DlSEASES OF AQUATIC ORGANISMS Vol. 22: 115-128.1995 1 Published June 15 Dis aquat Org

A -like infection of the Norway norvegicus: observations on pathology and progression of infection

R. H. Field, P. L. Appleton

Division of Environmentaland EvolutionaryBiology, Graham Kerr Building, Universityof Glasgow, Glasgow G12 800. Scotland, UK

ABSTRACT: The discovery of a Hematodinium-like dinoflagellateinfecting has led to a pathological investigation intothe effects on the host and the apparent progression of the dis- ease syndrome The parasite is systemic, invadingthe haemocoel and the connective tissues of most organs via the haemal spaces.An increase in the combined number of parasites and haemocytes inthe haemolymph was due to an increase in the relative proportion of and suggested a reduction in numbers of haernocytes. These parameters did not correlate d~rectlywith severity Comparison of the level to which tissues were invaded and the relationship between haemocyte and parasite numbers in the haemolymph suggests that at least some organs maybe invaded very early in infection, if not before parasites enter the Ohaemolymph.There was evidence of host cellular defence reactions, in the form of haemocyte encapsulationsin the gills and heart, and phagocytosis of dino- flagellates by the fixed phagocytes of the hepatopancreas.

KEY WORDS. Nephropsnorvegicl~s.Hematodini~~rn spp.. Parasitic dinoflagellate- Cl-ustacea- Pathology

INTRODUCTION Field (1992) and Field et al. (1992) have described preliminary investigations into the pathology and epi- In recent years, individuals of the commercially im- zootiology of Hematodinium-like infection of Nephrops portant Nephrops norvegicus (L.),caught on norvegicus. Following several years of high infection grounds around the west coast of Scotland have been prevalences, catches of N. norvegicus, and in par- observed to be infected by a parasitic dinoflagellate of ticular infected individuals, have been poor. In view of the botanical order (Field et al. 1992). In par- the commercial importance of N. norvegicus, and the ticular, this organism resembles Hematodinium spp., implication from previous work that infection may be first reported in and Liocarcinus depu- leading to significant mortalities, further research was rator by Chatton & Poisson (1931), and other similar instigated. Despite the paucity of infected material, types of parasitic dinoflagellate reported recently in reported here are further investigations into the an increasing number of over a wide pathology of infection and the course of the disease, geographic range [ sapidus in the eastern undertaken in conjunction with investigations into the Atlantic (Newman & Johnson 1975), several species of in vitro culture of the organism and its life cycle benthic amphipod (Johnson 1986), , (Vickerman et al. 1993). C. borealis and Ovalipes ocellatus (Maclean & Ruddell Diagnosis of infection and determination of severity 1978) in the western Atlantic, Necora puber and Cancer were made by pleopod examination as described by pagurus on the northern coast of and the west Field et al. (1992) (see Fig. 1).This technique estimates coast of Scotland (Latrouite et al. 1988, Wilhelm & Boulo the relative degree of parasite aggregation beneath 1988), bairdi and C. opilio in , USA the cuticle of the pleopods (and presumably through- (Meyers et al. 1987, 1990, Meyers 1990, 1990, Eaton et al. out the haemocoel), and makes the premise that as 1991, Love et al. 1993), and Portunus pelagicus (Shields infection progresses the number of parasitic cells, and 1992) and Trapezia sp. (Hudson et al. 1992) in Australia]. hence the thickness of the aggregation layer in the

0 Inter-Research1995 Dis aquat Org 22: 115-128, 1995

pleopod, increases. Field et al. (1992) conducted some Since it was not possible to reliably distinguish be- counts of haemocytes and parasites together to investi- tween parasites and haemocytes in fresh haemolymph, gate this hypothesis and to assess its potential as a field haemolymph smears were made from an additional 89 diagnostic method for infection and severity. We have infected and uninfected (again after pleopod now conducted combined paras~te and haemocyte examination) as follows. Haemolymph was withdrawn counts in fresh haemolymph and determined the rela- directly from the haemocoel into a disposable syringe tive proportions of haemocytes and parasites in stained containing chilled 5% formalin in sea water (33%o),in haemolymph smears, from both apparently uninfected a ratio of approximately 1:1, from the base of a fifth and infected lobsters, to assess the accuracy of this pereiopod. After 5 to 10 min fixation, samples were technique in disease and severity diagnosis. Since gently agitated to re-suspend cells and a small drop of individuals examined in the pathological investigation each was smeared on a clean glass slide. Smears were were assigned an infection status by this method, it is allowed to dry thoroughly, post-fixed in methanol and now possible to relate haemolymph parasite numbers stained with a 0.2% w/v solution of Leishman's stain and tissue changes. In this way it has been possible to (BDH Chemicals Ltd, Poole, England). Each smear was determine whether increasing parasite numbers in examined at x400 magnification, and the relative the ildellluiy~llpll d~Luu1iy~'efieii ihc pi-oyression of iiti~krsof host haernocy:es 2nd dinof!agc!!a:cs from a infection, or are merely the result of variation between total of 200 cells were counted. From these counts the individual infections. number of parasites was expressed as a percentage of the total of both haemocytes and dinoflagellates to- gether in the haemolymph of each lobster. MATERIALS AND METHODS Light and electron microscopy. Major tissues and organs were dissected from a total of 29 Nephrops Experimental lobsters. Nephrops norvegicus were norvegicus, previously staged by pleopod examina- caught by trawling on grounds around the Isle of Cum- tion. Of these, 9 were apparently uninfected, 5 showed brae, Clyde Sea area, Scotland, and transported to the stage I infection, 5 stage 11, 5 stage I11 and 5 stage IV. Zoology Department, University of Glasgow, where The organs and tissues removed were hepatopancreas, they were maintained in well-aerated sea water for up antenna1 gland, midgut, abdominal muscle, haemopoi- to 5 d. The aquarium water temperature ranged etic tissue, heart, gills, and, in some cases, brain and between 10 and 13°C and the salinity between 33 and eye/eyestalk. Prior to dissection, lobsters were narco- 34 %o. Lobsters were fed ad libitum on squid and mus- tised in ice for about 1 h. Immediately upon removal, sel flesh. All lobsters were in intermoult as defined by tissue samples for histopathology were fixed In Helly's the moult staging technique of Aiken (1980).Diagnosis mercuric chloride fixative (Johnson 1980) and embed- of infection was made by the pleopod assessment ded in paraffin wax. Thick sections (6 pm) were treated method of Field et al. (1992) (Fig. 1). with Lugol's iodine solution to remove mercury, and Counts of haemocytes and dinoflagellate parasites. stained with haematoxylin and eosin (H&E). After assessment of infection severity by pleopod Tissues removed for electron microscopy were fixed examination, counts were performed of numbers, in in 1 % glutaraldehyde, 2 % paraformaldehyde in 0.1 M fresh haemolymph, of haemocytes and parasites to- phosphate buffer, pH 7.4 with 2% sucrose and 1.5% gether in 28 infected and uninfected Nephrops sodium chloride for 2 h at room temperature. Speci- norvegicus, by the method given in Field et al. (1992), mens were then rinsed in 0.1 M phosphate buffer with based on that of Stewart et al. (1967). For statistical 4 % sucrose, then post-fixed in 1 % osmium tetroxide in analysis, these counts were combined with those phosphate buffer for 1 h. Specimens were washed in reported in Field et al. (1992). since the protocols several changes of distilled water and block stained in employed were identical. 0.5% aqueous uranyl acetate for 1 h. After dehydrating

norvegicus. , , '. Fig. 1. Nephrops Light

dinoflagellate-infected lobsters. & The density of the layer of aggre- .I gated haemocytes and parasites beneath the cuticle indicates the f severity of ~nfectionon an arbitrary p scale from I to IV 0: uninfected; I:slight infection; IV heavy infec- .- A , , lion. Scale bar = 0.5 mm Field & Appleton: A Hernatodiniurn-like infection of Nephrops norvegicus

through an ethanol series specimens were embedded Table 2. Nephrops norveglcus. Variation in percentage dino- in Spurr resin (Spurr 1969), using propylene oxide as a flagellates in the haemolymph in relation to severity of in- transitional solvent. fection as determined by pleopod examination. 'Significantly higher than the previous stage, p < 0.005 (l-way ANOVA) Thick sections (1 pm) for light microscopy were stained with 1 % toluidine blue. Suitable areas of tissue Pleopod No. of % Dinoflagellates were selected and thin sections were cut and mounted infection stage lobsters Mean on uncoated 300 mesh copper/palladium grids and stained with uranyl acetate (methanolic) and lead citrate. Thin sections were examined in a Zeiss 902 trans- mission electron microscope operating at 80 kV.

RESULTS Pathology and electron microscopy Numbers of haemocytes and dinoflagellate parasites in the haemolymph The initial report of this disease syndrome (Field et al. 1992) recognised 2 dinoflagellate cell forms in Table 1 shotvs the results of the counts of haemo- Nephrops norvegicus, uninucleate and multinucle- cytes and parasites together performed on haemo- ate/plasmodial cells in the haemolymph and vermi- lymph from staged Nephrops norvegicus. These results form cells attached in the tissues. Since publication of indicate that there was an increase in the total number this work, we have made much progress in the in vitro of haemocytes and dinoflagellates in the haemolymph culture and study of the life cycle of this organism, of infected N. norvegicus. This increase was significant revealing a more complex range of developmental only in individuals staged at 111 and IV. The in- forms (Vickerman et al. 1993) comprising both fila- crease was due to the higher proportion of dinoflagel- mentous and network syncytia, uninucleate and multi- lates within the haemolymph (Table 2). Stage I indi- nucleate/plasmodial cells, and 2 types of biflagellate viduals showed a slight increase above stage 0 motile dinospores. lobsters, but stage 11, 111 and IV individuals showed a During the current study we identified 4 separate further significant rise above this. Although all those parasite morphologies within Nephrops norvegicus. lobsters placed in stage 0 were diagnosed as unin- Filamentous syncytia (see Fig. 3), sometimes radiating fected by pleopod examination a small number were from a central mass, were attached to host tissue misdiagnosed. Three apparently uninfected lobsters bounding haemal spaces and lumina in several organs. were found to have dinoflagellate parasites in their These were , individual filaments con- haemolymph when smears were examined. This dis- taining up to 5 nuclei. Filament nuclei contained the crepancy accounts for the above zero percentage of prominent condensed chromosomes typical of the dinoflagellate parasites in group 0. Three uninfected dinoflagellate (see Figs. 3 & 13).This para- lobsters were also misdiagnosed as infected stage I, site form corresponds to the vermiform type described since their smears contained no detectable parasites. by Field et al. (1992), and resembles the filamentous syncytia observed in vitro by Vickerman et al. (1993). A separate network syncytial form was observed, ramify- Table 1. Nephrops norvegicus. Variation of the combined count of haemocytes and parasites in the haemolymph (com- ing between muscle fibres in abdominal muscle (see bined count) in relation to severity of infection as determined Fig. 12) and heart, which may correspond to the net- by pleopod examination. 'Significantly higher than the previ- work form described by Vickerman et al. (1993). ous stage, p < 0.005 (l-way ANOVA). (This table incorporates data from Field et al. 1992) Hepatopancreas Pleopod No, of Combined count infection stage lobsters (X 104 mm-7 Mean SD In infected individuals, the spacing between hepatopancreatic tubules was much enlarged com- pared with that of uninfected lobsters (Fig. 2). This was sometimes seen as an artefact of dissection and fixation, even in uninfected lobsters, but tubule sepa- ration increased with infection severity. The hepato- pancreas of infected individuals was difficult to 118 Dls aquat Org 22: 115-128, 1995

Fig. 2. Nephrops norvegicus. Light micrographs showing the enlargement of spacing between hepatopancreatic tubules in dinoflagellate-infected lobsters. (a) Hepatopancreas of uninfected individual. Toluidine blue. (b) Hepatopancreas of stage IV infected individual, showing increased spacing between the tubules, and the concurrent presence of many uninucleate parasites. H: haemal sinus; L: lumen of tubule; T: tubule epithelium; A: hepatopancreatic arteriole. H&E. Scale bars = 20 pm

remove because of its almost liquid state. Enlarged spaces contained normal dinoflagellates and parasite spaces within the hepatopancreatic ha.ema1 sinus debris (Fig. 6). were found to contain large numbers of uninucleate (and occasionally binucleate) parasites, but haemo- cytes were rarely seen. Occasionally, filamentous syncytia were seen in heavily infected lobsters, attached to the outside of tubules (Fig. 3). Tubule damage in the heavier infections was more profound, involving not only vacuolation of epithelial cells (Figs. 2 & 3) but the breach of tubule walls, as indi- cated by the presence of uninucleate parasites within the lumlna of tubules in both stage 111 and stage IV lobsters (Fig. 4). Activity of the fixed phagocytes surrounding the hepatic arterioles was observed in infected lobsters of all stages, as indicated by the hypertrophied state of these cells (Fig. 5) and the presence of an interrupted layer (Johnson 1987) (Fig. 6). Evidence was seen of retention of parasites in the extracellular space between the interrupted layer and the fixed phago- cytes. The majority of dinoflagellates associated with fixed phagocytes were retained in this manner. Phago- Fig. 3. Nephrops norvegicus. Light micrograph showing fila- cytosis was also observed, but less often, evidenced mentous dinoflagellate syncytium attached to the outer wall of a hepatopancreatic tubule of a stage 111 infected lobster. by the form of phagosomes containing degenerative H: haemal sinus of hepatopancreas; F. parasite syncytium; parasite material (Fig. 6). Fixed phagocytes containing T. epithelia1 cell of hepatopancreatic tubule. Tolu~dineblue. such phagosomes were often necrotic. Extracellular Scale bar = 15 pm Field & Appleton: A Hematod~n~~~nl-likeinfection of Nephrops norvegicus 119

Fig. 4 Nephrops norvegicus. Light micrograph showing the presence of a dinoflagellate with~nthe lumen of a hepatopancreatic tubule of a stage 1V infected individual. Note also the presence of flagellate spores in the haemal spacessurrounding the tubules, and a 'secretory packet' (Johnson 1980) in the tubule lumen, possibly a shed 'B'-cell. T: tubule epithelium; b: brush border of ep~thelialcells; Hs: haemal sinus of hepatopancreas;X: 'secretory packet'; P- biflagellate spores of the parasite; arrow: dino- flagellate within tubule lumen Toluidine blue. Scale bar = 20 pm

Fig. 5. Nephrops norvegicus. Light micrograph showing fixed phagocytes surroundingan hepatic arteriolein a stage IV infected lobster. Note the dinoflagellates associated with2 fixed phagocytes (arrows). Ph: parasites inhepatopancreatic haemalsinus; T: hepatopancreatic tubule;Fp: fixed phagocytes; E: endothellum of arteriole; P: parasites within lumen of arteriole. Toluidine blue. Scale bar = 10 urn 120 DISaquat Org 22: 115-128, 1995

Fig. 6. Nephrops norvegicus. Transmission electron micrograph showing detail of a dinoflagellate associated with an hepatopan- creatic fixed phagocyte of a stage IV infected lobster. Note the vesicular cytoplasm and the interrupted layer surrounding the fixed phagocyte, and the granular nature of the cytoplasm of the parasite. I: interrupted layer; Hs: haemal sinus of hepato- pancreas; Dn: nucleus of dinoflagellate; DC: cytoplasm of dinoflagellate; Pn: nucleus of adjacent phagocyte; Pc: cytoplasm of fixed phagocyte; Pp: phagosomes. Scale bar = 2.5 pm

Antenna1 gland was unchanged, except for the presence of dinoflagel- lates in the narrow haemal spaces surrounding it. The The structure of the antennal glands of infected labyrinthal epithelium showed some vacuolation, and Nephrops norvegicus was largely unaffected in all but parasites were present in the lumen of the labyrinth the most heavily lnfected individuals, despite the pres- itself (Fig. 7), indicating breach of the epithelium. The ence of many dinoflagellates within the haemal spaces labyrinthal epithelium of Infected and uninfected lob- and connective tlssue of the labyrinth and coelomosac, sters showed a high degree of secretory activity when and branches of the antennary artery. As in the hepa- compared to that of the grass Palaernonetes topancreas, the majority of the parasites were uninu- pugio, which only showed such activity levels after cleate, but filamentous multinucleate stages were also exposure to biocides (Doughtie & Rao 1983). This activ- present, especially attached to the basal side of the ity was evident even in the antennal gland of heavily labyrinthal epithelium (Fig. 7). There was an increase infected stage IV Nephrops norvegicus, with the api- in numbers of attached parasites with increasing infec- cal brush border of cells often showing extruded tion stage, though even light infections of stage I granular bodies (Fig. 8), similar to those seen in the showed well-established groups of attached parasite antennal gland of Astacus sp. (Parry 1960) and P pugio syncytia in the labyrinth. The coelomosac epithelium (Doughtie & Rao 1983). Field & Appleton: A Hen~atodjnjum-llkeinfection of Nephrops norvegjcus 121

Fig. 7. Nephrops norveyicus. Light micrograph of the labyrinthal epithelium of the ] gland of a stage III infected indi- vidual, showing attachment of filamentous parasite syncytia within the haemal spaces and the presence of dinoflagellates within the lumen of the labyrinth (arrows). Le: labyrinthal epithelium; L labyrinthal lumen; F: filamentous parasite syncytia H&E Scale bar = 20 um

Fig. 8. Nephrops norvegicus.Light micrograph showing detail of the labyrinthal epithelium of a stage IV infected lobster, and the presence of biflagellate spores of the parasite within the haemal spaces. L: labyrinthal lumen; Hs: haemal spaces of labyrinth; P: biflagellate spores of the parasite; E: epithelia1 cells; G. granular bodies. Toluidine blue. Scale bar = 5 pm Dis aquat Org 22: 115-128, 1995

Midgut is as yet uncertain, though they resemble granulocytes. Evidence of any alteration of the midgut epithelium Tissue of the midgut exhibited some of the most was scant, with both villi and epithelial corrugations marked changes as a result of infection by the parasite. still present in stage IV infected lobsters. Lysis and The midgut wall of lobsters from all pleopod infection vacuolation of epithelial cells by infection was not stages showed large-scale infiltration by the dino- readily distinguished from that caused by prefixation flagellates. The connective tissue and muscles of artefact. There was also no evidence of parasite inva- the outer midgut wall had been almost completely sion of the epithelium or any tissues on the lumen side replaced by parasites, even in stage I lobsters. The of the basement membrane. A concurrent gregarine muscle tissue still present in stage I and I1 individuals infection, tentatively identified as an eugregarine was (Fig. 9) appeared fragmentary and reduced when com- found in the midgut lumen of one of the stage IV pared with that of uninfected lobsters (Fig. 10). The lobsters (Fig. 11). haemal spaces were enlarged and partially occluded by large numbers of attached filamentous parasite syncytia. Few haemocytes were observed in these Abdominal muscle naemai spaces in infected iobsters, especiaiiy in stage 111 and IV individuals. There was limited evidence of a The abdominal muscle of healthy Nephrops norvegi- host cellular defence reaction within the gut wall of cus displayed histologic features typical of stage I lobsters, where small numbers of haemocytes homogeneous fast phasic skeletal muscle. The fibres were aggregated around dinoflagellates. had a short sarcomere length (2 to 4 pm) and lightly Parasites in the midgut tvall were predominantly fila- staining nuclei, with peripherally dense chromatin. In mentous syncytia, especially in light infections, where general, these features remained unchanged in the uninucleate forms were less apparent. Histologically, the abdominal deep muscles of dinoflagellate-infected basement membrane and the layers of granular cells un- individuals. In wax-embedded sections parasite inva- derlying it were unaffected by ~nfection.The frequency sion of muscle fibres was not apparent, even though of occurrence of these granular cells did not seem to cor- free uninucleate parasites were frequently encoun- relate with infection. The identity of these granular cells tered in interstices between muscle fibres in stage I

Fig. 9. Nephrops norvegicus. Light micrograph of the midgut wall of a stage I1 infected individual showing both attached filamen- tous syncytia and possibly un~nucleateparasites. P: possibly uninucleate parasites; F: filamentous syncytia; M: circular muscle layer of midgut tvall; b: basement membrane; E: epithelium; L: lumen. Toluldine blue. Scale bar = 20 pm Field & Appleton A t-lematodin~um-11keinfection of Nephrops norveglcus 123

Fig 10 Nephrops norveglcus L~ghtm~crograph of the inidgut wall of an unlnfected lobster showing the connective tlssue and inuscles of the outer wall Ct connective tlssue, L longitudinal inuscle C clrcular muscle B basement membrane, E ep~thelium, Lu lumen H&E Scale ba~= 20 p111

Fig. 11. Nephropsnorvegicus. Light inicrograph of the midgut wall of a stage 111lnfected lobster showing a simultaneous infection by dinoflagellates and an unident~fiedgreganne. P- dinoflagellates within the midgut wall; M: muscle layer; B: basement membrane; E: midgut epithelium; L midgut lumen; G: gregarines w~thinmidgut lumen. H&E.Scale bar = 20 pm Dis aquat Org 22: 115-128, 1995

lobsters. The interstitial connective tissues of skeletal In contrast, the haemopoietic tissue of dinoflagellate- muscle remained intact. In stage I11 and IV lobsters, infected Nephrops norvegicus showed a dramatic however, network-like parasite syncytia were present increase in size, evident even to the naked eye during within skeletal muscle interstices (Fig. 12), and con- dissection of stage I11 and IV lobsters. This was due, nective tissue was reduced. Furthermore, peripheral in part, to a much increased level of haemopoietic areas of some fibres were lysed, and uninucleate activity, with many nodes containing differentiating parasites were also present in the haemal spaces sur- cells, mitotic figures and what appeared to be stem rounding the fibres. Uninucleate and multinucleate cells. However, despite this vast increase in both the forms of the parasite, like those found in the haemo- number and activity of stem cells, very few newly dif- lymph, were often closely associated with the sarco- ferentiated haemocytes were visible in the haemal lemma of fibres (Fig. 13). spaces surrounding the haemopoietic nodes (Fig. 14). Instead these spaces were filled with many uninucle- ate and multinucleate dinoflagellates, even in lower Haemopoietic tissue level stage I and I1 infections. -. I ne haemopoietic tissue of I

Fig. 12. Nephrops norvegicus.Light micrograph of abdominal muscle of a stage I1infected individual showing network-like parasite syncytia within the muscle interstices. M: abdominal muscle fibres; N parasite nuclei; S: muscle nuclei; arrows: parasite syncytia within muscle interstices. H&€. Scale bar = 20 pm F~eld& Appleton: A Heniatodiniuni-like ~nfectionof Nephrops norvegicus

Fig. 13. Nephrops norvegicus. Transmission electron micrograph showing a dinoflagellate associated with the sarcolemmal membrane of an abdominal muscle fibre from a stage IV infected lobster. m: mitochondrion of parasite; T: trichocysts; Dn: dino- flagellate nucleus; L: lipid-like droplet; S- sarcolemma of abdominal n~usclefibre; M: muscle fibre; arrow: amphiesmal alveolus of dinoflagellate plasma membrane. Scale bar = 1 pm

Fig. 14. Nephrops norvegicus. Light micrograph showing apparently active haernopoietic tissue of a stage IV infected lobster, surrounded by many uninucleate and multinucleate dinoflagellates. N: haemopoietic node; S: probable stem cell; F: mitot~c figure; D: differentiating cells; arrow: host haemocyte. Toluidine blue. Scale bar = 20 pm Dis aquat Org 22: 115-128, 1995

Fig. 15. Nephrops norvegicus. Light micrograph of the myocardlum of the heart of a stage 11 infected individual, show~ngthe presence of both filamentous and network syncytia of parasites attached to myocardial muscle within the lumen. L: lumen of heart; M: myocardial muscle: F: filamentous dinoflagellate syncytia; S: network-like dinoflagellate syncytia; N: myocardial nuclei; Ct: connective tissue. H&E.Scale bar = 20 pm

cardial muscle in all stages (Fig. 15), similar to those seen in the narrower regions of the filaments. Free in abdominal muscle interstices. Some haemocyte haemocytes were rare, particularly in those lobsters with aggregations were seen in the lumen of the hearts of gills having large numbers of free parasites. stage I to 111 individuals. These aggregations were tightly packed and had nuclei of a karyolyhc appearance. These resembled haemocyte encapsulations observed in res- Other organs ponse to bacterial infections in Homarus amencanus (Johnson et al. 1981) and Callinectes sapidus (Johnson In brain and eyestalk tissues there were no overt 1976), and previously reported in Nephrops norvegicus signs of parasite infiltration or tissue change. Parasites gills (Field et al. 1992), and may represent a degree of were restricted to uninucleate forms in the haemal host response to parasites, although aggregations have sinuses and vessels of these tissues. not yet been confirmed as containing dinoflagellates. It should be noted that in some organs (particularly heart and hepatopancreas) the numbers of unattached parasites observed in haemal spaces and lumina may Gills be an under-representation of actual numbers, due to losses during processing for both light and electron The major effect on the gills was the occlusion of microscopy. haemal spaces by large numbers of dinoflagellates. This was more severe in higher infection stages, but still apparent in stage I and I1 lobsters. Attached parasite DISCUSSION syncytia were not observed in this tissue, but evidence of host reaction to infection was seen. Haemocyte aggre- The effects of dinoflagellate infection of Nephrops gations slmilar to those observed in the lumen of the norvegicus are typical of those reported for haemo- heart were seen in gLUfilaments (Field et al. 1992),some- coelic infections of decapods by both protistans and times apparently blocking them. Aggregations were of several groups. The general histologic fea- more frequent in the gills than the heart, but were only tures of tissue change of the digestive and excretory Field & Appleton: A Hematodinium -like infection of Nephrops norrregicus 127

organs and of interference with respiratory exchange, cytopoenia in dungeness infected with to haemopoiesis or metabolism (Field 1992) are char- consumption of haemocytes by the invading cells. acteristic of such haemocoelic infections caused by However, reduction in haemocyte numbers in F!perni- bacteria (Johnson 1976), dinoflagellates (Maclean ciosa infections was reported to be the result of host & Ruddell 1978, Meyers et al. 1987, Meyers 1990), cellular defence reactions, the posslble lysis of host Chlamydia-like organisms (Sparks et al. 1985) and cil- cells after phagocytosing parasite cells, and possible iates (Sparks et al. 1982). However, many protistan and disruption of haemopoietic tissue function (Johnson bacterial diseases, such as gaffkemia and the presently 1977). Similarly, in gaffkaemic lobsters, haemocyte reported dinoflagellate infection, may have notable aggregations are widely reported in response to the effects on the serum of the haemolymph, as well as on invading bacteria (Rittenburg et al. 1979, Johnson et the physiology and metabolism of their hosts. These al. 1981). It is, therefore, possible that in Nephrops aspects remain largely un-investigated. More informa- norvegicus infected with dinoflagellates, increased tion is available upon the histopathology of infected activity in the haemopoietic tissue and the presence of hosts, on haemocyte changes and responses and on haemocyte aggregations are indications of the initia- their progenitor, the haemopoietic tissue. tion of a host reaction to infection. The haemocytes The results presented here confirm that there is an may be removed from circulation by encapsulating increase in the combined numbel- of haemocytes and dinoflagellates, forming the aggregations observed. parasites in the haemolymph with infection, due to The increase in haemopoietic activity may be ex- increases in the proportion of dinoflagellates, and that plained, in part at least, by the time of year at which this increases with severity as determined by the pleo- infected individuals were available. The occurence of pod staging method. These increases did not correlate lobsters infected with the dinoflagellate is seasonal directly with the pleopod staging series; combined (Field 1992, Field et al. 1992), occurring in the spring haemocyte/parasite counts were not ralsed signifi- and early summer, a time when large-scale haemocyte cantly above the range of apparently uninfected levels production is known to occur in some decapods, e.g. until the pleopod assessment indicated stage III or C. sapidus (Johnson 1980). Even stage IV dinoflagel- stage IV infection. At this stage, dinoflagellate para- late-infected N. norvegicus retain apparently func- sites comprised over 70% of the combined haemocyte/ tional fixed phagocytes, but despite this and the dinoflagellate numbers. This also suggests a reduction increased haemopoietic activity, appear unable to con- in the number of haemocytes in the circulation, coinci- trol the parasites. The paucity of haemocytes may be dent with parasite proliferation. The staging technique explained by their sequestration into haemocyte cap- detects an increase in the severity of haernolymph/ sules and other defence reactions at an early stage haemocoel infection even in stage I lobsters. However, of infection. These reactions are apparently over- histopathological evidence suggests that dinoflagellate whelmed by the increasing parasite load. An alterna- infection is much more severe than indicated by tive view is that the host fails to respond due to some haemolymph infection alone, and that the pleopod action of the parasite. staging method is less sensitive than previously Another salient feature of the presence of so many in- believed. Most major organs and tissues were heavily vading parasites wlthin the haemocoel is the mechanical infiltrated with parasite stages In the stage I lobsters disruption they cause to blood circulation. Apart from the examined, raising the possibility that parasites in the hydrostatic effects of this overburden of circulating cells, haemolymph originate from earlier tissue forms. These clogging of blood vessels and sinuses may also occur. results indicate that the pleopod staging method is This will be particularly so in areas of restricted dia- relatively reliable as a field method for detecting the meter, such as the smaller capillaries and sinuses, and presence of dinoflagellates in the haemolymph, but the haemal spaces within organs. Restriction of blood may be less so as an indicator of absolute infection and flow is likely to be further exacerbated within areas the involvement of the tissues. where large numbers of filamentous parasite syncytia Haemocytopoenia has been noted as a sign of many are attached to host tissues. Clogging may also result haemocoelic infections of crustaceans caused by both from the formation of haemocyte aggregations as part of bacteria and . Disappearance of haemocytes a host response to infection, as seen in the gill filaments from circulation has been noted in gaffkemia of of Nephrops norvegjcus,and postulated by Rittenburg et Homarus americanus (Stewart et al. 1969, Johnson et al. (1979) to contribute to tissue hypoxia in gaffkaemic al. 1981), Paranophrys sp. infection of dungeness crabs lobsters. The aggregation of haemocytes, and the for- Cancer magister (Sparks et al. 1982), and mation of aceLlular haemolymph clots in blue crabs Calli- perniciosa infections (Johnson 1977) and bacterial nectes sapidus in response to bacterial infections, have infections (Johnson 1976) of blue crabs Callinectes also been implicated in impeding blood flow through the sapidus. Sparks et al. (1982) attribute the haemo- gills (Johnson 1976). Dis aquat Org 22. 115-128, 1995

As yet there is no irrefutable evidence that this con- dinoflagelle parasite: Hematodinium sp. Comm Meet int dition is always fatal to infected lobsters (Field et al. Coun Explor Sea CM-ICES 1988/K:32 1992), although the systemic nature of the infection Love DC, Rice SD, Moles DA, Eaton WD (1993) Seasonal prevalence and Intensity of R~tter dinoflagellate and its wide variety of effects upon the host suggest infection and host mortality in Alaskan Tanner crabs Chio- that severe debility and death probably result in many, noecetes bairdi from Auke Bay. Alaska, USA. Dis aquat if not all, cases Org 15:l-7 Maclean SA, Ruddell CL (1978) Three new crustacean hosts for the parasitic dinoflagellate Acknowledgements: This work was conducted with support (DlnoflageUata: Syndinidae). J Parasitol 64:158-160 partly from the Science and Engineenng and Research Meyers TR (1990) Disease of Crustacea Diseases caused by Council (CASE award to R.H.F.) and partly from the Ministry protistans and metazoans. In: Kinne 0 (ed) Diseases of of Agriculture, and Food. marine , Vol 3. Biologische Anstalt Helgoland, Hamburg, p 350-389 Meyers TR, Botelho C, Koeneman TM. Short S, lmamura K LITERATURE CITED (1990) Distribution of bitter crab dinoflagellate syndrome in southeast Alaskan tanner crabs . Aiken DE (1980) Moulting and growth. In. Cobb JS, Phillips Dis aquat Org 9:37-43 RF !eds! The bioloqy and manaqement of lobsters. Vol 1 Meyers TR. Koenamiin TM. Rotelho C: Short S (1987) Ritt~r Academic Press, New York, p 91-163 Crab Disease: a fatal dinoflagellate infection and market- Chatton E, Poisson R (1931) Sur 1' existence, dans le sang des ing problem for Alaskan tanner crabs Chionoecetes bairdi. crabs, de peridiniens parasites Hematodinium perezi n.g., Dis. aquat. 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Dis aquat Org 13:l-15 Sparks AK, Morado JF, Hawkes JW (1985) A systemic micro- Hudson DA, Hudson NB, Shields JD (1992) Infection of bial disease in the dungeness crab Cancer magister, Trapezia spp. (: Xanthidae) by Hematodinium caused by a Chlamydia-like organism J Invertebr Pathol sp. (Duboscquodinida: Syndinidae): a new family record 45~204-217 of infection. J Fish Dis 16:273-276 Spurr AR (1969) A low viscos~ty epoxy embedding resin for Johnson PT (1976) Bacterial infection in the blue crab. Calli- electron rnicroscopy. J Ultrastruct Res 26:31-43 nectes sapidus: course of infection and histopathology. Stewart JE, Ane B, Zwicker BM, Dingle JR (1969) Gaffkernia, J Invertebr Pathol 28:25-36 a bacterial disease of the lobster, Homarus amencanus: Johnson PT (1977) Paramoeblasis in the blue crab Calljnectes effects of the pathogen, Gaffkya homari, on the physiology sapidus. J Invertebr Pathol 29:308-320 of the host. Can J Microbiol 15:925-932 Johnson PT (1980) Histology of the blue crab, Callinectes Stewart JE, Cornick JW. Dingle JR (1967) An electronic sapidus. A model for the Decapoda. Praeger, New York method for counting lobster (Homarus americanus Milne Johnson PT (1986) Parasites of benthic arnphipods: dino- Edwards) hemocytes and the influence of diet on hemo- flagellates (Duboscquodinida: Syndinidae). Fish Bull 84: cyte numbers and hemolymph proteins. Can J Zoo1 45: 605-614 291-304 Johnson PT (1987) A revlew of fixed phagocytes and pinocy- Vickerman K, Appleton PL. Field RH (1993) Cultivation and totic cells of decapod crustaceans, with remarks on hemo- development in vifro of a parasitic dinoflagellate (Hemato- cytes Dev Comp lmmunol 11:679-704 dinium sp) from the Norway lobster (Nephrops norvegi- Johnson PT, Stewart JE, Arie B (1981) Histopathology of cus). In: Abstracts of the IX International Congress of Pro- Aerococcus viridans var. homari infection (Gaffkemia) in tozoolocy),. German Soc~etyof Protozoology and German the lobster, Homarus americanus, and a comparison with Society of Parasitology, Berlin, p 131 histological reactions to a Gram-negat~vespecies Pseudo- Wilhelm, G. & Boulo, V (1988). Infection de l'etrille Liocarci- monas perolens. J Invertebr Pathol38:127-148 nuspuber (L.) par un dinoflagelle parasite de type Hema- Latroulte D. Morizur Y. Iioel P. Chagot D, Wilhelm G (1988) todinium sp. Comm Meet int Coun Explor Sea CM-ICES Mortalite du torteau provoquee par le 1988/K:4 1

Responsible Subject Editor- J. E. Stewart, Dartmouth, Manuscript first received: August 24, 1994 Nova Scotia, Canada Revised version accepted: February 23, 1995