Unusual labial glands in snakes of the genus Geophis Wagler, 1830 ( Serpentes: Dipsadinae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Consuming viscous prey: a novel protein-secreting delivery system in neotropical snail-eating snakes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Platinosomíase em felinos domésticos: um diferencial para obstrução biliar

The platinosomiase is a disease the hepatobiliary system of domestic felines being caused by trematode Platynosomum fastosum. The life cycle of the parasite includes the presence of three intermediate hosts (snails, terrestrial isopodes, lizards or frogs). The cat is considered definitive host of the parasite and acquires the infection eating a vertebrate intermediate host containing metacercariae. The adult parasites generally inhabit the liver, gallbladder and bile ducts of cats. The severity of clinical manifestations depends mainly on the number of flukes in the biliary tract. Many animals are asymptomatic or exhibit nonspecific clinical manifestations such as anorexia, lethargy, weight loss, vomiting and diarrhea. In large infestations can occur biliary obstruction resulting in jaundice, cirrhosis, hepatitis cholangiohepatitis and even death. The definitive diagnosis is accomplished through by the detection of fluke eggs in feces or through laparotomy and liver biopsy. Treatment should be based on the use of anthelmintic for the elimination of the parasite and supportive therapy for the animal. Prevention is difficult due to predatory nature of the cat. The control can be accomplished through periodic fecal examinations

Ocorrência de Angiostrongylus cantonensis (Nematoda, Angiostrongylidae) em Achatina fulica (Mollusca, Gastropoda) na Baixada Santista

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação do potencial citotóxico, moluscicida e alelopático dos extratos hidroetanólicos das folhas de Stryphnodendron obovatum Benth

Stryphnodendron obovatum Benth. known as barbatimão de folha miúda, is a typical tree of the Midwest and Southeast “Cerrado” regions of Brazil. Several secondary metabolites have been identified in its barks, including tannins, described on the literature as allelochemicals. Beholding to identify a possible allelopathic activity in S. obovatum leaves, we tested hydroethanolic EtOH:H2 O (70% and 50% v/v) extracts, in different concentrations, on the initial development of cucumber (Cucumis sativus L.). Among the results, we highlight the effect of the 50% hydroethanolic extract in inhibiting the formation of the main and the secondary roots and the stimulation of the hypocotyl growth in C. sativus by the 70% hydroethanolic extract, both in all concentrations tested. The cytotoxic activity, evaluated by bioassay toxicity on Artemia salina Leach., was negative for the tested extracts (LC50> 1000 mg.mL-1). Concomitantly, the molluscicidal activity, evaluated against snails of Biomphalaria glabrata Say, presented low acute toxicity of solutions of 70% hydroethanolic extract at concentrations of 128.9 mg.mL-1 and 172.8 mg.mL-1 and values above 250 mg.mL-1 to solutions of 50% hydroalcoholic extract, being therefore considered inactive for these. The results suggest the existence of allelochemicals in the extracts tested and they have low toxicity.

IMPOSEX AS AN INDICATOR OF ORGANOTIN POLLUTION AT RIO DE JANEIRO SOUTH COAST: SEPETIBA AND ILHA GRANDE BAYS

Imposex is the development of male sexual characteristics, i.e., penis and/or vas deferens in females of dioecious gastropods. This phenomenon has been associated to the use of the organotin based marine antifouling paints and used worldwide as a bioindicator of pollution by the TBT (antifouling agent) as well as to evaluate the related ecological impact. This study reports imposex in Stramonita haemastoma at south coast of Rio de Janeiro State (Brazil), specifically at Sepetiba and Ilha Grande Bays. All the stations where snails were found have shown imposex. Ilha Grande Bay presented the highest values of imposex indices when compared to Sepetiba Bay. The results suggest that high concentration of shipping activities and coastal morphology are the principal causes at the most polluted sites.

Oral glands in dipsadine ""goo-eater"" snakes: Morphology and histochemistry of the infralabial glands in Atractus reticulatus, Dipsas indica, and Sibynomorphus mikanii

Although snake infralabial glands are generally constituted of mucous cells, among dipsadines, they are much more developed and predominantly serous in nature, possibly due to the peculiar feeding habits of some species of this group, the ""goo-eaters"", which feed on soft and viscous invertebrates. We compared the morphology and histochemistry of the infralabial glands of three goo-eater species of Southeast Brazil, Atractus reticulatus, Dipsas indica and Sibynomorphus mikanii. In A. reticulatus the glands are formed by mixed acini composed of mucous and seromucous cells and in D. indica, they are composed of mucous tubules and seromucous acini. In S. mikanii the glands are organized in seromucous acini; mucous cells are restricted to the gland anterior region and to the duct lining epithelium. Ultrastructurally, secretory granule electron density varies from low to moderate, depending on their mucous or seromucous nature. The results indicate a large morphological and histochemical variation in the infralabial glands, probably reflecting differences in the secretion chemical composition and in feeding specialization among the three species. The protein content in the secretory cells can be related with the presence of toxins that can be used in chemical prey immobilization or detaching of snails from their shells. (c) 2007 Elsevier Ltd. All rights reserved.

Extremely slow feeding in a tropical drilling ectoparasite, Vitularia salebrosa (King and Broderip, 1832) (Gastropoda: Muricidae), on molluscan hosts from Pacific Panama

This study documents one of the slowest feeding behaviors ever recorded for a muricid gastropod in one of the most biotically rigorous regions on the planet. In Pacific Panama, Vitularia salebrosa attacks mollusks by drilling through their shells. The duration of attacks estimated by isotope sclerochronology of oyster shells collected during attacks in progress range from 90 to 230 days, while experimental observation of interactions documented one attack greater than 103 days. The prolonged nature of attacks suggests that V. salebrosa is best characterized as an ectoparasite than as a predator, which is the ancestral condition in the Muricidae. An ectoparasitic lifestyle is also evident in the unusual interaction traces of this species, which include foot scars, feeding tunnels and feeding tubes, specialized soft anatomy, and in the formation of male-female Pairs, which is consistent with protandrous hermaphroditism, as is typical in sedentary gastropods. To delay death of its host, V. salebrosa targets renewable resources when feeding, such as blood and digestive glands. A congener, Vitularia miliaris from the Indo-Pacific, has an identical feeding biology The origin and persistence of extremely slow feeding in the tropics challenges our present understanding of selective pressures influencing the evolution of muricid feeding behaviors and morphological adaptations. Previously, it has been suggested that faster feeding is advantageous because it permits predators to spend a greater proportion of time hiding in enemy-free refugia or to take additional prey, the energetic benefits of which could be translated into increased fecundity or defenses. The benefits of exceptionally slow feeding have received little consideration. In the microhabitat preferred by V. salebrosa (beneath boulders), it is possible that prolonged interactions with hosts decrease vulnerability to enemies by reducing the frequency of risky foraging events between feedings . Ectoparasitic feeding through tunnels by V. salebrosa may also reduce competitive interactions with kleptoparasites (e.g., crabs, snails) that steal food through the gaped valves of dead or dying hosts.

Aspisoma lineatum (Gyllenhal) (Coleoptera: Lampyridae) Firefly: Description of the Immatures, Biological, and Ecological Aspects

(Gyllenhal) is a common firefly in the Southeastern region of Brazil. Adults and larvae were collected in the municipality of Campinas, state of So Paulo, Brazil, and the immature stages were described and reared in the laboratory. Four generations were reproduced in the laboratory, and a method for its rearing was established. The life cycle usually lasts 6 months, but under optimal laboratory conditions, it lasted from 2 to 4 months. Larvae were fed with and snails since the beginning of the larval stage. This species was found to be easily adapted to environments under anthropic influence, such as urban areas and farms.

Morphology and biological aspects of Gundlachia ticaga from SE Brazil (Gastropoda: Basommatophora: Ancylidae)

Some aspects of the biology, reproduction and morphology of Gundlachia ticaga (MARCUS & MARCUS 1962) were studied and compared with literature data. A sample of similar to 100 specimens, collected in a darn in Ribeirao Preto, were maintained in Petri dishes at the laboratory during a period no longer than a month. The oviposition, number of capsules, and incubation time of eggs were registered for each individual. A morphologic study was carried out in both, alive and fixed specimens. Although some morphological discrepancies were identified, the majority of data confirms what was previously described for the species. Regarding the biology, however, the results were somewhat different from those previously reported. The incubation time of the eggs varied from 10 to 13 days, and the number of embryos in the interior of the ovigerous capsules varied from 1 to 4. In addition, each individual laid 9 eggs during the first month of the reproductive period. The formation of septa occurred in some animals and intermediary phases are figured.

Molluscicidal activity of some marine substances against the snail Biomphalaria glabrata (Mollusca, Planorbidae)

Freshwater snails of the genus Biomphalaria play a major role as intermediate hosts of Schistosoma mansoni, the etiologic agent of schistosomiasis. While Biomphalaria spp. control by molluscicides is one of the main strategies to reduce the snail population in infected areas, there are few effective molluscicides commercially available. Natural products may be considered as potentially useful and safe molluscicides. We have evaluated the molluscicidal activity of 12 extracts from ten marine organisms on adult and embryonic stages of Biomphalaria glabrata. Only extracts of the red algae Liagora farinosa and of the sponge Amphimedon viridis presented molluscicidal activity. Lethal concentration (LC)(50) values obtained were 120 mu g/mL for L. farinosa CH2Cl2 extract (apolar fraction) and 20 mu g/mL for A. viridis extract and halitoxin. The polar alga fraction and halitoxin had no effect on B. glabrata embryos. The algae apolar fraction was active on B. glabrata in all embryonic development stages, with LC50 values for blastulae at 42 mu g/mL, gastrulae at 124 mu g/mL, trochophore at 180 mu g/mL, and veliger at 222 mu g/mL. This is the first report of extracts from marine organisms which presented molluscicidal activity.

Efectos potenciales del marisqueo sobre moluscos gasterópodos de interés comercial ("Osilinus" spp. y "Patella" spp.) en el Archipiélago Canario

Abstract.- This study assessed the effects of human influences over the abundance and size patterns of five species of gastropods (top-shell snails and limpets) commonly collected in the Canarian Archipelago: ‘burgado hembra’ (Osilinus atrata), ‘burgado macho’ (Osilinus sauciatus), ‘lapa blanca’ (Patella aspera), ‘lapa negra’ (Patella candei crenata) and ‘lapa de sol’ (Patella rustica). We studied patterns of abundance and size of these species across three islands (Lanzarote, La Graciosa and Alegranza) corresponding to three levels of human influence: high, medium and low, respectively; which were quantified through three indicators: human pressure (inhabitants km-1 d-1), accessibility to the coast (km of sealed and unsealed roads), and volumes of capture (kg). The abundances of O. atrata and P. aspera were statistically higher at Alegranza than at La Graciosa and Lanzarote, whereas the density was 10 to 15 times larger at Alegranza for four of the five studied species. Significant differences in the size structure of the species among islands (= levels of human influence) were also observed. Not only all large-sized individuals disappeared at Lanzarote and La Graciosa, yet there was a decrease in numbers for the majority of size ranges. Thought populations of top-shell snails and limpets were affected by natural variability, human activities turned out to be the major driver for the observed differences among islands. These results seriously question the effectiveness of the current shellfish regulations contained in the Regional Fish Law. Resumen.- Este estudio determinó los efectos de la influencia humana sobre la abundancia y la estructura de talla de cinco especies de moluscos gasterópodos comúnmente recolectados en el Archipiélago Canario: el ‘burgado hembra’ (Osilinus atrata), el ‘burgado macho’ (Osilinus sauciatus), la ‘lapa blanca’ (Patella aspera), la ‘lapa negra’ (Patella candei crenata) y la ‘lapa de sol’ (Patella rustica). Se analizaron tres islas (Lanzarote, La Graciosa y Alegranza) correspondientes a tres categorías de influencia humana: alta, media y baja, respectivamente; cuantificadas a través de tres indicadores: la presión humana (hab km-1 d-1), la accesibilidad a la costa (km de red viaria) y los volúmenes de extracción de los recursos (kg). La abundancia de O. atrata y P. aspera fue estadísticamente mayor en Alegranza que en La Graciosa y Lanzarote, mientras que los valores de densidad fueron de 10 a 15 veces superiores en Alegranza para cuatro de las cinco especies estudiadas. Para la estructura de talla, se detectaron notables diferencias entre islas (= niveles de influencia humana). En Lanzarote y La Graciosa no sólo desaparecieron los ejemplares de mayor talla, sino que disminuyeron los ejemplares para la mayoría de rangos de talla. A pesar que la variabilidad natural de las poblaciones de burgados y lapas es alta, la actividad humana parece ser la causa principal de las diferencias observadas entre islas. Estos resultados cuestionan seriamente la efectividad de las normas sobre marisqueo de la actual Ley de Pesca de Canarias.

Determination of trace elemental species in periostracum of Cypraea shells

n this work, three Cypraea species (C. talpa, C. tigris and C. zebra) were exhaustively studied. The shells have been separated in the structural layers. The mineralogy, ultra- and micro-structure of each layer were analyzed by Confocal Laser Scanning Microscopy (CLSM), Scanning Electron Microscopy (SEM), X-Ray Diffractometry (XRD) and Raman Spectroscopy (RS). The presence of biologically relevant trace metals (Mn, Co, Fe, Zn, Cr, etc.) has been investigated using Instrumental Neutron Activation Analysis (INAA) and Inductively Coupled Plasma – Mass Spectrometry (ICP-MS) as detection tool. A new method has been developed and optimized to extract and analyze the soluble organic matrix (SOM) of the shell. Although the molecular nature of the SOM is not really known, it contains at least large protein fraction, if not only consists of proteins. The extracted matrices were compared between layers and species using Size Exclusion High Performance Liquid Chromatography coupled with Ultra Violet Spectrometry (SE-HPLC-UV), Gel electrophoresis (GE) and protein quantification tests. For the first time to our knowledge the association of trace elements to the protein in the SOM of the shell was studied using hyphenated on line as well as combined off line techniques and validated through inter-comparison tests between the different methods applied. Interesting correlations between the trace element concentration, the microstructure and the protein content were directly and indirectly detected. The metals Cu, Ni, Co and Zn have shown to bind to the SOM extracted from C. talpa, C. tigris and C. zebra shells. Within the conclusions of this work it was demonstrated that these protein-metal-complexes (or metal containing proteins) change from one layer to the other and are different between the three snails analyzed. In addition, the complexes are clearly related only to certain protein fractions of the SOM, and not to the whole SOM observed. These fractions and show not to be very metal-specific (i.e. some of these fractions bind two or three different metals).

Molekularbiologische Charakterisierung der beiden Untereinheiten des Mega-Hämocyanins der Schnecke Melanoides tuberculata

Bei dem 2010 von unserer Arbeitsgruppe entdeckten Mega-Hämocyanin handelt es sich um einen stark abgewandelten Typ des respiratorischen Proteins Hämocyanin, bestehend aus zwei flankierenden regulären Dekameren und einem zentralen Mega-Dekamer. Diese sind aus zwei immunologisch verschiedenen Untereinheiten mit ~400 bzw. ~550 kDa aufgebaut, die in unserer Arbeitsgruppe bereits proteinbiochemisch charakterisiert wurden. Im Zuge dieser Untersuchungen konnte zudem eine 3D-Rekonstruktion des Oligomers (13,5 MDa) mit einer Auflösung von 13Å erstellt werden. Das Ziel der vorliegenden Arbeit war die Aufklärung der Primärstruktur beider Polypeptide bei der Schnecke Melanoides tuberculata (MtH). Es gelang, die cDNAs der beiden Untereinheiten vollständig zu sequenzieren. Die zu typischen Dekameren assemblierende MtH400-Untereinheit umfasst 3445 Aminosäuren und besitzt eine theoretische Molekularmasse von 390 kDa. Nach dem Signalpeptid von 23 Aminosäuren Länge folgen die für Gastropoden-Hämocyanine typischen funktionellen Einheiten FU-a bis FU-h. Insgesamt verfügt die MtH400-Untereinheit über sechs potentielle N-Glykosylierungsstellen. Die MtH550-Untereinheit, welche mit 10 Kopien das Mega-Dekamer bildet, umfasst 4999 Aminosäuren und besitzt eine theoretische Molekularmasse von 567 kDa. Damit handelt es sich bei dieser Untereinheit um die zweitgrößte jemals bei einem Protein detektierte Polypeptidkette. Die MtH550-Untereinheit besteht aus einem Signalpeptid von 20 Aminosäuren Länge und den typischen Wand-FUs (FU-a bis FU-f). Daran anschließend folgen sechs weitere Varianten der FU-f (FU-f1 bis FU-f6). Die MtH550-Untereinheit verfügt über insgesamt zwölf potentielle N-Glykosylierungsstellen. Anhand der ermittelten Primärstrukturdaten wird klar, dass der auffällig vergrößerte Kragenbereich des Mega-Dekamers aus je 10 Kopien der FU-f1 bis FU-f6 besteht. Die ermittelten Sequenzdaten der beiden MtH-Untereinheiten weisen im Vergleich zu anderen Hämocyanin Sequenzen einige sehr charakteristische Indels sowie unübliche N-Glykosylierungsstellen auf. Es war zudem möglich, anhand einer molekularen Uhr den Entstehungszeitpunkt des Mega-Hämocyanins zu datieren (145 ± 35 MYA). Sowohl die Topologie als auch die berechneten Trennungszeitpunkte des an allen Verzweigungen gut unterstützten Stammbaums stimmen mit den bisher publizierten und auf Hämocyanindaten basierenden molekularen Uhren überein.

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.