Ceratomyxa sparusaurati N. Sp. (Myxosporea: Bivalvulida), a New Parasite from Cultured Gilthead...

J. Euk. Microbiol., 42(5), 1995, pp. 529-539 0 1995 by the Society of Protozoologists

Ceratomyxa sparusaurati N. Sp. (Myxosporea: Bivalvulida), a New Parasite from Cultured Gilthead Seabream (Sparus aurata L.) (Teleostei: Sparidae):

Light and Electron Microscopic Description -

ARIADNA SITJA-BOBADILLA,' OSWALDO PALENZUELA and PILAR kLVAREZPELLITER0 Institulo de Acuicultura de Torre de la Sal (IATS) (C.S.I.C.), Ribera de Cabanes, 12595 Castellh, Spain

ABSTRACT. A new Myxosporea, Ceratornyxa sparusaurati n. sp., was found in the gall bladder and bile of cultured gilthead sea bream (Sparus uurata L.) from different Spanish fish farms. It is clearly different from all the previously reported Cerutomyxu from sparids, and it is distinguished from other members of the genus by the shape and size of the spores. Prevalence of infection was 2.15% in an Atlantic farm, 48.7% in a Mediterranean farm and 28.6% in the facilities of the Instituto de Acuicultura de Torre de la Sal. The cell-in-a-cell patlern was found through all the sporogenesis and the general ultrastructure resembled other Myxosporea. Primary cells with two developing spores, harbored other secondary cells. Disporous sporoblasts contained numerous membrane-bound inclusions. a few lipid droplets and polysaccharides as evidenced by cytochemistry. In mature spores, binucleate sporoplasmic cells contained abundant Thikry-negative sporoplasmosomes.

Supplementary key words. Cytochemistry, fish, parasites, protozoa, sporogenesis, taxonomy.

HE gilthead sea bream is a marine teleost successfully cul- T tured in the Mediterranean area. Nevertheless there is an increasing number of reports of pathological problems. Several protozoan parasites have been reported in this fish from Spanish facilities [ 11, and four myxosporean species have been described [ 1, 5 . 22, 31, 321 but, to the best of our knowledge, no Cera- tornyxa species is known from this host.

The genus Ceratomyxa ThClohan 1892, includes more than 130 species, mainly from marine fish with a world wide geographical distribution. Most of these species were described many years ago, with poor data and illustrations, and even recent reports do not follow the guidelines for species descriptions established by Lom & Arthur [9]. This situation makes specially difficult the task of taxonomic comparisons. Presently, there is an increasing number of myxosporean descriptions accompa- nied with ultrastructural information on spores and developmental stages. Nevertheless, this information is very scarce in the genus Ceratoinyxa.

In the present work, a new species of Ceratornyxa from Sparus aurata is described using light and electron microscopy, and information on sporogenesis is provided. Data on the epide- miology and the pathogenic action of this myxosporea will be presented in a subsequent paper.

MATERIALS AND METHODS Fish. A total of 65 1 cultured gilthead sea bream were sampled

from January 1990 to July 1994 from three different Spanish manne facilities. One hundred and eighty-six fish came from a semiintensive growing system at the South Atlantic Coast. Their length ranged between 4.6 and 29.6 cm. One hundred and thirty three were from a semiintensive growing farm at the Western Mediterranean Coast (River Ebro delta). Their length ranged between 6.3 and 31 cm. The third source (Instituto de Acui- cultura de Torre de la Salk intensive open system, at the Western Mediterranean Coast of Castellon) provided 332 fish, ranging from 7 to 33 cm long. Fish were killed by overexposure to the anesthetic MS-222 (Sigma, St. Louis, MO) necropsied and their organs excised for both fresh and histological examination for the presence of fungi and parasites.

Microscopic examination and histological procedure. Small tissue portions were fixed in 2.5% v/v glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2, 4" C), postfixed in 1% w/v cacodylic OsO,, dehydrated through a graded ethanol series, and embedded in Spurr's resin [33]. Ultrathin sections were double stained

I To whom correspondence should be addressed.

with uranyl acetate and lead citrate [23]. Some sections were stained by the ThiCry reaction for carbohydrates [34], and the technique for lipids (OTO) [24]. They were studied in a Philips CM-200 transmission electron microscope (TEM), operating at 60 kV.

Spore measurements were taken directly from fresh material, mainly bile, observed in a light microscope (LM), and, unless otherwise stated, they are expressed in micrometers. The description of the myxosporean species was made according to the criteria established by Lom & Arthur [9].

RESULTS Host. Sparus aurata L. family: Sparidae. Juveniles and adults

were infected. Mean prevalence of infection was 2.15% in an Atlantic farm, 48.7% in a Mediterranean farm, and 28.6% in the facilities of the Instituto de Acuicultura de Torre de la Sal, Spain.

Locality. Mediterranean Coast (River Ebro delta and Cas- tell6n Coast) and South Atlantic coast of Spain.

Habitat. Coelozoic: bile and gall bladder. Characteristics of spores (n = 30). Mature spores somewhat

crescentic in front view, with a convex anterior end and a flattened or slightly curved posterior one (Fig. 1-4), measuring 4.5- 7.5 (K = 5.65; SD = 0.74) long x 14-17.5 (R = 15.76; SD = 1.01) thick (in sutural view). Spore surface smooth, without mucous envelope. Sutural line visible but not conspicuous. Two equal subspherical polar capsules, opening at the anterior end of the spore, measuring 2.2-3.4 (2 = 2.79; SD = 0.27) diameter. Coils of polar filament = 6. Sporoplasm with two nuclei, oc- cupying most of the spore volume. Aberrant spores with 3 polar capsules and 3 valve projections were also observed (Fig. 5).

Developmental stages. Ameboid to round trophozoites. with numerous refractive bodies or granulated appearance and slug- gish movement (Fig. 6), sometimes displaying filopodial projections, frequently harboring inner secondary cells (Fig. 8, 9). Pseudoplasmodia with two developing spores lying close to each other (Fig. 10, 1 l), generally ellipsoidal and sometimes with the ameboid appearance of young trophozoites (Fig. 7). Some disporous trophozoites also harbored other secondary cells (Fig. 11).

Holotype deposited in the Museo Nacional de Ciencias Na- turales (Madrid, Spain): Colecci6n Invertebrados no Insectos, with acquisition number 3701.01/10a.

Ultrastructural observations on C. sparusaurati. The earliest stage observed was a small uninucleate spherical trophozoite or primary (P) cell, whose nucleus occupied most of the cell volume. Its cytoplasm contained numerous ribosomes, rough endoplasmic reticulum (ER), and large (up to 5.5 pm long), curved mitochondria, with numerous cristae (Fig. 12). Some tropho-

529

5 30 .I. EUK. MICROBIOL.. VOL. 42, NO. 5 . SEPTEMBER-OCTOBER 1995

Fig. 1. Line drawings of Ceratomyxa sparusaurari n. sp. spores. A . Flattened type. B. Curved and the most common type. Bar = 10 pm.

zoites appeared with two nuclei (Fig. 13). Larger trophozoites, either with one or two nuclei, contained a variable number of secondary (S) cells within a vacuolar membrane (Fig. 14 inset). The nucleus of P cells harboring S cells had dispersed hetero- chromatin. The cytoplasm had scattered ribosomes, but abundant mitochondria with numerous cristae and pinocytic vesicles at the periphery (Fig. 13, 14). The Thikry reaction showed the scarce content of polysaccharides in the P cell cytoplasm (Fig. 15). As P cells matured and S cells divided within them, the appearance of the former changed. The number of cytoplasmic organelles decreased and large. spherical. non electron-dense, membrane-bound inclusions increased. These inclusions, av- eraging 1.22 Fm of diameter, and containing very small vesicles. were similar to those observed in the cytoplasm of P cells (Fig. 13). The inclusions contained no polysaccharides (Fig. 15) nor lipids (Fig. 16), as they were negative with the ThiPry and OTO reactions respectively. These inclusions were present even in P cells with maturing spores (Fig. 25). Electron-dense inclusions without membrane boundaries were also observed. They were less abundant and contained lipoidal material, as shown by the OTO reaction (Fig. 16). Rhizoid-like projections were observed at one end of some P cells. but never in contact with epithelial cells of the host gall bladder (Fig. 17).

S cells represented the first step in the cell-in-a-cell pattern described for other Myxosporea. They resembled young P cells. in their size, their large nucleus with peripheral heterochro- matin. and their high content of ribosomes and large mitochondria with numerous cristae (Fig. 18. 19). Whorls and strips of rough ER were very common close to the nucleus. The presence of a vacuolar membrane surrounding the cytoplasmic membrane. suggests endogenous division. Up to four S cells, each one with its own vacuolar membrane. were detected in the same section (Fig. 18). Groups of a variable number of S cells surrounded by a common vacuolar membrane were also ob-

served (Fig. 19). On one occasion, a peripheral S cell was seen enveloping another S cell (Fig. 20), both having independent vacuolar membranes (Fig. 20 inset). The former was more electron-dense than the latter due to the abundance of ribosomes, but both had mitochondria and a not very well defined nucleus.

Tertiary (T) cells. representing the second step in the cell-in- a-cell pattern. were observed within some S cells, and surrounded by a vacuolar membrane, probably as a result of endogenous division of S cells as well (Fig. 2 I) . The cytoplasmic and nuclear appearance of T cells were similar to those of recently formed S cells (Fig. 22). Some T cells were initially identified as capsulogenic cells, as they harbored immature capsular primordia (Fig. 23). Nevertheless, no T cells were identified as valvogenic or sporoplasmic cells.

In immature spores, the cytoplasm of capsulogenic cells had high amounts of rough ER, which formed whorls or concentric layers close to the cytoplasmic membrane, and formed a uni- form matrix in which mitochondria, ribosomes, external tubuli and capsular primordium were embedded (Fig. 24). Capsulo- genic cells were completely surrounded by sporoplasmic cells (Fig. 25. 26). The nucleus, located laterally in the cell, was similar to that of other sporogonic cells, with peripheric hetero- chromatin (Fig. 25) , and visible almost until the completion of capsulogenesis (Fig. 28). It shrank progressively until its dis- appearance (Fig. 27). The cytoplasm also degenerated at the end of capsulogenesis (Fig. 27). Mature polar capsules contained a 6-times coiled polar filament and an apical plug (Fig. 27). Poly- saccharides were not detected in the capsular primordium nor in the external tubuli. but were found in the cytoplasm with the ThiCry staining method (Fig 29).

Young binucleate sporoplasmic cells were observed in groups of S cells surrounded by a common vacuolar membrane (Fig. 30). Their cytoplasm contained numerous ribosomes, mitochondria and whorls of rough ER close to the two nuclei (Fig. 3 I ) . In maturing spores they seemed to envelope capsulogenic cells (Fig. 25, 26). In mature spores, sporoplasmic cells, which occupied most of the spore volume (Fig. 32). were enriched in glycogen granules (confirmed by the ThiPry reaction) (Fig. 36). The nuclei were close to each other and often surrounded by rough ER and sporoplasmosomes (Fig. 32). The latter structures, of variable shape and size, had an electron-dense core which appears to be delimited by a unit membrane as the one which delimited the whole sporoplasmosome (Fig. 33,35) and ThiCry- negative staining (Fig. 36). On some occasions. the core appeared segmented (Fig. 35).

Valvogenic cells occupied an external position in relation to other sporogonic cells (Fig. 26). The nuclei of each cell was located laterally and the cytoplasm contained few organelles, mainly mitochondria and small vesicles. The cytoplasm of the valvogenic cell thinned progressively (Fig. 37) until the formation of valves and the appearance of the sutures or desmo- some-like junctions in mature spores (Fig. 33. 34). No polysaccharide content was detected by the ThiPry reaction (Fig. 38).

Each maturing spore, formed in the same P cell, was envel- oped by its own common membrane (Fig. 39, 40). The cytoplasm of disporous P cells containing mature spores, appeared almost completely degenerated. but occasionally it maintained

+

Fig. 2-11. Light microscopy images of Ceruiotny.rcz sparusauruii n. sp. from fresh smears of bile. 2, 3. Mature spores. 3. Most common type of spore. Nomarski interference optics. 4. Flattened type of spore. 5 . Aberrant spores. 6 . Young round trophozoites. 7. Flattened trophozoite harboring a mature spore (arrowhead). 8. Primary cell harboring one secondary cell. 9. P cell harboring two S cells. 10. Disporous sporoblasts and small round and ameboid trophozoites (arrows), Notice the sporoblast with two aberrant spores (arrowhead). Phase contrast. 11. Detail of disporous sporoblasts. Notice one of them with two spores and other S cells (arrowheads). Phase contrast. Bars = 10 pm.

SITJA-BOBADILLA ET AL.-DESCRIPTION OF CERATOMYXA SPARUSAURATI N. SP 53 I

532 J . EUK. MICROBIOL., VOL. 42, NO. 5. SEPTEMBERaCTOBER 1995

SITJA-BOBADILLA ET AL.-DESCRIPTION OF CERATOMYXA SPARUSAURATI N. SP. 533

the ameboid shape of young trophozoites and some small mitochondria (Fig. 41).

DISCUSSION Taxonomic remarks. Ceratomyxa sparusaurati clearly differs

from the three species reported from the gall bladder of members of the family Sparidae. Its spores are much smaller than those of C. pallidae ThClohan, 1895 [8] from Boops spp., C. span Awerinzew, 19 13 [8] from Sparus berdu and C. diplodae Lubat, Radujkovic, Marques & Bouix, 1989 from Diplodus annularis

The morphometrical features of C. sparusaurati are also different from those of other members of the genus parasitizing fish from different host families and, in most cases, with a very different geographical distribution. Although the ranges of spore thickness and length of some species overlap the ones of C. sparusaurati (Table l), they differ in other aspects: C. navicularia Davis, 1917 [3] is boat-shaped; C. recurvata Davis, 1917 [3] and C. beloneae Lubat, Radujkovic, Marques & Bouix, 1989 [ 171 are greatly curved, and the former is even sharply pointed with bigger polar capsules; C. gibba Meglitsh, 1960 [18] has unequal polar capsules and valves; C. recta Meglitsh, 1960 [ 181 and C. sprenti Moser, Kent & Denis, 1989 [21] are flattened or straight: C. asvnirnetrica Moser & Noble, 1976 [20] and Cer- atornyxa sp. 2 [ 171 have unequal valves; C. opisthocentri Dogie], 1948 [25] and C. castigatoides Meglitsh, 1960 [ I81 have smaller polar capsules; C. declivis Meglitsh, 1960 [18] has crescent rounded, truncated tips and smaller polar capsules; C. lobata Evdokimova, 1977 [6] has a left, lateral process. Therefore, in view of the differentiating characteristics, it is considered that the currently described species is new, and it is named after its host.

Ultrastructural aspects. A comparative study of the species within the genus Ceratorqxxa is specially difficult due to the paucity of information concerning ultrastructural knowledge.

~ 7 1 .

Most of the features of C. spurusaurati spores resemble those of other myxosporea. The binucleate sporoplasm has been reported in many Ceratomy.xa light microscopical descriptions and in the fine structure of C. shasta Noble, 1950 [35], C. glob- ulifera ThClohan, 1895 [4], and C. labracis [27]. Two uninucleate sporoplasms have also been cited [ 1 11, but electron microscopic confirmation of this feature has not been provided. In any case, both sporoplasmic configurations could be considered as diag- nostic features of the genus, as it happens in Sphaerospora (a close bivalvulid genus) [30].

The so-called sporoplasmosomes [ 151, found in the sporoplasm of mature C. sparusaurati spores, were Thikry-negative, as in all sporoplasmosomes described thus far [ 161. They resemble those in the sporoplasm of C. labracis [27], though they were more numerous and variable in shape and size. The origin and function of these structures remains unclear. Their absence in young sporoplasmic cells and in immature spores of C. spa- rusuurati suggests that they are deposition products, since the spore is the resistance stage of the organism. Furthermore, the presence of an inner core of variable appearance surrounded by a membrane, leads us to hypothesize a mitochondria1 origin. The different aspects of the inner core could be considered as different stages of the mitochondria1 transformation. This change into deposit structures has been reported in distantly-related cell types, such as ovocytes of different vertebrates. In these cells. mitochondria turn into vitelinic platelets [2].

Concerning sporogenesis of C. sparusaurati. disporous development, observed both at LM and TEM has also been reported in C. hungarica [19], C. shasta [35], C. diplodae and C. labracis [27]. In cases of disporoblastic and monosporoblastic development, as shown in C. shasta [35] and several Sphaeros- pora species [ 13, 141 no pansporoblast formation is supposed to occur. When a pansporoblast is formed, an outer generative or pericyte cell envelopes an inner generative or sporogonic cell. This process seems to be illustrated in Fig. 20. On the other hand, if two sporoblasts are to be formed inside the same per-

~

c

Fig. 12-17. Transmission electron microscopy images of C. sparusaurati n. sp. 12. Young primary cells. Bar = 1 ~ I T I . 13. Primary (P) cell with two nuclei (n,), mitochondria (m), numerous pinocytic vesicles (arrowheads) and inclusions (i). Bar = 1 pm. 14. P cell harboring a secondary (S) cell. Bar = 1 pm. Inset: detail of the vacuolar membrane surrounding the S cell. Bar = 0.25 pm. 15. Thitry staining of a P cell harboring two S cells. Notice the lack of staining of the inclusions (i). Bar = 2 pm. 16. Lack of OTO staining of the inclusions (i) of a P cell, and the positive reaction of a droplet which lacks a delimiting membrane (*). Bar = 1 pm. 17. P cell with short rhizoids close to the gall bladder epithelium (h). Bar = 1 pm.

+ Fig. 18-23. Transmission electron microscopy images of C. sparusaurati n. sp. 18. Four secondary (S) cells in a primary (P) cell. n,, RUCleUS

of P cell; n,,, nucleus of S cell. Bar = 1 pm. Inset: detail of vacuolar membranes surrounding two S cells. Bar = 0.25 pm. 19. Group of six S cells, five of them surrounded by a common vacuolar membrane. Bar = 1 pm. 20. A S cell enveloping another S cell. Bar = 1 pm. Inset: detail of the two independent vacuolar membranes. Bar = 0.25 pm. 21. P cell with four S cells, one of them harboring a tertiary (T) cell. Bar = 1 pm. Inset: detail of the four membranes at the junction of two S cells. Bar = 0.25 pm. 22. Detail of the T cell. Notice the vacuolar membrane (arrowhead). Bar = 0.5 pm. 23. T cell as a capsulogenic cell. Notice the capsular primordium (cp). Bar = 1 pm.

Fig. 24-29. Transmission electron microscopy images of C. sparusaurati n. sp. 24. Young capsulogenic cell with capsular primordium (cp), external tubuli (et), abundant rough endoplasmic reticulum (*) and mitochondria (m). Bar = 0.5 pm. 25. Maturing spores in a primary (P) cell. Valvogenic cells (v) embrace a sporoplasmic cell (sc), and the latter embraces capsulogenic cells (cc). Arrows indicate the incipient sutures between valvogenic cells. Bar = 1 pm. 26. Detail of the limiting membranes among cc, sc, vc and P cell. Bar = 0.5 pm. 27. Detail of a capsulogenic cell in an almost mature spore. Notice the coils of the polar filament in the polar capsule and the plug (arrow). Bar = 1 pm. 28. Detail of the degenerated nucleus (n) of a cc; mitochondria (m) and glycogen-like granules are the dominant cell structures. Bar = 0.5 pm. 29. Thitry-positive staining of the cytoplasm of a capsulogenic cell. Bar = 1 pm.

534 J . EUK. MICROBIOL, VOL. 42, NO. 5, SEPTEMBERaffOBER 1995

536 J. EUK. MICROBIOL.. VOL. 42. NO. 5. SEPTEMBER4ffOBER 1995


Fig. 37-41. Transmission electron microscopy images of C. sparusaurati n. sp. 37. Valvogenic cells with incipient suture (arrowhead). Bar = 0.25 pm. 38. Lack of Thitry stained structures in a valvogenic cell (v). Bar = 0.5 pm. 39. Primary (P) cell with two maturing spores. Bar = 2 pm. 40. Detail of the limiting membranes between the two spores of Fig. 39. Bar = 0.5 pm. 41. Two mature spores inside a degenerated P cell. Bar = 2 pm.

icyte cell, a common membrane should be seen encircling them, and this does not seem to be the case in C. sparusaurati; instead, two different membranes surrounded each maturing spore inside the P cell, as in Sphaerospora testicularis [28] and Zschokella rnugilis [29]. The absence of an ectoplasm in P cells [ 131 and the observation of groups of five cells (possibly the not yet differentiated sporogonic cells) surrounded by a common mem-

brane could also refute the perycitic nature. Thus, we are more inclined to suggest that a sequence ofendogenous cleavages and mitosis are probably involved in C. sparusaurati sporogenesis.

The presence of other non-sporogonic S cells in disporous P cells, detected both by LM and TEM, eludes our understanding. The similar size and appearance of these cells and T cells, t o small P cells, could indicate a possible role in the enhancement

t

Fig. 30-36. Transmission electron microscopy images of C. sparus aurati n. sp. 30. Group of five secondary (S) cells. Notice the centrally located binucleated cell, probably a sporoplasmic cell. Bar = 1 pm. 31. Detail of a sporoplasmic cell. Notice the abundant rough endoplasmic reticulum (arrows). Bar = 0.5 pm. 32. Mature spore in frontal view. Polar capsules (pc) are broken. Bar = 2 rm. 33. Basal section of a mature spore showing the sutures (arrowheads) and the numerous sporoplasmosomes (*). Bar = 1 pm. 34. Detail of an apical suture. Bar = 0.25 pm. 35. Detail of sporoplasmosomes. Bar = 0.5 Km. 36. Thitry staining of a mature spore. Bar = 1 pm.

538 J . EUK. MICROBIOL.. VOL. 42, NO. 5. SEPTEMBER-OCTOBER 1995

Table 1. Comparison of Ceraro/nj:vu sparztsaurari n. sp. with the most closely related species of Ceratomy.xa. The minimum and maximum range and/or average, in parentheses. are provided. when available from the literature. Measurements in micrometers. * = fixed spores.

Polar Spore Polar capsules tila-

nient Geographical Data Species Lenath Thickness Lenath Thickness coils Location Hosts distribution from

C. s~arusaurati

C. asymrnetricu

C heloneae

C tasirgaroides*

C declivis

C ghha

C lobaia

C nar.iciilariu

C. opisthocentri

C. rec-ra

C. recurvatu

C. sprenti

4.5-7.5 14-17.5 (5.65) ( 1 5.76)

4-6 12.5-17 (4.9) (14.1)

6.5-7.5 14-16 (7.25) (16.05)

(5.9) (14.7)

(5.9) ( 14.4)

(6.9) ( I 7.0) 6.3-7 I 4- 1 4.7

5.1-7.3 9.8-17.8

5.1-6.8 13.5-15.2

5.6-8 14.2-18.9

5-7 14-17

6.8-8.8 14.7-16.? (7.8) ( 1 5.6)

8-9 (16)

4-8 14.0-23.0 (5.7) ( I 6.3)

2.2-3.4 6 G. bladder (2.79)

2-3.5 4-5 G. bladder (2.9)

(2.75)

(2.0) 1.7-2.8 (2.4)

2.5-3.3 (2.8) 3.5

I .8-2.6

? G. bladder

? G. bladder

1.7-2.2 &5 G. bladder

1.8-2.5 ? G. bladder

? U. bladder

(2.5)

(2.0)

(2.3)

? G. bladder ( 2 )

1-1.5 ? G. bladder

2.0-3.4 4-5 U. bladder (2.6)

? G. bladder (4.5)

2-3 5-6 G. bladder (2.4)

Sparus aurata

Coqphaenoides

Ventrifosa macro-

Diplodus annularis

Pseudolabrus cocci-

Cjttus novaeielan-

Congiopodus Ieuco-

.-lustroatherina in-

Paralichthp den-

P. albiguttus Sphaeroidcs macu-

latus Opisthocentrus

ocellatus Genypterus hla-

codes C'esiracion ggaena

Chaerodon aureo- ,fasc'iatus

Ch. ruinfordi Choerodon w u s -

Lutjanus amabilis Platycephalus bos-

cireneus

pogon

neus

diae

paecilis

cisa

datus

trus

schei

Mediterranean and Atlantic Spanish coasts

Alaska, Venezuela

Adriatic Sea

New Zealand

New Zealand

New Zealand

Patagonian Shelf

Beaufort, USA

Sea of Japan

New Zealand

Beaufort, USA

Australia

of the proliferation of the parasite. and they could be capable of starting new sporogonic cycles. as reported for blood and SBI stages of Sphaerospora renicola [ 101.

Energy reserves are found in most trophic stages of myxosporea, either as polysaccharides [7] or lipids [38]. C. sparusaurati P cells had a low content of both types of reserves, as evidenced with cytochemistry. instead they had abundant membrane-bound inclusions of unknown cornposition. which correspond to the granulated aspect of some trophozoites at LM. Similar inclusions were also dctected in C. lahracis [27]. Further studies are underway to elucidate their composition and role in P cells.

Capsulogenesis is supposed to proceed along the pattern which was pointed out by Lorn & Puytorac [ 171 and which appears valid for most myxosporea. The high content in rough ER of capsulogenic cells of C. sparusaurati is similar to those of other bivalvulids thus far described. which reaches its maximum ex- pression in Po/~~sporop/asi i~a species [ 3 11. The polysaccharide content of capsulogenic cells. observed in this study. is higher than that of other sporogonic cells, but is lower than that observed in other Myxosporea. such as Sphaerospora dicentrarchi [26]. Otherwise, in C. sparu.suurati, the identification of capsulogenic cells as T cells (Fig. 23) , could lead to suggest that T

cells are the sporogonic ones, as it has been hypothesized in Sphaerospora testicularis [ 2 8 ] . Nevertheless, the examination of immature spores showed that this is not the case, as the putative vacuolar membrane is in fact the cytoplasmic membrane of a neighboring sporoplasmic cell, which is surrounding the capsulogenic cell (Fig. 25. 26). Thus, we incline to think that S cells are the sporogonic ones, as in pseudoplasmodia of some Sphae- rospora species [ 101.

In conclusion, we have to keep an open mind concerning the interpretation of the sporogenesis in C. sparusaurati in view of the LM and TEM images. It is essential to obtain more precise ultrastructural information concerning sporogenesis in this myxosporea. In vitro culture of this parasite, currently under- wal. should provide abundant material for study.

ACKNOWLEDGMENTS This uork was supported by a research grant from Spanish

CICYT no. AGF92-0199-C02-01. We are grateful to techni- cians from the Electron Microscopy Service of the University of Barcelona. 0. P. was recipient of a research grant from the Departamento de Educacibn, Universidades e Investigacih del Gobierno Vasco.

539 SITJA-BOBADILLA ET AL.-DESCRIPTION OF CERATOMYXA SPARUSAURATI N. SP.

LITERATURE CITED 1. Alvarez-Pellitero, P., SitjB-Bobadilla, A., Franco-Sierra, A. & Pal-

enzuela, 0. 1995. Protozoan parasites of gilthead sea bream (Sparus aurata L.) from different culture systems of Spain. J. Fish Dis., 18: 105- 115.

2. Berkaloff, A., Bourguet, J., Favard, P. & Guinnebault, M. 1977. Biologia y Fisiologia Celular. Ediciones Omega, S. A. Barcelona.

3. Davis, H. S. 1917. The myxosporidia of the Beaufort region, a systematic and biological study. Bull. Bur. Fish., 35: 199-252.

4. Desportes, I. & ThCodorides, J. 1982. Donnes ultrastructurales sur le sporogenhse de deux myxosporidies rapporties aux genres Lep- totheca et Ceratomyxa parasites de Merluccius rnerluccius (L.) (TCleos- tCen Merluccidae). Protistologica, 18:553-557.

5. Diamant, A. 1992. A new pathogenic histozoic Myxidium (Myxosporea) in cultured gilthead sea bream Sparus aurata. Bull. Eur. Ass. Fish Pathol.. 12~64-66.

6. Evdokimova, E. B. 1977. Myxosporidians of teleost fishes from the Patagonian shelf (the Atlantic coast of Argentina). Parazitologiva, 11:166-178. (in Russian.)

7. Hermanns, W. & Korting, W. 1985. Sphaerospora tincae Plehn, 1925 in tench, Tinca tinca L. fry. J. Fish Dis., 8:281-288.

8. Kudo, R. 19 19. Studies on Myxosporidia. A synopsis of genera and species of Myxosporidia. Ill. Biol. Monographs, 3-4: 1-265.

9. Lom, J. & Arthur, J. R. 1989. A guideline for preparation of species descriptions in Myxosporea. J. Fish Dis., 12:151-156.

10. Lom. J. & Dykova, I. 1986. Comments on myxosporea life cycle. Symp. Biol. Hirng., 33.

11. Lom, J. & Noble. E. R. 1984. Revised classification ofthe class Myxosporea Butschli. 188 1. Folia Parasitol., 31: 193-205.

12. Lom, J. & Puytorac, P. 1965. Studies on the myxosporidian ultrastructure and polar capsule development. Protistolgica, 153-65.

13. Lam. J.. DykovL, I. & Lothikova, S. 1982. Fine structure of Sphaerospora renicola Dykova & Lom, 1982 a myxosporean from carp kidney and comments on the origin of pansporoblasts. Protistologica.

14. Lom, J., Korting, W. & Dykova, I . 1985. Light and electron microscopic redescription of Sphaerospora tincae Plehn, 1925 and S. galinae Evlanov, 1981 (Myxosporea) from the tench, Tinca tinca L. Prof istologica. 21 : 4 8 7 4 9 7.

1986. Hoferellus gilsoni (Debaisieux, 1925) n. comb. (Myxozoa: Myxosporea): redescription and mode ofattachment to the epithelium of the urinary bladder of its host, the European eel. Protistologica, 22:405-4 13.

16. Lom, J., Feist, S. W., Dykova, I. & Kepr, T. 1989. Brain myxo- boliasis of bullhead, Cottusgobio L., due to Myxobolus jiroveci sp. nov.: light and electron microscope observations. J. Fish Dis., 12:15-27.

17. Lubat, J. , Radujkovic, B., Marques, A. & Bouix, G. 1989. Par- asites des poissons marins du Montenegro: Myxosporidies. Acta Adria- tica, 30:31-50.

18. Meglitsh, P. A. 1960. Some coelozoic myxosporidia from New Zealand fishes. I. General and family Ceratomyxidae. Trans. R0.v. SOC. New Zealand, 88: 26 5-36 5.

19. Molnlr, K. 1992. Ceraiomji.xa hungarica n. sp. and Chloro- myxurii proterorhini n. sp. (Myxozoa: Myxosporea) from the freshwater goby Proterorhinus marmoratus (Pallas). Systematic Parasitol.. 22:25- 31.

18:489-502.

15. Lom, J., Molnar, K. & Dykova, I.

20. Moser, M. & Noble, E. R. 1976. The genus Ceratornyya in macrourid fishes. Can. J. Zool., 54:1535-1537.

21. Moser, M., Kent, L. &Dennis, D. 1989. Gall bladder Myxospo- rea in coral reef fishes from Heron Island, Australia. Aust. J Zool., 37:

22. Paperna, I. 1982. Kudoa infections in the glomeruly, mesentery and peritoneum of cultured Sparus aurata L. J . Fish. Dis., 5539-534.

23. Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol.. 17:208-2 12.

24. Seligman, A. M., Wasserhrug, H. L. & Hanker, J. S. 1966. A new staining method (OTO) for enhancing contrast of lipid droplets in osmium tetroxide-fixed tissue osmiophilic thiocarbohydrazide (TCH). J. Cell Biol., 3W424-432.

25. Schulman, S. S. 1988. Myxosporidia of the USSR. Nauka Publ., Moscow-Leningrad. English translation for the U.S. Dept. Interior and Nat. Sci. Found., Washington, D.C. by Amerind. Publish. Co. Prt. Ltd., New Dehli.

26. Sitja-Bobadilla, A. & Alvarez-Pellitero, P. 1992. Light and electron microscopic description of Sphaerospora dicentrarchi n. sp. (Myxosporea: Sphaerosporidae) from wild and cultured sea bass, Di- centrarchus labrax L. J. Protozool., 39:273-28 I .

27. Sitji-Bobadilla, A. & Alvarez-Pellitero, P. 1993a. Light and electron microscopic description of Ceratornysa labracis n. sp. and a redescription of C. diplodae (Myxosporea: Bivalvulida) from wild and cultured Mediterranean sea bass Dicentrarchus labras (L.) (Teleostei: Serranidae). Systematic Parasitol.. 26:2 15-223.

28. Sitji-Bobadilla, A. & Alvarez-Pellitero, P. 1993b. Ultrastruc- tural and cytochemical observations on the sporogenesis of Sphaeros- pora testicularis (Protozoa: Myxosporea) from Mediterranean sea bass, Dicenlrarchtrs labrax (L.). Europ. J . Protistol., 29:2 19-229.

29. Sitja-Bobadilla, A. & Alvarez-Pellitero, P. 1993~ . Zschokkella mugilis n. sp. (Myxosporea: Bivalvulida) from mullets (Teleostei: Mu- gilidae) of Mediterranean Waters: Light and electron microscopic description. J. Euk. Microbiol., 40:755-764.

30. SitjB-Bobadilla. A. & Alvarez-Pellitero. P. 1994. Revised classification and key species to the genus Sphaerospora (Protozoa: Myxo- sporea). Res & Rev. in Parasitol., 54:67-80.

3 1. Sit$-Bobadilla, A. & Alvarez-Pellitero, P. 1995. Light and electron microscopic description of Polysporoplasrna n. g. (Myxosporea: Bivalvulida), Polysporoplasrna sparis n. sp. from Sparrts aurala (L.) and Pol~~sporoplasma mugilis n. sp. from Liza aurata L. Eitrop. J Protistol..

32. Sitja-Bobadilla, A., Franco-Sierra. A. & Alvarez-Pellitero, P. 1992. Sphaerospora (Myxosporea: Bivalvulida) infection in cultured gilthead sea bream (Sparus aurata L.): a preliminary report. J. Fish Dis.,

33. Spurr, A. R. 1969. A low-viscosity epoxy-resin embedding me- dium for electron microscopy. J. Ultrastruc. Res., 26:3 1-43.

34. Thiiry, J. P. 1967. Mise en Cvidence des polysaccharides sur coupes fines en microscopie Clectronique. J . Microsc.. 6:987-1018.

35. Yamamoto, T. & Sanders, J. E. 1979. Light and electron microscopic observations of sporogenesis in the myxosporidian. Cerato- rny-ya shasta (Noble, 1950). J. Fish Dis.. 2:41 1 4 2 8 .

1-13,

3 1 :7 7-89.

15:339-343.

Received 12-6-94, 3-23-95; accepted 5-2-95

Ceratomyxa sparusaurati N. Sp. (Myxosporea: Bivalvulida), a New Parasite from Cultured Gilthead...

Documents

Transcript of Ceratomyxa sparusaurati N. Sp. (Myxosporea: Bivalvulida), a New Parasite from Cultured Gilthead...