Biodiversity of brachyuran crabs (Crustacea: Decapoda) from non-consolidated sublittoral bottom on the northern coast of São Paulo State, Brazil

The brachyuran community of the coast of São Paulo State is represented by about 188 species of crabs inhabiting different kinds of coastal marine environments. The biodiversity of brachyurans found on non-consolidated sublittoral bottom was investigated. The Ubatuba region (Ubatumirim, Ubatuba and Mar Virado bays, Couves and Mar Virado Islands, offshore region) was sampled for 3 years (1998–2000), at depths of 2–40 m. All sampling was performed using a fishing boat equipped with two double-rig nets. We collected 79 brachyuran species representing 9 superfamilies (4 Dromioidea, 1 Homoloidea, 2 Calappoidea, 5 Leucosioidea, 20 Majoidea, 7 Parthenopoidea, 17 Portunoidea, 18 Xanthoidea, and 5 Pinnotheroidea) and 41 genera. Ubatuba bay showed the greatest species richness with 50 species, followed by Ubatumirim with 45 and Mar Virado with 29. The number of species collected represents about 57% of the known species of crabs already reported for the shore of São Paulo State. It is worth noticing that this percentage is restricted only to non-consolidated sublittoral bottom. This fact indicates a great biodiversity of the habitat in this studied region, probably one to the diversity of habitat types present in the bays.     

Sand fly vectors (Diptera, Psychodidae) of American visceral leishmaniasis areas in the Atlantic Forest, State of Espírito Santo, southeastern Brazil

The aim of this study was to evaluate the sand fly fauna of American visceral leishmaniasis (AVL) endemic areas within the Central Atlantic Forest Biodiversity Corridor, State of Espírito Santo, southeastern Brazil. The sand fly captures were performed between January, 1989 and December, 2003 in localities where autochthonous cases of AVL were recorded, as well as in their boundary areas. Sand flies were collected from surrounding houses and domestic animal shelters using two to five CDC automatic light traps, and manual captures were also performed using mouth aspirators in one illuminated Shannon trap during the first four hours of the night. We used cladistic analysis to determine the geographic relationships among the collected sand fly species as well as the index species for the occurrence of other sand flies. A total of 62,469 sand flies belonging to 17 species and eight genera was collected in 164 localities from nine municipalities with AVL records. The richness (S=17) and diversity (H=0.971) of sand flies were lower than in conservation areas and similar to modified environments in the Atlantic Forest of Espírito Santo. Lutzomyia longipalpis was identified in 79 localities. The cladistic analysis identified Evandromyia lenti as the index species for Lutzomyia longipalpis. The latter seems to be the main vector of AVL in the Central Atlantic Forest Biodiversity Corridor due to its high abundance and distribution matching the disease occurrence. Therefore, Evandromyia lenti may be used as an index species for the occurrence of Lutzomyia longipalpis.     

Chitooligosaccharides antagonize the cytotoxic effect of glucosamine

Chitooligosaccharides (COS) are partially hydrolyzed compounds derived from chitosan that exhibit a number of biological activities, including antitumor, antibacterial and antifungal properties. In this work, we examined the cytotoxicity of pure COS and oligomers A, B and C (solutions composed of different amounts of COS) produced by enzymatic hydrolysis using a crude enzyme extract produced by the fungus Metarhrizium anisopliae. The antiproliferative effect of these molecules was analyzed using tumor cell lines (HepG2 and HeLa cells) and in a normal cell line (3T3). The antioxidant activity was analyzed in several in vitro experiments. Glucosamine showed higher toxicity (approximately 92%) to all cell lines studied. However, the oligomers obtained after hydrolysis demonstrated no toxic effects on the normal cells (3T3). Furthermore, we showed that a small amount of other COS can decrease the cytotoxic effect of glucosamine against 3T3 cells, indicating that glucosamine could be used as an antitumor drug in the presence of other COS. In addition, different effects were found in antiproliferative assays, which depended on the COS composition in the oligomers (A, B and C), showing that a combination of them may be essential for developing antineoplastic drugs. Superoxide anion scavenging was the main antioxidant activity demonstrated by the COS and oligomers. This activity was also dependent on the oligomer composition of the chitosan hydrolysates. Further work will identify the ideal proportions of COS and glucosamine for maximizing the effects of these biological activities.     

Essential Oil from Bark of Aniba parviflora (Meisn.) Mez (Lauraceae) Reduces HepG2 Cell Proliferation and Inhibits Tumor Development in a Xenograft Model

Aniba parviflora (Meisn .) Mez (Lauraceae) is an aromatic plant of the Amazon rainforest, which has a tremendous commercial value in the perfumery industry; it is popularly used as flavoring sachets and aromatic baths. In Brazilian folk medicine, A. parviflora is used to treat victims of snakebites. Herein, we analyzed the chemical composition of A. parviflora bark essential oil (EO) and its effect on the growth of human hepatocellular carcinoma HepG2 cells in vitro and in vivo. EO was obtained by hydrodistillation and characterized by GC-MS and GC-FID. The main constituents of EO were linalool (16.3±3.15), α-humulene (14.5±2.41 %), δ-cadinene (10.2±1.09 %), α-copaene (9.51±1.12 %) and germacrene B (7.58±2.15 %). Initially, EO's cytotoxic effect was evaluated against five cancer cell lines (HepG2, MCF-7, HCT116, HL-60 and B16-F10) and one non-cancerous one (MRC-5), using the Alamar blue method after 72 h of treatment. The calculated IC₅₀ values were 9.05, 22.04, >50, 15.36, 17.57, and 30.46 μg/mL, respectively. The best selectivity was for HepG2 cells with a selective index of 3.4. DNA Fragmentation and cell cycle distribution were quantified in HepG2 cells by flow cytometry after a treatment period of 24 and 48 h. The effect of EO on tumor development in vivo was evaluated in a xenograft model using C.B-17 SCID mice engrafted with HepG2 cells. In vivo tumor growth inhibition of HepG2 xenograft at the doses of 40 and 80 mg/kg were 12.1 and 62.4 %, respectively.     

Humic acid and moderate hypoxia alter oxidative and physiological parameters in different tissues of silver catfish (Rhamdia quelen)

Aquatic animals are naturally exposed simultaneously to environments with different concentrations of humic acid (HA) and seasonal or daily variations of dissolved oxygen (DO) levels. This study investigated the effects of simultaneous exposure to different HA and DO levels on plasma ion levels and some hematological and oxidative parameters in different tissues of silver catfish (Rhamdia quelen). The fish were exposed to 0, 2.5 or 5 mg L^?1 HA for 120 h. After this period, each group was divided into two groups: normoxia and hypoxia. Exposure to the different DO levels lasted 96 h, totaling 216 h of experimentation. At the end of the experimental period, blood sampling was performed, and the fish were euthanized prior to the excision of the gills and the brain to evaluate hematological and oxidative parameters. To verify the antioxidant capacity of HA, total phenolic compounds were measured. In general, all tissues of silver catfish exposed simultaneously to hypoxia and different HA concentrations showed a reduction in lipid peroxidation levels, as well as a modulation of the antioxidant system. These effects occurred in an HA concentration-dependent manner. Thus, HA is beneficial to silver catfish exposed to hypoxia. These beneficial effects can be attributed, most likely, to the action of the different HA constituents and functional groups, including phenolic compounds, which have antioxidant properties.     

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.     

Adsorption in a Fixed-Bed Column and Stability of the Antibiotic Oxytetracycline Supported on Zn(II)-[2-Methylimidazolate] Frameworks in Aqueous Media

A metal-organic framework, Zn-[2-methylimidazolate] frameworks (ZIF-8), was used as adsorbent material to remove different concentrations of oxytetracycline (OTC) antibiotic in a fixed-bed column. The OTC was studied at concentrations of 10, 25 and 40 mg L^-1. At 40 mg L^-1, the breakthrough point was reached after approximately 10 minutes, while at 10 and 25 mg L^-1 this point was reached in about 30 minutes. The highest removal rate of 60% for the 10 mg L^-1 concentration was reached after 200 minutes. The highest adsorption capacity (28.3 mg g^-1) was attained for 25 mg L^-1 of OTC. After the adsorption process, a band shift was observed in the UV-Vis spectrum of the eluate. Additional studies were carried out to determine the cause of this band shift, involving a mass spectrometry (MS) analysis of the supernatant liquid during the process. This investigation revealed that the main route of adsorption consisted of the coordination of OTC with the metallic zinc centers of ZIF-8. The materials were characterized by thermal analysis (TA), scanning electron microscopy (SEM), powder X-ray diffraction (XRD), and infrared spectroscopy (IR) before and after adsorption, confirming the presence of OTC in the ZIF-8 and the latter’s structural stability after the adsorption process.     

Ecological aspects of hermit crabs (Crustacea,Anomura, Paguroidea) off the northern coast of São Paulo State,Brazil

The occurrence of species of hermit crabs and their ecological distribution in soft bottoms off Ubatuba, São Paulo, Brazil were analyzed. To better understand the distribution of the species in relation to environmental factors, the similarity and species-diversity indexes were calculated. Paguroideans were sampled monthly from January through December 2000. The trawls were made with two otter-trawl nets at 13 different sites, at depths of 2–40 m. Water temperature, salinity, sediment texture, and organic matter content were measured. Gastropod shells occupied by hermit crabs were also assessed. A total of 1,238 specimens was collected, belonging to the families Diogenidae and Paguridae, comprising seven genera and thirteen species. The most abundant hermit crab species were Dardanus insignis (761 specimens) and Loxopagurus loxochelis (351 specimens). Phimochirus holthuisi is newly reported from the São Paulo coast. The highest diversity index was found for the shallower sites near rocky shores. The results of the grouping analysis for sites and species indicated three distinct groups for sites, and four groups for species. This suggests that the occurrence of these anomurans is associated with the environmental and biotic factors analyzed.     

Vertical distribution by demographic groups of ghost crab Ocypode quadrata (Crustacea: Brachyura)

The ghost crab Ocypode quadrata plays an important role in energy transfer between trophic levels, and has been widely used in evaluations of impacted environments. In order to provide data on the biology of this potential bioindicator species, the population structure and vertical distribution of individuals were studied on two beaches in southeastern Brazil. Each beach was divided into quadrants of 1000 m² with boundaries of upper, middle and lower levels in relation to the waterline. Collected monthly by active searching through one year, the specimens of O. quadrata were sexed, measured for carapace width, and returned to the beach. Of the total of 1904 specimens collected, the largest proportion (46.2%) were males, followed by 31.4% juveniles. The vertical distribution of the ghost crabs differed among age groups: males mostly occupied the middle and upper levels; adult females, ovigerous or not, were more abundant in the lower level; and juveniles were evenly distributed in all levels, with a slight tendency toward the middle. The sex ratio favored males in a few months of the year and in the larger size classes. The abundance of O. quadrata is limited by low temperatures, and its spatial and temporal distribution is controlled by food availability and ease of reproduction. Knowledge of the biology of these crabs is essential in order to use them as a bioindicator species; the vertical distribution patterns may reflect changes in the beach hydrodynamics or other environmental factors.