Heat tolerance, behavioural temperature selection and temperature-dependent respiration in larval Octopus huttoni

This study reports temperature effects on paralarvae from a benthic octopus species, Octopus huttoni, found throughout New Zealand and temperate Australia. We quantified the thermal tolerance, thermal preference and temperature-dependent respiration rates in 1-5 days old paralarvae. Thermal stress (1 °C increase h⁻¹) and thermal selection (∼10-24 °C vertical gradient) experiments were conducted with paralarvae reared for 4 days at 16 °C. In addition, measurement of oxygen consumption at 10, 15, 20 and 25 °C was made for paralarvae aged 1, 4 and 5 days using microrespirometry. Onset of spasms, rigour (CT_max) and mortality (upper lethal limit) occurred for 50% of experimental animals at, respectively, 26.0±0.2 °C, 27.8±0.2 °C and 31.4±0.1 °C. The upper, 23.1±0.2 °C, and lower, 15.0±1.7 °C, temperatures actively avoided by paralarvae correspond with the temperature range over which normal behaviours were observed in the thermal stress experiments. Over the temperature range of 10 °C-25 °C, respiration rates, standardized for an individual larva, increased with age, from 54.0 to 165.2 nmol larvae⁻¹ h⁻¹ in one-day old larvae to 40.1-99.4 nmol h⁻¹ at five days. Older larvae showed a lesser response to increased temperature: the effect of increasing temperature from 20 to 25 °C (Q₁₀) on 5 days old larvae (Q₁₀=1.35) was lower when compared with the 1 day old larvae (Q₁₀=1.68). The lower Q₁₀ in older larvae may reflect age-related changes in metabolic processes or a greater scope of older larvae to respond to thermal stress such as by reducing activity. Collectively, our data indicate that temperatures >25 °C may be a critical temperature. Further studies on the population-level variation in thermal tolerance in this species are warranted to predict how continued increases in ocean temperature will limit O. huttoni at early larval stages across the range of this species.     

Differential responses of the floating-leaved aquatic plant Nymphoides peltata to gradual versus rapid increases in water levels

We compared the growth responses of the floating-leaved species Nymphoides peltata to gradual and rapid rising water levels under two nutrient concentrations (1 g and 12 g of slow released fertilizer (N-P-K: 16-8-12) per container filled with 8 kg washed sand), and predicted the population expansion after these floods. The results showed that the capacity for petiole elongation was dependent on leaf age, and only leaves that were no more than five days old had the capability to reach the water surface when the water level increased rapidly from 50 cm to 300 cm. Plants subjected to a gradual rising water level tracked the increase in water depth whose petioles elongated at 3.96 ± 1.70 cm per day and 4.80 ± 0.16 cm per day under low and high nutrient concentrations respectively throughout the experiment period. When water levels were rapidly raised, leaf petioles elongated rapidly at 25.48 ± 1.51 cm per day and 26.64 ± 2.24 cm per day under low and high nutrient concentrations respectively during the first ten days. Plants under a constant water level maintained highest mean leaf recruitment (mean 3.0 ± 0.33 leaves and 24.4 ± 5.87 leaves every ten days under low and high nutrient concentrations, respectively). Therefore, more young leaves existed in the canopy ensuring that when the water level increases, young leaves can rapidly emerge after submergence. Gradual water level rise did not significantly affect biomass and ramet production (4.75 ± 1.41 g and 5.50 ± 1.22 ramets in low nutrient; 48.49 ± 21.45 g and 35.67 ± 11.78 ramets in high nutrient), but rapid water level rise negatively affected ramet production in both nutrient concentrations (3.00 ± 1.26 ramets and 11.25 ± 4.19 ramets in low and high nutrients, respectively). The results indicated that continual leaf recruitment and rapid petiole elongation were both important ways in which N. peltata adapted to increasing water levels. Extreme flooding may be a disturbance factor that affects plant growth and the population expansion of N. peltata, while small gradual water level rise should not harm this species.     

Photonic characteristics and ex vivo imaging of Escherichia coli-Xen14 within the bovine reproductive tract

The objectives of this study were to (1) characterize the photonic properties of Escherichia coli-Xen14 and (2) conduct photonic imaging of E. coli-Xen14 within bovine reproductive tract segments (RTS) ex vivo (Bos indicus). E. coli-Xen14 was grown for 24 h in Luria Bertani medium (LB), with or without kanamycin (KAN). Every 24 h, for an 8-d interval, inoculums were imaged and photonic emissions (PE) collected. Inoculums were subcultured and plated daily to determine the colony forming units (CFU) and ratio of photon emitters to nonemitters. In the second objective, abattoir-derived bovine reproductive tracts (n = 9) were separated into posterior and anterior vagina, cervix, uterine body, and uterine horns. Two concentrations (3.2 × 10⁸ and 3.2 × 10⁶ CFU/200 μL for relative [High] and [Low], respectively) of E. coli-Xen14 were placed in translucent tubes for detection of PE through RTS. The CFU did not differ (P = 0.31) over time with or without KAN presence; they remained stable with 99.93% and 99.98% photon emitters, respectively. However, PE were lower (P < 0.0001) in cultures containing KAN than in those containing no KAN (629.8 ± 117.7 vs. 3012.0 ± 423.5 relative lights units per second [RLU/sec], respectively). On average, the percentage of PE between RTS, for both concentrations, was higher (P < 0.05) in the uterine body. In summary, E. coli-Xen14 remained stable with respect to the proportions of photon emitters with or without KAN (used to selectively culture E. coli-Xen14). However, KAN presence suppressed photonic activity. The ability to detect PE through various segments of the reproductive tract demonstrated the feasibility of monitoring the presence of E. coli-Xen14 in the bovine reproductive tract ex vivo.     

Hydrolysis of the soluble fluorescent molecule carboxyumbelliferyl-beta-D-glucuronide by E. coli beta-glucuronidase as applied in a rugged, in situ optical sensor

Techniques utilizing β-glucuronidase (GUS) activity as an indicator of Escherichia coli (E. coli) presence use labeled glucuronides to produce optical signals. Carboxyumbelliferyl-β-d-glucuronide (CUGlcU) is a fluorescent labeled glucuronide that is soluble and highly fluorescent at natural water pHs and temperatures and, therefore, may be an ideal reagent for use in an in situ optical sensor. This paper reports for the first time the Michaelis-Menten kinetic parameters for the binding of E. coli GUS with CUGlcU as K_m = 910 μM, V_max = 41.0 μM min⁻¹, V_max/K_m 45.0 μmol L⁻¹ min⁻¹, the optimal pH as 6.5 ± 1.0, optimal temperature as 38 °C, and the Gibb's free energy of activation as 61.40 kJ mol⁻¹. Additionally, it was found CUGlcU hydrolysis is not significantly affected by heavy solvents suggesting proton transfer and solvent addition that occur during hydrolysis are not limiting steps. Comparison studies were made with the more common fluorescent molecule methylumbelliferyl-β-d-glucuronide (MUGlcU). Experiments showed GUS preferentially binds to MUGlcU in comparison to CUGlcU. CUGlcU was also demonstrated in a prototype optical sensor for the detection of E. coli. Initial bench testing of the sensor produced detection of low concentrations of E. coli (1.00 × 10³ CFU/100 mL) in 230 ± 15.1 min and high concentrations (1.05 × 10⁵ CFU/100 mL) in 8.00 ± 1.01 min.     

Administration of sulpiride or domperidone for advancing the first ovulation in deep anestrous mares

Since results with using sulpiride and domperidone are conflicting and since both have not been tested at the same time, the aim of this study was to compare the efficacy of these substances for the induction of ovulation in deep anestrous mares in the same experimental conditions and to determine their fertility after artificial insemination (AI) at the induced estrus. Twenty-six non-pregnant, non-lactating standardbred anestrous mares were randomly assigned to three groups and treated daily for 25 days (from February 3rd to February 28th) with either sulpiride (1 mg/kg of body weight im SID, n = 10), or domperidone (1 mg/kg po SID, n = 10); 6 animals were used as control. The beginning of the transition period and the first ovulation were hastened in sulpiride (16.4 ± 0.8 days) but not in domperidone (46.0 ± 3.3 days) treated mares (P < 0.05). The diameter of the largest follicle was affected by treatment, time and interaction of treatment-by-day (P < 0.05) and significantly increased in the sulpiride group (P < 0.05). Although a main effect of treatment on plasma LH concentration was not observed (P = 0.06), time and interaction of treatment-by-day were statistically significant (P < 0.05). The interval from the beginning of treatment to first ovulation was shorter (P < 0.05) in the sulpiride group (36.9 ± 2.5 days) than in the domperidone (74.7 ± 3.3 days) and control (81.4 ± 3.1) groups. The establishment of pregnancy was significantly (P < 0.05) hastened in sulpiride (61.0 ± 35.2 days) but not in domperidone (83.0 ± 44.0 days) treated mares. Treated mares not pregnant after the first AI, showed normal estrous cycles with regular interovulatory intervals (P > 0.05). It was concluded that sulpiride is effective in advancing the beginning of transition period and the first ovulation whereas domperidone is successful only in some mares.     

The effect of raffinose and methionine on frozen/thawed Angora buck (Capra hircus ancryrensis) semen quality, lipid peroxidation and antioxidant enzyme activities

The aim of the present study was to determine the effects of different doses of raffinose and methionine on post-thawed semen quality, lipid peroxidation and antioxidant enzyme activities of Angora buck (Capra hircus ancryrensis) sperm following cryopreservation.Ejaculates collected from three Angora bucks were evaluated and pooled at 37 °C. Semen samples, which were diluted with a Tris-based extender containing the additives raffinose (2.5, 5, 10 mM) and methionine (2.5, 5, 10 mM) and an extender containing no antioxidants (control), were cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were thawed individually at 37 °C for 20 s in a water bath for evaluation. The freezing extender supplemented with 2.5 and 5 mM methionine led to higher percentages of CASA motility (63.6 ± 7.0; 63.4 ± 3.1%, respectively), in comparison to the controls (P < 0.01) following the freeze-thawing process. The addition of antioxidants did not provide any significant effect on the percentages of post-thaw subjective and CASA progressive motilities as well as sperm motion characteristics (VSL and VCL), compared to the control groups (P > 0.05). The freezing extender with raffinose (5 and 10 mM) and methionine at three different doses (2.5, 5 and 10 mM) led to lower percentages of acrosome abnormalities, in comparison to the controls (P < 0.001). In the comet test, raffinose (5 and 10 mM) and methionine (10 mM) gave scores lower than those of the controls, and thereby reduced DNA damage (P < 0.05). Malondialdehyde formation was found to be lower (1.8 ± 0.1 nmol/L) in the group of 5 mM raffinose, compared to the controls following the freeze-thawing process (P < 0.01). The additives did not show any effectiveness on the maintenance of SOD, GSH-PX and GSH activities, when compared to the controls (P > 0.05). In conclusion, methionine and raffinose play a cryoprotective role against sperm CASA motility, acrosome abnormality and DNA damage. Raffinose 5 mM exhibited antioxidative properties, decreasing MDA levels. Further studies are required to obtain more concrete results on the characterization of microscopic parameters and antioxidant activities in cryopreserved goat sperm with different additives.     

Treatment of industrial wastewater with two-stage constructed wetlands planted with Typha latifolia and Phragmites australis

Industrial wastewater treatment comprises several processes to fulfill the discharge permits or to enable the reuse of wastewater. For tannery wastewater, constructed wetlands (CWs) may be an interesting treatment option. Two-stage series of horizontal subsurface flow CWs with Phragmites australis (UP series) and Typha latifolia (UT series) provided high removal of organics from tannery wastewater, up to 88% of biochemical oxygen demand (BOD₅) (from an inlet of 420 to 1000 mg L⁻¹) and 92% of chemical oxygen demand (COD) (from an inlet of 808 to 2449 mg L⁻¹), and of other contaminants, such as nitrogen, operating at hydraulic retention times of 2, 5 and 7 days. No significant (P < 0.05) differences in performance were found between both the series. Overall mass removals of up to 1294 kg COD ha⁻¹ d⁻¹ and 529 kg BOD₅ ha⁻¹ d⁻¹ were achieved for a loading ranging from 242 to 1925 kg COD ha⁻¹ d⁻¹ and from 126 to 900 kg BOD₅ ha⁻¹ d⁻¹. Plants were resilient to the conditions imposed, however P. australis exceeded T. latifolia in terms of propagation.     

Spilanthes acmella ethanolic flower extract: LC-MS alkylamide profiling and its effects on sexual behavior in male rats

According to Indian Systems of Medicine, Spilanthes acmella (L.) Murr. (Family - Asteraceae), is considered effective in the treatment of sexual deficiencies especially due to ageing. In the present study, characterization of ethanolic extracts of the Spilanthes acmella flower and its effect on general mating pattern, penile erection and serum hormone levels of normal male Wistar albino rats were investigated and compared with sildenafil citrate. In vitro nitric oxide release was also investigated in human corpus cavernosum cell line. As N-alkylamides are a promising group, their profiling was performed using a gradient reversed phase high performance liquid chromatography/electrospray ionization ion trap mass spectrometry (HPLC/ESI-MS) method on an embedded polar column. MS¹ and MS² fragmentation data were used for identification purposes. For assessment of sexual behavior, animals were divided into five groups of eight male rats. The extracts (50, 100 and 150 mg/kg body weight/day) and sildenafil citrate (5 mg/kg body weight/day) (positive control) were administered orally for 28 days. The behavioral and sexual parameters were observed at days 0, 15, 28 and after a lapse of 7 and 14 days of discontinuance of drug treatment. Five N-isobutylamides, one 2-methylbutylamide and one 2-phenylethylamide were identified. The orally administered extract had a dose dependent positive effect on mounting frequency, intromission frequency and ejaculation frequency and the most significant effects (p < 0.05) were observed at 150 mg/kg treatment, even after a lapse of 7 and 14 days of discontinuance of drug treatment. A dose dependent effect was also observed on the FSH, LH and testosterone serum levels. With 150 mg/kg of ethanolic extract the values for FSH, LH and testosterone were 3.10 ± 0.25 mlU/ml, 6.87 ± 0.18 mlU/ml and 3.72 ± 0.12 ng/ml, respectively. In vitro nitric oxide release was 21.7 ± 2.9 μM, which was significantly higher compared to the control group (p < 0.01). Sildenafil citrate exhibited also a significant effect on NO release, but no effect on hormone levels of rats was observed. The aphrodisiac potential of an ethanolic Spilanthes acmella extract was demonstrated in vitro and in vivo. N-Alkylamides might attribute to the improved sexual potential. Study lends support to the traditional utilization of S. acmella as a sexual stimulating agent.     

Semen characteristics and sperm morphology of serow (Capricornis sumatraensis)

The serow (Capricornis sumatraensis) is a critically endangered species. The objectives of this study were to evaluate ejaculate quality in captive males, and to investigate and characterize sperm morphology. Semen was collected using electroejaculation. Mean (±S.D.) seminal characteristics were: semen volume 2.3 ± 0.8 mL, pH 7.8 ± 0.4, and osmolality 329.9 ± 32.9 mOsmol/kg; sperm concentration 515.8 ± 263.1 × 10⁶ cells/mL; wave motion score (1-5) 3.9 ± 0.4; motile sperm 60.5 ± 22%; viable sperm 68.3 ± 9.4%; morphologically normal sperm 70.8 ± 19.3%; and an opacity that was yellowish to milky-white. Sperm head length, width, degree of elongation, area, and perimeter were 6.0 ± 0.6 μm, 4.3 ± 0.3 μm, 71.7 ± 8.6%, 19.8 ± 2.5 μm², and 17.9 ± 2.1 μm. Based on these measurements, we categorized sperm head morphometry as small, medium, or large. In addition, sperm morphology was examined by light and scanning electron microscopy; overall, morphologically normal and abnormal sperm were similar to those reported for other bovidae. In summary, this study provided baseline data regarding semen characteristics of C. sumatraensis, which should be of value in the preservation of this endangered species.     

Effect of storage temperature on survival and infectivity of Steinernema rarum (OLI strain) (Rhabditida: Steinernematidae)

Nematode strains of the entomopathogenic family Steinernematidae differ in their ability to infect insects at different temperatures. Survival and infectivity of infective juveniles (IJs) of Steinernema rarum (OLI) were studied after their storage at 23 ± 2 °C and at 5 ± 1 °C. Survival at 23 ± 2 °C was always above 95%. At 5 ± 1 °C, survival decreased at week 5, but infectivity did the same after week 2. Unlike other steinernematids, both infectivity and survival of IJs would be higher for S. rarum (OLI) when stored at 23 ± 2 °C.     

Growth and annual production of the brown alga Laminaria japonica (Phaeophyta, Laminariales) introduced into the Uwa Sea in southern Japan

The brown alga Laminaria japonica is distributed from southern Hokkaido to the northeastern Honshu in Japan. Recently, aquaculture of L. japonica has expanded to the southern coast of Japan and to China along the East China Sea. In order to elucidate the growth, biomass and productivity of L. japonica in a subtropical area, we cultivated and examined it in the Uwa Sea, in southwestern Japan over a period of 2 years. The seawater temperature ranged from 13.8 to 26.8 °C in 2001/2002 and from 13.1 to 27.2 °C in 2002/2003. In 2001/2002, the maximum density, maximum mean length and maximum mean wet wt. of L. japonica were 59.7 ± 28.0 ind. 50 cm^− 1 (mean ± S.D.), 187.5 ± 82.7 cm (360 cm in the largest individual) and 130.1 ± 94.6 g wet wt., respectively. In 2002/2003, these values were 94.7 ± 22.2 ind. 50 cm^− 1, 159.3 ± 74.4 cm (300 cm in the largest individual) and 95.2 ± 69.5 g wet wt., respectively. Thus, the length and weight increased when the density was low (2001/2002), and the length and weight decreased when the density was high (2002/2003). The maximum biomass was estimated to be 7200 ± 3400 g wet wt. 50 cm^− 1 in 2001/2002 and 7300 ± 2000 g wet wt. 50 cm^− 1 in 2002/2003. Annual production was estimated to be 33.3 kg wet wt. m^− 1 year^− 1 in 2001/2002 and 34.0 kg wet wt. m^− 1 year^− 1 in 2002/2003. The present study indicates that the annual production of L. japonica per rope of 1 m at Uwajima Bay, the Uwa Sea corresponded to 1.1-2.2 m² of that of Hokkaido in their native area. Thus, the present study indicates that L. japonica is highly adaptable because it is able to keep a high level of productivity when grown in water with a high temperature.     

High-level expression of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas diminuta NK703 in Escherichia coli by combined optimization strategies

In this work, a glutaryl-7-aminocephalosporanic acid acylase (GLA) coding gene was cloned from Pseudomonas diminuta NK703 which was screened from oilfield. The concerted effects of the expression system, inducing condition and culture medium on the expression of NK703 GLA in E. coli were firstly investigated. The best combination was the recombinant E. coli strain of pET-28a + GLA/BL21(DE3) with 2.0% (w/v) lactose inducing in YT medium at 25 °C. Then, by optimizing the components of culture medium, a synthetic medium with dextrin and a feeding medium with glycerol as the carbon sources were developed to further enhance the GLA yield and improve the GLA solubility. In the end, the NK703 GLA activity increased about 50-fold, reached 14,470 ± 465 U/L, and the GLA productivity and the proportion of soluble GLA to the total soluble protein attained 206.0 ± 9.033 U L⁻¹ h⁻¹ and 60.13%, respectively. Scaling up the GLA production in 3.7 L fermenter under the optimized conditions identified in shake flask, the GLA activity also reached 12,406 ± 521 U/L, which was the highest report at fermenter level.     

Membrane phase behavior of Escherichia coli during desiccation, rehydration, and growth recovery

The membrane lipid bilayer is one of the primary cellular components affected by variations in hydration level, which cause changes in lipid packing that may have detrimental effects on cell viability. In this study, Fourier transform infrared (FTIR) spectroscopy was used to quantify changes in the membrane phase behavior, as identified by membrane phase transition temperature (T_m), of Escherichia coli during desiccation and rehydration. Extensive cell desiccation (1 week at 20%-40% RH) resulted in an increase in T_m from 8.4 ± 1.7 °C (in undried control samples) to 16.5 ± 1.3 °C. Fatty acid methyl ester analysis (FAME) on desiccated samples showed an increase in the percent composition of saturated fatty acids (FAs) and a decrease in unsaturated FAs in comparison to undried control samples. However, rehydration of E. coli resulted in a gradual regression in T_m, which began approximately 1 day after initial rehydration and plateaued at 12.5 ± 1.8 °C after approximately 2 days of rehydration. FAME analysis during progressive rehydration revealed an increase in the membrane percent composition of unsaturated FAs and a decrease in saturated FAs. Cell recovery analysis during rehydration supported the previous findings that showed that E. coli enter a viable but non-culturable (VBNC) state during desiccation and recover following prolonged rehydration. In addition, we found that the delay period of approximately 1 day of rehydration prior to membrane reconfiguration (i.e. decrease in T_m and increase in membrane percent composition of unsaturated FAs) also preceded cell recovery. These results suggest that changes in membrane structure and state related to greater membrane fluidity may be associated with cell proliferation capabilities.     

Comparison of embryo yield and pregnancy rate between in vivo and in vitro methods in the same Nelore (Bos indicus) donor cows

To investigate why the preferred means to produce bovine embryos in Brazil has changed from in vivo to in vitro, we compared these two approaches in the same Nelore cows (n = 30) and assessed total embryo production and pregnancy rates. Without a specific schedule, all cows were subjected to ultrasound-guided ovum pick up (OPU)/in vitro production (IVP) and MOET, with intervals ranging from 15 to 45 d between procedures, respectively. To produce in vivo embryos, cows were superovulated and embryos were recovered nonsurgically from 1 to 3 times (1.4 ± 0.6), whereas OPU/IVP was repeated from 1 to 5 times (3.2 ± 1.2) in each donor cow during a 12-mo interval. Embryos obtained from both methods were transferred to crossbred heifers. On average, 25.6 ± 15.3 immature oocytes were collected per OPU attempt. The average number of embryos produced by OPU/IVP (9.4 ± 5.3) was higher (P < 0.05) than the MOET method (6.7 ± 3.7). However, pregnancy rates were lower (P < 0.05) following transfer of IVP (33.5%) versus in vivo-derived embryos (41.5%) embryos. Embryonic losses between Days 30 and 60 and fetal sex ratio were similar (P > 0.05) between in vivo and in vitro-derived embryos. We concluded that in Nelore cows, with an interval of 15 d between OPU procedures, it was possible to produce more embryos and pregnancies compared to conventional MOET.     

Cryopreservation of sperm of an indigenous endangered fish species Nandus nandus (Hamilton, 1822) for ex-situ conservation

This study dealt with the development of cryopreservation protocol for Nandus nandus, which entailed a number of experiments. Sperm was collected by sacrificing males. The collected sperm was suspended in extenders. Activation of sperm motility was evaluated in different osmolalities of NaCl. Motility of sperm decreased as the osmolality of the extender increased and was completely inhibited at almost 319 mOsmol/kg. To evaluate the toxicity of cryoprotectant, sperm was incubated with DMSO, methanol and ethanol at 5%, 10% and 15% concentrations, respectively, for 5–35 min. Five and ten percent of cryoprotectants produced better motility during 5 and 10 min incubation. Sperm incubated with 15% cryoprotectant seemed to be toxic and this concentration was excluded in the subsequent trials. Three extenders, namely, Alsever’s solution, egg-yolk citrate and urea egg-yolk and three cryoprotectants, DMSO, methanol and ethanol were employed to preserve the sperm. Alsever’s solution with 10% DMSO showed best performance producing 90.0 ± 1.8% and 75.0 ± 2.5% equilibration and post-thaw motility followed by that of 82.5 ± 4.2% and 62.5 ± 5.5% with Alsever’s solution plus methanol, respectively. Between two diluents, sperm preserved with Alsever’s solution plus DMSO produced highest fertilization (76.7 ± 3.3%) and hatching (43.8 ± 7.9%) while fresh sperm yielded 83.3 ± 6.7% and 64.0 ± 10.4% fertilization and hatching, respectively. The protocol developed through the study can be applied for long-term conservation of genetic materials of the endangered fish N. nandus and the cryopreserved sperm can be used in artificial breeding for generating new individuals.     

Temperature-induced oviposition in the brachyuran crab Cancer setosus along a latitudinal cline: Aquaria experiments and analysis of field-data

Ovigerous females of Cancer setosus are present year-round throughout most of its wide range along the Peruvian/Chilean Pacific coast (2°S-46°S). However, their number of egg-masses produced per year remains speculative and as such has neither been considered in latitudinal comparisons of reproduction, nor for its fisheries management. In order to reveal the effect of temperature on egg-mass production and egg-development, female C. setosus were held in through-flow aquaria under natural seasonal temperature conditions (16-23 °C) in Antofagasta (23°S), Northern Chile (05/2005-03/2006; 10 months), and at three constant temperatures (12, 16, 19 °C) in Puerto Montt (41°S), Central Southern Chile (09/2006-02/2007; 5 months). Female crabs uniformly produced up to 3 viable egg-masses within 4 1/2 months in Antofagasta and in Puerto Montt (at 19 °C). The second egg-mass was observed 62.5 days (± 7.6; N = 7) after the oviposition of the first clutch and a third egg-mass followed 73.5 days (± 12.5; N = 11) later in Antofagasta (at 16-23 °C). Comparably, a second oviposition took place 64.4 days (± 9.8, N = 5) after the first clutch and a third, 67.0 days (± 2.8, N = 2), thereafter, at 19 °C in Puerto Montt. At the two lower temperatures (16 and 12 °C) in Puerto Montt a second egg-mass was extruded after 82.8 days (± 28.9; N = 4) and 137 days (N = 1), respectively. The duration of egg-development from oviposition until larval hatching decreased from 65 days at 12.5 °C to 22.7 days at the observed upper temperature threshold of 22 °C. Based on the derived relationship between temperature and the duration of egg-development (y = 239.3175e^{− 0.107x}; N = 21, r² = 0.83) and data on monthly percentages of ovigerous females from field studies, the annual number of egg-masses of C. setosus was calculated. This analysis revealed an annual output of about one egg-mass close to the species northern and southern distributional limits in Casma (9°S) and Ancud (43°S), respectively, while at Coquimbo (29°S) about two and in Concepción (36°S) more than 3 egg-masses are produced per year.     

Olive (Olea europaea) leaf extract effective in patients with stage-1 hypertension: Comparison with Captopril

A double-blind, randomized, parallel and active-controlled clinical study was conducted to evaluate the anti-hypertensive effect as well as the tolerability of Olive leaf extract in comparison with Captopril in patients with stage-1 hypertension. Additionally, this study also investigated the hypolipidemic effects of Olive leaf extract in such patients. It consisted of a run-in period of 4 weeks continued subsequently by an 8-week treatment period. Olive (Olea europaea L.) leaf extract (EFLA^®943) was given orally at the dose of 500 mg twice daily in a flat-dose manner throughout the 8 weeks. Captopril was given at the dosage regimen of 12.5 mg twice daily at start. After 2 weeks, if necessary, the dose of Captopril would be titrated to 25 mg twice daily, based on subject's response to treatment. The primary efficacy endpoint was reduction in systolic blood pressure (SBP) from baseline to week-8 of treatment. The secondary efficacy endpoints were SBP as well as diastolic blood pressure (DBP) changes at every time-point evaluation and lipid profile improvement. Evaluation of BP was performed every week for 8 weeks of treatment; while of lipid profile at a 4-week interval. Mean SBP at baseline was 149.3 ± 5.58 mm Hg in Olive group and 148.4 ± 5.56 mm Hg in Captopril group; and mean DBPs were 93.9 ± 4.51 and 93.8 ± 4.88 mm Hg, respectively. After 8 weeks of treatment, both groups experienced a significant reduction of SBP as well as DBP from baseline; while such reductions were not significantly different between groups. Means of SBP reduction from baseline to the end of study were −11.5 ± 8.5 and −13.7 ± 7.6 mm Hg in Olive and Captopril groups, respectively; and those of DBP were −4.8 ± 5.5 and −6.4 ± 5.2 mm Hg, respectively. A significant reduction of triglyceride level was observed in Olive group, but not in Captopril group. In conclusion, Olive (Olea europaea) leaf extract, at the dosage regimen of 500 mg twice daily, was similarly effective in lowering systolic and diastolic blood pressures in subjects with stage-1 hypertension as Captopril, given at its effective dose of 12.5-25 mg twice daily.     

Contaminant removal efficiency depending on primary treatment and operational strategy in horizontal subsurface flow treatment wetlands

This study aimed to evaluate the contaminant removal efficiency of shallow horizontal subsurface flow treatment wetlands (SSF TWs) as a function of (1) primary treatment (hydrolytic upflow sludge blanket (HUSB) reactor vs. conventional settling) and (2) operation strategy (alternation of saturated/unsaturated phases vs. permanently saturated). An experimental plant was constructed, operated and surveyed for the main water quality parameters over a period of 2.5 years. The plant had 3 treatment lines: a control line (settler-wetland permanently saturated), a batch line (settler-wetland operated with saturated/unsaturated phases) and an anaerobic line (HUSB reactor-wetland permanently saturated). In each line wetlands had a surface area of 2.80 m², a water depth of 25 cm and a granular medium D₆₀ = 7.3 mm, and were planted with common reed. During the study period the wetlands were operated at a hydraulic and organic load of 28.5 mm/d and about 4.7 g BOD/m² d, respectively. Effluent average redox potential was lower for the anaerobic line (−45 ± 78 mV) than for the other two lines (3 ± 92.7 and −5 ± 71 mV for control and batch, respectively). Overall, chemical oxygen demand (COD), biochemical oxygen demand (BOD₅) and ammonium mass removal efficiencies were slightly greater for the batch line (88%, 96% and 87%, respectively) than for the control line (83%, 94% and 80%) and the anaerobic line (80%, 87% and 73%). During cold seasons, COD and ammonium removal in the batch line was around 30% and 50% higher than in the control line, respectively. The results of this study indicate that the implementation of a HUSB reactor as primary treatment did not enhance the treatment capacity of the system (in comparison with a conventional settler). The efficiency of treatment wetland systems with horizontal subsurface flow can be improved using a batch operation strategy.     

Accumulation, speciation, and coordination of arsenic in an inbred line and a wild type cultivar of the desert plant species Chilopsis linearis (Desert willow)

This study investigated the absorption of arsenic (As), sulfur (S), and phosphorus (P) in the desert plant Chilopsis linearis (Desert willow). A comparison between an inbred line (red flowered) and wild type (white flowered) plants was performed to look for differential responses to As treatment. One month old seedlings were treated for 7 days with arsenate (As₂O₅, As^V) at 0, 20, and 40 mg As^V L⁻¹. Results from the ICP-OES analysis showed that at 20 mg As^V L⁻¹, red flowered plants had 280 ± 11 and 98 ± 7 mg As kg⁻¹ dry wt in roots and stems, respectively, while white flowered plants had 196 ± 30 and 103 ± 13 mg As kg⁻¹ dry wt for roots and stems. At this treatment level, the concentration of As in leaves was below detection limits for both plants. In red flowered plants treated with 40 mg As^V L⁻¹, As was at 290 ± 77 and 151 ± 60 mg As kg⁻¹ in roots and stems, respectively, and not detected in leaves, whereas white flowered plants had 406 ± 36, 213 ± 12, and 177 ± 40 mg As kg⁻¹ in roots, stems, and leaves. The concentration of S increased in all As treated plants, while the concentration of P decreased in roots and stems of both types of plants and in leaves of red flowered plants. X-ray absorption spectroscopy analyses demonstrated partial reduction of arsenate to arsenite in the form of As-(SX)₃ species in both types of plants.