Previously Unknown Apicomplexan Species Infecting Iceland Scallop, Chlamys Islandica

Journal of Invertebrate Pathology 108 (2011) 147–155

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Journal of Invertebrate Pathology

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Previously unknown apicomplexan species infecting Iceland scallop, Chlamys islandica (Müller, 1776), queen scallop, Aequipecten opercularis L., and king scallop, Pecten maximus L. ⇑ Árni Kristmundsson a, , Sigurður Helgason a, Slavko Helgi Bambir a, Matthías Eydal a, Mark A. Freeman b a Institute for Experimental Pathology at Keldur, University of Iceland, Reykjavik, Iceland b Institute of Biological Sciences & Institute of Ocean and Earth Sciences, University of Malaya, Kuala Lumpur, Malaysia article info abstract

Article history: Examination of three scallop species from three separate locations: Iceland scallop from Icelandic waters, Received 14 December 2010 king scallop from Scottish waters and queen scallop from Faroese and Scottish waters, revealed infections Accepted 19 August 2011 of a previously unknown apicomplexan parasite in all three scallop species. Developmental forms Available online 27 August 2011 observed in the shells appeared to include both sexual and asexual stages of the parasite, i.e. merogony, gametogony and sporogony, which suggests a monoxenous life cycle. Meronts, gamonts, zygotes and Keywords: mature oocysts were solely found in the muscular tissue. Zoites, which could be sporozoites and/or mer- Chlamys islandica ozoites, were observed in great numbers, most frequently in muscles, both intracellular and free in the Aequipecten opercularis extracellular space. Zoites were also common inside haemocytes. Examination of the ultrastructure Pecten maximus Iceland scallop showed that the zoites contained all the major structures characterizing apicomplexans. This apicom- Apicomplexa plexan parasite is morphologically different from other apicomplexan species previously described from Parasites bivalves. Presently, its systematic position within the phylum Apicomplexa cannot be ascertained. Bivalves Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction pelsineeri from Donax sp. and Tellina sp., they are poorly studied; this is especially so for scallops. Only few studies on parasites in the scal- Wild populations of Iceland scallop Chlamys islandica, queen lop species examined in the present paper exist in the literature, i.e. scallop Aequipecten opercularis (syn. Chlamys opercularis), and king the king scallop, the queen scallop and the Iceland scallop. One api- scallop Pecten maximus, are all commercially exploited shellfish complexan species, Pseudoklossia pectinis, has been reported from species. Iceland scallop has the most northern distribution, occur- king scallop off the French coast (Léger and Duboscq, 1917), and ring mainly in the sub-arctic transitional zone. In the Atlantic one from Iceland scallop in Icelandic waters, i.e. Margolisiella islan- Ocean it is found around Iceland, Greenland, in the Barents Sea, dica (Kristmundsson et al., 2011). The only parasitic examination off the northern coast of Norway south to Bergen and along the performed on queen scallop seems to be that of Lohrmann et al. east coast of Canada south towards Massachusetts. It has also been (2000), who reported a microsporidian infection in this species. recorded in the N-Pacific Ocean (Brand, 2006). King- and queen Mortenson (1993) published a health survey on selected bivalve scallops have a similar geographic distribution, which extends stocks in Norwegian waters. The king scallop was included in the more south than that of Iceland scallop. The queen scallop occurs survey, but no protozoan parasites were detected. To date, Argopec- in the NE-Atlantic from northern Norway and the Faroe Islands ten irradians is probably the most examined scallop species with re- south to the Iberian Peninsula, the Azores and the Canary Islands. gard to apicomplexan parasites, and several papers have been Its distribution also extends into the Mediterranean and Adriatic published (Karlsson, 1991; Cawthorn et al., 1992; Leibovitz et al., seas. The distribution of king scallop spans from northern Norway, 1984; Whyte et al., 1994). However, none of the apicomplexans along the west coast of Europe and south to North Africa (Nicolaj- encountered in these surveys have been identified to a species level. sen, 1997; Brand, 2006). Most apicomplexan species known to infect bivalves have been Although reports on apicomplexans infecting bivalves date back described from clams. These include Pseudoklossia (syn. Hyalloklos- to 1897, when Léger (1897) described the first species, Hyalloklossia sia) pelsineeri from Donax sp. and Tellina sp. (Léger, 1897), Pseudok- lossia glomerata, from Tapes floridus and Tapes virgineus (Léger and Duboscq, 1915), Pseudoklossia (syn. Merocystis) tellinovum from Tel- ⇑ Corresponding author. Address: Institute for Experimental Pathology, Univer- sity of Iceland, Reykjavik, Keldur v/Vesturlandsveg, IS-112 Reykjavik, Iceland. Fax: lina tenuis (Buchanan, 1979; Levine, 1988) and Margolisiella kabatai +354 567 3979. from the littleneck clam Protothaca staminae (Desser and Bower, E-mail address: [email protected] (Á. Kristmundsson). 1997). In addition, Morado et al. (1984) and Nakayama et al.

(1998) reported unidentified apicomplexans from the littleneck the following methods: (1) Fresh mounts: samples from each organ clam P. staminae and the giant clam Tridacna crocea, respectively. pressed between a glass slide and a coverslip, (2) Stained imprints: Some apicomplexan species infecting bivalves have direct life tissue imprints of organs (i.e. adductor muscle, heart and kidney) cycles, i.e. both asexual and sexual stages are present in the same were air dried, fixed in methanol for 3 min and stained with host (e.g. Desser and Bower, 1997), while in others, only sexual May–Grünwald–Giemsa, (3) Davidson’s fixed histological sections stages have been found, which could suggest the need for an addi- of all organs prepared by conventional methods and stained with tional host (Desser et al., 1998). Another possibility is that the Giemsa. Parasites present were described and their tissue distribu- asexual forms are present in the same host, but have not been de- tion analysed. All dimensions were measured (lm) and photo- tected. However, in some cases, such as Nematopsis spp., an indi- graphs taken using a Leica DMLB microscope equipped with a rect life cycle has been confirmed where sexual stages are found digital camera (Leica DC300F). in various bivalve species and schizogonic multiplication occurs For examination of parasite ultrastructure, small pieces in crustaceans (Lauckner, 1983; Soto et al., 1996; Darriba et al., (1mm3) of infected muscles were fixed in 2.5% buffered glutaral- 2010). Finally, there are documentations of apicomplexan parasites dehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 24 h at 4 °C, in bivalves where certain parasitic forms have been detected, washed three times in cacodylate buffer, post-fixed for 1 h in 1% which could not be identified as either asexual- or sexual forms OsO4, rinsed again with buffer, dehydrated in a graded alcohol ser- (Nakayama et al., 1998; Hine, 2002). According to papers published ies, sectioned, stained for 10 min in 5% uranyl acetate and for 6 min to date on apicomplexans of bivalves, it seems there are no exam- in 5% lead citrate. Ultrathin sections were examined using a FEI, ples where only the asexual reproduction, i.e. schizogony, occurs in Tecnai G2 Spirit Biotwin, Transmission Electron Microscope (at bivalves. 120 kV) at the Institute of Aquaculture, University of Stirling, In the year 2002 live scallops were sent to the Fish Disease Lab- Scotland. oratory at the Institute for Experimental Pathology at Keldur for examination due to abnormal mortality in the stock. Observations 3. Results revealed infections with two previously unknown apicomplexan species. All Iceland scallops and the queen scallops sampled in Faroese In this paper one of the apicomplexan species is described, but waters were infected with an apicomplexan parasite. Infections the second one is the subject of a separate paper (Kristmundsson were commonly intense. Identical developmental forms were also et al., 2011). Due to abnormal conditions of queen scallops off found in both queen scallops (detection frequency 40%) and king the Faroe Islands at that time, a Faroese company in the Scallop scallops (90%) from Scottish waters, but infections were very light fishery industry requested an observation of the scallops with re- in all cases. gard to diseases. In addition, queen- and king scallops from Scot- tish waters were sampled at a later stage and examined to further check the geographic distribution of the parasites. 3.1. Fresh mount observations

Various types of large cysts at different developmental stages 2. Materials and methods are present in the adductor muscle. Their size and outer and inner morphology is quite variable. Thin walled elongated cysts, 2.1. Scallop collection 320 ± 50 lm Â 75 ± 25 lm(n = 10) (Fig. 2a), with granular cytoplasm are common. Sometimes, these have membranous protru- Live Iceland scallops from two locations in the Bay of Breidafjör- sions at each end. In other cases, similar cysts contain a clear dur were sampled in November 2002 (n =2Â 30; size 3.0–9.0 cm). area, which resembles a formation of spindle apparatus, which be- Queen scallops (n = 60; size 6.0–7.0 cm) from the east coast of the comes evident when zygotes undergo the first nuclear division Faroe Islands were sampled in February 2005, and in September (Fig. 2b). Another type of cysts, commonly found, are usually more 2007 queen scallops (n = 10; size 4.5–7.0 cm) and king scallops slender and with pointed ends. These cysts, 285 ± 25 lm Â (n = 10; size 10.0–13.0 cm) from the west coast of Scotland 45 ± 10 lm(n = 10), are filled with numerous round spheres or nu- (Fig. 1). The scallops were sampled from natural beds by dredging, clei, 3.5–4.0 lm in diameter (Fig. 2c). Yet another type, 297 ± except the queen scallops from Scottish waters, which were col- 40 lm Â 98 ± 25 lm(n = 10), has a very thick wall (5–7 lm) lected by scuba diving. (Fig. 2d and e) with regular villar protrusions (Fig. 2f). Due to the The Iceland scallops were sampled from a population suffering thick wall, its inner structure is not clearly seen in wet mounts. extensive natural mortality leading to a collapse in the stock index Zoites, i.e. sporozoites and/or merozoites, are abundant in all (Anonymous, 2003). An examination of the queen scallops from muscular tissue of moderately and heavily infected individuals. Faroese waters was requested by a company in the Faroese Scallop The adductor muscle is generally most heavily infected. Live zoites fishery industry due to poor condition of the adductor muscle. No measure 17.5 ± 2.0 Â 6.5 ± 1.5 lm(n = 100), the size range most data on natural mortality of the scallop population off the Faroe Is- frequently encountered being 18–19 Â 6.5–7.5 lm. They are land was available. Macroscopic signs of disease were apparent in slightly curved with a distinct and large nucleus (Fig. 3a and b). adductor muscles of both Iceland scallops and queen scallops from the Faroe Islands. The muscles were small compared to shell size, 3.2. Cyst morphology had a brownish appearance, were less compact than normal and with a high fluid content. The scallops from Scottish waters were Histological examination showed numerous cysts intracellular sampled from an apparently healthy population experiencing no in the adductor muscle of infected individuals, presenting different abnormal mortality or signs of disease. developmental stages of the parasite. These include cysts with uniformly granular cytoplasm, cyst containing variable numbers of 2.2. Microscopic observations nuclei and zoite-forming cysts, apparently representing merogony, gametogony and sporogony. Syzygy, the pairing of two gametes Shells were dissected and all major organs (adductor muscle, prior to fertilization, was absent. Suggested developmental forms kidney, mantle, gonads, palps, gills, heart, digestive diverticula, for merogony, gametogony and sporogony are presented in Figs. 4 stomach, intestine) examined microscopically for parasites using and 5. Á. Kristmundsson et al. / Journal of Invertebrate Pathology 108 (2011) 147–155 149

Fig. 1. Sampling sites of scallops from Iceland, Scotland and the Faroe Islands.

3.2.1. Merogony found in great numbers, contain up to several hundred nuclei Apparently, the ﬁrst sign of merogony involves a formation of (Fig. 5d and e). With further development, these nuclei elongate cysts with few slightly elongated and peripherally arranged nuclei and a cytoplasm becomes evident (Fig. 5f). Mature oocysts, (Fig. 4a). With further development, these nuclei divide and be- 200 ± 80 Â 75 ± 25 lm(n = 20), enclose numerous regularly ar- come uniformly arranged inside the cyst (Fig. 4b). Subsequently, ranged sporozoites, 12–13 lm in length and 3–4 lm in width a series of nuclei cleavage occurs forming cysts, 75 ± 5 lm (diame- (n = 20), with a clear and distinct cytoplasm (Fig. 5g and h). ter), containing slightly elongated nuclei without obvious cyto- In some cases, developing cysts could be identiﬁed as belonging plasm, forming several elliptical forms within the cyst (Fig. 4c). to merogony (Fig. 4), gametogony or sporogony (Fig. 5), while in Mature meronts, 300 ± 25 Â 100 ± 20 lm(n = 20), contain merozo- other cases, their situation in the life cycle is more speculative. ites with obvious cytoplasm arranged in a rosette like fashion around residual bodies (Fig. 4d and e). 3.5. Characterization of zoites

3.2.2. Gametogony In histological sections, zoites were found in all muscular tis- Macrogamont, 330 ± 50 Â 75 ± 25 lm(n = 20), are the most sues. In the adductor muscle they were found in large clusters common type of cysts observed. They are elongated, with a granu- (Fig. 6a and b) in necrotic areas where they infected individual lar cytoplasm, commonly with pointed ends and enclosed with a muscle cells (Fig. 6c). They were also common in the intercellular thin membrane (Fig. 5a). Presumed microgamonts, 180 Â 100 ± space of various organs, but also inside haemocytes, most often 10 lm(n = 5), contain numerous curved and slender microga- one in each haemocyte, but in some cases up to four (Fig. 6d–f). metes, 13–14 Â 2.5 lm(n = 20), irregularly distributed inside the Zoites were not seen in the alimentary canal or its epithelium. cysts. The microgametes differ from zoites by their apparent lack Two zoite size groups were observed. The larger ones (14– of cytoplasm (Fig. 5b). 15 Â 7–8 lm) were most common, but the smaller ones (10– 12 Â 3–4 lm) were only occasionally seen. The smaller zoites ap- 3.2.3. Sporogony peared to have a more deﬁnite curled form with a nucleus covering Fertilization is detected by the formation of cysts with a spindle most of it and cytoplasm only at each end (Fig. 6g). like apparatus representing a zygote undergoing its ﬁrst nuclear Ultrastructurally, the larger zoites contained all major struc- division (Fig. 5c). The onset of oocyst formation seems to involve tures characterizing apicomplexan zoites (Fig. 7a–i). A large and numerous nuclei divisions resulting in cysts containing a variable round nucleus is located in the posterior half of the parasite occu- number of round uniformly spread nuclei. These cysts, which are pying almost the whole width of the cell and almost half of its 150 Á. Kristmundsson et al. / Journal of Invertebrate Pathology 108 (2011) 147–155

Fig. 2. Live specimens of cysts from adductor muscle. Slender cyst with granular cytoplasm (a). Cyst with granular cytoplasm and a visible fertilization spindle (b). Slender cyst with pointed ends containing numerous round spheres or nuclei (inserted picture) (c). Cyst where a thickening of the cyst wall becomes evident (d). Thick walled cyst with villar protrusions on the wall (e and f). Scale bar (a–c) = 25 lm; (inserted picture and f) = 5 lm.

the apical complex to near the anterior surface of the nucleus, but were occasionally posterior to the nucleus. Rhoptries were clearly seen but were not serially sectioned and counted (Fig. 7b,c and g). The Golgi cisternae were observed near the anterior surface of the nucleus (Fig. 7h) and the endoplasmic reticulum between the nucleus and the apical complex (Fig. 7e). Thick-walled structures were detected in the anterior part of the cell, which could possibly be apicoplasts (Fig. 7b). Mitochondria were seen in many sections at various placements in the cell and seemed to occupy a large space in the cytoplasm (Fig. 7a and i). Amylopectin granules were found in large numbers and almost exclusively in the anterior part (Fig. 7a and h).

4. Discussion

Only few papers have been published on apicomplexans infecting scallops and only two species have been identified to a species level, i.e. P. pectinis from Pecten maximus (Léger and Duboscq, Fig. 3. Cluster of free zoites (a) and a single zoite at high magnification (b) Live 1917) and M. islandica from Iceland scallop (Kristmundsson et al., specimens. Scale bar: (a) = 20 lm; (b) = 5 lm. 2011). Other reports of apicomplexans in scallops are from A. irradians, such as P. pectinis-like apicomplexan (Karlsson, 1991), Pseud- oklossia sp. (Cawthorn et al., 1992), and few other unnamed length (Fig. 7a). The cell boundary, the pellicle, consists of an outer examples (Leibovitz et al., 1984; Whyte et al., 1994). unit membrane and an inner layer, which is composed of two unit Apart from M. islandica, described by Kristmundsson et al. membranes closely attached to another. The outer unit membrane (2011) from the same material as in the present paper, the only re- and the inner membranous layer are separated by an intermediate port on parasites of Iceland scallop is that of Giguere et al. (1995), osmiophobic space (Fig. 7f). Approximately 80–85 sub-pellicular who found Iceland scallops and sea scallops Placopecten magellan- microtubules extended from the anterior margin of the nucleus icus on the lower shore of Quebec infected with unidentified and to the outermost front of the cell (Fig. 7g). The conoid was de- turbellarians and ciliates as well as rickettsia-like organisms. tected but not the polar rings (Fig. 7e). Micronemes spanned from Regarding the queen scallop, the only reported parasite is a Á. Kristmundsson et al. / Journal of Invertebrate Pathology 108 (2011) 147–155 151

Fig. 4. Meront development. Giemsa stained histological sections showing: Formation of multiple, slightly elongated, nuclei at the periphery signalling the onset of meront formation (a). Further development; nuclei multiplication (b and c). Mature meront with merozoites arranged in a rosette fashion around a residual body (d). Rosette arranged merozoites (e). (a and b) = longitudinal sections, (c–e) transverse sections. Scale bar: a–d = 20 lm; e = 5 lm. microsporidian infecting the connective tissue of the digestive forms whose position within the developmental cycle cannot be gland (Lohrmann et al., 2000). The only parasite described from definitely ascertained (Fig. 5b,d,e). Pecten maximus is the aforementioned species P. pectinis (Léger The presence of both sexual and asexual stages in individuals of and Duboscq, 1917). all three scallop species examined in the present study suggests a The apicomplexan parasite described in this paper was found in monoxenous life cycle, whereby a direct horizontal transmission three scallop species of three different, but related genera, which occurs between individuals with the possibility of autoinfection. indicates a relatively low host specificity of this parasite. The host All stages, i.e. zoites and cysts, of the parasite are present inside specificity of apicomplexans infecting bivalves is poorly known, adductor muscle cells. Zoites were also present inside muscle cells but those infecting fish differ considerably in their host specificity of other organs, inside haemocytes and widely distributed in the (Molnár, 1995). The study confirms the presence of this parasite off extracellular space of the host. No zoites were detected in the intes- the coast of Iceland, Scotland and the Faroe Islands. Infections were tinal lumen or intestinal mucosa. The route of entry of the parasite, detected in both queen- and king scallop off the Scottish coast de- its complete development within the host and the release of the spite only 10 shells being examined of each species. Although there infective stages is not known at present. Whether this is through ac- are no reports of this parasite from the continental coast of Europe tive filter feeding of the host, subsequent parasite penetration and the east coast of Canada, our results might indicate that this through the gastrointestinal mucosa, spread with haemocytes or parasite is common among scallops at least in the eastern part of via hemolymph, and release of the infective stage with faeces the North Atlantic. and/or decomposing dead host, remains speculative. Infection trials The authors feel confident that all life stages are present in in- with serial sampling would be a feasible approach to analyze the fected shells. The merogonic stages, presented in Fig. 4a–e, resem- successive entry and development of the parasite. ble comparable life stages reported by e.g. Desser and Bower (1997, Presently, the systematic position of this new species within the Fig. 2) and Lindsay et al. (2000, Figs. 1i and 2a and b). Similarly, the phylum Apicomplexa cannot be determined. It is morphologically presence of macrogamonts and a fertilization spindle (Fig. 5a and different from all other apicomplexan species previously described c) confirms the gametogony. Microgamonts were not observed from bivalves and apparently the only one infecting muscle cells. with certainty. An early development of microgamont resembles The gamonts and oocysts of this parasite are elongated and much those of young meront (Fig. 4a) (see Morado et al., 1984, Fig. 13). larger than observed in other known species infecting bivalves. The cyst in Fig. 5b, which we speculate might be microgamonts, The huge amount of free sporozoites inside each oocyst (hundreds) contains many curved elongated bodies, which, because of no and the lack of sporocysts also differ significantly from previously apparent cytoplasm, differ from zoite-forming cysts (Fig. 4d,e and described species. Although the sporozoites are crescent shaped, a 5a,h). Apart from these, there are various other developmental common morphology of apicomplexan zoites, they do not 152 Á. Kristmundsson et al. / Journal of Invertebrate Pathology 108 (2011) 147–155

Fig. 5. Giemsa stained histological section showing: Macrogamonts with granular cytoplasm closely packed in muscular tissue (a). Numerous presumable microgametes emerging from a mature microgamont (b). Fertilization spindle becoming visible whereby the chromatids migrate to the periphery – indication of ﬁrst nuclear division of the zygote (c). Developing oocyst tightly packed with numerous small spherical nuclei derived from series of cleavage (d and e). Oocyst with multiple nuclei which are beginning to elongate and a cytoplasm becoming evident (arrows) (f). Mature oocyst containing numerous sporozoites (g). Mature sporozoites inside an oocyst (h). (a and f) = longitudinal sections; (g and h) = transverse section. Scale bars: (a–g) = 25 lm;h=5lm.

resemble sporozoites of other known species, i.e. Pseudoklossia sp. various muscles of herbivores and omnivores (Rommel et al., and Margolisiella sp., which are common apicomplexan species 2000). There is, however, also a big difference as the developmen- found in bivalves (see Kristmundsson et al., 2011, Table 1). They tal forms of Sarcocystis species found in the muscles of herbivores are larger and furthermore, the nucleus is relatively bigger, occu- and omnivores are only asexual stages, but those found in our pying a signiﬁcant part of the cytoplasm. Of other apicomplexans material are both sexual and asexual. Furthermore, the oocysts in reported from bivalves, the zoites found in dredge oysters Ostrea the present study contain no sporocysts but numerous free spor- chilensis around New Zealand (Hine, 2002) show the most resem- ozoites. Sarcocystis spp., however, have oocysts with four sporozo- blance with the zoites of those found in the present study, such ites in each sporocyst (Rommel et al., 2000). as the great number (>80) of sub-pellicular microtubules in both Possible negative health effects on the Iceland scallop popula- these species. However, Hine (2002) merely detected zoites and tion caused by this undeﬁned apicomplexan parasite, described did not observe any sign of other developmental forms. Regarding in this paper along with M. islandica (Kristmundsson et al., 2011), the cysts observed in our material, which were exclusively found in also detected in the Icelandic scallop, cannot be ruled out. Long muscular tissue, they apparently show considerable similarity with term studies, which are presently in process, will shed light on this Sarcocystis spp., an apicomplexan species commonly found in issue. These studies will be discussed in a separate paper. Á. Kristmundsson et al. / Journal of Invertebrate Pathology 108 (2011) 147–155 153

Fig. 6. Histological section of zoite clusters in the adductor muscle (a and b). Numerous zoites in a necrotic area of adductor muscle. Host nuclei (thin arrow). Zoites within degenerating muscle cells (broad arrow). Zoites in necrotic muscle cell debris (arrowhead) (c). A single zoite inside haemocytes (d). Multiple zoites inside haemocytes (May– Grünwald–Giemsa stained imprints). Note host cell nucleus (arrow) (e and f). Cluster of smaller size zoites (g). Scale bars: (a and b) = 100 lm; (c and g) = 5 lm. 154 Á. Kristmundsson et al. / Journal of Invertebrate Pathology 108 (2011) 147–155

Fig. 7. Longitudinal section through a sporozoite in muscle showing many of the major components of a zoite (a). Section through the anterior part of a sporozoite showing rhoptries, micronemes, amylopectin granules and a thick walled structure (arrowhead), possibly apicoplasts (b). Anterior part of a sporozoite showing various cell structures, i.e. endoplasmic reticulum (arrowhead) (c). A large lipid vacuole (arrowhead) (d). Section through edge of the conoid (e). The pellicle, with an outer membrane (OM) and an inner membrane (IM) and middle membrane (MM). Between IM and OM is an osmiophobic space (f). Transverse section through the anterior part of the sporozoite showing approximately 80–85 sub-pellicular microtubules (Mt) as well as micronemes and rhoptries (R) (g). The Golgi apparatus (G) along with nucleus (N), micronemes (Mn) and amylopectin granules (A) (h). Mitochondria (arrow) (i). Scale bars: a = 2 lm, b = 1 lm, c = 0.4 lm, d = 1 lm, e = 0.2 lm, f = 0.05 lm, g = 0.3 lm, h = 0.4 lm, I = 0.5. Á. Kristmundsson et al. / Journal of Invertebrate Pathology 108 (2011) 147–155 155

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