Morphological and molecular study of Microphallus primas (Digenea: Microphallidae) metacercaria, infecting the shore crab Carcinus maenas from northern Portugal.

The present study describes the anatomy and surface topography of the metacercaria of Microphallusprimas (Jägerskiöld, 1909) infecting the shore crab Carcinus maenas (L.) in Aveiro estuary, northern Portugal. The metacercaria species identification resulted from the combined use of morphological and molecular data, particularly the 28S rDNA gene. The metacercariae encysted preferentially in the host's hepatopancreas and also in the gonads. Isolated cysts were present in two distinct forms, spherical and oval, and were shown to be the identical species by the internal transcribed spacer 1 (ITS1) sequence. Chemically excysted metacercariae were studied by light (LM) and scanning electron microscopy (SEM). Their specific characteristics observed include the particular aspect of the vesiculo-prostatic pouch surrounded by a very thin membrane, the presence of a prominent muscular papilla, and an obvious metraterm. The dorsal and ventral tegumental surfaces of the metacercaria were densely packed with similar squamous spines, which decreased in number and size towards the hindbody. The edges of the posterior and ventral face of the body were coated with numerous microvilli, whose function remains unknown. In order to identify the species of metacercariae, we compared a 28S partial rDNA sequence of the two forms of cysts with the same 28S partial region of M. primas available in GenBank. With this comparison, we determined that the sequences had a 100% similarity and therefore belonged to the same species, i.e., M. primas.

Identification and description of Bucephalus minimus (Digenea: Bucephalidae) life cycle in Portugal: morphological, histopathological, and molecular data.

The cercaria of Bucephalus minimus infects the digestive gland and gonads of its first intermediate host, the edible cockle, Cerastoderma edule. Light microscopy (LM) and scanning electron microscopy (SEM) of the cercaria showed a tail formed by a central stem, with 2 long contractile arms presenting distinct morphological surfaces. The encysted metacercaria naturally infected the flathead grey mullet, Mugil cephalus. The cysts found in the heart, liver, and spleen were shown to be identical by the internal transcribed spacer (ITS 1) sequence and morphological features and were associated with encapsulation, recruitment of cell infiltrates, and presence of melanomacrophages and adipose tissue. To establish the life cycle, we compared the ITS1 sequence in an adult from the known definitive host, Dicentrarchus labrax; encysted metacercariae from the liver, heart, and spleen of M. cephalus; and a cercaria from C. edule. With this comparison, we determined that they had a 100% similarity. Therefore, the ITSI sequence data clearly indicate that these 3 parasitic stages belong to the same species, i.e., B. minimus.

Clarification of Cercaria sevillana (Digenea: Microphallidae) life cycle using morphological and molecular data.

Cercaria sevillana is the cercaria larval stage that infects the gonads and the digestive gland of its first intermediate host, Nassarius reticulatus. In this study the decapodous crustacean Carcinus maenas was used to determine if it would serve as second intermediate host in the parasite's life cycle. The latter hypothesis was based on the knowledge that C. maenas is the second intermediate host of several other digenean species. After dissection, it was possible to observe encysted metacercariae in the antennal glands of the green crab. After biochemical excystment, the metacercariae were processed for light and scanning electron microscopy. The morphological features observed led us to conclude that this species was a microphallid fluke, and it was identified as Gynaecotyla longiintestinata. To establish a possible relationship between C. sevillana and this metacercariae, the ITS1 region was analyzed. Thus, DNA was extracted from C. sevillana and from the cysts isolated from the antennal glands. The ITS1 region was amplified and sequenced, and the alignment clearly demonstrated that the cercaria and the metacercariae belonged to the same species, G. longiintestinata.