DlSEASES OF AQUATIC ORGANISMS Vol. 22: 115-128.1995 Published June 15 Dis aquat Org 1 A Hematodinium-like dinoflagellate infection of the Norway lobster Nephrops norvegicus: observations on pathology and progression of infection R. H. Field, P. L. Appleton Division of Environmental and Evolutionary Biology, Graham Kerr Building, University of Glasgow, Glasgow G12 800. Scotland, UK ABSTRACT: The discovery of a Hematodinium-like dinoflagellate infecting Nephrops norvegicus has led to a pathological investigation into the effects on the host and the apparent progression of the dis- ease syndrome The parasite is systemic, invading the haemocoel and the connective tissues of most organs via the haemal spaces. An increase in the combined number of parasites and haemocytes in the haemolymph was due to an increase in the relative proportion of dinoflagellates 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 may be 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 encapsulations in 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 species 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 Syndiniales (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 Carcinus maenas 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 crustaceans over a wide pathology of infection and the course of the disease, geographic range [Callinectes 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), Cancer irroratus, (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 France 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), Chionoecetes bairdi and C. opilio in Alaska, 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-Research 1995 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 lobsters (again after pleopod now conducted combined paras~teand 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 ~'efieiiihc 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 , , '. Fig. 1. Nephrops norvegicus.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.