Genetic characterization of the invasive mosquitofish (Gambusia spp.) introduced to Europe: population structure and colonization routes

Biological invasions are considered one of the main anthropogenic factors that reduce the abundance of native species. Understanding the patterns of population structure and behavior of introduced species is important to determine invasion sources and pathways, in addition to improving the protective management of native species. Thus, we set out to advance our knowledge about the mosquitofish Gambusia spp., which is an invasive species that was introduced to southern Europe in 1921 to control mosquito populations. We assessed the genetic diversity and population structure of this species at 13 European locations, by screening variation at six microsatellite loci. We also evaluated six American samples (four of G. holbrooki and two of G. affinis) to identify the most likely source of the populations that established in Europe, and to determine whether G. affinis is also present. The results showed that, while there was evidence of recent bottleneck events in a few isolated locations, most introduced populations harbored a considerable amount of gene diversity, probably because of multiple introductions and secondary contacts. Populations displayed strong genetic differentiation that was mainly associated with geographical distance. At least two main routes of colonization of G. holbrooki seem to have occurred in Europe. The first, and more ancient colonization, was consistent with historical records, with the species invading the Iberian Peninsula. A second and more recent colonization probably occurred in Greece and, from there, France. The presence of G. affinis was not detected in any of the European samples.     

Lovastatin inhibits the extracellular-signal-regulated kinase pathway in immortalized rat brain neuroblasts

Arachidonic acid is a potential paracrine agent released by the uterine endometrial epithelium to induce PTGS2 [PG (prostaglandin)-endoperoxide synthase 2] in the stroma. In the present study, bovine endometrial stromal cells were used to determine whether PTGS2 is induced by arachidonic acid in stromal cells, and to investigate the potential role of PPARs (peroxisome-proliferator-activated receptors) in this effect. Arachidonic acid increased PTGS2 levels up to 7.5-fold within 6 h. The cells expressed PPARalpha and PPARdelta (also known as PPARbeta) (but not PPARgamma). PTGS2 protein level was increased by PPAR agonists, including polyunsaturated fatty acids, synthetic PPAR ligands, PGA1 and NSAIDs (non-steroidal anti-inflammatory drugs) with a time course resembling that of arachidonic acid. Use of agonists and antagonists indicated PPARalpha (but not PPARdelta or PPARgamma) was responsible for PTGS2 induction. PTGS2 induction by arachidonic acid did not require PG synthesis. PTGS2 levels were increased by the PKC (protein kinase C) activators 4beta-PMA and PGF(2alpha), and the effects of arachidonic acid, NSAIDs, synthetic PPAR ligands and 4beta-PMA were blocked by PKC inhibitors. This is consistent with PPAR phosphorylation by PKC. Induction of PTGS2 protein by 4beta-PMA in the absence of a PPAR ligand was decreased by the NF-kappaB (nuclear factor kappaB) inhibitors MG132 and parthenolide, suggesting that PKC acted through NF-kappaB in addition to PPAR phosphorylation. Use of NF-kappaB inhibitors allowed the action of arachidonic acid as a PPAR agonist to be dissociated from an effect through PKC. The results are consistent with the hypothesis that arachidonic acid acts via PPARalpha to increase PTGS2 levels in bovine endometrial stromal cells.     

Boar sperm velocity and motility patterns under capacitating and non-capacitating incubation conditions

This study compares the velocity and motility of boar sperm under capacitating and non-capacitating incubation conditions. Aliquots of pooled, washed boar sperm were incubated in either Tyrode's complete medium (TCM; a capacitating medium), Ca2+-free TCM (TCM-Ca2+), or Ca2+ and NaHCO3-free TCM (Tyrode's basal medium [TBM]; a non-capacitating medium). Motility patterns were determined every hour over a 3h period of incubation at 38 degrees C. Capacitation status was assessed by the chlortetracycline assay after 1 and 3h of incubation. Experiments were repeated five times. Compared to the TBM control, a significant increase was seen in the percentage of capacitated sperm after 1h of incubation in TCM: the kinematics of these sperm cells were favorably modified. However, the motility patterns of sperm cells incubated in TCM and TCM-Ca2+ were very similar. Under capacitating conditions (TCM), the coefficients of linearity (LIN) and straightness (STR) significantly increased over time (LIN values were significantly different after 3h of incubation, while STR values were significantly different after only 2 h). Significant correlations were seen between LIN and the percentage of cells showing the B pattern (r = 0.334, P < 0.05) and the number of acrosome reacted spermatozoa (r = 0.301, P < 0.05). This suggests that capacitated boar spermatozoa may have a species-specific motility pattern.     

Production of a Mixture of Fengycins with Surfactant and Antifungal Activities by <Emphasis Type="Italic">Bacillus</Emphasis> sp. MA04, a Versatile PGPR

Bacillus sp. strain MA04 a plant growth-promoting rhizobacteria (PGPR) showed hemolytic activity on blood agar plates, and the supernatant from liquid culture in nutrient broth at 24 h exhibited emulsification activity, suggesting the production of biosurfactants. In antagonist assays, the supernatant showed antifungal activity against phytopathogenic fungi such as Penicillium expansum, Fusarium stilboides, Sclerotium rolfsii y Rhizoctonia solani, finding a reduction of mycelial growth of all fungi tested, ranging from 35 to 69%, this activity was increased with time of culture, accomplishing percentages of inhibition up to 85% with supernatants obtained at 72 h. Then, the crude biorsurfactant (CB) was isolated from the supernatant in order to assay its antagonistic effect on the phytopathogens previously tested, finding an increase in the inhibition up to 97% at 500 mg/L of CB. The composition of CB was determined by infrared spectroscopy, identifying various functional groups related to lipopeptides, which were purified by high-performance liquid chromatography and analyzed by MALDI-TOF/TOF–MS, revealing a mixture of fengycins A and B whose high antifungal activity is been widely recognized. These results show that PGPR Bacillus sp. MA04 could also contribute to plant health status through the production of metabolites with antimicrobial activity.     

Temporal genetic dynamics among mosquitofish (<Emphasis Type="Italic">Gambusia holbrooki</Emphasis>) populations in invaded watersheds

The temporal components of genetic diversity and geographical structure of invasive mosquitofish populations are poorly known. Through the genetic monitoring of four consecutive cohorts of Gambusia holbrooki from three different river basins we aimed to determine temporal patterns of regional genetic variation and dispersal rates within invasive populations. Despite showing evidence of strong population size fluctuations, genetic diversity levels were maintained among local cohorts. We only detected temporal allele frequency changes associated with seasonal flooding that did not modify major trends on population structure among cohorts. Downstream gene flow coupled with increased connectivity at lowland locations to increase genetic diversity levels in these areas. A large proportion of local fish (up to 50 %) were dispersers, often originated from locations within the same river basin. High dispersal capability, ecological tolerance, and reproductive traits likely promote river colonization. Finally, our results also confirmed that human-assisted translocations promote within and among basin gene flow and maintained levels of genetic diversity, particularly in upstream locations.     

c-Jun N-terminal protein kinase signalling pathway mediates lovastatin-induced rat brain neuroblast apoptosis

We have previously shown that lovastatin, an HMG-CoA reductase inhibitor, induces apoptosis in rat brain neuroblasts. c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) are implicated in regulation of neuronal apoptosis. In this work, we investigated the role of JNK and p38 MAPK in neuroblast apoptosis induced by lovastatin. We found that lovastatin induced the activation of JNK, but not p38 MAPK. It also induced c-Jun phosphorylation with a subsequent increase in activator protein-1 (AP-1) binding, AP-1-mediated gene expression and BimEL protein levels. The effects of lovastatin were prevented by mevalonate. Pre-treatment with iJNK-I (a selective JNK inhibitor) prevented the effect of lovastatin on both neuroblast apoptosis and the activation of the JNK cascade. Furthermore, we found that the activation of the JNK signalling pathway triggered by lovastatin is accompanied by caspase-3 activation which is also inhibited by iJNK-I pre-treatment. Finally, a specific inhibitor of p38 MAPK, SB203580, had no effect on lovastatin-induced neuroblast apoptosis. Taken together, our data suggest that the activation of the JNK/c-Jun/BimEL signalling pathway plays a crucial role in lovastatin-induced neuroblast apoptosis. Our findings may also contribute to elucidate the intracellular mechanisms involved in the central nervous system side effects associated with statin therapy.     

The effect of melatonin on the quality of extended boar semen after long-term storage at 17 °C

Melatonin (MLT) is an efficient antioxidant that protects cells and tissues and initiates a host of receptor-mediated effects. In order to enhance the life span of refrigerated boar semen, our aim was to evaluate the effects of addition of 1 μM MLT to commercially produced pig semen (33 seminal doses from 14 boars) that had been preserved at 17 °C for 7 days. Samples without MLT served as controls. On Days 1, 4 and 7, we evaluated motility parameters and the percentage of total motile and progressively motile spermatozoa by a computer-aided sperm analysis system. Viability (SYBR-14/PI), acrosomal status (FITC-PNA/PI), membrane fluidity (M-540/YoPro-1) and mitochondrial membrane potential status (JC-1) were evaluated by flow cytometry. MLT treatment significantly enhanced the percentage of static spermatozoa after 7 days of storage and significantly reduced the percentage of progressively motile spermatozoa on Day 7. The velocity characteristics (VCL, VSL and VAP) were significantly higher for MLT-treated samples on Day 1 and were their lowest on Day 7. With regard to flow cytometry results, the percentage of viable spermatozoa with an intact acrosome was higher in MLT samples throughout the entire storage period. In addition, there was a significantly higher proportion of live spermatozoa on Day 7 in the samples that had not been treated with MLT. The proportion of spermatozoa showing a high mitochondrial membrane potential remained at similar levels (P > 0.05) throughout the trial. Although the findings of the present study revealed that 1 μM MLT increased the proportion of live sperm with an intact acrosome, this treatment did not enhance the spermatic quality of refrigerated boar semen.     

Rottlerin inhibits stimulated enzymatic secretion and several intracellular signaling transduction pathways in pancreatic acinar cells by a non-PKC-delta-dependent mechanism

Protein kinase C-delta (PKC-delta) becomes activated in pancreatic acini in response to cholecystokinin (CCK) and plays a pivotal role in the exocrine pancreatic secretion. Rottlerin, a polyphenolic compound, has been widely used as a potent and specific PKC-delta inhibitor. However, some recent studies showed that rottlerin was not effective in inhibiting PKCdelta activity in vitro and that may display unspecific effects. The aims of this work were to investigate the specificity of rottlerin as an inhibitor of PKC-delta activity in intact cells and to elucidate the biochemical causes of its unspecificity. Preincubation of pancreatic acini with rottlerin (6 microM) inhibited CCK-stimulated translocation, tyrosine phosphorylation (TyrP) and activation of PKC-delta in pancreatic acini in a time-dependent manner. Rottlerin inhibited amylase secretion stimulated by both PKC-dependent pathways (CCK, bombesin, carbachol, TPA) and also by PKC-independent pathways (secretin, VIP, cAMP analogue). CCK-stimulation of MAPK activation and p125(FAK) TyrP which are mediated by PKC-dependent and -independent pathways were also inhibited by rottlerin. Moreover, rottlerin rapidly depleted ATP content in pancreatic acini in a similar way as the mitochondrial uncouplers CCCP and FCCP. All studied inhibitory effects of rottlerin in pancreatic acini were mimicked by FCCP (agonists-stimulated amylase secretion, p125(FAK) TyrP, MAPK activation and PKC-delta TyrP and translocation). Finally, rottlerin as well as FCCP display a potent inhibitory effect on the activation of other PKC isoforms present in pancreatic acini. Our results suggest that rottlerin effects in pancreatic acini are not due to a specific PKC-delta blockade, but likely due to its negative effect on acini energy resulting in ATP depletion. Therefore, to study the role of PKC-delta in cellular processes using rottlerin it is essential to keep in mind that may deplete ATP levels and inhibit different PKC isoforms. Our results give reasons for a more careful choice of rottlerin for PKC-delta investigation.     

Partial characterization of digestive proteases in Totoaba (Totoaba macdonaldi)

ABSTRACT

The objective of this study was to characterize the digestive proteases of totoaba (Totoaba macdonaldi). Fish were sacrificed to obtain the multienzymatic extracts from the stomach and intestine, and determine the stability and optimum pH and temperature values. Residual activity and number of isoforms were determined with some inhibitors. Optimal pH of stomach proteases was 2, with stability above 100% at that same pH. Optimum pH of intestinal proteases was between 9 and 11, with stability above 100% between 8-12. Optimum temperature for stomach proteases was 35°C and remained highly stable, while optimum temperature for intestinal proteases was 45°C, with high stability between 35-55°C. Pepstatin A totally inhibited acid protease activity and revealed a single band. SDS-PAGE zymogram revealed 8 bands in the intestine, where phenanthroline inhibited 80% of the total activity. The digestive capacity of T. macdonaldi is characteristic of a strict carnivore, similar to other marine fish species.