Population Growth of Six Iranian Brachionus Rotifer Strains in Response to Salinity and Food Type

Intrinsic rates of population increase (r) were evaluated as a measure of population dynamics of four strains of Brachionus plicatilis and two strains of B. urceolaris from Iran in response to different salinities and feeding algae. Each rotifer strain was cultured at four salinities: 5, 20, 25 and 30‰ and fed with two microalgal species: Chlorella vulgaris and Nannochloropsis oculata. Salinity of 5‰ was critical for all the examined strains, at which r was at minimum and was different from the other salinities (P < 0.05). For B. plicatilis strains, the maximum r was observed in those fed on Chlorella at salinities of 10 and 30‰ (64 ± 0.01 day⁻¹). While, in B. urceolaris, maximum r was for Nannochloropsis fed rotifers at salinity of 20‰ (0.69 ± 0.01 day⁻¹). Maximum final population density (FD) was obtained for a strain of B. urceolaris fed on Nannochloropsis at 20‰ (329.3 ± 10.9 ind.mL⁻¹). FD was relatively lower in B. plicatilis strains among all examined salinities. ANOVA showed the significant effect of salinity and rotifer strain, and algae × rotifer strain on both r and FD, and salinity × rotifer × algae on FD (P < 0.05). (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Interactions between red tide microalgae and herbivorous zooplankton: effects of two bloom-forming species on the rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> (O.F. Muller)

Experiments were carried out to investigate interspecific interactions between the rotifer Brachionus plicatilis and two harmful algal bloom (HAB) species using single and mixed culture methods. B. plicatilis populations and the growth of two algae were compared at different algal cell densities. The results demonstrate that B. plicatilis obtained sufficient nutrition from Alexandrium tamarense to support net population increase. When exposed to a density of 8 × 10⁴ cells ml⁻¹ A. tamarense, the number of B. plicatilis increased faster than it did when exposed to other four algal densities (16 × 10⁴, 24 × 10⁴, 32 × 10⁴, and 40 × 10⁴ cells ml⁻¹). Cell densities of A. tamarense decreased due to the grazing of B. plicatilis. In contrast, Heterosigma akashiwo had an adverse effect on the B. plicatilis population and its growth was largely unaffected by rotifer grazing. In this case, the B. plicatilis population decreased and H. akashiwo grew at a rate similar to that of a control without addition of rotifers. Mixed culture experiments showed that A. tamarense could partly counteract the effect of H. akashiwo in limiting the rate of population increase of rotifer. In addition, the effect of different initial cell densities on interspecific competition between A. tamarense and H. akashiwo in mixed culture(s) was also investigated. The results show that A. tamarense competed very successfully when the inoculation proportions of A. tamarense and H. akashiwo were 40:5 and 40:30. Handling editor: D. Hamilton     

Production and purification of 2-phenylethanol by Saccharomyces cerevisiae using tobacco waste extract as a substrate

2-phenylethanol (2-PE), which is extracted naturally from plant or biotechnology processing, is widely used in the food and cosmetics industries. Due to the high cost of 2-PE production, the valorization of waste carbon to produce 2-PE has gained increasing attention. Here, 2-PE was produced by Saccharomyces cerevisiae using tobacco waste extract (TWE) as the substrate. Considering the toxicity of nicotine and its inhibition of 2-PE, the tolerance of S. cerevisiae was first evaluated. The results suggested that the production of 2-PE by S. cerevisiae in TWEs could be carried out at 2·0 mg ml⁻¹ nicotine concentrations and may be inhibited by 1·0 mg ml⁻¹ 2-PE. Thus, the compounds in the TWEs prepared at different temperatures were detected, and the results revealed that the TWEs prepared at 140°C contained 2·18 mg ml⁻¹ of nicotine, had total sugar concentrations of 26·8 mg ml⁻¹ and were suitable for 2-PE production. Due to feedback regulation, the 2-PE production was only 1·11 mg ml⁻¹, and the remaining glucose concentration remained at 13·78 mg ml⁻¹, which indicated insufficient glucose utilization. Then, in situ product recovery was further implemented to remove this inhibition; the glucose utilization (the remaining concentration decreased to 3·64 mg ml⁻¹) increased, and the 2-PE production increased to 1·65 mg ml⁻¹. The 2-PE produced in the fermentation broth was first isolated by elution from the resin with 75% ethanol and then by removing the impurities with 2·5% activated charcoal, and pure 2-PE was identified by gas chromatography mass spectrometry. The results of this study suggest that TWE could be an alternative carbon source for 2-PE production. This could provide an outlet tobacco waste as well as reducing the price of natural 2-PE, although more strategies need to be explored to improve the production yield of 2-PE by using TWE.     

Determination of Eubacterial and Cyanobacterial Size and Number in Lake Baikal Using Epifluorescence

Chroococcoid cyanobacteria, (mean size = 0.79 μm, likely Synchetocystis limnetica Popovsk) and total eubacteria (mean size = 0.33 μm), from Lake Baikal, USSR, were enumerated using epifluorescence microscopy and sized with image analysis. Bacterial densities ranged from 0.44 · 10⁶ cells ml⁻¹ at 250 m to 2.3 · 10⁶ cells ml⁻¹ at the surface. Mean eubacterial abundance was 1.3 · 10⁶ cells ml⁻¹. Cyanobacterial densities were more variable, ranging from 0.42 · 10⁴ cells ml⁻¹ at 250 m to 9.8 · 10⁴ cells ml⁻¹ at the surface, with a mean abundance of 2.7 · 10⁴ cells ml⁻¹. The cyanobacteria, in particular, occurred in clusters resembling “marine snow”. Our results indicate that Lake Baikal picoplankton size and density are similar to other large lakes but may have a more diverse community structure than in other large oligotrophic lakes.     

Lifetable demography and population growth of the rotifer Brachionus angularis in Kenya: influence of temperature and food density

Lifetable demography and reproductive traits of a Kenyan strain of the rotifer Brachionus angularis were investigated using individual and small batch culture approaches. The rotifer was identified morphologically before conducting studies at 20, 25 and 30 °C, using Chlorella vulgaris at 2.5 × 10⁵ to 2.5 × 10⁷ cells ml^–1. The rotifers were highly fecund, producing 2.11 ± 0.07 offspring female^–1 day^–1 and reproductive, producing 8.43 ± 0.24 offspring female^–1 at 25 °C with 2.5 × 10⁶ algal cells ml^–1. The highest intrinsic rate of natural increase (0.74 ± 0.02 d^–1), specific population growth rate (0.49 ± 0.01), longest life expectancy at hatching (12.41 ± 0.28 d) and shortest generation time (2.87 ± 0.03 d) also occurred at 25 °C with 2.5 × 10⁶ algal cells ml^–1. The duration of hatching to first spawning was shortest (2.86 ± 0.21 h) at 30 °C with 2.5 × 10⁷ algal cells ml^–1 and longest (8.83 ± 0.39 h) at 20 °C with 2.5 × 10⁵ algal cells ml^–1. The highest population density (255.7 ± 12.6 ind. ml^–1) was realised at 25 °C with 2.5 × 10⁶ cells ml^–1 on Day 8, whereas the lowest population density (122.0 ± 3.6 ind. ml^–1) was realised at 20 °C with 2.5 × 10⁵ cells ml^–1 on Day 8. The lorica length and width of the Kenyan strain of B. angularis are 85.6 ± 3.1 µm and 75.4 ± 3.6 µm, respectively. The rotifer optimally reproduces at 25 °C when fed with 2.5 × 10⁶ algal cells ml^–1.     

Temporal variability in egg production rates of Acartia tonsa Dana in Long Island Sound

The relationship between week-by-week variations in the in situ egg production rates of Acartia tonsa Dana and changes in chlorophyll concentration in several size fractions was investigated by incubating adult females in natural sea water for 24-h periods. Our results indicate that the egg production of A. tonsa in Long Island Sound was better related to the 10 μm chlorophyll size fraction than to the total chlorophyll concentration. The < 10 μm size fraction comprised the greatest percentage of the chlorophyll during July and August when the water column was stratified. Egg production rates were lowest (8.7 eggs · female⁻¹ · day⁻¹) in early August when less than 0.5 μg chlorophyll 1 ⁻¹ was observed in the 10 μm chlorophyll a size fraction. Following destratification in late August, the “fall” diatom bloom occurred and egg production rates increased to the maximum observed rate of 56.6 eggs · female⁻¹ · day⁻¹. At this time, the concentration of the 10μm chlorophyll size fraction was 5.5 μg 1⁻¹. Maximum egg production rates were observed at chlorophyll concentrations as low as 0.8 μg 1⁻¹ in the 10 μm size fraction.     

Influence of ethanol and ethylene on the seed germination of three nymphaeid water plants

1. We offered Scenedesmus obliquus in five densities, from 0.5 to 8 ± 10⁶ cells ml^?1, to the rotifer Anuraeopsis fissa. Growth rates (r) during the exponential phase (first 7 days) were significantly and positively related to food density. The r values (mean ± SD) varied between 0.454 ± 0.067 and 0,856 ± 0.090, from the lowest to the highest food concentration, respectively. Population growth went through a phase of exponential increase which lasted from 7 to 10 days before a plateau and, in some cases, a decrease occurred. 2. There was a linear relation between food density and rotifer plateau density. At the highest food density, a peak abundance (mean ± SD) of 2312 ± 226 individuals ml^?1 was reached, while at the lowest food density there was no identifiable single peak but a plateau, at a density of 361 ± 62 ind. ml^?1. 3. The egg ratio decreased with increasing population density. The ratio of loose eggs to eggs attached to females indicated that more eggs became detached at higher food densities. 4. As in many other rotifer species studied so far, population density of A. fissa was less stable at higher algal food concentrations. Numerically, A. fissa could be grown at twice the density achieved in Brachionus.     

The Cost of Reproduction in the Rotifer Brachionus calyciflorus Pallas

A study of lifespan, fecundity, and reproductive schedules was carried out with the rotifer Brachionus calyciflorus under different food concentrations. At high food densities (6.1 to 12.4 μg · ml⁻¹, dry mass) there was an increase in offspring number but the reproductive investment remained constantly. This was possible because Brachionus calyciflorus invested a smaller amount of energy one egg volume being able to increase offspring number. Although egg volume was reduced, this was compensated by the greater amount of energy available to the neonates. The length of the prereproductive period remained constant throughout this range of food concentrations. Within this range a reduction in lifespan was observed. This, however, was not due to a total higher reproductive investment, but to an increased offspring rate production resulting in a shortening of the reproductive period. The length of the post-reproductive period was not different at all food concentrations.     

The essential omega-3 fatty acid EPA affects expression of genes involved in the metabolism of omega-6-derived eicosanoids in Daphnia magna

We quantified the predation of Acanthocyclops americanus from the shallow Mediterranean lake Albufera, using gut contents from field collections and laboratory feeding tests. For functional response studies, we used Brachionus plicatilis (at 6 concentrations, 400–4000 ind. 40 ml⁻¹) and Diaphanosoma mongolianum (at 2–20 ind. 40 ml⁻¹). Copepod feeding rates were also estimated using different proportions of rotifer prey and lake seston (0–67.5% of seston + 40 individuals of B. plicatilis). Prey selection studies were conducted using five zooplankton species: Brachionus angularis, Brachionus plicatilis, Keratella tropica, Daphnia magna and Diaphanosoma mongolianum. Gut contents of field-collected adult Acanthocyclops contained filamentous algae and cyanobacteria and 16 zooplankton species (Keratella cochlearis, unspined and spined forms, K. tropica, Brachionus plicatilis, Brachionus calyciflorus, Brachionus angularis, Brachionus variabilis, Asplanchna girodi, Polyarthra vulgaris, Synchaeta pectinata, Lepadella rhomboides, unidentified bdelloids, Alona rectangula, Chydorus sphaericus, Bosmina longirostris, D. magna, Ceriodaphnia dubia and copepod nauplii). When fed B. plicatilis or D. mongolianum, female A. americanus had higher prey consumption rates than males. Increased proportion of lake seston caused reduced consumption of brachionid prey. Our data suggest that A. americanus is omnivorous in nature.

     

In vitro assays on the susceptibility of four species of nematophagous fungi to anthelmintics and chemical fungicides/antifungal drug

Using nematophagous fungi for the biological control of animal parasitic nematodes will become one of the most promising strategies in the search for alternative chemical drugs. The purpose of this study was to check the in vitro activity of four anthelmintics, four chemical fungicides and two antifungal drugs on the spore germination of nematophagous fungi: Duddingtonia flagrans (SF170), Arthrobotrys oligospora (447), Arthrobotrys superba (435) and Arthrobotrys sp. (PS011). A modified 24-well cell culture plate assay was conducted to evaluate the susceptibility of nematophagous fungi against drugs tested by calculating the effective middle concentrations (EC₅₀) of each tested drug to inhibit the germination of fungal spores. EC₅₀ ranged between 0·7 and 47·2 μg ml⁻¹ for fenbendazole, thiabendazole and ivermectin, except levamisole (546·5–4057·8 μg ml⁻¹). EC₅₀ of tested fungicides was 0·6–2·3 μg ml⁻¹ for carbendazim, 55·9–247·4 μg ml⁻¹ for metalaxyl, 24·4–45·2 μg ml⁻¹ for difenoconazole, and 555·9–1438·3 μg ml⁻¹ for pentachloronitrobenzene (PCNB). EC₅₀ of two antifungal drugs was 0·03–3·4 μg ml⁻¹ for amphotericin B and 0·3–10·9 μg ml⁻¹ for ketoconazole. The results showed that 10 tested drugs, except for levamisole and PCNB, had in vitro inhibitory effects on nematophagous fungi. The chlamydospores of D. flagrans had the highest sensitivity to nine tested drugs, except for ketoconazole.     

Production and nutritional quality of the rotifer Brachionus plicatilis fed marine Chlorella sp. at different cell densities

The effect of different cell densities of marine Chlorella sp. on the growth rate, doubling time and production of the rotifer Brachionus plicatilis was investigated. A significant increase in rotifer production was achieved at a density of 50 × 10⁶ Chlorella cells ml^–1. The nutritional quality of rotifers grown at different concentrations of Chlorella is discussed.     

Cultivation of Lemna gibba under desert conditions. I: Twelve months of continuous cultivation in open ponds

Duckweed, Lemna gibba, was grown in 12 m² shallow ponds in the Negev desert, during 12 months of continuous cultivation, beginning April 1984. Average monthly growth rates varied with the season of the year. The lowest daily yield, 2·6±0·4 g dry weight m⁻² day⁻¹, was obtained during January. Highest daily yields, 7·9±2·6 g dry weight m⁻² day⁻¹ and 7·0±1·2 g dry weight m⁻² day⁻¹, were obtained during September and May. A 35% decline of the yield was seen during midsummer (July), 4·8±1·2 g dry weight m⁻² day⁻¹. The average rate for the year was 5·15±1·7 g dry weight m⁻² day⁻¹. The protein content of the plants ranged from 30 to 38% per unit dry weight.Growth performance is discussed in relation to the prevailing climatic conditions.     

Effects of thymol and diphenyliodonium chloride against Campylobacter spp. during pure and mixed culture in vitro

Aims: To determine if the purported deaminase inhibitors diphenyliodonium chloride (DIC) and thymol reduce the growth and survivability of Campylobacter. Methods and Results: Growth rates of Campylobacter jejuni and Camp. coli were reduced compared to unsupplemented controls during culture in Muellar–Hinton broth supplemented with 0·25 μmol DIC or thymol ml^?1 but not with 0·01 μmol monensin ml^?1 or 1% ethanol. Recovery of Camp. jejuni and Camp. coli was reduced >5 log₁₀ CFU from controls after 24 h pure culture in Bolton broth supplemented with 0·25 or 1·0 μmol DIC ml^?1 or with 1·0 μmol thymol ml^?1. Similarly, each test Campylobacter strain was reduced >3 log₁₀ CFU from controls after 24 h mixed culture with porcine faecal microbes in Bolton broth supplemented with 0·25 or 1·0 μmol DIC ml^?1 or with 1·0 μmol thymol ml^?1. Treatments with 0·25 μmol thymol ml^?1, 0·01 μmol monensin ml^?1 or 1% ethanol were less effective. Ammonia production during culture or incubation of cell lysates was reduced by 0·25 or 1·0 μmol DIC ml^?1 but only intermittently reduced, if at all, by the other treatments. Conclusions: Diphenyliodonium chloride and thymol reduced growth, survivability and ammonia production of Camp. jejuni and Camp. coli. Significance and Impact of the Study: Results suggest a potential physiological characteristic that may be exploited to develop interventions.     

Synergistic antibacterial activity of the essential oil of Cuminum cyminum L. seed and nisin in a food model

Aims: To investigate effects of various concentrations of the essential oil of Cuminum cyminum L. seed alone and in combination with nisin on survival of vegetative forms of Bacillus cereus and Bacillus subtilis in a food model (commercial barley soup) and their ultrastructure. Methods and Results: Gas chromatography–mass spectrometry analysis indicated that cumin aldehyde (29·02%) and α‐terpinen‐7‐al (20·70%) constituted the highest amount of the essential oil. The lowest concentration of the essential oil significantly affected the growth of the bacteria at 8°C but not at 25°C. Synergistic effect of the essential oil in combination with the lowest concentration of nisin was observed on the bacteria at 8°C. Evaluation of the sensory properties showed that concentration of 0·15 μl ml^?1 of the essential oil was the most acceptable. Conclusions: The essential oil of C. cyminum L. seed showed the most bactericidal effects on B. cereus at 8°C. Ultrastructural studies of vegetative cells confirmed the synergistic destructive effects of the essential oil and nisin on membrane and cell wall of the bacteria. Significance and Impact of the Study: Utilization of essential oil of C. cyminum L. seed in combination with nisin can inhibit growth of food‐borne pathogens in food.     

Life cycles and growth rates of Baetis spp. (Ephemeroptera: Baetidae) in the laboratory and in two stony streams in Austria

SUMMARY. Larvae of Baetis alpinus, B. lutheri and B. rhodani were reared in a stream channel (water temperature range 4.2–11.4°C) in the laboratory. The larval growth was exponential and the mean specific growth rate varied from 1.93 to 2.24% day^?1 for B. alpinus, 1.49 to 3.41% day^?1 for B. lutheri and 0.79 to 3.11% day^?1 for B. rhodani. These variations in growth rate were related to variations in mean temperature and this was the major factor affecting growth in the laboratory. Non-quantitative samples of the benthos in the Seebach and Unterseebach, two stony streams near Lunz, Austria, were taken at approximately monthly intervals from November 1965 to May 1968. In each year, there were two winter and three summer cohorts for B. alpinus from Seebach and two or three winter and one to three summer cohorts for B. lutheri and B. rhodani from Unterseebach. Over the study period of 30 months, eleven cohorts were recorded for B. alpinus and B. lutheri, and ten cohorts for B. rhodani. The life cycle of a cohort varied from 3 to 8 months in B. alpinus, from 2.5 to 9 months in B. lutheri and from 2.5 to 8 months in B. rhodani. Mean specific growth rate in length varied from 0.82 to 2.97% day^?1 for B. alpinus, 0.96 to 3.33% day^?1 for B. lutheri and 0.65 to 3.01% day^?1 for B. rhodani. The percentage of the variability in growth rate accounted for by variations in mean temperature was 63% for B. alpinus, 91% for B. lutheri and 82% for B. rhodani. Therefore mean temperature was clearly the major factor affecting the growth rates in the field. An agreement was found between the growth rates of B. alpinus in the field and the laboratory. The growth rates of B. lutheri and B. rhodani were slower in the field than in the laboratory at higher temperatures. The possible reasons for this latter discrepancy are discussed, and the growth rates of the three Baetis spp. are compared with those of other species of Ephemeroptera.     

Inactivation of Cronobacter spp. (Enterobacter sakazakii) in infant formula using lactic acid,copper sulfate and monolaurin

Aims: To investigate the effect of lactic acid (LA), copper (II), and monolaurin as natural antimicrobials against Cronobacter in infant formula. Methods and Results: The effect of LA (0·1, 0·2 and 0·3% v/v), copper (II) (10, 50 and 100 μg ml^?1) and monolaurin (1000, 2000, and 3000 μg ml^?1) suspended into tween‐80? or dissolved in ethanol against Cronobacter in infant formula was investigated. Reconstituted infant formula and powdered infant formula were inoculated with five strains of Cronobacter spp. at the levels of c. 1 × 10⁶ CFU ml^?1 and 1 × 10³ CFU g^?1, respectively. LA at 0·2% v/v had a bacteriostatic effect on Cronobacter growth, whereas 0·3% v/v LA resulted in c. 3 log₁₀ reduction. Copper (II) at the levels of 50 μg ml^?1 and 100 μg ml^?1 elicited c. 1 and 2 log₁₀ reductions, respectively. The combination of 0·2% LA and 50 μg ml^?1 copper (II) resulted in a complete elimination of the organism. Monolaurin exhibited a slight inhibitory activity against Cronobacter (c. 1·5 log₁₀ difference) compared to the control when ethanol was used to deliver monolaurin. Conclusions: A complete elimination of Cronobacter was obtained when a combination of sublethal concentrations of LA (0·2%) and copper (II) (50 μg ml^?1) was used. Significance and Impact of the Study: The use of the synergistic interactive combination of LA and copper (II) could be beneficial to control Cronobacter in the infant formula industry.     

The effects of CO2, temperature and their interaction on the growth and yield of carrot (Daucus carota L.)

Stands of carrot (Daucus carota L.) were grown in the field within polyethylene-covered tunnels at a range of soil temperatures (from a mean of 7·5°C to 10·9°C) at either 348 (SE = 4·7) or 551 (SE = 7·7) μmol mol⁻¹ CO₂. The effect of increased atmospheric CO₂ concentration on root yield was greater than that on total biomass. At the last harvest (137d from sowing), total biomass was 16% (95% CI = 6%, 27%) greater at 551 than at 348 μmol mol⁻¹ CO₂, and 37% (95% CI = 30%, 44%) greater as a result of a 1°C rise in soil temperature. Enrichment with CO₂ or a 1°C rise in soil temperature increased root yield by 31% (95% CI = 19%, 45%) and 34% (95% CI = 27%, 42%), respectively, at this harvest. No effect on total biomass or root yield of an interaction between temperature and atmospheric CO₂ concentration at 137 DAS was detected. When compared at a given leaf number (seven leaves), CO₂ enrichment increased total biomass by 25% and root yields by 80%, but no effect of differences in temperature on plant weights was found. Thus, increases in total biomass and root yield observed in the warmer crops were a result of the effects of temperature on the timing of crop growth and development. Partitioning to the storage roots during early root expansion was greater at 551 than at 348 μmol mol⁻¹ CO₂. The root to total weight ratio was unaffected by differences in temperature at 551 μmol mol⁻¹CO₂, but was reduced by cooler temperatures at 348 μmol mol⁻¹ CO₂. At a given thermal time from sowing, CO₂ enrichment increased the leaf area per plant, particularly during early root growth, primarily as a result of an increase in the rate of leaf area expansion, and not an increase in leaf number.     

Feeding and growth of juvenile sea bass: the effect of ration and temperature on growth rate and efficiency

The effect of ration on the growth of pairs of juvenile sea bass Dicentrarchus labrax fed squid mantle was recorded at four temperatures: 6, 10, 14 and 18) C, covering the range typical of Welsh coastal waters. Initial weight of the fish ranged from 2.8 to 15.9 g. A predictive model for the maximum meal size (M_max) at temperatures between 10 and 18) C, accounted for 95% of the variance in lnM_max. Even when offered excess food, bass at 6) C had a low rate of food consumption [0.19% body weight (BW) day^?1] and lost weight (G=?0.04% day^?1). Predictive regression models for specific growth rate (G) accounted for 86% of the variance at reduced rations and 70% at maximum meals. The relationship between G (calculated for total biomass per tank) and ration was a decelerating curve. G at maximum meals increased with temperature, at lower rations G decreased with temperature. For a pair of bass with a combined weight of 15 g, predicted maintenance ration ranged between 0.7 and 2.3% BW day^?1 and increased with temperature. Maximum meal size was more sensitive to temperature than maintenance ration. At 18) C optimum ration was 7.4% BW day^?1. At lower temperatures, the optimum ration was the maximum meal. The maximum gross growth efficiency was 17.4% at 18) C. Mean absorption efficiency was 94.8%. Ration level had no significant effect on absorption efficiency, which was lowest at 6) C. Condition indices (Fulton condition factor, wet and dry liver—somatic indices and body depth index) increased with meal size at all temperatures except 6) C. An increase in temperature between 10 and 18) C generally resulted in a decrease in condition indices at a given ration. When comparisons were made at a given standard length, gut and carcass weight increased with ration. Visceral fat and gut weight decreased with increased temperature.