Susana Pina

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.

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.

Optic Neuritis in an Adult Patient with Chickenpox

Central nervous system involvement in a patient with primary infection with Varicella zoster virus is rare, especially in the immunocompetent adult. In particular, isolated optic neuritis has been described in a small number of cases. The authors present a case of optic neuritis in an immunocompetent patient. A 28-year-old woman presented to the emergency room with a history of headaches during the previous week, without visual symptoms. The examination was unremarkable, except for a rash suggestive of chickenpox and hyperemic and edematous optic disc, bilaterally. Visual acuity and neurological examination were normal. Two days later, she complained of pain on eye movement and decreased visual acuity, which was 20/32 in her right eye and 20/60 in her left eye. Four days after admission, her visual acuity started to improve, and two months later, she had 20/20 visual acuity in both eyes. To our knowledge, this is the first reported case of an immunocompetent adult in which a Varicella zoster virus associated optic neuritis presented with fundoscopic changes before decreased visual acuity. This suggests that this condition may be underdiagnosed in asymptomatic patients.

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.

A new species of gymnophallid (Digenea) and an amended diagnosis of the genus Gymnophalloides fujita, 1925.

Abstract:  Morphological and molecular evidence suggest that specimens formerly described as Lacunovermis sp. from Nacella (Patinigera) spp. (Patellogastropoda: Patellidae) belong to a new species of Gymnophalloides Fujita, 1925. Based on the new information, they are identified as Gymnophalloides nacellae n. sp. The new species differs from Gymnophalloides tokiensis, Gymnophalloides seoi, and Gymnophalloides heardi mainly through the presence of a group of papillae located on the ventral surface between oral and ventral suckers. A detailed morphological study revealed the lack of pars prostatica, a character previously reported in G. seoi, which is why it was formerly placed in the Gymnophallinae. Molecular information proved that G. nacellae is close to G. seoi, being nestled together with Parvatrema representatives. This molecular information, along with the absence of pars prostatica, allows these 2 genera to be placed in Parvatrematinae. An amended diagnosis of Gymnophalloides is provided. Histological sections of mantle epithelium of the limpet show metacercariae attached by their oral and ventral suckers in a similar manner to G. seoi in its host, the oyster Crassostrea gigas. Tissue reaction includes cells of outer mantle epithelium being stretched by sucker attachment, hemocyte infiltration of connective tissue between mantle epitheliums, and abnormal calcareous deposition on the inner surface of the shell.

Revision of the family Gymnophallidae Odhner, 1905 (Digenea) based on morphological and molecular data

This paper reviews the family Gymnophallidae, recognizing as valid seven genera; four within the subfamily Gymnophallinae: Gymnophallus Odhner, 1900 (syn. Meiogymnophallus Ching, 1965), Paragymnophallus Ching, 1973, Pseudogymnophallus Hoberg, 1981, and Bartolius Cremonte, 2001, and three in the Parvatrematinae: Parvatrema Cable, 1953, Lacunovermis Ching, 1965, and Gymnophalloides Fujita, 1925. Specimens representing one species of each available genus were chosen from those well-described and non controversial species, for which strong morphological information was available, and used for molecular studies (ITS1-5.8S-ITS2-28S rDNA strands were sequenced). The presence or absence of a pars prostatica differentiates between the 2 subfamilies, Gymnophallinae and Parvatrematinae. The characters used to differentiate genera are: location of the ovary (pre-, post- or inter-testicular), size and location of the genital pore (inconspicuous and located at the anterior margin of the ventral sucker, or conspicuous and located at some distance from the anterior margin of ventral sucker), presence of caecal pockets, and presence or absence of ventral pit (a muscular structure which can be either well-developed, similar in size and musculature to the ventral sucker, or be poorly developed). The characters previously used to distinguish among genera that actually should be considered to separate species include: shape of tegument spines (broad, sharp or serrated), presence of lateral projections on the oral sucker (also called papillae or lips), shape of the seminal vesicle (unipartite or bipartite), shape of the prostatic duct (elongate or oval), presence of papillae on the genital pore, shape of the genital atrium (tubular, wide, oval), shape of the vitellaria (follicular in a variable degree, paired or single), shape of the excretory vesicle (V or Y), and extension of uterus (restricted to forebody, at hindbody or extending in both). Additionally, some of these characters may vary with the age of worm. The morphological and molecular information obtained in this study provided strong support for recognizing seven valid genera in the family Gymnophallidae.

Ocular Toxicity Secondary to Asclepias physocarpa: The Balloon Plant

We report a case of a 65-year-old woman with symptoms of blurred vision and ocular irritation a few hours after accidental contact of the right eye with Asclepias physocarpa milky latex. Observation showed a diffuse conjunctival hyperemia and stromal corneal edema with Descemets membrane folds. Recovery was fast and apparently complete in less than one month. However, specular microscopy at 6-months follow-up showed an abnormal endothelial morphology as sequelae, suggesting this condition is not as innocuous as it has been suggested.