Cell Structure of Shellfish Pathogens and Hyperparasites in the Genera Minchinia, Urosporidium, Haplosporidium, and Marteilia-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 spore structure beyond phylogeny and taxonomy. 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 spores 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 organelle 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, Ostrea edulis, 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 organelles 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 mitochondrion (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). Golgi apparatus (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