MATING TYPES IN Glaucoma 375

5. Guttman, H. N. & Wallace, F. G. 1964. Nutrition and physiology of the Trypanosomatidae. In Hutner, S. H. (Ed.) Biochemistry and Physiology of Protozoa, Academic Press, N. Y.

6. Kaufman, S. 1967. Pteridine cofactors. Ann. Rev. Biochem.

7. Kidder, G. W. 1967. Nitrogen: distribution, nutrition and metabolism. In Florkin, M. & Scheer, B. T. (Eds.), Chemical Zoology, (Kidder, G. W., Ed.), Academic Press, N. Y. 1, 93-159.

8. Kidder, G. W. & Dewey, V. C. 1968. A new pteridine from Tetrahymena. J . Biol. Chem. 243, 826-33.

9. Kidder, G. W. & Dutta, B. M. 1958. The growth and nutrition of Crithidia fasciculata. J . Gen. Microbiol. 18, 62 1-38.

10. Nathan, H. A. & Cowperthwaite, J. 1955. “Crithidia fac- tor”-a new member of the folic acid group of vitamins. J. Pro- torool. 2, 37-42.

3, 459-494.

36, 171-84.

1 1 . Patterson, E. L., Milstrey, R. & Stokstad, E. L. R. 1956. The synthesis of a pteridine required for the growth of Crithidio fasciculata. J . A m . Chem. SOC. 78, 5868-71.

12. Sakyrai, A. & Goto, M. 1967. Neopterin: isolation from human urine. J . Biochem. (Tokyo) 61, 142-5.

13. Simpson, L. 1968. Effect of acriflavin on the kinetoplast of Leishmania tarentolae. J . Cell Biol. 37, 660-82.

14. Trager, W. 1957. Nutrition of a hemoflagellate (Leishmania tarentolae) having an interchangeable requirement for choline or pyridoxal. I. Protorool. 4, 269-76.

15. -___ 1968. Cultivation and nutritional requirements. Chap. 8 in, Ristic, M. & Weinman, D. (Eds.) Infectious Blood Diseases, Academic Press, N. Y. 1, 149-74.

16. Trager, W. & Rudzinska, M. A. 1964. The riboflavin re- quirement and the effects of acriflavin on the fine structure of the kinetoplast of Leishmania tarentolae. J . Protorool. 11, 133-45.

J . PROTOZOOL. 16(2), 375-377 (1969).

Mating Type Inheritance in Glaucoma”

RUTH PHILLIPS and IRENE ABRAHAM2

Zoology Dept., Uniw. of Illinois, Urbana

SYNOPSIS. Mating was observed in collections of Glaucoma from 2 localities in Illinois. The collections could be divided into 2 syngens on the basis of mating type reactions. Corticotype analysis showed that syngen 1 was intermediate in meridian num- ber between Glaucoma chattoni and Glaucoma scintillans, while syngen 2 had the same range as Glaucoma scintillans (Lee, unpub- lished).

The major features of mating type inheritance in syngen 1 are that exconjugant clones usually have identical mating types and

LTHO a considerable literature exists on the genetics A of Tetrahymena (Nanney, 1968), very little informa- tion is available concerning even mating in the related genus Glaucoma. Chatton (1929) observed mating in Glaucoma scintillans and some of the older observations on Tetrahymena may have involved Glaucoma since only re- cently have the 2 genera been clearly differentiated (Cor- liss, 1953).

Recently several new strains in Glaucoma have been sub- jected to corticotypic analysis (Klug, 1968; Lee, unpub- lished) and mating types have been established in strains from 2 localities in Illinois. The Illinois strains were di- vided into 2 different breeding groups or syngens on the basis of mating type reactions. Corticotype analysis showed that the Ottawa strains were Glaucoma scintillans, while the Urbana strains were intermediate in meridian number between Glaucoma chattoni and Glaucoma scintillans (Lee, unpublished). Two different modes of mating type inheri- tance are indicated.

MATERIALS AND METHODS Two collections of Glaucoma, one from the vicinity of Urbana,

Illinois (Glaucoma sp.) and one from Ottawa, Illinois (Glaucoma scintillans) were grown in Cerophyl medium (0.15 %, w/v) inocu-

i t Supported by grant GM-0779 from the U. S. Public Health

Postdoctoral Trainee in Cell Biology, Training Grant GM- Service to D. L. Nanney.

0094 1 . ’ Undergraduate Honors Project.

that mating types I and I1 appear in an approximately 1 : 1 ratio in successive generations. This is the result expected in genic mat- ing type inheritance if one of the original parents was a homozy- gote and the other a heterozygote a t the mating type locus. No significant immaturity period was found in these strains.

Only 2 viable pairs were obtained from syngen 2 crosses, but re- sults from these suggest that mating type inheritance may be epi- genetic and a longer immaturity period may characterize this syn- gen.

lated with Aerobacter aerogenes. The cells were mated by mixing an equal volume of each culture (0.1 ml of each) plus 0.6 ml of bacterized Cerophyl diluted 50% in depression slides. Conjugating pairs were isolated in a depression slide approximately 24-30 hours after mixing the cultures in a small volume of bacterized Cerophyl diluted to 25%. Approximately 24 hours after isolation of the pairs the exconjugants were isolated into separate depressions. The depression from which the exconjugant cells were isolated was ob- served for cells, which would indicate that one or both of the ex- conjugants had undergone division before isolation of the supposed exconjugants. The same methods were employed in the 1st and 2nd generations.

Non-conjugants were defined as isolated pairs which after 24-30 hours had produced great numbers of cells in the depression. In pairs defined as true conjugants only 2 cells (or very rarely 3) were present in the pair isolation depression at this time since the act of conjugation delays cell division. Selfing cultures were de- fined as exconjugant clones within which mating occurred.

RESULTS

Two mating types were found in the Urbana collection (Glaucoma sp.) , and 3 mating types in the Ottawa collec- tion (Glaucoma scintillans) . Sporadic mating occurred be- tween strains of different collections, but these reactions were very weak and no viable progeny were obtained from these inter-crosses (40 pairs isolated). The Urbana collec- tion was designated Syngen 1, mating types I and 11; the Ottawa collection was designated Syngen 2, mating types I, I1 and 111. Mating occurs regularly between all com- binations of different mating types within the same syngen.

Syngen 1 . The viability of isolated pairs was followed

376 MATING TYPES IN Glaucoma

TABLE 1 . Crosses of Glaucoma, syngen I ,

Pairs Pairs Percent Pairs Pairs non- viable viable

Cross isolated died conjugants conjunants coniunants

TABLE 2 . Mating type of progeny of crosses PI x P I , F I X FI and F, x F, of Glaucoma syngen I .

Both Both 1 Selfer 1 Selfer Both Cross Mt I Mt I1 1 Mt I 1 Mt I1 Selfer Total

~~

Pi X Pi 144 101 26 1 7 12 Fi X Fi 150 89 26 35 23 Fz X F? 96 53 13 30 31 Total 390 243 65 82

Pi x Pi 9 5 1 1 17* FI X Fi 12 17 4 2 35 Fz X F, 10 16 4 30 Total 31 38 8 3 1 82

thru 3 generations of inbreeding and increased (Table 1) from 12% to 31% in this interval. Exconjugant clones from these crosses were initially tested for mating type after approximately 10- 15 cell divisions. One-hundred and sixty- four exconjugant clones representing 82 viable pairs were tested; of these all except 15 mated at the 1st test and some of the cultures selfed. The 15 clones which failed to react were taken thru 2-3 isolation transfers, aging the clones by an additional 20-40 cell divisions. Upon testing for mating after these transfers, all were sexually reactive.

Excluding selfers and with one exception, exconjugant clones from the same pair had identical mating types (Ta- ble 2). Unfortunately the exceptional pair, which was ob- tained in the 1st cross, was lost and could not be re-tested. Since the delay of cell division test is not conclusive proof of conjugation, this pair may have been a non-conjugant. All of the other exconjugant pairs had either the same mating type (84%) or else the pairs were mixed, with one selfing and one mating type I or 11, or both selfing. In no case did a mating type other than I or I1 appear in the progeny. These results would suggest direct genic mating type determination since the exconjugants appear to be genetically identical (Doerder, preliminary cytologic obser- vations). In all 3 generations the 2 mating types were found in approximately a 1 : 1 ratio. This is the result ex- pected if mating type is controlled by a single locus and if one of the original parents was a heterozygote and one a homozygote at the mating type locus.

Selfers were found in 16% of the pairs (8% of the total exconjugants) . To determine whether selfing cultures con- tained 2 stable mating types or if selfing was due to a per- sistent instability, 3 of the exconjugant clones that were observed selfing were expanded to 36 and transferred in daily isolation lines. The “left-over’’ isolation lines were examined for selfing. Most of the initial sublines selfed, a few were pure for mating type 11, and subsequently more sublines became pure for mating type 11. The data were complicated by death in the transfers, but strongly selfing cultures usually gave rise to sublines which selfed, and weakly selfing cultures gave rise to sublines which were either pure type I1 or weak selfers. Thus, the selfing in- volves a semi-persistent unstable condition of some kind and is not due to mixed cultures. If the selfing were a re- sult of the original exconjugant dividing and producing cells of different mating types, then the single cell isolations would not have given rise to selfing cultures. The Glau- coma selfers behave much like Tetrahymena pyriformis syngen 1 selfers (Nanney and Allen, 1958) even tho the system of mating type determination is different.

* I n one pair the exconjugants were of different mating types, I and 11.

Syngen 2. Only 2 pairs produced viable exconjugant clones in a cross in syngen 2 of mating types I X I1 (44 pairs isolated). These clones were not sexually reactive in the 1st transfer culture, but were reactive at 40-50 fissions. The exconjugant clones were mating types I1 and I11 from one pair and mating type I1 and selfer from the other pair. Thus, in the only cases available, exconjugant clones (or caryonides) from the same pair were different from each other and were not the same as the parents. This suggests that epigenetic rather than genic mating type inheritance may be involved in syngen 2. Unfortunately only 2 pairs were viable and subsequent crosses yielded no viable pairs (200 pairs isolated).

DISCUSSION The 2 sets of Glaucoma strains included in this study

were divided into 2 syngens on the basis of several signifi- cant differences. Corticotype studies on strains of syngen 1 and syngen 2 (Lee, unpublished) showed that syngen 1 was intermediate in its meridian range between Glaucoma chattoni and Glaucoma scintillans, while syngen 2 appeared to have the same range as Glaucoma scintillans. Thus, it was unclear from corticotype analysis whether syngen 1 and 2 were members of the same species. Mating affinity showed clearly that they were in separate breeding groups. Mass matings occurred within the sets, but only sporadic pairs were found in mixtures between the sets. Two differ- ent mating types were found in syngen 1, and 3 different mating types in syngen 2.

A 3rd difference between the syngens was in viability of crosses. Survival of pairs from crosses within syngen 1 in- creased from 12% to 31% with inbreeding. Within syngen 2 only 570 of pairs survived from one cross. No viable pairs were recovered from crosses between the syngens. New more vigorous strains will be required for a meaningful comparison between the 2 syngens.

A 4th possible difference was in maturity period. At the 1st test for maturity, 91% (including selfers) of the excon- jugant clones in syngen 1 crosses were sexually reactive and 9% failed to mate. The non-reactive clones mated when tested again after 3 isolation transfers. Thus, if an imma- turity period exists for these strains, it is very short. The 4 viable clones obtained from the syngen 2 cross were not sexually reactive in the 1st 2 transfer cultures, but were reactive at 40 fissions. Thus, there may be a significant im- maturity period for this syngen.

A 5th difference between the 2 syngens was in mating

GROWTH OF

type determination. In syngen 1 excluding selfers and with one exception, exconjugant clones from the same pair had identical mating types. The exconjugants appear to be genetically identical since their macronuclei and micronu- clei come from a common zygotic fusion nucleus as in Tet- rahymena pyriformis (Doerder, unpublished observations) . Mating types I and I1 appeared in an approximately 1 : 1 ratio in all crosses as would be expected of a monofactorial segregation. The data are compatible with genic mating type determination, if one of the original parents was a homozygote and the other a heterozygote at the mating type locus. In the only case available from syngen 2, excon- jugant clones from the same pair were different from each other and not the same as the parents. Since the cyto- genetic events appear to be the same as syngen 1 (Doerder, unpublished observations) this suggests that epigenetic mating type inheritance may be involved in syngen 2.

J . PROTOZOOI.. 16(2), 377-395 (19G9).

Quantitative Studies on the Growth of

Allogromia 377

Since both mating type tests and corticotype analysis are simple to perform, future investigators of Glaucoma may use these tools to help identify their organisms and add to our knowledge of the genetics of this ciliate.

REFERENCES 1 . Chatton, E. & Chatton, M. 1929. Les conditions de la con-

jugaison du Glaucoma scintillans en cultures lethobaceriennes. AC- tion directe et specifique deu certains agents zygogenes. C . R. Acad. Sci. 188, 1315-7.

2. Corliss, J. 0. 1953. Comparative studies on holotrichous ciliates in the Colpidium - Glaucoma - Leucophrys - Tetrahymena group. 11. Morphology life cycles and systematic status of strains in pure culture. Parasitology 43, 49-87.

3. Doerder, P. (Unpublished). 4. Lee, P. (Unpublished). 5. Klug, S. 1968. Cortical studies on Glaucoma. I . Protorool.

6. Nanney, D. L. 1968. Ciliate genetics: Patterns and pro- grams of gene action. Ann. Rev. Gen. 2, 121-40.

7. Nanney, D. L. & Allen, S. L. 1959. An analysis of nuclear differentiation in the selfers of Tetrahymena. Am. Nut . 98, 139-60.

15, 321-7.

Allogromia laticollaris (Foraminifera)

JOHN J. LEE, MARIE E. McENERY and HOWARD RUBIN

Def)t. of Biology, City College of the City Univ. of New York, Convent Avenue at 138th Street, New York, N.Y. 10031

SYNOPSIS. The growth and reproduction of Allogromia lati- collaris was studied. More schizozoites were generally produced in mixtures of food organisms than on single algal foods. In the presence of moderate numbers of bacteria, cultures with Phae- odactylum tricornutum, Chlorococcum sp., Nannochloris sp., and an unidentified chlorophyte (BL-1 ), added singly, were also highly productive. Schizogony was the dominant asexual form of reproduction. Binary fission and cytotomy also occurred in bac- terized otherwise unfed controls. =S and 32P are convenient labels for measuring growth of A . laticollaris when introduced into the

TRESS on stratigraphic applications of foraminifera re- S sulted in neglect of the anatomically simple, presum- ably primitive forms. Taxonomy of the monothalmous foraminifera is still confused because they make poor fossils and because so little is known about living forms. The standard taxonomic work is still Rhumbler's (33 ) . Re- cently Loeblich & Tappan (25-27) advocated a broadly based taxonomy which would include comprehensive ana- tomic knowledge (including features of the test and cyto- plasm), life cycles, and ecology. For the monothalms one would want to examine type of pseudopodia; pseudopodial peduncle; structure and position of the aperture (s) ; tex- ture, formation, cytochemistry and ultrastructure of the wall; nucleus (-i) , structure and fine structure; and life cycle details (1-8, 13-16, 19, 21, 23, 29-32). Taxonomic di- vision of one of the larger groups of monothalms, the su- perfamily Lagynacea, is based on test composition and the speculated possession of either flagellated or ameboid ga- metes. Because few of them have been kept in continuous culture, the life cycles of most forms are either unknown or only partly known (including the structure of the gamete)

system in the range of 1 x lo4-1 x lo' dpm/ml ("P specific activity - 2.03 MCi/g; "S specific activity - 95 pCi/g). Small allogromiids grew faster than did larger ones. By means of the Taylor series modification of the classical least-squares method, a continuous life-cycle representation was calculated for A. lati- collaris for the conditions of the experiment. Four points of cell volume growth were maxima for reproduction: 1.0 X 10' p3 per organism for curve I ; 2.2 X 10' p3 and 1.2 x 10' p3 for curve 11; and 6.7 X lo' p3 for curve 111.

and taxonomic separation is still based on test structure alone. This taxonomic system, largely ignoring the living organism seems at 1st artificial and alien to the biologist. Nevertheless, Loeblich & Tappan (27) rejoiced that ". . . each bit of new information as to life cycles, the char- acter of gametes, nuclear characters, etc., had aided in re- fining the classifications based on shells alone, but in no case has conflicted with them." To cite an example, in a recent key paper ( 1 6 ) , placement of the large, naked, rarely found and studied Boderia turneri in the family Lagynidae was supported by a study of its gametes. These are the 1st foraminiferan gametes to be studied by electron microscopy. The heterokont flagellation of these gametes is of particular interest since it is restricted to brown-stock algae, including Phaeophyceae, and some groups of lower fungi, and may indicate a link to an ancestral chrysophyte group, perhaps the Rhizochrysidales.

The giant nuclei (>lo0 p ) (5, 6, 30) of some of the monothalms challenge cellular and molecular biologists as well as students of foraminifera. Few species have been studied by cytochemical and ultrastructural technics altho