Comparative Genomics of Field Isolates of Mycobacterium bovis and M. caprae Provides Evidence for Possible Correlates with Bacterial Viability and Virulence

Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly affect humans and animals worldwide. The life cycle of mycobacteria is complex and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Recently, comparative genomics analyses have provided new insights into the evolution and adaptation of the MTBC to survive inside the host. However, most of this information has been obtained using M. tuberculosis but not other members of the MTBC such as M. bovis and M. caprae. In this study, the genome of three M. bovis (MB1, MB3, MB4) and one M. caprae (MB2) field isolates with different lesion score, prevalence and host distribution phenotypes were sequenced. Genome sequence information was used for whole-genome and protein-targeted comparative genomics analysis with the aim of finding correlates with phenotypic variation with potential implications for tuberculosis (TB) disease risk assessment and control. At the whole-genome level the results of the first comparative genomics study of field isolates of M. bovis including M. caprae showed that as previously reported for M. tuberculosis, sequential chromosomal nucleotide substitutions were the main driver of the M. bovis genome evolution. The phylogenetic analysis provided a strong support for the M. bovis/M. caprae clade, but supported M. caprae as a separate species. The comparison of the MB1 and MB4 isolates revealed differences in genome sequence, including gene families that are important for bacterial infection and transmission, thus highlighting differences with functional implications between isolates otherwise classified with the same spoligotype. Strategic protein-targeted analysis using the ESX or type VII secretion system, proteins linking stress response with lipid metabolism, host T cell epitopes of mycobacteria, antigens and peptidoglycan assembly protein identified new genetic markers and candidate vaccine antigens that warrant further study to develop tools to evaluate risks for TB disease caused by M. bovis/M.caprae and for TB control in humans and animals.

First molecular detection and characterization of herpesvirus and poxvirus in a Pacific walrus (Odobenus rosmarus divergens)

BACKGROUND Herpesvirus and poxvirus can infect a wide range of species: herpesvirus genetic material has been detected and amplified in five species of the superfamily Pinnipedia; poxvirus genetic material, in eight species of Pinnipedia. To date, however, genetic material of these viruses has not been detected in walrus (Odobenus rosmarus), another marine mammal of the Pinnipedia clade, even though anti-herpesvirus antibodies have been detected in these animals. CASE PRESENTATION In February 2013, a 9-year-old healthy captive female Pacific walrus died unexpectedly at L'Oceanografic (Valencia, Spain). Herpesvirus was detected in pharyngeal tonsil tissue by PCR. Phylogenetic analysis revealed that the virus belongs to the subfamily Gammaherpesvirinae. Poxvirus was also detected by PCR in skin, pre-scapular and tracheobronchial lymph nodes and tonsils. Gross lesions were not detected in any tissue, but histopathological analyses of pharyngeal tonsils and lymph nodes revealed remarkable lymphoid depletion and lymphocytolysis. Similar histopathological lesions have been previously described in bovine calves infected with an alphaherpesvirus, and in northern elephant seals infected with a gammaherpesvirus that is closely related to the herpesvirus found in this case. Intracytoplasmic eosinophilic inclusion bodies, consistent with poxviral infection, were also observed in the epithelium of the tonsilar mucosa. CONCLUSION To our knowledge, this is the first molecular identification of herpesvirus and poxvirus in a walrus. Neither virus was likely to have contributed directly to the death of our animal.

Molecular Systematics and Traits Evolution in Phasmatodea Leach, 1815 (Hexapoda; Insecta)

Phasmatodea Leach, 1815 (Hexapoda; Insecta) is a polyneopteran order which counts approximately 3000 described species, often known for their remarkable forms of mimicry. In this thesis, I provide a comprehensive systematic framework which includes over 180 species never considered in a phylogenetic framework: the latter can facilitate a better understanding of the processes underlying phasmids evolutionary history. The clade represents in fact an incredible testing ground to study trait evolution and its striking disparity of reproductive strategies and wing morphologies have been of great interest to the evolutionary biology community. Phasmids wings represent one of the first and most notable rejection of Dollo’s law and they played a central role in initiating a long- standing debate on the irreversibility of complex traits loss. Macroevolutionary analyses presented here confirm that wings evolution in phasmids is a reversible process even when possible biases - such as systematic uncertainty and trait-dependent diversification rates - are considered. These findings remark how complex traits can evolve in a dynamic, reversible manner and imply that their molecular groundplan can be preserved despite its phenotypical absence. This concept has been further tested with phylogenetic and transcriptomic approaches in two phasmids parthenogenetic lineages and a bisexual congeneric of the European Bacillus species complex. Leveraging a gene co-expression network approach, male gonad associated genes were retrieved in the bisexual species and then their modifications in the parthenogens were charachterized. Pleiotropy appears to constrain gene modifications associated to male reproductive structures after their loss in parthenogens, so that the lost trait molecular groundplan can be largely preserved in both transcription patterns and sequence evolution. Overall, the results presented in this thesis contribute to shape our understanding of the interplay between the phenotypic and molecular levels in trait evolution.

Fusarium head blight: characterization of secondary Fusarium species, evaluation of their pathogenetic role and ability to produce mycotoxins

Fusarium head blight (FHB) is a worldwide cereal disease caused by a complex of Fusarium species resulting in high yield losses, reduction in quality and mycotoxin contamination of grain. A shift in Fusarium head blight community has been observed worldwide. The present work aimed to analyze the evolution of Italian FHB community focusing the attention on species considered “secondary” in the past years such as members of Fusarium tricinctum species complex (FTSC) and F. proliferatum. The first goal of the study was to analyze the fungal community associated with Italian durum wheat in two different years. F. poae, F. avenaceum and F. proliferatum were the main species detected on Italian durum kernels. A variable mycotoxins contamination was observed in the analyzed samples. Considering, the increased incidence of F. avenaceum and other members of FTSC in Italian FHB, the second aim was to investigate genetic diversity among the FTSC and estimate the mycotoxin risk related to these species. Phylogenetic analyses revealed that F. avenaceum (FTSC 4) was the most common species in Italy, followed by an unnamed Fusarium sp., F. tricinctum and F. acuminatum. In addition to these four phylospecies, five other F. tricinctum clade species were sampled. These included strains of four newly discovered species (Fusarium spp. FTSC 11, 13, 14, 15) and F. iranicum (FTSC 6). Most isolates tested for mycotoxin production on rice cultures were able to produce quantitative levels of enniatins and moniliformin. In addition, a preliminary study was conducted to evaluate the ability of a selected F. proliferatum isolate to produce fumonisins on wheat in open field and under natural climatic conditions. The three analogues (FB1, FB2 and FB3) were quantified by HPLC-FLD analysis on kernels, chaff and rachis. Fumonisins were detected in all the three investigated fractions without significant differences.

La relazione tra simbiosi e plasticità fisiologica nel corallo temperato Balanophyllia europaea in un ambiente naturalmente acidificato

L’alga dinoflagellata Symbiodinium spp. è il simbionte di Balanophyllia europaea. Lo scopo di questo Studio è valutare l’acclimatamento e il grado di plasticità fisiologica di B. europaea in condizioni naturalmente acidificate in funzione della simbiosi con Symbiodinium spp. I polipi derivano da popolazioni naturali che vivono in prossimità del cratere sottomarino di Panarea, caratterizzato da emissioni costituite per il 99% da anidride carbonica, condizioni che acidificano l’acqua creando un gradiente stabile di pH/pCO2. Sono stati valutati parametri correlati con la fotosintesi algale ed è stata misurata la variazione della diversità relativa dei cladi di Symbiodinium spp. I risultati mostrano che l’aumento di pCO2 e la riduzione di pH agevolano l’incremento della densità delle cellule algali nel corallo, mantenendo invariata la capacità fotosintetica della singola cellula algale. È stata poi evidenziata una riduzione della diversità di cladi presenti nei campioni a pH acido. Gli esemplari di B. europaea che vivono in un ambiente con valori pH previsti per il 2100 hanno raggiunto un nuovo equilibrio omeostatico in cui la maggiore densità algale garantisce un (maggiore) apporto nutrizionale per sostenere la fitness dell’olobionte. L’aumento dell’attività fotosintetica delle alghe potrebbe mitigare gli effetti dell’acidosi nelle cellule dell’ospite esposte ad elevate pCO2 perché sottrae CO2 all’ambiente intracellulare. La riduzione della diversità di Symbiodinium spp. mostra che il 99% delle alghe presenti nel tessuto del corallo appartiene a un clade estremamente resistente (A1 Med) e quindi in grado di fornire una maggiore tolleranza allo stress ambientale. Però, la conseguente perdita di flessibilità della partnership simbiotica potrebbe essere dannosa nel caso di future condizioni ambientali mutate. I risultati ottenuti devono essere interpretati nell’insieme delle modificazioni fisiologiche e morfologiche osservate in B. europaea lungo il gradiente di Panarea.

New insights into vaginal environment during pregnancy: a multi-omics approach

The present thesis aims to provide a thorough comprehension of the vaginal ecosystem of pregnant women and enhance the knowledge of pregnancy pathophysiology. The first study emphasized the importance of limiting protein intake from animal sources, consuming carbohydrates, and avoiding starting pregnancy overweight to maintain a healthy vaginal environment characterized by lactobacilli and related metabolites. In the second paper, a reduction in bacterial diversity, an increase in Lactobacillus abundance, and a decrease in bacterial vaginosis-related genera were observed during pregnancy. Lactobacillus abundance correlated with higher levels of lactate, sarcosine, and amino acids, while bacterial vaginosis-related genera were associated with amines, formate, acetate, alcohols, and short-chain fatty acids. An association between intrapartum antibiotic prophylaxis for Group B Streptococcus and higher vaginal abundance of Prevotella was found. Moreover, women experiencing a first-trimester miscarriage displayed a higher abundance of Fusobacterium. The third study explored the presence of macrolides and tetracyclines resistance genes in the vaginal environment, highlighting that different vaginal microbiota types were associated with distinct resistance profiles. Lactobacilli-dominated ecosystems showed fewer or no resistance genes, while women with increased bacterial vaginosis-related genera were positive for resistance genes. The last two papers aimed to identify potential biomarkers of vaginal health or disease status. The fourth paper showed that positivity for Torquetenovirus decreased from the first to the third trimester, being more prevalent in women with higher vaginal leukocyte counts. Torquetenovirus-positive samples showed higher levels of cytokines, propionate, and cadaverine. Lactobacillus species decreased in Torquetenovirus-positive samples, while Sneathia and Shuttleworthia increased. The last work pointed out the association between clade 2 of Gardnerella vaginalis and bacterial vaginosis. Moreover, as the number of simultaneously detected G. vaginalis clades increased, bacterial vaginosis-associated bacteria also tended to increase. Additionally, sialidase gene levels negatively correlated with Lactobacillus and positively correlated with Gardnerella, Atopobium, Prevotella, Megasphaera, and Sneathia.