Organization of Sporulated Oocysts of Eimeria Funduli in the Gulf Killifish, Fundulus Grandis

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Organization of Sporulated Oocysts of Eimeria funduli in the Gulf Killifish, Fundulus grandis

William E. Hawkins Gulf Coast Research Laboratory, [email protected]

John W. Fournie United States Environmental Protection Agency, [email protected]

Robin M. Overstreet Gulf Coast Research Laboratory, [email protected]

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Hawkins, William E.; Fournie, John W.; and Overstreet, Robin M., "Organization of Sporulated Oocysts of Eimeria funduli in the Gulf Killifish, Fundulus grandis" (1983). Faculty Publications from the Harold W. Manter Laboratory of Parasitology. 470. https://digitalcommons.unl.edu/parasitologyfacpubs/470

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ORGANIZATIONOF SPORULATEDOOCYSTS OF EIMERIAFUNDULI IN THEGULF KILLIFISH, FUNDULUS GRANDIS

William E. Hawkins, John W. Fournie, and Robin M. Overstreet Gulf Coast Research Laboratory,Ocean Springs, Mississippi 39564

ABSTRACT: Oocysts of Eimeriafunduli were studied by transmissionelectron microscopy in naturally-infected livers of the Gulf killifish, Fundulusgrandis. Tissues were cryo-processedbecause membranousstructures in the oocyst appearto hinderroutine fixation and embedment.The oocyst wall (about 25 nm thick) was adjacent to the host cell and consistedof an outer membrane thi e host cell cytoplasmand an inner membrane separatedfrom the outer membraneby a narrowspace. In some specimens,dense materialwas applied to the inner face of the inner membrane.Individual sporocysts were surroundedby a membranous"veil" (about 25 nm thick) that consisted of two unit membranes.Sporopodia, projections of the sporocystwall, supportedthe veil. The sporocystwall (130-150 nm thick) consisted of two layers, a thin electron-lucentouter layer (about 10 nm thick) and a thick electrondense inner layer (about 130 nm thick). Dependingon the plane of section, the inner layer had transversestriations with periods of 3 to 4 nm or 12 to 15 nm. A narrowfissure, broadest at the anteriorpole of the sporocyst,extended about one-thirdthe length of the sporocystwall. The posterior pole of the sporocystwas characterizedby a bulbous swelling.Although this swellingresembled a Stieda body in light microscopicpreparations, ultrastructurally, the swellingwas a knoblikethickening in the sporocystwall and did not plug a gap in this wall.

Compared with eimerians in terrestrial hosts, tine TEM. This study reports that E. funduli lacks piscine eimerians have received little attention a true oocyst wall, that the sporocyst lacks a Stie- despite their possible importance in coccidian da body, and that the sporocyst wall has a narrow evolution and systematics (Desser, 1981; Over- fissure through which sporozoites probably ex- street, 1981) and in the health of commercially cyst. important fishes (Dykovla and Lom, 1981; Over- MATERIALSAND METHODS street, 1981). In these coccidians, the oocyst rep- resents the most commonly encountered life cycle Naturally-infectedF. grandiswere collectedin min- now trapsfrom Halstead Bayou in OceanSprings, Mis- and the feature most used to stage frequently sissippi.After removing livers, we squashedfresh prep- separate species. The ultrastructure of piscine arationsbetween glass slides to confirm the presence coccidian oocysts, however, has been studied of sporulatedoocysts. In initial stages of the study, rarely. oocyst-containinglivers were minced with a razorblade, fixed in 0.1 M 3% In the present study, oocysts of Eimeria fun- phosphate-buffered glutaraldehyde for 1 to 3 hr, rinsed 1 hr to overnight in phosphate duli Duszynski, Solangi, and Overstreet, 1979, buffer,postfixed in phosphate-buffered1% osmium te- were examined by transmission electron mi- troxidefor 2 hr, rinsedbriefly in buffer,and dehydrated croscopy (TEM) in the naturally-infected Gulf in a gradedseries of ethanol.Following treatment with killifish, Fundulus grandis Baird and Girard. propylene oxide, tissues were embedded in Embed 812. infects of (Epon) Eimeria funduli primarily hepatocytes Becauseof resistanceof the oocysts to fixation and killifishes, requires the grass shrimp Palaemo- embedment,we employeda modificationof the double netes pugio Holthuis to complete its life cycle, sectioningtechnique of Birch-Andersenet al. (1976). and sporulates within host tissues (Solangi and Oocyst-containinglivers, mounted with Ames O.C.T. on a metal chuck and frozen Overstreet, 1980). Duszynski et al. (1979) de- embeddingcompound with liquifiedcarbon dioxide, were minced with a sin- scribed some ultrastructural features of E. fun- gle-edgedrazor blade that had been cooled with carbon duli oocysts isolated by sugar flotation from dioxide and held with a pair of locking pliers. Minced homogenized tissues. To preserve in situ rela- tissues were allowedto thaw in 0.1 M phosphate-buff- then fixed in the same tionships of oocysts and at the same time over- ered 3% glutaraldehydeand solution for 1 to 3 hr. Tissues were subsequentlypro- of to fixation and come the resistance oocysts cessed as describedabove. To find suitableoocysts for embedment, we cryo-processed infected livers thin sectioning,thick (1-2 ,um)sections were cut with and then fixed and embedded the tissues for rou- an LKBUltratome, mounted on glassslides, and stained with toluidineblue. Thin sections were cut with a dia- mondknife, mounted on uncoated,copper grids, stained Received 19 April 1982; revised 9 August 1982; ac- with uranylacetate and lead citrate,and examined in cepted 20 October 1982. a Siemens Elmiskop 1A electronmicroscope.

496 HAWKINSET AL.-TEM OF SPORULATED OOCYSTS OF E FUNDULI 497

RESULTS branes appeared nearly equal in thickness. Often, all three layers of the oocyst wall separated from In squash preparations, most sporocysts and the host cell, especially in routinely-processed sporozoites appeared fully developed. Sporo- tissues. The space between the inner membrane cysts usually collapsed in tissues processed by of the oocyst wall and the outer membrane of conventional techniques, probably because of the sporocysts sometimes contained cellular de- poor infiltration of embedding medium. How- bris or flocculent material. A three-layered mem- ever, cryo-processing gave much better results. branous "veil" (about 10-20 nm thick) that con- Whereas some sporocysts collapsed, many spo- sisted of two unit membranes surrounded each rocysts, as well as host cells and noninfected liver sporocyst (Figs. 3, 8, 9). Clear, homogeneous ma- parenchyma, were preserved satisfactorily. In terial that in fresh preparations appeared to fill epoxy-embedded thick sections, oocysts ap- the space between the veil and sporocyst wall did peared spherical (about 21 ,um in diameter), with not appear in electron micrographs, apparently the oocyst wall usually abutting the host cell. having been lost during preparation. However, the oocyst wall was often torn away The sporocyst wall (130-150 nm thick) was from the host cell (Fig. 1). In some sporocysts regularly organized and uniform in thickness ex- with uncollapsed walls, the sporozoites appeared cept where it gave rise to sporopodia and where poorly preserved and shrunken, whereas in oth- it thickened at the posterior pole. The sporocyst ers the sporozoites were well-preserved and com- wall consisted of a thick, electron-dense, inner pletely filled the sporocyst. Sporocysts with un- layer (about 120-130 nm thick) lying beneath a collapsed walls were ovoid (about 7 A,mX 4 Am) thin, electron-lucent, outer layer (about 10 nm and contained two sporozoites. A bulge occurred thick) that had a thin, superficial, electron-dense at the posterior pole of the sporocyst (Figs. 1, 2). coating (Figs. 5, 6). In sections through the long This bulge resembled a Stieda body but, as de- axis of the sporocyst, the inner layer of the spo- scribed below, it did not have the ultrastructural rocyst wall appeared homogeneous. However, in characteristics of a true Stieda body. Depending some planes the inner layer had regular trans- on the plane of section, the posterior pole ap- verse striations with a period of 12 to 15 nm peared either sharply pointed (Figs. 1, 2) or (Fig. 5). In other planes, transverse striations with somewhat rounded (Fig. 2). The anterior end of a period of 3 to 4 nm could be seen (Fig. 6). the sporocyst was rounded, regardless of the plane Sporopodia, which had the same layers as the of section (Figs. 1, 2). Projections of the sporocyst sporocyst wall, but which did not appear trans- wall, the sporopodia, appeared to support a thin versely striated, terminated in knoblike swellings membrane that approached the surface of the (Fig. 7) that often apposed the sporocyst veil. sporocyst at the anterior end. In some views of Various planes of section through the sporo- sporocysts, a gap could be seen at the anterior cyst revealed an opening or fissure that appeared pole (Fig. 1). In heavily infected livers, oocysts to extend about one-third the length of the long occurred in groups separated from uninfected axis of the sporocyst. Longitudinal sections per- liver by empty spaces or enclosed by a connective pendicular to the fissure showed that it was wid- tissue capsule. est (about 0.3 um) at the rounded, anterior pole A thin ring of host cell cytoplasm completely of the sporocyst (Fig. 8). There, the fissure was surrounded each oocyst (Fig. 3). The host cell bridged by the sporocyst veil which was attached nucleus was compressed to conform to the spher- to the outer lips of the sporocyst wall at the edges ical oocyst. The oocyst wall (about 25 nm thick) of the fissure and by a thin deposit of dense ma- consisted of two unit membranes separated by a terial that accumulated on the inner aspect of the space adjacent to the host cell cytoplasm (Fig. fissure (Fig. 9). In other regions, sporopodia held 4). The outer membrane of the oocyst wall lim- the membranous veil away from the surface of ited the host cell cytoplasm. A narrow space sep- the sporocyst. In cross-sections of the sporocyst, arated this membrane from the inner membrane the fissure gradually narrowed to a suture often which faced the oocyst contents. In some spec- with some intervening dense material that ap- imens, especially those processed by conven- peared to be derived from sporocyst contents tional techniques, dense material adhered to the (Fig. 10). Superficially, a short projection of the oocyst side of the inner membrane, making it sporocyst wall often indicated the location of the variably thicker than the outer membrane. In suture (Fig. 10). There was no evidence for a cryo-processed oocysts, however, the two mem- Stieda body or subStieda body associated with 498 THEJOURNAL OF PARASITOLOGY,VOL. 69, NO. 3, JUNE 1983

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FIGURES 1-4. Eimeria funduli. 1. Light micrograph of epoxy-embedded 1-,m-thick section. Note gap in anteriorpole (arrowhead).Oocyst wall (OW) is torn away from host cell cytoplasm.Sporocyst veil approaches surfaceof sporocystat anteriorpole (arrow).X2,000. 2. Lightmicrograph of epoxy-embedded1 -,m-thick section with two oocysts. Note "Stiedalikebody" (arrowhead)at posteriorpole of one sporocystand cross-sectionsof sporozoitesin sporocystin right of figure.X 1,900. 3. Low power TEM micrographshowing oocyst wall (OW), sporocyst veil (SV), and sporocyst wall (SW). Nucleus (N) belongs to a connective tissue cell adjacent to hepatocyte.An area comparableto that inside the rectangleis shown at highermagnification in Figure4. Host cell (HC), sporopodium(Sp). X6,100. 4. The oocyst wall is the boundarybetween the host cell (HC) cytoplasm and the oocyst (0). Note some ribosomelike granulesline the outer membrane of oocyst wall (arrowhead) whereasthe inner membraneis smooth and somewhatthicker than outer membrane.X92, 100.

the fissure. In some sporocysts, sporozoites pro- layer of electron-dense granular material over- truded out of the anteriormost wide portion of laid a layer of less electron-dense material. In the fissure. sections that paralleled the fissure, the bulge ap- In sections perpendicular to the fissure in the peared as a ridged structure (Fig. 12). Sporopodia sporocyst wall, the bulge at the posterior pole occurred at the boundaries of the ridge (Figs. 12, appeared pointed (Figs. 8, 11). This bulge was 13). mainly a thickening in the inner layer of the spo- We did not achieve optimal preservation of rocyst wall (Fig. 1 1). Beneath this thickening, a sporozoites, but nevertheless have provided some * s N t

HAWKINSET AL.-TEM OF SPORULATEDOOCYSTS OF E. FUNDULI 499

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FIGURES5-9. Eimeria funduli. 5. In some planes of section, sporocyst wall has transverse striations with 12- to 15-nm period. Sporocystveil (SV). X66,700. 6. Sporocystwall showing periodic substructureof 3 to 4 nm. X76,200. 7. Sporopodiumending in knoblike swelling and having same layers as sporocyst wall (SW). Sporocystveil (SV). X3,500. 8. Sporocystsectioned through fissure (F) at anteriorend. Note pointed thickening of sporocystwall at posterior end. Oocyst wall (OW), sporocyst veil (SV), sporocyst wall (SW). X13,500. 9. Highermagnification of anteriorend of sporocystin Figure8. Sporocystwall composed of outer,electron-lucent layerand inner, electron-densearea. Sporocystveil (SV) attachesto sporocystwall at marginsof fissure(arrow- heads). Note dense material applied to inner aspect of fissure. Oocyst wall (OW). X47,600.

information on their organization. The absence veloped. Each sporocyst contained two sporo- of certain organelles might be artifactual or might zoites curled around one another. A nucleolem- indicate that the sporozoites were not fully de- ma did not enclose the nuclear material (Fig. 13) 500 THEJOURNAL OF PARASITOLOGY,VOL. 69, NO. 3, JUNE 1983

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FIGURES10, 11. Eimeriafunduli. 10. Diagonal section of sporocyst with the two sporozoites each cut in two sections. Note suture (S) with intervening dense material on one side of oocyst wall and papillalike projection (P) on opposite side. Subpellicular microtubules (arrow), sporocyst residuum (R), lipoid bodies (L). X1 9,700. 11. Posterior pole of sporocyst is pointed in longitudinal sections perpendicular to the fissure. Three layers make up this region of sporocyst: an outer thickening of the sporocyst wall (0), a middle electron-dense layer (M), and an inner electron-lucent area (I). Sporocyst refractile body (SR), amylopectin (A), fissure (F), foci of ribosomelike granules (arrows). X16,400. and there were no anlagen of rhoptries or mi- zoites showed approximately 32 subpellicular cronemes. Sporozoites had a conoid and an an- microtubules (Fig. 10). In addition to the two terior polar ring system, numerous small amy- sporozoites, some sporocysts contained residual lopectin (polysaccharide) granules (about 90 nm material consisting of lipidlike bodies and amy- diameter) (Fig. 11), a few lipidlike bodies (about lopectin granules (Fig. 11). 0.4 Am diameter), and a large (about 2 ,tm di- DISCUSSION ameter) refractile body (Figs. 8, 11, 12, 13). Foci of ribosomelike granules occurred throughout the Few detailed studies on coccidian oocysts were sporozoite (Fig. 11). Cross-sections of sporo- available before Birch-Andersen et al. (1976) in- HAWKINSET AL.-TEM OF SPORULATEDOOCYSTS OF E. FUNDULI 501

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FIGURES12, 13. Eimeriafunduli.12. Knoblikeappearance of posteriorpole of sporocystin sections parallel to the fissure.Sporocyst refractile body (SR). X1 5,500. 13. Knoblikeposterior pole in longitudinalsection through sporocystparallel to fissure.Note sporopodia(Sp) at each end of ridge. Sporozoitenucleus (N). XI 8,700. troduced a cryo-processing technique in which to thick-walled coccidian oocysts in terrestrial isolated oocysts are embedded in cross-linked hosts remains to be determined. bovine serum albumin, cryostat-sectioned, fixed Some findings in the present study do not agree in glutaraldehyde, and embedded in epoxy resin with those of Duszynski et al. (1979) who ex- for routine TEM. Whether the simpler technique amined oocysts of E. funduli isolated by sugar used in the present study will be useful if applied flotation from homogenized tissues. Apparently, 502 THEJOURNAL OF PARASITOLOGY,VOL. 69, NO. 3, JUNE 1983

most of the discrepancies in the two studies, as of species in the genus Eimeria Schneider, 1875, discussed below, result from differences in meth- has a single polar opening plugged by a Stieda ods of preparation. body and an associated subStieda body (Roberts The oocyst wall of E. funduli consists of two et al., 1970). Excystation occurs when digestive unit membranes separated by a narrow space and fluids dissolve the Stieda and subStieda bodies, possibly some electron-dense material that lines allowing the sporozoites to escape (Roberts et the inner membrane. Ultrastructurally-similar al., 1970). The other type of wall, represented in oocyst walls consisting of one or two unit mem- some members of the genus Isospora Schneider, branes have been reported in the piscine eime- 1881 (see Speer et al., 1973; Speer et al., 1976) rians Goussia degiustii (Molnafr and Fernando, and by Toxoplasma gondii (Nicolle and Man- 1974) by Lorn (1971, reported as E. spleni), G. ceaux, 1908) (see Ferguson et al., 1979b) consists gadi (Fiebiger, 1913) by Odense and Logan of two layers, the inner of which is comprised of (1976), and E. variabilis (Th6lohan, 1893) by up to four separate plates. Digestive juices act Davies (1978). Oocyst walls in most coccidia on material at the sites of apposition of the plates, having terrestrial hosts have one to three layers causing the plates to separate and release the spo- and form from deposition of wall-forming bodies rozoites (Ferguson et al., 1979a; Speer et al., 1973; at the limiting membrane of the macrogamont Speer et al., 1976). The mechanism for excys- or zygote (reviewed by Speer et al., 1979). The tation in E. funduli must differ from either of oocyst wall in E. funduli, and probably other these because the sporocyst does not have a Stie- piscine coccidia possibly is not equivalent to that da body and the sporocyst wall does not consist of coccidia that infect terrestrial hosts because it of joined plates although some dense material lies adjacent to the host cell and part of it appears that originates from sporocyst contents occurs in to some investigators as the limiting membrane the gap near its posterior extent. of the host cell cytoplasm. Structures resembling The sporocyst wall of E. funduli apparently wall-forming bodies in E. funduli macrogamonts consists of a single shell, or valve, with a thick- (Hawkins et al., 1983a) are either vestigial or ening at one end and a narrow fissure bridged by function in some other manner, possibly in the the membranous veil at the other end. The Stie- formation of the sporocyst wall or the thin layer dalike thickening in E. funduli cannot be con- of electron-dense material that lines the inner sidered a true Stieda body as suggested by Dus- membrane of the oocyst wall. Dykovaf and Lorn zynski et al. (1979) because it does not close an (1981) suggested that oocyst walls in piscine coc- opening in the sporocyst wall. Dykovaf and Lom cidians are remnants of the limiting membrane (1981) proposed that piscine coccidians be clas- of the macrogamont. This is probably not true sified according to, among other characteristics, in E. funduli. Macrogamonts (Hawkins et al., the organization of the sporocyst wall. They 1983a), microgamonts (Hawkins et al., 1983b), placed species in which sporocysts lack a Stieda and other developmental stages (preliminary ob- body and have a wall consisting of two valves servations) of E. funduli are all situated in a par- joined along a meridional suture within the genus asitophorous vacuole limited by a cisterna of Goussia Labbe, 1896. The three piscine coccid- rough endoplasmic reticulum that apparently de- ians, E. variabilis, G. gadi, and G. degiustii, that velops from host membranes. Because of the lo- have been studied ultrastructurally generally cation and organization of the oocyst wall in E. conform to these characteristics. In E. variabilis, funduli, the limiting membranes of the parasi- Davies (1978) identified Stieda bodies by light tophorous vacuole probably become the oocyst microscopy and attributed their absence in TEM wall. Lom (1971) suggested that the oocyst walls preparations to the early developmental stage of in fish coccidia need not be thick because the the examined sporocysts. However, the TEM mi- aquatic environment protects the oocyst from crograph by Davies showed sporocyst walls to mechanical damage and desiccation. In prelim- have a short papillalike projection similar to the inary studies, the oocyst wall of E. funduli ap- one that occurs where the lips of the fissure in pears remarkably strong in spite of its thinness. the sporocyst wall of E. funduli join. Therefore, In fact, it remains intact for at least 12 hr in the possibility exists that the Stiedalike body seen the gastric mill of its intermediate host, Palae- with LM in E. variabilis is actually similar to the monetes pugio. posteriorly-located structure in E. funduli. Nei- Two primary types of sporocyst walls occur in ther Stieda nor Stiedalike bodies were reported coccidians. One type, represented in sporocysts in sporocysts of G. gadi by Odense and Logan HAWKINSET AL.-TEM OF SPORULATED OOCYSTS OF E. FUNDULI 503

(1976) or in sporocysts of G. degiustii by Lom DESSER,S. S. 1981. The challenge of fish coccidia. J. (1971). Sporocyst walls in both G. gadi and G. Protozool. 28: 260-261. DUSZYNSKI,D. W., M. A. ANDR. M. OVER- degiustii have fissures that might completely sep- SOLANGI, STREET.1979. A new and unusual eimerian (Pro- arate those sporocysts into two valves, although tozoa: Eimeriidae) from the liver of the Gulf kil- a scanning electron micrograph of G. gadi by lifish, Fundulus grandis. J. Wildl. Dis. 15: 543- Odense and Logan (1976) showed what might be 552. a sporocyst with incompletely-separated valves DYKOVA,I., ANDJ. LOM. 1981. Fish coccidia: critical notes on life cycles, classification and and thus to the of E. pathogen- similar sporocyst funduli. icity. J. Fish Dis. 4: 487-505. The sporocyst wall of G. gadi has a transverse FERGUSON,D. J. P., A. BIRCH-ANDERSEN,J. C. SIIM, periodic substructure similar to that in E. fun- AND W. M. HUTCHISON.1979a. An ultrastruc- duli, but its substructure in G. gadi was inter- tural study on the excystation of the sporozoites fracture lines. Lom of Toxoplasma gondii. Acta Path. Microbiol. rupted by (1971) reported Scand. Sect. B 87: 227-283. that G. degiustii had a "membranaceous collar" , , , AND . 1979b. Ultra- that attached to the sporocyst wall along the su- structural studies on the sporulation of oocysts of ture line similar to the attachment of the veil Toxoplasma gondii. II. Formation of the sporo- along the fissure in the sporocyst wall in E. fun- cyst and structure of the sporocyst wall. Acta Path. Microbiol. Scand. Sect. B 87: 183-190. of E. duli. Once the oocyst wall funduli becomes HAWKINS,W. E., M. A. SOLANGI,AND R. M. OVER- disrupted in its intermediate host, the membra- STREET. 1983a. Ultrastructure of the macroga- nous veil appears to be the only structure re- mont of Eimeriafunduli, a coccidium parasitizing taining the sporozoites in the sporocyst. This killifishes. J. Fish Dis. 6: 33-43. , AND . 1983b. Ultrastructure membrane in E. funduli and G. degiustii might of the microgamont and microgamete of Eimeria correspond to the outer, double-membrane layer funduli, a coccidium parasitizing killifishes. J. Fish of the sporocyst wall which is derived from the Dis. 6: 45-57. limiting membranes of the sporoblast in coccidia LOM, J. 1971. Remarks on the spore envelopes in fish coccidia. with terrestrial hosts such as Toxoplasma gondii Folia Parasitol. (Prague) 18: 289- 293. (see Ferguson et al., 1979b). ODENSE,P. H., ANDV. H. LOGAN. 1976. Prevalence In any event, until the genus Goussia is re- and morphology of Eimeria gadi (Fiebiger, 1913) viewed adequately, with a type-species estab- in the haddock. J. Protozool. 23: 564-571. lished, its status will be uncertain. That review OVERSTREET,R. M. 1981. Species of Eimeria in non- sites. J. Protozool. 28: 258-260. should include ultrastructural aspects of the spo- epithelial ROBERTS,W. L., C. A. SPEER,AND D. M. HAMMOND. rocysts of some, if not all, the nominal species. 1970. Electron and light microscopic studies of the oocyst walls, sporocysts, and excysting spo- ACKNOWLEDGMENTS rozoites of Eimeria callospermophili and E. lari- We thank Mrs. Eve Wells and Mr. Robert J. merensis. J. Parasitol. 56: 918-926. M. AND R. M. OVERSTREET.1980. Bi- Allen for their technical assistance. This SOLANGI, A., study ology and pathogenesis of the coccidium Eimeria was conducted in cooperation with the U.S. De- funduli infecting killifishes. J. Parasitol. 66: 513- partment of Commerce, NOAA, National Ma- 526. rine Fisheries Service, under PL 88-309 Project SPEER,C. A., D. M. HAMMOND,J. L. MAHRT,AND W. No. 2-382-R. L. ROBERTS. 1973. Structure of the oocyst and sporocyst walls and excystation of sporozoites of LITERATURECITED Isospora canis. J. Parasitol. 59: 35-40. , A. A. MARCHIONDO,D. W. DUSZYNSKI,AND BIRCH-ANDERSEN,A., D. J. P. FERGUSON,AND R. D. S. K. FILE. 1976. Ultrastructure of the sporocyst PONTEFRACT.1976. A technique for obtaining wall during excystation of Isospora endocallimici. thin sections of coccidian oocysts. Acta Path. Mi- J. Parasitol. 62: 984-987. crobiol. Scand. Sect. B 84: 235-239. , , B. MUELLER, AND D. W. DUSZYNSKI. DAVIES,A. J. 1978. Coccidianparasites of intertidal 1979. Scanning and transmission electron mi- fishesfrom Wales:Systematics, development, and croscopy of the oocyst wall of Isospora lacazei. Z. cytochemistry.J. Protozool. 25: 15-21. Parasitenkd. 59: 219-225.