J. PROTOZOOL. 13(1), 5-8 (1966).

The Endocommensal Ciliates of Venezuelan Sea Urchins

SERVIO URDANETA-MORALES and M. T E N G L E R DE McLURE

Department of Microbiology and Parasitology, School of Biology, Faculty of Sciences, Universidad Central de Venezuela, Caracas, Venezuela

5

SYNOPSIS, Two hundred and fifty-nine specimens of the sea urchins Echinometra lucunter (201), Diadema antillarum (52), Tripneustes ventricosus ( 5 ) and Eucidaris tribuloides ( I ) , collected on the littoral of the Federal District of Venezuela, were examined for intestinal ciliates.

INTRODUCTION

HE most common species of sea urchins on the littoral T of the Federal District of Venezuela are Echinometra lucunter, Diadema antillarum, Tripneustes ventricosus and Eucidaris tribuloides.

The endocommensal protozoa of these species have been investigated by several workers. Ball(2) worked on Dia- demo sp. and Echinometra sp. of Bermuda. Biggar & Wen- rich( 11) completed these observations, noting the species of sea urchins previously studied by Ball. These authors worked only with living material.

Lucas( 13) described new species of intestinal ciliates in D . antillarum and E. lucunter of Bermuda. Powers(l9) worked on E . lucunter, D . antillarum, T . ventricosus and E. tribuloides of Tortugas, redescribing the species already published and adding others. Lucas( 14) increased the data on Cryptochilidium bermudense (Biggar & Wenrich)- synonym of Biggaria bermudensis ( Biggar & Wenrich, 1932) -found in D . antillarum of Bermuda. Berger(8) published a list of endocommensal ciliates found in D . antillarum, E . lucunter and T . ventricosus of the Bahamas.

In this paper, we shall present the results obtained after examining 259 specimens of sea urchins belonging to the four species collected on the coast of the Federal District (Venezuela).

ECHINOIDEA A N D LOCALITIES STUDIED

Sea urchins were collected from May until December 1962 from zones of shallow water on the littoral of the Federal District of Venezuela. Both of the major localities were divided into east and west zones on the basis of differences between the populations of sea urchins and of their ciliates. One locality, “Playa Grande,” lies 8 Km. west of the port of La Guaira; its two zones are separated by a narrow breakwater. The two zones of the other locality, “El Caribe,” lie 13 and 14 Km. east of La Guaira.

The two hundred and fifty-nine specimens of sea urchins were identified as follows: 201 Echinometra lucunter, 52 Diadema antil- larum, 5 Tripneustes ventricosus and one Eucidaris tribuloides. The last-named specimen, from “El Carihe,” had no ciliates.

Table 1 shows the distribution of the specimens collected by lo- cality and zone.

METHODS

1) Collection and maintenance of the sea urchins Upon collection, the sea urchins were placed in polyethylene con-

tainers with sea water and taken to the laboratory (920 m. above sea level) where they were put in an aerated sea-water aquarium.

Iron hematoxylin and silver-impregnation staining technics per- mitted determination of eight of the nine ciliates found: Anophrys aglycus, A . elongata, Cohnilembus caeci, Biggaria bermudensis, B . echirtometris, Cyclidium rhabdotectum, Metopus circumlabens and M . rotundus. An unidentified species of Euplotes was also found.

TABLE 1. Distribution of sea urchins studied b y locality and Bone, showing number of specimens examined.

Locality ‘ E l Caribe ’ ’ “Playa Grande” Zones East West Total Eas t West Total

Echinometra liicunter ti8 44 112 35 54 89 Diadema antillarum 51 - 51 1 - 1 Tripneustes ventricosus - - - 5 - 5 Eucidaris tribuloides 1 - 1 - - -

2 ) Dissection To obtain samples of the liquid contents of the intestine we have

used the technics of Lynch(l5) and Beers(3) ; we prefer the latter, modified by the recommendation of Powers( 19) to concentrate the intestinal material by centrifugation. We have centrifuged the ma- terial at 2 5 X g for 5 minutes, with good results.

We have only occasionally seen ciliates outside the intestinal tract, in the perivisceral fluid, probably due to contamination.

3 ) Fixation and staining Bouin’s fixative was used prior to staining with Heidenhain’s iron

hematoxylin or with silver impregnation according to Tengler (25) who substituted silver-gelatin for “Protargol” ; impregnation at 56- 60°C gave better results than at 37°C.

Champy-Da Fano fixative was used prior to staining by the technic of Chatton and Lwoff according to Corliss(l2), and with some sug- gestions by Berger(9).

,

RESULTS

We found nine different species of ciliates in the ma- terial studied.

The following percentages of sea urchins were infected: E . lucunter, 80.6; D . antillarum, 90.4; T . ventricosus, 100.0; E . tribuloides, 0.

The nine species of intestinal ciliates identified are the following:

Anophrys aglycus Powers, 1935 Powers( 19) found this species in Diadema antillurum,

Echinometra lucunter and Tripneustes ventricosus of Tor tugas.

We found this species endocommensal in the hosts men- tioned by Powers. The dimensions obtained from 92 ciliates were: 83 (57-118) p X 33 (23-50) p, with standard devia- tions of 12.0 p for length and 5.8 p for width. Eighty-three ciliates from E . lucunter were 82 (57-118) p X 33 (23-47) p, standard deviations being 11.8 p and 5.6 p for length and width respectively. Nine ciliates from D . antillarum were 95 (82-105) p x 37 (28-50) p. with standard devi-

6 CILIATES OF SEA URCHINS

TABLE 2 . Dimensions of Biggaria echinometris.

Sea urchin Length in microns Width in microns Authors hosts Locality n mean mnx min s mean inax min s

Present paper Echinometra “ E l Caribe” 23 57 75 42 9.2 22 36 16 4.8

Present paper E . lucunter “Playa Grande” 107 74 108 55 10.5 29 42 20 4.6

lucunter (east zone)

l’ripnewtes “ E l Caribe” ventricosus (west zone)

Biggar t% Wen- E . lucunter Bermuda - 7 3 - - - 2 6 - - - rich (1932)

Powers (1933) L?/lwhinus North Carolina - 122 195 80 - 46 70 33 - variegatus

n = No. of ciliates measured max = maximum min I minimum s z standard deviation

ations of 7.8 p in length and 6.9 p in width. No specimens from T. ventricosus were measured, due to lack of stained material.

Anophrys elongata Biggar & Wenrich, 1932 This species was first mentioned by Ball(2) and later

described by Biggar & Wenrich( 11). I t has been reported from a number of hosts(6,8,16,19,20,21,26).

We found it in E. lucunter and T . ventricosus, but not in D . antillarum. The dimensions of 125 ciliates were: 87 (65-108) p X 26 (16-39) p, with standard deviations of 9.3 p for length and 4.7 p for width. One hundred ciliates from E . lucunter were 88 (65-108) p X 25 (16-39) p with standard deviations of 9.1 p and 4.5 p for length and width respectively. Twenty-five from T . ventricosus were 85 (65-101) p X 28 (20-39) p with standard deviations of 10.3 p and 4.8 p.

Cohnilembus caeci Powers, 1935 Describing this species, Powers(l9) reported i t from E .

lucunter, D . antillarum, T. ventricosus and Lytechinus vari- egatus. We have also found it in the first three species, percentages of infection being 0.570, 3.8% and 60.0%, respectively.

Dimensions of 23 ciliates (20 from T . ventricosus and 3 from E . lucunter) were 48 (28-73) p X 10 (7-16) p, with standard deviations of 14.5 p for length and 2.2 p for width. The width/length ratio was 0.225 (0.170-0.300) with a standard deviation of 0.04. Width was measured through the middle of the body. Biggaria bermudensis (Biggar & Wenrich, 1932) Kahl, 1934

This species was first mentioned by Ball(2) and later described by Biggar & Wenrich( 11). I t has been reported from several hosts( 8,14,16,18,19,20,2 1).

We have found this species abundant in every infected D . antillarum (90.4% of those studied), in 80.0% of T. ventricosus and in 7.5% of E . lucunter. Dimensions of 209 ciliates were 126 (75-183) p X 63 (39-98) p ; standard deviations were 20.9 p for length and 10.7 p for width; 100 ciliates from D. antillarum were 131 (75-183) p X 63 (39-98) p, with standard deviations of 22.3 p and 12.2 p, respectively; 100 from T. ventricosus were 120 (78-150) p j( 62 (42-85) p, with standard deviations of 18.4 p and

9 . 0 ~ ; 9 from E . lucunter were 135 (108-156) p X 70 (59- 78) p, with standard deviations of 14.1 p and 5.9 p.

Biggaria echinometris (Biggar & Wenrich, 1932) Kahl, 1934 This species was first mentioned by Ball(2) and later

described by Biggar & Wenrich(l1) from E . lucunter. It has since been reported from various hosts (8,18).

We have found B . echinometris in E . lucunter of “Playa Grande” and of “El Caribe” and in T . ventricosus of “Playa Grande.” There appear to be two distinct populations- possibly, two varieties. Our sample of the first population consisted of 23 specimens found in two E . lucunter from the east zone of “El Caribe.” We found none of these ciliates in the abundant D. antillarum from the same zone. The other population was represented by 100 specimens from E . lucunter of “Playa Grande” and the west zone of “El Caribe,” plus 7 specimens from T. ventricosus of “Playa Grande.”

Table 2 shows measurements of the two populations, compared with those given by Biggar and Wenrich( 11 ) and Powers(l8). According to the t-test (Snedecor, 23) the difference between the means of the two groups was sig- nificant a t the 1% level.

Cyclidium rhabdotectum Powers, 1935 Powers( 19) described this ciliate from D. entillarum, E .

lucunter, T. ventricosus and Clypeaster rosaceus. Berger ( 8 ) found it in C . rosaceus. We found it in the first three species. Dimensions of 165 ciliates were 26 (17-36) p X 17 (12-24) ,A with standard deviations of 3.1 p for length and 2.6 p for width. By host species, 100 ciliates from E . lucunter were 26 (22-34) p X 16 (12-23) p with standard deviations of 2.7 p for length and 2.3 p for width; and 65 specimens from T . ventricosus were 27 (17-36) p X 18 (12-24) p with standard deviations of 3.7 p and 2.6 p.

Metopus circumlabens Biggar & Wenrich, 1932 This species was first mentioned by Ball(2) and later

described by Biggar and Wenrich( 11). It has been reported from various hosts (8,13,16,19,20,2 1,2 6) . We have found it in D. antillarum, E . lucunter and T . ventricosus. The dimensions of 166 specimens were 92 (65-143) ,U X 64 (42- 114) p with standard deviations of 14.4 p for length and 14.2 p for width. By host species, 100 specimens from E .

CILIATES OF SEA URCHINS I

Zucunter were 94 (65-143) p X 68 (46-114) p with stand- ard deviations of 16.3 p and 15.5 p ; 49 specimens from D. antillarum were 91 (68-108) p x 54 (42-68) p with stand- ard deviations of 10.6 p and 7.9 p ; and 17 specimens from T. ventricosus were 86 (65-104) p X 64 (55-72) p with standard deviations of 9.3 p and 5.0 p.

Metopus rotundus Lucas, 1934 Lucas ( 13 ) described this species from D. antillarum ;

Powers(l9) also found it only in D. antillarum. We found it in E. lucunter of the west zone of “El Caribe” and of “Playa Grande,” but not in D. antillarum. The dimensions of 68 specimens were 119 (78-235) p X 94 (55-196) p with standard deviations of 32.8 p and 28.4 p.

Powers(l9) found only small numbers of M . rotundus (5-15 specimens to 100 M . circumlabens). We found much higher infections in some sea urchins (100 M . rotundus to 10 M . circumlabens, or equal numbers of both species).

Euplotes sp. AndrC(1)) in 1910, reported finding Euplotes charon in

Echinus esculentus. There has been doubt as to the status of this genus as a true endocommensal in sea urchins, al- though it has been reported on occasion(3,4,5,7,8,10,17,18, 24)) usually in low infections. It may be a genus that, in- gested on occasion by sea urchins together with their food, can survive in their intestines and is adapting itself to endo- commensal life( 5).

We have found ciliates of the genus Euplotes in 5 speci- mens of E. lucunter from the east zone of “Playa Grande” (2.5% of the total number of E. lurunter). Less than 10 individuals were found in any infected sea urchin. We can- not give dimensions as we have no stained material.

According to Scorza(ZZ), the two marine species of Eu- plotes so far identified in Venezuela are E. charon and E . taylori. Our species may be one of these.

Table 3 shows the distribution of the nine species of ciliates in the three most common species of sea urchins in the two localities studied.

VARIATION I N THE IKCIDENCE OF CILIATE IA\TFECTIOATS I N E. LUCUNTER I N RELATION

TO T H E COLLECTIOllr ZONE

Table 4 shows the percentage infection by each ciliate species in E. lucunter of each of the four collection zones. Comparable numbers of the sea urchins were taken from

T.ZI{IJ 3. F r ~ q i r ~ n i ~ ~ i of eiZitr/r,s in s r m trr.chin.s strtrlicd.

h’clr ino ntctrn Diudrnia Tripnrztstes lncrtn Icr art t iZZuririn vmtricostis

Cilia tcts % infectioii -, dnop l i ry s nglyct~.s 3 1.8 80.8 20.0

100.0 d?topltrys rlon,ijiitq. 6 l . i - c01m i l p n i b u s curci 0.5 3.8 60.0 Biggnria berrnnrlorsis I .a 90.4 80.0

20.0 Biggaria eeli ino~rrtrtri.s 19.4 - Cyclitlitrni ,hnbdotr.ctii t i t 2i.4 15.4 80.0 Metoprts cirerr nrlubots 34.3 23.1 60.0 Yetopzra rot~ll tdrrs 15.9 - E’Upl0lP.s S1’. 2.5

r r

- - -

TARLE 4. I’arinfion of tltc poptilation of ciliates in relation to the /7lz.stribution of Ecliiiionic+r:i 1ueulIter b y locality and zone (figures

show % infect ion) .

“Playa Grande” “El Cnrihe” Cilintcs Eas t zone

A n opRry.9 aglqcus 60.3 Anopltrys rlongata 32.4 Coknzlemb?ts caecz 1.5 Rz ggar ta berm uden sis 2.9 Btpgnrza rchinomrtris 2.9 C!/eltt7rnnt rhabdotcctiim i . 4 Mrtopttn cirrttmlabens 22.1 Nr.loprr.$ rotnndns - IC’rrpZoles ”I. -

West zone E a s t zone West zone

9.1 42.9 7.4 88.6 94.3 55.6

- 34.3 1.9 63.6 20.0 7.4 4i.7 51.4 20.4 61.4 28.6 31.5 38.6 17.1 16.7

- - -

- - 14.3

each zone, but their intestinal faunas show a large varia- tion in incidence. None of the ciliate species is evenly distributed through the whole four zones, and two species (Cohnile?nbus caeci and Euplotes sp.) were found in only one zone each. It is clear that the ciliates prefer to infect the sea urchins of particular areas. The reasons for this preference are not understood.

The authors wish to express their gratitude to Prof. JosC V. Scorza, head of the Laboratory of Parasitology of the Department of Micro- biology and Parasitology of the Universidad Central de Venezuela, and Dr. Jacques Berger of the University of Illinois for their valuable advice on technics and help in the preparation of this paper. They also gratefully acknowledge the aid of Dr. Kojiro Kato and Prof. Evelyn Zoppi of the Universidad Central de Venezuela in the identi- fication of the sea urchin hosts. Finally they wish to thank Mr. Ian McLure for his help in the translation and preparation of the English version of this paper.

REFERENCES

1. Andre, R. 1910. Sur quelques infusoires marins parasites et commensaux. R e v . Suisse 2001. 18, 173-87.

2. Ball, R. J. 1924. Some new parasites of the Bermuda Echi- noidea. (Abstr.) Anat. Rec. 29, 125.

3. Beers, C. D. 1948. The ciliates of Strongylocentrotus drubachi- ensis: incidence, distribution in the host, and division. Biol. Bull. 94,

4. - 1954. Plagiopyla minuta and Euplotes balleatus, ciliates of the sea urchin Strongylocentrotus dr6bachiensis. J . Protozool. 1,

5 . ~ 1961. Is the ciliate Euplotes balleatus adapting to com- mensal life in the sea urchin Strongylocentrotus drobachiensis? J . Parasit. 47, 478.

6. Berger, J. 196Oa. Holotrich ciliates entocommensal in the sea urchin Strongylocentrotus echinoides from San Juan County, Wash- ington. J . Parasit. 46, 164.

7. __ 1960b. The entocommensal ciliate fauna of Strongylo- centrotus spp. from the Northeast Pacific. (Abstr.) 1. Protozoi 1. 7 (Suppl.), 17.

8. - 1961. Additional records of entocommensal ciliates from nearctic echinoids. (Abstr.) J . Protozool. 8 (Suppl.), 14.

9. - 1962. (Personal communication.) 10. Berger, J . & Profant, R. J . 1961. The entocommensal ciliate

fauna of the pink sea urchin, Allocentrotus fragilis. J . Parasit. 47,

11. Biggar, R . B. & Wenrich, D. H. 1932. Studies on ciliates from Bermuda sea urchins. 1. Parasit. 18, 252-7.

12. Corliss, J . 0. 1953. Silver impregnation of ciliated protozoa by the Chatton-Lwoff technique. Stain Tech. 28, 97-100.

13. Lucas, M. S. 1934. Ciliates from Bermuda sea urchins. I . Metopus. J . R o y . Micr. Soc. 54, 79-93.

14. - 1940. Cryptochilidium (Cryptochi lum) bermudense, a ciliate from Bermuda sea urchins. J . Morph. 66, 369-90.

15. Lynch, J . E. 1929. Studies on the ciliates from the intestine of Strongylocentrotus. I. Entorhipidium gen. nov. Univ. Calif. Publ. 2001. 33, 27-56,

16. M e , D. 1934. Studies of the intestinal ciliates of sea urchin

99-112.

86-92.

417-8.

8 PLASMODIUM IN THE FAR EAST

from Amoy. Ann. Rep. Mar. Biol. Assoc. China 3, 81-90. 17. Poliansky, G. I. & Golikova, M. N. 1959. [On the infusoria

dwelling in the intestine of sea urchins. ,Part 111. Infusoria of sea urchins of the Barents Sea.] 2001. Zhur. 38, 1138-45.

18. Powers, P. B. A. 1933. Studies on the ciliates from sea urchins. I. General Taxonomy. Biol. Bull. 65, 106-21.

19. - 1935. Studies on the ciliates of sea urchins. A general survey of the infestations occurring in Tortugas echinoids. Pap. Tortugas Lab., Carnegie Inst. Wash. 29, 293-326.

20. - 1936. Ciliates infesting Acapulco sea urchins. (Abstr.) J. Parasit. 22, 541.

21. - 1937. Studies of the ciliates of sea urchins. Yearbook Carnegie Inst. Wash. 36, 101-3.

22. Scona, J. V. 1963. (Personal communication.) 23. Snedecor, G. W. 1957. Statistical Methods, 5th ed. The Iowa

State College ,Press, Ames, Iowa. 24. Strelkov, A. 1959. [Parasitic infusoria of sea urchins from the

sea shores of southern Kuril Isles.] 2001. Zhurn. 38, 23-30. 25. Tengler, M. 1957. Un metodo ripido de impregnacihn de la

plata para protozoarios enterozoicos. Actu Cient. Venez. 8, 131-3. 26. Yagiu, R. 1935. Studies on the ciliates from sea urchins of

Yaku Island, with a description of a new species, Cryptochilidium ozakii sp. nov. J . Sci. Hiroshima Univ. (B, 1) 3, 139-47.

J. PROTOZOOL. 13(1), 8-11 (1966).

Plasmodium japonicum, P . juxtanucleare and P . nucleophilum in the Far East*

REGINALD D. MANWELL

Department of Zoology, Syracuse University, Syracuse, New York

SYNOPSIS. Plasmodium japonicum and P. juxtanucleare are two very similar species of avian malaria parasites. The former was dis- covered in domestic fowl in Japan, and the latter in the same host species in Brazil; it has since been found in chickens in Uruguay, Mexico, and Ceylon. The present study, based on a Ceylon strain of P . juxtanucleare and slides from the Bamboo Partridge (Bambusi- cola thoracica sonorivon) of Taiwan indicates that the latter host is a natural reservoir of P . juxtanucleare, since this species appears t o differ in no significant way from P . japonicum. Infection is com-

LTHOUGH interest in avian malaria has declined some- A what in recent years, partly because of the lessening importance of human malaria and partly because of the availability of Plasmodium berghei as an experimental tool, new species of avian Plasmodium continue to be found and it is apparent that what once seemed a relatively small genus of Haemosporidina is in fact a large one. Since it is important to know just how large, a critical comparison of morphologically similar species is essential. Ideally, this should mean not simply a careful study of morphology in stained blood films but one of mosquito transmission, exoerythrocytic forms, susceptibility of various host spe- cies, parasite physiology, and all other aspects subject to laboratory or field investigation. Circumstances often make so complete a study impractical however, and this is pres- ently the case with Plasmodium juxtanucleare, P. japonicum, and the probably closely related species P. nucleophilum. Since the first two may well be identical, and the third could easily be mistaken for them if only the younger stages were seen, a comparison seems important.

P . japonicum was described by Ishiguro in 1957 from the common fowl in Japan, and P. juxtanucleare by Versi- ani and Gomes(9) in 1941 from the same host species in Brazil. Ishiguro may not have been aware of this earlier work since he did not mention it. A critical comparison WQUM also have been difficult, since Versiani and Gomes published no figures. Plasmodium nucleophilum was de- scribed by the present author from a catbird (Dumetella carolinensis) caught in Syracuse, N. Y. ( 7 ) .

*Aided in part by Grant AI-05182, National Institutes of Health.

mon in the Bamboo Partridge (5 of 26 birds showed i t ) , but it has not so far been found in any other avian species, although a total of 973 birds, belonging to 17 orders, 45 families, 110 genera, and 183 species, have been examined from this area. Since the younger stages of Plasmodium nucleophilum look very much like similar stages of P. juxtanucleare, they are compared. The older stages however are usually easily distinguishable, and the former species is so far known only from non-gallinaceous birds whereas the latter appears to be limited to gallinaceous ones.

MATERIALS AND METHODS

Parasites at first identified as Plasmodium japonicum were available for study on blbod films from the Bamboo Partridge (Bambusicola thorucica sonorivox) of Taiwan (Formosa). This is a gallinaceous bird resident in low mountainous areas in the central part of the island. The films were made from birds captured or killed either by members of Naval Medical Research Unit No. 2 or by collectors employed by the unit, fixed, stained with Giemsa, and later shipped in batches to the author’s laboratory.1 Examination was always under an oil immersion apochromatic objective and a 1OX ocular.

A strain of Plasmodium juxtanucleare for comparison was secured ,through the courtesy of Dr. P. C. C. Garnham, of the London School of Hygiene and Tropical Medicine. It was originally isolated by Dhanapala in 1959 from a domestic chicken in Ceylon. Since receiving infected blood in the fall of 1964 we have maintained it in adult White Leghorn chickens.

Plasmodium nucleophilum was maintained in our laboratory for a number of years after its isolation in 1935. Both canaries and White Pekin ducklings were used as hosts. Unfortunately i t was lost after the author entered the army in World War 11, and despite the examination of blood films from thousands of birds of various species in the years since it has never been seen again. It is appar- ently very uncommon at least in the eastern United States, although it has been reported from various parts of the world by a number of investigators, among them Prof. A. A. Sandosham, who recently sent the author a slide from the Glossy Tree Starling ( A p h i s panayensis strigatus) of Malaysia, in the blood of which were numer- ous very typical parasites.

1 The slides are from a collection of Dr. Robert E. Kuntz (Captain, MSC, USN), of the U. S. Naval Medical Research Unit No. 2 . The collector (REK) is indebted to Mr. Zuh-ming Dien, Curator of Birds and Mammals, Taiwan Museum, for assistance in the preliminary identification of hosts, and to Mr. H. G. Deignan, Curator, Division of Birds, U. S. National Museum, Washington, D. C., for later con- firmation. He also wishes to acknowledge the capable technical assistance given by George M. Malakatis, HMC, USN, and Mr. Lo Chin-tsong of NAMRU No. 2.