Seasonal variation of peptidase activities in the reproductive tract of Crotalus durissus terrificus

Seasonal quantitative patterns of acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive neutral (APN-PI), cystyl (CAP), dipeptidyl IV (DPPIV), type-1 pyroglutamyl (PAP-I) and prolylimino (PIP) aminopeptidases and prolyl oligopeptidase (POP) activities in soluble (SF) and solubilized membrane-bound (MF) fractions from ductus deferens, vagina and uterus were studied to evaluate their relationships with the reproductive cycle and the extensive long-term spermatozoa storage (LTSS) of the Neotropical rattlesnake Crotalus durissus terrificus. APB, PIP and POP were detected only in SF, while other peptidases were detected in SF and MF. APB, APN-PI and APN-PS were predominant in most tissues in all seasons. Peptidase activities had a common pattern of increment during the dry season (winter/autumn), which coincides with the mating period (autumn) and LTSS in the female (winter), as well as the reduction of spermatozoa motility and maintenance of fertilization capacity of spermatozoa. The high CAP activity in the soluble fraction of the vagina during winter, compared to summer (time of parturition) and spring, coincides with the relaxation of this tissue. In the soluble fraction, the low PAP-1 activity of the ductus deferens coincided with its high activity in the vagina during the winter; and the inverse occurred in summer, which is consistent with the physiological process of preserving spermatozoon viability. In conclusion, the studied peptidase activities had seasonal and tissue-specific characteristics, which suggest a relevant role in the reproductive physiology of C. d. terrificus. (C) 2008 Elsevier Inc. All rights reserved.

Peptidase activities in Crotalus durissus terrificus semen

To understand the role of peptidases in seminal physiology of Crotalus durissus terrificus, activity levels of representative enzymes in semen and their sensitivities to inhibitors, cofactors, and peptide hormones were evaluated. The existence of seminal fractions and the association of peptidases with these fractions were also characterized for the first time in snakes. The prominent inhibitors of aminopeptidases (APs) were amastatin for acid, basic, and neutral; bestatin for basic; and diprotin A for dipeptidyl-IV. Cystyl and prolylimino AN were similarly susceptible to the majority of these inhibitors. The basic and neutral were characterized as metallo-peptidases, acid AP was activated by MnCl(2), and cystyl, prolyl-imino, and type I pyroglutamyl were characterized as sulphydryl-dependent APs. Angiotensin II, vasotocin, bradykinin, fertilization-promoting peptide, and TRH altered the majority of these peptidase activities; these peptides are possible substrates and/or modulators of these peptidases. Peptidase activities were found in all seminal fractions: seminal plasma (SP), prostasome-like (PR) structures, and soluble (S-) and membrane-bound fractions (MFs) of spermatozoa. The levels of activity of each peptidase varied among different seminal fractions. In SP, the higher activities were puromycin-insensitive neutral and basic APs. in PR, the higher activity was puromycin-insensitive neutral AP. In spermatozoa, the higher activity in subcellular SF was puromycin-sensitive neutral, while in MF both puromycin-sensitive and -insensitive neutral AN were equally higher than the other examined peptidases. Data suggested that these peptidases, mainly basic and neutral, have a high relevance in regulating seminal functions of C. d. terrificus.

Biogas production and valorization by means of a two-step biological process

The scope of this research work was to investigate biogas production and purification by a two-step bench-scale biological system, consisting of fed-batch pulse-feeding anaerobic digestion of mixed sludge, followed by methane enrichment of biogas by the use of the cyanobacterium Arthrospira platensis. The composition of biogas was nearly constant, and methane and carbon dioxide percentages ranged between 70.5-76.0% and 13.2-19.5%, respectively. Biogas yield reached a maximum value (about 0.4 m(biogas)(3)/kgCOD(i)) at 50 days-retention time and then gradually decreased with a decrease in the retention time. Biogas CO(2) was then used as a carbon source for A. platensis cultivation either under batch or fed-batch conditions. The mean cell productivity of fed-batch cultivation was about 15% higher than that observed during the last batch phase (0.035 +/- 0.006 g(DM)/L/d), likely due to the occurrence of some shading effect under batch growth conditions. The data of carbon dioxide removal from biogas revealed the existence of a linear relationship between the rates of A. platensis growth and carbon dioxide removal from biogas and allowed calculating carbon utilization efficiency for biomass production of almost 95%. (C) 2009 Elsevier Ltd. All rights reserved.

Chlorination treatment of aqueous samples reduces, but does not eliminate, the mutagenic effect of the azo dyes Disperse Red 1, Disperse Red 13 and Disperse Orange 1

The treatment of textile effluents by the conventional method based on activated sludge followed by a chlorination step is not usually an effective method to remove azo dyes, and can generate products more mutagenic than the untreated dyes. The present work evaluated the efficiency of conventional chlorination to remove the genotoxicity/mutagenicity of the azo dyes Disperse Red 1, Disperse Orange 1, and Disperse Red 13 from aqueous solutions. The comet and micronucleus assays with HepG2 cells and the Salmonella mutagenicity assay were used. The degradation of the dye molecules after the same treatment was also evaluated, using ultraviolet and visible absorption spectrum measurements (UV-vis), high performance liquid chromatography coupled to a diode-array detector (HPLC-DAD), and total organic carbon removal (TOC) analysis. The comet assay showed that the three dyes studied induced damage in the DNA of the HepG2 cells in a dose-dependent manner. After chlorination, these dyes remained genotoxic, although with a lower damage index (DI). The micronucleus test showed that the mutagenic activity of the dyes investigated was completely removed by chlorination, under the conditions tested. The Salmonella assay showed that chlorination reduced the mutagenicity of all three dyes in strain YG1041, but increased the mutagenicity of Disperse Red 1 and Disperse Orange 1 in strain TA98. With respect to chemical analysis, all the solutions showed rapid discoloration and a reduction in the absorbance bands characteristic of the chromophore group of each dye. However, the TOC was not completely removed, showing that chlorination of these dyes is not efficient in mineralizing them. It was concluded that conventional chlorination should be used with caution for the treatment of aqueous samples contaminated with azo dyes. (C) 2010 Elsevier B.V. All rights reserved.

Geographic variation and positive selection on M7 lysin, an acrosomal sperm protein in mussels (Mytilus spp.)

Successful fertilization in free-spawning marine organisms depends on the interactions between genes expressed on the surfaces of eggs and sperm. Positive selection frequently characterizes the molecular evolution of such genes, raising the possibility that some common deterministic process drives the evolution of gamete recognition genes and may even be important for understanding the evolution of prezygotic isolation and speciation in the marine realm. One hypothesis is that gamete recognition genes are subject to selection for prezygotic isolation, namely reinforcement. In a previous study, positive selection on the gene coding for the acrosomal sperm protein M7 lysin was demonstrated among allopatric populations of mussels in the Mytilus edulis species group (M. edulis, M. galloprovincialis, and M. trossulus). Here, we expand sampling to include M7 lysin haplotypes from populations where mussel species are sympatric and hybridize to determine whether there is a pattern of reproductive character displacement, which would be consistent with reinforcement driving selection on this gene. We do not detect a strong pattern of reproductive character displacement; there are no unique haplotypes in sympatry nor is there consistently greater population structure in comparisons involving sympatric populations. One distinct group of haplotypes, however, is strongly affected by natural selection and this group of haplotypes is found within M. galloprovincialis populations throughout the Northern Hemisphere concurrent with haplotypes common to M. galloprovincialis and M. edulis. We suggest that balancing selection, perhaps resulting from sexual conflicts between sperm and eggs, maintains old allelic diversity within M. galloprovincialis.

Wolbachia: why these bacteria are important to genome research

Intracellular bacteria of the genus Wolbachia were first discovered in mosquitoes in the 1920s. Their superficial similarity to pathogenic rickettsia initially raised interest in them as potential human pathogens. However, injection experiments with mice showed that they were non-pathogenic, and they were subsequently classified as symbionts of insects. Until the 1970s, Wolbachia was considered to infect a limited number of species of mosquitoes. It is now clear that Wolbachia is an extremely common intracellular agent of invertebrates, infecting nearly all the major groups of arthropods and other terrestrial invertebrates. Its wide host range and abundance can be attributed partly to the unusual phenotypes it exerts on the host it infects. These include the induction of parthenogenesis (the production of female offspring from unmated mothers) in certain insects, the feminization of genetic male crustaceans to functional phenotypic females, and the failure of fertilization in hosts when males and females have a different infection status (cytoplasmic incompatibility). All of these phenotypes favor maternal transmission of the intracellular Wolbachia. In the last year, Wolbachia has also been shown to be a widespread symbiont of filarial nematodes. It appears that Wolbachia is needed by the adult worm for normal fertility, indicating that Wolbachia is behaving like a classic mutualist in this case. This discovery exemplifies that the extent of the host range of Wolbachia and its associated phenotypes is still far from fully understood.

Bidirectional incompatibility between conspecific populations of Drosophila simulans

Cytoplasmic incompatibility (CI) describes the phenomenon whereby eggs fertilized by sperm from insects infected with a rickettsial endosymbiont fail to hatch. Unidirectional CI between conspecific populations of insects is a well documented phenomenon. Bidirectional CI has, however, only been described in mosquito populations, and recently between closely related species of parasitic wasps, where it is of interest as both an unusual form of reproductive isolation and as a potential means of insect population suppression. Here we report on the first known example of bidirectional CI between conspecific populations of Drosophila simulans. Further, we show that defects as early as the first cleavage division are associated with CI. This observation suggests that the cellular basis of CI involves disruption of processes before or during zygote formation and that CI arises from defects in the structure and/or function of the sperm during fertilization.

Positive selection on an acrosomal sperm protein, M7 lysin, in three species of the mussel genus Mytilus

Marine invertebrate sperm proteins are particularly interesting because they are characterized by positive selection and are likely to be involved in prezyogotic isolation and, thus, speciation. Here, we present the first survey of inter and intraspecific variation of a bivalve sperm protein among a group of species that regularly hybridize in nature. M7 lysin is found in sperm acrosomes of mussels and dissolves the egg vitelline coat, permitting fertilization. We sequenced multiple alleles of the mature protein-coding region of M7 lysin from allopatric populations of mussels in the Mytilus edulis species group (M. edulis, M. galloprovincialis, and M. trossulus). A significant McDonald-Kreitman test showed an excess of fixed amino acid replacing substitutions between species, consistent with positive selection. In addition, Kolmogorov-Smirnov tests showed significant heterogeneity in polymorphism to divergence ratios for both synonymous variation and combined synonymous and non-synonymous variation within M. galloprovincialis. These results indicate that there has been adaptive evolution at M7 lysin and, furthermore, shows that positive selection on sperm proteins can occur even when post-zygotic reproductive isolation is incomplete.

Engineered ecosystem for sustainable on-site wastewater treatment

A Pilot-Scale Engineered Ecosystem (PSEE) operated for over two years in sub-tropical conditions, produced an effluent with COD (median 38 mg/L) and TSS (median 3 mg/L) levels comparable to that required by the AS/NZS 1547:2000 Onsite Domestic Wastewater Management standard. Only partial nitrification was achieved as dissimilatory nitrate reduction to ammonia occurred; however the level of NH4-N was reduced by 75% and total inorganic nitrogen by 53%. Phosphorus was not removed by the system due to the lack of regular sludge removal. Mass balances around the system showed that bacteria removed 36% of the influent nitrogen and 76% of the influent COD. Algae and plants were shown to remove 5% of the influent nitrogen, and 6% of the influent phosphorus. Challenges in developing a sustainable on-site wastewater treatment system were largely met by minimising chemical, energy and labour inputs, eliminating the need for frequent sludge handling, and creating an effluent quality suitable for re-use in non-potable applications. However, the sludge removal from the system needs to be adequately managed to avoid excessive accumulation as this can cause a range of negative impacts.

Evaluation of extractants for estimation of the phytoavailable trace metals in soils

Despite its environmental (and financial) importance, there is no agreement in the literature as to which extractant most accurately estimates the phytoavailability of trace metals in soils. A large dataset was taken from the literature, and the effectiveness of various extractants to predict the phytoavailability of Cd, Zn, Ni, Cu, and Pb examined across a range of soil types and contamination levels. The data suggest that generally, the total soil trace metal content, and trace metal concentrations determined by complexing agents (such as the widely used DTPA and EDTA extractants) or acid extractants (such as 0.1 M HCl and the Mehlich 1 extractant) are only poorly correlated to plant phytoavailability. Whilst there is no consensus, it would appear that neutral salt extractants (such as 0.01 M CaCl2 and 0.1 M NaNO3) provide the most useful indication of metal phytoavailability across a range of metals of interest, although further research is required.

Adjustment to a low-control situation: Reexamining the role of coping responses

The aim of the study was to test a revised conceptualization of the role of coping in adjustment to a low-control stressor-women's adjustment to a failed in vitro fertilization (NF) attempt. Data were collected prior to the IVF treatment (Time I) and twice after the failed NF attempt (1 to 2 weeks after finding out the results, n = 171, and fi weeks later, n = 139). Initial adjustment was assessed at Time I, whereas measures of coping and both self-report and partner ratings of adjustment were obtained at Times ? and 3. As predicted, escapist strategies and problem-management strategies (mainly at Time ?) were associated with poor adjustment, whereas problem-appraisal coping was associated with better adjustment., There was also support for the proposed positive relationship between adjustment and emotional approach coping (on self-report adjustment).

Bacteria in shrimp pond sediments: their role in mineralizing nutrients and some suggested sampling strategies

Strategies for sampling sediment bacteria were examined in intensive shrimp, Penaeus monodon (Fabricius), ponds in tropical Australia. Stratified sampling of bacteria at the end of the production season showed that the pond centre, containing flocculated sludge, had significantly higher bacterial counts (15.5 X 10(9) g(-1) dw) than the pond periphery (8.1 X 10(9) g(-1) dw), where the action of aerators had swept the pond floor. The variation in bacterial counts between these two zones within a pond was higher than that between sites within each zone or between ponds. Therefore, sampling effort should be focused within these zones: for example, sampling two ponds at six locations within each of the two zones resulted in a coefficient of variation of approximate to 5%. Bacterial numbers in the sediment were highly correlated with sediment grain size, probably because eroded soil particles and organic waste both accumulated in the centre of the pond. Despite high inputs of organic matter added to the ponds, principally as pelleted feeds, the mean bacterial numbers and nutrient concentrations (i.e. organic carbon, nitrogen and phosphorus) in the sediment were similar to those found in mangrove sediments. This suggests that bacteria are rapidly remineralizing particulates into soluble compounds. Bacterial numbers were highly correlated with organic carbon and total kjeldahl nitrogen in the sediment, suggesting that these were limiting factors to bacterial growth.

Intraspecific co-variation between egg and body size affects fertilisation kinetics of free-spawning marine invertebrates

Fertilisation of eggs of free-spawning marine invertebrates depends on factors affecting sperm concentration in the field and also on gamete characteristics such as egg size. In the free-spawning intertidal ascidian Pyura stolonifera mean egg size increased with maternal size in 2 separate populations. The largest ascidian produced eggs that were, on average, 50% greater in volume than the eggs produced by the smallest individual studied. There was no evidence to suggest that egg density varied with adult size and egg dry organic weight increased with maternal size. The fertilisation kinetics of this species were strongly affected by the variation in egg size, with the eggs of large individuals requiring much less concentrated sperm to achieve maximal levels of fertilisation success than the eggs of small individuals. We suggest that variation in egg size between individuals of different sizes and ages may be an important factor in determining fertilisation success for ascidians of this species.

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA

Two small RNAs regulate the timing of Caenorhabditis elegans development(1,2). Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA(1,3,4), and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA 2. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs1,2,5,6. Here we have detected let-7 RNAs of similar to 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

Sperm environment affects offspring quality in broadcast spawning marine invertebrates

The provisioning of offspring can have far-reaching consequences for later life in a wide range of organisms and generally this provisioning is thought to be under maternal influence or control. In experiments with a broadcast-spawning ascidian, we found that the size of offspring was determined by egg size and the abundance of sperm present during fertilization. Larger eggs were fertilized at low sperm concentrations, whilst smaller eggs were successfully fertilized at high sperm concentrations. These differences in fertilized egg size resulted in differences in the development rate, hatching success and mean size of the subsequent larvae. Our results suggest that, in contrast to females that reproduce by other mating systems, free-spawning mothers lack some control over the provisioning of offspring. Furthermore, because males can alter the sperm environment, they can exert paternal (non-genetic) control over key offspring characteristics.