Fernanda Russell-Pinto

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.

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)

Abstract 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.

Digenean trematode species in the cockle Cerastoderma edule: identification key and distribution along the north-eastern Atlantic shoreline.

We describe the digenean fauna of one of the dominant intertidal hosts, the common cockle Cerastoderma edule , in terms of biomass, off north-eastern Atlantic shores. Using published and unpublished literature we have prepared an identification key and provide an up-date of the large-scale distributional patterns of digenean species of the common cockle. At least sixteen digenean species, belonging to seven families, use cockles as intermediate host. Among these species two utilize cockles as first intermediate host only, whereas two species utilize cockles as both first and second intermediate host. The remaining eleven species have cockles as their second intermediate host. Water birds and fish are the definitive hosts to twelve and four species, respectively. Cockles are infected with digeneans along the latitudinal gradient from southern Morocco to the western region of the Barents Sea often with high infection levels. Whereas some of these digenean species occur along most of the latitudinal gradient others show a more restricted northern or southern distribution mostly caused by an underlying latitudinal gradient of host species. Knowledge of digenean species and their large-scale distribution pattern may serve as a baseline for future studies dealing with the effects of climate change on parasite–host systems. For such studies the cockle and its digenean community could be an ideal model system.

IDENTIFICATION AND DESCRIPTION OF BUCEPHALUS MINIMUS (DIGENEA: BUCEPHALIDAE) LIFE CYCLE IN PORTUGAL: MORPHOLOGICAL, HISTOPATHOLOGICAL, AND MOLECULAR DATA

Abstract 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 (ITS1) 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 ITS1 sequence data clearly indicate that these 3 parasitic stages belong to the same species, i.e., B. minimus.

Morphology, ultrastructure, genetics, and morphometrics of Diplostomum sp. (Digenea: Diplostomidae) metacercariae infecting the European flounder, Platichthys flesus (L.) (Teleostei: Pleuronectidae), off the northwest coast of Portugal

The morphology, ultrastructure, genetics, and morphometrics of a species of Diplostomum von Nordmann 1832 (Digenea: Diplostomidae), isolated from the European flounder (Platichthys flesus (L.)) caught off the northwest coast of Portugal, are characterized. The metacercarial stage was found unencysted in the lens capsule of the eye. Light microscopical observations revealed the existence of some variability in specimen shape and size, with two morphotypes, referred to as “round” and “long”, being apparent. Scanning electron microscopy revealed a smooth, unarmed tegument, with the lappet region being the most irregular and porose. Both the oral and ventral suckers were provided with a series of papillae, which presented very distinctive ultrastructural features and were particularly conspicuous in the case of the ventral sucker. The two morphotypes detected were found to have 100% genetic correspondence in the 18S + ITS1 + 5.8S region of the rDNA. Since the genetic data for this metacercaria differed from those of the species of Diplostomum available in GenBank, a description of a new genotype (accession number GQ370809) is provided. The molecular phylogenetic analyses, in conjunction with principal components and cluster analyses based on morphometric data, revealed the existence of consistent differences between the Diplostomum sp. metacercariae from flounder compared with Diplostomum spathaceum, Diplostomum mergi, Diplostomum pseudospathaceum, and Diplostomum paracaudum. The latter of these species was found to be the most similar to the present material. Our results do not support an evolutionary separation of the European and North American species of Diplostomum.

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.

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 electron-dense 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.

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 parasites 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.

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.

Description of Maritrema portucalensis sp. nov. (Digenea, Microphallidae) parasite of Carcinus maenas (Crustacea, Decapoda) from Aveiro estuary, Northern Portugal

Maritrema portucalensis sp. nov. (Digenea, Microphallidae) from Aveiro estuary, northern Portugal, is described on the basis of excysted metacercariae obtained from the gill lamellae of the shore crab Carcinus maenas (L.). M. portucalensis sp. nov. most closely resembles Maritrema subdolum Jägerskiöld, 1909, a species that also frequently uses C. maenas as second intermediate host. The new species differs from M. subdolum in having a narrower body at the level of testes and a smaller ovary and testes. Also, the digestive caeca presented various folds along their length, thickening in the terminal region. Moreover, the body surface was fully covered by tegumental spines provided with several teeth. To complement our morphological characterization and to identify M. portucalensis metacercariae, the ITS1 rDNA region of the cysts isolated from C. maenas was sequenced and compared with the corresponding available sequences of digenean trematodes belonging to the family Microphallidae. Alignments revealed 28 base-pair differences between the query-ITS1 and that of M. subdolum, and more importantly a 100% similarity with the sequence of Microphallidae sp. no. 15 cercaria from the snail Hydrobia ulvae. The last observation allowed us to establish an unequivocal association between cercaria known as Microphallidae sp. no. 15, and the metacercaria of M. portucalensis sp. nov. characterized in this work, contributing decisively to the clarification of its life cycle.