l DISEASES OF AQUATIC ORGANISMS Vol. 32: 75-78,1998 Published February 26 Dis Aquat Org 1 NOTE Palaemon elegans, an intermediate host in the life-cycle of Aggregata octopiana C. Arias, C. Gestal, H. Rodriguez, M. Soto, J. Estevez* Laboratorio de Parasitologia, Facultad de Ciencias del Mar, Universidad de Vigo, Apartado 874, E-36200 Vigo, Spain ABSTRACT: Based on immunological methods, the prawn elegans (Decapoda: Palaemonidae) (Haywar & Ryland Palaemon elegans apparently acts as the natural intermediate 1985) were caught in the Ria de Vigo (NW Spain) and host for Aggregata octopiana, a coccidian that infects the dissected. Their digestive tracts were extracted and cephalopod Octopus vulgaris. However, cross-reactivity with another coccidian, A. eberthi, was present in both sporogonic examined under the stereo microscope in order to and merogonic stages. detect cysts. Oocysts of Aggregata octopiana and A. eberthi were isolated from the digestive tracts of KEY WORDS: Aggregata octopiana . Life cycle Palaemon the naturally infected molluscan cephalopods Octopus elegans vulgaris and Sepia officinalis, respectively, in the Ria de Vigo (Estevez et al. 1996. Pascual et al. 1996). Purification ofsporocysts and preparation ofsporo- cyst extracts: Sporocysts of Aggregata octopiana Coccidians of the genus Aggregata cause major dis- and A. eberthi were purified from oocysts. Briefly, eases in cephalopods. The merozoites migrate through sporocysts were obtained by maceration of infected, the epithelium of the digestive tract causing degenera- cephalopod host tissues in phosphate-buffered saline tion and death of the parasitized cells and leading to (PBS), pH 7.2. The resulting suspension was then detachment of necrotic fragments of the intestine filtered through increasingly fine meshes to remove (Hochberg 1990). Despite their importance, reliable tissue fragments. The filtrate was then centrifuged at information currently exists only about the life-cycle 2000 X g for 15 min. Filtration-centrifugation of the of the species A. eberthi Labbe, 1895, a coccidian pellet was repeated several times until a pure sample parasite of crabs and cuttlefish. As in some other of sporocysts was obtained. Sporocysts were lysed by coccidians, there is alternation of generations and sonication on ice (60 W in 1 min pulses for 45 min) and alternation of hosts. In both hosts, the parasites are centrifuged at 10000 X g for 30 min at 4OC. The super- found in the intestinal epithelium or wall. The asexual natant (used in all analyses) was stored at -30°C until phase is in the peri-intestinal tissue of Portunus depu- used (Leiro et al. 1993). rator L., 1758 while the sexual phase occurs within Collection of sera: Ten female Balb/c mice were cells of the submucous connective tissue of the caecum immunized by subcutaneous injection of a 0.2 m1 mix- and intestine of Sepia officinalis L. 1758 (Dobell 1925). ture of Freund's complete adjuvant (Sigma Chemical The cephalopod Octopus vulgaris Cuvier, 1789 is the Co., St. Louis, MO, USA) and PBS containing about definitive host for the species Aggregata octopiana 5 X 106 sporocysts of Aggregata octopiana. They were Schneider, 1875 (Estevez et al. 1996). This paper pre- boostered 30 d later by intraperitoneal (i.p.)injection of sents the first report of the prawn Palaemon elegans the same number of sporocysts in 0.2 m1 of PBS without Rathke, 1837 as intermediate host in the life-cycle of adjuvant. On Day 45 a third dose (without adjuvant) this coccidian. was administered by i.p, injection. Mice were bled 60 d Materials and methods. Prawns andparasites: Forty- after primary immunization through the retroorbital eight crustaceans belonging to the species Palaemon route. Serum (anti- Aggregata octopiana) was sepa- rated by centrifugation at 2000 X g for 10 min, mixed 1:l with glycerol and stored at -30°C until use. A simi- 'Addressee for correspondence. E-mail: [email protected] lar protocol was used to obtain anti - A. eberthi serum. O Inter-Research 1998 Resale of full article not permitted 76 Dis Aquat Org 32: 75-78, 1998 Arias et al.: Life-cycle of Aggregata octop~ana 77 Fig. 1. Palaemon elegans. (A) Segment of the gut of the prawn Palaemon elegans showing the intestine (*) and cysts with multiple nuclei arranged in the cytoplasm. Scale bar = 100 pm. (B-D) Section of a cyst in prawn gut tested with: (B) normal mouse serum, background level, (arrows indicate the cyst location): (C) anti-Aggregatd octopiana mouse serum (arrows indicated the area of strong positive reaction), and (D)anti-A. eberthi mouse serum (arrows indicate the area of slight reaction). Haematoxylin was used as the counterstain. Scale bar = 10 pm Slide pre-processing: Infected intestines from the Enzyme-linked immunosorbent assay (ELISA):Titra- crustacean Palaenlon elegans were kept in 10'X tion curves were derlved and the cross-reactivity of formaldehyde in PBS, pH 7.2, for 12 to 24 h. The the antisera assayed was tested using the indirect samples were dehydrated in increasing concentrations ELISA technique, as described previously (Estevez et of ethanol and embedded in paraffin wax, after which al. 1994). 4 pm microtome sections were cut, dewaxed and Results. Immunohistochemistry: At least l cyst was mounted. found to be present in the peri-intestinal tissues of 63 % Immunohistochemistry: Immunohistochemistry was of the natural Palaemon elegans examined. Fig. 1A performed by the indirect immunoperoxidase method shows these cysts, inside of which multiple nuclei can (Tijsen 1987). Mounted sections were incubated for be seen, corresponding to the merozoites. 30 min in 10% H202 in Tris-buffered saline (TBS: Intestinal sections of Palaemon elegans containing 50 mM Tris, 0.15 M NaC1, pH 7.4) to inhibit endoge- cysts were chosen for immunoassay comparisons. Neg- nous peroxidase. They were then washed with TBS ative controls used normal mouse serum (background and incubated for 1 h with 5 % non-fat dry calf milk in level) which gave no reaction (Fig. 1B).In all the coccid- TBS containing 0.2 % (v/v) Tween-20 (TBS-Tween) at ian-infected prawns tested, the cysts containing mero- room temperature (RT) to block non-specific reactions. zoites reacted positively to anti-Aggregata octopiana After being washed with TBS-Tween, the slides were sporocyst mouse serum (Fig. 1C) but showed little reac- incubated with a 1:20 dilution of the primary sera (anti- tion to anti- A. eberthi sporocyst mouse serum (Fig. ID). Aggregata octopiana or anti-A. eberthi) in TBS-Tween Cross-reactivity study: ELISA was used to deter- for 4 h at RT in a humid chamber. Next, they were mine the titer of the 2 polyclonal antisera, which was washed with TBS-Tween and incubated for 1 h with estimated at over 1:106 for both (Fig. 2A). Cross- polyclonal rabbit anti-mouse immunoglobulin serum reactivity was also observed. Antibodies raised in mice (Dakopats A/S, Glostrup, Denmark) diluted 1:100 in with Aggregata eberthi purified sporocysts cross- TBS-Tween. The slides were then washed with TBS reacted with sporocyst extracts of A. octopiana (hetero- and incubated with 100 1-11 substrate solution of TBS logous antigen) (Fig. 2B). containing 0.003 % H202, 0.06('/b diaminobenzidine Discussion. Polyclonal antisera are effectively ap- tetrahydrochloride (Sigma) and 0.03% NiC1,. After a plied in taxonomic studies on protozoa (McGovern & brown colour had developed, the reaction was stopped Burreson 1989, Martin-Gonzalez et al. 1991) and here by washing the slides with TBS. The sections were we used them to identify the intermediate host in the then mounted in TBS-glycerol (1:l)and covered with a cycle of Aggregata octopiana. cover glass for photography. Appropiate negative con- Immunoassay of the prawn tissues indicated that trols were included in each case. Haematoxylin was mouse anti - Aggregata octopiana sporocyst serum gave used as the counterstain (Culling et al. 1985). a strong positive reaction to cysts (merozoites) found in Fig. 2. (A) Titration curves for ELISA as- says of sporocyst antisera. (a)Anti-Aggre- gata octopiana sporocyst mouse serum; (0) anti-A. eberthi sporocyst mouse serum. Background level (normal mouse serum) is represented by the dotted line. Values are expressed as logz of the anti- body dilution with initial dilution 1500. (B) Cross-reactivity of the antisera as- sayed (at dilution 1:20000) with antigen extracted from A. octopiana sporocysts. White bar: anti-A. octopiana sporocyst serum; black bar. anti-A. eberthi serum; 9 10 l1 12 13 14 15 16 17 18 19 20 hatched bar: normal mouse serum Antibody dilution A.octopiana antigen 78 Dis Aquat Org 32: 75-78, 1998 the digestive tract of Palaemon elegans. Nevertheless, Estevez J, Leiro J, Santamarina MT. Dominguez J, Ubeira FM a slight reaction was also observed between these (1994) Monoclonal antibodies to turbot (Scophthalmus cysts and mouse anti A. eberthi sporocyst serum. maximus) immunoglobulins: characterization and applica- - bility in irnmunoassays. Vet Immunol Immunopathol 41: Cross-reactivity between species of the same genus 353-366 has already been recorded in coccidians of the genus Estevez J, Pascual S, Gestal C, Soto M, Rodnguez H, Arias C Eimeria by Xie et al. (1992).Here, the cross-reactivity (1996) Aggregata octopiana Frenzel 1885 (Apicomplexa: between the 2 species of Aggregata was shown to Aggregatidae) from Octopus vulgaris (Cuvier, 1798) off NW Spain. Dis Aquat Org 27:227-231 occur for 2 stages of the biological cycle (i.e. the mero- Haywar PJ, Ryland JS (1995) Handbook of the marine gony stage in the crustacean gut and the sporogony fauna of North-West Europe. Oxford University Press, stage in the cephalopod gut). Oxford The prevalence of cysts of Aggregata octopiana in Hochberg FG (1990) Diseases of Mollusca: Cephalopoda. Dis- eases caused protistans and metazoans. In: Kinne 0 the gut of Palaemon elegans was approximately 63 %, by (ed) Diseases of marine animals, Vol 111.