Freshwater Euplotes Species with a 9 Type 1 Cirrus Pattern Depend upon Endosymbionts

J Pro lurord 30(?). 1983. pp. 284-289 C 1983 b) the Societ) of Protoroologlsls

Freshwater Euplotes Species with a 9 Type 1 Cirrus Pattern Depend upon Endosymbionts1,2

KLAC S HECKMSNN, RUDOLF TEN HAGEN, and HANS-DIETER GORTZ Zoological Insrrriite L n i ~ e t s i f i ~ of Vunster, 0-4400 Wunster, FRG

ABSTRACT. The hypothesis is advanced that all freshwater Euplores species with a 9 type 1 fronto-ventral cirrus pattern (E. patella type) depend upon bacteria-like endosymbionts. Aposymbiotic cells of these species are unable to divide. The hypothesis is based on the investigation of 40 different freshwater Eiiplores stocks collected in Germany. France. the USA, and Japan. No symbionts were found in E. crenosus and E. pnlusrris. freshwater species with 10 fronto-ventral cirri, nor in E. muxicola, a representative of the freshwater Euplotes group with a 9 type 2 fronto-ventral cirrus pattern ( E . qfinis type). Characteristic for the essential endosymbionts are multiple nucleoids, a feature described earlier for ornikron. an indispensable symbiont of E. aediculatus. Although the symbionts differ from omikron and among each other in size. shape. and their average number per host, they are believed to be related to omikron. In two stocks a different type of bacterium was found in which no defined nucleoids can be detected. Transfer of this symbiont into aposymbiotic cells, originally cawing ornikron. revealed that it can restore the ability to multiply. Similarly, omikron was also able to restore the ability to divide in cells freed of this symbiont. It is assumed that this different type of symbiont is a secondary invader of Euplotes which displaced the original oi~iikron-like endosymbiont. Some of the stocks were found to carry, in addition to omikron-like symbionts, other symbiotic bacteria: E. duidal~os carries in addition an alga. The findings suggest that the freshwater Euplotes species with a 9 type 1 cirrus pattern are closely related to each other and evolved from an ancestor (probably of cirrotype 10) which already was dependent upon endosymbionts of the ornikron type. It supports the view that the two subgroups of freshwater Euplotes forms with a cirrotype of 9 have evolved independently from each other from species with 10 fronto-ventral cirri by losing a cirrus at different positions

NDOSYMBIOTIC bacteria have often been noticed in pro- E tozoa (for earlier accounts. see 1. 3. 17) and received par- ticular attention in ciliates after it was discovered that a killer- phenotype is frequently associated with their presence ( I 9. 24). Although it is generally believed that they are of some advantage for their hosts (14, 18), strains which have lost their symbionts can usually be maintained in the laboratory. and symbiont-free strains can be isolated from nature as well.

In Euplotes, bacteria-like symbionts were first reported by FaurC-Fremiet in 1952 ( 5 ) in the cytoplasm of several strains of E. patella Ehrenberg, 1838, and E. ~ U I ~ I I S ~ O I ~ ~ Z ~ S Kahl. 1932: he suggested that they might be essential for these species be- cause he observed that small doses of penicillin not only led to a loss of symbionts but also to the death of the hosts. The possibility that death was due to sensitivity to penicillin rather than the loss of symbionts was. however. not excluded. More recently, a symbiont named otnikron was described by Heck- mann (12) for the cytoplasm of E. aediculatris Pierson. 1943. H e reported that the ciliate. upon removal of this symbiont. ceases to divide and eventually dies. Heckmann achieved rein- fection with omikron in a small percentage of aposymbiotic cells. whereupon they resumed multiplication. This proves that E. aediculatus indeed depends upon ornikr.ori.

One of the features which distinguishes omikron from other symbiotic bacteria was the presence of many nucleoids per bac- terium. Since Heckmann found similar looking symbionts in other freshwater Euplotes species ( E . patella, E . euqxfomzis. E. plumipes, E. woodryfn, he raised the question whether perhaps all freshwater Euplotes depend upon bacterial endosymbionts (13). We are investigating this question here by examining a total of 40 stocks distributed among 10 different freshwater Euplotes species. Our results show that the postulated depen- dence upon endosymbionts must be limited to freshwater forms with a 9 type 1 fronto-ventral cirrus pattern ( E . patella type).

’ We are grateful to Mme G. Fryd-Versavel, Dr. J . J . Ruffolo. Jr.. Dr. B. F. Hill. and Dr. T. Kosaka for supplying us with cultures. We thank Dr. J . Frankel. Dr. A. Miyake. Dr. K. Muller and Dr. J. R. Preer. Jr. for valuable comments and criticisms of the manuscript and Mrs. G. Newels for secretarial help.

Part of this work has been reported in abstract form ( 1 5 ) . Dedicated to Karl G. Grell on the occasion of his 70th birthda).

It does not apply to freshwater species with a 9 type 2 cirrus pattern (E. afinis type) nor to species with 10 fronto-ventral cirri. Our results suggest that freshwater Euplotes species with a 9 type 1 fronto-ventral cirrus pattern evolved from a form which already was dependent upon bacterial symbionts and support the view that the two subgroups of freshwater Euplotes forms with a cirrotype of 9 have evolved independently of each other from species with 10 fronto-ventral cirri by losing a cirrus a t different positions.

MATERIALS A N D METHODS The term “stock” is used in this paper in accordance with

Sonneborn (23) to designate the progeny of a single individual, usually an individual isolated from a pond or a stream. This does not exclude the possibility of sexual reproduction among the progeny of such a cell; in some stocks conjugation has fre- quently been observed.

Most stocks originated from collections a t Steinfurt, a town near Miinster (Westf.), Germany. To avoid duplications, diverse isolates from the same place were only maintained when they belonged to different species. We also tested stocks from Dr. John J. Ruffolo, Jr., Department of Biophysics, Virginia Com- monwealth University, Richmond, USA, Dr. Bruce F. Hill, University of New Hampshire, Durham, USA, Dr. T. Kosaka, Hiroshima University, Japan, and M m e G. Fryd-Versavel, Uni- versitC Paris-Sud, Orsay, France. All stocks were grown a t room temperature (21-23°C) in a diluted soil medium (for details of preparation, see 20) and fed either with Chlorogonium elon- garutn or Chilomonas paramecium. Chlorogonium was culti- vated in soil medium under artificial illumination, Chilomonas in soil medium containing 2-3 rice grains per 40 ml. Some stocks grew better after enriching the medium with 2.5 g Cerophyl (Cerophyl Laboratories, Inc., Kansas City, Missouri, USA) and 0.25 g yeast extract (Difco Laboratories, Detroit, Michigan, USA) per 1000 ml. Good results were also achieved with a medium prepared according to Hill & Reilly (16).

T o identify the species o f t h e various stocks, fixed and stained cells as well as living cells were observed. Cells were stained by the silver impregnation technique of Chatton & Lwoff as de- scribed by Frankel & Heckmann (7), by the nigrosin-HgC1,- formalin stain of Borror (2) and with a staining procedure spe- cific for the nucleus, using aceto-carmin or the reagents for the

284

HECKMANN ET AL.--ELJPLOTES ENDOSYMBIONTS 285

Feulgen reaction. With the help of Curds’s monograph (4), iden- tification was then in most cases possible. Stock 36, however, differed from all species so far described and is now named Euplotes palustris (for description, see 11). We disagree with Curds in that we consider E. plumipes to be a separate species because our stock clearly differs in morphology from E. eurys- tomus. It actually resembles E. aedzculatus more closely but seems to be sexually isolated from this species as well. Our stock of E. plumipes shows intraclonal mating but was never found to react with our mating types of E. aediculatus.

For observations of endosymbionts, cells in a small drop on a glass slide were lightly fixed by exposure to OsO, vapor for a few seconds. Then a coverslip with vaseline smeared around the edges was placed over the drop and lightly pressed down, flattening but not disrupting the animals. The preparations were examined under phase contrast with a X 100 oil-immersion ob- jective. To stain the symbionts, a small drop of aceto-carmin or aceto-orcein was added to the fixed cells before they were covered with a coverslip.

For penicillin treatment, rapidly growing cells were trans- ferred into food-containing culture medium to which penicillin had been added. Four to five days later, cells were washed free of penicillin and transferred back to normal culture medium. Single cell isolates were then maintained until they died. A large fraction of treated cells was stained and squashed and examined for the presence of symbionts. In most cases concentrations of 250-500 U/ml penicillin turned out to be sufficient to free the Euplotes cells from their symbionts. In two stocks the symbionts could not be removed by the method; they are probably peni- cillin-resistant.

RESULTS A total of 40 different stocks of freshwater Euplotes species

were collected, grown, identified, and checked for the presence of bacteria-like endosymbionts. They are listed in Table I which also gives the fronto-ventral cirrus pattern of the species to which they belong and the origin of the various stocks. The table shows that all stocks of species with a 9 type 1 fronto-ventral cirrus pattern that we have observed did contain symbionts. No symbionts could be found in two stocks of E. crenosus Tuffrau, 1960 and one stock of E. palustris ten Hagen, 1980 (a recently described species, see 1 I ) . Both species belong to the group of freshwater Euplotes with 10 fronto-ventral cirri. We have also not been able to find symbionts in two stocks of E. muscicola Kahl, 1932, the only species with a 9 type 2 fronto-ventral cirrus pattern which we have been able to obtain.

We have subjected our symbiont-bearing stocks to various doses of penicillin for various lengths of time in order to test their dependence upon endosymbionts. We found that a con- centration of 250-500 U/ml normally sufficed to reduce the number of symbionts sharply in treated cells. Such a treatment often eliminated them completely if it was extended over 5-6 days. At the end of such a treatment, Euplotes was washed free of the drug and then maintained as single cell isolates in regular culture medium to which the food organism Chlorogonium was added in small quantities. We found that cells which had lost their symbionts by the penicillin treatment stopped dividing and finally died after approximately 15 days as had been found previously for E. aediculatus (1 2). In some cells a few symbionts were still present when the penicillin treatment was ended. The symbionts multiplied quickly and reestablished the host’s ability to multiply after a lag period of a few days. Whenever we ex- amined cells that started multiplying again after such treatment, they were found to contain symbionts. We therefore conclude that all these various Euplotes stocks do depend on symbionts.

Two stocks (stock 1 of E. aediculatus and stock 2 of E. oc-

tocarinatus) could not be freed of their symbionts. Since peni- cillin concentrations up to 500 U/ml turned out to be ineffective in these stocks, they were finally treated with a concentration of 750 U/ml. This relatively high dose, however, was found to harm the Euplotes cells without reducing their number of sym- bionts. It is conceivable that the symbionts of these two stocks are penicillin-resistant.

We have also treated the stocks ofthe symbiont-free Euplotes species (E. crenosus, E. palustris, and E. muscicola) with var- ious concentrations of penicillin and found that they continued to multiply at doses up to 500 U/ml. At 500 U/ml and higher, E. muscicola started to encyst, and at 750 U/ml, E. crenosus and E. palustris showed signs of being harmed soon after initial exposure to this concentration. Since a search for symbionts with the help of the electron microscope did not reveal sym- bionts in these species either, we believe that, in contrast to the species with a 9 type 1 fronto-ventral cirrus pattern, they do not depend on symbionts.

Close examination of the symbionts of the various Euplotes stocks revealed characteristic differences. In some stocks they are long and thin, in others, short forms prevail. Most of the stocks carry symbionts which are slightly curved, but in some stocks the symbionts form straight rods. Table I1 summarizes the shape, size, and other characteristics of the symbionts. Fig- ures 1-8 illustrate the various forms. We found that these fea- tures show little variation between cells of a given clone, if the cells examined are in a physiologically comparable stage. The number of symbionts per ciliate and their average length, how- ever, may change when, for example, the temperature at which a culture is maintained is raised or lowered and if cells become starved instead of being well nourished. The data given in Table I1 were obtained from well fed cells maintained at 21-23°C.

The symbionts of stocks 7 and 10 do not reveal nucleoids either by light or electron microscopy. They are indistinguishable from each other but differ from the essential symbionts of the other stocks in so many features that we are convinced that they belong to an entirely different class of bacteria. They have ta- pered ends (Fig. 3), are about twice as wide as the other sym- bionts, and average only 500 per host, which is about half the population size found for the other symbionts. We observed that this type of symbiont, which shall henceforth be called type 7 symbiont, can also be removed with the help of penicillin and that its hosts-two stocks of E. aediculatus- then also stop mul- tiplying. In order to examine whether type 7 symbionts serve the same function as omikron, which is present in stock 15, we have tried to infect aposymbiotic cells of stock 15 with type 7 symbionts. In an experiment in which 90 cells were provided with a cell homogenate containing type 7 symbionts, a procedure successfully used earlier in reinfection experiments with omi- kron (1 2), we observed a resumption ofgrowth in one cell, which was caused by type 7 symbionts as revealed by microscopy. The experiment was recently repeated but employing a much more efficient microinjection procedure for the transfer of symbionts, and many growing lines with type 7 symbionts were obtained (unpublished work, Fujishima & Heckmann). With the same method also, the reverse experiment was carried out. It estab- lished omikron in stock 7 cytoplasm. The results show that both types of symbionts can restore in E. aedzculatus the ability to multiply and therefore probably provide the same kind of func- tion for this ciliate.

In stock 40 of E. eurystomus, stock 14 of E. patella, and stocks 2, 3, 11, and 32 of E . octocarinatus, we found another type of symbiont in addition to omikron-like particles. This additional symbiont is very small and of the coccobacilli type. Since it was not found in other stocks of these species, we do not think that it serves an essential function for its hosts but

286 J . PROTOZOOL.. VOL. 30. NO. 2 . MAY 1983

Figs. 1-8. Bacteria-like endosymbionts of various freshwater Euplofes species. Nucleoids can be recognized in omikron-like symbionts as dark dots (arrows). Double arrows indicate a second type of symbiont found in addition to ornrkron-like symbionts in certain stocks. Mi = micronucleus, Ma = macronucleus. Z = zoochlorellae. All figures are photomicrographs of aceto-orcein stained Euplotes cells by phase contrast illumination. X2000. 1. E. aedrculatus stock 15. The symbionts of this stock have been named ornrkron (see 12). 2. E. aediculatus stock 27. The symbionts are longer and have more nucleoids than owzikron. 3. E. aedrculurus stock 7. The symbionts are not of the omikron-like type, no nuckoids can be recognized. 4. E. woodrufi stock 22. The symbionts are of the ornikron-like type. 5. E. eurysromus stock 40. The omikron-like symbionts form long threads and are often twisted. An additional second type of symbiont (double arrow) coexists with them. 6. E. eurystomus stock 25. The symbionts are similar to omikron. 7. E. dnidnleos stock 13. Here zoochlorellae are found in addition to omikron-like symbionts. 8. E. ocfocarinatus stock 1 1. The ornikron-like symbionts are straight. In addition, a small coccobacilli type of symbiont (double arrow) is present.

HECKMANN ET AL.-EUPLOTES ENDOSYMBIONTS 287

TABLE I. Stocks examined for the presence of Omikron-like symbionts

Symbionts Species Stock Provenance Cirrotype present

E. aediculatus Pierson, 1943 I Steinfurt, Germany 9 type 1 Yes E. aediculatus Pierson, 1943 6 Steinfurt, Germany 9 type 1 Yes

E. aediculatus Pierson, 1943 8 Steinfurt, Germany 9 type 1 Yes

E. aediculatus Pierson, 1943 15 Marseille, France 9 type 1 Yes E. aediculatus Pierson, 1943 21 Ohio, USA 9 type 1 Yes E. aediculatus Pierson, 1943 31 Colorado, USA 9 type 1 Yes E. aediculatus Pierson, 1943 33 New York, USA 9 type 1 yes E. aediculatus Pierson, 1943 34 New York, USA 9 type 1 Yes

E. aediculatus Pierson, 1943 1 Steinfurt, Germany 9 type 1 noa

E. aediculatus Pierson, 1943 10 Miinster, Germany 9 type 1 noa

E. euystomus Kahl, 1932 E. eurystomus Kahl, 1932 E. eurystomus Kahl, 1932 E. eurystomus Kahl, 1932 E. eurystomus Kahl, 1932

19 Miinster, Germany 20 Maryland, USA 25 Ohio. USA 26 N. Carolina, USA 40 Marl, Germany

9 type 1 Yes 9 type 1 Yes 9 type 1 Yes 9 type 1 Yes 9 type 1 Yes

E . plumipes Stockes, 1884 24 Varenne, France 9 type 1 yes E. daidaleos Diller and Kounaris, 1966 13 Steinfurt, Germany 9 type I Yes E. daidaleos Diller and Kounaris, 1966 16 Miinster, Germany 9 type 1 Yes E. daidaleos Diller and Kounaris, 1966 11 Miinster, Germany 9 type 1 Yes E. daidaleos Diller and Kounaris, 1966 18 Miinster, Germany 9 type 1 yes E. daidaleos Diller and Kounaris, 1966 37 Steinfurt, Germany 9 type 1 yes E. octocarinatus Carter, 1972 E. octocarinatus Carter, 1912 E. octocarinatus Carter, 1912 E. octocarinatus Carter, 1912 E. octocarinatus Carter, 1912 E. octocarinatus Carter, 1912 E. octocarinatus Carter, 1972 E. octocarinatus Carter, 1972 E. patella Ehrenberg, 1838 E. patella Ehrenberg, 1838 E. patella Ehrenberg, 1838 E. woodruji Caw, 1939 E. woodruji Caw, 1939 E. woodrufi Caw, 1939 E. muscicola Kahl, 1932 E. muscicola Kahl, 1932 E. crenosus Tuffrau, 1960 E. crenosus Tuffrau, 1960 E. palustris ten Hagen, 1980

2 3 4 9

11 28 32 38

5 12 14 21 22 23 35 39

Steinfurt, Germany Steinfurt, Germany Steinfurt, Germany New Hampshire, USA Miinster, Germany Steinfurt, Germany Ohio, USA Steinfurt, Germany Steinfurt, Germany Steinfurt, Germany Steinfurt, Germany Hiroshima, Japan Hiroshima, Japan Hiroshima, Japan Steinfurt, Germany Steinfurt, Germany

9 type 1 9 type I 9 type 1 9 type I 9 type 1 9 type I 9 type 1 9 type 1 9 type 1 9 type 1 9 type I 9 type 1 9 type 1 9 type 1 9 type 2 9 type 2

29 Steinfurt, Germany 10 30 Steinfurt, Germany 10 36 Emsdetten, Germany 10

a Bears another bacterial symbiont which probably has displaced the original omikron-like symbiont.

rather consider it as one of the dispensable symbionts frequently encountered in ciliates.

Finally, it should be mentioned that stocks 13, 17, and I8 of E. duiduleos contain symbiotic algae in addition to omikron- like symbionts. That the zoochlorellae are not essential for them becomes obvious from the fact that stocks 16 and 37 of this species were free of zoochlorellae originally but could easily be infected with them. An interesting observation was that the number of omikron-like symbionts per cell dropped from ap- proximately 400 to about 200 after infection with zoochlorellae. We also observed that cells with low numbers of zoochlorellae tend to have high numbers of bacteria-like symbionts and vice versa. This suggests competition between the two types of sym- bionts. It should, however, be stressed, that E. duiduleos can live perfectly well without zoochlorellae, but is dependent on its omikron-like symbionts.

DISCUSSION Currently, 22 freshwater Euplotes species are recognized. They

can be subdivided into three major groups: one characterized

by 10 fronto-ventral cim, one by a 9 type 1 or E. patella cirrus pattern, and one by a 9 type 2 or E. afinis cirrus pattern. Our data show that species with a cirrotype of 10 and those with a cirrus pattern of 9 type 2 can live and multiply without the help of endosymbionts. Hence an earlier suggestion by one of us (1 3) that perhaps all freshwater Euplotes depend upon symbiotic bacteria has to be limited to the group of species with a 9 type 1 cirrus pattern.

The 40 stocks which we investigated belong to 10 of the 22 known freshwater Euplotes species (Table I). Seven of the 10 species are characterized by a 9 type 1 cirrus pattern. From this group only E. umieti Dragesco, 1970, a form described from the Cameroun in Africa, is missing. It is so similar to E. eu- rystomus Kahl, 1932 that the suggestion has been made that i t might be a geographic variation of this species (4). Therefore we expect that it also depends upon endosymbiotic bacteria as was found to be the case for the 35 stocks of the other 7 species of this cirrotype 9 subgroup. Out of the 9 described freshwater species with a 9 type 2 cirrus pattern, we have been able to examine only E. muscicola Kahl, 1932. It was represented in

288 J. PROTOZOOL.. VOL. 30. NO. 2 . MAY 1983

TABLE 11 Shape 5rze. and other churacterrstrcc of the Omikron-like winhionis of the znvestigated stocks

Width Length No of nucleoids No. of symbionts Species Stock Shape of slrnbionts ( rm) (rm) per symbiont per host

E. aediculatus E. aediculatus E. aediculatus E. aediculat us E. aediculatus E. aediculatus E. aediculatus E. aediculat us E. aediculatus E. aediculatus E. eurystomus E. eurystornus E. eurystomus E. eurystornus E. eurystomus E. plumipes E. woodruji E. woodruffi E. woodrufi E. patella E. patella E. patella E. daidaleos E. daidaleos E. daidaleos E. daidaleos E. daidaleos E. octocarinatus E. octocarinatus E. octocarinatus E. octocarinatus E. octocarinatus E. ortocarinatus E. octocarinatus E. octocarinatus

15 27

8 33 34

I 6

31 7

10 25 40 19 26 20 24 21 22 23 14 12 5

13 16 17 18 31 2 3 4 9

32 1 1 38 28

slightly curved slightly curved slightly curved slightly curved slightly curved slightly curved slightly curved slightly curved straight straight slightly curved slightly curved slightly curved slightly curved curved slightly curved straight straight straight slightly curved curved curved slightly curved slightly curved slightly curved slightly curved slightly curved straight straight straight straight straight straight slightly curved slightly curved

0.3 0.4 0.4 0.5 0.5 0.6 0.6 0.6 1 .o 1 .o 0.4 0.5 0.5 0.5 0.5 0.6 0.6 0.6 0.6 0.4 0.6 0.7 0.4 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.6 0.6

2.5-9 2.5-13

2-10 2.5-12 2.5-8 2.5-13 2.5-13 2.5-15 2.5-8 2.5-10

2-7 2.5-12 2.5-15

3-9 2.5-9

3-25 2-12

2.5-15 2.5-7

3-19 2.5-8 2.5-5 1.5-12

3-8 2.5-12

2-8 2-10 2-10 2-8 3-1 1 2-7.5

2.5-8 1.5-10 2.7-7.5

2-9

3-12 2-18 2-6 3-20 2-10 2-1 5 3-1 5 2-20

none visible none visible

2-10 3-1 5 3-19 3-12 3-10 3-28 2-1 5 2-1 5 2-10 3-16 2-8 2-8 3-1 1 3-9 4-1 3 2-7 3-9 2-9 2-6 2-8 2-9 2-5 2-6 2-8 2-9

800-1000 900-1 000 900-1 000 900-1000 900-1 100 900-1 100 900-1 100 600-800 400-600 400-600

1000-1 200 1000-1 200 900-1 100 900-1000 900- 1 100 650-750

1000-I 100 1000- I 100 1000-1 1 00 600-650 400-500 350-450

350-400 2 5 0-3 00

200-250 200-250 350-400 150-300 200-300 200-300 150-200

300-400 200-300

100-150 80-1 50

our collection by two stocks which were found to be free of symbionts. Gates (9) has also reported that this species is free of symbionts. The group of species with a 9 type 2 cirrus pattern is not well represented in our collection. But on the other hand, several species of this group have only been reported once and one cannot expect to find them easily. From the group of species with a cirrotype of 10, E. inkystance Tuffrau, 1960 and E. moe- biusi Kahl, 1932 are missing.

The symbionts of most stocks of species with a 9 type 1 cirrus pattern resemble each other in that they have multiple nucleoids which are easily revealed by light microscopy. This feature was first recognized for omikron-particles and it has been pointed out that this character separates oinikron from all other known symbiotic bacteria (1 2). The new symbionts briefly described in Table I1 share with omikron the possession of multiple nu- cleoids. They will hence be called omikron-like. The name om- ikron will be restricted to the symbionts of stock I S . It is at present the only symbiont of the omikron-like group for which the GC content of its DNA and the genome size are known (21. 22). We believe that the symbionts with multiple nucleoids are closely related to each other and interpret differences between them as due to mutations which occurred after their original host species broke up into the species recognized today.

Morphological differences between the symbionts are not spe- cific for the host species. Characteristic differences are observed also between stocks classified as belonging to the same taxo-

nomic unit (Table 11). Since we, however, are dealing with “species” defined on morphological grounds and not by breed- ing analysis, the possibility must be kept in mind that stocks classified for the same taxonomic species might actually belong to different sibling species or syngens which have not yet been recognized and set apart.

The symbionts which we found in stocks 7 and 10 do not show nucleoids and differ from the omikron-like symbionts in so many features that we have to conclude that they belong to an entirely different class of bacteria. This, however, raises prob- lems concerning the view that all the Euplotes species with a 9 type 1 cirrotype descended from an ancestral Euplotes which already was dependent on endosymbionts of the omikron-type. The finding that type 7 symbionts can restore the ability to divide in aposymbiotic cells of stock 15, and that omikron can do the same when introduced into type 7 cytoplasm shows that both types of symbionts provide for Euplotes the same essential functions. It may be argued that one of the two types of sym- bionts is a secondary infectant which has displaced the original endosymbiont from its ecological niche. Since we find omikron- like symbionts in stocks from different continents, belonging to different species, it seems to be reasonable to assume that they are the original inhabitants of the Euplotes species with a 9 type 1 cirrus pattern and that the type 7 symbionts are the later acquisition. This view is also supported by the finding that otnikron has a very small genome (2 1, 22) which we interpret

HECKMANN ET AL.-EUPLOTES ENDOSYMBIONTS 289

as a consequence of a long and well adapted life in an intracel- lular environment. So far no data with respect to the genome size of the type 7 symbionts are available. W e also do not know whether the type 7 symbionts depend upon Euplotes as it is the case for omikron (1 2).

Gates (8, 9) has shown by morphometric measurements that the two Euplotes cirrotype 9 subtypes form internally coherent groups. He demonstrated that they can artificially be derived from the pattern of a species with 10 fronto-ventral cirri by removal of cirrus 3/IV (9 type 1 pattern) or cirrus 2/IV (9 type 2 pattern). This suggests, that cirral pattern evolution may have occurred in Euplotes by the loss of cirri and that the two cir- rotype 9 subtypes have evolved independently (1 1). Our data support this view. We can rationalize them best by assuming that the freshwater species with a 9 type 1 cirrus pattern form a natural group which evolved from a n ancestral form which was already dependent upon omikron-like endosymbionts. We have not found species with a 9 type 1 cirrus pattern which can multiply when freed of symbionts. This could be used to argue in favor of the idea that the ancestral form of this group was probably still of cirrotype 10, i.e., the dependence upon sym- bionts evolved before the loss of cirrus 3/IV.

Among the Euplotes species with a cirrotype of 9 there are also a few marine forms. Some of them have been classified by Gates (9) as belonging to the E. patella subgroup (E. dogieli Agamaliev, 1967; E. latus Agamaliev, 1967); others are placed into the E. afinis group (E. bisulcatus Kahl, 1932; E. apsher- onicus Agamaliev, 1966; E. zenkewitchi Burkovsky, 1970). We have not investigated any of these marine forms and therefore are not able t o tell whether marine forms with a 9 type 1 cir- rotype do or do not depend on omikron-like symbionts. Pro- tozoologists who happen to come across these species should look into this matter. If omikron-like symbionts were found, it would indicate that the marine 9 type 1 species and the fresh- water 9 type 1 species are closely related to each other. It seems possible, however, that the transition from the marine habitat to the freshwater habitat was taken by Euplotes only once and that the marine forms with a cirrotype of 9 have arisen inde- pendently from those in the fresh water. In that case dependence on symbionts would not be expected for them. Since Gates (9) has not included marine forms in his analysis of vanations in the distribution ofcirri in species with a cirrotype of 9, we cannot use his data as support for either of these views.

Foissner (6) reports that he has observed omikron-like sym- bionts in E . moebiusi f. quadricirratus Kahl, 1932. This is a freshwater form with 1 0 fronto-ventral cirri. Unfortunately, he has not tested whether this ciliate depends on its symbionts. As long as such dependence has not been demonstrated it seems futile to speculate on relationships between this species and those with a 9 type 1 cirrus pattern.

LITERATURE CITED 1. Ball, G. H. 1969. Organisms living on and in Protozoa, in Chen,

T. T., ed., Research in Protozoology, Pergamon Press, Oxford, Vol. 111:

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Received 12 V 82; accepted 19 X 82