Gema Alama-Bermejo

Understanding myxozoan infection dynamics in the sea: Seasonality and transmission of Ceratomyxa puntazzi

Ceratomyxa puntazzi affects the sharpsnout seabream, Diplodus puntazzo, a recently explored aquaculture species in the Mediterranean. Little is known about the transmission and seasonality of marine myxozoans, although this knowledge is of considerable importance for the design of management strategies for aquaculture. In the present study on C. puntazzi we investigated the potential pathways of transmission as well as the parasite abundance in fish and its density in environmental water samples, throughout a full year. We performed monthly sentinel fish exposures in a C. puntazzi enzootic environment and quantified waterborne stages in seawater. Two novel C. puntazzi-specific PCR and quantitative PCR assays were developed to determine infection levels in fish and water samples. Ceratomyxa puntazzi presents marked seasonal changes in parasite density, with a double-peaked prevalence of infection in sentinel fish in spring and late summer/autumn, at 16-24°C, and a covert infection during the winter months. Invasive blood stages were detected all year round by PCR. The combination of sentinel fish exposure with the quantification of waterborne stages allowed us to attribute this pattern in C. puntazzi density to higher numbers of actinospores in the water, while myxospores are predominant in summer and winter. We demonstrated that temperature increase triggered actinospore production in the invertebrate host in a benthic habitat and we suggest that the life cycle dynamics of the invertebrate host explain the double-peaked infection prevalence in fish. Experimental transmission of different C. puntazzi developmental stages in seawater or by oral and intracoelomic injection was unsuccessful which indicates fish-to-fish transmission is unlikely to occur in aquaculture systems. This is the first model studying seasonality and infection dynamics of a marine myxozoan.

Skoulekia meningialis n. gen., n. sp. (Digenea: Aporocotylidae Odhner, 1912) a parasite surrounding the brain of the Mediterranean common two-banded seabream Diplodus vulgaris (Geoffroy Saint-Hilaire, 1817) (Teleostei: Sparidae): description, molecular phylogeny, habitat and pathology.

This study describes a new aporocotylid genus and species, Skoulekia meningialis n. gen., n. sp. which was detected in the ectomeningeal veins surrounding the optic lobes of the brain of the common two-banded seabream Diplodus vulgaris (Geoffroy Saint-Hilaire, 1817) from the Gulf of Valencia (Mediterranean Sea). A detailed morphological description of S. meningialis is provided, including drawings, measurements and scanning electron microscopy images as well as a phylogenetic study of S. meningialis and closely related taxa using DNA sequence data obtained from whole ITS2 and partial 18S and 28S rDNA regions. Morphology as well as molecular phylogeny strongly support the erection of a new genus and demonstrate its close relationship with the genera Psettarium (Goto & Ozaki, 1930) and Pearsonellum Overstreet & Køie, 1989. Skoulekia is the second aporocotylid genus described in the Sparidae, a family including economical important fishes. In contrast to the majority of the aporocotylids, which inhabit the heart or the blood vessels of the gills, S. meningialis inhabits the ectomeningeal veins surrounding the optic lobes. Eggs were found trapped within the gill vessels. Normally, blood fluke pathology is almost exclusively related to the eggs. However, in the case of S. meningialis, main histopathological alterations were related to the adult blood flukes which were found to cause mild localised meningitis.

Host-parasite relationship of Ceratomyxa puntazzi n. sp. (Myxozoa: Myxosporea) and sharpsnout seabream Diplodus puntazzo (Walbaum, 1792) from the Mediterranean with first data on ceratomyxid host specificity in sparids.

Sparidae are economically important fishes to both, fisheries and aquaculture in the Mediterranean. Species diversification is an important strategy for the development of Mediterranean aquaculture. One of the species recently introduced is the sharpsnout seabream Diplodus puntazzo (Walbaum, 1792). During a parasitological study of fish from the Gulf of Valencia and the Mar Menor (Spain), myxozoan spores belonging to the genus Ceratomyxa were found in the gall bladder of D. puntazzo. A morphological description of the spores, which includes histology and transmission electron microscopy (TEM) as well as molecular (SSU ribosomal DNA) data resulted in the erection of a new species, Ceratomyxa puntazzi n. sp. A histopathological study of C. puntazzi n. sp. infection in D. puntazzo showed that the parasite causes necrosis and loss of epithelial cells in the gall bladder, and provokes a pericholangitis in the liver tissue surrounding the bile ducts. Furthermore, molecular data obtained from C. puntazzi n. sp. and three other ceratomyxids from the closely related fish species Diplodus annularis L. and Sparus aurata L. which share the same habitat suggest that the genus Ceratomyxa is host-specific in sparids, which agrees with data previously obtained from Serranidae, Labridae and Pomacentridae, and that ceratomyxid species from sparids in the Mediterranean originated from a common ancestor.

3D Morphology, Ultrastructure and Development of Ceratomyxa puntazzi Stages: First Insights into the Mechanisms of Motility and Budding in the Myxozoa

Free, amoeboid movement of organisms within media as well as substrate-dependent cellular crawling processes of cells and organisms require an actin cytoskeleton. This system is also involved in the cytokinetic processes of all eukaryotic cells. Myxozoan parasites are known for the disease they cause in economical important fishes. Usually, their pathology is related to rapid proliferation in the host. However, the sequences of their development are still poorly understood, especially with regard to pre-sporogonic proliferation mechanisms. The present work employs light microscopy (LM), electron microscopy (SEM, TEM) and confocal laser scanning microscopy (CLSM) in combination with specific stains (Nile Red, DAPI, Phalloidin), to study the three-dimensional morphology, motility, ultrastructure and cellular composition of Ceratomyxa puntazzi, a myxozoan inhabiting the bile of the sharpsnout seabream. Our results demonstrate the occurrence of two C. puntazzi developmental cycles in the bile, i.e. pre-sporogonic proliferation including frequent budding as well as sporogony, resulting in the formation of durable spore stages and we provide unique details on the ultrastructure and the developmental sequence of bile inhabiting myxozoans. The present study describes, for the first time, the cellular components and mechanisms involved in the motility of myxozoan proliferative stages, and reveals how the same elements are implicated in the processes of budding and cytokinesis in the Myxozoa. We demonstrate that F-actin rich cytoskeletal elements polarize at one end of the parasites and in the filopodia which are rapidly de novo created and re-absorbed, thus facilitating unidirectional parasite motility in the bile. We furthermore discover the myxozoan mechanism of budding as an active, polarization process of cytokinesis, which is independent from a contractile ring and thus differs from the mechanism, generally observed in eurkaryotic cells. We hereby demonstrate that CLSM is a powerful tool for myxozoan research with a great potential for exploitation, and we strongly recommend its future use in combination with in vivo stains.

Morphological and molecular redescription of the myxozoan Unicapsula pflugfelderi Schubert, Sprague & Reinboth 1975 from two teleost hosts in the Mediterranean. A review of the genus Unicapsula Davis 1924

Elongate plasmodia with myxosporean spores belonging to the genus Unicapsula, Davis, 1924 were found in the skeletal muscle of the striped seabream, Lithognathus mormyrus (L.), a candidate for the mediterranean aquaculture. The only species of Unicapsula described from the Mediterranean is Unicapsula pflugfelderi Schubert et al. 1975, which occurs in the picarel, Spicara smaris (L.). For morphological and molecular comparison of U. pflugfelderi from S. smaris with Unicapsula sp. from L. mormyrus measurements of plasmodia and spores, ultrastructural details and 18S and 28S rDNA sequences were analysed. Whereas plasmodia were 2-3 times larger in S. smaris than in L. mormyrus (length 2.47-0.81 mm; width 0.22-0.09 mm; P = 0.000), spore morphology showed minor differences and both 18S and 28S rDNA sequences were 100% identical identifying the myxozoan as U. pflugfelderi. Scanning electron microscopy of the spores revealed a different shell valve distribution than the one used for the diagnosis of the genus Unicapsula. This resulted in a review of the genus Unicapsula dividing it into two morphological groups of different spore valve arrangement. TEM revealed the presence of a yet undescribed crystalline structure in the sporoplasm of the spores.

Cellular Processes in Myxozoans

This chapter describes general cellular processes associated with myxozoan development and includes the first review of motility at the cellular level. We consider modes of cell proliferation by endogeny and division by mitosis and meiosis. We also describe cellular processes associated with sporogony, including the formation of plasmodia and spores. We review cellular motility by amoeboid movement in host invasion and ‘dancing’ or twitching of presporogonic proliferative stages in some myxosporeans. The key role of F-actin and myosin in cellular motility is highlighted.

Species complexes and phylogenetic lineages of Hoferellus (Myxozoa, Cnidaria) including revision of the genus: A problematic case for taxonomy

BackgroundMyxozoans are metazoan parasites whose traditional spore morphology-based taxonomy conflicts DNA based phylogenies. Freshwater species of the genus Hoferellus are parasites of the excretory system, with several members infecting food and ornamental fish species, as well as amphibians. This study aims to increase our understanding of their molecular diversity and development, aspects about which little is known, and to generate a molecular diagnostic tool to discriminate between different pathogenic and non-pathogenic Hoferellus spp.MethodsSSU and ITS rDNA phylogeny, along with morphological descriptions using light and electron microscopy were used to identify and characterize Hoferellus species collected from the urinary system of fishes and frogs. A PCR-based diagnostic assay was designed to differentiate between cryptic Hoferellus spp in cyprinid fishes commonly cultured in Central Europe.ResultsOur phylogenetic results separate the species of Hoferellus into two phylogenetic sublineages which are indistinguishable on the basis of generic morphological traits: 1) The Hoferellus sensu stricto sublineage, which is composed of the type species Hoferellus cyprini, Hoferellus carassii and a cryptic species, Hoferellus sp. detected only molecularly in common carp. 2) The Hoferellus sensu lato sublineage into which the new species we described in this study, Hoferellus gnathonemi sp. n. from the kidney of the elephantnose fish and Hoferellus anurae from reed frogs, are placed together with Hoferellus gilsoni previously sequenced from European eel. Apart from phylogenetic analyses, we also provide novel ultrastructural data on the phagocytotic nature of some Hoferellus plasmodia and on the elusive intracellular stages ascribed to the presporogonic development of this genus.ConclusionsWe provide molecular evidence of the polyphyly of the genus Hoferellus and provide novel morphological details of its members. Based on the presented data, we revise and propose emendation of the genus Hoferellus.

A revision of the genus Neogrubea Dillon & Hargis, 1968 (Monogenea: Mazocraeidae): new morphological and molecular data from off the Patagonian coast of Argentina.

The genus Neogrubea Dillon & Hargis, 1968 (syn. Asymmetria Suriano, 1975) is revised based on examination of type- and voucher material, and from new specimens collected from the gills of Seriolella porosa Guichenot and Stromateus brasiliensis Fowler from off Patagonia, Argentina. Morphological comparisons based on light and scanning electron microscopy and molecular data (partial SSU and LSU rDNA sequences) of the monogeneans from off Patagonia suggest that N. seriolellae Dillon & Hargis, 1968 (syns N. stromateae Gibson, 1976, A. asymmetria Suriano, 1975 and A. platensis Rey & Meneses, 1985) is currently the only species of the genus. Neogrubea soni Evdokimova, 1969 is considered a species inquirenda. An emended diagnosis of Neogrubea is presented, and new host and locality records for N. seriolellae are given in detail. Morphological characters of the members of the mazocraeid subfamily Grubeinae Price, 1961 are also discussed.

Morphological and molecular characterisation of Aporocotyle margolisi Smith, 1967 (Digenea: Aporocotylidae) from the North Pacific hake Merluccius productus (Ayres) (Gadiformes: Merlucciidae) off Oregon, USA

Abstract Aporocotylid blood flukes conspecific with Aporocotyle margolisi Smith, 1967 were collected from the bulbus arteriosus of the North Pacific hake Merluccius productus (Ayres). This study revisits the morphology of A. margolisi, including drawings, measurements and scanning electron microscopy images, and provides for the first time molecular data for the large subunit of the ribosomal RNA (28S rDNA) and the mitochondrial cytochrome c oxidase subunit 1 (cox1) genes for this species. A 28S rDNA phylogenetic study of A. margolisi, and all available Aporocotyle spp., was also performed. The distribution range of A. margolisi is extended to the Pacific coast of the USA. We provide a morphological comparison of Aporocotyle spp. from the Pacific coast in North America as well as other Aporocotyle spp. infecting hake. Comparisons with the original description revealed that the new specimens of A. margolisi were considerably larger with respect to all morphological features, except for shorter spines. Molecular results showed a close relationship between A. margolisi and A. argentinensis Smith, 1969 from the Argentine hake Merluccius hubbsi Marini. The phylogenetic relationships of Aporocotyle spp. point to a possible co-speciation of hakes species and these blood fluke parasites.