TESI DI DOTTORATO Sede Amministrativa: Università degli Studi di Padova Dipartimento di Medicina Animale, Produzioni e Salute CORSO DI DOTTORATO DI RICERCA IN: SCIENZE VETERINARIE CICLO XXXI PARASITIC INFECTIONS IN SEA TURTLES AND CETACEANS IN MEDITERRANEAN SEA WATERS, WITH A FOCUS ON THE ADRIATIC SEA Coordinatore: Ch.mo Prof. Valentina Zappulli Supervisore: Dott.ssa Federica Marcer Dottorando : Erica Marchiori Index Abstract pg. 1 Background pg. 3 SECTION I – SEA TURTLES 1. Blood flukes (Spirorchiidae, Stunkard 1921) pg. 7 2. Gastrointestinal helminth community of Caretta caretta pg. 11 3. Aims of the project and outputs pg. 14 4. Paper 1 pg. 24 Cardiovascular flukes (Trematoda: Spirorchiidae) in Caretta caretta Linnaeus, 1758 from the Mediterranean Sea 5. Paper 2 pg. 53 A comprehensive mitochondrial DNA mixed-stock analysis clarifies the composition of loggerhead turtle aggregates in the Adriatic Sea 6. Paper 3 pg. 75 Qualitative and quantitative methods to estimate spirorchidiasis burden in sea turtles 7. Scientific communications pg. 92 SECTION II - CETACEANS 1 Parasitofauna of cetaceans: metazoan parasites pg. 109 2. Protozoan parasites of cetaceans: Toxoplasma gondii pg. 117 3. Aims of the project and outputs pg. 121 4. Paper 4 pg. 133 Parasitological and pathological findings in fin whales (Balaenoptera physalus) stranded along Italian coastlines 5. Paper 5 pg. 159 Morphological and molecular characterization of adults and larvae of Crassicauda spp. (Nematoda: Spirurida) from Mediterranean fin whales Balenoptera physalus (Linnaeus, 1758) 6. Scientific communications pg. 177 Conclusions and future perspectives pg. 185 Abstract Parasitic infections contribute to natural mortality in population of free ranging sea turtles and cetaceans worldwide. Notwithstanding all host-dependant factors influencing the outcomes of parasitic infections, some parasites, spanning from protozoan to helminthic taxa, have a high pathogenic potential and can cause fatal disease in these animals. Sea turtles blood flukes (Digenea: Spirorchiidae) are a main factor in mortality of populations of green and loggerhead turtles in Atlantic and Pacific Ocean. In order to explore presence and pathology of spirorchiidiasis in the Mediterranean area, data from 168 loggerhead turtles (Caretta caretta) stranded dead along North Western Adriatic coast were collected during a six-years period. Parasitological study and pathological findings revealed the presence of eggs and adult flukes of Hapalotrema mistroides and Neospirorchis – Neogen 11, with prevalence of 15.5% and 6.0% respectively. Mild lesions were observed, including multifocal granulomatous enteritis and chronic focal vasculitis of the major vessels. Egg emboli were found widespread in the tissues, mostly attributable to H. mistroides. Sequencing of rDNA markers (28S and ITS-2) and phylogenetic analyses revealed the identity of the Mediterranean isolates with Floridian specimens. Genetic analysis on the hosts led to the conclusion that the life cycles of both genera can be entirely supported by the Mediterranean ecosystem. To estimate the infection intensity, a new rapid method was set up. Correlation between spleen egg burden and fecal egg output was studied by means of statistical tests applied to splenic tissue and fecal material, revealing the absence of correlation and the unreliability of fecal burden to assess severity of the disease in vivo. Gastrointestinal helminth community of loggerheads was also studied, revealing a species diversity typical of demersal stage turtles and similar to that observed in other neritic areas of the Mediterranean. As for cetaceans, crassicaudosis is considered one of most severe helminthic infections in these animals. Nevertheless, knowledge on the epidemiology of Crassicauda species is scarce, due to constraints of sampling free-ranging wild host species and to the difficulty of obtaining entire specimens suitable for morphologic studies. Presence and pathology of Crassicauda boopis were investigated in fin whales (Balaenoptera physalus) stranded along Italian coasts. Severe lesions linked to the presence of adult nematodes in circulatory system and kidneys were detected in five rorquals (5/7), associated with renal function impairment and arteritis of the mesenteric vessels linked to spirurid larvae migrans. Morphological studies enabled the identification of the adult nematodes as C. boopis. Sequencing of molecular barcode regions was performed on adult and larval nematodes from the whales; additionally, specimens of Crassicauda spp. isolated from toothed whales were morphologically and molecularly studied. A multigene analysis made on the barcode regions of rDNA and on the mtDNA revealed the ITS-2 spacer to be the most efficient 1 marker in species differentiation inside the genus Crassicauda. This analysis opened new insights on the identity of the isolated larvae migrans and on the life cycles of these poorly known nematodes. Furthermore, during the parasitological examination, immature elements of the genus Pennella were recovered from one fin whale and described from a morphological point of view for the first time. Preliminary molecular data were obtained, with the aim of clarifying the taxonomy of the genus Pennella. The coccidian Toxoplasma gondii was detected in the tissues of one rorqual as well, likely associated with a chronic infection. In conclusion, these parasitological surveys highlighted the importance of a continue monitoring of the health of these endangered species avoiding underestimating the potential impact of parasitic diseases in natural mortality. 2 Background The Mediterranean Sea, including the Adriatic basin, are the home of several species of cetaceans and sea turtles. Due to the migratory nature of these animals, most of the populations within the Mediterranean cannot be considered completely isolated from the adjacent Atlantic populations; nevertheless, genetic tools have demonstrated that Mediterranean populations of several species of cetaceans and sea turtles actually diverged in different ages from the Atlantic ones (Berubé et al., 1998; Gaspari et al., 2007). Loggerheads (Caretta caretta) and green turtles (Chelonia mydas) regularly nest along Mediterranean coasts, the second species being much more abundant in the Eastern region. Loggerheads are instead regularly present in all the basin, and are found in the Adriatic Sea all over the year, with seasonal variations in their abundance and distribution (Margaritoulis et al., 2003; Casale et al., 2004; Lazar et al., 2004; Casale and Margaritoulis, 2010; Casale and Simone, 2017). The opportunistic nature of this species and the richness and variety of preys inside this arm of the Mediterranean Sea make it an important foraging area for loggerheads at the neritic stage. The deeper waters of the Ionian Sea and Balearic Sea provide a suitable habitat for smaller turtles at the pelagic stage. Rookeries from Greece and Crete, with Cyprus and Turkey, provide the most important contribution to the Adriatic stock; other important nesting sites for the Mediterranean population are included in the North African coasts and Southern Italy, nevertheless, the large-scale migratory habits of these reptiles enables individuals originating in the Western Atlantic coasts to reach the Mediterranean waters through the Gibraltar strait. Their presence has been occasionally registered also in the Adriatic Sea (Clusa et al., 2014). Common bottlenose dolphins (Tursiops truncatus, Montagu, 1821), striped dolphins (Stenella ceruleoalba, Meyen, 1833), Risso's dolphins (Grampus griseus [Cuvier, 1812]) and Cuvier's beaked whales (Ziphius cavirostris, Cuvier, 1823) are regularly present in the Adriatic Sea. With the exception of the bottlenose dolphin, which is adapted to live also on the continental shelf of the Northern region of the Adriatic, all other species of odontocetes prefer the deep waters of the Southernmost sub-region and are most frequently sighted in other areas of the Mediterranean, such as in the Pélagos Sanctuary and in Western Mediterranean until the Alboran Sea. The fin whale (Balaenoptera physalus), the only mysticete regularly present in the Mediterranean, is considered also a seasonally regular species in the central Southern Adriatic. Recent surveys, carried out in the framework of the European Project “Network for the conservation of Cetaceans and Sea Turtles” (NETCET), confirmed the presence of fin whale specimens in the central Adriatic, likely 3 related to the presence of masses of krill in a limited part of the year (Fortuna et al., 2015). In the Mediterranean, it is more abundant in highly productive pelagic regions, such as the Ligurian- Corsical-Provençal Basin, where high whale densities, comparable to those of rich oceanic habitats, are described (Notarbartolo di Sciara et al. 2007). Relying on genetic analyses, the Mediterranean fin whales have been defined as a sub-population, the animals being mainly resident inside the basin. Total estimated abundance of this species in the Mediterranean reaches no more than 5,000 individuals (Fortuna et al., 2015). Human-induced mortality is a well-known threat to the conservation of sea turtles and cetaceans worldwide; bycatch, ship strikes and pollution by both chemicals and marine debris are considered causes of major concern, and mitigation strategies are under researchers’ attention to ensure conservation of marine species. Environmental stressors, here including immunotoxicity of chemical substances, direct and indirect