself, cause the egg to open. In his experiments several conditions caused expansion of the cushion without release of the operculum. Heating the eggs to 70°C or submerging them in dilute solutions of silver nitrate, mercuric chloride, formalin and many other killing agents had this effect. The cause of expansion of the cushion was not determined at that time. The viscous cushion also has been described as playing a role in hatching after the release of the operculum. Onorato and Stunkard (1931), working with the operculate egg of Spirorchis sp., observed that the material of the viscous cushion flows out of the egg before the escape of t,he miracidium and forms a flexible envelope in the aqueous medium outside the egg. They noted that at first the wall of this envelope merely gives under the probing art,ion of the emerging miracidium in the envelope, but, with repeated application of pressure by the miracidium “the elast,icity of the membrane is gradually reduced until it ruptures and the larva is able to escape.” They found that “if miracidia are liberated into water as soon as they emerge from the shell they usually disintegrate,” and they believed it, probable that during the time the larvae are in the envelope “they become adjusted to life in water.” Rowan (1956) has observed also the formation of a flexible envelope from the material of the viscous cushion of the F. hepatica egg when it hatches either in tap water, dilute NaCl or certain other dilute salt solutions. He found, however, that when an egg hatches in Pyrex-redistilled water the cushion material simply disperses as it flows from the egg. An understanding of the volume and viscosity changes of the cushion material is essential for a full appreciation of the mode of hatching of the trematode egg, and forms the subject of this paper. The source of materials and the general methods used are the same as those used previously. New methods relating specifically t,o individual experiments will be described more fully in the text. THE
hVELOPE
It has just been stated that when an egg of F. hepatica is put into Pyrex-redistilled water and allowed to hatch the material of the viscous cushion flows out of the egg and disperses in the water. On the other hand, if an egg is put into either tap water, dilute NaCl, or other salt solut8ions the material of the viscous cushion does not disperse as it flows from the egg, but forms a flexible envelope with a capacity of approximately one-half that of the egg. The fully formed envelope is thick walled,
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FIG. 1. Hatched egg of F. hepatica with the “envelope” outlined in ink. The shell, thick wall of the envelope, which tapers to t,he point, of att,aehment to the .E is indicated by parallel lines.
the thickness tapering to an opening at the point of attachment to the shell (Figs. 1, 2). The wall of the envelope has the consistency of 1% agar or stiff gelatin. It is resilient, flexible and homogeneous through out. The wall can be torn with a dissecting needle without causing the col-
FIG. 2. Hatched egg of F. hepatica showing the envelope as it actually appears.
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B.
ROWAN
lapse or contraction of the enclosed cavity. The miracidium partly enters t,he envelope as it emerges from the shell. It gently probes the inner wall in an hnsuccessful effort to escape, and then usually turns back on itself and breaks out near the point where the envelope contacts the shell. If the envelope is undisturbed after hatching has occurred, it may remain int,act for several hours. Since the envelope forms when an egg hatches in dilute salt solutions but does not form when an egg hatches in Pyrex-redistilled water, experiments were undertaken to observe the effect of alternately exposing the envelope first to dilute salt solution and then to Pyrex-redistilled water. These experiments were done in the following manner. Fifty or more fully developed eggs were drawn with a medicine dropper from a darkened culture bottle and put on a glass slide in a drop of 0.15 M phosphate buffer solution (pH 7.2) between two parallel ridges of Vaseline. A cover slip was placed over the drop and pressed down to form a seal with the Vaseline. Thus the cover slip, slide and Vaseline ridges enclosed a shallow channel running parallel with the length of the slide and measuring lo- to 15-mm wide by 0.5- to l-mm deep. Fluids could be made to flow slowly and continuously through this channel by adding them dropwise at the right of the channel and removing the overflow with absorbent filter paper at the left. The eggs usually remained fixed in place beneath the cover and were bathed by the fluids which flowed past. After this preparation each slide was placed beneath a fluorescent light to stimulate hatching of the eggs. The eggs began to hatch usually within a few minutes and as each egg opened the material of the viscous cushion flowed out into the buffer solution to form a typical envelope. After a few minutes the slide was placed on the microscope stage and a single hatched egg with its attached envelope was brought into focus at a magnificat,ion of 100X. Then a drop of Pyrex-redistilled water (pH 7.2) was put on the slide at the right of the channel and this water was caused to flow through the channel by placing absorbent filter paper at the left. Within seconds after the addition of the pure water the wall of the envelope in the field of vision began to expand and as it expanded it lost its refractive properties and became almost invisible. Expansion of the envelope wall continued until it was two or three times its original size. The volume of the cavity enclosed by the wall of the envelope changed only slightly. After exposing the envelope momentarily to a stream of the Pyrex-redistilled water, a drop of the 0.15 M buffer solution was added to the right of the channel and a fresh piece of filter paper
HATCHING OF FASCIOLA HEPATICb
EGG. II
135
was placed at the left. The buffer solution replaced the pure water in the vicinity of the egg and as it did so the wall of the envelope contracted again to its original size. The experiment just described was repeated many times and with several variations, In several instances nigrosin was used in the buffer solution to surface stain t,he envelope wall and render it] visible during the expansion in pure water. Another variation tried involved freeing the envelope from the egg before subjecting it t,o the solutions. The envelope was caused t,o break away from the egg by tapping t,he ~OWI slip directly above the egg with a dissecting needle. The freed envelope, torn open at t’he side, was t,hen exposed alternately to water and buffer solution. All of these experiments produced similar results. Pyrex-redistilled water caused the wall of the envelope to expand and phosphate buffer solution caused it to contract,. However, in every case it was essential that the buffer solution be replaced by Pyrex-redistilled water soon after expansion of t,he envelope wall, for when pure water was allowed to remain in contact with the envelope for longer t,han a fen minutes the wall of the envelope dispersed and failed to return t)o t,hc contracted condition following the addition of buffer solution. The results of these experiments suggest that’ the wall of the envelope has the properties of a colloidal system that contracts to form a gel on contact, with either the ions of t,ap water, phosphat,e buffer solut’ion, or dilute NaCl solution, and expands to form a sol when these ions are removed or greatly diluted by the addition of pure water. 11 THEORETIC!.~L EXPLAXATIOX FOR Ex~a~sro~ OF THE VISCOUS CUSHIOK DURIXG HATCHING
The observations suggest a plausible explanation for the phenomenon of expansion of the viscous cushion during normal hatching, and for expansion of the viscous cushion when t,he unhatched egg is treated with heat or exposed to various toxic solutions. If the material of the cushion has the properties of a colloidal system after hatching, it probably exists also as a colloidal system while it is within t*he egg. It follows that if the concentration of ions wit,hin the egg were to be lowered markedly in some manner, such as through the osmosis of salts or other materials from the egg to the surrounding medium, the viscous cushion within the egg would be expected to change from a gel to a sol, and the change would he accompanied by expansion. The writ’er believes that expansion of the cushion is accompanied by au exosmosis of salt’s or ot,her ma-
136
WILLIAM
B.
ROWAN
terials. Hereafter “salts” will refer to those undetermined materials. It is suggested that the exosmosis of “salts” occurs as a result of naturally or artificially induced damage to the vitelline membrane. Exposure of eggs to heat or to toxic chemicals could readily damage the selective permeability of the vitelline membrane and initiate an exosmosis of “salts.” This assumption is supported by work reviewed by Needham (1931, p. 798) indicating that when the trout egg is injured or poisoned the permeability of its membranes increases and salts diffuse out of the egg in measurable amounts. During normal hatching, damage to the vitelline membrane of the egg with subsequent exosmosis of “salts” and expansion of the viscous cushion, could result from either of two causes or from a combination of them. In an earlier paper (1956) the writer demonstrated that the miracidium releases a proteolytic enzyme prior to hatching and that this enzyme destroys an opercular cement. Possibly this enzyme also damages or destroys the vitelline membrane. In this connection it can be said that fishery workers according to Needham (1931, p. 1598) are familiar with the fact that “the membranes of the trout egg, while at first thick and parchment-like, become at hatching thin and brittle.” It is stated that the hatching enzyme released by the trout embryo is powerful enough to kill the embryo and there is “literally a race as to whether hatching or death will take place first.” Many embryos are “so weakened or necrosed at hatching that they cannot long survive it . . . and such necrosed embryos have the tips of their tails and fins eaten away.” A second factor may account for the proposed naturally-induced damage to the vitelline membrane with an accompanying exosmosis of “salts.” The enzymatic release of the operculum is undoubtedly a gradual process. Leakage probably occurs about the digesting opercular border as the operculum gradually splits away from the remainder of the shell. Since both the shell and the vitelline membrane serve normally as differentially permeable barriers to the passage of salts, leakage would upset the particular osmotic balance between the vitelline membrane and the shell. Such a change could produce damage to the vitelline membrane and cause an exosmosis of “salts” from the egg. In his statements concerning the egg of C. cotylophorum, Bennett (1936) notes that rough handling of an egg in one case loosened its operculum and allowed the entrance of water, and that this change resulted in immediate expansion of the viscous cushion. Similar rough handling was found occasionally to cause expansion of the viscous cushion of the unhatched F. hepatica egg.
HATCHING
OF
FASCIOLA
HEPATICA
EGG.
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137
THE EXOSMOSIS OF “SALTS”
In support of the hypothesis that has been presented a series of 20 experiments were done over a period of several weeks which demonstrate that there is a decrease in the vapor pressure of wat,er surrounding unhatched eggs of F. hepatica following the expansion of t,he viscous cushion. By virtue of the conditions of each experiment,, it is presumed that the decrease in vapor pressure is the result, of t,he osmosis of “salts” from the eggs into the surrounding water. In each experiment over 100 fully developed, but unhat’ched eggs of F. hepatica were removed from a darkened culture bottle and plunged into Pyrex-redistilled water at a t,emperature of 45°C. They were held at that temperature for 30 minutes. This exposure to heat killed the miracidia within t)he eggs but. did not, cause either expansion of the viscous cushion, release of t,he operculum, or any other visible change in the eggs. Exposure to 45°C apparently inactivates at least temporarily any hatching enzyme present in the eggs, since those which had undergone this exposure frequent,ly remained unchanged for periods of as long as 72 hours. After the exposure to heat, these fully developed, unhatched eggs containing dead larvae were transferred immediately to a Syraruse watch glass containing Pyrex-redistilled water and washed thoroughly with several changes. Any damaged, abnormal or undeveloped eggs were removed and the remaining ones were transferred to another Syracuse watch glass of Pyrex-redistilled water. These eggs were caused to collect, on the bottom of the watch glass towards the center by gently swirling the dish. One end of a fine capillary tube (600 microns outside diameter, 400 microns inside diameter) was int’roduced into the watch glass, and using the method of manipulation of the capillary tube dewrihed prcviously by the writ.er (19.56) three droplets of water were drawn into the tube. The droplets in the tube measured approximately 900 microns in length and were separated from each other by short air spaces (GO0microns in length). Following the introduction of t,he three water droplets, approximately one-half of the eggs on the bot,tom of the watch glass were drawn into the capillary tube as a part of a fourth droplet of water. The egg-containing droplet was adjusted to a length of approximately 900 microns, the adjustment being accomplished without removing t,he capillary tube from beneath the surface of the water. A fourth air space was interposed and three additional water droplets separated by air spaces were drawn into the t,ube. Aft.er t’he seven separate droplets of wat.er were centered in t’he tube both ends were sealed by fire. During
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ROWAN
the sealing procedure the droplets were protected from the heat by covering the section with the fingers. A second tube was filled and sealed in exactly the same manner using the remaining eggs in the watch glass. The first tube was immersed in a water bath, temperature 65”C, for 10 minutes, and after removal was fixed to a microscope slide. The second tube was not exposed to the hot water, but was fixed beside the first one on the slide. Microscopic examination revealed that the hot water had caused the expansion of the viscous cushion in each of the eggs in the egg-containing droplet of the heat treated tube. The eggs in the eggcontaining droplet of the untreated tube remained unchanged. As previously stated, 20 experiments were done and in each two capillary tubes prepared as described were used. Each tube contained seven droplets of Pyrex-redistilled water of nearly equal length separated by air spaces and the central droplet in each tube contained 25 to 75 eggs, The two tubes in each experiment differed in that one had been exposed to a temperature of 65°C for 10 minutes while the other had not. The eggs in the experimental tube contained fully developed, dead larvae and the viscous cushion of each egg was expanded. The eggs in the control tube also contained fully developed, dead larvae and the viscous cushion of each egg was unexpanded-unchanged in volume from the normal. The temperature of the room was recorded and the length of each of the droplets in each tube was measured microscopically, using an ocular micrometer at a magnification of 52.5X. After the measurements had been taken the glass slide bearing the two tubes was placed in an incubator and maintained at a temperature of 25 to 27°C for 24 hours. The slide was removed from the incubator, to a room temperature adjusted to the temperature of the previous day, and the droplets in each tube again were measured. Then both tubes were opened with a glass file and their contents expelled into separate water glasses containing Pyrex-redistilled water. A record was made of both the number of eggs in each tube and the number of these in each tube which contained a viscous cushion in the expanded condition. The results of the 20 experiments are recorded in Table I. It is evident, from the data in Table I that in each experiment the egg-containing droplet of the tube subjected to heat increased measurably in length, while the length of the egg-containing droplet in the control tube changed only slightly or not at all. Likewise, the water droplets on either side of the egg-containing droplet, decreased in length in each experimental
HATCHING
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FASCIOLA
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II
TABLE I Changes in Droplet Length in Capillary Tubes Used as a Measure of Vapor Pressure Changes Within the Droplets Experimental
-. T otal number of eggs -
Experiment
,
25 37 28 35 42 65 53 58 39 75 46 84 68 44 54 57 52 61 53 67 52
1
2 3 4 5 6 7 8 9 10 11
12 13 14 15 16 17 18 19 20 Average -
--
25 36 28 35 42 65 53 58 39 75 46 84 68 44 54 56 52 61 53 67 52
Control tube
Percent change in length of the three center droplets
I! 5
tube
4 5% rd’c1 3
5 5% cd-c 3
-12.0 -10.2 -8.6 0 -7.6 -6.8 -10.8 -7.6 -3.5 -4.8 -11.7 -3.5 -7.1 -3.1 -5.7 -5.8 -1.4 -6.1 -4.0 -10.7 -6.5
rota1 num,er of eggs
f21.4 +18.9 +8.2 +16.4 +16.8 +12.7 f7.5 +10.0
+8.8 $5.9 +6.4 +4.6 +10.5 +10.0 +10.0
$8.8 +4.8 +9.4 +8.1 $8.9 -i-10.4
-11.3 -10.0
-7.5 -20.0 -8.7 -11.4 -11.5 -11.3 -6.2 -6.4 -4.9 -4.5 -4.4 -6.1 -3.4 -4.2 -1.6 -5.8 -2.5 -3.2 -7.2
-
- T 5 4 8 B 31 30 29 48 34 45 48 57 64 43 68 46 55 28 70 39 53 55 53 69 48
-
- ~.%Z ;gp !ge 3 0 0 0 3 0 2 3 4 0 0 2 2 3 3 0 5 2 2 3 2
1Percent change in length of the three center droplets
-1.6 0 0 -3.1 -1.9 0 -5.7 -4.8 0 0 0 0 0 0 0 0 f2.4 0 0 0 -0.7 -
-
$1.9 i-1.7 -0.9 f5.4 +2.7 0 +1.4 +4.9 0 f2.9 0 +1.3 0 +1.6 f1.6 0 0 0 $0.9 0 $1.3
-1.9
-4.4 i-2.1 -5.7 -2.9 -0.8 0 0 -4.9 -4.6 0 0 -1.9
-2.2 0
-
-1.0 0 0 0 0 -1.4
tube, but their lengths changed very little or not at all in the control tubes. The procedure described above is a modification of the capillary tube method of Halket (1913) for determining the osmotic pressure of unknown solutions. It has been used recently by Hayes (1953) to find the osmotic pressure of the blood of the mosquito, Aedes aegypti. The method incorporates a very simple and useful physical principle. If two solutions of different salt concentration are separated from each other by an air space, but are hermetically sealed in a tube, water molecules will diffuse through the air from the solution of lower concentration
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WILLIAM
B. ROWAN
which, however, has a higher vapor pressure, to the solution of higher concentration (lower vapor pressure). Applied to the experiment just described it means that in each case the egg-containing droplet of the experimental tube must have had, during the incubation period, a higher “salt” concentration (lower vapor pressure) than the bracketing but airseparated droplets of Pyrex-redistilled water. The ‘$alt” concentration of the egg-containing droplet of each control tube, on the other hand, must have been essentially the same as that of the bracketing droplets of Pyrex-redistilled water. Since the experimental and control tubes in each experiment were prepared in the same manner the egg-containing droplets in both tubes had the same “salt” concentration (vapor pressure) at the time they were sealed. The change in concentration in the egg-containing droplet of the experimental tubes can readily be accounted for by assuming an osmosis of “salts” from the experimental eggs into the egg-containing droplet. The fact that the experimental eggs differ visibly from the control eggs only in having an expanded viscous cushion leads one to believe that the exosmosis of “salts” is associated with and perhaps accounts for the expansion of the cushion. THE CHEMICAL
NATURE
OF THE VISCOUS CUSHION
Results of attempts to determine the chemical nature of the viscous cushion were inconclusive. The colloidal nature of the cushion material suggested tests for protein. In fact Onorato and Stunkard (1931) stated that the cushion material from hatched Spirorchis eggs gives a protein reaction. The envelope formed from the cushion material of hatched 8’. hepatica eggs failed to give the characteristic reaction for protein when heated in 2% ninhydrin reagent. On the other hand, the potassium ferrocyanide reaction for protein of Hartig-Zacharias (Gomori, 1952) was positive. Furthermore, the envelope was rapidly destroyed by artificial digestive fluid (1 g dry pepsin, 1 ml HCl, 100 ml water) and by a solution of trypsin (10 mg per ml in 0.15 M phosphate buffer solution, pH &LO), but the envelope was not destroyed by either hyaluronidase from bovine testes (10 mg per ml in 0.15 M phosphate buffer solution, pH 6.4), by malt diastase at the same concentration (pH (i.9), or by control solutions of any of these four enzymes. These results would seem to indicate that the cushion material is at least in part composed of protein. The negative ninhydrin reaction, however, casts some doubt on this conclusion.
HATCHING OF FASCIOLA HEPATICA EGG. II
141
DISCUSSION
The viscous cushion within the egg of F. hepatica is described as a colloid which changes from a gel to a sol with changes in the ion constitution of the egg contents. The writer believes that any expansion of the viscous cushion within the egg results from damage to the vitelline membrane-damage which permits an osmosis of salts or other materials from the egg into the surrounding medium. If this explanation is correct then expansion of the viscous cushion plays no active role in egg hatching. The phenomenon simple accompanies hatching as a natural result of chemical and physical changes that are in process within the egg at this time. It will be recalled that Onorato and Stunkard (1931) were the first to mention the formation of a flexible envelope from the material of the viscous cushion of a trematode egg as the material flows out during normal hatching. The envelope traps the emerging miracidium and delays its escape, and they suggest that in the case of Spirorchis sp. the envelope gives t,hc miracidium a necessary period in which to adjust gradually to the new environment outside the egg. The writer does not believe that the miracidium of F. hepatica requires an adjustment period after release of the operculum. He has watched repeatedly perfectly normal miracidia emerge in a matter of seconds from eggs hatching in Pyrex-redistilled water, and swim energetically away to live for several hours, and yet it will be recalled that the envelope does not form when an egg hatches in Pyrex-redistilled water. The developing miracidium within the trematode egg is surrounded by two delicate, differentially permeable membranes and is therefore vulnerable to many physical and chemical factors in the medium surrounding these membranes. It should be productive to determine the effect of various penetrable materials on the development of the miracidium and on its subsequent ability to infect an intermediate host. The possibility of using a safe, inexpensive prophylactic drug against the miracidium should be explored. SUMMARY
1. A viscous and granular cushion of material at the opercular end of the fully developed egg of Fasciola hepatica expands within the egg just prior to the release of the operculum during normal hatching. This expansion is shown to play a passive role in the hatching phenomenon.
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2. The cushion resemblesa colloid and consists at least in part of protein. It changes from a contracted gel to an expanded sol with changes in the ion constitution of the fluid bathing it. 3. Evidence is presented to show that expansion of the cushion within the unhatched egg is accompanied by osmosis of salts or other materials from the egg. This exosmosis is believed to result under natural conditions from damage to or destruction of the vitelline membrane by the direct or indirect action of the hatching enzyme. 4. Expansion of the cushion within the egg can be artificially induced by exposing the egg to heat or to toxic solutions. This change provides a valuable visual clue to the penetrability to toxic materials and may play a useful role in ovicide research. ACKNOWLEDGMENT The writer is grateful to Dr. Wendell H. Krull for his helpful criticism of the manuscript.
(Oklahoma A and M College)
HXPERENCES BARLOW, C. H. 1925. The life cycle of the human intestinal fluke Fasciolopsis buski (Lankester). Am. J. Hyg. Monogr. Ser. 4, July, 98 pp. BENNETT, H. J. 1936. The life history of Cotylophoron cotylophorum, a trematode from ruminants. Illinois Biol. Monogr. 14, 1-119. GOMORI, G. 1952. Microscopic Histochemistry. The Univ. of Chicago Press,
Chicago, 273 pp. HALKET, A. D. 1913. On various methods for determining osmotic pressures. New Phytologist 12, 164-176. HAYES, R. 0. 1953. Determination of a physiological saline solution for Aedes aegypti (L.). J. Econ. Entomol. 46, 624-627. MATTES, 0. 1926. Zur Biologie der Larvenentwichlung von Fasciola hepatica,
besonders tiber den Einfluss der Wasserstoffionenkonsentration auf das Ausschltipfen der Miracidien. Zool. Anz. 69, 133-156. NEEDHAM, J. 1931. Chemical Embryology. Cambridge Univ. Press. Cambridge, 2921 pp. ONORATO, A. R., AND STUNKARD, H. W. 1931. The effect of certain environmental factors on the development and hatching of the eggs of blood flukes. Biol. Bull. 61. 129132. ROWAN, W. B. 1956. The mode of hatching of the egg of Fasciola hepatica. Exptl. Parasitol. 6, 118-137.