The role of macroinvertebrates for conservation of freshwater systems

Freshwater ecosystems are the most threatened ecosystems worldwide. Argentinian‐protected areas have been established mainly to protect vertebrates and plants in terrestrial ecosystems. In order to create a comprehensive biodiverse conservation plan, it is crucial to integrate both aquatic and terrestrial systems and to include macroinvertebrates. Here, we address this topic by proposing priority areas of conservation including invertebrates, aquatic ecosystems, and their connectivity and land uses. Location: Northwest of Argentina. We modeled the ecological niches of different taxa of macroinvertebrates such as Coleoptera, Ephemeroptera, Hemiptera, Megaloptera, Lepidoptera, Odonata, Plecoptera, Trichoptera, Acari, and Mollusca. Based on these models, we analyzed the contribution of currently established protected areas in the conservation of the aquatic biodiversity and we propose a spatial prioritization taking into account possible conflict regarding different land uses. Our analysis units were the real watersheds, to which were added longitudinal connectivity up and down the rivers. A total of 132 species were modeled in the priority area analyses. The analysis 1 showed that only an insignificant percentage of the macroinvertebrates distribution is within the protected areas in the North West of Argentina. The analyses 2 and 3 recovered similar values of protection for the macroinvertebrate species. The upper part of Bermejo, Salí‐Dulce, San Francisco, and the Upper part of Juramento basins were identified as priority areas of conservation. The aquatic ecosystems need special protection and 10% or even as much as 17% of land conservation is insufficient for species of macroinvertebrates. In turn the protected areas need to combine the aquatic and terrestrial systems and need to include macroinvertebrates as a key group to sustain the biodiversity. In many cases, the land uses are in conflict with the conservation of biodiversity; however, it is possible to apply the connectivity of the watersheds and create multiple‐use modules.     

Isolation,purification and physicochemical characterization of water‐soluble Bacillus thuringiensis melanin

Melanins are widely used in medicine, pharmacology, cosmetics and other fields. Although several technologies for the purification of water‐insoluble dioxyphenylalanine (DOPA) melanins have been described, a source of water‐soluble melanin is highly desirable. Here we describe an effective procedure for the isolation and purification of water‐soluble melanin using the culture medium of Bacillus thuringiensis subsp. galleriae strain K1. Water‐soluble melanin from this organism has an isoelectric point (pI = 3.0–3.2) and was purified optimally by adsorbtion using the IA‐1r resin and elution as a concentrated solution. The purified melanin obtained exhibited a similar infra‐red absorbtion spectrum to synthetic melanin and contained quinolic and phenolic structures and an amino acid content of around 20% after acid hydrolysis. The molecular weight of the purified melanin determined by SDS‐PAGE was 4 kDa and the electromagnetic spin resonance spectrum of the purified microbial melanin was a slightly asymmetric singlet without hyperfine structure with about 7 Gauss width of the line between points of the maximum incline and g = 2.006. The concentration of paramagnetic centers in melanin is 0.21 × 10¹⁸ spin/g. The results obtained provide a rapid, simple and inexpensive method for the large scale purification of water soluble melanin that may have widespread applications.     

Oxidative stress in digestive gland and gill of the brown mussel (Perna perna) exposed to air and re-submersed

Mussels Perna perna were exposed to air for 24 h showing a clear increase in the levels of lipid peroxidation and oxidative DNA damage, measured as 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). The levels of lipid peroxidation increased both in the digestive gland and gills, while oxidative DNA damage increased only in the gills. After the 24 h of air exposure, mussels were re-submersed for a period of 3 h, leading values to return to a pre-aerial exposure levels. Control animals were kept immersed during the whole period. Several antioxidant and complementary enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), glutathione S-transferase (GST) and the levels of total glutathione (Total GSH) were assayed in a second set of experiments where one group of mussels were exposed to air for 18 h and other to 1 h re-submersion after 18 h aerial exposure. Only a 52% increase in the glutathione S-transferase activity was observed in the digestive gland, which remained elevated to about 40% after 1 h re-submersion, showing that defense systems can be modulated even during oxygen deprivation in P. perna. The DNA and lipid oxidative damage observed after aerial exposure indicates that mussels face an oxidative challenge, and are able to counteract such an “insult” as values of lipid peroxidation and DNA damage returned to control values after 3 h re-submersion.     

Cortisol response and immune-related effects of Atlantic salmon (Salmo salar Linnaeus) subjected to short- and long-term stress

It is generally considered that stress causes decreased immune function in fish. In this study we examined in Atlantic salmon (Salmo salar Linnaeus) the effects of both short- (a single 15s out of water) and long-term (4 weeks of daily handling 15s out of water) stress on plasma cortisol (free and total) and glucose levels, expression of interleukin-1beta (IL-1beta) and survival of head kidney (HK) macrophages under culture with Aeromonas salmonicida. In the short-term study, samples were collected prior to the application of the stressor, and at 1, 3, 6, 12 and 24h post stress. Free and total plasma cortisol levels and the percentage of free cortisol increased significantly in the stressed group at 1 and 3h post stress. Plasma glucose levels were significantly higher than those of control fish at 1, 3 and 6h post stress. Constitutive expression of IL-1beta in macrophages isolated from head kidneys in stressed fish was significantly higher at 1 and 3h post stress. However, lipopolysaccharide (LPS) stimulated expression of IL-1beta in HK macrophages, exhibited significantly higher fold increases in unstressed fish compared to stressed fish. In the long-term study, with the exception of an increase in plasma glucose levels at 1 week, there were no significant differences in stress parameters between groups. There was a significantly higher constitutive IL-1beta expression in macrophages isolated from stressed fish over the first 2 weeks. At weeks 1, 2 and 3 the magnitude of IL-1beta response of isolated HK macrophages to LPS stimulation was reduced in >90% of the stressed fish. At 4 weeks there was no significant difference in inducible IL-1beta expression between the groups. Macrophages isolated from stressed fish also showed significantly decreased survival when exposed to A. salmonicida. This study shows a clear pattern from repeated handling stress, whereby effects on immune cells begin with increased constitutive expression of IL-1beta, followed by decreased stimulation of leucocytes by extracellular antigen, and finally decreased leukocyte survival when exposed to A. salmonicida. The implications of these changes in the immune system will be discussed with respect to the use of classical indicators of stress to predict possible effects on the immune system of fish.     

Phylogenetic relationships of Middle American cichlids (Cichlidae, Heroini) based on combined evidence from nuclear genes, mtDNA, and morphology

Heroine cichlids are the second largest and very diverse tribe of Neotropical cichlids, and the only cichlid group that inhabits Mesoamerica. The taxonomy of heroines is complex because monophyly of most genera has never been demonstrated, and many species groups are without applicable generic names after their removal from the catch-all genus Cichlasoma (sensu Regan, 1905). Hence, a robust phylogeny for the group is largely wanting. A rather complete heroine phylogeny based on cytb sequence data is available [Concheiro Pérez, G.A., Říčan O., Ortí G., Bermingham, E., Doadrio, I., Zardoya, R. 2007. Phylogeny and biogeography of 91 species of heroine cichlids (Teleostei: Cichlidae) based on sequences of the cytochrome b gene. Mol. Phylogenet. Evol. 43, 91–110], and in the present study, we have added and analyzed independent data sets (nuclear and morphological) to further confirm and strengthen the cytb-phylogenetic hypothesis. We have analyzed a combined cytb-nuclear (RAG1 and two S7 introns) data set of 48 species representing main heroine lineages to achieve further resolution of heroine higher taxonomic levels and a combined cytb-morphological data set of 92 species to stabilize generic taxonomy. The recovered phylogenies supported the circumamazonian—CAM—Heroini (sensu Concheiro Peréz et al., 2007) as a monophyletic group, that could be divided into six main clades: (1) australoheroines (the southernmost heroine genus Australoheros), (2) nandopsines (the Antillean genus Nandopsis), (3) caquetaines (including the north western Amazonian genera Caquetaia and Heroina), (4) astatheroines (including Astatheros, Herotilapia and Rocio), (5) amphilophines (including Amphilophus and related genera), and (6) herichthyines (including Herichthyis and related genera). Nuclear and mitochondrial data partitions arrived at highly congruent topologies. Suprageneric relationships were influenced mainly by the nuclear signal, as well as the most basal phylogenetic position of Australoheros within CAM heroines. The new phylogeny of the tribe Heroini provides robust framework to stabilize the taxonomy of the group and for future comparative studies on these morphologically and ecologically diverse freshwater fishes. Morphology was mostly informative at the genus level and aid in determining the monophyly and composition of heroine genera. Upon acceptance of all putative genera, as recovered in this study, the Heroini would be with 35 genera the most genus-rich clade of Neotropical cichlids.     

Maximal lactate steady-state prediction through quadratic modeling of selected stages of the lactate minimum test

In this study, we compared the maximal lactate steady state (MLSS) with lactate minimal (LM) intensities determined visually and through a quadratic polynomial function of selected stages of LM test. Eleven male recreational cyclists (27.7 +/- 4.5 years, 175.7 +/- 5.6 cm, 69.5 +/- 10.8 kg, and 12.0 +/- 5.5% body fat) performed one LM test under previous induction of hyperlactaemia with an initial intensity of 75 W with 30-W increments every 3 minutes with blood lactate concentration (HLa) and rating of perceived exertion (RPE) measurements. The LM intensity was determined visually (VLM) and by modeling the lactate response through polynomial function by using: 1) all stages (LMP); 2) the first stage, the stage corresponding to RPE-13 and the last stage/exhaustion (LMP3max); 3) the three lowest lactate concentration stages (LMP3adj); and 4) the initial, RPE-13, and RPE-16 stages (LMP3sub). The MLSS was determined as the highest intensity at a variation not greater than 0.05 mmol.l.min of HLa during the last 20 minutes of a 30-minute exercise session. The MLSS (204.0 +/- 16.0 W), VLM (198.6 +/- 15.2 W), LMP3adj (190.4 +/- 12.9 W), and LMP3sub (192.1 +/- 27.2 W) were not different, well correlated, and in agreement to each other. In conclusion, the polynomial modeling of HLa response to three submaximal stages produced exercise intensities that did not differ from MLSS.     

Differential effects of retinol and retinoic acid on cell proliferation: a role for reactive species and redox-dependent mechanisms in retinol supplementation

While some authors suggest that retinoids are potential anti-proliferative and antioxidant agents, evidence has suggested those present pro-oxidant properties, which might lead to malignant proliferation. These discordances stimulated one to investigate the proliferative/anti-proliferative properties of two major retinoids, retinol (ROH) and retinoic acid (RA). In Sertoli cells, ROH increased proliferation while RA was anti-proliferative. ROH increased DNA synthesis, decreased p21 levels and induced cell cycle progression. ROH increased reactive species (RS) production and stimulated p38, JNK1/2 and ERK1/2 MAPKs activation. Antioxidant treatment with Trolox blocked ROH-induced RS production, MAPKs activation and proliferation; MAPKs inhibition blocked proliferation. The potential sites of RS indicate that ROH-induced RS is promoted via mitochondria and xanthine oxidase. In contrast, RA induced neither RS production nor MAPKs activation. RA decreased DNA synthesis and increased p21 leading to cell arrest. Overall, data show that ROH, but not RA, is able to induce proliferation through non-classical and redox-dependent mechanisms.     

Oxidative damage-induced inflammation initiates age-related macular degeneration

Oxidative damage and inflammation are postulated to be involved in age-related macular degeneration (AMD). However, the molecular signal(s) linking oxidation to inflammation in this late-onset disease is unknown. Here we describe AMD-like lesions in mice after immunization with mouse serum albumin adducted with carboxyethylpyrrole, a unique oxidation fragment of docosahexaenoic acid that has previously been found adducting proteins in drusen from AMD donor eye tissues and in plasma samples from individuals with AMD. Immunized mice develop antibodies to this hapten, fix complement component-3 in Bruch's membrane, accumulate drusen below the retinal pigment epithelium during aging, and develop lesions in the retinal pigment epithelium mimicking geographic atrophy, the blinding end-stage condition characteristic of the dry form of AMD. We hypothesize that these mice are sensitized to the generation of carboxyethylpyrrole adducts in the outer retina, where docosahexaenoic acid is abundant and conditions for oxidative damage are permissive. This new model provides a platform for dissecting the molecular pathology of oxidative damage in the outer retina and the immune response contributing to AMD.     

Failure of Helicobacter pylori Treatment After Regimes Containing Clarithromycin: New Practical Therapeutic Options

Failure of Helicobacter pylori treatment is a growing problem in daily practice. Aim: To evaluate the efficacy of two new regimes as second‐line options in a randomized and prospective study. Methods: Patients in whom a first eradication regime containing clarithromycin had failed were included. After performing gastroscopy and a 13C‐urea breath test (UBT), the patients were randomized to receive a combination of 20 mg of rabeprazole, 500 mg of levofloxacin, and 200 mg (two tablets) of furazolidone administered once daily for 10 days (RLF) or the combination of 20 mg of rabeprazole, 120 mg (two tablets) of bismuth subcitrate, 100 mg of doxycycline, and 200 mg of furazolidone, administered twice daily for 10 days (RBDF). Clinical examinations and new UBT were performed 60 days after therapy. Results: Sixty patients were included (mean age, 46 years, 57% females). Two patients were excluded: one because of adverse effects and another as a result of protocol violation. Compliance was similar in both groups (90% took all medications correctly). Side‐effects (96% mild) were observed in 87% of the patients and were comparable between groups, except diarrhea, which was more frequent in group RLF (p= .025). Intention‐to‐treat cure rates were 77% (95% confidence interval (CI): 62–93%) in the RLF group and 83% (95% CI: 68–97%) in the RBDF group (p= .750). Per‐protocol cure rates were 80% (95% CI: 65–95%) in the RLF group and 82% (95% CI: 67–96%) in the RBDF group (p= 1.0). Conclusions: Both once‐daily triple (rabeprazole, levofloxacin, and furazolidone) and twice‐daily quadruple therapy (rabeprazole, bismuth subcitrate, doxycycline, and furazolidone) for 10 days achieved encouraging results. Subsequent studies should be performed to evaluate antibiotic resistance, doses, dosing intervals, duration of treatment, and safety of these two regimes.