Funktionelle, strukturelle und phylogenetische Untersuchungen an Lipoproteinen des Flusskrebses Astacus leptodactylus

Diese Arbeit untersucht zwei Lipoproteine, das discoidale High density-Lipoprotein (dHDL) und das β-Glukan-Bindeprotein (BGBP) aus dem Flusskrebs Astacus leptodactylus in funktioneller, struktureller und phylogenetischer Hinsicht. Die Nukleotid-Sequenz des BGBP konnte nahezu vollständig entschlüsselt werden. Dabei errechnet sich aus der abgeleiteten Aminosäure-Sequenz ein Molekulargewicht von 153 kDa. Das reife BGBP hat nur eine molekulare Masse von 105 kDa. Vermutlich kommt es durch eine Furin-ähnliche Protease zu einer post-translationalen N- und C-terminalen Prozessierung: zwei bisher nicht beschriebene, aber auch in der BGBP-Sequenz von anderen höheren Krebsen vorhandene, typische Furin-Schnittstellen (RAKR, bzw. RARR) wurden anhand von Sequenzvergleichen identifiziert. BGBP hat zwei Funktionen: zum Einen ist es für den Transport und die Aktivierung des proPhenoloxidase-Systems zuständig, zum Anderen für die Versorgung der Organe mit Lipiden, welche vermutlich der Energiegewinnung dienen. Eine 100 kDa große, BGBP-bindende Rezeptor-Fraktion konnte in Hämocyten-Membranen identifiziert werden. Das Vorkommen von dHDL war aus eigenen Befunden bisher ausschließlich in Astacus leptodactylus bekannt, doch konnte in dieser Arbeit ein mit dem dHDL-Antikörper reagierendes Protein erstmalig auch in anderen Arthropoden-Spezies nachgewiesen werden. Die discoidale Form und das Untereinheiten-Muster (240 + 85 kDa) sind typisch für die bei Vertretern ursprünglicher Tiergruppen gefundenen Lipoproteine (z.B. beim Cheliceraten Limulus und beim Polychaeten Nereis). Eventuell handelt es sich bei dHDL also um einen ‚Prototypen’ in der Lipoprotein-Evolution. Obwohl die Sequenz des dHDL auf Nukleotid-Ebene unbekannt ist, wurden die Sequenzen einiger dHDL-Peptide aus massenspektroskopischen Analysen gewonnen. Überraschenderweise befinden sich diese Sequenzen in der Aminosäuresequenz des BGBP. Dabei liegen alle Peptide am N- und/oder am C-Terminus der abgeleiteten BGBP-Aminosäure-Sequenz, und zwar in den Bereichen, die vermutlich durch das erwähnte Furin vom BGBP abgeschnitten werden, im reifen BGBP also gar nicht mehr vorkommen. Deshalb ist zu vermuten, dass BGBP und dHDL ein gemeinsames Vorläuferprotein haben und durch Genduplikation entstanden sind, oder dass es sich beim dHDL- und beim BGBP-Gen um ein und dasselbe Gen handelt. Das Genprodukt wird dann auf unterschiedliche Weise prozessiert und es entstehen die beiden Proteine dHDL und BGBP. Die Funktion von dHDL ist noch nicht eindeutig geklärt, es ließen sich aber dHDL-bindende Rezeptor-Fraktionen mit einer molekularen Masse von 160 kDa in somatischen Geweben (Muskel, Darm, Hepatopankreas, Kiemen und Samenleiter) sowie in Oocyten und Hämocyten nachweisen. Deshalb wird vermutet, dass dHDL als Energielieferant in Stoffwechsel-aktiven Organen und als Speicherprotein in Oocyten dient. Eine endocytotische Aufnahme konnte gezeigt werden.

The experimentally pathogenesis of Aeromonas hydrophila in Astacus leptodactylus with emphasis on temperature in bacterial pathogenesis

In this study, four hundred freshwater Crayfish (A. leptodactylus) with average weight of 25-40g were purchased from Aras dam reservoirs in west Azerbayjan province and transported to Iranian Artemia Research Center of Uromya province in September 2010. (One hundred crayfish extra purchased for probably mortality). Before implement of experiment the Crayfish were acclimated for ten days. These experiments was designed in four group treatments (Number, 1,2,3,4) and one control group (Number 5) in triplicate with 20 Crayfish in each repetition prepared of glass aquarium with size (50x40x30cm). Many of infected Crayfish were used for isolation of bacteria. Haemolymph sample had been gathered from infected Crayfish with cutting their antennules and transferred to TSA medium (tryptic soy agar) and then A. hydrophila were determined in order to biochemical test. The treatments and repetitions has exposed to A. hydrophila. The concentrations of the bacteria in 4 treatments were respectively 3 x 108 (T=10-15°C), 3x106 (T=25°C), 3 x 106 and 3 x 104 Cfu mL-1 (T=10- i5oc) (4, 2, 3 and 1) that were prepared in individual containers for exposure of treatments. The control (5) prepared without any bacteria and disinfected by oxytetracycline antibiotic with concentration 100 ppm for 24 hours. The hemolymph samples were withdrawn from abdominal second segments of Crayfish for measuring of THC and TPC in interval hours (2, 6, 12, 24, 48, 96, 144, 240 and 336). For histopatological studies the crayfish samples fixed in Davidson fixative. The results indicated that interval 2 hours after experiment the difference of THC value between treatment 4 with control and treatments 1,2, and 3 was significant (P< 0.05). After 48 hours of experiment the difference of THC value between control group with treatment 1 ,2 and 3 was significant (P< 0.05). The interval 336 hours after experiment also the difference of THC value between treatment 2 with treatments 1, 3 and 4 was significant (P< 0.05). The finding of TPP value showed that the last time after challenge (336 h) there was significant difference between treatment 2 with treatment 4 and control group (P< 0.05). In histopathology studying, in hepatopancreas observed hemocyte aggregated and necrosis withof peknosis nucleus that with increased concentration of bacteria and temperature, The value of hemocyte has increased. Gill revealed necrosis and cell death especially with increased concentration of bacteria and temperature. In lower concentration of bacteria in heart no difference observed, but with increased concentration of bacteria (3 x 108) the low aggregation of hemocyte observed in heart. In treatment 3 x 106 with high temperature also distributed of high hemocyte in heart was observed. In digestive system didn't appear any difference in treatments land 3 but in concentration of 3 x 108 Cfu m1-1 and 3 x 106 (T=25°C) in digestive system was revealed the low aggregation of hemocyte.

Intra- and extracellular osmotic regulation in the hololimnetic Caridea and Anomura: a phylogenetic perspective on the conquest of fresh water by the decapod Crustacea

We investigate extra- and intracellular osmoregulatory capability in two species of hololimnetic Caridea and Anomura: Macrobrachium brasiliense, a palaemonid shrimp, and Aegla franca, an aeglid anomuran, both restricted to continental waters. We also appraise the sharing of physiological characteristics by the hololimnetic Decapoda, and their origins and role in the conquest of fresh water. Both species survive salinity exposure well. While overall hyperosmoregulatory capability is weak in A. franca and moderate in M. brasiliense, both species strongly hyporegulate hemolymph [Cl(-)] but not osmolality. Muscle total free amino acids (FAA) increase slowly but markedly in response to the rapid rise in hemolymph osmolality consequent to hyperosmotic challenge: 3.5-fold in A. franca and 1.9-fold in M. brasiliense. Glycine, taurine, arginine, alanine and proline constitute a parts per thousand 85% of muscle FAA pools in fresh water; taurine, arginine, alanine each contribute a parts per thousand 22% in A. franca, while glycine predominates (70%) in M. brasiliense. These FAA also show the greatest increases on salinity challenge. Muscle FAA titers correlate strongly (R = 0.82) with hemolymph osmolalities across the main decapod sub/infraorders, revealing that marine species with high hemolymph osmolalities achieve isosmoticity of the intra- and extracellular fluids partly through elevated intracellular FAA concentrations; freshwater species show low hemolymph osmolalities and exhibit reduced intracellular FAA titers, consistent with isosmoticity at a far lower external osmolality. Given the decapod phylogeny adopted here and their multiple, independent invasions of fresh water, particularly by the Caridea and Anomura, our findings suggest that homoplastic strategies underlie osmotic and ionic homeostasis in the extant freshwater Decapoda.

3D-Elektronenmikroskopie dreier respiratorischer Proteine unter Verbesserung der bioinformatischen Abläufe

Die Transmissionselektronenmikroskopie gepaart mit bioinformatischen Methoden zur digitalen Bildverarbeitung ist ein schneller Weg zur Erstellung dreidimensionaler Rekonstruktionen großer Proteinkomplexe. Durch die Kombination der 3D-Elektronenmikroskopie mit der Röntgenstruktur von Untereinheiten erhält man ein pseudoatomares Modell der Quartärstruktur.rnIn dieser Arbeit wurden auf diese Weise die Quartärstrukturen von drei unterschiedlichen respiratorischen Proteinen analysiert (einem Hämoglobin und zwei Hämocyaninen). Zudem wurden spezielle Software-Tools entwickelt, um vorhandene Softwarepakete besser miteinander kombinieren zu können.rnDie ca. 15Å 3D-Rekonstruktion des Hämoglobins vom Wasserfloh Daphnia pulex klärt die umstrittene Frage, wie viele Untereinheiten die Quartärstruktur aufbauen: Es sind 16 (mit je zwei Häm-Domänen), angeordnet in zwei Schichten als D4-symmetrisches Sandwich. Die ca. 15 Å 3D-Rekonstruktion des 2x6meren Hämocyanins des Flusskrebses Astacus leptodactylus gibt neue Einblicke in die Kontaktstelle zwischen den beiden Hexameren; sie liegt im Bereich der Domäne 3. Bei dem aus 48 Untereinheiten bestehenden Hämocyanin des Pfeilschwanzes Limulus polyphemus wurde eine Auflösung von ca. 7 Å erreicht. Die Homologiemodelle der Untereinheiten wurden flexibel gefittet. An einer der Kontaktstellen zwischen den beiden Halbmolekülen wurden Molekulardynamik (MD)-Simulationen durchgeführt, um mehr über die Art der chemischen Bindung an dieser Kontaktstelle zu erfahren.rnSpeziell für die Kombination von 3D-Elektronenmikroskopie und MD-Simulation wurden verschiedene bioinformatische Werkzeuge und eine leicht zu erweiternde universelle grafische Benutzeroberfläche (GUI) entwickelt.

A systemic parvo-like virus in the freshwater crayfish Cherax destructor

Systemic Cowdry Type A inclusions (CAs) were observed in a moribund Cherax destructor collected at an aquaculture farm in South Australia. Inclusions were most common in the gills and were associated with multifocal necrosis of the main gill axis and lamellae. The hepatopancreas was necrotic; however, only one CA was observed in the interstitial tissues. CAs were associated with necrosis in the abdominal and gut musculature. CAs were also observed in the spongy connective tissues and the epicardium. Empty capsids (17.5 +/- 0.5 nm) and microfilaments were most commonly observed within these inclusions by transmission electron microscopy. Complete icosahedral viral particles (20.8 +/- 1.2 nm) were difficult to distinguish within the viroplasm, but were visualised better in aggregates between the viroplasm and the inner nuclear membrane. The nucleolus was closely associated with the developing viroplasm, and was hypertrophied and segregated into its fibrillar and granular components. The virus was named Cherax destructor systemic parvo-like virus (CdSPV) on the basis of its histopathology, cytopathology and morphology. CdSPV is the first systemic virus described in a freshwater crayfish.

Characterization and Biological Activities of Ocellatin Peptides from the Skin Secretion of the Frog Leptodactylus pustulatus

Eight new peptides were isolated from the skin secretion of the frog Leptodactylus pustulatus and their amino acid sequences determined by de novo sequencing and by cDNA cloning. Structural similarities between them and other antimicrobial peptides from the skin secretion of Leptodactylus genus frogs were found. Ocellatins-PT1 to -PT5 (25 amino acid residues) are amidated at the C-terminus, while ocellatins-PT6 to -PT8 (32 amino acid residues) have free carboxylates. Antimicrobial activity, hemolytic tests, and cytotoxicity against a murine fibroblast cell line were investigated. All peptides, except for ocellatin-PT2, have antimicrobial activity against at least one Gram negative strain. Ocellatin-PT8 inhibited the growth of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Salmonella choleraesuis strains with MICs in the 60−240 μM range. No significant effect was observed in human erythrocytes and in a murine fibroblast cell line after exposure to the peptides at MICs. A comparison between sequences obtained by both direct HPLC-MS de novo sequencing and cDNA cloning demonstrates the secretion of mature peptides derived from a pre-pro-peptide structure.

Evaluation of the exploitation potential of red swamp crayfish (Procambarus clarkii) in Portugal

Dissertação para obtenção do Grau de Mestre em Engenharia do Ambiente, perfil de Ordenamento do Território e Impactes Ambientais

The larva of the frog leptodactylus hylaedactylus (Leptodactylidae) [by] W. Ronald Heyer and Philip A. Silverstone.

v.51:no.11(1969)

Diet of the South American frog Leptodactylus ocellatus (Anura, Leptodactylidae) in Uruguay

The diet of Leptodactylus ocellatus (L., 1758) was studied in specimens collected at Maldonado Department (Uruguay), where monthly surveys were made between August 1998 and March 2000. A total of 143 frogs were collected, measured, sexed, and dissected, to assess stomach contents. The anurans were grouped into three age classes and four categories (considering sex and capture date). The trophic amplitude for each age class and sex-season category was quantified using Shannon-Weaver Index, and the trophic niche overlap between ages and categories by using the Pianka Index. Comparisons among treatments were made by G-tests. The most important prey items were coleopterans (IRI = 2547), and significant correlations were found between predator and prey sizes. The major differences on diet composition were found between extreme age classes (froglets and adults). Significant differences were also detected between sexes in the cold season but not in the warm season. Any of these changes in diet may be related with the availability of prey.

Molecular systematics of the frog genus Leptodactylus (Amphibia: Leptodactylidae) / Linda R. Maxson, W. Ronald Heyer.

n.s. no.41(1988)

Cuidado parental em Leptodactylus natalensis (Amphibia, Anura, Leptodactylidae)

O presente trabalho registra mais uma espécie pertencente à família Leptodactylidae apresentando cuidado parental, Leptodactylus natalensis Lutz, 1930. Fêmeas foram observadas em assistência a ovos e cardumes de girinos em duas poças temporárias na mata Atlântica do Estado de Pernambuco, Brasil.

Seasonal variations in the intermediate metabolism of the crayfish Parastacus brasiliensis (Crustacea, Decapoda, Parastacidae) in the natural environment and experimental culture

The goal of this study was to evaluate the effects of seasonal variations on energy metabolism in different tissues of the freshwater crayfish Parastacus brasiliensis (von Martens, 1869). Crayfish were collected monthly from January 2001 to January 2003 in São Francisco de Paula, Rio Grande do Sul, Brazil, in a stream and in a culture tank. Haemolymph samples were collected from each crayfish in the field with a syringe, by puncturing the membrane at the base of the chelipeds. Hepatopancreas, gills, and abdominal muscle were removed for determination of free glucose, glycogen, total lipids, and triglycerides. The haemolymph samples were used for determination of glucose, total proteins, total lipids, and triglycerides. Statistical analysis revealed significant differences in biochemical composition in crayfish collected in the stream compared to the experimental tank during the year, principally in glucose and triglycerides in haemolymph, glycogen and total lipids in all tissues study, and triglycerides only in abdominal muscle. The regular food intake partially modified these seasonal variations of the metabolic pattern. Environmental conditions (e.g., food availability and water temperature) and reproductive period appeared to be the main factors influencing the seasonal patterns of variation in energy metabolism.

Reproductive behavior of Leptodactylus mystacinus (Anura, Leptodactylidae) with notes on courtship call of other Leptodactylus species

Here we present data on the reproductive behavior of Leptodactylus mystacinus (Burmeister, 1861), including details on courtship behavior. We also describe and compared the courtship calls of L. mystacinus, L. furnarius Sazima & Bokermann, 1978 and Leptodactylus sp. (L. aff. andreae). Field works were conducted in Uberlândia (central Brazil). During courtship, a female approaches a calling male and is led to a previously excavated chamber; a female can approach a silent male that beat his hands and/or feet on the ground as well. The courtship call of L. mystacinus consists of one single arch-shaped note (duration = 0.04 s) repeated 258 times per minute; the courtship calls of L. furnarius (0.06 s, 84 times per minute) and Leptodactylus sp. (0.15 s, 5 times per minute) also are arch-shaped. The courtship behavior of L. mystacinus is similar to that of other species of the L. fuscus (Schneider, 1799) group; unique to it is that males can beat his hands and/or feet on the ground while courting. The male behavior of conducting the female to a previously excavates chamber and the arch-shaped courtship call may represent other shared derived features of members of the L. fuscus group, including the former Adenomera species.

Reproductive biology of Leptodactylus fuscus (Anura, Leptodactylidae) in the subtropical climate, Rio Grande do Sul, Brazil

The aim of this study was to characterize, for the central region of the State of Rio Grande do Sul, Brazil, the reproductive biology of Leptodactylus fuscus (Schneider, 1799), based on the analysis of gonadal development of males and females, reproductive effort, size-fecundity relationships, and occurrence of sexual dimorphism in body size. Mature individuals were found from October 1996 to February 1997 and from October 1997 to December 1997. The highest input of juveniles in the population was recorded in March 1997. There was a positive and significant correlation between the number of mature individuals and the mean monthly temperature. The population did not present sexual dimorphism in size. Males presented significant correlation only between snout-vent length and testes length. All females had oocytes at four different maturation stages and there were no significant correlations regarding size-fecundity variables. The correlation between ovarian size factor and females snout-vent length was not significant either. The main difference between this population and those that inhabit tropical climate was that temperature was responsible for stimulating the reproduction activity, instead of rainfall.

Seasonality, age structure and reproduction of Leptodactylus (Lithodytes) lineatus (Anura, Leptodactylidae) in Rondônia state, southwestern Amazon, Brazil

Leptodactylus(Lithodytes) lineatus (Schneider, 1799) is an Amazonian leaf litter frog considered rare or uncommon in several studies on anuran communities. Despite being a widely distributed frog in Amazonian forests, knowledge of the biology and ecology of this species is relatively scarce. This species has been reported to live in association with leaf-cutter ant nests (Atta spp.) during the breeding period. In this paper we present data on the seasonality of this species and some reproductive information gathered at a locality of Rondônia state, northwestern Brazil. Field work was carried out between April 2001 and March 2002, with the use of pitfall traps with drift fences as a survey method. Leptodactylus (L.) lineatus had a higher capture frequency in this locality compared to that of other studies carried out in other Amazonian localities, possibly because this species has secretive habits, such as calling and breeding from nests of leaf-cutting ants, and are difficult to find during visual encounter surveys. The breeding period occurs between October and March. Calling males and egg-bearing females were found between September and February and juvenile recruitment occurred mainly from the end of the rainy season to the beginning of the dry season (February to June). Males and females show sexual dimorphism in SVL, females being significantly larger than males. The number of ovarian eggs per female varies from 110 to 328 and analyses indicate that there is a significant correlation with SVL.