Immaturity substances in paramecium primaurelia and their specificity

An exconjugant clone of Paramecium, as with a variety of other ciliates, has a well-defined life cycle, consisting of the periods of immaturity, maturity and senility. During the immaturity period, Paramecium is unable to mate even under appropriate conditions. The duration of the immaturity period is measured in terms of the number of fissions after conjugation (Sonneborn, 1957; Miwa & Hiwatashi, 1970, and others). As already reported, cells of P. caudatum in the immaturity period contain immaturity substances which repress the expression of mating ability when introduced into mature cells by microinjection (Miwa, Haga & Hiwatashi, 1975). Immature cells of P. bursaria also contain immaturity substances but with different specificity to those of P. caudatum (Miwa, 1979). The duration of the immature period has been shown to be controlled genetically in P. primaurelia (Siegel, 1961) and some mutants with altered length of immaturity have been reported in Tetra-hymena thermophila (Bleyman & Simon, 1967) and in P. caudatum (Myohara & Hiwatashi, 1978). Thus, the phenomenon of immaturity in Paramecium provides one of the best systems for studying the genetics of biological timing because of the possibility of identifying its material basis through immaturity substances.

In the present article, I will show that the immature cells of P. primaurelia contain immaturity substances which are effective in inducing immaturity when injected into P. tetraurelia, P. caudatum and P. multimicronucleatum but are ineffective in P. bursaria.

Immature cells used as donor for the microinjection of cytoplasm were progeny from the cross between stocks 16 and 513, which belong to mating types I and II respectively in P. primaurelia (one of the sibling species in the P. aurelia complex (Sonneborn, 1975)). The stocks used as recipients and testers for mating reactivity were as follows. Stocks hr^d of mating types VII and VIII in P. tetraurelia (another sibling species in P. aurelia complex) were highly mating-reactive mutants derived from stock 51 (Sonneborn, 1974) supplied by S. Koizumi. Stocks d^m -21 (mating type V) and d^m -23 (mating type VI) belonging to syngen 3 of P. caudatum were F₃ progeny from a cross between natural stocks Kok 1 and Koj. Stocks 53B (mating type III) and 49B (mating type IV) of syngen 2 in P. multicronucleatum were acyclic strains obtained from Y. Takagi. Stocks B^w and c^w (complementary mating types belonging to an unknown syngen) in P. bursaria were Chlorella-free variants induced by rapid growth in the dark from natural stocks B^g and C^g collected by Y. Tsukii.

All stocks in the present experiment were cultured in lettuce medium which was 1 · 25 % (w/v) fresh lettuce juice in Dryl’s solution (Dryl, 1959), pH 7· 0, inoculated with Klebsiella pneumoniae I day before use (Hiwatashi, 1968). Cultures were kept at 25 °C.

Microinjection of cytoplasm was performed with a micromanipulator of de Fonbrune type (Narisige, Tokyo) with 2 needles after the method of Knowles (1974). Recipient cells after injection were kept in the cell-free culture supernatant from the stationary phase culture of the recipient for 3 h and then grown in a microcapillary (Drummond) with a certain volume of culture medium, and mating reactivity was observed in depression slides by adding about 100 highly reactive living cells of the complementary mating type.

About 5 x 10¹ µm¹ cytoplasm from immature cells (8 fissions after conjugation between stock 16 and 513) were injected into mating-reactive cells of stock 16 (mating type I), which had undergone 20 fissions since the last autogamy. As a control, the same volume of mature cytoplasm from mating-reactive cells of stock 16 (20 fissions after autogamy) was injected into cells of the same stock at the same stage. About 3 h after injection, each recipient cell was transferred into 1 µl of fresh culture medium in a microcapillary. Each injected cell undergoes 1-2 fissions 1 day after injection, and 3 fissions 2 days after injection in the microcapillary. Mating reactivity of each recipient clone was tested 2 days after injection in a depression slide by adding 10 µl of mating-reactive culture (contains about 100 cells) of stock 513 (mating type II). The number of mating-reactive cells in the recipient clone, which usually contains 8 cells, was calculated. The results are shown in Table 1. In 19 out of 32 clones injected with immature cytoplasm, no cells expressed mating reactivity when tested 3 fissions after injection, but in the control, which was injected with mature cytoplasm, all clones showed mating reactivity. The immature cyto-plasm of P. primaurelia can induce immaturity when injected into mature cells.

In order to know whether immature cytoplasm of P. primaurelia has an ability to induce immaturity in the mating-reactive cells of P. tetraurelia, which is known to have no immaturity period after conjugation, stock hr^d of P. tetraurelia (mating type VII) was used as the recipient cells. Stock hr^d cells are suitable for such experiments since mating reactivity of this stock is strong and is retained for a long time. About 5 x 10¹µm¹ cytoplasm from immature cells (8 fissions after conjugation between stock 16 and 513) were injected into mating-reactive cells of stock hid (VII) 5-6 fissions after autogamy. The injected cells were transferred into I µI fresh culture medium after being kept for 3 h in the cell-free culture supernatant and were grown for 2 days, during which the cells divided 3 times. Mating reactivity of each recipient clone was then tested by adding 10 µl mating-reactive culture of stock hr^d (VIII) 6-7 fissions after autogamy. In the control, mating-reactive cells of hr^d (VII) 5-6 fissions after autogamy were injected with the mature cytoplasm from mating-reactive cells of stock 16 in P. primaurelia. Mating reactivity of the control was tested in the same way. As seen in Table 2, cells of P. tetraurelia which have no immaturity period after conjugation showed immaturity after the injection of immature cytoplasm from P. primaurelia. The results suggest that the effect of immature cytoplasm of the P. aurelia complex has no species specificity within the complex.

To examine further the species specificity of the effect of immature cytoplasm in P. primaurelia, immature cytoplasm of P. primaurelia was injected into mature cells of P. caudatum, P. multimicronucleatum and P. bursaria. Immature cells 8 fissions after conjugation between stock 16 and 513 (P. primaurelia) were used as donors and mating-reactive cells of d^m -23 (P. caudatum), 49B (P. multimicronucleatum) and B^w(P. bursaria) were used as the recipients. About I x 10¹ µm¹ cytoplasm from the immature cells of P. primaurelia were injected into mating-reactive cells of the recipient stocks mentioned above. About 3 h after injection, each recipient cell was transferred into 2 µl fresh culture medium in a microcapillary and was grown for 2 days, during which period most cells divided twice. Mating reactivity of each recipient clone was tested 2 days after injection by adding 20 µI mating-reactive cultures of complementary mating types, stocks d^m -21, 53B and c^w respectively. In the controls, mating-reactive cells of d^m -21, 53B and n^w were injected with cytoplasm from mating-reactive cells of stock 16. The results are shown in Table 3. Many recipient clones of P. caudatum and P. multimicronucleatum which were injected with P. primaurelia immature cytoplasm failed to express mating reactivity when tested 2 fissions after injection, while normal mating reactivity was observed in the controls. In P. bursaria, however, no significant difference was observed between the experimental and the control groups. The immature cytoplasm of P. primaurelia is effective in inducing immaturity when injected into P. caudatum and P. multimicronucleatum but ineffective in P. bursaria.

Next, experiments were performed to discover the reciprocal effects of immature cytoplasm. Immature cells 15 fissions after conjugation between d^m -21 and d^m-23 of P. caudatum were used as the donor. Mating-reactive cells of stock 16 (mating type I) 20 fissions after autogamy in P. primaurelia and of hr^d (VII) 5-6 fissions after autogamy in P. tetraurelia were used as the recipients. The cells injected with about 5 x 10¹ ;1,m¹ donor cytoplasm were kept in the cell-free supernatant for about 3 h and then grown in 1 µ l fresh culture medium for 2 days, during which period most cells divided 3 times. Mating reactivity of each recipient clone was tested 2 days after injection by adding 10 µ l mating-reactive culture of stock 513 (mating type II) in P. primaurelia or of hr^d (VIII) in P. tetraurelia. In the control, mating reactive cells of stocks 16 and hr^d (VII) were injected with cytoplasm from mating reactive cells of d^m-23. The results are shown in Table 4. As seen in the table, many clones not only of P. primaurelia but also of P. tetraurelia failed to express mating reactivity when tested 3 fissions after injecting immature cytoplasm of P. caudatum, while no significant loss of mating reactivity was observed in the controls. The immature cytoplasm of P. caudatum is effective in inducing immaturity when injected into P. primaurelia and P. tetraurelia.

Almost the same experiment was done with immature cells of P. bursaria as the donor. About 5 x 10¹µ1,m¹ cytoplasm from immature cells of P. bursaria (15 fissions after conjugation between n^w and c^w) were injected into mating-reactive cells of stock 16 (mating type I) in P. primaurelia 20 fissions after autogamy and of stock hr^d (VII) in P. tetraurelia 5-6 fissions after autogamy. In the control, mature cytoplasm of stock n^w was used for injection. About 3 h after injection, each recipient cell was grown in 1 µl fresh culture medium for 2 days, during which period cells underwent 3 fissions. Mating reactivity of each clone was tested 2 days after injection by adding 10 µ l mating-reactive culture of stock 513 (mating type II) in P. primaurelia or of stock hrd (VIII) in P. tetraurelia. The results are shown in Table 5. Essentially normal mating reactivity was observed in either the experimental or in the control groups. The immature cytoplasm of P. bursaria is ineffective in inducing immaturity in P. primaurelia or in P. tetraurelia.

In the previous paper (Miwa, 1979), I reported that the immature cells of P. caudatum and P. bursaria contain immaturity substances that repress the mating ability when introduced into mature cells by microinjection, and that a difference in specificity of the immaturity substances exists between those 2 species. The present experiments extend those observations to the P. aurelia complex. Immature cells of P. primaurelia also contain an immaturity substance and its specificity is the same as that of P. caudatum; immature cytoplasms of the 2 species are reciprocally effective in inducing immaturity in mature cells. Though reciprocal injections were unable to be done with P. multimicronucleatum, because immature cells were not obtained in the available stocks of this species, the available evidence suggests that the specificity of the immaturity substances is common in 3 species complexes of the ‘aurelia group’: P. caudatum, P. aurelia and P. multimicronucleatum. Only one species, P. bursaria, among those in the’ bursaria group’ was tested and its immaturity substance is different from that of the ‘aurelia group’. Other species of the ‘bursaria group’ should also be examined.

P. tetraurelia is a sibling species in P. aurelia complex which has no immaturity period after conjugation but nevertheless responds to immaturity substances from P. primaurelia and from P. caudatum. The cells of P. tetraurelia probably share with P. primaurelia and P. caudatum a common receptor to the substances.

In the late part of the period of maturity in the P. aurelia complex, autogamy is induced in response to food depletion (see Sonneborn, 1974). Whether the induction of autogamy is delayed by the injection of the immaturity substances is an interesting problem which is now under investigation.

The author wishes to thank Dr K. Hiwatashi for help in preparation of the manuscript. This work was supported by a grant from the Japanese Ministry of Education, Science and Culture.