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Fabio Crocetta Marine Biodiversity: a Multidisciplinary Journey

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Fabio Crocetta Marine Biodiversity: a Multidisciplinary Journey UNIVERSITY OF NAPLES “FEDERICO II” Ph.D. Thesis by Fabio Crocetta Marine biodiversity: a multidisciplinary journey from genes to species Ph.D. Programme Model organisms in the biomedical and veterinary research XXVI cycle Tutor Coordinator Dr. Paolo Sordino Prof. Paolo De Girolamo 2011-2014 1 2 TABLE OF CONTENTS INTRODUCTION..................................................................................................................................... 7 CHAPTER 1 - Mutation research in tunicates: tools for studying development and evolution....... 11 1. N-ETHYL-N-NITROSOUREA (ENU) MUTANTS……………………………………………….. 12 2. RANDOM INSERTIONAL MUTAGENESIS...................................................................................... 13 3. TARGETED INSERTIONAL MUTAGENESIS................................................................................... 13 4. NATURALLY OCCURRING MUTATIONS....................................................................................... 14 4.1. TOWARDS A TOOLBOX FOR FORWARD GENETICS................................................................ 14 4.1.1. Where, which and when of cryptic species and populations............................................................. 15 4.1.2. From the field to the lab: sampling and maintainance...................................................................... 16 4.1.3. From the field to the lab: self-fertilization procedures...................................................................... 16 4.1.4. Saving ascidian mutations by cryopreservation................................................................................ 17 4.1.5. Sorting mutations by morphology..................................................................................................... 17 4.1.6. Searching for subtle mutants by automated whole mount in situ hybridization (ISH)..................... 18 4.1.7. Back to facilities: raising the ascidian mutation............................................................................... 19 4.2. TESTING THE PROTOCOL.............................................................................................................. 20 4.2.1. Field observations, sampling and self-fertilization data................................................................... 21 4.2.2. Searching for mutants: morphological and automated ISH screening procedures........................... 21 4.2.3. Growing ascidian mutant lines.......................................................................................................... 22 4.2.4. ISH and fluorescent in situ hybridization (FISH) on sensory organ mutants................................... 23 4.2.5. ISH on a trunk mutant....................................................................................................................... 25 5. REFERENCES........................................................................................................................................ 26 CHAPTER 2 - Magoh in basal chordates: insights into the evolution of the brain............................ 40 1. INTRODUCTION……………………………………………………………………………………... 40 2. MATERIALS AND METHODS……………………………………………………………………… 41 2.1 Bioinformatics………………………………………………………………………………………... 41 2.1.1. Phylogenetic analysis……………………………………………………………………………… 42 2.1.2. Synteny conservation analysis in Chordates………………………………………………………. 42 2.2. Biological material…………………………………………………………………………………... 42 2.2.1. Sampling and acclimatization procedures…………………………………………………………. 42 2.2.2. Gamete sampling and embryo collection………………………………………………………….. 43 2.3. Magoh transcription and translation…………………………………………………………………. 44 2.3.1. Digoxigenin-labeled antisense and sense riboprobe synthesis…………………………………….. 44 3 2.3.2. In situ hybridization (ISH) and immunohistochemistry (IHC) analyses………………………... 45 3. RESULTS…………………………………………………………………………………………… 46 3.1. Sequence conservation and gene duplication……………………………………………………… 46 3.2. Synteny conservation in Vertebrates………………………………………………………………. 46 3.3. Early polarization and neural restriction of Magoh mRNA and protein in Ciona intestinalis sp. A... 47 3.4. Magoh mRNA expression in Branchiostoma lanceolatum……………………………………….. 48 4. DISCUSSION……………………………………………………………………………………….. 48 5. REFERENCES………………………………………………………………………………………… 51 CHAPTER 3 - Flagging a Mediterranean icon: Bursa scrobilator (Linné, 1758)……………..….. 61 1. INTRODUCTION……………………………………………………………………………………... 61 2. MATERIAL AND METHODS……………………………………………………………….………. 62 2.1. Sample data and morphological/anatomical features………………………………………………... 62 2.2. DNA sequencing and alignment…………………………………………………………………... 63 2.3. Phylogenetic Analyses…………………………………………………………………………….. 64 3. RESULTS…………………………………………………………………………………………… 64 3.1. Morphological analyses…………………………………………………………………………… 65 3.1.1. Protoconch-teleoconch features…………………………………………………………………. 65 3.1.2. Radular features…………………………………………………………………………………. 65 3.1.3. Anatomy…………………………………………………………………………………………… 65 3.2. Molecular analyses…………………………………………………………………………………... 66 4. DISCUSSION……………………………………………………………………………………….. 67 5. REFERENCES………………………………………………………………………………………… 69 CHAPTER 4 - Appendix: papers and short communication published (or in press) during the Ph.D. programme (chronological order by 2014 to 2011)..................................................................... 83 2014 (early view). Long-term demographic and reproductive trends in Ciona intestinalis sp. A.............. 84 2014. Investigating sperm cryopreservation in a model tunicate, Ciona intestinalis sp. A........................ 85 2013. The origin and dispersal pathway of the spotted sea hare Aplysia dactylomela (Mollusca: Opisthobranchia) in the Mediterranean Sea................................................................................................ 86 2013. Molecular data reveal cryptic lineages within the northeastern Atlantic and Mediterranean small mussel drills of the Ocinebrina edwardsii complex (Mollusca: Gastropoda: Muricidae).......................... 87 2013. Alien molluscan species established along the Italian shores: an update, with discussions on some Mediterranean alien species categories.............................................................................................. 88 2013. Biogeographical homogeneity in the eastern Mediterranean Sea - I: the opisthobranchs (Mollusca: Gastropoda) from Lebanon....................................................................................................... 89 2013. Biogeographical homogeneity in the eastern Mediterranean Sea. II. Temporal variation in 4 Lebanese bivalve biota................................................................................................................................ 90 2013. Slipping through the cracks: The taxonomic impediment conceals the origin and dispersal of Haminoea japonica, an invasive species with impacts to human health.................................................... 91 2013. The evolution of the Molluscan biota of the Sabaudia Lake: a matter of human history................. 92 2013. The alien spreading of Chama pacifica Broderip, 1835 (Mollusca: Bivalvia: Chamidae) in the Mediterranean Sea....................................................................................................................................... 93 2013. Spreading ways of an Atlantic fish species, Pomadasys incisus (Bowdich, 1825) (Osteichthyes: Haemulidae), within the Mediterranean Sea with new additional records from the French Mediterranean coast.................................................................................................................................... 94 2012. The status of the northeastern Atlantic and Mediterranean small mussel drills of the Ocinebrina aciculata complex (Mollusca: Gastropoda: Muricidae), with the description of a new species................ 95 2012. Marine alien Mollusca in Italy: a critical review and state of the knowledge................................. 96 2012. The invasive spotted sea hare Aplysia dactylomela (Mollusca: Gastropoda: Aplysiidae) - new records and spread pattern in the Mediterranean........................................................................................ 97 2012. Dyspanopeus sayi (Brachyura: Xanthoidea: Panopeidae) in Lago Fusaro (SW Italy)..................... 98 2011. Marine alien Mollusca in the Gulf of Trieste and neighbouring areas: a critical review and state of knowledge (updated in 2011)................................................................................................................. 99 2011. Marine alien species in Greek Seas: Additions and amendments by 2010....................................... 100 2011. New records of the genus Pachygrapsus (Crustacea: Decapoda) from the central Mediterranean Sea with a review of its Mediterranean zoogeography............................................................................... 101 2011. Mya arenaria Linné, 1758 (Mollusca: Bivalvia) in the Mediterranean: its distribution revisited.... 102 ACKNOWLEDGMENTS........................................................................................................................ 104 5 6 INTRODUCTION The present dissertation is divided into an introduction and three different chapters. Its general organization is intended to facilitate future publications, and therefore Chapters 1, 2 and 3 are hereby reported in journal-like style and should lay the basis for independent papers to be submitted to international peer-reviewed journals
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