Fasciola hepatica: A light and electron microscope study of sustentacular tissue and heterophagy in the testis

In order to investigate cytolytic activity in the testis of Fasciola hepatica, flukes belonging to several different triclabendazole (TCBZ)-sensitive and TCBZ-resistant isolates, and wildtype flukes from field infections, were studied by light and electron microscopy with a view to identifying sites of heterophagy and macromolecular hydrolysis. At the periphery of the testis tubules in all the flukes examined, large euchromatic nuclei, each bearing a prominent nucleolus, were seen. These were invested with a thin cytoplasmic layer, extensions of which partially enveloped and probably supported the neighbouring spermatogonia. No lateral cell boundaries were identified in this tissue, possibly indicating syncytial organisation. The tissue, considered to be analogous to Sertoli cells in vertebrate testis, was identified as sustentacular tissue. It displayed cytoplasmic features consistent with protein/glycoprotein synthesis (through a granular endoplasmic reticulum-Golgi mediated mechanism) and intracellular digestion/heterophagy (through a lysosomal system). The intracytoplasmic hydrolytic activity of the sustentacular tissue probably serves to scavenge effete cells and cytoplasmic debris, to recycle useful molecules, to promote spermatozoon maturation and possibly to aid osmoregulation within the tubules. Certain protein-containing macromolecules synthesised in the sustentacular tissue may contribute to the seminiferous fluid, or have pheromonal activity. The presence of numerous mitochondria and abundant smooth endoplasmic reticulum is consistent with facilitation of inward and outward movement of micromolecular nutrients, metabolites including excretory products and water. In the sustentacular tissue of certain flukes with dysfunctional spermiogenesis, there was increased heterophagic and cytolytic scavenging activity. The cytoplasmic residual vacuoles remaining after the release of spermatids were also identified as possible sites for lysosome-mediated intracellular digestion and osmoregulation in the testis tubules of F. hepatica. (C) 2012 Elsevier B.V. All rights reserved.

The effects of chemicals in isopods : a multi-organizational evaluation

The global aim of this thesis was to evaluate and assess the effects of a pesticide (dimethoate) and a metal (nickel), as model chemicals, within different organization levels, starting at the detoxification pathways (enzymatic biomarkers) and energy costs associated (energy content quantification, energy consumption and CEA) along with the physiological alterations at the individual and population level (mortality), leading to a metabolomic analysis (using liquid 1H-NMR) and finally a gene expression analysis (transcriptome and RT-qPCR analysis). To better understand potential variations in response to stressors, abiotic factors were also assessed in terrestrial isopods such as temperature, soil moisture and UV radiation. The evaluation performed using biochemical biomarkers and energy related parameters showed that increases in temperature might negatively affect the organisms by generating oxidative stress. It also showed that this species is acclimated to environments with low soil moisture, and that in high moisture scenarios there was a short gap between the optimal and adverse conditions that led to increased mortality. As for UV-R, doses nowadays present have shown to induce significant negative impact on these organisms. The long-term exposure to dimethoate showed that besides the neurotoxicity resulting from acetylcholinesterase inhibition, this stressor also caused oxidative stress. This effect was observed for both concentrations used (recommended field dose application and a below EC50 value) and that its combination with different temperatures (20ºC and 25ºC) showed different response patterns. It was also observed that dimethoate’s degradation rate in soils was higher in the presence of isopods. In a similar study performed with nickel, oxidative stress was also observed. But, in the case of this stressor exposure, organisms showed a strategy where the energetic costs necessary for detoxification (biomarkers) seemed to be compensated by positive alterations in the energy related parameters. In this work we presented for the first time a metabolomic profile of terrestrial isopods exposed to stressors (dimethoate and niquel), since until the moment only a previous study was performed on a metabolomic evaluation in nonexposed isopods. In the first part of the study we identify 24 new metabolites that had not been described previously. On the second part of the study a metabolomic profile variation of abstract non-exposed organism throughout the exposure was presented and finally the metabolomic profile of organisms exposed to dimethoate and nickel. The exposure to nickel suggested alteration in growth, moult, haemocyanin and glutathione synthesis, energy pathways and in osmoregulation. As for the exposure to dimethoate alterations in osmoregulation, energy pathways, moult and neurotransmission were also suggested. In this work it was also presented the first full body transcriptome of a terrestrial isopod from the species Porcellionides pruinosus, which will complement the scarce information available for this group of organisms. This transcriptome also served as base for a RNA-Seq and a RT-qPCR analysis. The results of the RNA-Seq analysis performed in organisms exposed to nickel showed that this stressor negatively impacted at the genetic and epigenetic levels, in the trafficking, storage and elimination of metals, generates oxidative stress, inducing neurotoxicity and also affecting reproduction. These results were confirmed through RT-qPCR. As for the impact of dimethoate on these organisms it was only accessed through RT-qPCR and showed oxidative stress, an impact in neurotransmission, in epigenetic markers, DNA repair and cell cycle impairment. This study allowed the design of an Adverse Outcome Pathway draft that can be used further on for legislative purposes.

Caracterização endócrina da PTH-L e calcitonina no transporte de cálcio e na secreção de bicarbonato no intestino da dourada (Sparus auratus)

Dissertação de Mestrado, Biologia Marinha, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2009

Role of insulin and insulin-like peptides in bone formation: identification of bone-specific target genes and regulatory mechanisms, and characterization of the insulin-mimetic effect of vanadium

Tese de Doutoramento em Biologia, Especialidade em Biologia Molecular, Universidade do Algarve, 2008

Five gonadotrophin-releasing hormone receptors in a teleost fish: isolation, tissue distribution and phylogenetic relationships

Gonadotrophin-releasing hormone (GnRH) is the main neurohormone controlling gonadotrophin release in all vertebrates, and in teleost fish also of growth hormone and possibly of other adenohypophyseal hormones. Over 20 GnRHs have been identified in vertebrates and protochoordates and shown to bind cognate G-protein couple receptors (GnRHR). We have searched the puffer fish, Fugu rubripes, genome sequencing database, identified five GnRHR genes and proceeded to isolate the corresponding complementary DNAs in European sea bass, Dicentrachus labrax. Phylogenetic analysis clusters the European sea bass, puffer fish and all other vertebrate receptors into two main lineages corresponding to the mammalian type I and II receptors. The fish receptors could be subdivided in two GnRHR1 (A and B) and three GnRHR2 (A, B and C) subtypes. Amino acid sequence identity within receptor subtypes varies between 70 and 90% but only 50–55% among the two main lineages in fish. All European sea bass receptor mRNAs are expressed in the anterior and mid brain, and all but one are expressed in the pituitary gland. There is differential expression of the receptors in peripheral tissues related to reproduction (gonads), chemical senses (eye and olfactory epithelium) and osmoregulation (kidney and gill). This is the first report showing five GnRH receptors in a vertebrate species and the gene expression patterns support the concept that GnRH and GnRHRs play highly diverse functional roles in the regulation of cellular functions, besides the ‘‘classical’’ role of pituitary function regulation.

Contamination of farmed gilthead sea bream (Sparus aurata, Linnaeus, 1758) by the parasite Amyloodinium ocellatum: a proteomic approach

Dissertação de mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Uniersidade do Algarve, 2015

Cation transporters/channels in plants: Tools for nutrient biofortification

Cation transporters/channels are key players in a wide range of physiological functions in plants, including cell signaling, osmoregulation, plant nutrition and metal tolerance. The recent identification of genes encoding some of these transport systems has allowed new studies toward further understanding of their integrated roles in plant. This review summarizes recent discoveries regarding the function and regulation of the multiple systems involved in cation transport in plant cells. The role of membrane transport in the uptake, distribution and accumulation of cations in plant tissues, cell types and subcellular compartments is described. We also discuss how the knowledge of inter- and intra-species variation in cation uptake, transport and accumulation as well as the molecular mechanisms responsible for these processes can be used to increase nutrient phytoavailability and nutrients accumulation in the edible tissues of plants. The main trends for future research in the field of biofortification are proposed.

The effect of extracellular osmolality on cell volume and resting skeletal muscle metabolism

The purpose of the current investigation was to establish an in-l'itro skeletal muscle model to study acute alterations in resting skeletal muscle cell volume. Isolated. whole muscle (SOL and EDL) was dissected from Long Evans rats and incubated for 60 min in Sigma Medium-199 (resting tension (lg). bubbled with 95:5% 02:C02, 30 ± 2°C, and pH 7.4). Media osmolality was altered to simulate hypo-osmotic (190 ± 10 Osm) (HYPO) or hyper-osmotic conditions (400 ± 10 Osm) (HYPER) while an iso-osmotic condition (290± 1 0 Osm) (CON) served as a control (n= 17.19.17). Following incubation, relative muscle water content decreased with HYPER and increased with HYPO in both muscle types (p<0.05). The cross-sectional area of HYPO SOL type I and type II fibres increased (p<0.05) while the EDL type 11 fibre area decreased in HYPER and increascd from HYPO exposure. Furthermore, HYPER exposure in both muscles lead to decreased ATP and phosphocreatine (p<0.05) and increased creatine and lactate (p<0.05) compared to CON. This isolated skeletal muscle model proved viable and demonstrated that altering extracellular osmolality could cause acutc alterations in muscle water content and resting muscle metabolism.

Biochemical effects of cadmium, salinity and temperature on the catabolism of proteins and purines in oreochromis mossambicus (Peters)

In the present investigation, three important stressors: cadmium ion (Cd++), salinity and temperature were selected to study their effects on protein and purine catabolism of O. mossambicus. Cadmium (Cd) is a biologically nonessential metal that can be toxic to aquatic animals. Cadmium is a trace element which is a common constituent of industrial effluents. It is a non-nutrient metal and toxic to fish even at low concentrations. Cadmium ions accumulate in sensitive organs like gills, liver, and kidney of fish in an unregulated manner . Thus; the toxic effects of cadmium are related to changes in natural physiological and biochemical processes in organism. The mechanics of osmoregulation (i.e. total solute and water regulation) are reasonably well understood (Evans, 1984, 1993), and most researchers agree that salinities that differ from the internal osmotic concentration of the fish must impose energetic regulatory costs for active ion transport. There is limited information on protein and purine catabolism of euryhaline fish during salinity adaptation. Within a range of non-lethal temperatures, fishes are generally able to cope with gradual temperature changes that are common in natural systems. However, rapid increases or decreases in ambient temperature may result in sub lethal physiological and behavioral responses. The catabolic pathways of proteins and purines are important biochemical processes. The results obtained signifies that O. mossambicus when exposed to different levels of cadmium ion, salinity and temperature show great variation in the catabolism of proteins and purines. The organism is trying to attain homeostasis in the presence of stressors by increasing or decreasing the activity of certain enzymes. The present study revealed that the protein and purine catabolism in O. mossambicus is sensitive to environmental stressors.

Separation and viability of gill and hepatopancreatic cells of a mangrove crab Ucides cordatus

The gills contain essential cells for respiration and osmoregulation, whereas the hepatopancreas is the site of digestion, absorption, and nutrients storage. The aim of this work was to separate and characterize gill and hepatopancreatic cells of the mangrove crab, Ucides cordatus. For gills, the methodology consisted of an enzymatic cellular dissociation using Trypsin at 0.5%, observation of cellular viability with Tripan Blue, and separation of cells using discontinuous sucrose gradient at concentrations of 10%, 20%, 30%, and 40%. The hepatopancreatic cells were dissociated by magnetic stirring, with posterior separation by sucrose gradient at the same concentrations above. For gills, a high cellular viability was observed (92.5 +/- 2.1%), with hemocyte cells in 10% sucrose layer (57.99 +/- 0.17%, *P < 0.05), principal cells in the 20% sucrose layer (57.33 +/- 0.18, *P < 0.05), and thick cells and pillar cells in the 30% and 40% sucrose layers, respectively (39.54 +/- 0.05%, *P < 0.05; and 41.81 +/- 0.04%, *P < 0.05). The hepatopancreatic cells also showed good viability (79.22 +/- 0.02%), with the observation of embryonic (E) cells in the 10% sucrose layer (67.87 +/- 0.06%, **P < 0.001), resorptive (R) and fibrillar (F) cells in the 20% and 30% sucrose layers (44.71 +/- 0.06%, **P < 0.001, and 43.25 +/- 0.01%, *P < 0.05; respectively), and blister (B) cells in the 40% sucrose layer (63.09 +/- 0.03%, **P < 0.001). The results are a starting point for in vitro studies of heavy metal transport in isolated cells of the mangrove crab U. cordatus, subjected to contamination by metals in the mangrove habitat where they are found.

Purification and partial characterization of a midgut membrane-bound alpha-glucosidase from Quesada gigas (Hemiptera: Cicadidae)

Adults of Quesada gigas (Hemiptera: Cicadidae) have a major alpha-glucosidase bound to the perimicrovillar membranes, which are lipoprotein membranes that surround the midgut cell microvilli in Hemiptera and Thysanoptera. Determination of the spatial distribution of alpha-glucosidases in Q. gigas midgut showed that this activity is not equally distributed between soluble and membrane-bound isoforms. The major membrane-bound enzyme was solubilized in the detergent Triton X-100 and purified to homogeneity by means of gel filtration on Sephacryl S-100, and ion-exchange on High Q and Mono Q columns. The purified alpha-glucosidase is a protein with a pH optimum of 6.0 against the synthetic substrate p-nitrophenyl alpha-D-glucoside and M(r) of 61,000 (SDS-PAGE). Taking into account V(Max)/K(M) ratios, the enzyme is more active on maltose than sucrose and prefers oligomaltodextrins up to maltopentaose, with lower efficiency for longer chain maltodextrins. The Q gigas alpha-glucosidase was immunolocalized in perimicrovillar membranes by using a monospecific polyclonal antibody raised against the purified enzyme from Dysdercus peruvianus. The role of this enzyme in xylem fluid digestion and its possible involvement in osmoregulation is discussed. (C) 2009 Elsevier Inc. All rights reserved.

Functional analysis of natriuretic peptide receptors in the bladder of the toad, bufo marinus

This study aimed to localize and characterize natriuretic peptide binding sites in the urinary bladder of Bufo marinus and to then examine the effect of natriuretic peptides on the bladder vascular tone and water reabsorption in isolated perfused bladder preparations. Specific ¹²⁵I-rat atrial natriuretic peptide (¹²⁵I-rANP) binding sites were present on blood vessels, muscle, and epithelium. In tissue sections and/or isolated membranes, the binding was completely displaced by frog ANP, rat ANP, and porcine C-type natriuretic peptide (CNP; membranes only). However, a reduction in binding was observed after incubation with ¹²⁵I-rANP and 1 μM of the natriuretic peptide receptor-C (NPR-C) ligand C-ANF, but residual binding remained suggesting the presence of two distinct binding sites. Electrophoresis of bladder membranes cross-linked to ¹²⁵I-rANP identified two bands at approximately 70 and 140 kDa that correspond to the monomeric mass of NPR-C and the guanylate cyclase receptors, respectively. Furthermore, the presence of natriuretic peptide receptor-A and NPR-C mRNA in the bladder was demonstrated with reverse transcription–polymerase chain reaction. In addition, rat ANP, frog ANP, and porcine CNP stimulated a significant increase in cGMP generation in bladder membrane preparations, which indicated the presence of guanylate cyclase-linked receptors. In perfused bladder preparations, arginine vasotocin increased perfusion pressure and water permeability. The infusion of frog ANP or porcine CNP failed to alter perfusion pressure or water reabsorption in the presence or absence of arginine vasotocin. This study identified a well-developed natriuretic peptide receptor system in the urinary bladder of B. marinus but the function of the receptors remains unclear.

Comparative aspects of natriuretic peptide physiology in non-mammalian vertebrates: a review

The natriuretic peptide system is a complex family of peptides and receptors that is primarily linked to the maintenance of osmotic and cardiovascular homeostasis. A natriuretic peptide system is present in each vertebrate class but there are varying degrees of complexity in the system. In agnathans and chondrichthyians, only one natriuretic peptide has been identified, while new data has revealed that multiple types of natriuretic peptides are present in bony fish. However, it seems in tetrapods that there has been a reduction in the number of natriuretic peptide genes, such that only three natriuretic peptides are present in mammals. The peptides act via a family of guanylyl cyclase receptors to generate the second messenger cGMP, which  mediates a range of physiological effects at key targets such as the gills, kidney and the cardiovascular system. This review summarises the current knowledge of the natriuretic peptide system in non-mammalian vertebrates and discusses the physiological actions of the peptides.

Natriuretic peptide guanylyl cyclase receptors in the kidney of the Japanese eel, Anguilla japonica

Natriuretic peptides are linked to osmoregulation, cardiovascular and volume regulation in fishes. The peptides bind to two guanylyl-cyclase-linked receptors, natriuretic peptide receptor-A (NPR-A) and NPR-B, to elicit their effects. Atrial natriuretic peptide (ANP) binds principally to NPR-A, whereas C-type natriuretic peptide (CNP) binds to NPR-B. The teleost kidney has an important role in the maintenance of fluid and electrolyte balance; therefore, the location of NPR-A and NPR-B in the kidney could provide insights into the functions of natriuretic peptides. This study used homologous, affinity purified, polyclonal antibodies to NPR-A and NPR-B to determine their location in the kidney of the Japanese eel, Anguilla japonica. Kidneys from freshwater and seawater acclimated animals were fixed overnight in 4% paraformaldehyde before being paraffin-embedded and immunostained. NPR-A immunoreactivity was found on the apical membrane of proximal tubule 1 and the vascular endothelium including the glomerular capillaries. In contrast, NPR-B immunoreactivity was located on the smooth muscle of blood vessels including the glomerular afferent and efferent arterioles, and on smooth muscle tissue surrounding the collecting ducts. No difference in the distribution of NPR-A and NPR-B was observed between freshwater and seawater kidneys. Immunoreactivity was not observed in any tissue in which the antibodies had been preabsorbed. In addition, there was no difference in NPR-A and NPR-B mRNA expression between freshwater-acclimated and seawater-acclimated eels. These results suggest that, although utilizing the same second messenger system, ANP and CNP act on different targets within the kidney and presumably elicit different effects.

Identification and expression of natriuretic peptide receptor Type-A and-B mRNA in freshwater and seawater rainbow trout

Natriuretic peptide receptors mediate the physiological response of  natriuretic peptide hormones. One of the natriuretic peptide receptor types is the particulate guanylyl cyclase receptors, of which there are two identified: NPR-A and NPR-B. In fishes, these have been sequenced and characterized in eels, medaka, and dogfish shark (NPR-B only). The euryhaline rainbow trout provides an opportunity to further pursue examination of the system in teleosts. In this study, partial rainbow trout NPR-A-like and NPR-B-like mRNA sequences were identified via PCR and cloning. The sequence information was used in real-time PCR to examine mRNA expression in a variety of tissues of freshwater rainbow trout and rainbow trout acclimated to 35 parts per thousand seawater for a period of 10 days. In the excretory kidney and posterior intestine, real-time PCR analysis showed greater expression of NPR-B in freshwater fish than in those adapted to seawater; otherwise, there was no difference in the expression of the individual receptors in fresh water or seawater. In general, the expression of the NPR-A and NPR-B type receptors was quite low. These findings indicate that NPR-A and NPR-B mRNA expression is minimally altered under the experimental regime used in this study.