Ecophysiological studies on citrate-synthase: (I) enzyme regulation of selected crustaceans with regard to temperature adaptation

The characteristics and properties chromatographically purified citrate synthase from the euphausiids Euphausia superba (Antarctica) and Meganyctiphanes norvegica (Scandinavian Kattegat and Mediterranean Sea) and from the isopods Serolis polita (Antarctica) and Idotea baltica (Baltic Sea) were used to elucidate biochemical mechanisms of temperature adaptation. Additionally, maintenance experiments were carried out on the euphausiids to determine mechanisms of short term acclimation. Temperature optima (between 37 and 45°C) were unrelated to genotypic cold adaptation, but the activation energy of the Antarctic krill E. superba (10.9 kJ · mol^-1) was only a quarter of that in other species (41.8–45.1 kJ · mol^-1). The minima of apparent Michaelis constants (total range: 4–20 μmol · 1^-1 oxaloacetate; 7–45 μmol · 1^-1 acetyl-coenzyme A) showed no relation to natural conditions, and no distinct pH optimum occurred at ambient temperatures. In contrast, apparent Michaelis constants and specific enzyme activities were related to maintenance temperatures in M. norvegica, but not in E. superba. The differences between M. norvegica and E. superba can be interpreted as adaptations to the changes in ambient temperature with regard to the respective steno- and eurythermic tolerances of these crustaceans.     

Partial characterization of chitin degrading enzymes from two euphausiids,Euphausia superba and Meganyctiphanes norvegica

Summary Chitinase and N-acetyl--D-glucosaminidase have been demonstrated in Meganyctiphanes norvegica and in Euphausia superba and partly characterized. The enzymes from both species have broad pH-optima (maxima around pH 5.0) and temperature optima between 40 and 50°C. The enzymes are relatively stable; even at about 45°C half of the enzyme activity is retained after 30 min incubation. The presence of fluoride does not affeet enzymatic activity. Chitinase activity appears in three different molecular masses, N-acetyl--D-glucosaminidases in two different forms. pH and temperature optima, thermal stability and kinetic properties of the two enzymes are strikingly similar in the polar E. superba versus the boreal euphausiid M. norvegica. Enzyme activity in the lower temperature range is still high, whereas activation energies are low in both euphausiids. This suggests a functional adaptation to a low temperature range in seawater.     

Experiments on the physiology of southern and northern krill,Euphausia superba and Meganyctiphanes norvegica,with emphasis on moult and growth – a review

Physiological data are needed for life history studies on krill, and as parameters for input into energy budgets and models. In conjunction with moult and growth data, these may also prove useful for assessing the fishable biomass of krill. Here, the development of physiological concepts in experimental krill research is briefly evaluated, with emphasis on the gaps to be filled. Krill growth is very flexible, as well as strongly temperature and nutrition dependent. The polar Antarctic krill Euphausia superba grows as fast as the boreal species Meganyctiphanes norvegica, at least during the first 2.5 years, and the species are comparable in terms of physiological plasticity. Accordingly, as krill appear to adjust quickly to specific laboratory conditions, short-term experiments are essential if field conditions are to be reflected as closely as possible. Furthermore, direct comparisons between laboratory experiments and swarming studies in the field are advantageous. For these, M. norvegica is particularly well-suited, as swarms can be followed over longer times and more easily than in E. superba. For example, processes of moult and reproduction were found to be highly coordinated in swarms and populations of Northern krill. For this species a conceptual model of reproduction was developed based on a combination of short-term laboratory observations coupled with field data on moult and ovary stages. In further physiological experiments krill should be studied as groups when swarming. Using proxies, that is applying physiological and/or biochemical methods side by side, is a promising way to enhance the reliability of life history data.     

A comparison of Antarctic euphausiids sampled by net and from geothermally heated waters: insights into sampling bias

The Antarctic euphausiids Euphausia superba and E. crystallorophias were sampled using an 8 m² rectangular midwater trawl inside the caldera of Deception Island. Specimens of the same species were collected on the same day from the strandline and sublittoral fringe within the caldera at a bay where these euphausiids had been incapacitated by high temperature geothermally heated waters. Differences between length frequency distributions of samples obtained by both methods were investigated once net-caught specimens had been subject to the simulated effects of geothermally heated waters. Differences between maturity stage/sex ratios for E. superba samples were also assessed. No significant differences between sizes of net-caught or beach-sampled E. crystallorophias were detected, but significant differences were found between both the sizes and sex/maturity stages for E. superba samples. Possible explanations for these differences are discussed. Accepted: 15 March 1999     

Moult cycle and seasonal activities of chitinolytic enzymes in the integument and digestive tract of the Antarctic krill,Euphausia superba

Summary Chitinolytic activity was quantified in euphausiid integuments in relation to moulting. In Euphausia superba, shortly before moult the activity increased in chitinase and N-acetyl--D-glucosaminidase to pronounced maxima indicating the onset of massive resorption of cuticular material. Enzymatic activity of E. superba corresponded to values in Meganyctiphanes norvegica, a boreal euphausiid which was investigated for comparison, as well as in insecta. Antarctic krill from winter catches displayed activities comparable to summer material suggesting physiological preparation for moulting. Accordingly, moulting did not cease during winter. Both enzymes were also active in the digestive tract in summer as well as in winter krill: chitin containing food of phyto-and zooplankton origin is digestable. Seasonally stable activities did not point to changes in nutritional preference. In contrast to other crustacea, digestive enzyme activity was not reduced around moult, suggesting a high capacity to continuously utilize food sources including chitin. This property can be linked directly to the high energy need caused by the necessity of constant active swimming in both krill species.Supported by German Research Counsil (DFG), grant-nos. Ad 24/9 and Bu 548/1Dedicated to Professor Dr. G. Hempel on the occasion of his 60th birthday     

Biological monitoring of exposure to sevoflurane in operating room personnel by the measurement of hexafluoroisopropanol and fluoride in urine

The objectives of this study were to evaluate the value of urinary hexafluoroisopropanol (HFIP) and fluoride (F^-) measurement for the biological monitoring of operating room personnel exposed to sevoflurane. Fifty members of operating room staffs from eight different hospitals took part in the study. To assess external exposure to sevoflurane, air samples were collected during the whole anaesthesia period by a passive sampling device (3M 3500 organic vapour monitor) attached close to the breathing zone of each subject. Urine was collected before (BA) and at the end of anaesthesia (EA) for the determination of HFIP, fluoride and creatinine. Average airborne concentration of sevoflurane was 19.0 ppm (range: ND-139.9 ppm) with a mean duration of anaesthesia of 221 min (range: 60-435 min). There was a better correlation between external and internal exposure as estimated by EA urinary HFIP (r = 0.78; p <0.0001) compared with EA urinary F^- (r = 0.41; p = 0.0031). Furthermore determination of urinary HFIP seemed more suited than that of F^- for the assessment of sevoflurane exposure because of lower background in BA samples (86 % of BA HFIP values were under the limit of detection). Based on these results, values of 9.6 and 4.3 mg HFIP g^-1 creatinine correspond to airborne concentrations of 50 and 20 ppm of sevoflurane, respectively. Among the confounding parameters investigated (body mass index (BMI), sex, cytochrome P450 polymorphism) only BMI showed statistically significant influence on sevoflurane metabolism at these low levels of exposure. The measurement of HFIP in urine at the end of the surgical procedure constitutes a good index to assess occupational exposure to sevoflurane. Further studies will be necessary to propose an health-based limit value which remains to be determined from the relationship between effects and internal dose as can be assessed by HFIP measurement in urine.     

Calcium translocations during the moulting cycle of the semiterrestrial isopod <Emphasis Type="Italic">Ligia hawaiiensis</Emphasis> (Oniscidea,Crustacea)

Terrestrial isopods moult first the posterior and then the anterior half of the body. During the moulting cycle they retain a significant fraction of cuticular calcium partly by storing it in sternal CaCO₃ deposits. We analysed the calcium content in whole Ligia hawaiiensis and the calcium distribution between the posterior, the anterior ventral, and the anterior dorsal cuticle during four stages of the moulting cycle. The results indicate that: (1) overall, about 80% of the calcium is retained and 20% is lost with the exuviae, (2) in premoult 68% of the calcium in the posterior cuticle is resorbed (23% moved to the anterior ventral cuticle, 17% to the anterior dorsal cuticle, and the remaining 28% to internal tissues), (3) after the posterior moult 83% of the calcium in the anterior cuticle is shifted to the posterior cuticle and possibly to internal storage sites, (4) following the anterior moult up to 54% of the calcium in the posterior cuticle is resorbed and used to mineralise the new anterior cuticle. ⁴⁵Ca-uptake experiments suggest that up to 80% of calcium lost with the anterior exuviae may be regained after its ingestion. Whole body calcium of Ligia hawaiiensis is only 0.7 times that of the fully terrestrial isopods. These terrestrial species can retain only 48% of whole body calcium, suggesting that the amount of calcium that can be retained by shifting it between the anterior and posterior integument is limited. We propose that fully terrestrial Oniscidea rely to a larger degree on other calcium sources like internal stores and uptake from the ingested exuviae.     

Hydrocarbon accumulation and lipid biosynthesis during larval development in the cabbage looper,Trichoplusia ni

Larvae of the cabbage looper, Trichoplusia ni, were analyzed for the accumulation and biosynthesis of cuticular and internal hydrocarbon at closely spaced and accurately timed intervals during the fourth and fifth stadia. Large differences in the incorporation of [1-¹⁴C]acetate into hydrocarbon were observed at different times during larval development. Much higher incorporation was observed during feeding stages as compared to wandering stages, while lowest rates of biosynthesis occurred just prior to ecdysis. Fourth stadia wanderers accumulated increased amounts of internal hydrocarbon, which is apparently used to cover the newly forming cuticle. During the fourth to fifth stadium moult insects lost all cuticular hydrocarbon that was present on the old cuticle (about 8 μg/insect) and had about 8 μg/insect on the surface of the newly exposed cuticle. During the fourth stadium incorporation of [1-¹⁴C]acetate into total lipid declined between feeding and wandering stages from 24% of injected radiolabel to 7%. Similar decreases in lipid biosynthesis were observed between feeders and wanderers in fifth stadium larvae with the greatest decrease found in the triacylglycerol fraction. These results document dramatic changes in the accumulation and biosynthesis of hydrocarbon and other lipids during larval development.     

Enzyme kinetics in cold water: characteristics of N-acetyl-β-d-glucosaminidase activity in the Antarctic krill,Euphausia superba,compared with other crustacean species

N-acetyl--d-glucosaminidase, a chitin-degrading enzyme, is highly active in the integument and digestive tract of euphausiids. The enzyme was used as a model to compare temperature-dependent enzymatic parameters of Antarctic krill, Euphausia superba, with those of a euphausiid species (Meganyctiphanes norvegica) found in both the Scandinavian Kattegat and the Mediterranean. Other species examined were an Antarctic isopod, Serolis polita, and a tropical crab, Ocypode ryderi. Enzyme isoforms of NAGase were isolated chromatographically. Temperature optimum (between 30 and 53 °C) and activation-energy (47–59 kJ·mol^-1) of isoenzymes were generally unrelated to genotypic cold adaptation. Although pH profiles were temperature-dependent, there was no apparent temperature-related control of activities by pH in the experienced physiological range. In contrast, apparent Michaelis constants showed minima at ambient water temperatures (total range: 0.1–0.6 mol·l^-1). Potentially, enzyme variants play a role in acclimatisation regulated by Michaelis constants. Apparently, the rate-limiting effects of polar temperatures are partly compensated in the Antarctic crustaceans by construction of enzymes with substrate affinities similar to those of species from warmer climates. The significance of apparent Michaelis constants in evaluating mechanisms of metabolic cold compensation is discussed. Necessary additional experimental approaches are highlighted.Abbreviations CPB citrate-phosphate buffer - FPLC fast protein liquid chromatography - K _m ^app apparent Michaelis constant - LC liquid chromatography - MW molecular weight - NAGase N-acetyl--d-glucosaminidase     

Ecophysiological studies on citrate synthase: (II) enzyme regulation of selected crustaceans with regard to life-style and the climatic zone

Citrate synthase is a regulatory enzyme of the energy metabolism pathway controlling the citric acid cycle. It was studied in order to determine modes of enzyme regulation with regard to the life-style of the investigated species. Citrate synthase from crustaceans with different life-styles were compared: the pelagic euphausiids Euphausia superba from the Antarctic and Meganyctiphanes norvegica from the Scandinavian Kattegat and the Mediterranean were compared to the benthic isopods Serolis polita from the Antarctic and Idotea baltica from the Baltic. Citrate synthase was partly purified chromatographically and the influence of adenosine 5′-triphosphate on enzyme activity was examined. Mechanisms of inhibition and inhibitor constants were determined. Two different mechanisms of enzyme regulation by ATP were found. Citrate synthase from isopods was only competitively inhibited, while citrate synthase from euphausiids showed not only competitive inhibition but also activation by low concentrations of ATP. This activation is equivalent to the reversed methanism of uncompetitive inhibition. The ecophysiological relevances of the coupling of these mechanisms are discussed. The degree of competitive inhibition was different in the two groups of investigated crustaceans. Inhibitor constants were similar within the euphausiids but not in isopods, which showed higher or lower inhibition depending on the climatic zone: the colder the ambient temperature the lower the ATP inhibition. A possible mechanism of temperature adaptation through effects of varying inhibition constants is concluded.     

The simultaneous effect of soil-borne NaF and air pollutant SO2 on CO2-uptake and pollutant accumulation

Summary Spruce cuttings were potted in quartz sand. One half was watered with tap water, the other with deionized water containing 100 ppm F^- as NaF, during winter till bud break. After flushing the plants were exposed continuously for five weeks (June/July) either to normal air or to air with 0.025 and 0.075 ppm SO₂, respectively. CO₂-uptake was measured in the laboratory (40,000 lux) with an IRGA.Even in the absence of visible symptoms of injury to last year's needles, root-applied fluoride did suppress CO₂-uptake significantly. Also the fumigation with 0.075 ppm SO₂ caused a depression of CO₂-uptake, whereas 0.025 ppm SO₂ remained without significant reaction within five weeks. Fluorides infiltrating through roots and airborne SO₂ acting together may depress CO₂-uptake synergistically.Soil applied fluoride accumulated particularly in roots and twigs, whereas very little was found in the stem. The F^--accumulation even increased with increasing SO₂-concentrations but was without effect on S-accumulation.     

Proteomic analysis of urine in rats chronically exposed to fluoride

Urine is an ideal source of materials to search for potential disease‐related biomarkers as it is produced by the affected tissues and can be easily obtained by noninvasive methods. 2‐DE‐based proteomic approach was used to better understand the molecular mechanisms of injury induced by fluoride (F^?) and define potential biomarkers of dental fluorosis. Three groups of weanling male Wistar rats were treated with drinking water containing 0 (control), 5, or 50 ppm F^? for 60 days (n = 15/group). During the experimental period, the animals were kept individually in metabolic cages, to analyze the water and food consumption, as well as fecal and urinary F^? excretion. Urinary proteome profiles were examined using 2‐DE and Colloidal Coomassie Brilliant Blue staining. A dose‐response regarding F^? intake and excretion was detected. Quantitative intensity analysis revealed 8, 11, and 8 significantly altered proteins between control vs. 5 ppm F^?, control vs. 50 ppm F^? and 5 ppm F^? vs. 50 ppm F^? groups, respectively. Two proteins regulated by androgens (androgen‐regulated 20‐KDa protein and α‐2μ‐globulin) and one related to detoxification (aflatoxin‐B1‐aldehyde‐reductase) were identified by MALDI‐TOF‐TOF MS/MS. Thus, proteomic analysis can help to better understand the mechanisms underlying F^? toxicity, even in low doses. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 25:8–14 2011; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20353     

Horizontal and vertical distribution and demography of euphausiids in the Ross Sea and its adjacent waters in 2004/2005

The horizontal and vertical distribution and population structure of euphausiids in the Ross Sea and its adjacent waters were investigated during the summers of 2004/2005 using stratified towed samples. Nine species of euphausiids occurred in the survey area. Among them, Euphausia triacantha was dominant in biomass north of the southern boundary of the Antarctic circumpolar current (SB). Thysanoessa spp. was widely distributed north of the continental slope, while E. superba was distributed from the SB to the slope, where it showed the highest biomass. Juvenile E. superba was distributed offshore near the SB and remained at the surface, but gravid females were dominant in the slope and mainly occurred in the middle layers (400–600 m). Adult and juvenile E. crystallorophias were found at 200–300 m in the colder water of the continental shelf. In general, the peak biomass of euphausiids was found in the mid layers of the Ross Sea area. The life span and the number of spawns for major species are also discussed.     

Fluoride turnover in adélie penguins (Pygoscelis adeliae) and other brid species

Summary Fluoride concentrations found in soft tissues of free ranging birds are below 10 mg/kg d.w. In ducks (Anas platyrhynchos) 99.5% of all fluoride is bound in the skeleton. Krill-eating adélie penguins (Pygoscelis adeliae) reached equilibrium concentrations in the order of 10000 mg F/kg d.w. in femur. Fluoride uptake with the diet is buffered by the skeleton where it has a half-life of 3–4 weeks. Fluoride from fresh krill does not reach the organs of adélie penguins to the same extent as NaF. Most fluoride remains bound to the krill cuticle and is not absorbed by the penguin gut during digestion. The availability of krill fluoride was in the range of 21%–30% for muscle and 48%–68% for femur of adélie penguins as compared to NaF (100%). The role of the salt glands in fluoride excretion is neglegible. The uptake of approx. 240 mg F daily with the krill diet is balanced by excretion via the cloaca.This work was supported by a grant from the DFG Nr. MZ-AD 24/9 and by a stipend from the Studienstiftung des deutschen Volkes     

Density- and sound speed contrasts in sub-Arctic zooplankton

Summary The sound speed was determined for Meganyctiphanes norvegica, for a mixture of Thysanoessa raschii and Thysanoessa inermis and for a mixture of Calanus finmarchicus and Calanus hyperboreus. The sound speed contrasts ranged from 1.014 to 1.044. Seasonal variations in specific density were measured for Thysanoessa inermis, Thysanoessa raschii, Meganyctiphanes norvegica, Calanus finmarchicus and Calanus hyperboreus. The density of 20 mm T. inermis was lowest in November (1.052 g/cm³) and highest in February–March (1.065 g/cm³). For a 20 mm T. raschii the minimal density was determined in December (1.059 g/cm³) and the maximum in February–March (1.074 g/cm³). M. norvegica individuals of 35 mm also had their lowest density in December (1.060 g/cm³), but reached their maximum density in July (1.076 g/cm³).The density of the euphausiids was found to be size dependent. The density increases as the size decreases. C. finmarchicus and C. hyperboreus had densities less than seawater (1.026 g/cm³) during most of the year. Just before spawning the density increased to 1.028 g/cm³ and 1.036 g/cm³ for C. finmarchicus and C. hyperboreus respectively. The seasonal variations of the density were closely related to the lipid content of the animals.     

13C CP/MAS NMR Spectral Analysis of 6-O-Tosyl, 6-Deoxy-6-iodo,and 6-Deoxy Derivatives of N-Acetylchitosan in a Solid State

6-O-Tosyl (1, d.s. 0.94, 80% yield), 6-deoxy-6-iodo (2, d.s. 0.49, 86% yield) and 6-deoxy (3, d.s. 0.49, 50% yield) derivatives of N-acetylchitosan were prepared, and a ¹³C CP/MAS NMR spectral analysis was performed because no suitable solvent for 3 was found. The ¹³C signal for CH₃ at C-6 in 3 was detected at 18.9 ppm, and that for C-4 in 1–3 appeared at 72.2–72.7 ppm, which is in a higher magnetic field than those (82.5–86.0 ppm) in N-acetylchitosan, 6-O- (ethylthio), 6-O-(benzylthio)- and 6-O-(methylthio)-thiocarbonyl derivatives, chitosan, and chitin. This strongly suggests a different molecular conformation for 1–3.     

Synchronous moulting of krill,<Emphasis Type="Italic"> Euphausia superba</Emphasis>, in the Bransfield Strait (Antarctica)

A large mass of cast exoskeletons of the Antarctic krill Euphausia superba was fished in February 2001 at one station in the Bransfield Strait between 411 and 153 m. We assume that the mass of cast exoskeletons originated from a synchronous moult of thousands of individuals. Depending on the size of the krill moulted, the amount of exoskeletons corresponded to a krill abundance of 1,480–48,000 individuals per 1,000 m³.     

Role of surface exclusion genes in lethal zygosis in Escherichia coli K12 mating

Summary We find that diaminopimelic acid in the recipient membrane is released into the medium during bacterial matings, indicating that membrane damage was inflicted on the recipient by the donor, probably for forming a channel for DNA transfer. When the damage is extensive, as in matings with an excess of Hfr bacteria, the F^- bacteria are killed (lethal zygosis). The transfer of a large amount of DNA in Hfr matings appears to enhance the killing. In analogous F⁺xF^- (Nal^r) matings, on the other hand, killing of F^- bacteria does not occur unless F plasmid transfer is inhibited by a substance like nalidixic acid. The F^- bacteria are killed, suggesting that F plasmids contain genes that express immunity to lethal zygosis in the recipient. For example, bacteria containing surface exclusion-deficient mutants of F plasmids, such as traS ^- and traT ^-, induce lethal zygosis in F^- bacteria and are susceptible to it. Various tra ^- polar mutants that abolish surface exclusion are also susceptible to lethal zygosis when mated with Hfr bacteria. Kinetic experiments indicate that in F⁺ (wild type) x F^- matings, immunity to lethal zygosis is expressed in the F^- recipient within 1/4 division time, whereas a complete expression of surface exclusion requires more than 1 division time. Thus, a complete change in all receptor sites seems to be required for the expression of surface exclusion.     

Feeding behaviour in Meganyctiphanes norvegica (M. Sars) (Crustacea: Euphausiacea)

Meganyctiphanes norvegica (M. Sars) will feed upon the centric diatom Thalassiosira weissflogii (Grunow) G. Fryxell & Hasle but cannot fulfil its energy requirement for metabolism on this food. Its daily metabolic requirement can be exceeded when the euphausiid feeds upon the copepods Calanus finmarchicus Gunner or Centropages typicus Krøeyer, but not when feeding upon the smaller copepods Pseudocalanus spp. or Acartia spp. When feeding upon a natural copepod assemblage Meganyctiphanes norvegica requires high concentrations of copepods to achieve its metabolic requirements, suggesting that the euphausiid may exploit vertically patchy concentrations of prey. Short-term predation rates on Pseudocalanus spp. were also used to estimate feeding rates. Feeding in Meganyctiphanes norvegica appears to be adapted to a spatially variable food supply and rapid exploitation of food sources concentrated into patches or layers. The filter area of the feeding basket of M. norvegica is proportionally smaller than the filter area of Euphausia superba Dana, but has the same allometric length exponent. The filter area probably reflects the difference between the primarily carnivorous diet of Meganyctiphanes norvegica and herbivorous diet of Euphausia superba.     

Influence of sodium fluoride and caffeine on the concentration of fluoride ions, glucose, and urea in blood serum and activity of protein metabolism enzymes in rat liver

The aim of the study was examining the effect of fluoride ions and caffeine administration on glucose and urea concentration in blood serum and the activity of protein metabolism enzymes and selected enzymes of the urea cycle in rat liver. The study was carried out using 18 male Sprague-Daowley rats (4.5 mo old). Rats were divided into three groups. Group I received distilled water ad libitum. Group II received 4.9 mg F⁻/kg body mass/d of sodium fluoride in the water, and group III received sodium fluoride (in the above-mentioned dose) and 3 mg/kg body mass/d of caffeine in the water. After 50 d, the rats were anesthetized with thiopental and fluoride ions, glucose, and urea concentration in blood serum were determined. Also determined were the activities of aspartate aminotransferase, alanine aminotransferase glutamate dehydrogenase, ornithine carbamoylotransferase and arginase in liver homogenates. Liver was taken for pathomorphological examinations. The applied doses of F⁻ (4.9 mg/kg body mass/d) and F⁻+ caffeine (4.9 mg F⁻/kg body mass/d+3 mg caffeine/kg body mass/d) resulted in a statistically significant increase of fluoride ion concentration in blood serum, a slight increase of the glucose concentration, and no changes in the concentration of urea in blood serum. This might testify to the absence of kidney lesions for the applied concentrations of F⁻. No change in the functioning of hepatocytes was observed; however, slight disturbances have been noted in the functioning of the liver, connected with the activation of urea cycle, increase of arginase activity, and accumulation of F⁻ in this organ. There was no observed significant influence of caffeine supplementation on the obtained results.