Morphological and molecular analysis of metacercariae of Diphtherostomum brusinae (Stossich, 1888) Stossich, 1903 from a new bivalve host Mytilus galloprovincialis.

The digenean trematode Diphtherostomum brusinae (Stossich, 1888) Stossich, 1903 presents a complex life cycle that may involve more than one intermediate host. The present study represents the first description of the metacercariae from D. brusinae infecting the labial palps of a new intermediate host, Mytilus galloprovincialis, in the Aveiro estuary, Portugal. The morphology of this parasitic stage was studied by light (LM) and scanning electron microscopy (SEM) and some differences were reported (body and sucker sizes, and spine distribution and shape). In this work, the 18S partial region of the ribosomal DNA was sequenced from D. brusinae metacercariae isolated from M. galloprovincialis collected in different localities of the Aveiro estuary. In addition, sequences from the same region of the 18S rDNA were obtained from D. brusinae cercariae and metacercariae, hosted by Nassarius reticulatus and Cerastoderma edule, respectively. No intraspecific polymorphism was detected in the 18S partial region, since there was 100% homology among all the sequences analysed. The same comparison was made for the ITS1, and we observed intraspecific polymorphism in this region. To our knowledge, this is the first report of D. brusinae metacercariae infecting the mussel M. galloprovincialis with support from morphological and molecular data.

Morphological and molecular studies on life cycle stages of Diphtherostomum brusinae (Digenea: Zoogonidae) from northern Portugal.

Diphtherostomum brusinae was first recorded by the present study in the north of Portugal. Sporocysts, containing cercariae and encysted metacercariae, were observed in the gonads and digestive gland of the gastropod Nassarius reticulatus. Metacercariae were also found infecting the foot, mantle border and gills of the cockle Cerastoderma edule. The adult form was lodged in the rectum of the definitive host Diplodus sargus. The morphology of the three parasitic stages was studied by light (LM) and scanning electron microscopy (SEM). Despite the close similarity between cercaria and metacercaria, SEM data provided information that allowed their differentiation, namely the presence of a dense crown of microvilli around the oral cavity of the cercariae, which was absent in the metacercariae. In addition, the metacercariae presented a specific pre-acetabular rectangular band with conspicuous triangular spines. The adult showed characteristics of D. brusinae species, in particular the presence of acetabular lips, compact vitellaria and large elliptical eggs. Sequenced ITS1 data clearly demonstrated that the cercariae and metacercarial cysts from N. reticulatus, the cysts from C. edule and the adult isolated from D. sargus were life cycle stages that belonged to the same species, i.e. D. brusinae. Two transmission strategies in the life cycle of this species were observed: (1) cercariae encyst within the sporocysts of N. reticulatus and await ingestion by the definitive host; and (2) N. reticulatus naturally emits cercariae; they encyst in C. edule or the environment and are ingested by the definitive host.

Ultrastructural studies on the tegument of the metacercariae of Meiogymnophallus minutus and Meiogymnophallus fossarum (Digenea: Gymnophallidae) in Cerastoderma edule (Bivalvia) from Portugal.

Unencysted metacercariae of Meiogymnophallus minutus (Cobbold, 1859) and the sibling species Meiogymnophallus fossarum (Bartoli, 1965) infect the edible cockle, Cerastoderma edule (L.), with free metacercariae occurring simultaneously in the same host specimens in Portugal. Observations on the tegument of both species show that changes in the morphology of the body surface occurs in the intermediate host. Scanning and transmission electron microscopy revealed an alveoli-like tegument with palmate, digitated spines protruding from each individual alveolus. Uniciliated papillate receptors, which may or not may be recessed, were abundant, surrounding the suckers as well as being distributed on the body surface. Transmission electron microscopy revealed a significant number of 3 types of densely packed secretory vesicles, thin cysternae, and mitochondria in the surface tegument of the body, oral cavity, and esophagus. No distinguishing differences were observed in the surface ultrastructure of M. minutus and M. fossarum metacercariae.

The life cycle of Monorchis parvus (Digenea: Monorchiidae) demonstrated by developmental and molecular data.

Cercaria cerastodermae I, a digenean parasite of Cerastoderma edule, was recorded for the first time in the Atlantic Ocean off the Iberian peninsula. Sporocysts were present in the hemolymph of the digestive gland, gonad, gills, and foot of the mollusc. Most of the cercariae present within sporocysts were encysted as metacercariae. The corresponding adult stages were obtained after experimental infection of several Diplodus sargus artificially reared in fish farms and that had previously been protected against natural infections. Numerous adult specimens of Monorchis parvus were collected in Diplodus annularis along the French Mediterranean coast. Comparison of wild and experimental adults allowed the adult stage of Cercaria cerastodermae I to be identified as M. parvus. Another monorchid, Monorchis monorchis, a parasite of Spondyliosoma cantharus, was found in the same Mediterranean area and compared with M. parvus. Additionally, ITS1 nuclear ribosomal DNA sequences of C. cerastodermae I and of the adults collected in naturally infected D. annularis and S. cantharus were obtained. Sequence data indicate that C. cerastodermae I corresponds to the adult of M. parvus found in D. annularis and is clearly distinct from M. monorchis found in S. cantharus.

Ultrastructure study of the intramolluscan stages of Meiogymnophallus minutus (digenea: gymnophallidae) in Scrobicularia plana (bivalvia) from Portugal.

The ultrastructure of the tegument of the sporocyst and contained cercariae of Meiogymnophallus minutus found in Scrobicularia plana collected in Ria de Aveiro estuary (Portugal) were studied by scanning (SEM) and transmission electron microscopy (TEM). TEM revealed microvilli extending from the surface of the sporocyst tegument that contributed to the lacetrimmed surface morphology observed by SEM. The cercarial surface was papilliform and covered densely with single-pointed tegumental spines. On TEM, besides the spines and several mitochondria, translucid and electrondense spherical secretory vesicles were evident in the matrix of the tegument but were not seen on the sporocyst tegument. Uniciliate sensory receptors found in the cercaria of M. minutus were examined under SEM and TEM. Structural differences between the oral and the ventral sucker were observed. On the lip of the ventral sucker, large and strong spines were arranged in concentric rows, and two groups of six sensory papillae were placed concentrically and equidistantly. The oral sucker, surrounded by a crown of dense and long microvilli, exhibited a ring of uniciliate sensory papillae and some tegumental spines along the lip. The surface morphology of the M. minutus cercaria is compared with that of other Gymnophallidae cercariae.

The metacercariae of sibling species of Meiogymnophallus, including M. rebecqui comb. nov. (Digenea: Gymnophallidae), and their effects on closely related Cerastoderma host species (Mollusca: bivalvia).

The morphology and ecology are described for four sibling species of digenean metacercariae: Meiogymnophallus minutus (Cobbold 1859), M. strigatus (Lebour 1908) from the estuarine cockle Cerastoderma edule L. and M. fossarum (Bartoli, 1965) and M. rebecqui (Bartoli, 1983) comb. nov. from the brackish water lagoon cockle C. glaucum (Poiret). Although occupying different sites within the two species of cockle, both M. minutus and M. fossarum infections result in pathological effects that may cause heavy host mortality. Remarkably, they also result in an inversion of the cockles in situ in the sand such that predation by the final host species is enhanced.