Vol. 106: 163–172, 2013 DISEASES OF AQUATIC ORGANISMS Published October 11 doi: 10.3354/dao02650 Dis Aquat Org Postmonorchis sp. inq. (Digenea: Monorchiidae) metacercariae infecting natural beds of wedge clam Donax trunculus in Italy F. Carella1,*, J. Culurgioni2, S. Aceto1, G. Fichi3, T. Pretto4, D. Luise1, A. Gustinelli5, G. De Vico1 1Department of Biology, University of Naples Federico II, 80134 Napoli, Italy 2Department of Life and Environmental Sciences, University of Cagliari, 09126 Cagliari, Italy 3Laboratory of Ichthyopathology, Experimental Zooprophylactic Institute of Lazio and Tuscany, 56123 Pisa, Italy 4Experimental Zooprophylactic Institute of Venice, 35020 Legnaro, Padua, Italy 5Department of Veterinary Public Health and Animal Pathology, University of Bologna, 40064 Ozzano dell’ Emilia, Italy ABSTRACT: The wedge clam Donax trunculus Linnaeus, 1758 is one of the most common bivalve molluscs inhabiting the sandy shores of the Mediterranean Sea and is considered an important commercial resource. In this study, we report the first molecular, morphological and histopatho- logical descriptions of metacercariae from a trematode belonging to the genus Postmonorchis (Digenea: Monorchiidae) that infects D. trunculus in natural beds of the Italian Tyrrhenian coast (Campania, Lazio and Tuscany). Morphological analysis of the parasite revealed a combination of features that exist in the 3 previously identified species of Postmonorchis, viz. P. donacis, P. vari- abilis and P. orthopristis, with the addition of new, distinctive morphological characteristics. The pathogen exhibited a predilection for the gill; however, it was also present in the labial palp and mantle in addition to the gut, kidney epithelium and foot. The inflammatory response was charac- terised by either a focal or diffuse haemocyte infiltration followed by the formation of multiple, large multi-layered capsules associated with tissue destruction. The prevalence of the pathogen ranged from 75 to 100%, while the infection intensity fluctuated among the study areas. Further studies regarding the life cycle of this parasite and the identification of other larval and adult stages and their respective hosts may confirm the identification of a new species of Postmonorchis that infects wedge clams in Mediterranean waters. The study of the parasite is completed by molecular analysis of the ITS1 and ITS2 rDNA sequences. KEY WORDS: Donacidae · Bivalvia · Metacercariae · Monorchiidae · Postmonorchis · Trematoda · Parasite · Mediterranean · ITS Resale or republication not permitted without written consent of the publisher INTRODUCTION Trematodes have complex life cycles with asexual forms parasitizing the internal organs of molluscs, During the last several years, research regarding whereas the sexual stages occur mainly in verte- bivalve diseases has significantly increased, particu- brates and only occasionally in invertebrates. De - larly with respect to the bacterial and protozoan pending on the species, the larvae of most trema- agents in cultured species that are listed by the todes need a second intermediate host, which may be World Organisation for Animal Health (OIE) (Berthe either an invertebrate or a vertebrate (Cribb et al. 2008). Studies of Platyhelminthes have focused pri- 2003, Madhavi 2008). marily on taxonomy and rarely on the lesions that are In the Mediterranean Sea, parasitosis in bivalve linked to the host−parasite interaction (Doanh et al. molluscs is caused mainly by faustulid, fellodistomid, 2007, de Montaudouin et al. 2009). gymnophallid and bucephalid trematodes (Palombi *Email: [email protected] © Inter-Research 2013 · www.int-res.com 164 Dis Aquat Org 106: 163–172, 2013 1934, Lauckner 1983, Ramòn et al. 1999, Culurgioni et Campania region were the mouth of the Volturno al. 2006). In contrast, reports on the Monorchiidae River (41° 0’ 57.24’’ N, 13° 56’ 49.56’’ E) during July family have mostly described species in the Atlantic or 2009/ 2010 (20 clams) and Litorale Domizio (41° 4’ Pacific Ocean, where the cercariae develop within 8.39’’ N, 13° 51’ 3.65’’ E) during July 2012 (20 clams). spo ro cysts of bivalve species such as Cumingia telli- In September 2012, samples were collected at Formia noides, Abra ovata, Macoma nasuta (Martin 1940, (41° 14’ 20.73’’ N, 13° 36’ 43.42’’ E) in the Lazio region Maillard 1975, Bartoli 1984) and Donax gouldii (Young (20 clams) and at sites in the Tirrenia locality 1953, Winter & Hatch 2010). The only re ported case (10° 17’ 04’’ N, 43° 37’ 28’’ E) in Tuscany (100 clams), in the Mediterranean Sea is Cerastoderma edule while samples were also collected at Feraxi Beach infected by Monorchis parvus (Bartoli et al. 2000). (39° 19’ 07’’ N, 9° 13’ 19’ E) in eastern Sardinia (30 The wedge clam Donax trunculus is an important clams; Fig. 1). The clams were collected from the marine resource obtained by the exploitation of natu- shore using a hand-held semi-circular dredge as used ral beds, distributed from the Atlantic coast of France by the local fishermen. The clams were immediately to Senegal and the shallow bottoms of the Mediter- transferred into ~15 l of water collected from the sam- ranean Sea (Gaspar et al. 2002, Manca Zeichen et al. pling location and maintained in a cool environment 2002, La Valle 2006). Marketable landings of this bi- in an insulated sealed container for transit to the labo- valve are in the range of 535 to 970 t yr−1 in Europe ratory with a maximum transit time of 2 h. The sam- (Food and Agriculture Organisation of the United ples were opened in the laboratory and examined for Nations — Fisheries Global Information System: www. external signs under a dissecting microscope (Nikon fao.org), and although fishing has slightly declined in SMZ-10). To obtain a positive identification of the recent years, the wedge clam remains a significant Donax species as the Mediterra nean D. trunculus, 5 commercial species for Italy. Despite the importance samples were submitted to expert taxonomists at the of the wedge clam, little is known about the patho- Zoological Museum of Naples. The observed meta - gens and parasites that affect the health of D. truncu- cercarial cysts were scraped off the surface of gills lus populations. Recent studies suggest the use of this and labial palps, and the wet mounts were examined species as a bioindicator of physiological and cellular by light micro scopy. The cysts were stored in 70 and alterations caused by pollution (Fishelson et al. 1999, 100% alcohol for morphological identification and Yawetz et al. 2010). Moreover, some reports refer to further molecular analysis, respectively. Specimens the parasitic castration of D. trunculus caused by the were also de posited in the Scientific Catalogue of the faustulid Bacciger bacciger and damage from the Museum Collection of the Department of Biology, gymnophallid Meiogymnophallus strigatus or a Rick- University of Naples Federico II under reference ettsia-like organism (Comps 1979, Bartoli 1983, Ra mòn number DBCUnina-Plat00057. et al. 1999), with no record of Monorchiidae infections. In this report, we present the first study of Post- monorchis sp. inq. (Digenea: Monorchidae) metacer- Light microscopy cariae infecting the wedge clam Donax trunculus along the Italian coast of the Mediterranean Sea. The The bivalves were fixed in Davidson’s fixative for parasite was detected in the years 2009, 2010 and 24 to 48 h and then processed for routine histopathol- 2012. After a histopathological study on wedge ogy. Two transverse sections, approximately 5 mm clams, in order to assess the taxonomic position of the thick, including the mantle, gonad, digestive gland, parasite, it was characterised morphologically and gills, kidney and foot, were excised from each clam analysed at the molecular level by sequencing the and placed into histological cassettes. To obtain a internal transcribed spacer regions 1 and 2 of the more complete view of the parasitosis, a 5 mm piece ribosomal DNA. of the gill was also removed from each animal and included with the sample in the longitudinal orienta- tion. The tissue samples were embedded in paraffin MATERIALS AND METHODS wax, sliced in 5 µm sections and subsequently stained with haematoxylin and eosin (H&E). Additional stains, Sampling such as PAS-BA and Masson’s Trichrome (Mazzi 1977), were also used. Donax trunculus specimens were collected during Parasite prevalence, infection intensity and immune the summer from different geographical areas over a response efficiency were also evaluated at the sites of period of several years. The sampling sites in the infection, including the gill, mantle and labial palps. Carella et al.: Postmonorchis sp. infecting Donax trunculus 165 Fig. 1. Clam collection sites in the areas of Tuscany (Tirrenia, T), Lazio (Formia, F), Campania (Litorale Domizio, LD; Volturno River mouth, V) and Sardinia (Feraxi, FE) For the gill tissues, the data were ob tained by histo- For morphological observations, the metacercarial logical examination (histological infection density, cysts were scraped from the infected tissues and exa - HID; Bush et al. 1997). In particular, the value of in- mined as wet mounts. A number of fixed specimens tensity was determined on a fixed area of 8 mm2 in 2 were stained with acetic carmine to highlight the consecutive slides under 10× magnification. For the developing organs. Measurements were obtained labial palp and mantle the infection intensity (macro- from 10 well-developed metacercariae using an scopic infection intensity,