2b-RAD genotyping for population genomic studies of Chagas disease vectors: Rhodnius ecuadoriensis in Ecuador

Background: Rhodnius ecuadoriensis is the main triatomine vector of Chagas disease, American trypanosomiasis, in Southern Ecuador and Northern Peru. Genomic approaches and next generation sequencing technologies have become powerful tools for investigating population diversity and structure which is a key consideration for vector control. Here we assess the effectiveness of three different 2b restriction site-associated DNA (2b-RAD) genotyping strategies in R. ecuadoriensis to provide sufficient genomic resolution to tease apart microevolutionary processes and undertake some pilot population genomic analyses. Methodology/Principal findings: The 2b-RAD protocol was carried out in-house at a non-specialized laboratory using 20 R. ecuadoriensis adults collected from the central coast and southern Andean region of Ecuador, from June 2006 to July 2013. 2b-RAD sequencing data was performed on an Illumina MiSeq instrument and analyzed with the STACKS de novo pipeline for loci assembly and Single Nucleotide Polymorphism (SNP) discovery. Preliminary population genomic analyses (global AMOVA and Bayesian clustering) were implemented. Our results showed that the 2b-RAD genotyping protocol is effective for R. ecuadoriensis and likely for other triatomine species. However, only BcgI and CspCI restriction enzymes provided a number of markers suitable for population genomic analysis at the read depth we generated. Our preliminary genomic analyses detected a signal of genetic structuring across the study area. Conclusions/Significance: Our findings suggest that 2b-RAD genotyping is both a cost effective and methodologically simple approach for generating high resolution genomic data for Chagas disease vectors with the power to distinguish between different vector populations at epidemiologically relevant scales. As such, 2b-RAD represents a powerful tool in the hands of medical entomologists with limited access to specialized molecular biological equipment. Author summary: Understanding Chagas disease vector (triatomine) population dispersal is key for the design of control measures tailored for the epidemiological situation of a particular region. In Ecuador, Rhodnius ecuadoriensis is a cause of concern for Chagas disease transmission, since it is widely distributed from the central coast to southern Ecuador. Here, a genome-wide sequencing (2b-RAD) approach was performed in 20 specimens from four communities from Manabí (central coast) and Loja (southern) provinces of Ecuador, and the effectiveness of three type IIB restriction enzymes was assessed. The findings of this study show that this genotyping methodology is cost effective in R. ecuadoriensis and likely in other triatomine species. In addition, preliminary population genomic analysis results detected a signal of population structure among geographically distinct communities and genetic variability within communities. As such, 2b-RAD shows significant promise as a relatively low-tech solution for determination of vector population genomics, dynamics, and spread

Functional transcriptome analysis in ARSACS KO cell model reveals a role of sacsin in autophagy

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare early-onset neurological disease caused by mutations in SACS, which encodes sacsin. The complex architecture of sacsin suggests that it could be a key player in cellular protein quality control system. Molecular chaperones that operate in protein folding/unfolding and assembly/disassembly patterns have been described as essential modulators of selectivity during the autophagy process. We performed RNA-sequencing analysis to generate a whole-genome molecular signature profile of sacsin knockout cells. Using data analysis of biological processes significantly disrupted due to loss of sacsin, we confirmed the presence of decreased mitochondrial function associated with increased oxidative stress, and also provided a demonstration of a defective autophagic pathway in sacsin-depleted cells. Western blotting assays revealed decreased expression of LC3 and increased levels of p62 even after treatment with the lysosomal inhibitor bafilomycin A1, indicating impairment of the autophagic flux. Moreover, we found reduced co-immunolocalization of the autophagosome marker LC3 with lysosomal and mitochondrial markers suggesting fusion inhibition of autophagic compartments and subsequent failed cargo degradation, in particular failed degradation of damaged mitochondria. Pharmacological up-regulation of autophagy restored correct autophagic flux in sacsin knockout cells. These results corroborate the hypothesis that sacsin may play a role in autophagy. Chemical manipulation of this pathway might represent a new target to alleviate clinical and pathological symptoms, delaying the processes of neurodegeneration in ARSACS

A Cationic Contrast Agent in X-ray Imaging of Articular Cartilage: Pre-Clinical Evaluation of Diffusion and Attenuation Properties

The aim of this study was the preliminary assessment of a new cationic contrast agent, the CA4+, via the analysis of spatial distribution in cartilage of ex vivo bovine samples, at micrometer and millimeter scale. Osteochondral plugs (n = 18) extracted from bovine stifle joints (n = 2) were immersed in CA4+ solution up to 26 h. Planar images were acquired at different time points, using a microCT apparatus. The CA4+ distribution in cartilage and saturation time were evaluated. Tibial plates from bovine stifle joints (n = 3) were imaged with CT, before and after 24 h-CA4+ bath immersion, at different concentrations. Afterward, potential CA4+ washout from cartilage was investigated. From microCT acquisitions, the CA4+ distribution differentiated into three distinct layers inside the cartilage, reflecting the spatial distribution of proteoglycans. After 24 h of diffusion, the iodine concentration reached in cartilage was approximately seven times that of the CA4+ bath. The resulting saturation time was 1.9 ± 0.9 h and 2.6 ± 2.9 h for femoral and tibial samples, respectively. Analysis of clinical CT acquisitions confirmed overall contrast enhancement of cartilage after 24 h immersion, observed for each CA4+ concentration. Distinct contrast enhancement was reached in different cartilage regions, depending on tissue's local features. Incomplete but remarkable washout of cartilage was observed. CA4+ significantly improved cartilage visualization and its qualitative analysis

Genetic fingerprinting of salmon louse (Lepeophtheirus salmonis) populations in the North-East Atlantic using a random forest classification approach

Caligid sea lice represent a significant threat to salmonid aquaculture worldwide. Population genetic analyses have consistently shown minimal population genetic structure in North Atlantic Lepeophtheirus salmonis, frustrating efforts to track louse populations and improve targeted control measures. The aim of this study was to test the power of reduced representation library sequencing (IIb-RAD sequencing) coupled with random forest machine learning algorithms to define markers for fine-scale discrimination of louse populations. We identified 1286 robustly supported SNPs among four L. salmonis populations from Ireland, Scotland and Northern Norway. Only weak global structure was observed based on the full SNP dataset. The application of a random forest machine-learning algorithm identified 98 discriminatory SNPs that dramatically improved population assignment, increased global genetic structure and resulted in significant genetic population differentiation. A large proportion of SNPs found to be under directional selection were also identified to be highly discriminatory. Our data suggest that it is possible to discriminate between nearby L. salmonis populations given suitable marker selection approaches, and that such differences might have an adaptive basis. We discuss these data in light of sea lice adaption to anthropogenic and environmental pressures as well as novel approaches to track and predict sea louse dispersal

A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field

Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION sequencer. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure a reliable and robust performance under suboptimal field conditions without increasing costs. Here, we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding, and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 1000 reads per sample. ONTrack-derived consensus barcodes have a high accuracy, ranging from 99.8 to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions

Hydraena (s.str.) dinarica, new species (Coleoptera: Hydraenidae) along with further records of Hydraena spp. from Durmitor National Park, Montenegro and comments on the DNA barcoding problem with the genus

Background Long-palped Water Beetles were collected during a taxon expedition in Montenegro which involved citizen scientists, students and taxonomists. The material was collected from springs, brooks, fens and the Tara River, at altitudes between 600 m and 1450 m above sea level, using fine-meshed hand-nets and by manual checking of submerged substrates. The morphological species delimitation was supplemented and congruent with mtDNA sequences mainly obtained in the field using the newly-developed MinION-based ONTrack pipeline. New information The new species Hydraena dinarica Freitag & de Vries, sp. n. from Durmitor Mt. is described, illustrated and compared in detail to closely-related congeners of the H. saga d\u27Orchymont, 1930/H. emarginata Rey, 1885 species complex. Five additional species and female specimens of two unidentified morphospecies of the genus were also recorded in the vicinity of Durmitor National Park. New records and the first DNA barcodes for Hydraena biltoni Jäch & Díaz, 2012 (endemic to Montenegro) and H. morio Kiesenwetter, 1849 are provided. Further records of H. nigrita Germar, 1824, H. minutissima Stephens, 1829, H. subintegra Ganglbauer, 1901 and females of two unidentified morphospecies are commented upon. The resulting inter- and intraspecific genetic distances and some observations of low or zero sequence divergence between recently-diverged species of Hydraena Kugelann, 1794 are briefly discussed

A population genomics insight by 2b‐RAD reveals populations' uniqueness along the Italian coastline in Leptopsammia pruvoti (Scleractinia, Dendrophylliidae)

Aim Marine bioconstructions such as coralligenous formations are hotspot of biodiversity and play a relevant ecological role in the preservation of biodiversity by providing carbon regulation, protection and nursery areas for several marine species. For this reason, the European Union Habitat Directive included them among priority habitats to be preserved. Although their ecological role is well established, connectivity patterns are still poorly investigated, representing a limit in conservation planning. The present study pioneers a novel approach for the analysis of connectivity in marine bioconstructor species, which often lack suitable genetic markers, by taking advantage of next‐generation sequencing techniques. We assess the geographical patterns of genomic variation of the sunset cup coral Leptopsammia pruvoti Lacaze‐Duthiers, 1897, an ahermatypic, non‐zooxanthellate and solitary scleractinian coral species common in coralligenous habitats and distributed across the Mediterranean Sea. Location The Italian coastline (Western and Central Mediterranean). Methods We applied the restriction site‐associated 2b‐RAD approach to genotype over 1,000 high‐quality and filtered single nucleotide polymorphisms in 10 population samples. Results The results revealed the existence of a strongly supported genetic structure, with highly significant pairwise FST values between all the population samples, including those collected about 5 km apart from each other. Moreover, genomic data indicate that the strongest barriers to gene flow are between the western (Ligurian–Tyrrhenian Sea) and the eastern side (Adriatic Sea) of the Italian peninsula. Main conclusions The strong differentiation found in L. pruvoti is similar to that found in other species of marine bioconstructors investigated in this area, but it strongly contrasts with the small differences found in many fish and invertebrates at the same geographical scale. All in one, our results highlight the importance of assessing connectivity in species belonging to coralligenous habitats as, due to their limited dispersal ability, they might require specific spatial conservation measures

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

POPULATION GENOMICS APPROACH FOR THE STUDY OF CONNECTIVITY IN MARINE SPECIES

MARINE CONNECTIVITY, SEASCAPE GENETICS and IMPLICATIONS for PROTECTED AREAS (MPAs) Population connectivity is a keystone in a wide range of processes, including the shaping of population genetic structure, local and meta-population dynamics and the design of protected areas. Understanding the extent at which connectivity occurs is critical for the conservation of marine species and for the improvement of biological and ecological resources management. Genetic approaches are widely used as a powerful tool for the indirect inference of connectivity among populations of marine organisms, especially when the working-conditions makes the direct measure of dispersal pattern hard to quantify, such as the tracking of larval stage of benthic invertebrates. Despite their power, genetic isolation patterns can be difficult to explain, especially when the genetic differentiation is weak. The integration of genetic data with oceanographic feautures, the biology of species and the modelling of larval dispersal in a multidisciplinary approach, the so-called seascape genetics, can provide new insights into drivers shaping the spatial genetic variability of species. Worldwide, the anthropogenic activities have a strong negative impact to the marine ecosystem, causing coastal-habitat loss or fragmentation and the biodiversity depletion. Given the global extent of these phenomena, restoration of marine ecosystem is actually top conservation priority. Marine protected areas (MPAs), especially the no-take zones, have been increasingly identified as one of the most effective tool for conservation and management in reducing the alteration of marine ecosystem and mitigating the negative effect of human activities. The implementation of network of MPAs, where the individual protected areas can operate in synergy, could offer a greater protection at a wider spatial scale, leading more effectively the conservation goals than individual sites alone. The European Commission funded CoCoNET project aimed to the identification of groups of putatively connected MPAs in the Mediterranean and the Black Seas, shifting from local to regional (Networks of MPAs) and basin (network of networks) scales. It adopted an interdisciplinary approach based on population genetics of 13 selected species, the modelling of the dispersal and the beta diversity. In this thesis, 2 CoCoNET species were studied: the edible common sea urchin Paracentrotus lividus (Lamarck, 1816) and the Mediterranean mussel Mytilus galloprovincialis (Lamarck, 1819). The 2b-RAD protocol was successfully used to processing hundreds samples as well as for genotyping of hundreds SNPs, that were used to evaluating the genetic structure of these 2 species in the CoCoNET study areas. • A seascape genomics approach was applied to explore connectivity patterns of the sea urchin Paracentrotus lividus in the Mediterranean pilot area: the potential larval connectivity and retention (Lagrangian simulations) and the realized connectivity (genomic analyses) were assessed and compared with a focus on the Adriatic and Ionian seas. A total of 275 samples from 10 population samples of Western and Central Mediterranean Sea were processed with the 2b-RAD protocol, and 1122 Single Nucleotide Polymorphisms (SNPs) were genotyped and used for population genomics analysis. Lagrangian simulations was performed with a biophysical model of larval dispersal focused on the Adriatic-Ionian basins and it was integrated with features of the P. lividus larval phase (PLD, temperature constrain, reproductive timing). Genetic homogeneity was found among the eight population samples collected in the focal Adriatic-Ionian area, whereas a weak but significant differentiation was found with respect to two samples from the Western Mediterranean (France and Tunisia). This result was not affected by the few putative outlier loci identified in our dataset. Lagrangian simulations found a significant potential for larval exchange among the eight Adriatic-Ionian locations through a relatively persistent flux of propagules from each sites to its closest neighbor, mainly in a counterclockwise direction. Overall, these findings supported the hypothesis of connectivity of P. lividus populations in this area. We also compared our findings with previously published mitochondrial genetic data (cytochrome b gene) showing that our genome-scale SNPs dataset had a greater power in resolving the population structure of this species, providing solid and reliable indirect measures of connectivity, broadly in line with the predictions of Lagrangian simulations. • The population genomics structure of the mussel Mytilus galloprovincialis was explored at different geographic scales (local and wide) in the Mediterranean and Black seas. The application of a genome-wide approach based on single nucleotide polymorphism (SNP) markers, a wide sampling design and a good sample size, allowed to overcome the previous limitations providing the first reliable biogeographic data concerning this species throughout its native range. A total of 19 population samples were analysed, 10 from the Mediterranean Sea and 9 from the Black Sea. To obtain the best resolution for the wide and the local scale, we obtained and investigated 3 different datasets: 1) the overall dataset (461 individuals, 512 polymorphic loci); 2) the Adriatic-Ionian dataset (201 individuals, 811 polymorphic loci) and 3) the Black Sea’s dataset (228 individuals, 998 polymorphic loci). A marked structuring between the Mediterranean and Blacks Seas was found, revealing that the Mediterranean and Black seas are genetically distinct, likely due to oceanographic constrains. In the Black Sea, a completely lack of genetic structure was detected, where the currents pattern and the long pelagic larval duration of this species probably ensure a good connection among the sampled locations. On the other hand, in the Mediterranean Sea, no signal of differentiation was found between the Western and Central, whereas the Central Mediterranean (the Adriatic-Ionian population samples) showed a weak genetic heterogeneity, that underlined an East-West differentiation in the Central Adriatic Sea, and a general homogeneity in the Southern Adriatic and Ionian seas among the Italian, Greek and Albanian localities. This pattern of genetic differentiation likely depends on the Adriatic gyral circulation, that prevents or allows the exchange of larvae, acting as barrier among some localities and determing a certain level of self retention in others. Overall, connectivity data obtained so far in the CoCoNET framework revealed a heterogeneous mosaic of species-specific connectivity patterns throughout the targeted areas in Mediterranean and Black seas, ranging from the total absence of genetic differentiation (P. lividus) to the strong genetic structure even at a local scale (in the habitat former P. oceanica). Overall, the heterogeneity of connectivity patterns depends on species-specific peculiarities in relation to environmental features, and connectivity results so far obtained warns against any generalization in defining conservation units based on the results of the single species. The wide heterogeneity of species-specific connectivity outcomes suggests that the implementation of ecological effective networks of MPAs should standing on meta-community information across a range of spatial scales and a representative set of species to obtain a “collection of networks of genetic variation of all species within a community”. LANDSCAPE GENETICS IN PARASITIC DISEASES Neglected Tropical Diseases (NTDs) are a group of parasitic infectious diseases affecting worldwide approximately 2.7 billion people living in poverty, especially in the tropical areas. The NTDs pathogens have often a complex life-cycle, involving at least one host, including humans. A great heterogeneity exists in the dispersal ability, distribution and transmission routes of parasitic species, that are strongly influenced by ecological settings and landscape variation. Understanding how abiotic and biotic variables interact and affect the movements and distribution of parasites, hosts and vector species is key for predicting and controlling the disease spread. Trypanosoma cruzi is the most important parasite in Latin America, the infectious agent of Chagas disease, a NTD that caused around 14000 deaths annually. In Ecuador, where the Chagas disease is endemic, one of main insect vectors is the kissin bug Rhodnius ecuadoriensis. In this thesis we worked on a pilot study regarding the vector Rhodnius ecuadoriensis based on 20 specimens collected from four communities in Ecuador, with the aims of i) testings the 2b-RAD protocol by assessing and comparing the effectiveness of three type IIB Restriction Enzymes: ii) generating several hundred SNPs markers for this species and iii) performing preliminary genomic analysis to test the efficacy of the identified SNP markers. The three type IIB Restriction Enzymes (AlfI, BcgI, and CspCI) resulted to be effective for processing Rhodnius ecuadoriensis genomic DNA samples and, as expected, the enzyme showing the most abundance of cutting sites (estimated via an in silico digestion of the Rhodnius prolixus genome) provided the smaller number fo usable molecular markers (AlfI). CspCI enzyme produced the largest amount of polymorphic markers whatever the read depth, thus evidencing its good experimental performance in R. ecuadoriensis and likely to other Rhodnius sp. Vectors. Preliminary population genomic analyses were performed with BgcI and CspCI datasets, and a signal of structuring among populations was detected. Specifically, Bayesian clustering detected two distinct clusters without previous location information, one of them was the most distant population (Bejuco) from the others 3 from Loja region, that clustered together. Our findings suggest that 2b-RAD genotyping is both a cost-effective and methodologically simple approach for generating high resolution genomic data for Chagas disease vectors with the power to distinguish between different vector populations at epidemiologically relevant scales.CONNETTIVITA’ MARINA, SEASCAPE GENETICS E IMPLICAZIONI PER LE AREE MARINE PROTETTE La connettività di popolazione è la chiave di volta di un’ampia varietà di processi, tra i quali la determinazione della struttura genetica di popolazione delle specie, le dinamiche di popolazioni locali e meta-popolazioni e la progettazione di aree protette. Capire e definire le modalità con cui le popolazioni sono connesse e a che scala geografica la connessione avviene, è critico per la conservazione delle specie marine e per migliorare la gestione delle risorse marine economiche e biologiche. Gli approcci genetici sono ampiamente utilizzati come mezzo efficace per la stima indiretta della connettività tra popolazioni di organismi marini, specialmente quando le condizioni di lavoro rendono difficile lo studio diretto dei pattern di dispersione, come nel caso del tracking delle larve negli invertebrati bentonici. Nonostante il potere della genetica, i pattern di isolamento rilevati possono risultare di difficile interpretazione, soprattutto quando il differenziamento genetico osservato è debole. Integrare i dati genetici con le caratteristiche oceanografiche dell’area di studio, dati riguardanti la biologia delle specie e il modelling della dispersione larvale in un approccio multidisciplinare chiamato seascape genetics, può chiarire quali siano i fattori che determinano la distribuzione spaziale della variabilità genetica delle specie. In tutto il mondo, le attività antropiche hanno effetti estremamente negativi sull’ecosistema marino, causando la perdita o la frammentazione degli habitat costieri e il depauperamento della biodiversità ad essi associata. Data l’estensione globale di questi fenomeni, il ripristino dell’ecosistema marino è una delle principali priorità target nell’ambito della conservazione. Le aree marine protette (MPAs) sono state riconosciute come uno dei mezzi più efficaci per conservare e gestire l’intero ecosistema marino, in quanto sono in grado di ridurre e mitigare gli effetti negativi causati delle attività umane. L’implementazione di network di MPAs, all’interno dei quali le singole aree marine protette possono agire in sinergia, potrebbe offrire una maggior protezione su ampia scala spaziale, agendo in modo più efficace rispetto a quanto possano fare le singole aree da sole. Il progetto CoCoNET, finanziato dalla Commissione Europea, è un progetto che mirava all’identificazione di gruppi di MPAs putativamente connesse nel Mediterraneo e nel Mar Nero, sia su scala locale sia considerando l’intero bacino. Il progetto CoCoNET si proponeva di adottare un approccio multidisciplinare basato sulla genetica di popolazione di 13 specie, il modelling della dispersione larvale e la beta diversity. In questa tesi sono state studiate 2 specie del progetto CoCoNET, il riccio di mare Paracentrotus lividus (Lamarck, 1816) e il mitilo del Mediterraneo Mytilus galloprovincialis (Lamarck, 1819). Per il processamento di centinaia di campioni e il genotyping di altrettanti polimorfismi a singolo nucleotide (SNPs) utilizzati per studiare la struttura genetica di popolazione delle 2 specie, è stato utilizzato il protocollo 2b-RAD. • Per il riccio di mare P. lividus è stato utilizzato un approccio di seascape genomics nel Mediterraneo centrale: sono stati stimati e confrontati il potenziale di dispersione larvale e di retention (ottenuti con simulazioni di Lagrange) e la connettività realizzata (ottenuta con i dati genetici) con focus a livello dell’Adriatico e dello Ionio. Sono stati processati un totale di 275 individui da 10 campioni di popolazione provenienti dal Mediterraneo occidentale e centrale, e 1122 SNPs sonno stati genotipizzati ed utilizzati per le analisi di genomica di popolazione. Le simulazioni di Lagrange sono state condotte utilizzando un modello biofisico di dispersione larvale focalizzato nel bacino Adriatico-Ionico, che è stato integrato con caratteristiche dello stadio larvale di P. lividus (durata delle larve, influenza della temnperatura, periodo riproduttivo). Gli 8 campioni di popolazione campionati in Adriatico-Ionio sono risultati essere geneticamente omogenei, mentre è stato rilevato un lieve ma significativo differenziamento di queste 8 rispetto ai 2 campioni di popolazione provenienti dal Mediterraneo occidentale (Francia e Tunisia). Questo risultato non è stato influenzato dalla presenza di alcuni loci probabilmente sotto selezione direzionale. Le simulazioni di Lagrange hanno predetto uno scambio larvale potenziale tra gli 8 campioni di popolazione Adritico-Ionici attraverso un flusso di propaguli relativamente persistente da ogni sito, prevalentemente in direzione oraria. Complessivamente, i risultati ottenuti dalla genetica di popolazione e dalle simulazioni supportano ampiamente l’ipotesi di una buona connessione tra le popolazioni di P. lividus in quest’area. • La struttura genomica di popolazione del mitilo M. galloprovincialis è stata esplorata su diverse scale geografiche nel Mediterraneo e Mar Nero. L’utilizzo di un approccio genome-wide basato su SNPs, un ampio campionamento e una buona dimensione dei campioni di popolazione hanno permesso di superare le limitazioni finora incontrate nell’ottenere dati biogeografici affidabili per questa specie all’interno del suo range di distribuziona nativo. Sono state analizzati 19 campioni di popolazione, 10 provenienti dal Mar Mediterraneo e 9 dal Mar Nero. Al fine di ottenere il miglior potere di risoluzione possibile sia su ampia scala sia su scala ridotta, sono stati utilizzati per le analisi 3 diversi dataset: 1) un dataset complessivo (461 individui, 512 loci); 2) un dataset adriatico-ionico (201 individui, 811 loci) e 3) un dataset per il Mar Nero (228 individui, 998 loci). È stato evidenziato un marcato differenziamento genetico tra il Mar Mediterraneo e il Mar Nero, probabilmente a causa di costrain oceoanografici. Nel Mar Nero è evidente una totale mancanza di struttura, probabilmente dovuta al pattern delle correnti e alla durata dello stadio larvale che permettono una buona connessione tra le popolazioni campionate. Dall’altro lato invece, non è stato notato differenziamento tra il Mediterraneo occidentale e quello centrale, mentre un certo grado di eterogeneità genetica è stato rilevata tra le popolazioni adriatico-ioniche, con un differenziamento est-ovest nell’Adriatico centrale e una relativa omogeneità tra le popolazone dell’Adriatico meridionale e dello Ionio. Il pattern di differenziamento osservato dipende probabilmente dalla presenza di gyres minori in aggiunta alla circolazione adriatica principale, che può favorire o impedire lo scambio di propaguli, fungendo da barriera al flusso genico tra alcune località e favorendo la self-retention in altre. Complessivamente, i risultati ottenuti finora nel progetto CoCoNET hanno messo in luce un mosaico eterogeneo di pattern di connettività specifici per le diverse specie, che vanno dalla totale assenza di differenziamento come nel caso del riccio, alla forte struttura come nel caso dell’habitat former Posidonia oceanica. Questa eterogeneità può dipendere da peculiarità specie-specifiche messe in relazione a caratteristiche ambientali, e i dati di connettività finora ottenuti sconsigliano qualsiasi forma di generalizzazione basandosi sui risultati ottentui in una singola specie. Il quadro che sta emergendo suggerisce che la progettazione di network di MPAs ecologicamente efficaci debbano basarsi su informazioni ottenute a livello di comunità considerando un ampio range spaziale, e un set rappresentativo delle specie che vivono nell’area target. LANDSCAPE GENETICS NELLE PARASSITOSI Le malattie tropicali sono un gruppo di malattie infettive causate da parassiti che infettano quasi 3 milioni di persone in tutto il mondo, specialmente nei paesi poveri delle zone tropicali. I patogeni che causano questa malattie hanno solitamente un ciclo vitale complesso, che coinvolge almeno un ospite, compresi gli esseri umani. Esiste una grande variabilità nelle capacità di dispersione, nella distribuzione e nelle vie di trasmisisone delle specie parassite. Questa variabilità è fortemente influenzata dalle caratteristiche ambientali e del paesaggio. Capire come le variabili biotiche e abiotiche interagiscono e influenzano la distribuzione del parassita, dei loro ospiti e delle specie vettore è un fattore chiave per la predizione ed il controllo della diffusione di queste malattie. Il Tripanozoma cruzi è il parassita più importante del Sud America ed è l’agente infettivo del morbo di Chagas, una malattia tropicale endemica in molte regione dell’Ecuador dove uno dei principali vettori è la cimice ematofaga Rhodnius ecuadoriensis. In questa tesi è stato prodotto uno studio pilota sul vettore R. ecuadoriensis utilizzando 20 individui campionati in 4 comunità dell’Ecuador, con lo scopo di i) testare il protocollo 2b-RAD utilizzando e confrontanto l’efficacia di 3 diversi enzimi di restrizione di tipo IIb, ii) ottenere un numero di marcatori SNPs sufficiente per iii) fornire delle analisi preliminari di genetica di popolazione per questa specie. I 3 enzimi testati (AlfI, BcgI, and CspCI) sono risultati efficaci nel processare il DNA genomico di questa specie e, come era atteso, l’enzima con il maggior numero di siti di riconosciemento (stimato tramite una restrizione in silico del genoma di Rhodnius prolixus) ha prodotto il minor numero di loci utilizzabili (AlfI). L’enzima CspCI ha prodotto il maggior numero di loci polimorfici a qualsiasi read depth considerata evidenziando così l’ottima performance di questo protocollo in questa specie e presumibilmente anche in altre specie appartenenti allo stesso genere. Le analisi preliminari di genetica di popolazione sono state condotte utilizzando i dataset ottenuti dagli enzimi BcgI e CspCI che sono stati in grade di evidenziare un certo grado di struttura genetica tra I 4 campioni di popolazione. Un clustering bayesiano, condotto senza l’informazione a priori riguardante le località, ha identificato la presenza di 2 cluster geneticamente distinti: uno rappresentato dalla popolazione geograficamente più distante rispetto alle altre (Bejuco), l’altro invece formato dalle altre 3 popolazioni che provenivano tutte dalla regione di Loja. Questi risultati evidenziano come il protocollo 2b-RAD sia una metodologia semplice ed efficace nel generare marcatori su scala genomica in uno dei principali vettori del morbo di Chagas, e come permetta di ottenere informazioni sulla struttura di popolazione di questa specie ad una scala rilevante a livello epidemiologico

POPULATION GENOMICS APPROACH FOR THE STUDY OF CONNECTIVITY IN MARINE SPECIES

MARINE CONNECTIVITY, SEASCAPE GENETICS and IMPLICATIONS for PROTECTED AREAS (MPAs) Population connectivity is a keystone in a wide range of processes, including the shaping of population genetic structure, local and meta-population dynamics and the design of protected areas. Understanding the extent at which connectivity occurs is critical for the conservation of marine species and for the improvement of biological and ecological resources management. Genetic approaches are widely used as a powerful tool for the indirect inference of connectivity among populations of marine organisms, especially when the working-conditions makes the direct measure of dispersal pattern hard to quantify, such as the tracking of larval stage of benthic invertebrates. Despite their power, genetic isolation patterns can be difficult to explain, especially when the genetic differentiation is weak. The integration of genetic data with oceanographic feautures, the biology of species and the modelling of larval dispersal in a multidisciplinary approach, the so-called seascape genetics, can provide new insights into drivers shaping the spatial genetic variability of species. Worldwide, the anthropogenic activities have a strong negative impact to the marine ecosystem, causing coastal-habitat loss or fragmentation and the biodiversity depletion. Given the global extent of these phenomena, restoration of marine ecosystem is actually top conservation priority. Marine protected areas (MPAs), especially the no-take zones, have been increasingly identified as one of the most effective tool for conservation and management in reducing the alteration of marine ecosystem and mitigating the negative effect of human activities. The implementation of network of MPAs, where the individual protected areas can operate in synergy, could offer a greater protection at a wider spatial scale, leading more effectively the conservation goals than individual sites alone. The European Commission funded CoCoNET project aimed to the identification of groups of putatively connected MPAs in the Mediterranean and the Black Seas, shifting from local to regional (Networks of MPAs) and basin (network of networks) scales. It adopted an interdisciplinary approach based on population genetics of 13 selected species, the modelling of the dispersal and the beta diversity. In this thesis, 2 CoCoNET species were studied: the edible common sea urchin Paracentrotus lividus (Lamarck, 1816) and the Mediterranean mussel Mytilus galloprovincialis (Lamarck, 1819). The 2b-RAD protocol was successfully used to processing hundreds samples as well as for genotyping of hundreds SNPs, that were used to evaluating the genetic structure of these 2 species in the CoCoNET study areas. • A seascape genomics approach was applied to explore connectivity patterns of the sea urchin Paracentrotus lividus in the Mediterranean pilot area: the potential larval connectivity and retention (Lagrangian simulations) and the realized connectivity (genomic analyses) were assessed and compared with a focus on the Adriatic and Ionian seas. A total of 275 samples from 10 population samples of Western and Central Mediterranean Sea were processed with the 2b-RAD protocol, and 1122 Single Nucleotide Polymorphisms (SNPs) were genotyped and used for population genomics analysis. Lagrangian simulations was performed with a biophysical model of larval dispersal focused on the Adriatic-Ionian basins and it was integrated with features of the P. lividus larval phase (PLD, temperature constrain, reproductive timing). Genetic homogeneity was found among the eight population samples collected in the focal Adriatic-Ionian area, whereas a weak but significant differentiation was found with respect to two samples from the Western Mediterranean (France and Tunisia). This result was not affected by the few putative outlier loci identified in our dataset. Lagrangian simulations found a significant potential for larval exchange among the eight Adriatic-Ionian locations through a relatively persistent flux of propagules from each sites to its closest neighbor, mainly in a counterclockwise direction. Overall, these findings supported the hypothesis of connectivity of P. lividus populations in this area. We also compared our findings with previously published mitochondrial genetic data (cytochrome b gene) showing that our genome-scale SNPs dataset had a greater power in resolving the population structure of this species, providing solid and reliable indirect measures of connectivity, broadly in line with the predictions of Lagrangian simulations. • The population genomics structure of the mussel Mytilus galloprovincialis was explored at different geographic scales (local and wide) in the Mediterranean and Black seas. The application of a genome-wide approach based on single nucleotide polymorphism (SNP) markers, a wide sampling design and a good sample size, allowed to overcome the previous limitations providing the first reliable biogeographic data concerning this species throughout its native range. A total of 19 population samples were analysed, 10 from the Mediterranean Sea and 9 from the Black Sea. To obtain the best resolution for the wide and the local scale, we obtained and investigated 3 different datasets: 1) the overall dataset (461 individuals, 512 polymorphic loci); 2) the Adriatic-Ionian dataset (201 individuals, 811 polymorphic loci) and 3) the Black Sea’s dataset (228 individuals, 998 polymorphic loci). A marked structuring between the Mediterranean and Blacks Seas was found, revealing that the Mediterranean and Black seas are genetically distinct, likely due to oceanographic constrains. In the Black Sea, a completely lack of genetic structure was detected, where the currents pattern and the long pelagic larval duration of this species probably ensure a good connection among the sampled locations. On the other hand, in the Mediterranean Sea, no signal of differentiation was found between the Western and Central, whereas the Central Mediterranean (the Adriatic-Ionian population samples) showed a weak genetic heterogeneity, that underlined an East-West differentiation in the Central Adriatic Sea, and a general homogeneity in the Southern Adriatic and Ionian seas among the Italian, Greek and Albanian localities. This pattern of genetic differentiation likely depends on the Adriatic gyral circulation, that prevents or allows the exchange of larvae, acting as barrier among some localities and determing a certain level of self retention in others. Overall, connectivity data obtained so far in the CoCoNET framework revealed a heterogeneous mosaic of species-specific connectivity patterns throughout the targeted areas in Mediterranean and Black seas, ranging from the total absence of genetic differentiation (P. lividus) to the strong genetic structure even at a local scale (in the habitat former P. oceanica). Overall, the heterogeneity of connectivity patterns depends on species-specific peculiarities in relation to environmental features, and connectivity results so far obtained warns against any generalization in defining conservation units based on the results of the single species. The wide heterogeneity of species-specific connectivity outcomes suggests that the implementation of ecological effective networks of MPAs should standing on meta-community information across a range of spatial scales and a representative set of species to obtain a “collection of networks of genetic variation of all species within a community”. LANDSCAPE GENETICS IN PARASITIC DISEASES Neglected Tropical Diseases (NTDs) are a group of parasitic infectious diseases affecting worldwide approximately 2.7 billion people living in poverty, especially in the tropical areas. The NTDs pathogens have often a complex life-cycle, involving at least one host, including humans. A great heterogeneity exists in the dispersal ability, distribution and transmission routes of parasitic species, that are strongly influenced by ecological settings and landscape variation. Understanding how abiotic and biotic variables interact and affect the movements and distribution of parasites, hosts and vector species is key for predicting and controlling the disease spread. Trypanosoma cruzi is the most important parasite in Latin America, the infectious agent of Chagas disease, a NTD that caused around 14000 deaths annually. In Ecuador, where the Chagas disease is endemic, one of main insect vectors is the kissin bug Rhodnius ecuadoriensis. In this thesis we worked on a pilot study regarding the vector Rhodnius ecuadoriensis based on 20 specimens collected from four communities in Ecuador, with the aims of i) testings the 2b-RAD protocol by assessing and comparing the effectiveness of three type IIB Restriction Enzymes: ii) generating several hundred SNPs markers for this species and iii) performing preliminary genomic analysis to test the efficacy of the identified SNP markers. The three type IIB Restriction Enzymes (AlfI, BcgI, and CspCI) resulted to be effective for processing Rhodnius ecuadoriensis genomic DNA samples and, as expected, the enzyme showing the most abundance of cutting sites (estimated via an in silico digestion of the Rhodnius prolixus genome) provided the smaller number fo usable molecular markers (AlfI). CspCI enzyme produced the largest amount of polymorphic markers whatever the read depth, thus evidencing its good experimental performance in R. ecuadoriensis and likely to other Rhodnius sp. Vectors. Preliminary population genomic analyses were performed with BgcI and CspCI datasets, and a signal of structuring among populations was detected. Specifically, Bayesian clustering detected two distinct clusters without previous location information, one of them was the most distant population (Bejuco) from the others 3 from Loja region, that clustered together. Our findings suggest that 2b-RAD genotyping is both a cost-effective and methodologically simple approach for generating high resolution genomic data for Chagas disease vectors with the power to distinguish between different vector populations at epidemiologically relevant scales