Caratterizzazione genetica di emoparassiti (hepatozoon) e valutazione dell’impatto sulle popolazioni di iguana terrestre delle isole galápagos (conolophus)

The Phylum Apicomplexa includes obligate intracellular parasites that are extremely relevant in human and veterinary medicine as responsible for a wide variety of diseases. Infections by Apicomplexa may severely impact species in the wild and this causes further concern for endangered wildlife. Among Apicomplexa, species of the genus Hepatozoon (Miller, 1908) infect a wide variety of mammal, bird, reptile and amphibian hosts, The infection by this parasite causes anemia or blood cell abnormalities, resulting in immunosuppression and also may lead to reduction of growth, reproductive fitness or juvenile’s survival. The impact and specificity of Hepatozoon infections on wildlife natural populations are poorly known, especially in the case of reptiles. In this regard, parasites of the genus Hepatozoon have been found in the blood of three species of Galápagos land iguanas (Conolophus subcristatus, C. pallidus and C. marthae), endemic to that archipelago and recognized as endangered as result of direct and indirect human impact on the wildlife of the islands. Currently there are no basic information about levels and pattern of genetic variability, dispersal patterns and the dynamics of evolutionary processes of Hepatozoon associated with Conolophus. In this thesis, the genealogical relationships (phylogeny) and the geographical structure of genetic divergence (phylogeography) between the different lineages of Hepatozoon found in Galápagos iguanas has been investigated. A molecular characterization of the parasite was conducted by amplification of the 18S rRNA. This study revealed a high degree of haplotype variability in Hepatozoon associated with iguanas of the Galápagos Islands. This variability is geographically structured and originated through multiple events of differentiation within separate lineages. The level of divergence between these lineages is comparable to what is observed between nominal species and suggests that more than one species of Hepatozoon occur in Galápagos. Data suggest that this protozoan has entered the archipelago more than once, but it is not yet clear which lineage of Hepatozoon in these islands can be considered ancestral. Hepatozoon shows a complex evolutionary pattern, with two main clades, one of which contains lines that seem to have specialized to placental mammals hosts, while the other (which includes the lineages from Galápagos) comprises lines that have retained the ability to infect different hosts, including amphibians, reptiles, mammals, both marsupials (more than once, independently) and placentals. Further studies are needed to clarify the tempo and mode of the evolution of this interesting haemoparasite. To clarify the status and characteristics of Hepatozoon infection associated with Galapagos land iguanas, prevalence, parasitemia and haematological values were assessed for the three species of land iguanas. Prevalence and parasitemia were determined by using both a traditional, microscopy-based and a PCR approach. The results indicated that the PCR-based technique developed in the study have a better power of resolution, compared to the microscopic examination. In this study, the PCR technique has established the presence of Hepatozoon as the only parasite in the blood of Colonophus and by sequencing it also allowed the clarification of the extent and modalities of co-infection. In this study, different populations of Conolophus exhibit different degrees of prevalence and parasite loads varies from medium to low level. Comparison between values of parasitemia and prevalence for the two populations of volcano Wolf (WS and WM) shows that C. subcristatus is characterized by a high number of infected individuals and a medium parasite load, C. marthae shows a reversed pattern, with very high prevalence and low parasitemia. The analysis of white blood cell count for individuals parasitized shows significant differences between WS and WM and the other populations, in eosinophils and monocytes. The altered leucocytic formula observed in WS and WM does not correlate to the presence of Hepatozoon in the blood of iguanas. Probabily, special environmental conditions that causes the same pattern of variation of the parameters of white cell in C. subcristatus and C. marthae occur in volcano Wolf. This study provide the first original hematological and parasitological data set on wild populations of Galapagos land iguanas, contributing a physiological range of normal hematological values for C. subcristatus. Microsatellite data were used to estimate Ne and the level of genetic variability of C. marthae. Levels of genetic variability and structure of Galapagos land iguana populations were also confirmed. Ultimately, no correlation was found between levels of genetic variation and parameters indirectly related to fitness.