Structure of Shellfish Pathogens and Hyperparasites in the Genera Minchinia, Urosporidium, Haplosporidium, and -Taxonomic Implications

FRANK O. PERKINS

Introduction Frank O. Perkins is Head. Division of Biological from along the eastern continental shelf Oceanography, Virginia Institute of Marine Sci­ of the United States, a species of imma­ Light microscope studies of species ence and School of Marine Science, The College ture anisakid worm (possibly in the genera Minchinia Labbe, 1896, of William and Mary, Gloucester Point, VA 23062. This paper is Contribution No. 897 of the Paranisakiopsis pectinis Cobb, 1930; Haplosporidium Caullery and Mesnil, Virginia Institute of Marine Science. see Lichtenfels et aI., 1977) becomes 1899, and Urosporidium Caullery and black when the hyperparasite, Urospo­ Mesnil, 1905, showed that they are re­ ridium spisuli Perkins, Zwerner, and lated and belong in the order Balano­ Kins, 1975, but in the same class, Stel­ Dias, 1975, sporulates. This causes sporida (Caullery and Mesnil, 1899) latosporea (Caullery, (953) Sprague, consternation in the seafood industry Sprague, 1979, formerly termed the 1979. since the worms become highly visible Haplosporida and herein referred to as It is believed that considerations of against the light-colored clam tissues. the balanosporidans. The judgment was the kind presented herein are of interest The clam, Abra ovata, from the Rhone based primarily on structure beyond phylogeny and . If delta in France is parasitized by the (Caullery, 1953; Sprague, 1963) and the various pathogens and parasites trematode, Gymnophallus nereicola, has, since then, been confirmed by under discussion are establ ished to be which is hyperparasitized by Urospo­ studies of fine structure (Ormieres and closely related, it should be recognized, ridium jiroveci Ormieres, Sprague, de Puytorac, 1968; Ormieres et aI., because information gained from and Bartoli, 1973. The trematode also 1973; Perkins, 1968, 1969, 1971, studies of the life history, ecology, becomes black when of the 1975a; Perkins et aI., 1975, 1977; Ro­ physiology, disease control, etc. of one balanosporidan are formed. senfield et aI., 1969). Also related to species may then be expected to yield Minchinia nelsoni Haskin, Stauber, the Balanosporida are the oyster patho­ insight into the biology of any of the and Mackin, 1966 is a serious pathogen gens, Marteilia refringens Grizel, other species. of oysters (Crassostrea virginica) Comps, Bonami, Cousserans, Duthoit, The following comparisons of Uro­ along the eastern mid-Atlantic Coast of and Le Pennec, 1974, and Marteilia sporidium, Minchinia, Haplospori­ the United States and Minchinia cos­ sydneyi Perkins and Wolf, 1976. The dium, and Marteilia species are made talis (Wood and Andrews, 1962) available structural information on considering systems, then Sprague, 1963 causes severe localized species of the four genera is reviewed sporulation. Urosporidium crescens mortalities of C. virginica in Virginia herein and arguments presented for De Turk, 1940 is found in the metacer­ waters. Minchinia louisiana Sprague, considering them to be interrelated. cariae of Carneophallus sp. which 1963 causes mortalities in one species Marteilia spp. have been placed in a parasitizes the blue crab, Callinectes of mud crab, Panopeus herbstii, in separate order, Occlusosporida Per- sapidus, of the eastern and southern Gulf of Mexico and Atlantic Ocean U.S. estuaries causing the syndrome coastal populations. Minchinia sp. as called "pepper crab disease" by work­ described by Perkins (1975a) is now ABSTRACT-The ultrastructure of shell­ ers in the seafood industry. The en­ considered to be M. louisiana since the fish pathogens and hyperparasites in the cysted metacercariae become black, only difference, a slight differential in genera Minchinia, Urosporidium, Haplos­ thus resembling small peppercorns spore length, is not considered to be poridium, and Marteilia is reviewed and new structural information provided. Em­ when the hyperparasite sporulates. This significant. Final proof that the species phasis is placed on the variations in size and blackening also occurs in Microphallus are identical will depend upon results of structure ofhaplosporosomes, a unique or­ sp. metacercariae from grass shrimp, ultrastructural studies to be done on the ganelle common to all species in the group. Palaemonetes pugio, of the southern Louisiana species. There is not enough Arguments for allying Marteilia spp. with Atlantic Coast of the United States, in­ the other species are presented based on information to judge whether Min­ observations of haplosporosomes and in­ fected with Urosporidium sp. Likewise chinia sp. as described by Rosenfield et ternal cleavage during sporulation. in the surf clam, Spisula solidissima, al. (1969) is the same as M. louisiana.

January-February /979 25 26 Marine Fisheries Review Minchinia armoricana van Banning, sporulation of the above-mentioned cuneiform, or club-shaped units in the 1977 parasitizes the European flat oys­ species of Minchinia and Marteilia fully differentiated state (Fig. 1-13),29 ter, , from Dutch and does not result in blackening of the host to 249 nm in the shortest axis and up to French waters. Haplosporidium as­ tissues. Color change mayor may not 650 nm in the longest axis (Table I). cidiarum Doboscq and Harrant, 1923 occur in the host tissues. If it does, the Spherical or spheroidal (Fig. 2a-d, 4), has been found in three species of tuni­ tissues become slightly yellow or vermiform or club-shaped (Fig. 3, 6), cates in European coastal waters (Or­ green. and pyriform (Fig. 5) configurations are mieres and de Puytorac, 1968). found in the plasmodia, whereas Materials and Methods Marteilia refringens, the lethal agent spheroidal (Fig. II), pyriform (Fig. of Aber disease in European flat oys­ Techniques 4sed in specimen prep­ 10), vermiform or club, and truncated ters, O. edulis, is well described in this aration may be found in the relevant club or cuneiform (Fig. 12, 13) types symposium (Alderman, 1979; Balouet, papers reviewed herein. Unpublished are found in the spores. During dif­ 1979; Cahour, )979; Grizel, 1979). Its data on Urosporidium sp. in metacer­ ferentiation the may be closely related counterpart, M. sydneyi cariae of Microphallus sp. found in P. highly polymorphoric. Despite their Perkins and Wolf, 1976, in Australian pugio were derived from specimens varied shapes and sizes, when mature east coast waters causes severe mor­ collected under Folly Bridge in the their substructure is similar, consisting talities of Crassostrea commercialis. Charleston, S.c. area. They were fixed of a delimiting unit membrane and a As opposed to Urosporidium spp., using the glutaraldehyde and osmium continuous internal membrane which tetroxide techniques described in Per­ separates the organelle into a cortex and kins (1975'1). medulla both of high electron density (Fig. 2a-d, 3-6, 10-/3). The interface Results membrane may assume a pyriform, Figure I.-Plasmodium of Minchinia nel­ cup, or spherical shape (Fig. 2a-d, 14) soni in oyster hepatopancreas. Nucleus (N); Haplosporosomes (M); multivesicular body in spherical or spheroidal haplosporo­ (MY) where haplosporosomes are presum­ The most striking and most consis­ somes or may simply follow the profile ably formed; pseudopodium e"l) (P); mature tent similarity among the species of the organelle equidistant from the haplosporosome (H). (G). examined is the presence of organelles del imiting organelle membrane (Fig. 3, 15,000x. found in the plasmodia (Fig. I) which 6, 12). either disappear from the protoplast Plasmodial haplosporosomes appear (Minchinia spp., Urosporidium spp.) to be formed from multivesicular Figures 2a-d. -Serial sections through ma­ or from that part of the protoplast which bodies (MVB) (Fig. 1,7,8, 14). I have ture (H) and forming (F) haplosporosomes differentiates into spores (Marteilia now seen such formative regions in in Minchinia nelsoni plasmodium. The spp.) during sporulation. They reap­ plasmodia of M. refringens, M. syd­ forming organelles are components of a multivesicular body. Note cup-like configu­ pear in developing spores and become neyi, Minchinia nelsoni, and U. cres­ ration of internal membrane and spherical prominent in mature spores. The or­ cens, but not M. costalis, M. louisiana, shape of lower. free haplosporosome ganelles, termed haplosporosomes, U. spisuli, and Urosporidium sp. They 84.000x. consist of spheroidal, vermiform, were also not reported from U. jiroveci

Figures 3-6. -Plasmodial haplosporosomes of Minchinia louisiana (Fig. 3) M. nelsoni (Fig. 4), M. costalis (Fig. 5), and Marteilia refringens (Fig. 6) Note internal membrane between cortex and medulla and variations in shape: Club-like (Fig. 3), spherical (Fig. 4), pyriform (Fig. 5). and vermiform (Fig 6) 120.000X, 116,000x, 77.000 x, and 175,000x, respectively.

January-February 1979 27 Figure 9.-Nearly mature spore of Marreilia refringens. Nucleus lll... ( 2) of intermediate sporoplasm; spore wall (W); haplosporosome ,.. (H) in outermost sporoplasm; double membrane-limited vesicles (V). 47.000 x.

Figures 7, 8.-Multivesicular bodies of Minchinia nelsoni (Fig. 7) and Marreilia refringens (Fig. 8) believed to be organelles for synthesis of haplosporosomes. Figures 10-13. -Spore haplosporosomes of Urosporidium spisuli ... The probable maturation se­ (Fig. ID), Marteilia sydneyi (Fig. I I), Minchinia costalis (Fig. 12, ,.. quence is indicated by 1-'4. 13). Note delimiting membrane and membrane between cortex and See Figure 14 also. Figure 7, medulla. Terminology used in text to denote shape: Pyriform (Fig. 85,000 x; Figure 8, 108,000 x. 10), spherical (Fig. II), cuneiform (Fig. 12), and truncated club (Fig. 13). 123,000 x, 215.000 x, 135,000 x, and 42,000 x, respec­ tively.

28 Marine Fisheries Review January-February /979 29 (Ormieres et aI., 1973), M. armoricana detected; however, loss of the haplo­ the unit membrane which lies between (van Banning, 1977, 1979), and the sporosome formative areas (M VB's) the cortex and medulla is the membrane blue crab balanosporidan (Newman et may occur before then. In Marteilia of the former vesicle. The inner mem­ aI., 1976). However, I suspect that the spp., delimiting walls are only formed brane appears only as an electron light formative regions will eventually be around those parts of the protoplast zone in glutaraldehyde fixed prepara­ demonstrated in the other species, be­ which form spores. Haplosporosomes tions but can be resolved in KMn04­ cause in all studies, except those of and their formative regions (Fig. 8) are fixed cells (figure 5 of Perkins, 1975a). Newman et al. ( 1976), sporulation was found only in the portion of the proto­ Generally, the medulla acquires mate­ occurring in the specimens being ob­ plast lying outside the walls and persist rial of high electron density first during served. Possibly the plasmodia ob­ through sporulation. As with Uro­ development followed by the cortex. served by the latter workers had ceased sporidium spp. and Minchinia spp., the Individual vesicles within the to synthesize haplosporosomes in prep­ within the wall does not ac­ MVB's vary greatly in size and shape aration for spore formation. quire haplosporosomes until they ap­ (Fig. 14). Presumably, subdivisions Plasmodia of Minchinia spp. and pear in spores (Perkins, 1976; Perkins and enlargements occur to yield units of Urosporidium spp., which are convert­ and Wolf, 1976). a narrow size range prior to being in­ ing to sporonts, form a delimiting thin Vesicles or haplosporosome primor­ corporated into the haplosporosome wall (ca. 20 nm thick in most species, dia within the plasmodial M VB's ap­ which is budded from the MVB but up to 131 nm in U. crescens) around pear to bud from the periphery of the periphery. Fibrillar substructure can be the protoplast which persists through MVB's thereby forming free units (Fig. seen in the medulla of immature hap­ sporocyst maturation (Perkins, 1969, 14). The delimiting membrane of hap­ losporosomes (see fig. 13 e, f-Per­ 1971, 1975a; van Banning, 1979). losporosomes is thus derived from the kins, 1968). Their identity is not Thus, initiation of sporulation can be delimiting membrane of the M VB, and known, but may be related to the fact that M VB's of Minchinia nelsoni plasmodia are Feulgen positive. Such Table 1. Sizes of mature haplosporosomes in different species of balanosPQridans'. staining characteristics have not been Diameter or noted in other stellatosporeans, possi­ Species Cell type Shape Length (nm) Width (nm) Citation bly because the organelle densities and Minchinia nelson; Plasmodium Spherical 137-217 Ix ~175) New data (N~20) mass have not been great enough to Oblate 151-288 (x=201) 130-249 IX=168) New data detect the stain. spheroid (N~30) (N=30) Spore Oblate 162-239 IX=214) 130-217 (X=156) New data Two basic mechanisms may be spheroid (N~25) (N=25) utilized for haplosporosome formation Vermiform 214-391 (x=267) 66-174 (x=134) New data or club IN=25) 1!'J=25) in spores, one represented by Min­ M. costa/is Plasmodium Oblate 214-272 Ix ~235) 162-235 (x=201) Perkins. 1969 spheroid (N=20) (N=20)' and new data chinia spp. and Urosporidium spp. and Pyritorm 218-336 IX~289) 154-215 (x=181) the other by Marteilia spp. In Min­ IN=30) (N~30) Spore Truncated 350-650 (x=480) 140-220 Ix=180) chinia louisiana spores, haplosporo­ club or IN=20) (N=20) somes appear to arise from MVB's in cuneiform M. louisiana Plasmodium Club 300-586 (x=456) 129-186 (x=155) Perkins. 1975a much the same way as in M. nelsoni (~=7) 1~=11)1 plasmodia. The MVB's are derived Spore Pyriform 133-200 (x~174) 104-151 (x=124) Perkins. 1975a (~=15) (N=20) and new data from a Golgi apparatus-like organelle M. sp. Plasmodium Spherical 150-200 (x~175) Newman et aL, (~=?) 1976 ("spherule" of classical literature) at Urosporidium Plasmodium Spherical 123-159 IX=139) Perkins, 1971 the anterior end of the spore (Perkins, crescens (~=10) Oblate 133-200 (x=158) 110-133 Ix=1H)) New data 1975a). Haplosporosome origins in M. spheroid (~=10) (~~10~ nelsoni and M. costalis spores are less Spore Pyriform 172-218 (x=199) 126-178 (x=152) New data (~=15) (N=15) well known, but appear to arise directly U. spisuli Spore Oblate 97-164 (x=121) 83-149 (x=101) Perkins et ai., from the Golgi apparatus-like cisternae spheroid IN=25) (N=75), 1975 and new data Pyriform 114-190 (x=I53) 86-139 (x= 117) New data as evidenced by accumulation of elec­ (N=10) (N=10)., U. sp. (tram Micro­ Spore Spherical 69-115 (x=88) New data tron dense material (Perkins, unpub­ phallUS sp. in IN=45) '- lished data). In Urosporidium sp. and Pa/aemonetes pugio) Marteilia refringens Plasmodium Vermiform 130-490 (x=240) 43-130 (x=60)-: Perkins, 1976 U. crescens evidence for the or club (N=30) (~=40) and new data "spherule" being a Golgi apparatus Spore Oblate 175-203 (x=189) 71-158 Ix~11.11 Perkins, 1976 spheroid (N=16) (N=34) and the site of haplosporosome forma­ Spherical 98-196 IX=113) New data (N=18) , tion is strongest since haplosporosomes M. sydneyi Plasmodium Vermiform 146-603 (x=312) 29-65 (x=52)~ Perkins and Wolf, were found in the cisternae (Perkins, or oblate (N=28) (N=28)· 1976 and new data spheroid 1971; unpublished data). In U. spisuli a Spore Vermdorm 148-288 (x=187) 44-163 (x~96)' Perkins and Wolf. similar sequence was suggested, al­ or oblate (N =30) (N~30) 1976 and new data spheroid though cisternae were not organized 'Widths ot club-shaped and pyriform haplosporosomes were measured through the most enlarged portion of the organelle. into an anastomosing network like a

30 Marine Fisheries Review H

® Figure 14. -Diagrammatic representation of haplosporosome (H) formation from multivesicular body (M VB) as seen in Minchinia nelsoni plasmodia. Within the M VB large polymorphic vesicles (I) pinch off spherical vesicles (2) both with low density contents. Fibrillar and granular material is added to the interior of the vesicles (3), they migrate to the MYB periphery, and pinch off the MYB periphery thereby acquiring an additional, delimiting membrane (4). Additional electron-dense material is subsequently added to the cortex. Small, dense bodies (arrow) in the MYB may enlarge to participate in haplosporosome formation.

Golgi apparatus (Perkins et a!., 1975). "spherule," but no involvement in between the plasmodium and host cell Although it was not mentioned in Or­ haplosporosome formation was men­ (Fig. 16). In each case the cortex mate­ ml'eres et a!. (1973), U. juroveci may tioned (Ormleres and de Puytorac, rial appears to decrease first in electron also form haplosporosomes in cisternae 1968). density indicating loss of or chemical of the Golgi apparatus-like organelle as In Marteilia refringens and M. syd­ change in the cortical material. Haplo­ is suspected from examination of Fig­ neyi spores there are no anastomosing sporosomes in which cortex and ure 13 where at least one haplosporo­ cisternae resembling Golgi apparati nor medulla had become less dense were some-like structure can be seen in a are there MVB's which could give rise not recognized. The organelles may cisterna. Minchinia armoricana spores to haplosporosomes. They appear to also be deposited between the host cell have a "spherule" and truncated, arise individually in the outermost and early sporont or plasmodium in a club-shaped haplosporosomes which sporoplasm (Perkins and Wolf, 1976) population of sporulating cells of U. resemble those of M. costalis (Perkins, and are never found in the middle or crescens (Perkins, 1971). Since sporu­ 1969; van Banning, 1977); however, inner sporoplasms (Fig. 9). lation is associated with extensive host no evidence for formation of haplospo­ Haplosporosomes are known to be cell damage in most species it is rosomes in the cisternae of the Euro­ liberated from plasmodia of Minchinia suggested that haplosporosome release pean parasite were presented. Haplo­ nelsoni and enter oyster cells intact and dispersion may be related to host sporidium ascidiarum spores have a (Fig. 15) or to be emptied into the space cell lysis.

January-February 1979 31 . u+4l 'If;Ar: ..;:"~ ~

Figure 15. -Haplosporosomes (H) in hepatopancreas cell of Minchinia nelsoni-infected oyster. Note loss of cortex material from labeled haplosporosome. 72,000 x. Figure 16. -Haplosporosome (H) dispersing cortex material into intercellular region between one oyster cell (0,) and plasmodium (PI). Note continuity of intercellular region and cul-de-sac in which haplosporosome is situated. Second oyster cell (02). 54,000 x. Figure 17. -Golgi apparatus (G) in spore of Urosporidium spisuli. a species which lacks the "spherule" or Golgi apparatus normally found at the anterior end of Minchinia. Hapiosporidium. and other Urosporidium spores. 58,000 x.

Other Organelles sparse arrays of flattened cisternae each cidiarium, Urosporidium sp., U. cres­ of which has an anastomosing substruc­ cens, and U. jiroveci. They appear to Mitochondria of the Stellatosporea ture typical of Golgi apparati. Budding be Golgi apparati in that anastomosing are either tubulo-vesicular in substruc­ of vesicles from the nuclear envelope cisternae comprise the substructure and ture as in Minchinia spp. and Urospo­ and fusion with the proximal face of the haplosporosomes have been observed ridium spp. (Fig. I) (Perkins 1969, organelle are observed (Fig. 17, 18). to be formed therein; however, the typ­ 1975a) or are vesicular with shelf-like On the distal face of M. nelsoni Golgi ical stacked layers of flattened vesicles cristae as in Marteilia spp. (Perkins, apparati, cisternae curl into nearly cir­ are never visualized. It is interesting to 1976; Perkins and Wolf, 1976). Cristae cular profiles (Fig. 19). On the inner note that U. spisuli spores lack a were numerous and easily visualized in face of the curve electron dense mate­ "spherule," but contain a typical Golgi Minchinia spp., less so in Urosporid­ rial is deposited. Whether these struc­ apparatus (Fig. 17). Neither Golgi ap­ ium spp., and difficult to find in Mar­ tures become spherical and then meta­ parati nor "spherules" have been ob­ teilia spp. A paucity ofcristae is typical morphose into haplosporosomes has served in Marteilia spp. of many parasitic (Tandler not been determined. If so, it is not Only in Minchinia nelsoni plasmodia and Hoppel, 1972). In all cases known how they might interact with the have nuclear structure and been mitochondria are easily located because multivesicular bodies suspected to be observed in detail. Nuclei are typically the electron light areas of the vesicular the haplosporosome formative regions found in pairs with a concavity in the mitochondria reveal the DNA nucleoid (see previous "Haplosporosome" sec­ surface ofeach nucleus where they face which distinguishes the organelle from tion). Golgi apparati of the other each other (figure 10 in Perkins, cytoplasmic vesicles (Perkins, 1969, balanosporidan plasmodia have not 1975b). There is a persistent mitotic 1976; Perkins and Wolf, 1976). been observed if they exist. apparatus, found during interphase and Although questioned in previous The "spherule" or mass of anas­ in mitotic nuclei, which consists of two papers (Perkins, 1968, 1975a), Golgi tomosing cisternae appears in the an­ spindle pole bodies free in the nucleo­ apparati are now known to be present in terior end ofthe sporoplasm ofdevelop­ plasm and not attached to the nuclear Minchinia nelsoni, M. louisiana, and ing spores of M. nelsoni, M. costalis, envelope with a bundle of 33-53 micro­ U. spisuli plasmodia. They appear as M. louisiana, M. armoricana, H. as- tubules between them (Perkins,

32 Marine Fisheries Review Figure 18.-Golgi apparatus (G) in Minchinia nelsoni plas­ Figure 19.-Golgi apparatus (G) of Minchinia nelsoni plas­ modium. Note budding (arrow) of nuclear envelope toward modium showing development of vesicle (V) from recurved proximal face of apparatus and paucity of appratus cisternae. apparatus cisterna. Electron-dense material (E) is added to (SP), mitotic apparatus inner face and ultimately fills the medulla region. Despite the (MI). 65.000 x. resemblance to developing haplosporosomes. such structures are believed to be unrelated to haplosporosomes. 50 ,000 x.

1975b). When mitosis occurs the nu­ tubules in Marteilia sp. from am­ were about 3.8 I1-ffi diameter. Further clear envelope remains intact and all phipods; thus a reexamination of the evidence for meiosis lies in the observa­ mitotic microtubules are contained oyster pathogens for is war­ tion of synaptonemal complex-like and within the envelope. The nuclear ranted. polycomplex-like structures in sporont medial profile goes from circular at in­ nuclei. Polycomplex-like structures Sporulation terphase to a spindle shape at meta­ have also been seen in immature spore phase then a dumbbell shape at telo­ Spore formation in Minchinia spp. nuclei of Marteilia refringens. phase. The remains peripher­ and Urosporidium spp. appears to con­ There are two proposals to explain ally located throughout and appears to sist of enlargement of plasmodia, for­ spore differentiation from sporoblasts. pull apart during division. In M. mation of a wall around the cells, From studies of U. crescens, Perkins louisiana and M. costalis, nuclear divi­ increase in numbers of nuclei, then (1971) suggested that invagination of sion occurs in the same manner, but condensation of cytoplasm around each the sporoblast periphery carved out the whether the interphase nucleus retains nucleus to yield uninucleate sporo­ sporoplasm thus yielding the anucleate the mitotic apparatus has not been de­ blasts. However, nuclear fusion, fol­ extraspore cytoplasm and the uninu­ termined. In Marteilia refringens and lowed by meiosis, may occur in the cleate sporoplasm. Ormieres et al. M. sydneyi, mitosis was not observed sequence as evidenced by studies of M. (1973) suggested that in U. jiroveci a nor were centrioles or spindle pole louisiana (Perkins, 1975a) where pair­ binucleate sporoblast formed the ma­ bodies seen. Ginsburger- Vogel and ing of small (ca. 3.0 Il-m diameter) nu­ ture spore as a result of one half par­ Desportes (1979) have seen centrioles clei and large (>4 11-ffi) nuclei were tially engulfing the other half, followed consisting of a singlet ring of micro- observed in sporonts. Sporoblast nuclei by degeneration of the nucleus of the

January-February 1979 33 outennost protoplast. Separation of the From the binucleate, endogenously now known as the balanosporidans, and innermost protoplast then occurred to cleaved stage, sporulation is initiated Sprague (1979) has erected the family form the sporoplasm, free within the by enlargement of the cells and multi­ Marteiliidae in the order Occluso­ extraspore cytoplasm. Whether both or plication of the internal cells which then sporida to accommodate them. It ap­ one of the mechanisms occurs in Min­ serve as sporangia. Thus the complex pears reasonable to ally Marteilia spp. chinia spp. and Urosporidium spp. re­ becomes a sporangiosorus (i.e., a cell with the balanosporidans, because hap­ mains to be detennined. containing several sporangia). Spores losporosomes, with their unique sub­ After delimitation of the sporoplasm are formed in the sporangia and consist structure, are fou nd in all species the spore wall is formed in the extra­ of three uninucleate sporoplasms, an studied and not in other species of mi­ spore cytoplasm and consists of a cup intermediate one containing an inner croorganisms. The organelles are found with the anterior end occluded by a sporoplasm, all of which are contained only in plasmodia and spores, not in the tongue of wall material, termed the Iin­ in an outer sporoplasm (Fig. 20). As intermediate cell stages leading to spore gua, in Urosporidium spp. (Perkins et they approach maturity, the spores are formation. The suspected mode of hap­ aI., 1977; Perkins, 1971; Onnieres et fully delimited by a thin wall which losporosome formation from mul­ al., 1973) and by a cap of wall material lacks any lingua or cap. Grizel et al. tivesicular bodies occurs in at least one in Minchinia spp. (Perkins, 1968, (1974) used the tenns "primary cell" indisputable balanosporidan, Min­ 1969, 1975a). for sporangiosorus, "secondary cell" chinia nelsoni, as well as Marteilia The above-described sequence for for sporangia, and "tertiary cell" for spp. Internal cleavage during spore sporoblast formation predominates; the spores. Internal delimitation of all fonnation is found in at least one estab­ however, at least in M. louisiana, in­ nucleated units (sporangia, spores, lished balanosporidan, Minchinia ternal cleavage of sporoblasts occurs sporoplasms) during sporulation is ac­ louisiana, as well as Marteilia spp. within the sporont protoplast without complished by vesicle fusion (Perkins, One problem in accepting balanospori­ cytoplasmic condensation (Perkins, 1976; Perkins and Wolf, 1976). After dan affinities for Marteilia spp. lies in 1975a). The mechanism of sporoplasm spore maturation the protoplasm, not the multicellular sporoplasm. Whether delimination within the sporoblast was included within the spore wall, degen­ the extraspore cytoplasm has a nucleus not determined; however, fully mature erates. during differentiation which is later lost spores are known to be formed as a Wall ornamentation around spores of as suggested by Onnieres et al. (1973) result of this type of sporoblast forma­ Minchinia spp. and Urosporidium spp. remains to be proven. If so, those tion. is formed in the extraspore cytoplasm spores could also be called multicellu­ Internal cleavage also occurs in Mar­ which then disperses in the case of lar in origin (Sprague, 1979), particu­ teilia refringens, Marteilia sp., and M. Minchinia spp. leaving the ornaments larly since the ornaments formed in the sydneyi during formation of sporangia which are threads (Fig. 21) (Perkins, extraspore cytoplasm are an integral and spores (Ginsburger- Vogel and De­ 1968, 1969, 1975a) or ribbons (Per­ part of the spore. sportes, 1979; Perkins, 1976; Perkins kins, 1969). In Urosporidium spp., Another problem lies in the general and Wolf, 1976), but condensation of ribbons are formed in U. crescens (Per­ multicellularity of Marteilia spp. with cytoplasm to fonn sporoblasts does not kins, 1971) and U. jiroved (Ormieres cells engaged in sporulation (i.e., occur. The earliest cell type observed in et aI., 1973) and a labyrinthine complex sporangia within a sporangiosorus and newly infected hosts consists of a uni­ in U. spisuli (Perkins et aI., 1977) and spores within sporangia). In balano­ or binucleate cell (Grizel et al., 1974). I Urosporidium sp. (Perkins, unpub­ sporidans there are only spores within a observed no less than two nuclei per lished data). The extraspore cytoplasm sporont, not an intermediate cell type. cell in M. refringens and M. sydneyi. probably disperses revealing the orna­ Whether one should consider such a Because the cells were without walls ments, but this has not yet been ob­ difference of enough importance to and had more than one nucleus, they served. With the possible exception of warrant placement of Marteilia spp. in were termed plasmodia. Whether they U. crescens and U. jiroveci, substruc­ a class separate from the balanospori­ always consist of an uninucleate cell ture of the ornaments appears to be dans should await further ultrastruc­ within an uninucleate cell (see figure 5 species specific. Marteilia spp. form no tural studies of other species resembl­ of Perkins and Wolf, 1976) from the ornaments around the spores. Only ing the Marteilia spp. already studied. earliest stage of infection or may con­ membrane whorls resulting from de­ The centrioles found in Marteilia sp. sist of a binucleate cell is problemati­ generation of extraspore cytoplasm in by Ginsburger-Vogel et al. (1976) and cal. Cells which we interpreted (Per­ the sporangium are found wrapped Ginsburger- Vogel and Desportes kins, 1976; Perkins and Wolf, 1976) to around the wall. (1979) are of potential significance in be simple binucleated ones could have efforts to determine the taxonomic Discussion been endogenously separated. Never­ affinities of Marteilia spp. since pres­ theless, the term plasmodium has been In attempting to establish the ence or absence of microtubular cen­ used in protozoology for multinucleate taxonomic affinities ofMarteilia spp., I trioles is considered by many workers cells with endogenous subdivisions have suggested that they are related to as a marker of phylogenic significance (Poisson, 1953). the haplosporidans (Perkins, 1976), (Pickett-Heaps, 1969; Fulton, 1971).

34 Marine Fisheries Review Figure 20.-Two developing Minchinia refringens spores in sporangium. Sporangia) nucleus (Sn) and cytoplasm (CYt)

which is not incorporated into spores; intermediate sporoplasm nuclei (Nt). inner sporoplasm nucleus (N3 ). cytoplasm of outer sporoplasm (C I), sporangial wall (W). multi vesicular body (MY) of sporangiosorus (Cy I) which is not incorporated into sporangia. 9.000 x.

January-February /979 J5 Coccidian Protozoa of the subphylum have microtubular cen­ trioles arranged in a singlet ring with ninefold symmetry (Dubremetz, 1973) as was found in Marteilia sp. However, the Apicomplexa also in­ clude species which form spindle pole bodies (SPB's) (no microtubular sub­ structure) as for example, Plasmodium spp. (Aikawa et aI., 1972). Thus the existence of SPB 's in balanosporidans (Perkins, 1975b) and microtubular cen­ trioles in Marteilia sp. does not neces­ sarily serve as evidence that the two are not closely related. It will be interesting to determine which organelle type is found in M. refringens and M. sydneyi. Since numerous biochemicals are available today for control of protozoan diseases of humans and farm animals, considerations of ultrastructure and phylogenetic affinities have particular significance. For example, it is known that the antimalarial drug, pyrimetha­ mine, has an inhibitory effect on nucle­ ar division in Plasmodium berghei nigeriensis (Peters, 1974). Since the mitotic apparati of Plasmodium spp. and Minchinia spp. are similar, one Figure 21.-Scanning electron micrograph of Minchinia might expect the drug to inhibit nuclear louisiana spore showing spore wall cap (c) and thread-like division in the oyster pathogens. This spore wall ornaments. 10,000 x. hypothesis needs to be tested for pyrimethamine as well as for other chemotherapeutic agents which inhibit mitosis in species of the Apicomplexa where both spindle pole bodies and cen­ reinfection. Such an approach needs to the Stellatosporea must be reconsid­ trioles consisting of singlet rings of be explored. ered. ] microtubules are found. [Note added in proof. Two publica­ Even when the mode of action of a tions have appeared since this paper Acknowledgments drug is not known, the drug should be was presented which have information The author wishes to acknowledge considered as a possible control for a relevant to the taxonomic position of the expert assistance ofPatsy Berry and shellfish disease when the shellfish dis­ Marteilia spp. Desportes and Gins­ Judy Parrish. Phillip A. Madden is ease agent can be demonstrated to be burger-Vogel (1977) have suggested thanked for providing the scanning closely related to the species known to that Marteilia spp. should be consid­ electron micrograph. be inhibited by the drug. It is obvious ered as members of a new order, Mar­ that estuaries or oceans cannot be effec­ teiliida, in the Cnidosporidia, because Literature Cited tively treated with drugs due to the large they have a pluricellular structure. Cur­ Aikawa. M., C. R. Sterling, and J. Rabbege. volumes; however, if drugs effective rent and Janovy (1977) have observed 1972. Cytochemistry of the nucleus of malarial against shellfish diseases can be found, inclusions in the sporoplasm of Hen­ parasites. In E. H. Sadun and A. P. Moon they could be used under holding tank neguya exilis. one ofthe Myxosporidia, (editors). Basic research in malaria. Proc. or aquaculture conditions where a lim­ which resemble haplosporosomes; Helminthol. Soc. Wash. 39: 174-194. Alderman, D. J. 1979. Epizootiology of Mar­ ited volume of seawater would be in­ however, the resolution was not teilia refringens. In F. O. Perkins (editor), volved for selected time periods. If the adequate to make definitive judge­ Haplosporidian and haplosporidian-like dis­ shellfish acquired immunity after being ments. Therefore, affinities of Mar­ eases of shellfish. Mar. Fish. Rev. 41(1­ 2):67-69. "cured" then subsequent addition to teilia spp. with the balanosporidans and Balouel, G. 1979. Martei/ia refringens-con­ the estuary or ocean would not result in the uniqueness of haplosporosomes for siderations of the life cycle and development of

36 Marine Fisheries Review Abers disease in Ostrea edulis. In F. O. Per­ serans, 1. L. Duthoit, and M. A. Le Pennec. tozool. 23:64-74. kins (editor), Haplosporidian and haplospo­ 1974. Recherche sur I'agent de la maladie de la ____ , P. A. Madden, and T. K. Sawyer. ridian-like diseases of shellfish. Mar. Fish. glande digestive de Ostrea edulis Linne. Sci. 1977. Ultrastructural study ofthe spore surface Rev. 41(1-2):64-66. Peche 240:7-30. of the haplosporidian Urosporidium spisuli. Cahour, A. 1979. Marteilia refringens and Cras­ Lichtenfels, J. R., J. F. A. Sprent, J. W. Bier, P. Trans. Am. Microsc. Soc. 96:376-382. sostrea gigas. In F. O. Perkins (editor), Haplo­ A. Madden, T. K. Sawyer, and L. R. G. Can­ ____ , and P. H. Wolf. 1976. Fine struc­ sporidian and haplosporidian-like diseases of non. 1977. New information on identity of ture of Marteilia sydneyi sp. n. - haplospori­ shellfish. Mar. Fish. Rev. 41(1-2):19-20. anasakid nematode in shellfish of Atlantic dan pathogen of Australian oyster. J. Parasitol. Caullery, M. 1953. Appendice aux Sporozoaires. Coast of North America. Prog. Abstr. 52nd 62:528-538. Classe des haplosporidies (Haplosporidia Annu. Meet. Soc. Protozool., p. 53 (Abstr. ____ , D. E. Zwerner, and R. K. Dias. Caullery et Mesnil, 1899). In P. P. Grasse #100). 1975. The hyperparasite, Urosporidium (editor), Traite de Zoologie 1(2):922-934. Newman, M. W., C. A. Johnson III, and G. B. spisuli sp. n. (Haplosporea), and its effects on Masson et Cie, Paris. Pauley. 1976. A Minchinia-like haplosporidan the surf clam industry. J. Parasitol. 61 :944­ Current, W. L., and J. Janovy, Jr. 1977. parasitizing blue crabs, Callinectes sapidus. J. 949. Sporogenesis in Henneguya exilis infecting the Invertebr. Pathol. 27:311-315. Peters, W. 1974. Recent advances in antimalarial channel catfish: an ultrastructural study. Protis­ Ormieres, R., and P. de Puytorac. 1968. Ultra­ chemotherapy and drug resistance. In B. tologica 13: 157-167. structure des spores de I'Haplosporidie Haplo­ Dawes (editor), Advances in parasitology, Vol. 12, p. 87. Academic Press, N.Y. Desportes, I., and T. Ginsburger-Vogel. 1977. sporidium ascidiarum endoparasite du Pickett-Heaps, 1. D. 1969. The evolution of the Affinites du genre Marteilia, parasite Tunicier Sydnium elegans Giard. C. R. Acad. mitotic apparatus: an attempt at comparative d'Huitres (maladie des Abers) et du Crustace Sci., Paris 266: 1134-1136. ultrastructural cytology in dividing plant cells. Orchestia gammarellus (Pallas), avec les ____ , V. Sprague, and P. Bartoli. 1973. Cytobios 1:257-280. Myxosporidies, Actinomyxidies et Paramyxi­ Light and electron microscope study of a new Poisson, R. 1953. Sous-embranchement des dies. C. R. Acad. Sci., Paris 285:1111-1114. species of Urosporidium (Haplosporida), hyperparasite of trematode sporocysts in the Cnidosporidies.ln P. P. Grasse (editor), Traite Dubremetz, J. F. 1973. Etude ultrastructurale de de Zoologie:Anatomie, Systematique, biolgie la mitose schizogonique chez la coccidie clam Abra ovata. J. Invertebr. Palhol. 21 :71­ 86 1(2):1006-1008. Masson et Cie, Paris. Eimeria necatrix (Johnson 1930). J. Ultra­ Rosenfield, A., L. Buchanan, and G. B. Chap­ struct. Res. 42:354-376. Perkins, F. O. 1968. Fine structure of the oyster pathogen, Minchinia nelsoni (Haplosporida, man. 1969. Comparison of the fine structure of Fulton, C. 1971. Centrioles. In 1. Reinert and H. spores of three species of Minchinia (Haplo­ Haplosporidiidae). J. Invertebr. Pathol. Ursprung (editors), Origin and continuity of sporida, Haplosporidiidae). J. Parasitol. 10:287-305. cell organelles, Vol. 2, p. 170-221. 55:921-941. 1969. Electron microscope studies Springer-Verlag, N.Y. Sprague, V. 1963. Revision of genus Haplo­ of sporulation in the oyster pathogen, Min­ Ginsburger- Vogel, T., and I. Desportes. 1979. sporidium and restoration of genus Minchinia chinia costalis (Sporozoa: Haplosporida). 1. Structure and biology of Marteilia sp. in the (Haplosporidia, Haplosporidiidae). J. Pro­ Parasitol. 55:897-920. amphipod Orchestia gammarellus. In F. O. tozool. 10:263-266. Perkins (editor), Haplosporidian and 1971. Sporulation in the trematode 1979. Classification of the haplo­ haplosporidian-like diseases of shellfish. Mar. hyperparasite Urosporidium crescens De sporidia. In F. O. Perkins (editor), Haplospo­ Fish. Rev. 41(1-2):3-7. Turk, 1940 (Haplosporida: Haplosporidi­ ridian and haplosporidian-like diseases of ____ , , and C. Zerbib. 1976. idae)-an electron microscope study. J. shelifish. Mar. Fish. Rev. 41(1):40-44. Presence chez I' Amphipode Orchestia gam­ Parasitol. 57:9-23. Tandler, B., and C. L. Hoppel. 1972. Mitochon­ marellus (Pallas) d'un Protiste parasite; ses 1975a. Fine structure of Minchinia dria. Academic Press, N. Y., 59 p. affinites avec Marteilia refringens agent de sp. (Haplosporida) sporulation in the mud van Banning, P. 1977. Minchinia armoricana sp. I'epizootie de I'Hultre plate. C. R. Acad. Sci., crab, Panopeus herbstii. Mar. Fish. Rev. nov. (Haplosporida), a parasite of the Euro­ Paris 283:939-942. 37(5-6):46-60. pean flat oyster, Ostrea edulis. J. Invertebr. Grizel, H. 1979. Marteilia refringens and oyster 1975b. Fine structure of the haplo­ Pathol. 30: 199-206. disease-recent observations. In F. O. Perkins sporidan Kernstab, a persistent, intranuclear 1979. Haplosporidian diseases of (editor), Haplosporidian and haplosporidian­ mitotic apparatus. J. Cell. Sci. 18:327-346. imported oysters (Ostrea edulis) in Dutch es­ like diseases of shellfish. Mar. Fish. Rev. 1976. Ultrastructure of sporulation tuaries. In F. O. Perkins (editor), Haplospori­ 41 (1-2):38-39. in the European flat oyster pathogen, Marteilia dian and haplosporidian-like diseases of ____ , M. Comps, J. R. Bonami, F. Cous- refringens - taxonomic implications. J. Pro- shellfish. Mar. Fish. Rev. 41(1-2):8-18.

January-February f 979 37