FOLIA PARASITOLOGICA 46: 117-122, 1999 Amphipod intermediate host of Polymorphus minutus (Acanthocephala), parasite of water birds, with notes on ultrastructure of host-parasite interface Bahram Sayyaf Dezfuli and Luisa Giari Dipartimento di Biologia, Università di Ferrara, Via Borsari, 46, 44100 Ferrara, Italy Key words: intermediate host, acanthocephalan, freshwater birds, host-parasite interface Abstract. From November 1997 to June 1998, 3,118 specimens of Echinogammarus stammeri (Karaman, 1931) (Amphipoda) were collected from the River Brenta (Northern Italy) and examined for larval helminths. Larvae of Polymorphus minutus (Goeze, 1782) singly infected the hemocoel of 23 (0.74%) crustaceans; all these larvae were cystacanth stages. This is the first record of Polymorphus minutus in E. stammeri. Some cystacanths had their forebody and hindbody fully inverted. Parasites were bright orange in colour and each was surrounded by a thin acellular envelope. This envelope likely protects the developing parasite larva from cellular responses of the amphipod. Hemocytes were seen adherent to the outer surface of the envelope. The sex ratio among the parasitised E. stammeri was almost 1:1. All Polymorphus minutus larvae were central in the amphipod body, made intimate contact with host internal organs, and frequently induced a marked displacement of them. None of the infected females of E. stammeri carried eggs or juveniles in their brood pouch. In five hosts, Polymorphus minutus co-occurred with the cystacanth of another acanthocephalan, Pomphorhynchus laevis (Müller, 1776), a parasite of fish. Species of Polymorphus are parasites of the alimen- MATERIALS AND METHODS tary canal of aquatic birds which act as their definitive From November 1997 to June 1998, monthly samples of hosts (Nicholas and Hynes 1958). According to Amin 310 to 543 specimens of Echinogammarus stammeri (1992), this genus was divided in two subgenera, (Karaman, 1931) were collected from the River Brenta near respectively, Polymorphus Lühe, 1911 with 36 species, Grantorto (Northern Italy, Province of Padua). Amphipods and Profilicollis Meyer, 1931 with 10 species. The life were sampled with a dip net (mesh size, 3 mm) in shallow cycle of a Polymorphus species was first worked out by waters near the bank, both from overhanging vegetation and Greeff (1864), working on P. minutus (see Nicholas and below the waterline, then fixed in 6% formaldehyde for Hynes 1958). Later, Le Roux (1933) and Hynes (1955) subsequent investigations. In the laboratory, the amphipods provided data on the effects of this acanthocephalan on were placed in Petri dishes with lactic acid to clear the its intermediate host, freshwater amphipods. Larvae of crustaceans, then measured and sexed. The number and position of parasites in the host hemocoel were noted and helminths are known to have a variety of effects on some infected crustaceans were dissected, and the parasites arthropod intermediate host. isolated and mounted in lactophenol for measurement and Within the hemocoel of the arthropod host, larval identification of larval stages. acanthocephalans are encircled by an envelope or Twenty-three Polymorphus minutus larvae form the basis for capsule. The origin of this envelope or capsule has been the following description. In addition, in May 1998, seven the subject of controversy among many researchers (see adult specimens of an acanthocephalan were encountered in Wanson and Nickol 1973). The evidence from the intestines of two moribund wild ducks, Anas transplantation, cytochemical, and biochemical platyrhynchos, at Grantorto. On three occasions, some investigations suggests that the envelope protects the crustaceans were examined while alive, then processed for a parasite from the arthropod immune response (Lackie host-parasite interface. Six E. stammeri infected with and Holt 1988). Polymorphus minutus and four uninfected ones were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, post- This paper is the first record of an intermediate host fixed in 1% osmium tetroxide in the same buffer for 1.5 h, for Polymorphus minutus in Italy. Herein we provide dehydrated in a graded ethanol series and embedded in a data on: parasite loads, infection rates, co-occurrence of Epon-Araldite mixture. Sections of 7µm in thickness were P. minutus with Pomphorhynchus laevis larva stained with azur A-methylene blue. Ultra-thin sections were (Acanthocephala) in the same host individual, and a stained with uranyl acetate and lead citrate and observed with description of the parasite larvae. Ultrastructural a Zeiss EM9. Light micrographs were obtained using a Leitz observations are also included. photomicroscope. Address for correspondence: B. S. Dezfuli, Dipartimento di Biologia, Università di Ferrara, Via Borsari, 46, 44100 Ferrara, Italy. Phone: ++39 532 291334; Fax: ++39 532 249761; E-mail: [email protected] 117 RESULTS electron-dense “line” 0.1 to 0.2 µm thick (Figs. 6a,b). In six amphipods in which the host-parasite interface was Acanthocephalan larvae were identified as cystacanth examined, often the envelope of Polymorphus minutus stages, based on larval shape and dimensions, and had been lifted off the surface of the parasite by a space development of internal organs. Features of the called by Crompton (1964) capsular fluid (Figs. 5, 6a,b, proboscis (e. g. number of rows of hooks and the 7). This space rarely was filled with a homogeneous numbers of hooks in each row) in the cystacanth stage granular substance of low electron density (Figs. 5, 6b, are the same as in an adult worm (Schmidt 1985). Thus, 7). This substance could have been a residual artifact fully extruded proboscides of several larvae were left when the envelope was separated from the surface compared with the same organ of seven adult specimens of the cystacanth tegument. of Polymorphus minutus recovered in May 1998 from Concerning the host-parasite interface, P. minutus the intestines of two moribund wild ducks, A. was encountered mainly in a central position in its host platyrhynchos, found at the sampling site. Accordingly, (Fig. 1). The parasite made intimate contact with host the 23 parasite larvae found during the present survey alimentary canal, associated organs (e.g. digestive were found to be cystacanths of Polymorphus minutus. caeca) (Figs. 5, 7), gonads, and the nervous system. The Over eight months, a total of 3,118 specimens of E. acanthocephalan larva induced a marked displacement stammeri was examined (Table 1). Twenty-three of host internal structures (Fig. 5). Host hemocytes were (0.74%) individuals (average total length ± SD: 7.03 ± observed in contact with the outer part of the cystacanth 0.94 mm), 12 females and 11 males, were infected with envelope; they appeared sparsely or in clumps (Fig. 7) Polymorphus minutus (Fig. 1). Sex ratio among the 2 around the P. minutus larva. Based on the quantity of infected E. stammeri was not significant (χ = 0.04; p > granules within the cytoplasm of these cells and their 0.10). ultrastructural features, three types of hemocytes were None of the infected females carried eggs or juve- recognised around acanthocephalan larva, namely niles in their brood pouch. Prevalence of infection granular (Fig. 8), semi-granular, and hyaline (Barracco ranged from 0.26 in April 1998 to 1.88 in February of and Amirante 1992). Hemocytes were intact (Fig. 7) or the same year (Table 1). No more than one P. minutus in many instances were partially (Fig. 8) or completely larva was encountered in a single host. In five E. stam- disintegrated. meri, Polymorphus minutus co-occurred with Pompho- rhynchus laevis cystacanths (Fig. 2). The larvae of P. minutus had a much more intense orange colour in both Table 1. Occurrence of Polymorphus minutus in Echino- fixed and live amphipods than those of P. laevis. gammarus stammeri of the River Brenta. Infected Larvae Month Examined Description of larva of Polymorphus minutus (prevalence) recovered Figs. 3,4 Nov. ’97 543 4 (0.74) 4 Small size cystacanths ranged from 0.80 to 0.84 mm Dec. ’97 363 1 (0.27) 1 in length and from 0.52 to 0.55 mm in width (Fig. 3). Jan. ’98 361 2 (0.55) 2 The length of withdrawn proboscides ranged from 0.40 Feb. ’98 319 6 (1.88) 6 to 0.42 mm. The body length of big-size cystacanths Mar. ’98 484 2 (0.41) 2 ranged from 2.08 to 2.18 mm, and the width of the Apr. ’98 388 1 (0.26) 1 middle trunk region was 0.58 mm (Fig. 4). Extruded May ’98 350 3 (0.86) 3 Jun. ’98 310 4 (1.29) 4 proboscides were short, cylindrical or ovoid (0.45 × Total 3,118 23 (0.74) 23 0.20 mm) (Fig. 4), with similar armature in both sexes: 16-18 longitudinal rows of 9 hooks each. Hooks increased in size (46 µm) from the apex of the proboscis, but were smaller (35 µm) toward the base. DISCUSSION Neck was distinct, 0.34 mm long by 0.20 mm wide. The recognition of parasitised E. stammeri was made Trunk was more or less plump and spined anteriorly easier by the intense orange colour of the acantho- (spines 22 µm long) (Fig. 4). In males, testes were cephalan larva. The colour of parasite larvae is due to ovoid to spheroid (0.12 × 0.09 mm), tandem or slightly the presence of carotenoids that are taken from the host overlapping, anterior in the second quarter of the trunk. hemolymph (Barrett and Butterworth 1968). Each acanthocephalan larva was covered with a thin Only 23 out of 3,118 E. stammeri were infected with acellular envelope, 1.2-1.3 µm in thickness (Figs. 3, 5, Polymorphus minutus; in 5 months out of 8, the 6). The envelope was moderately electron-dense, and infection rates did not exceed 0.8%. A similar finding was formed by granular and filamentous materials was reported for P.