Tesis doctoral de Gema Alama-bermejo
Aquaculture is a growing agricultural sector that supplies nearly half of the food fish consumed by humans worldwide. In the mediterranean, the aquaculture market is dominated by two species, the sea bass (dicentrarchus labrax, moronidae) and the gilthead sea bream (sparus aurata, sparidae). The excess of production has led to a market saturation and an important decrease in price, which has provoked an urgent need for establishing new fish species in aquaculture in order to diversify the market. Species of the family sparidae are important candidates for species diversification in the mediterranean aquaculture because they are highly appreciated for commercial use due to their excellent flesh. One of the main handicaps in the commercial culture of fish is disease. Infectious diseases are produced by a variety of pathogens, i.E. Bacteria, viruses or parasites. While parasites generally occur in low numbers in the natural environment, under culture conditions, they may multiply more rapidly, cause disease and mortalities and have an important impact on the production of fish. Some of the most problematic parasites in the mediterranean aquaculture belong to the myxozoa (cnidaria), the monogenea (platyhelminthes) and to the aporocotylidae (trematoda). this thesis aims to improve our knowledge of myxozoan and aporocotylid parasites in sparids which are new candidates or existing mediterranean aquaculture species. The species studied are the sand steenbras lithognathus mormyrus, the picarel spicara smaris, the annular seabream diplodus annularis, the common two-banded seabream diplodus vulgaris, the sharpsnout seabream diplodus puntazzo and the gilthead seabream sparus aurata. The present study seeks to identify, describe and clarify the taxonomic position of the parasite taxa present in these hosts by using morphological and molecular based approaches, and by defining their phylogenetical position and relationship with previously described species. Furthermore, development and habitat selection in the host, as well as pathology are analysed in order to evaluate the pathogenic potential of the parasites described in the present work. Another aspect of this thesis focuses on the study of parasite life cycles, seasonality, transmission and routes of infection in the fish host, with the final aim to provide information allowing for the development of management strategies for aquaculture systems. the study led to the following findings and conclusions: 1) a new genus and species of blood fluke, skoulekia meningialis n. Gen., N. Sp. Was described from the common two-banded seabream d. Vulgaris. The erection of the new genus was strongly supported by morphological data as well as molecular phylogeny (ssu, its2 and lsu rdna). Skoulekia was demonstrated to have a close relationship with the genera psettarium and pearsonellum. S. Meningialis was detected in a special location, the ectomeningeal veins surrounding the optic lobes of the brain of d. Vulgaris. This contrasts studies on the majority of aporocotylids, which usually inhabit the heart or the blood vessels of the gills. While blood fluke pathology is normally almost exclusively related to the eggs trapped within the gill vessels, it is the adults of s. Meningialis which were found to cause mild, chronic, localised meningitis. S. Meningialis did not seem to cause an important pathological effect on wild d. Vulgaris. However, its special location surrounding a vital organ should be kept in mind when establishing d. Vulgaris in aquaculture systems. 2) macroscopic, elongate plasmodia containing myxosporean spores belonging to the genus unicapsula were found in the skeletal muscle of the striped seabream, l. Mormyrus. The only species of unicapsula described from the mediterranean to date is unicapsula pflugfelderi from the picarel spicara smaris. A morphological and molecular comparison (ssu rdna) was carried out between u. Pflugfelderi from s. Smaris and unicapsula sp. From l. Mormyrus. Despite differences related to the size of plasmodia, spore morphology showed minor differences and ssu rdna sequences were identical, thus determining conspecificity of the myxozoans from the two hosts. Ultrastructural data revealed the presence of two previously undescribed structures in the sporoplasm of u. Pflugfelderi spores: a crystalline structure and a vesicular body, which may be related to osmotic changes and the production of material for polar capsule formation. Morphological comparison of all species of the genus unicapsula described to date revealed that, according to shell valve distributions in the spores, two basic types can be defined, dividing the genus unicapsula into two groups. As molecular data are missing, it is unclear whether this separation is artificial or is mirrored by phylogenetic clustering. Disruption of the muscle tissue (myoliquefaction) is common for species of unicapsula and kudoa and may render fillets liquefied and unfit for marketing. This condition does not occur in u. Pflugfelderi-infected s. Smaris, however, the massive occurence of macroscopic plasmodia in the fillets of l. Mormyrus could produce marketing problems due to rejection by the consumer. 3) myxosporean spores belonging to the genus ceratomyxa were detected in the gall bladder of the sharpsnout seabream d. Puntazzo. A detailed morphological and molecular (ssu rdna) characterisation of these spores and a comparison with existing descriptions of ceratomyxids resulted in the erection of a new species, ceratomyxa puntazzi n. Sp. C. Puntazzi was found to cause necrosis and epithelial sloughing in the gall bladder, and to provoke mild pericholangitis in the liver tissue surrounding the bile ducts of d. Puntazzo. Whereas no direct mortalities were observed, c. Puntazzi may be considered an opportunistic parasite as shown in a previous study on ceratomyxid infection in steroid-treated, potentially immunosuppressed d. Puntazzo. Ssu rdna data of three other ceratomyxids from two sparids sharing the habitat with d. Puntazzo, i.E. D. Annularis and s. Aurata were also collected and indicate that the genus ceratomyxa is very host-specific in sparids this agrees with data on ceratomyxids previously obtained from other fish families. The phylogenetical results revealed that all available ceratomyxid species from sparids in the mediterranean arise from a common ancestor. The present results indicate that a high species diversity can be expected from ceratomyxids in mediterranean sparids. 4) the combination of light microscopy, confocal laser scanning microscopy using specific stains and electron microscopy was used for a detailed study of the three-dimensional morphology, ultrastructure and cellular composition of c. Puntazzi parasite stages in the bile. It was shown that the combined use of several microscopic methods can considerably improve the morphological characterization of myxozoan microparasites and allows for unique insights into the organisation of different cellular components. C. Puntazzi showed two developmental pathways in the bile of d. Puntazzo, i.E. Pre-sporogonic proliferation and sporogony. In the pre-sporogonic development proliferation occurred by means of plasmotomy with frequent budding. Pre-sporogonic stages were also shown to be highly motile. For the first time, myxozoan locomotive behaviour and the distribution and potential effectors of motility were analysed providing unique information on the processes of directional locomotion and budding: f-actin rich cytoskeletal elements were found to concentrate at one end of the parasite, which produces localised filopodia, thus facilitating parasite motility. Furthermore, the mechanism of budding was identified, for the first time, as a process dependent on f-actin concentration at opposite ends of the separating stages, thus causing their active separation. Other structures detected in the present study were lipid droplets and external blebs which might facilitate buoyancy of the parasites. In sporogonic stages, f-actin was more evenly disperse, coinciding with the end of the swimming period of the stages and the release of the mature spores. 5) monthly natural exposures of sharpsnout seabream d. Puntazzo to infective actinosporean stages of c. Puntazzi throughout a full year was conducted during the present study and demonstrate the first attempt of a seasonal infection study in marine myxozoans. Natural exposure of fish in a small cage and subsequent maintenance of fish in the laboratory for 60 days at 20-23ºc resulted in c. Puntazzi infection in the receptor fish. The parasite presented a marked temperature-related seasonality, with microscopical detection of proliferative stages and spores in the bile exclusively from april until november. Two peaks of infection prevalence were registered during this period: in april-may (80%), when seawater temperature first reached 16ºc, and in october (86%), while prevalences were lower from june to september (46-64%). These two peaks of infection prevalence could be related to actinospore production and/or viability. However, pcr detection in the bile was possible all year round, which indicates that the parasite can infect and stay in the fish in low numbers (latent infection) during the winter months. Blood stages were detected by pcr in sentinel fish and presented a very high prevalence after exposure and all year round, but the infection was sometimes cleared or lost over time, as not all fish initially infected successfully established in the bile. This may be dependent on the infective parasite dose or on the immunological condition of host in the winter months. The combination of pcr results obtained from blood, gall bladder and liver suggests the following route of infection: entry into the blood system in the gills, as suggested for other myxozoans, exit into the liver tissue, entry into the bile ducts, through which the parasite reaches the gall bladder, where it starts pre-sporogonic proliferation and sporogony with the consequent release of spores with the bile via the intestine into the environment. Artificial transmission of different developmental stages, i.E. Blood stages as well as pre-sporogonic proliferative and sporogonic stages of c. Puntazzi via oral, intracoelomic, bath application or cohabitation was not successful. This indicates that fish-to-fish transmission as demonstrated previously for enteromyxum spp. Is unlikely to occur in c. Puntazzi. The natural exposure study implicates that initial exposure of fish/transfer from the hatchery to sea cages is best done in late autumn/early winter, as the parasite would then establish in low number and potentially cause some kind of immunization, potentially resulting in higher resistance to re-infection with high numbers of parasites in the following summer. This has been demonstrated in other myxozoans and is an important implication for control measures of d. Puntazzo.
Datos académicos de la tesis doctoral «Parasitological study of cultured and wild sparids in the mediterranean«
- Título de la tesis: Parasitological study of cultured and wild sparids in the mediterranean
- Autor: Gema Alama-bermejo
- Universidad: Universitat de valéncia (estudi general)
- Fecha de lectura de la tesis: 05/12/2011
Dirección y tribunal
- Director de la tesis
- Juan Antonio Raga Esteve
- Tribunal
- Presidente del tribunal: Juan Antonio Balbuena díaz-pinés
- pavla Bartosova (vocal)
- ariadna Sitjí bobadilla (vocal)
- monica Caffara (vocal)