Charakterisierung und Expression zweier multimerer Hämolympheproteine der Schnecke Biomphalaria glabrata

Die tropische Süsswasserschnecke Biomphalaria glabrata gehört zu der Familie der Planorbidae, welche als einziges Taxon der Gastropoden Hämoglobin als Sauerstofftransportprotein verwenden. Als Zwischenwirt des Bilharzioseerregers Schistosoma mansoni ist B. glabrata von tropenmedizinischer Interesse. Das extrazelluläre BgHb zeigt sich mit einem Anteil von 95% als Hauptprotein in der Hämolymphe. Dieses setzt sich aus Polypeptidketten mit je 240kDa zusammen. Diese wiederrum lassen sich in 13-Häm-Domänen und eine deutlich kleinere N-terminalen nicht Häm-Domäne untergliedern. Die Sequenzierung von zwei der drei Untereinheiten des BgHb (BgHb1, BgHb2) ermöglichte die rekombinante Expression ganzer Untereinheiten in Insektenzellen, und die Expression einiger BgHb2-Konstrukte in E. coli Zellen. Im Rahmen meiner Arbeit gelang es, BgHb1 in biologisch aktiver Form in Insektenzellen zu exprimieren. Das aus dem Überstand der Insektenzellen aufgereinigte rekombinante BgHb1 zeigte eine immunologische Identität mit nativen BgHb. Strukturelle Analysen belegten zudem die Assemblierung des rekombinanten BgHb1 zu einer dem nativen Protein gleichenden Quartärstruktur. Demnach konnte in meiner Arbeit der Nachweis erbracht werden, dass eine einzelne Isoform in der Lage ist, zur Quartärstruktur zu assemblieren. Zusätzlich ergaben Sauerstoffbindungsanalysen, dass das rekombinante BgHb1 reversibel Sauerstoff binden kann.rnIn den restlichen 5% der B. glabrata Hämolymphe zeigt sich ein rudimentäres Hämocyanin, welches für den Sauerstofftransport keine Rolle zu spielen scheint, und ein rosettenförmiges Protein, das es aufzuklären galt. Durch massenspektrometrische Analysen erhaltene Peptidfragmente zeigten eine hohe Sequenzähnlichkeit zu den löslichen Acetylcholin -Bindeproteinen anderer Mollusken. Diese AChBP zeigen eine hohe Sequenzähnlichkeit zur Ligandenbindedomäne von Rezeptoren der Cys-Loop-Proteinfamilie.rnDatenbankrecherchen deckten die Existenz zweier Isoformen auf

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.

Steroid Androgen Exposure during Development Has No Effect on Reproductive Physiology of Biomphalaria glabrata

Funding: The research presented in this manuscript was wholly funded by National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), https://www.nc3rs.org.uk/. ‘The Snail Assay as an Alternative to the Rodent Hershberger Assay for Detecting Androgens and Anti-androgens’ funding reference: G0900802/1 to SJ, EJR, CSJ, and LRN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Evaluación del estado poblaciónal del Cangrejo violáceo Platyxanthus orbignyi (Milne Edwards y Lucas, 1843) del área de Lambayeque por medio de un modelo dinámico de Biomasa

Se hace un análisis del estado poblacional del cangrejo violáceo Platyxanthus orbignyi (Milne Edwards y Lucas, 1843) del litoral de Lambayeque – Perú para el periodo 2001-2010 por medio de: 1) El modelo dinámico de biomasa de Schaefer en su versión de error de observación; a este modelo se le introdujo la variable ambiental anomalía de la temperatura superficial del mar (ATSM) del área de San José (Lambayeque) y se obtuvo 2) el modelo dinámico con variable ambiental, ambos basados en datos de captura, esfuerzo y CPUE. Se utilizó el método de máxima verosimilitud en el proceso de ajuste y el bootstrap para determinar los intervalos de confianza de los parámetros. Los parámetros poblacionales y pesqueros estimados por el modelo dinámico de biomasa de Schaefer (MDB) fueron: K: 750 000 kg, r : 0,21 y q: 8,36 x 10-6 y por el modelo dinámico con variable ambiental (MDVA) los parámetros fueron K: 765 000 kg, r: 0,23 y q: 8,02 x 10-6. Con los valores de los parámetros estimados mediante el MDB y el MDVA se calcularon los principales puntos biológicos de referencia (PBR) los cuales fueron: MRS: 39 822 kg, BMRS: 375 000 kg, fMRS: 12 561 nasas, FMRS: 0,11, F0.1: 0,10 para el MDB; y MRS: 44 069 kg, BMRS: 382 500 kg, fMRS: 13 782 nasas, FMRS: 0,12, F0.1: 0,10 para el MDVA. Los resultados indican que el estado actual de la pesquería del cangrejo violáceo del Litoral de Lambayeque se encuentra muy cerca al nivel óptimo. En vista de que no se dispone de información de evaluaciones directas de este recurso que confirme o no los resultados del MDB y MDVA y en virtud de la calidad de datos, se sugiere que el manejo de la pesquería sea del tipo adaptativo alrededor del punto de referencia F0.1 y teniendo en cuenta las condiciones ambientales.

Advances in gastropod immunity from the study of the interaction between the snail Biomphalaria glabrata and its parasites: A review of research progress over the last decade

This review summarizes the research progress made over the past decade in the field of gastropod immunity resulting from investigations of the interaction between the snail Biomphalaria glabrata and its trematode parasites. A combination of integrated approaches, including cellular, genetic and comparative molecular and proteomic approaches have revealed novel molecular components involved in mediating Biomphalaria immune responses that provide insights into the nature of host-parasite compatibility and the mechanisms involved in parasite recognition and killing. The current overview emphasizes that the interaction between B. glabrata and its trematode parasites involves a complex molecular crosstalk between numerous antigens, immune receptors, effectors and anti-effector systems that are highly diverse structurally and extremely variable in expression between and within host and parasite populations. Ultimately, integration of these molecular signals will determine the outcome of a specific interaction between a B. glabrata individual and its interacting trematodes. Understanding these complex molecular interactions and identifying key factors that may be targeted to impairment of schistosome development in the snail host is crucial to generating new alternative schistosomiasis control strategies.

Effects od y-Fe²O³ nanoparticles on the survival and reproduction of Biomphalaria glabrata (Say, 1818) and their elimination from this benthic aquatic snail.

2016

A new species of Neotropical freshwater stingray of the genus Potamotrygon Garman, 1877 from the Río Madrede Díos, Peru (Chondrichthyes: Potamotrygonidae)

Potamotrygon tatianae sp. nov., is described from Río Madre de Díos, Peru, upper Rio Madeira basin. The new species is distinguished from all congeners by a unique combination of characters, including its dorsal color pattern formed by a relatively slender, highly convoluted, beige to dark brown vermicular pattern, a single row of dorsal tail spines, and a relatively longer tail posterior to caudal stings. Potamotrygon tatianae sp. nov., occurs sympatrically with other species of Potamotrygon (P. falkneri, P. orbignyi and P. motoro). From the similar species P. falkneri, P. tatianae sp. nov., is further distinguished by the absence of circular, reniform, and oval spots, by its proportionally much longer tail, by having dorsal tail spines in one irregular row, and by features of the ventral lateral-line canal, dermal denticles and neurocranium. From P. orbignyi, the new species is distinct by lacking a reticulate pattern on dorsal disc and by the presence of two angular cartilages. From P. motoro, P. tatianae sp. nov., is further separated by the lack of ocelli formed by strong black concentric rings, by the more flattened aspect of its head and disc, and by having smaller and more numerous teeth. The discovery of a new species that so closely resembles a congeneric form in color pattern, a feature highly variable within the latter, highlights the importance of examining large series of individuals and of detailed morphological analyses in revealing the potentially highly cryptic nature of the diversity within the family.

Muscle-specific regulation of tropomyosin gene expression and myofibrillogenesis differs among muscle systems examined at metamorphosis of the gastropod Haliotis rufescens

The spatial and temporal association of muscle-specific tropomyosin gene expression, and myofibril assembly and degradation during metamorphosis is analyzed in the gastropod mollusc. Haliotis rufescens. Metamorphosis of tile planktonic larva to the benthic juvenile includes rearrangement and atrophy of specific larval muscles, and biogenesis of the new juvenile muscle system. The major muscle of the larva - the larval retractor muscle - reorganizes at metamorphosis, with two suites of cells having different fates. The ventral cells degenerate, while the dorsal cells become part of the developing juvenile mantle musculature. Prior to these changes in myofibrillar structure, tropomyosin mRNA prevalence declines until undetectable in the ventral cells, while increasing markedly in the dorsal cells. In the foot muscle and right shell muscle, tropomyosin mRNA levels remain relatively stable, even trough myofibril content increases. In a population of median mesoderm cells destined to form de novo the major muscle of the juvenile and adult (the columellar muscle), tropomyosin expression is initiated at 45 h after induction of metamorphosis. Myofibrillar filamentous actin is not detected in these cells until about 7 days later. Given that patterns of tropomyosin mRNA accumulation in relation to myofibril assembly and disassembly differ significantly among the four major muscle systems examined, we suggest that different regulatory mechanisms, probably operating at both transcriptional and post-transcriptional levels, control the biogenesis and atrophy of different larval and postlarval muscles at metamorphosis.

Histopathological changes induced by extracts from the tissue covering the stingers of Potamotrygon falkneri freshwater stingrays

Pain is the most conspicuous symptom observed in patients wounded by stingrays, and skin necrosis is common in accidents by freshwater stingrays. The extract from the stinger integumentary tissue of Potamotrygon falkneri containing toxic components (venom) was tested for its ability to induce histopathological changes in the dorsal skin of mice at different times. 3-6 h after injection, foci of necrosis in isolated basal epidermal cells were observed. Full coagulative necrosis of the skin, subcutaneous tissue and skeletal muscle was evident as soon as 24 h after venom exposure, with a clear demarcation from the normal skin. After 48 h, round collections of necrotic cells start to coalesce originating extensive skin necrotic plaques that detach from viable tissue after 72-96 h. Inflammatory infiltrate was observed after 6 h, but was always mild. Acute vascular thrombosis was rare, and hemorrhage was not present at any time. Superficial bacterial infection was present in two of the examined cases. In conclusion, the venom of P. falkneri is responsible for the development of an early necrosis with mild inflammatory reaction, probably due to direct action of the venom. The severe local damage is probably worsened by the mechanical trauma caused by the stinger. (c) 2010 Elsevier Ltd. All rights reserved.

Focos ativos de esquistossomose mansoni no Vale do Ribeira, Estado de São Paulo, Brasil

São apresentados os resultados das investigações sobre os focos de esquistossomose nas localidades de Pedro de Toledo e Itariri, com referência especial aos índices de infecção natural obtidos para a espécie Biomphalaria tenagophila. Êste molusco, transmissor de esquistossomose no Vale do Ribeira já foi assinalado nos seguintes municípios daquela região: Eldorado, Iguape, Itariri, Jacupiranga, Juquiá, Miracatu, Pariqueraçu, Pedro de Toledo, Registro e Sete Barras. Os índices cercáricos de 68,1%, 75,0% e 81,3% obtidos em alguns lotes de B. tenagophila, os mais altos já verificados para esta espécie, vem mais uma vez comprovar sua grande suscetibilidade à infecção pelo Schistosoma mansoni.

Contribuição ao levantamento da carta planorbídica do Estado de São Paulo: Pesquisa de focos com formas evolutivas do Schistosoma mansoni. II. Município de Peruibe (Litoral Sul do Estado de São Paulo)

Em levantamento realizado em Peruibe, em janeiro de 1966, foram identificados focos de Biomphalaria tenagophila infestados com formas evolutivas do Schistosoma mansoni.

Levantamentos parasitológicos, em particular a esquistossomose mansônica, nas cidades de Itanhaém e Mongaguá (Litoral Sul do Estado de São Paulo)

São apresentados os resultados dos exames parasitológicos obtidos em levantamentos realizados na cidade de Itanhaém, em 1968, e de Mongaguá, em 1969. Entre os casos de esquistossomose registrados não houve nenhum identificado como autóctone das áreas estudadas; os exemplares de Biomphalaria tenagophila coletados estavam negativos para formas evolutivas do Schistosoma mansoni.

Aspectos epidemiológicos da esquistossomose mansônica na região da Represa de Americana, estado de São Paulo, Brasil

Após conhecimento de casos humanos de esquistossomose mansônica supostamente autóctones da Represa de Americana (São Paulo, Brasil) procedeu-se ao estudo epidemiológico da região. Foram constatados seis focos localizados junto à Represa. Foram encontradas as seguintes espécies de moluscos: Biomphalaria tenagophila; B. straminea; B. peregrina; Drepanotrema cimex; D. lucidum; |Lymnaeidae; Ancylidae e Physidae. Exemplares de Biomphalaria tenagophila coletados nos focos apresentaram índices de infecção para cercárias de S. mansoni que variaram de 0,9 a 45%. Mus musculus albinos foram infectados com cercárias no laboratório e nos focos, sendo reproduzido o ciclo do S. mansoni em ambas as condições. Foram registrados 82 casos humanos autóctones de esquistossomose mansônica, na região da Represa de Americana.

Alimentação e fecundidade de planorbídeos criados em laboratórios: IV - Helisoma duryi (Wetherby, 1879). (Pulmonata, Planorbidae)

Utilizando dois tipos distintos de alimentos: "Aquariol" (alimento utilizado para alimentar peixes ornamentais) e alface, isolados e associados, na criação de Helisoma (Seminolina) duryi (Wetherby, 1879) em laboratório, foi verificado que; 1. Os caramujos alimentados com alface fresca liberaram, em 17 semanas, 826 desovas on 17.707 ovos. 2. Os especimens alimentados com "aquariol" depuseram 534 desovas on 11.361 ovos. 3. O oferecimento dos dois materiais associados resultou no aumento da fertilidade, obtendo-se, assim, 1.355 desovas ou 34.645 ovos. 4. Em termos médios, cada conjunto de quinze exemplares (com 10 a 14 mm de diâmetro) liberou, diariamente, 95,5 - 148,8 e 291,1 ovos/dia, segundo o tipo de alimento consumido: "aquariol", alface fresca, ou ambos associados. O número de desovas/dia, na mesma ordem enunciada, foi de 4,5 - 6,9 e 11,4, respectivamente. 5, Os exemplares utilizados descendiam de uma cepa supostamente recém introduzida no município de Formosa, GO (Brasil)e originária da Região Neártica (EUA). Foi comentada a possibilidade de controle biológico de diferentes espécies de Biomphalaria através de H. duryi - questão sobre a qual vêm desenvolvendo investigações biológicas e ecológicas.