Behavioural and neural correlates of operant conditioning in Lymnaea stagnalis : role of previous experience during development /

The freshwater mollusc Lymnaea stagnalis was utilized in this study to further the understanding of how network properties change as a result of associative learning, and to determine whether or not this plasticity is dependent on previous experience during development. The respiratory and neural correlates of operant conditioning were first determined in normally reared Lymnaea. The same procedure was then applied to differentially reared Lymnaea, that is, animals that had never experienced aerial respiration during their development. The aim was to determine whether these animals would demonstrate the same responses to the training paradigm. In normally reared animals, a behavioural reduction in aerial respiration was accompanied by numerous changes within the neural network. Specifically, I provide evidence of changes at the level of the respiratory central pattern generator and the motor output. In the differentially reared animals, there was little behavioural data to suggest learning and memory. There were, however, significant differences in the network parameters, similar to those observed in normally reared animals. This demonstrated an effect of operant conditioning on differentially reared animals. In this thesis, I have identified additional correlates of operant conditioning in normally reared animals and provide evidence of associative learning in differentially reared animals. I conclude plasticity is not dependent on previous experience, but is rather ontogenetically programmed within the neural network.

Elucidation of the Retinoid Signalling Pathway Involved in Axon Guidance in Lymnaea stagnalis and Xenopus laevis

The vitamin A metabolite, retinoic acid (RA), is known to play a crucial role in several developmental processes including axial patterning and differentiation. More recently, RA has been implicated in the regenerative process acting through its classical signaling pathway, the nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), to mediate gene transcription. Moreover, RA has been shown to act as a guidance molecule for growth cones of regenerating motorneurons of the pond snail, Lymnaea stagnalis. Our lab has recently shown that RA can induce this morphological response independent of nuclear transcription, however, the role of the retinoid receptors in RA-induced chemoattraction is still unknown. Here, I show that the retinoid receptors, RXR and RAR, may mediate the growth cones response to the metabolically active retinoic acid isomers, all-trans and 9-cis RA, in Lymnaea stagnalis. Data presented here show that both an RXR and RAR antagonist can block growth cone turning in response to application of both isomers. Because no prior investigations have shown growth cone turning of individual vertebrate neurons, I aimed to show that both retinoic acid isomers were capable of inducing growth cone turning of embryonic spinal cord neurons in the frog, Xenopus laevis. For the first time in Xenopus, I showed that both all-trans and 9-cis RA were able to induce significantly more neurite outgrowth from cultured embryonic spinal cord neurons and induce positive growth cone turning of individual growth cones. In addition, I showed that the presence of the RXR antagonist, HX531, blocked 9-cis RA-induced growth cone turning and the RARβ antagonist, LE135, blocked all-trans RA-induced growth cone turning in this species. Evidence provided here shows for the first time, conservation of retinoic acid-induced growth cone turning in a vertebrate model system. In addition, these data show that the receptors involved in this morphological response may be the same in vertebrates and invertebrates.

The role of retinoic acid in long-term memory formation and synaptic plasticity in the mollusc Lymnaea stagnalis

The active metabolite of vitamin A, retinoic acid (RA), is involved in memory formation and hippocampal plasticity in vertebrates. A similar role for retinoid signaling in learning and memory formation has not previously been examined in an invertebrate species. However, the conservation of retinoid signaling between vertebrates and invertebrates is supported by the presence of retinoid signaling machinery in invertebrates. For example, in the mollusc Lymnaea stagnalis the metabolic enzymes and retinoid receptors have been cloned from the CNS. In this study I demonstrated that impairing retinoid signaling in Lymnaea by either inhibiting RALDH activity or using retinoid receptor antagonists, prevented the formation of long-term memory (LTM). However, learning and intermediate-term memory were not affected. An additional finding was that exposure to constant darkness (due to the light-sensitive nature of RA) itself enhanced memory formation. This memory-promoting effect of darkness was sufficient to overcome the inhibitory effects of RALDH inhibition, but not that of a retinoid receptor antagonist, suggesting that environmental light conditions may influence retinoid signaling. Since RA also influences synaptic plasticity underlying hippocampal-dependent memory formation, I also examined whether RA would act in a trophic manner to influence synapse formation and/or synaptic transmission between invertebrate neurons. However, I found no evidence to support an effect of RA on post-tetanic potentiation of a chemical synapse. Retinoic acid did, however, reduce transmission at electrical synapses in a cell-specific manner. Overall, these studies provide the first evidence for a role of RA in the formation of implicit long-term memories in an invertebrate species and suggest that the role of retinoid signaling in memory formation has an ancient origin.

3D-ultrastructure, functions and stress responses of rhogocytes from the freshwater gastropods Biomphalaria glabrata and Lymnaea stagnalis

Rhogocytes, also termed ‘pore cells’, exist free in the hemolymph or embedded in the connective tissue of different body parts of molluscs, notably gastropods. These unique cells can be round, elongated or irregularly shaped, and up to 30 μm in diameter. Their hallmark is the so-called slit apparatus: i.e. pocket-like invaginations of the plasma membrane creating extracellular lacunae, bridged by cytoplasmic bars. These bars form distinctive slits of ca. 20 nm width. A slit diaphragm composed of proteins establishes a molecular sieve with holes of 20 x 20 nm. Different functions have been assigned to this special molluscan cell type, notably biosynthesis of the hemolymph respiratory protein hemocyanin. It has further been proposed, but not proven, that in the case of red-blooded snail species rhogocytes might synthesize the hemoglobin. However, the secretion pathway of these hemolymph proteins, and the functional role of the enigmatic slit apparatus remained unclear. Additionally proposed functions of rhogocytes, such as heavy metal detoxification or hemolymph protein degradation, are also not well studied. This work provides more detailed electron microscopical, histological and immunobiochemical information on the structure and function of rhogocytes of the freshwater snails Biomphalaria glabrata and Lymnaea stagnalis. By in situ hybridization on mantle tissues, it proves that B. glabrata rhogocytes synthesize hemoglobin and L. stagnalis rhogocytes synthesize hemocyanin. Hemocyanin is present, in endoplasmic reticulum lacunae and in vesicles, as individual molecules or pseudo-crystalline arrays. The first 3D reconstructions of rhogocytes are provided by means of electron tomography and show unprecedented details of the slit apparatus. A highly dense material in the cytoplasmic bars close to the diaphragmatic slits was shown, by immunogold labeling, to contain actin. By immunofluorescence microscopy, the protein nephrin was localized at the periphery of rhogocytes. The presence of both proteins in the slit apparatus supports the previous hypothesis, hitherto solely based on similarities of the ultrastructure, that the molluscan rhogocytes are phylogenetically related to mammalian podocytes and insect nephrocytes. A possible secretion pathway of respiratory proteins that includes a transfer mechanism of vesicles through the diaphragmatic slits is proposed and discussed. We also studied, by electron microscopy, the reaction of rhogocytes in situ to two forms of animal stress: deprivation of food and cadmium contamination of the tank water. Significant cellular reactions to both stressors were observed and documented. Notably, the slit apparatus surface and the number of electron-dense cytoplasmic vesicles increased in response to cadmium stress. Food deprivation led to an increase in hemocyanin production. These observations are also discussed in the framework of using such animals as potential environmental biomarkers.

Alpha-lactalbumin affects the acceptor specificity of Lymnaea stagnalis albumen gland UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalactosaminyltransferase: synthesis of GalNAc beta 1-->4Glc.

The N,N'-diacetyllactosediamine (lacdiNAc) pathway of complex-type oligosaccharide synthesis is controlled by a UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalac-tesaminyltransferase (beta 4-GalNAcT) that acts analogously to the common UDP-Gal:GlcNAc beta-R beta 1-->4-galactosyltransferase (beta 4-GalT). LacdiNAc-based chains particularly occur in invertebrates and cognate beta 4-GalNAcTs have been identified in the snail Lymnaea stagnalis, in two schistosomal species, and in several lepldopteran insect cell lines. Because of the similarity in reactions catalyzed by both enzymes, we investigated whether L. stagnalis albumen gland beta 4-GalNAcT would share with mammalian beta 4-GalT the property of interacting with alpha-lactalbumin (alpha-LA), a protein that only occurs in the lactating mammary gland, to form a complex in which the specificity of the enzyme is changed. It was found that, under conditions where beta 4-GalT forms the lactose synthase complex with alpha-LA, the snail beta 4-GalNAcT was induced by this protein to act on Glc with a > 100-fold increased efficiency, resulting in the formation of the lactose analog GalNAc beta 1-->4Glc. This forms the second example of a glycosyltransferase, the specificity of which can be altered by a modifier protein. So far, however, no protein fraction could be isolated from L. stagnalis that could likewise interact with the beta 4-GalNAcT. Neither had lysozyme c, a protein that is homologous to alpha-LA, an effect on the specificity of the enzyme. These results raise the question of how the capability to interact with alpha-LA has been conserved in the snail enzyme during evolution without any apparent selective pressure. They also suggest that snail beta 4-GalNAcT and mammalian beta 4-GalT show similarity at a molecular level and allows the identification of the beta 4-GalNAcT as a candidate member of the beta 4-GalT family.

Perturbing the impulse activity of a single identified neuron augments the formation of long-term memory in a molluscan semi-intact preparation /

The aim of this study was to investigate the neural correlates of operant conditioning in a semi-intact preparation of the pond snail, Lymnaea stagnalis. Lymnaea learns, via operant conditioning, to reduce its aerial respiratory behaviour in response to an aversive tactile stimulus to its open pneumostome. This thesis demonstrates the successful conditioning of na'ive semiintact preparations to show learning in the dish. Furthermore, these conditioned preparations show long-term memory that persists for at least 18 hours. As the neurons that generate this behaviour have been previously identified I can, for the first time, monitor neural activity during both learning and long-term memory consolidation in the same preparation. In particular, I record from the respiratory neuron Right Pedal Dorsal 1 (RPeD 1) which is part of the respiratory central pattern generator. In this study, I demonstrate that preventing RPeDl impulse activity between training sessions reduces the number of sessions needed to produce long-term memory in the present semi-intact preparation.

Mechanisms underlying Retinoic acid-induced chemoattraction in molluscan neurons

Retinoic acid, a derivative of vitamin A, is known to play diverse roles in development and regeneration. Previous research in the mollusc Lymnaea stagnalis has shown that a gradient of all-trans retinoic acid attracts the growth cones of cultured neurons. The present study investigates the sub-cellular mechanisms within the growth cones of Lymnaea pedal A neurons which mediate the attractive response to a gradient of alltrans retinoic acid. In this study, the mechanism of growth cone turning is shown to be local, as neurites mechanically isolated from their cell body retain the capacity to turn towards an exogenous gradient of all-trans retinoic acid. The turning response is dependent on the initiation of protein synthesis and calcium influx, but does not appear to involve signaling through protein kinase C (PKC). The retinoid X receptor (RXR), which classically functions as a transcription factor, was also shown to be involved in the turning response, functioning locally through a non-genomic pathway. These data show, for the first time in any species, that all-trans retinoic acid's chemotropic action involves a local mechanism involving non-genomic signaling through the RXR. As retinoic acid is known to playa role in regeneration, understanding the mechanisms underlying retinoic acid signaling may lead to further advances in regenerative neuroscience.

Identification of novel retinoid receptors and their roles in vertebrate and invertebrate nervous systems

In vertebrates, signaling by retinoic acid (RA) is known to play an important role in embryonic development, as well as organ homeostasis in the adult. In organisms such as adult axolotls and newts, RA is also important for regeneration of the CNS, limb, tail, and many other organ systems. RA mediates many of its effects in development and regeneration through nuclear receptors, known as retinoic acid receptors (RARs) and retinoid X receptors (RXRs). This study provides evidence for an important role of the RA receptor, RAR~2, in ,( '. regeneration ofthe spinal cord and tail of the adult newt. It has previously been proposed that the ability of the nervous system to regenerate might depend on the presence or absence of this RAR~2 isoform. Here, I show for the very first time, that the regenerating spinal cord of the adult newt expresses this ~2 receptor isoform, and inhibition of retinoid signaling through this specific receptor with a selective antagonist inhibits tail and spinal cord regeneration. This provides the first evidence for a role of this receptor in this process. Another species capable of CNS ~~generation in the adult is the invertebrate, " Lymnaea stagnalis. Although RA has been detected in a small number of invertebrates (including Lymnaea), the existence and functional roles of the retinoid receptors in most invertebrate non-chordates, have not been previously studied. It has been widely believed, however, that invertebrate non-chordates only possess the RXR class of retinoid receptors, but not the RARs. In this study, a full-length RXR cDNA has been cloned, which was the first retinoid receptor to be discovered in Lymnaea. I then went on to clone the very first full-length RAR eDNA from any non-chordate, invertebrate species. The functional role of these receptors was examined, and it was shown that normal molluscan development was altered, to varying degrees, by the presence of various RXR and RAR agonists or antagonists. The resulting disruptions in embryogenesis ranged from eye and shell defects, to complete lysis of the early embryo. These studies strongly suggest an important role for both the RXR and RAR in non-chordate development. The molluscan RXR and RAR were also shown to be expressed in the adult, nonregenerating eNS, as well as in individual motor neurons regenerating in culture. More specifically, their expression displayed a non-nuclear distfibution, suggesting a possible non-genomic role for these 'nuclear' receptors. It was shown that immunoreactivity for the RXR was present in almost all regenerating growth cones, and (together with N. Farrar) it was shown that this RXR played a novel, non-genomic role in mediating growth cone turning toward retinoic acid. Immunoreactivity for the novel invertebrate RAR was also found in the regenerating growth cones, but future work will be required to determine its functional role in nerve cell regeneration. Taken together, these data provide evidence for the importance of these novel '. retinoid receptors in development and regeneration, particularly in the adult nervous system, and the conservation of their effects in mediating RA signaling from invertebrates to vertebrates.

Identification and characterization of retinoic acid-induced morphological and electrophysiological changes in an invertebrate nervous system

The vitamin A metabolite, retinoic acid (RA) is known to play an important role in the development, patterning and regeneration of nervous tissue, both in the embryo and in the adult. Classically, RA is known to mediate the transcription of target genes through the binding and activation ofits nuclear receptors: the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Recently, mounting evidence from many animal models has implicated a number of RA-mediated effects operating independently of gene transcription, and thus highlights nove~ nongenornic actions of RA. For example, recent work utilizing cultured neurons from the pond snaa Lymnaea stagnalis, has shown that RA can elicit a regenerative response, growth cone turning, independently of "classical" transcriptional activation While this work illustrates a novel regeneration-inducing effect in culture, it is currently -unknown whether RA also induces regeneration in situ. This study has sought to determine RA's regenerative effucts at the morphological and molecular levels by utilizing an in situ approach focusing on a single identified dopaminergic neuron which possesses a known "mapped" morphology within the CNS. These studies show, for the first time in an invertebrate, that RA can increase neurite outgrowth of dopaminergic cells that have undergone a nerve-crush injury. Utilizing Western blot analysis, it was shown that this effect appears to be independent of any changes in whole CNS expression levels of either the RAR or RXR. Additionally, utilizing immunohistochemistry, to examine protein localization, there does not appear to be any obvious changes in the RXR expression level at the crush site. Changes in cell morphology such as neurity extension are known to be modulated by changes in neuronal firing activity. It has been previously shown that exposure to RA over many days can lead to changes in the electrophysiological properties of cultured Lymnaea neurons; however, no studies have investigated whether short-term exposure to RA can elicit electrophysiological changes and/or changes in firing pattern of neurons in Lymnaea or any other species. The studies performed here show, for the first time in any species, that short-tenn treatment with RA can elicit significant changes in the firing properties of both identified dopaminergic neurons and peptidergic neurons. This effect appears to be independent of protein synthesis, activation of protein kinase A or phospholipase C, and calcium influx but is both dose-dependent and isomer-dependent. These studies provide evidence that the RXR, but not RAR, may be involved, and that intracellular calcium concentrations decrease upon RAexposure with a time course, dose-dependency and isomer-dependency that coincide with the RA-induced electrophysiological changes. Taken together, these studies provide important evidence highlighting RA as a multifunctional molecule, inducing morphological, molecular and electrophysiological changes within the CNS, and highlight the many pathways through which RA may operate to elicit its effects.

Análise ultraestrutural da glândula corpo dorsal do caracol pulmonado Megalobulimus abbreviatus Becquaert 1948

O corpo dorsal (CD) é uma glândula endócrina presente junto aos gânglios cerebrais de todos os moluscos pulmonados (sinapomorfia) e, em Megalobulimus abbreviatus, o CD estende-se até o complexo de gânglios subesofageais. Tem sido atribuído o papel de produtor de hormônio gonadotrópico feminino para os pulmonados mais estudados (Helix pomatia, Helix aspersa e Lymnaea stagnalis). Em trabalho anterior foram descritos o aspecto histológico de glândula esteroidogênica e as variações citológicas sazonais do CD de Megalobulimus; o período de maior atividade sintética glandular coincide com o período de maior atividade reprodutiva. Um dos objetivos desse trabalho é identificar ultra-estruturalmente a presença e o tipo de terminações nervosas junto às células do CD, nas diferentes porções desta glândula, parte supraesofageal, junto aos gânglios cerebrais (GC) e subesofageal, junto aos gânglios pleurais (PL). Nesse estudo também pretende-se investigar a imunorreatividade ao neuropeptídeo FMRFamida no CD, supondo que este peptídeo, existente em grande quantidade em numerosos neurônios cerebrais, possa estar envolvido no controle inibitório destes gânglios sobre a atividade sintética glandular. Para o trabalho, diferentes regiões do CD (Fig. 2) foram fixadas em diferentes meses (5 animais/estação) em paraformaldeído 2% e glutaraldeído 1%, diluídos em tampão fosfato 0,1M, pH 7,4 (para imunoistoquímica, a fixação foi em paraformaldeído 4% e glutaraldeído 0,1%); pós-fixados em OsO4 1% diluído em TF; desidratado em álcool e acetona e incluído em resina Durcupan. As secções ultrafinas foram analisadas no Centro de Microscopia Eletrônica da UFRGS (microscópio eletrônico de transmissão JEOL JEM1200ExII). Para a imunoistoquímica, antes da pós-fixação com OsO4, os cortes (vibrátomo, 100 µm) foram processados para imunoistoquímica ao anticorpo anti-FMRFamida (Chemicon, 1:900) pelo procedimento peroxidase-anti-peroxidase. O CD se estende dos GC aos PL, acompanhando os conetivos cérebro-pleurais. Tanto na região subesofageal como supraesofageal, as células do CD de Megalobulimus abbreviatus estão inervadas por terminações axonais que fazem estreito contato com as células secretoras, semelhantes a sinapses “en passant”, contendo grânulos eletrodensos. Na porção dorsal à comissura cerebral são encontrados pequenos feixes de axônios, indicando que a inervação do CD provém da comissura cerebral. As terminações axonais encontradas no CD mostram algumas terminações axonais imunnorreativas a FMRFamida, sugerindo a participação deste neuropeptídeo no controle do CD. As células do CD apresentam características evidentes de tecido esteroidogênico.

Altered gene expression in the host brain caused by a trematode parasite: Neuropeptide genes are preferentially affected during parasitosis

Schistosome parasites adjust the physiology and behavior of their intermediate molluscan hosts to their own benefit. Previous studies demonstrated effects of the avian-schistosome Trichobilharzia ocellata on peptidergic centers in the brain of the intermediate snail host Lymnaea stagnalis. In particular, electrophysiological properties and peptide release of growth- and reproduction-controlling neuroendocrine neurons were affected. We now have examined the possibility that the expression of genes that control physiology and behavior of the host might be altered during parasitosis. A cDNA library of the brain of parasitized Lymnaea was constructed and differentially screened by using mRNA from the brain of both parasitized and nonparasitized snails. This screening yielded a number of clones, including previously identified cDNAs as well as novel neuronal transcripts, which appear to be differentially regulated. The majority of these transcripts encode neuropeptides. Reverse Northern blot analysis confirmed that neuropeptide gene expression is indeed affected in parasitized animals. Moreover, the expression profiles of 10 transcripts tested showed a differential, parasitic stage-specific regulation. Changes in expression could in many cases already be observed between 1.5 and 5 hr postinfection, suggesting that changes in gene expression are a direct effect of parasitosis. We suggest that direct regulation of neuropeptide gene expression is a strategy of parasites to induce physiological and behavioral changes in the host.

Parasitic castration by the digenian trematode Allopodocotyle sp alters gene expression in the brain of the host mollusc Haliotis asinina

Infection of molluscs by digenean trematode parasites typically results in the repression of reproduction - the so-called parasitic castration. This is known to occur by altering the expression of a range of host neuropeptide genes. Here we analyse the expression levels of 10 members of POU, Pax, Sox and Hox transcription factor gene families, along with genes encoding FNIRFamide, prohormone convertase and P-tubulin, in the brain ganglia of actively reproducing (summer), non-reproducing (winter) and infected Haliotis asinina (a vetigastropod mollusc). A number of the regulatory genes are differentially expressed in parasitised H. asinina, but in only a few cases do expression patterns in infected animals match those occurring in animals where reproduction is normally repressed. (c) 2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

The heteromeric 5-HT3A/B receptor:the effect of 5-HT3B subunits on receptor structure and ligand recognition

The 5-HT3 receptors are members of the cys-loop family of ligand-gated ion channels. Two functional subtypes are known, the homomeric 5HT3A and the heteromeric 5HT3A/B receptors, which exhibit distinct biophysical characteristics but are difficult to differentiate pharmacologically. Atomic force microscopy has been used to determine the stoichiometry and architecture of the heteromeric 5HT3A/B receptor. Each subunit was engineered to express a unique C-terminal epitope tag, together with six sequential histidine residues to facilitate nickel affinity purification. The 5-HT3 receptors, ectopically expressed in HEK293 cells, were solubilised, purified and decorated with antibodies to the subunit specific epitope tags. Imaging of individual receptors by atomic force microscopy revealed a pentameric arrangement of subunits in the order BBABA, reading anti-clockwise when viewed from the extracellular face. Homology models for the heteromeric receptor were then constructed using both the electron microscopic structure of the nicotinic acetylcholine receptor, from Torpedo marmorata, and the X-ray crystallographic structure of the soluble acetylcholine binding protein, from Lymnaea stagnalis, as templates. These homology models were used, together with equivalent models constructed for the homomeric receptor, to interpret mutagenesis experiments designed to explore the minimal recognition differences of both the natural agonist, 5-HT, and the competitive antagonist, granisetron, for the two human receptor subtypes. The results of this work revealed that the 5-HT3B subunit residues within the ligand binding site, for both the agonist and antagonist, are accommodating to conservative mutations. They are consistent with the view that the 5-HT3A subunit provides the principal and the 5-HT38 subunit the complementary recognition interactions at the binding interface.

Lymnaea truncatula em Portugal: contribuição para o estudo da bioecologia e da variação genética

Lymnaea truncatula é um gastrópode de água doce com importância em medicina por ser hospedeiro intermediário do tremátode parasita Fasciola hepatica. É o único hospedeiro intermediário desta espécie encontrado até agora em Portugal. A fasciolose é responsável por perdas de produtividade em gado. Nos humanos, é uma parasitose emergente com relevo em saúde pública em várias regiões do globo. Portugal é o segundo país europeu com maior prevalência. L. truncatula é de difícil controlo por ser anfíbia e ter boa capacidade de sobrevivência e adaptação. A eficácia dos programas de controlo e monitorização depende da correcta identificação das espécies de hospedeiros intermediários, dado que nem todas as espécies apresentam a mesma sensibilidade à infecção por F. hepatica. A morfologia da concha e a anatomia dos órgãos são insuficientes na identificação das espécies, sendo necessário usar técnicas de biologia molecular. Os objectivos deste estudo foram: estudar a distribuição, a variação da densidade populacional ao longo do ano, a diversidade genética, os habitats e a influência de parâmetros físicos, químicos e biológicos, na densidade populacional de L. truncatula em cinco distritos portugueses (Coimbra, Évora, Leiria, Lisboa e Funchal). Realizaram-se inquéritos malacológicos bimestrais durante 2 anos, entre Janeiro de 2006 e Dezembro de 2007 em Portugal continental e dois (Julho e Novembro de 2009) na ilha da Madeira. No continente, encontrou-se L. truncatula em: ribeiros temporários, com pouca vegetação, substrato de argila e matéria em decomposição e com água límpida, incolor e inodora, e com concentração de cálcio e de sulfatos até 50 mg/l e 267mg/l, respectivamente. A presença de outras espécies de moluscos, como Planorbarius metidjensis, Lymnaea peregra e da subclasse Prosobronchiata, assim como concentrações elevadas de nitratos, estão associados a uma menor densidade populacional. Na ilha da Madeira, os habitats foram predominantemente: escorrimentos de encosta, permanentes, com fraca exposição solar, pouca vegetação, substrato de rocha, argila e matéria em decomposição e com água límpida, incolor e inodora, acima dos 14,4ºC. A densidade populacional diminui com o aumento dos valores de nitratos e aumenta com a concentração de cálcio na água. As fezes de animais presentes junto às colecções de água não apresentaram ovos de F. hepatica. Foi encontrado um exemplar de F. hepatica no fígado de um gamo da Tapada Nacional de Mafra (distrito de Lisboa)Estudou-se a diversidade genética de L. truncatula através de RAPD-PCR e sequenciação do gene ribossomal 18S e da região ITS-2 (este também por PCR-RFLP). Identificou-se pela primeira vez em Portugal continental e na ilha da Madeira uma espécie, geneticamente diferente mas morfologicamente muito semelhante a L. truncatula – Lymnaea schirazensis. Na ilha da Madeira, foi detectado um haplotipo distinto do presente no continente. O marcador de RAPD - OPA2 e PCR-RFLP com HpaII, são bons marcadores para distinção entre L. truncatula e L. schirazensis. Adicionalmente, detectou-se pela primeira vez na ilha da Madeira L. (Pseudosuccinea) columella, conhecido hospedeiro intermediário de F. hepatica. Este estudo permitiu melhorar o conhecimento sobre hospedeiros intermediários de F. hepatica em Portugal, o que poderá melhorar o controlo e monitorização da fasciolose.