The field slug Deroceras reticulatum is a major pest in UK agriculture and amidst growing concern and regulatory pressures surrounding chemical molluscicides, innovation is required to advance the current repertoire of slug controls. This study set out to investigate the bacteria associated with D. reticulatum to assess their importance to the slug and potential as a target for biological control. Slug gut bacterial isolates identified using the phenotypical API system (BioMérieux) and 16S rRNA gene sequencing, were mainly soil-dwelling organisms of the phyla Proteobacteria and Bacteriodetes some of which may be important in human or plant disease. A ribosomal intergenic spacer analysis (RISA) was developed to study microbial communities in the slug gut. Slugs had an average species richness of 12 and comparing the bacterial communities in slugs from different locations yielded a mean similarity of 0.159 (Jaccard index) which was significantly lower than similarity indices of slugs collected within a single location (Mean Jaccard index 0.205, p<0.001, ANOVA). Cloning and sequencing of RISA bands common to slugs and slug eggs, but absent from the surrounding soil and plants identified bacteria for future investigation as potential beneficial symbionts. Bacteria extracted from the slug gut were tested for sensitivity to 16 antibiotics and greatest inhibition of growth was observed for chloramphenicol, gentamicin and tetracycline. These antibiotics administered to slugs by feeding and injection caused a reduction in gut-associated bacteria in plate counts, and in bacterial 16S rDNA quantities estimated by real-time quantitative PCR. Field collected D. reticulatum has a large transient gut bacterial population which is reduced upon starvation to a low background level. No significant detrimental effect of antibiotic treatment on the fitness and survival of the slugs was seen, in some instances control slugs suffered greater mortality than slugs that had been injected with antibiotic. Slugs that died during bioassays had a significantly greater amount of bacterial 16S rDNA in their gut than slugs that were sacrificed as healthy individuals suggesting the presence of a bacterial pathogen. This study has found little evidence that a bacterial symbiont may exist and be important for optimal fitness and survival of D. reticulatum, but insight into slug associated bacteria will be valuable in the direction of future studies in this field.

Ph.D. thesis "The complex chacterisation of the nematode Phasmarhabditis hermpahrodita" is focused on the study of ecology of slug parasitic nematode P. hermaphrodita, its general characterisation in the context of all nowadays knowledge. The thesis consists the general introduction, that introduces the whole nowadays research of P. hermaphrodita, and four original works, that follow the introductory chapter. The first work deals with the orientation of P. hermaphrodita in 2D and 3D space and reaction on the presence of different hosts or nutritive substrates. The other study deals with the reaction of P. hermaphrodita on various animal and plant nutritive substrates and in the first place the influence of these substrates on the development of nematodes and quality and quantity of progeny. The third scope of this thesis is the influence of intraspecific competition on development, quality and quantity of nematodes and the last part of this text comprises a short study of the persistence of P. hermaphrodita in different substrates, from mineral to organic.

One of the goals of neuroscience is to classify all of the neurons in the brain. Neuronal types can be defined using a combination of morphology, electrophysiology, and gene expression profiles. Gene expression profiles allow differentiation between cells that share similar characteristics. Leveraging the advantage of Berghia stephanieae (Gastropoda; Nudibranchia), which has around 28,000 neurons, I constructed high-throughput single-neuron transcriptomes for its whole brain. I produced a single-cell dissociation protocol and a custom data analysis pipeline for data of this nature. Around 129,000 cells were collected from 18 rhinophore ganglia and 20 circumesophageal ring ganglia (brain), consisting of the cerebropleural, pedal, and buccal ganglia. Messenger RNA libraries were constructed using the 10X Genomics’ Chromium platform. After library preparation, around 1,000 cells were recovered and sequenced. The HTStream package was utilized to trim off unwanted sequences from the raw reads and remove PCR duplicates and other contamination, then the salmon alevin package was employed to construct gene-by-cell matrices containing all the transcripts for each gene in each cell. The Seurat pipeline was used to extract this expression data from the matrices, normalize it, and perform dimensionality reduction. The cells were clustered based on similarities in their gene expression profiles. The cells formed eight clusters on a UMAP graph, each having distinct marker genes. Additionally, one cluster was composed of almost exclusively cells from the rhinophore ganglia, accounting for 30% of all rhinophore ganglion cells in the sample. Cells from the rhinophore ganglia are as heteregenous as cells from the rest of the brain, with cells forming six clusters. Cell populations that express the same neurotransmitter were identified for a wide range of both small-molecule neurotransmitters and neuropeptides. In a separate project, the locomotion of Berghia was recorded over 9 days with 2 lighting regimes: LD first and DD first. The results suggest that locomotion of Berghia is governed by circadian clock and that Berghia is nocturnal. Hunger state likely plays a role in modulating this circadian rhythm.

Yes / The extensive use of mollusc shell as a versatile raw material is testament to its importance in prehistoric times. The consistent choice of certain species for different purposes, including the making of ornaments, is a direct representation of how humans viewed and exploited their environment. The necessary taxonomic information, however, is often impossible to obtain from objects that are small, heavily worked or degraded. Here we propose a novel biogeochemical approach to track the biological origin of prehistoric mollusc shell. We conducted an in-depth study of archaeological ornaments using microstructural, geochemical and biomolecular analyses, including ‘palaeoshellomics’, the first application of palaeoproteomics to mollusc shells (and indeed to any invertebrate calcified tissue). We reveal the consistent use of locally-sourced freshwater mother-of-pearl for the standardized manufacture of ‘double-buttons’. This craft is found throughout Europe between 4200–3800 BCE, highlighting the ornament-makers’ profound knowledge of the biogeosphere and the existence of cross-cultural traditions. / Ministry of Education, Universities and Research Young Researcher: European Commission PERG-GA-2010-26842: Leverhulme Trust: Centre National de la Recherche Scientifique: Campus France, Universita` Italo-Francese PHC Galile´ programme

Developing robust behavioral assays to study olfactory-driven behaviors allows for greater insight into the neural mechanisms behind them. Oftentimes, olfactory behavioral assays require a two-choice design, consistent variables, and controlled stimulus application. This can be challenging when working with marine dwelling organisms such as nudibranchs. Extensive work shows the importance of olfaction in both pre-metamorphic larval development and adult stage nudibranchs (Gastropoda, Mollusca). However, there is little research investigating how olfaction plays a role in rapidly developing, post-metamorphic juvenile nudibranchs such as Berghia stephanieae. To study olfactory-associated behaviors in juvenile Berghia, a novel behavioral microfluidic chip was designed which met the requirements for a reliable olfaction assay. Baseline motor behaviors such as general locomotion, turns, and contractions were observed. Despite individuals being raised in the same cohort, animal-to-animal variability was found in relation to their baseline behaviors. Xylene cyanol, a commonly used dye in microfluidics, was found to be aversive at a range of concentrations (0.075% - 0.025%) causing the animal to tightly contract and turn away from the stimulus. Juveniles showed no significant behavioral response to a 0.00125% dilution which was necessary to confirm a constant flow in the microfluidic chip. Juveniles showed a strong preference towards fluid that had been conditioned with their preferred food source odor, the sea anemone Exaiptasia diaphana. This was judged to be an innate preference because it occurred upon the first exposure of these animals to the odor. Serial dilution of Exaiptasia conditioned seawater (ECS) uncovered a threshold for behavioral preference of 60%. In summary, the development of this behavioral provides an opportunity to present controlled olfactory stimuli while observing the juvenile’s behaviors. This will allow future experiments to examine the neural mechanisms behind both aversive and attractive stimuli and provides a method for testing olfactory learning and memory in this species.

This thesis centres around two genera of large peltospirid gastropods (Mollusca: Neomphalina: Peltospiridae) endemic to hydrothermal vent ecosystems. One is the 'scaly-foot gastropod', an emblematic species of the Indian Ocean vents with unique dermal sclerites covering the foot like roof tiles. The other was recently discovered from expeditions to the Southern and Indian oceans, lacks sclerites and possesses large opercula. As both genera and their assigned species remained undescribed, they were formally described herein which forms a basis to understanding their biology. The 'scaly-foot gastropod' from both the Central Indian Ridge (CIR) and the Southwest Indian Ridge (SWIR) were confirmed to represent a single species and is formally named as Chrysomallon squamiferum. Through molecular genetic analyses using the COI gene, genetic differentiation between SWIR and CIR populations was detected for the 'scaly-foot gastropod'. In contrast, the peltospirids with large opercula from the East Scotia Ridge (ESR) and the SWIR proved to be two distinct species within an undescribed genus. The ESR species was formally described as Gigantopelta chessoia and the SWIR species as G. aegis. The molecular genetic analyses of the COI gene, confirmed the genetic isolation of the two and consolidated their status as separate species. A 3D tomographic model of Chrysomallon squamiferum was generated to characterise the soft anatomy and morphology as well as to understand its internal anatomy and adaptation which remained little-studied. Further to the enlarged esophageal gland already known to house chemosynthetic endosymbionts, C. squamiferum was discovered to have a hypertrophied circulatory system with a gigantic, muscular heart and large ctenidium to adapt to life in a hypoxic environment and to supply the endosymbionts with necessary chemicals. Histological examinations of the sclerites and operculum showed that it was unlikely that the sclerites originated from operculum duplication. Comparisons with polyplacophoran scales revealed starkly different secretion mechanisms despite the superficial similarity, which has implications on the placement of sclerite-bearing Cambrian taxa. Overall, the results from this thesis ascertained the systematic positions of these large-sized, enigmatic peltospirids, and led to improved understanding of their ecology and evolution. The important role of larval dispersal in maintaining metapopulations across the distribution of a vent-endemic taxa is highlighted. The adaptations of vent-endemic taxa remains little-known even in well-studied species, warranting future studies on these and other species.

Nematodes of the genus Phasmarhabditis (Nematoda: Rhabditidae) are world-wide distributed molluscs´ parasites. Species P. hermaphrodita is the only commercially produced bio-agent for slug control. The aim of this thesis is to evaluate some ecologic characteristics of three newly described species of the genus Phasmarhabditis (P. bohemica, P. bonaquaense a P. apuliae) in polyxenic and monoxenic cultures. Three bacterial isolates, Pseudomonas sp., Acinetobacter sp. and Flavobacterium sp. were chosen for testing the monoxenic cultures. The growing ability of tested nematodes, the effect on the quality of progeny and the rate of development were examined on various organic substrates (compost, faeces of Deroceras slugs, homogenized Deroceras slugs, homogenized larvae of Galleria mellonella and homogenized pig kidney). Subsequently, the growth of nematodes was tested in solid and liquid media. The last experiment was focused on evaluating the impact of polyxenic and monoxenic nematode cultures on the mortality and feeding activity of Deroceras reticulatum and Cepaea hortensis. As is assumed in the results chapter, all examined species of nematodes are lethal facultative parasites of Deroceras spp. They are able to grow in broad spectre of organic substrates and bacteria which influence the production of the progeny, but the qualitative parameters of dauer juveniles remain more or less unaffected.

Mollusc shells are unique adaptations that serve to protect the organisms that make them, and are a defining feature of the phylum. However the molecular underpinnings of shell forming processes are still largely unexplored. To further understand mollusc shell formation, I studied three bivalve species in this project: the blue mussel Mytilus edulis, the Pacific oyster Crassostrea gigas, and the king scallop Pecten maximus. While previous analyses of the shell proteomes showed species specificity, transcriptomes of the mantle tissues revealed more commonalities. To reconcile these differences, I studied differential gene expression in shell damage-repair experiments and during the formation of the first larval shell, to produce a comprehensive overview of shell formation processes. Expression data showed large biological variability between individuals, requiring matched-pair experimental designs to detect differential gene expression during shell repair. Loci differentially expressed during shell repair and in the larvae encoded shell matrix proteins, transmembrane transporters, and novel transcripts. A large number of shell matrix proteins, encoded in differentially expressed loci, were common in all three species during shell formation, indicating that shell forming proteins between different species may be more common than previously thought. Differential expression of transmembrane transporters during shell repair indicated that the animals may be regulating bicarbonate ions during shell formation. Finally, the experiments revealed novel transcripts, with unknown annotations to public datasets, that may putatively be involved in shell formation.

À partir des ovocytes de la palourde Spisula solidissima, un ADNc codant un récepteur nommé Spi-OAR a été cloné et séquencé. Une analyse de la séquence en acides aminés a indiqué que ce nouveau récepteur possède une forte similarité avec les récepteurs β-adrénergiques et les récepteurs à octopamine. En effet, il est étroitement lié à la classe des récepteurs à octopamine « β-adrénergique-like » couplés à une protéine Gs. L’ADNc de Spi-OAR a été introduit dans un vecteur d'expression (pCEP4) et un épitope reconnaissable par un anticorps commercial a été ajouté au segment N-terminal. Cette construction a été transfectée dans des cellules hôtes (HEK 293) et des études d’immunofluorescence ont montré une expression efficace du récepteur au niveau membranaire. Également, des mesures d'AMPc pour les cellules exprimant Spi-OAR ont révélé une augmentation de ce messager secondaire lors de l'ajout de l'octopamine, et dans une moindre mesure, la tyramine, tandis que la dopamine, la sérotonine et l'histamine n’ont engendré aucun effet. Une légère activité constitutive de ce récepteur dans les cellules hôtes a été observée. De plus, une analyse RT-PCR avec des oligonucléotides spécifiques a révélé l'ARNm de Spi-OAR non seulement dans les ovocytes, mais aussi dans les gonades, le cœur, les muscles adducteurs, les branchies et les ganglions suggérant que ce récepteur soit exprimé de façon ubiquitaire dans divers tissus et dans différents stades embryonnaires chez la palourde. En outre, des études avec des ovocytes isolés n'ont montré aucun effet de l’octopamine sur la réactivation méiotique. Des études éventuelles pourront finalement confirmer le rôle fonctionnel de Spi-OAR. / A cDNA encoding for an octopamine receptor named Spi-OAR was cloned and sequenced from the surf clam Spisula solidissima oocytes. An analysis of its predicted amino acid sequence showed a high degree of similarity with β-adrenergic and octopamine receptors. This receptor qualifies as a novel receptor closely related to the proposed class of insect octopamine « β-adrenergic–like » receptors coupled to Gs protein. This cDNA was introduced into an expression vector (pCEP4), with an added N-terminal FLAG tag sequence, and transfected in host cells (HEK 293). Immunofluorescence studies showed expression of the receptor with a proper localization to the plasma membrane. Measurements of cAMP in transfected cells revealed that addition of octopamine, and to a lower extent, tyramine induced a rise in cAMP while dopamine, serotonine and histamine had no effect. Overexpression of Spi-OAR in mammalian cells induced slight constitutive increase of cAMP. An RT-PCR analysis with specific oligonucleotides revealed the presence of the receptor mRNA not only in oocytes but also in whole gonads, heart, adductor muscle, gills and ganglia suggesting that this receptor is likely ubiquitously expressed. Expression of Spi-OAR was also detected at different embryonic stages. Despite the demonstrated expression of Spi-OAR in oocytes, octopamine had no effect on meiotic reinitiation. Further studies will examine the function of Spi-OAR.

The molluscs are very suitable model of invertebrate group for ecological and palaeoecological studies for their specific traits. In the first part of the thesis, we showed based on available fossil mollusc successions that development of the floodplain mollusc fauna took place quite different way in various river floodplains, depending on their specifics and geographical location, because especially the ones situated in the chernozem area of the Czech Republic had very different history. Detailed processing of five fossil mollusc successions in the lower Ohře River floodplain confirmed the impoverishment of recent forest malacofauna does not relate to natural processes only (chapters 3 and 10), but it arises from historical development of this area which was under permanent stress of the human settlement and agricultural utilization (chapter 1) since the coming of the Neolithic people. We made similar conclusions in case of another 11 fossil mollusc successions in the neighbouring České Středohoří Mts. (chapter 2). In the second part of the thesis (chapter 3), we investigated the ecological patterns responsible for species richness and composition of the floodplain mollusc fauna are especially the elevation and humidity gradient, and then vegetation type and its biomass, light conditions of the...