J. Euk Microhiol., 44(5). 1997 pp. 420-426 0 1997 by the Society of Protozoologists

Trichodina ctenophorii N. Sp., a Novel Symbiont of Ctenophores of the Northern Coast of the Gulf of Mexico

ANNE M. ESTES,* BARBARA S. REYNOLDS** and ANTHONY G. MOSS*,' *Department of Zoology and Wildlife Sciences, I01 Cary Hall, Auburn University, Auburn, Alabama 36849, USA, and

**Opp High School, 502 North Muloy Street, Opp, Alabama 36467, USA

ABSTRACT. Peritrich ciliates of the genus Trichodina are internal or external symbionts of invertebrate and vertebrate hosts. We describe here Trichodina ctenophorii n. sp., a symbiont of Mnemiopsis mccraydii and Beroe ovata (Phylum Ctenophora). The mor- phology of fixed and living specimens is revealed by silver impregnation, scanning electron microscopy, and differential interference microscopy. Distinguishing features of Trichodina ctenophorii include a denticular morphology composed of falcate, blunt-tipped blades, and long, straight thorns, with five pins per denticle. Trichodina ctenophorii is found only on the comb plates of these ctenophores. To the best of our knowledge, this is the first report of a trichodinid from the Gulf of Mexico and the first associated with ctenophores.

Supplementary key words. Comb jellies, comb plate, marine invertebrate parasites, peritrich ciliate.

FUCHODINIDS are peritrichous ciliates found worldwide T on a variety of hosts [ l l , 161. The majority of trichodinids are described for freshwater environments [ l , 9, 15, 181. How- ever, they are also found as ecto- and endoparasites or com- mensals of marine organisms, primarily teleost fishes 12, 121. Trichodinids are endoparasites of the urogenital tract of am- phibians [S], chondrichthys [17], and teleosts [lo].

Trichodinids are endo- and ectoparasites of invertebrates as well as vertebrates [8, 10, 161. Two specific cases of Trichodina infestations of invertebrates are early observations by James- Clark of Trichodina on freshwater Hydra [4] and, almost a cen- tury later, the interaction of Trichodina myicola and the marine bivalve Mya 1161.

Mnemiopsis is an extremely common New World coastal ctenophore of approximately 5 cm in length that occurs from the Gulf of Mexico to as far north as Cape Anne in the Gulf of Maine. In the northern Gulf of Mexico, the predominant coastal species is Mnemiopsis mccraydii. A voracious filter feeder, Mnemiopsis, like many ctenophores, plays a critical role in the coastal ecology of the region (Edmiston, L. 1979. The zooplankton of the Apalachicola Bay system. Dissertation. Flor- ida State University, Tallahassee, FL). Beroe ovuta feeds upon Mnemiopsis, and occurs as large individuals late in the summer, when it encounters high prey concentrations, often greater than 24 mum3 (Edmiston, 1979. Dissertation).

Here we describe Trichodina ctenophorii n. sp., a moderate- sized ciliate, which is, to the best of our knowledge, the first trichodinid described for the Gulf Coast region, and the first ever described as being symbiotic on ctenophores. Denticle morphology, small number of pins per denticle, geographic dis- tribution, and the unique host, collectively indicate that this is a previously undescribed species.

MATERIALS AND METHODS Specimen collection. Mnemiopsis mccruydii and Beroe ova-

ta were collected in the vicinity of docks, jetties and pilings located at the Dauphin Island Sea Lab on the eastern end of Dauphin Island, AL from July to November, 1995 and 1996. Ctenophores were also collected from a causeway connecting the mainland to St. George's Island, FL, which divides Apa- lachicola Bay and St. George Sound near the towns of East Point and Apalachicola. Animals were scooped up in small buckets or with one-liter plastic cups attached to sticks as they swam near the surface.

Reagents. All reagents used were from Fisher Scientific (Pittsburgh, PA) except where indicated. Unless otherwise not- ed, all solutions were made up in 0.1 Fm-filtered Type I water.

' To whom correspondence should be addressed. Telephone: 334-844- 9257 ; Fax: 3 34-844-4065 ; Email: mossant @ mail. auburn.edu

Light microscopy. Low power light microscopy of intact ctenophores, and removal of comb plates was performed under a dissecting microscope (Model SZ11, Olympus Corporation, New York). Brightfield, phase contrast and video-enhanced dif- ferential interference contrast (VE-DIC) microscopy of living and fixed, stained specimens was carried out with a compound light microscope (model BHS, Olympus Corporation).

Silver staining. Preparations were subjected to silver im- pregnation after the method of Klein, modified for use with marine specimens (Schaffer, pers. comm.). Comb rows were cut into sections bearing five comb plates and placed onto a clean slide. All excess sea water was drained from the slide prepa- ration with a piece of No. 1 Whatman filter paper. The slide was suspended over a 20-ml scintillation vial containing 4% OsO, at 23" C. Slides were then dried in an open plastic box overnight at 4" C. Sea salts were rinsed from the slide with Type I water and again dried for one to two hours at 4" C. Slide preparations were stained with 2% AgNO, for 14 min at 23" C and again rinsed with Type I water to remove excess silver. Stained slides were placed into a small UV sterilization cham- ber containing two-15 cm UV tubes and irradiated for 20 min. Preparations were then mounted in Permount and after several weeks sealed with model airplane dope (Aero-Gloss, Pacta Corp.). Trichodinids were examined with a X 100, 1.4 n.a. plan- apochromatic oil immersion objective (Olympus).

Video-based imaging and image capture. VE-DIC images were generated by a Newvicon camera (model VE1000, Dage- MTI, Indianapolis, IN). Images were saved on S-VHS tape on a video recorder equipped for still-field playback (model AG7355, Panasonic Industries, Tokyo, Japan), in some cases using a real-time image processor (model Argus-10, Hamama- tsu Industries, Hamamatsu, Japan). Still-field images were cap- tured live or from tape playback with a frame-grabber (model 3030 Flashpoint PCI, Integral Technologies, Indianapolis, IN) and image processing software (model Image-Pro Plus, Media Cybernetics, Bethesda, MD). Images were printed on a dye- sublimation printer (model Primera, Fargo Electronics, Eden Prairie, MN).

Scanning electron microscopy. Ctenophore comb plates were dissected free from the animal with fine iridectomy scis- sors (model E3388GW, Storz Co, St. Louis, MO), stripped of mesoglea, and fixed after the method of Tamm and Tamm [14]. Fixative osmolarity was adjusted with NaCl to the sea salt con- centration at the collecting sites. Scrupulous care was taken to ensure that all solutions were kept on ice at all times to prevent an osmiudglutaraldehyde precipitate. Comb plates were fixed for one hour and washed thoroughly with 0.2 M sodium cac- odylate, pH 7.8, and 0.3 M NaCl. Samples were post-fixed in 1% OsO,, 0.2 M sodium cacodylate pH 7.8, and 0.3 M NaCl for 30 min.

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2

Fig. 3. Brightfield transmitted laser scanning (nonconfocal) view of auricular plates infested with Trichodina ctenophorii.

Fig. 1-2. The location of Trichodina ctenophorii n. sp. on Mne- miopsis mccraydii. 1. Diagram of Mnemiopsis mccraydii. Only one lobe and one of the two pair of auricles are shown for simplicity. The cten- ophore is viewed in the sagittal (= stomodeal) plane. 2. Diagrammatic view of trichodinas on comb plates. Note aboral ("outside surface") location.

Samples were then rinsed twice for 15 min with ice-cold Type I water and dehydrated through a 30-100 % ethanol se- ries. They were then exchanged four times with 100 70 pro- pylene oxide (Electron Microscopy Sciences), and received three exchanges of 2-3 ml of hexamethyldisilizane (Electron Microscopy Sciences) and allowed to dry on filter paper. These were then immobilized on aluminum stubs with double-sided tape and sputter-coated for 2 min with a gold-palladium mixture (model SC-7 auto sputter coater, Ted Pella, Inc., Redding, CA). Samples were viewed on a Zeiss DSM 940 scanning electron microscope (Zeiss Corporation, Germany).

Type specimens. One silver-stained holotype slide specimen has been deposited with the International Protozoan Type Slide Collection, National Museum of Natural History, Smithsonian Institution, Washington, D.C., accession number USNM #47907. Remaining holotype specimens currently remain in the collection of AGM describing the microbial communities of ctenophores.

RESULTS Silver stain analysis was performed on thirty-one Trichodina

ctenophorii n. sp. from five Mnemiopsis mccruydii. The internal structure of fifty living cells were examined by VE-DIC mi- croscopy.

Host association. Trichodina ctenophorii was observed to be attached primarily to the aboral side of auricular, subsaggital, and subtentacular comb plates (Fig. 1, 2). Prior to 1996, we observed the symbionts to be attached solely to the aboral side of plates of the eight meridional rows. During the summer of 1996 at both collection sites, we observed greatly increased concentrations of trichodinids on Mnemiopsis, with some being found on the oral side of the comb plates. T. ctenophorii were observed on auricular plates at a lower frequency (Fig. 3), but in some cases were seen only on the auricular plates. To date, we have never observed T. ctenophorii on the ectodermal sur- face of any ctenophore.

Few trichodinids (n = 3) were attached to B. ovata comb

plates. A portion of the denticular ring could be seen in each specimen which revealed, as best as could be determined, that they were identical to the trichodinids found on M. mccradyii comb plates. Beroid specimens were not included in our mor- phometric analysis because few individuals were collected.

T. ctenophorii was observed to swim freely or remain at- tached to the comb plate. It often attached loosely and spun clockwise in place (as viewed from the oral side of the cell using DIC), or tightly, without spinning, presumably as it might during the comb plate's vigorous effective stroke. The basal ciliary wreath propagated clockwise metachronal waves. In contrast, the adoral ciliary wreath propagated counterclockwise ciliary waves, again as viewed from the oral surface.

VE-DIC was used to obtain cellular detail in living and fixed specimens. Through-focusing clearly revealed the position, ori- entation, and morphology of the macronucleus relative to other organelles, such as the adoral basal bodies and the contractile vacuole. Although the silver stain penetrated the trichodinids well, the radial pins were more easily observed by high-mag- nification DIC of living cells. Details of the denticular ring were also clearly revealed by through-focusing while observing by VE-DIC. We observed numerous bacteria throughout the cy- toplasm of many T. ctenophorii.

Description. Hosts. Mnemiopsis mccradyii and Beroe ovata Locale. Northern coast of the Gulf of Mexico, specifically

Mobile Bay off Forts Gaines and Morgan, and from the main- land-to-island causeway that separates Apalachicola Bay and St. George Sound.

Type population. Dauphin Island, Mobile Bay, AL Phylum. Ciliophora Dorflein, 1901 Class. Oligohymenophora de Puytorac et al., 1974 Subclass. Peritricha Stein, 1859 Order. Peritrichida Stein, 1859 Suborder. Mobilina Kahl, 1933 Family. Trichodinidae Claus, 1874 Genus. Trichodina Ehrenberg [3] Species. Trichodina ctenophorii n. sp. Overall size and shape. In profile, Trichodina ctenophorii

varied in shape from a flat disc to a dome in each population and at all collection sites. We suspect that this variability de- pends on the physiological state of the ciliate, for instance, whether it is attached tightly to the substrate, is about to release from the comb plate, or is loosely attached (Fig. 4-9). The adoral ciliary wreath wrapped - 390 (range: 380-415") about the oral surface (Fig. 4). Silver staining revealed a clear adhe-

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Table 1. Trichodina ctenophorii n. sp. morphology measurements. Measurements of Trichodina ctenophorii n. sp. from two populations in the Gulf of Mexico from 1995 and 1996. All morphological measurements are in microns: average valuelrange. DISL: Dauphin Island Sea Lab (Mobile Bay, eastern extreme of Dauphin Island); AP95, AP96: Apalachicola Bay area, 1995 and 1996 respectively. Border Memb.: border membrane; Ad. Disc: adhesive disc; Dent. ring: denticular ring; # Dent.: number of denticles; PinsDent.: no. pinsldenticle.

Locale Body Border memb. Ad. disc Dent. ring Blade Thorn Knob # Dent. Pinddent.

DISL 27120-32 1.8104.5 25120-30 1511 1-18 2.711.9-3.3 2.811.8-3.5 0.9410.6-1.3 26123-30 515-6 AP95 38.5127-50 1.711.5-2 26.2125-28.5 16.5116-17 NIA NIA NIA 27126-28 NIA AP96 35130-43 1.811.1-2.8 30.2125-35 17.8115-21 NIA 2.811-3.5 1.711.1-2.8 26123-28 515-6

sive disk center with irregular dark granules. The adhesive disc in all cells was flat and circular in shape (Fig. 5). T. ctenophorii varied somewhat in size with regard to its locality [Table 11. Denticular morphology and associated structures were the same at all locations.

Denticular anatomy. The structures comprising the denti- cular ring displayed a unique morphology (Fig. 10-14). The blades were falcate, forming an overall triangular shape with a blunt or rounded tip. There were no notches or indentations on the posterior of the blade. The thorns were long, thin, and straight with a blunt tip. The knobs of the denticular ring were also blunt-tipped [Fig 151.

Young cells. Two smaller specimens with extremely short thorns were not included in these overall measurements (Fig. 14). These trichodinids however, had values of other morpho- logical measurements such as the number of denticles, pin num- ber, blade, thorn, and knob sizes similar to the above specimens. By these criteria we identified them as young cells. Uzmann and Stickney have described such cells as the recent products of cell division that had yet to lengthen and thicken their thorns [16]. Their measurements were as follows (mean dimension [range]): body size: 28.9 pm [28.8-291; adhesive disk: 24.5 pm [24-251; denticle ring: 17.4 p,m [17.3-17.51; blades: 2.8 pm [2.6-31; thorns: 1.2 pm; knob: 0.85 pm [0.84.9]; border mem- brane: 1.9 pm [1.3-2.41; denticle number: 26, and there were five pins per denticle.

Macronucleus. VE-DIC and silver-stained preparations re- vealed a 5 pm wide, 27.8 pm diameter horseshoe-shaped mac- ronucleus (Fig. 16). The plane of the macronucleus was parallel to the plane of the denticular ring with its open end directly below and rotated CCW - 5" from the counterclockwise-most edge of the adoral ciliary ring.

DISCUSSION Trichodina ctenophorii n. sp: a unique symbiont of Gulf

Coast ctenophores. We describe herein a new species of tri- chodinid, Trichodina ctenophorii, found on the comb plates of the coastal ctenophores Mnemiopsis mccraydii and Beroe ovata. This symbiont is distinct from similar trichodinids by a number of ultrastructural details, host, and geographic distribution. To the best of our knowledge, this is the first report of a trichodinid associated with ctenophores.

Affinities. Trichodina ctenophorii is similar in denticular morphology to T. mutabilis, T. nigra, T. hypsilepis and T. mi- crodenticula. T. mutabilis, from the gills of the carp Cyprinus carpio L. [7, 91 had a similar number of denticles (29 [26-301)

and similar denticle morphology to that of T. ctenophorii; how- ever, it was much larger with an overall diameter of 77.6 pm [60.4-1061, border membrane size of 6 pm and 9-10 radial pins per denticle. Blades were spade-shaped and the center of the adhesive disk uniformly dark. The macronucleus was 55.7 pm [45-63.51 in diameter while it was 10.5 Fm [7.3-13.51 wide, twice that of T. ctenophorii.

T. nigra [7, Lom, J . 1960. Urceolariid ciliates from fresh water fish in Czechoslovakia. J. Protozool., 7, suppl.90.], from the skin of the host Cyprinus carpio, were reported to be larger (55.6 pm cell diameter) and had a larger border membrane ( 5 pm) and more pins per denticle (9-ll), but fewer denticles (24.5 [21-30]), and again a dark center. The T. nigra macro- nucleus was 37 pm [31.2-42.61 in diameter but 5.7 km [4.2- 7.31 wide. Its denticles were similar to T. ctenophorii in size and shape.

Denticular morphology in T. hypsilepis [18] from the body and fins of a high scale shiner, Notropis hypsilepsis, was quite distinct from that of T. ctenophorii. It too had falcate blades and long, narrow thorns; however, both were pointed instead of blunt-tipped, and larger. The blades were 5.3 pm [5-61, the thorns 8 pm [7-91, and the knob 2.6 pm 12-31, In addition the number of pins per denticle (lo), the overall body, denticular ring, and adhesive disk were all approximately twice the size. However, the adhesive disk of T. hypsilepis is cup-shaped in- stead of flat and the border membrane was also 4-4.5 pm wide. The macronucleus was 40 pm [35-461 in diameter and its width 5-6 pm. The contractile vacuole was also located in the center of the oral surface in T. hypsilepis, whereas it was located inside the overlap of the adoral ciliary ring in T. ctenophorii. Finally, the adoral ciliary wreath overlaps to a lesser extent (370-380') than in T. ctenophorii.

The only other trichodinid we are aware of with five pins per denticle is T. microdenticula from the body and gills of a shad host, Dorosoma petenense [18]. It was smaller, with a 26 pm [22-371 diameter body, but a border membrane of 2 pm [1.5- 2.51, much like T. ctenophorii. The T. microdenticula adhesive disk was only 13 pm diameter [12-15.51 with fewer denticles (16 [15-181). Denticular morphology was truncated instead of falcate and the thorns unusually short (1.9 p m [1.5-2.0]), and again its center stained darkly with Klein's silver stain. One of the most distinct differences between the two species was the oral ciliary wreath, which in T. microdenticula wrapped only 330-350". The macronucleus was 17 pm [13-221 in diameter and 2.5 pm [2-31 wide.

It is important to note that all of the previously mentioned

t

Fig. &9. SEM views of Trichodina ctenophorii; different aspects; all are of the symbionts attached to comb plates. Bars = 5 pm. 4. SEM of Trichodina ctenophorii. Oral view. 5. Aboral view of Trichodina ctenophorii. The blades are clearly seen; the aboral surface is very flat. 6. Profile view-disk configuration. The adoral and medial cilia are expanded; the contractile vacuole is open. 7. Profile viewdisk conformation of Trichodina ctenophorii. Mouth and contractile vacuole are on opposite aspect. Adoral ciliature is closed, as are the cilia of the medial ring. 8. Dome profile with mouth open. All cilia are flat. 9. Oblique view of Trichodina ctenophorii showing extended adoral and medial ciliary wreaths and obvious contractile vacuole opening.

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Fig. 10-11. Decriptions of measurements made on silver-stained Trichodina ctenophorii using a X 100 planapochromatic oil immersion lens. 10. Silver impregnation of Trichodina ctenophorii. 11. Video en- hanced differential interference contrast light micrograph of Trichodina ctenophorii. One half of the ring is shown to display the pin structure and denticle arrangement. Note that the pins alternate in intensity of appearance. B: overall body, M: border membrane, A: adhesive disk, D: denticular ring, P: pins, L: blade, K: knob, T. thorn.

trichodinas were reported from freshwater locations, further supporting our claim that Trichodina ctenophorii described herein is indeed a distinct, new species.

Population variation between our collection sites. We ob- served only minor overall size differences between collection sites. The proportions and relative ultrastructural details of all of the measured indices were invariant between populations ir- regardless of the overall size of the cell.

We have observed that ctenophores collected in similar sa- linity conditions at Dauphin Island and Apalachicola appear to be quite different with regard to their overall health. Cteno- phores and trichodinas from the Mobile Bay area are less likely to thrive in laboratory conditions after transport inland. In con- trast, we are able to maintain Apalachicola Bay animals with ciliates up to a month or two post-collection. It is likely that

Fig. 12-14. Comparison of mature and young Trichodina cteno- phorii, silver impregnated. 12-13. Mature specimens. 14. Young cell. Note the short, stubby thorns.

this is the result of water-quality differences. Such variables are likely to have some impact on the physiology and/or nutritional state of the symbiotic trichodinids, which could translate to mi- nor differences in size.

Size discrepencies in trichodinid populations elsewhere [5-

ESTES, REYNOLDS & MOSS-TRICHODINA CTENOPHORII N. SP. 425

Fig. 15. Line drawing of a single complete denticle taken from a silver-stained preparation.

71 have been attributed to temperature fluctuations as a result of seasonal variation. Trichodinids are most common on cten- ophores during the summer. However, we do not observe any systematic variation in the size of the cells that could be attrib- uted to such seasonal differences. We therefore ascribe any small changes in cell size to environmental variables other than temperature. The only systematic environmental variable we could detect other than water quality characteristics was the generally less variable salinity of Apalachicola Bay. Analysis of the populational characteristics of ctenophore trichodinids at the two locations awaits further study.

Host association. We cannot say whether the ctenophore is the primary or intermediate host in the life cycle of Trichodina ctenophorii. Trichodinid populations decline during the cooler months. It is quite possible that the trichodinids are associated with another host during the winter. Alternatively, they may live as solitary cells during that time.

A number of fish species routinely prey upon Mnemiopsis and Beroe. We have directly observed Menidia peninsulae, Pe- trilus burti and Harengula jaguana all attack Mnemiopsis and Beroe, biting out pieces of the comb rows and body, and some cases, swallowing the entire animal (AGM & AME, pers. ob- serv.). Menhaden are known to swim through heavy concentra- tions of ctenophores and ingest large quantities of these animals in northeastern US waters (AGM, pers. observ.) and it is quite likely that the Gulf Menhaden (Brevoortia patronus) also eats Mnerniopsis. More strikingly, we have directly observed and videotaped the Atlantic spadefish, Chaetodipterus faber, selec- tively strip away entire comb rows that are heavily infested with trichodinids, leaving the ctenophore to hang motionless in the water column. Insofar as any of these small fish are likely prey for game species, Mnemiopsis mccraydii might be an important vector for transmission to an as-yet-unknown, possibly econom- ically important, fish host.

Beroe eats Mnemiopsis; indeed it appears to prefer the ten- taculate ctenophores over other food sources, and it is generally recognized as an important controlling predator of ctenophore populations in the coastal regime [13]. Beroe could obtain its light parasite load by virtue of ingesting Mnemiopsis. The tri- chodinids might abandon the lobate prey for the predator. We have observed that T. ctenophorii swims freely around the Mne- rniopsis host. It could therefore easily become associated with Beroe as it feeds upon Mnemiopsis. It seems unlikely, however, that Beroe constitutes the normal host for Trichodina cteno- phorii, because despite easy access to a new host, it is rarely

Fig. 16. VE-DIC image of horseshoe-shaped macronucleus of live Trichodina ctenophorii. Ma: Macronucleus.

seen on Beroe. This seems to hold true for even very large Beroe, which must have ingested many Mnemiopsis. Beroe fed in the lab with heavily infested M. mccraydii never themselves became infested. We have never observed trichdinids within the stomodeum, other regions of the ctenophore digestive tract, or canal structures of either M. mccraydii or B. ovata, nor have we seen them on the ectodermal surface. In these species it appears to be confined solely to the surface of the comb plates, where it is part of a complex community of microorganisms (Moss, Reynolds, Morgan & Estes, unpubl. data).

ACKNOWLEDGMENTS We are indebted to Scott Schaffer for providing us with his

modification of Klein’s silver stain for use with marine speci- mens, to Dr. Christine Sundermann for much useful advice and encouragement through many helpful discussions, Dr. John Clamp for his advice, and Dr. Roland Dute, Darrell Morgan, Maria Toivio-Kinnucan, and Portia Lane, who helped with elec- tron microscopy. The manuscript was greatly improved by the suggestions of two anonymous reviewers. Dr. Lee Edmiston and Chip Bailey of the Apalachicola National Estuarine Research Reserve helped greatly with animal collection, while Rita George and Dr. George Crozier freely provided facilities at the Dauphin Island Sea Lab. Funded by a grant to Drs. Lawrence Wit and Robert Lishak from the Howard Hughes Foundation and grants to AGM from Auburn University, the Auburn Uni- versity Department of Zoology, and the National Institutes of Health.

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Received 2-17-97, 3-31-97: accepted 5-I-97