1. Various flagellates and small ciliates stick to the axopods of Actinophrys. Contact with the base of an axopod or with the body surface leads to the outgrowth of a food funnel, by which the prey is ultimately surrounded.

  2. If a fine probe or micropipette touches the body surface or the base of an axopod, a small cup may grow out over it, investing it closely, but the instrument is soon released. A squirt of tap water from a micropipette may also provoke the outgrowth of a small lobe or cup, or local pinocytosis.

  3. Contact with, or a squirt from, a micropipette containing a solution of egg albumin provokes a more extensive reaction. The micropipette usually becomes invested extensively. The micropipette is drawn into the body and held there for up to an hour.

  4. Immersion in egg albumin solution leads to a temporary spreading and lobulation of the axopod bases, and later a ‘skin’ may separate from the body surface. Skin formation is more pronounced in serum albumin solution, and may also be induced by y-globulins and gelatin.

  5. On treatment in vivo with toluidine blue or thionine a violet layer in the body surface separates as a pinkish violet or violet skin, leaving the body surface unstained.

  6. There is evidence that the skin-forming substance is associated directly or indirectly with the maintenance of cell shape.

The Heliozoa are carnivorous. Suitable microscopic oragnisms which swim onto the axopods of Actinophrys and Actinosphaerium are in some way held fast; a membranous funnel grows out and surrounds the prey; and digestion proceeds in the resulting food vacuole, which is drawn gradually into the body of the captor (Looper, 1928).

This study began with an investigation of the feeding reactions of Actinophrys, and in particular with the conditions which lead to the outgrowth of the membranous funnels. This led to observations on the curious phenomenon of the elevation and separation of a sheath or ‘skin’ from the body surface, which takes place in response to the presence of certain substances in solution in the outside medium.

A clone of Actinophrys sol was grown on a diet of Colpidium and Astasia in Bristol tap water. The stock cultures were normally fed once a week, and were used 5–10 days after feeding.

Bovine gamma globulins fraction II, bovine plasma albumin fraction V, crystallized bovine plasma albumin, and crystallized egg albumin were obtained from the Armour Laboratories, flake egg albumin from B.D.H., and bacteriological peptone from Hopkins and Williams.

Material

The ciliates Tetrahymena pyriformis, Colpidium sp., Colpoda cucullas, and the flagellates Peranema trichophorum, Astasia longa, Chlamydomonas pulsatilla and Chlamydomonas globosa, were used as test food. Tetrahymena pyriformis was originally received from Dr Muriel Robertson, F.R.S., and all the remainder except Colpidium sp. were supplied by Mr E. A. George from the Cambridge culture collection.

Methods

Usually a drop of Actinophrys culture and a drop of the culture to be tested as food were mounted together under a cover-glass supported by filter-paper. Sometimes the Heliozoa were mounted first and the food was added by irrigation. When Tetrahymena pyriformis was given as food, this ciliate was first transferred gradually to Bristol tap water since its normal culture medium had a rather high osmotic concentration.

Capture of prey

Organisms which happened to touch the axopods of Actinophrys were seen to stick. Sometimes they only stuck momentarily, sometimes they broke loose after further swimming movements, and sometimes they remained stuck. Flagellates usually got caught on the distal parts of axopods and gradually moved along the axopods towards the body of the Actinophrys until engulfed by a membranous funnel. It is not clear whether the movement towards the body was due to the prey itself or to the axopods. All the flagellates tested underwent this process except Chlamydomonas globosa which adhered to the axopods and usually (but not always) escaped. Small ciliates swam between the axopods and were possibly guided by these, until they hit the body surface proper. Fast-swimming ciliates often broke loose again. Flagellates normally remained stuck long enough to be enveloped by a funnel, and small ciliates also, provided they were not excited.

Swimming movements normally continued in ciliates, and often in flagellates, after they had stuck ; writhing or euglenoid movements were often made by euglenoid flagellates at this stage. There was no evidence of any rapidly spreading paralysis. Adhesion was by the flagella and cilia, and it appeared that this adhesion would explain the limitation of the movements of the prey in the early stages of captivity. Flagellates and ciliates which had broken loose often had small beads or droplets on the flagella or cilia which had been held fast. Sometimes they swam away, but usually their swimming movements were abnormal, and sometimes they stopped moving.

Outgrowth of membranous funnels

Stages in the engulfment of prey are illustrated in Text-fig. 1. Outgrowth of a membranous funnel only occurred soon after the prey had touched the body surface (excluding the axopods) of the heliozoon, or the thickened base of an axopod. Thus it was often delayed for flagellates, but with ciliates it often happened very quickly after first contact of prey with predator. Outgrowth sometimes happened even if the ciliate was moving very fast and failed to stick; and sometimes a series of collisions provoked the formation of numerous unsuccessful funnels.

Text-fig. 1.

Diagrams showing the capture of (a) Tetrahymena pyriformis, (b) Astasia langa, and (c) Chlamydomonas pulsatilla, by Actinophrys sol.

Text-fig. 1.

Diagrams showing the capture of (a) Tetrahymena pyriformis, (b) Astasia langa, and (c) Chlamydomonas pulsatilla, by Actinophrys sol.

All the species of flagellates mentioned as test food were at some time eaten by Actinophrys sol. Of the ciliates, Colpoda cucullas was captured most readily, probably because it is a rather weak swimmer. Of the flagellates, Chlamydomonas globosa was only occasionally eaten; it stuck to the axopods but usually failed to move along them, so that no funnels grew out.

Food vacuoles

As soon as a funnel had closed to form a food vacuole, the prey became more active, probably because it was no longer held by an axopod. Ciliates turned round and round, and flagellates writhed. Movements of the prey continued for a period ranging from a few minutes to half an hour ; and the contractile vacuole of the prey was seen to continue operating. Ultimately all activity ceased, and the surface membrane of the prey suddenly disintegrated.

Meanwhile the food vacuole was drawn closer to the body of the Actinophrys and became partly embedded in it Small protuberances grew out from the outer edge of the food vacuole.

General methods

Actinophrys was mounted in a hanging drop of its own culture medium, sealed from evaporation with mineral oil (Boots’s medicinal paraffin BP). A de Fonbrune micromanipulator and microforge were used. Pipettes normally had an internal diameter of 1–2 μ. Test solutions were made in Bristol tap water.

Results

If a glass probe or a micropipette filled with tap water was placed with the tip in contact with the body surface or with an axopod base, it was usually held immediately as though stuck. Within a minute or two a small cup grew out around it, investing it closely (Text-fig. 3). The cup then withdrew, and the instrument was usually released within 10–20 min. Sometimes a vacuole was formed by closure of the cytoplasmic cup, and this was absorbed into the body.

Text-fig. 3.

Diagram showing the effects of (a) contact with a glass probe at the body surface, and irrigation with (b) 0·05 % γ-globulms, (c) 5 % serum albumin fraction V, and (d) 1 % egg albumin (flake), in four separate experiments.

Text-fig. 3.

Diagram showing the effects of (a) contact with a glass probe at the body surface, and irrigation with (b) 0·05 % γ-globulms, (c) 5 % serum albumin fraction V, and (d) 1 % egg albumin (flake), in four separate experiments.

In many cases if an axopod was stroked there was a slight spreading of the base, with formation of lobes and protuberances and sometimes the appearance of small vacuoles. This response was much more easily elicited by mechanical stimulation of the proximal half of an axopod; usually mechanical stimulation of the distal half had little or no effect.

As a result of a considerable number of preliminary experiments, it was found that individual specimens of Actinophrys varied greatly in their response to a fine squirt of water from a micropipette, and also that the use of serum albumin in such experiments led to complications which will be discussed later. Accordingly, methods were standardized as far as possible and the later results obtained with egg albumin will be described first.

A comparison was made of the stimulating action of a micropipette filled with tap water and (immediately afterwards) of the same pipette filled with a solution of crystallized egg albumin . In any one experiment the same Actinophrys was used throughout, and the stimulus took the same form. Either the pipette was placed and left in contact with the body surface proper (without any squirting) or a squirt was given from a distance of 12 or 34 of the radius of the body (without axopods), and the pipette was left in position. If there was no response to the pipette filled with tap water the stimulus was repeated within a few minutes.

The results of these experiments are summarized in Table 1, and an example is illustrated in Text-fig. 2. With tap water in the pipette, contact with the body surface sometimes provoked the formation of a small cup around the pipette tip, but this was soon withdrawn; the response to squirting was no greater, and usually less. Egg albumin, whether administered by contact or by squirting, provoked a greater response; usually a cup held the pipette and spread a considerable distance up the outside of it, as illustrated in Text-fig. 2. The pipette was then drawn deep into the body. Finally it was extruded, usually 121 hr. after the initial stimulus.

Table 1.

Pokes and squirts with pipette loaded with (a) tap water, then (b) crystallized egg albumin dissolved in tap water

Pokes and squirts with pipette loaded with (a) tap water, then (b) crystallized egg albumin dissolved in tap water
Pokes and squirts with pipette loaded with (a) tap water, then (b) crystallized egg albumin dissolved in tap water
Text-fig. 2.

Diagrams showing the response of Actinophrys sol to a squirt of tap water and then of crystallized egg albumin (0·5 % in tap water).

Text-fig. 2.

Diagrams showing the response of Actinophrys sol to a squirt of tap water and then of crystallized egg albumin (0·5 % in tap water).

After preliminary work, the effects of squirting tap water and then bovine serum albumins fraction V (0·1% or 0·2% in tap water) were compared in fourteen experiments carried out otherwise as described above. Although the response was usually greater to the serum albumin than to tap water, the difference was not enough to carry conviction in view of the variation. The response to serum albumin ranged from the extension of a few small protuberances to the outgrowth of a large and extensive funnel which spread up the outside of the pipette. In two experiments no lobes were formed, but a skin was lifted up from the body surface in the region of the squirting and moved outwards along its axopods. This phenomenon is described in the next section. Owing to skin-lifting it was not profitable to use higher concentrations of serum albumin.

Methods

A number of Actinophrys and a little debris from the bottom of the culture were mounted under a cover-glass supported by filter-paper leads. With careful irrigation specimens having a few axopods lodged in debris were left undisturbed; and with extreme care it was possible to use individuals resting only on the glass. Irrigation was carried out by hand throughout an experiment, first with tap water, then with a solution of the test substance, and occasionally with tap water again. All solutions were filtered, in view of the fact that the y-globulin was found not to dissolve completely.

Results

Egg albumin (0·05–2·5 % ; 7 experiments with crystallized egg albumin, 11 experiments with flake)

There was an immediate shortening of the axopods, and an expansion and lobulation of their bases (Text-fig. 3), more marked at the higher concentrations. Within a few minutes or less, small vacuoles appeared at the surface of the axopod bases. I did not see where they came from, but I interpret them as pinocytic. After a few minutes the lobulation subsided and no more vacuoles appeared. Often, however, aggregations of material moved outwards along the axopods and were cast off at the tips (Text-fig. 3). In 0·5 % egg albumin and upwards, a coherent sheath or skin of similar material often separated from the body surface and moved outwards along the axopods.

No difference was seen in the responses to crystallized and to flake egg albumin. Serum albumin fraction V. (0·1–5%, 14 experiments, excluding preliminary work) There was little immediate change in the appearance of the axopods. ‘Skinlifting’ (Pl. 1) took place at all concentrations, though not in all individuals in the 0·1% solution. Skin-lifting was usually first apparent within a few minutes of irrigation with serum albumin solution, although sometimes it was delayed.

A sheath or skin appeared to form outside the body surface and began to separate from it, moving outwards along the axopods. Usually the separation started on one side but spread all round the body. The skin was then lifted completely off the body, and travelled outwards along a group of axopods, which were themselves drawn together in the process. At this stage the skin and axopods looked like a parachute. The skin was subsequently shed completely. The first stages of skinlifting proceeded rather quickly. A skin could move half way out along the axopods within a minute or two, but the later stages occupied 121 hr. or longer. With the higher concentrations (212 and 5 %) the skin was very much thicker (Text-fig. 3) and did not completely separate from the body during observations ranging up to 5 hr.

After separation of a skin the body surface was much more smoothly rounded, and the axopod bases appeared thinner. The organisms appeared perfectly healthy. When given food immediately after skin-lifting, many captured and ate Astasia and Colpidium, and some did so even though still partly surrounded by skins.

Normally after mass feeding of a culture of Actinophrys, groups of partially fused individuals are found sharing one or more common food vacuoles. On treatment with serum albumin such groups lifted skins, and afterwards the individuals of a group fused more completely, so that the angles between them disappeared and it was no longer possible to distinguish one individual from another.

Crystallized serum albumin has proved just as effective in provoking the lifting of skins, but the experiments concerned with this will be described in a later paper.

γ-Globulins fraction II (0·01 and 0 ·05%, 11 experiments)

In some cases local thickenings developed on the axopods, as though axopod substance had aggregated, and the tips of the axopods became bent. In some cases also an incomplete thin skin was lifted from the body surface, or small clumps of material appeared at the body surface and travelled quite rapidly outwards along the axopods. In one case a number of funnels grew out on irrigation with a 0·05 % solution.

Gelatin (Belgian ‘Gold Leaf’, 0·1%, 3 experiments)

A few specimens definitely formed skins. No extensive investigation was made. Peptone (Bacteriological, 0·1–3%, 9 experiments)

Occasionally the axopod bases expanded somewhat, and small vacuoles appeared in them. Out of numerous individuals three developed thin but recognizable skins.

Sucrose (0·01–0·1M, 4 experiments)

No skins were formed, and pinocytic vacuoles were rare.

Toluidine blue (0·5 × 10–2% and 0·25 × 10–2%, 10 experiments with toluidine blue only, 4 experiments after skin formation in 0·2% serum albumin fraction V) Direct treatment with toluidine blue solution led to a dense violet staining of the body surface. In many individuals a violet or violet pink skin was lifted off, leaving the body surface unstained. The body surface was smooth and rounded after the skin had separated.

Skins already induced by serum albumin stained blue on addition of toluidine blue, but later became more violet.

Thionine (1–5 % saturate, 9 experiments)

The body surface stained violet, and in some experiments a violet or violet pink skin then lifted off it, leaving it unstained. Sometimes there was no continuous skin, but a diffuse pinkish violet mass or a line of granules. The body surface was rounded and smooth after the separation of this material.

The formation of food cups by amoebae in response to the vibrations of a needle or to a capillary containing egg albumin has been described by Schaeffer (1917). It is clear that the outgrowth of a food funnel from Actinophrys sol can be caused by mechanical stimulation, and it appears also that a solution of egg albumin can assist the process. Immersion in a solution of egg albumin causes an expansion and a lobulation of the axopod bases, like that seen locally when dilute egg albumin is squirted at Actinophrys from a micropipette. It is not to be expected that combined chemical and mechanical stimulation with a micropipette will produce exactly the same pattern of response as that produced by a captured but still active ciliate. The beating of cilia probably holds the membranes off from the body surface of the prey and results in a much larger food vacuole, and the chemical stimulus provided by the ciliary surface may also be very different in its effects. Nevertheless, it is interesting to compare the mechanism of stimulation in Actinophrys with that determining the discharge of nematocysts, for which a combination of the two kinds of stimuli is much more effective (Pantin, 1942). It is possible that in Actinophrys also the dual mechanism of stimulation helps to prevent errors in response, although according to Looper (1928) Actinophrys eats inanimate particles.

The outgrowth of a food funnel in Actinophrys must represent a local excitation, and it is therefore reasonable to suggest that the advancing pseudopod of an amoeba is also in a state of excitation. In view of the efficacy of a biochemical stimulus it is likely that excitation occurs in the surface membrane. The small vacuoles which appear in the surface of the outgrowing lobes and protrusions are no doubt pinocytic. It is therefore likely that chemical stimulation and pinocytosis are closely related, and it is possible that both are brought about by surface adsorption (see Holter, 1959). It would be particularly interesting to know whether any local depolarization is associated with the outgrowth of a funnel of a pseudopod, and if so how this is affected by substances which promote pinocytosis or the formation of funnels.

The nature of the substance which forms the sheath or skin is not yet known, although its metachromatic staining is suggestive. Its origin and nature have not yet been traced. It emerges or separates from the body surface in response to the presence of certain substances, which include the proteins already mentioned; presumably it reacts with proteins. It is probably of the same general nature as that described for Tetrahymena by Bresslau (1922) and more especially by Robertson (1939), who showed that a thick sheath is developed in response to an antiserum. However, the skin-lifting of Actinophrys is far from specific. Its normal function is not known; it might perhaps serve to stick flagella and cilia, or to inactivate harmful proteins or other substances in the prey, or to hold on to debris during removal of these along the axopods. The rounding up of Actinophrys after skinlifting is particularly interesting, as it suggests that the skin material or layer may normally contribute to cell shape, although it is also possible that the influence is an indirect one.

I am much indebted to Mr E. A Livingstone for a steady supply of Actinophrys and for cultivating the various Protozoa used as food.

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Actinophrys sol was irrigated with 0·1 % serum albumin fraction V. The first photograph was taken 1 min. before, and the others at the time stated after irrigation with serum albumin began. Note the lifting and separation of the ‘skin’.

Plate 7

Kitching—responses of the heliozoon Actinophrys sol to prey, to mechanical stimulation, and to solutions of proteins and certain other substances

Plate 7

Kitching—responses of the heliozoon Actinophrys sol to prey, to mechanical stimulation, and to solutions of proteins and certain other substances