Collagenolytic serine proteinase from Euphausia superba Dana (Antarctic krill).

1. A serine proteinase isolated from E. superba shows collagenolytic properties: it acts on collagens from Achilles tendon (type I and V) and reconstituted fibrils of calf skin collagen under conditions that do not denature the substrates. 2. At 25 degrees C and pH 7.5 the enzyme both splits the calf skin collagen in solution to the fragments TCA and TCB and catalyses the conversion of dimeric molecules to monomeric chains. 3. The enzyme exhibits strong chymotrypsin-like and lower trypsin-like activities. 4. All the enzyme activities are inhibited to the same degree by diisopropylfluorophosphate (DFP), phenylmethylsulphonyl fluoride (PMSF), N alpha-tosyl-L-lysine chloromethyl ketone (TLCK), soybean trypsin inhibitor (SBTI), chicken ovomucoid (CHOM), chymostatin and leupeptin. None of the activities is inhibited by chelating agents and L-cysteine. 5. pH-Optima of the proteinase in protein substrates hydrolysis (6.0-6.2) are lower than those of synthetic substrates cleavage (7.8-8.0 in the case of BzTyrOEt and 8.7-8.9 for BzArgOEt). 6. Four from nine cysteine residues present in the enzyme molecule possess free thiol-groups. Since the enzyme is inhibited by p-chloromercuribenzoate (pCMB), N-ethylmaleimide (NEM) and iodoacetic acid (IAA), the role of its thiol-groups has been discussed.     

Purification and characterization of a proteinase from Euphausia superba Dana (Antarctic krill)

The thiol-dependent serine proteinase (inhibited by DFP, PMSF, pCMB and iodoacetate) was isolated from the whole krill specimens and from the content of the krill digestive tract. The enzyme was purified to homogeneity using a seven-step procedure. Its specific activity with denatured haemoglobin as a substrate was about 6.0 unit/mg. The molecular weight of the enzyme, as determined by gel exclusion chromatography was 33 000 and by polyacrylamide gel electrophoresis with SDS 31 600 (12.5% gel) and 27 000 (7.5% gel). The enzyme is an acidic glycoprotein (pI below 2.9) containing about 5% of carbohydrate. The pH optimum of the enzyme with haemoglobin was 6.0 at the optimal temperature of 40 degrees C in 15-min reaction. The enzyme showed the esterase activity (hydrolysis of BAEE) and was inactive with carbobenzoxy- and benzoyl-dipeptides with the following C-terminal amino acids: Phe, Tyr, Lys, Gly and Leu.     

Antarctic krill (Euphausia superba Dana) eat salps

 Feeding behaviour of Antarctic krill (Euphausia superba) on salps was observed in shipboard experiments during the 1994/1995 Kaiyo Maru Antarctic Ocean research cruise. The feeding rate was more than 0.5 salp/krill per day. When offered ethanol extracts of four prey types, salps, phytoplankton, krill and polychaetes, krill preferred the salp extracts. This evidence implies that the substances extracted from salps were most attractive to krill. These results might indicate a tight ecological relationship between krill and salps. Received: 24 May 1995/Accepted: 8 October 1995     

Susceptibility of Antarctic krill (Euphausia superba Dana) to ultraviolet radiation

We irradiated captive juvenile Euphausia superba in the laboratory with lower than spring surface levels of ultraviolet-B, ultraviolet-A and photosynthetically active radiation, in order to examine their response in terms of mortality and generalised activity. Levels of photosynthetically active radiation 3–5 times below surface irradiance caused krill to die within a week, while animals in the dark survived. Addition of ultraviolet-B typical of depths up to 15 m were found to significantly accelerate mortality and lead to a drop in activity in all experiments. A drop in activity in krill exposed to ultraviolet-A wavelengths was evident without an increase in mortality. The protein content of animals from various treatments was found not to vary. Accepted: 10 January 1999     

Identification and quantification of prostaglandins in Antarctic krill (Euphausia superba Dana)

Summary The amount and types of prostaglandins present in Antarctic krill (Euphausia superba Dana.) were estimated. Samples of fresh krill were collected during III Antarctic Cruise of RV Polarstern in November 1984. Prostaglandins were extracted, separated by column and thin-layer chromatography and identified as PGA₂, PGB₂, PGE₂, PGF₂. Quantitative measurements were made by a biological method (Vane cascade), concentrations of the most abundant prostaglandins PGE₂ and PGF₂ being 1.6 and 4 ng/1 g of fresh tissue, respectively. Such low level of prostaglandin would not be harmful when using krill as a food supplement.     

Characterization of proteinases from Antarctic krill (Euphausia superba)

Fractions of three trypsin-like proteinases, TL I, TL II, and TL III, a chymotrypsin-like proteinase, CL, two carboxypeptidase A enzymes, CPA I and CPA II and two carboxypeptidase B enzymes, CPB I and CPB II, from Antarctic krill (Euphausia superba) have been characterized with respect to purity by the means of capillary electrophoresis, CE, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The masses of the trypsin-like and chymotrypsin-like proteinases were determined to be 25,020, 25,070, 25,060, and 26,260Da for TL I, TL II, TL III, and CL, respectively. The masses of the CPA enzymes are likely 23,170 and 23,260Da, whereas the CPB enzyme masses likely are 33,730 and 33,900Da. The degradation efficiency and cleavage pattern of the trypsin-like proteinases were studied with native myoglobin as a model substrate using CE, MALDI-TOF-MS, and nanoelectrospray mass spectrometry (nESI-MS). The degradation efficiency of the trypsin-like proteinases was found to be approximately 12 and 60 times higher compared to bovine trypsin at 37 degrees C and 1-3 degrees C, respectively. All three fractions of trypsin-like proteinases showed a carboxypeptidase activity in combination with their trypsin activity.     

Spatial and temporal variability in reproductive timing of Antarctic krill (Euphausia superba Dana)

Spawning dates of Antarctic krill, Euphausia superba Dana, were calculated from larval stage compositions, and corrected using data on maturity stage composition of the adult krill. Both original and literature data obtained from the Antarctic Peninsula-Bellingshausen Sea area and around the Antarctic continent were used. A time series (1975/76–1986/87) for several subareas of the Antarctic Peninsula-Bellingshausen Sea area indicates considerable variation in the krill spawning start, maxima and completion. In particular years (1975/76, 1980/81), krill spawning in the western Atlantic sector began relatively early, was intensive, and completed early. Some years (1977/78, 1981/82) were characterised by long and non-synchronised krill spawning. Compiled data sets for the Atlantic sector (1980/81), the entire Antarctic (1983/84) and the east Indian-west Pacific Antarctic waters (1981–85) reveal some spatial patterns in krill reproductive timing. In relation to spawning timing variation, the habitats of the krill population fall into five categories: (1) areas with an early beginning (late Novemberearly December) and a variable, but normally long, duration (3–3.5 months) of krill spawning; this is generally the southern boundary of the Antarctic Circumpolar Current, (2) areas with an early beginning, but a short duration of krill spawning (Gerlache Strait), (3) areas with a highly variable (within 1–1.5 months) beginning and a relatively long duration (ca. 3 months) of krill spawning (Bransfield Strait, Palmer Archipelago), (4) areas with a late beginning (late December–January) and a long duration of krill spawning (Bellingshausen Sea, D'Urville Sea, and Balleny Islands area), and (5) areas with a delayed beginning, but a very short duration (ca. 1.5 months) of krill spawning (Ross Sea slope, probably the Coastal Current area off the Lasarev Sea shelf and in the south-eastern Weddell Sea. These patterns can be partly explained by peculiarities of the ice regime in particular areas and by routes of krill movement within water circulation systems.     

Molecular evidence for genetic subdivision of Antarctic krill (Euphausia superba Dana) populations.

Antarctic krill (Euphausia superba Dana) is a key species in the Antarctic food web and occurs on a circumcontinental scale. Population genetic structure of this species was investigated by sequence analysis of the ND1 mitochondrial gene in four population samples collected at different geographical localities around the Antarctic continent. Results indicate the existence of significant genetic differences between samples, and we suggest that oceanographic barriers could be sufficiently strong and temporally stable to restrict gene flow between distinct areas. Moreover, our data indicate that Antarctic krill is not at mutation-drift equilibrium and that the species possibly has a low effective population size as compared to the census size.     

Laboratory observations of moulting,growth and maturation in Antarctic krill (Euphausia superba Dana)

Summary Observations of intermoult period, growth and maturation were made on krill which were transported from Antarctic waters and maintained in the laboratory in Australia over a three year period. The mean intermoult period (IP) for each of 10 specimens, with initial body lengths of 24.7=46.8 mm, kept at -0.5° C varied from 22.0 to 29.8 days (overall mean = 26.6 days). These measurements of IP are significantly longer than those obtained in some previous studies. Differences in experimental temperatures, light, body sizes and growth patterns of the specimens between studies are unlikely to be causes of these dissimilar results. The pattern of changes in body length (BL) varies from one individual to the next. The greatest increase in BL over a series of 4–5 moults ranged from 0.024 to 0.070 mm/day, which is equivalent to 0.0020 to 0.0086/day in body weight, assuming exponential growth. This maximum growth rate is about half the rate predicted from the growth scheme of Mauchline (1980) for wild krill. Comparison of growth data for other euphausiids suggests that Mauchline's scheme produces anomalous growth rate. The slower growth rate observed in the present study would extend the estimated life span of krill from 3–4 years, as calculated by Mauchline (1980), to 4–7 years. If krill undergo body shrinkage during the Antarctic winter the estimated life span might be even longer. Examination of the external sexual characters of moults showed both progression and regression of maturity stage in association with changes in BL.     

The influence of feeding on the metabolic activity of Antarctic krill (Euphausia superba Dana)

Summary The influence of feeding on the metabolic activity of juvenile krill was assessed from 24h experiments in which krill were incubated with various concentrations of diatoms (Chaetoceros calcitrans, Phaeodactylum tricornutum, Thalassiosira eccentrica, Fragilariopsis vanheurkii), newly hatched Artemia nauplii and latex beads. Krill fed on the larger food more efficiently, with reluctant feeding on latex beads. Feeding of krill expressed as clearance rates was poorly correlated with their oxygen uptake rates. Instead, a positive correlation was found between the oxygen uptake rates and ingestion rate (except for latex beads). The result implies that the specific dynamic action is the major cause of the increased oxygen uptake of krill. Krill fed diatoms increased both ammonia and phosphate excretion with increasing ingestion rate, but only phosphate excretion was increased in parallel with ingestion rate for those fed Artemia nauplii. Assuming the daily ration of krill in the field is 5% of the body weight, and the major food source is phytoplankton, oxygen uptake, ammonia excretion and phosphate excretion rates of wild krill are estimated to be 1.6, 4.5 and 7.8, respectively, times the rates of non-feeding krill in 24h laboratory experiments. Krill offered various kinds of food showed different metabolic quotients (O/N, N/P and O/P ratios). While no functional relationship was seen between the metabolic quotient and the ingestion rate of krill fed Artemia nauplii, those fed Fragilariopsis showed a progressive decrease in O/N, N/P, and O/P ratios as their ingestion rates increased.     

Apparent independence of the spawning and moulting cycles in female Antarctic krill (Euphausia superba Dana)

Summary Sixty female Antarctic krill (Euphausia superba Dana) spawned in shipboard experiments and the interval between egg-laying and ecdysis was noted. The number of eggs laid per female ranged from 263–3662, most females produced only one batch of eggs before moulting, and the post-spawn ovaries of all females contained few, if any, mature oocytes. As reported in other studies, the total number of eggs produced per female was not well correlated with body size. Females appeared to spawn at all times during the moulting cycle and although no diurnal rhythm in spawning was observed, moulting occurred mainly at night-time despite the animals being kept in near-constant darkness. No evidence of synchronous moutling was detected.     

Isolation and immunological characterization of three highly purified serine proteinases from Antarctic krill (Euphausia superba)

Summary Three individual serine proteinases (I, II, III) originating from Antarctic krill (E. superba) were separated and highly purified using a combination of affinity and high resolution ion exchange chromatography. Each enzyme showed a single protein band (30 000 Daltons) in sodium dodecyl sulphate polyacrylamide gradient gel electrophoresis indicating high purity and identical molecular weights. Moreover, each enzyme demonstrated one immunoprecipitate on crossed immunoelectrophoresis (two-dimensional agarose gel electrophoresis) using polyclonal rabbit antibodies which confirmed the high purity of the individual enzymes. A mixture of the three enzymes (I, II, III) revealed two immunoprecipitates, not one or three which should have been the case for identical or non-identical immunological properties. Double immunodiffusion test according to Ouchterlony exhibited immunological identity between enzyme II and III. Enzyme I showed only partial identity with II/III. These findings correlated well with biochemical data on the three serine proteinases. Enzyme I is able to liberate free amino acids from polypeptides in comparison with enzyme II and III (classical true endopeptidases), which do not. We suggest that these unique biochemical properties also have their immunological counterpart expressed as other antigenic determinants of the molecular structure.     

Mature antarctic krill (Euphausia superba Dana) grown from eggs in the laboratory

Fertilized eggs obained from female krill in Antarctic waterswere transported to the Australian Antarctic Division in Tasmania,the eggs were successfully developed to larval, juvenile, subadultand finally mature adult stages in the laboratory. Under experimentalconditions of unlimited food supply, at 0°C and in completedarkness, the length of the life cycle from egg to egg was 3years. ^*Present address: Japan Sea Regional Fisheries Research Laboratory,5939–22, 1 Suido-Cho, Niigata 951, Japan     

Crossed immunoelectrophoretic analysis of proteins from Antarctic krill (Euphausia superba) with special reference to serine proteinases

Summary Immunochemical characterization of proteins from Antarctic krill (E. superba) and particularly serine proteinases was performed by crossed immunoelectrophoresis (CIE). A reference pattern with 34 arbitrarly numbered immunoprecipitates was established for a crude krill extract using IgG rabbit antiserum. A zymographic technique for detection of proteolytic activity was applied and revealed 4 casein-positive antigens. Introduction of different affinity media as intermediate gels in CIE demonstrated several proteins which strongly interacted with Sepharose-bound arginine and Con A. These results suggest the presence of trypsin-like enzymes and other serine proteases and a high content of glycoproteins with alfa-D-mannopyranoside and alfa-D-glycopyranoside residues. The CIE method thus offers a reliable and reproducible tool for identification and characterization of different krill enzymes as well as application in process control, when purifying individual proteins/enzymes. Moreover this technology may serve as a fingerprinting method suitable for biological studies such as classification of geographical subpopulations, genotyping a specific species as well as comparisons between different species.     

Purification and Characterization of Cold Adapted Trypsins from Antarctic krill (Euphausia superba)

Three trypsins (TRY-ES) were purified from Antarctic krill (Euphausia superba) by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography, with relative molecular mass of 28.7, 28.8 and 29.2 kDa respectively. The TRY-ES was inhibited by specific trypsin inhibitors (benzamidine, STI, CHOM and TLCK), with optimum temperature at 40 (Trypsin I), 45 (Trypsin II) and 40 °C (Trypsin III) repetitively. The TRY-ES was stabled between 5 and 40 °C, which was consistent with the red shift in fluorescence intensity peak at 40 °C (Trypsin I) and 45 °C (Trypsin II and Trypsin III) and blue shift at 40 °C (Trypsin II and Trypsin III). The K _cat/K _m values of the TRY-ES was 14.28, 9.46 and 5.93 mM^?1s^?1 respectively, 1.1–10.2 folds higher than trypsins from other crustacean and mammal, which was supported by the differences in thermodynamics parameters, the free energy, enthalpy, and entropy of benzamidine and the TRY-ES system.     

Genetic homogeneity of krill (Euphausia superba Dana) in the Southern Ocean

Summary Development of a comprehensive picture of the genetic population structure of the Antarctic krill (Euphausia superba) has been hampered by a lack of genetic data from two major areas of the species' distribution, the Bellingshausen Sea and the Ross Sea. Evidence from earlier studies of a discrete Bellingshausen Sea population was based on anomalous allele frequencies in two sample sets that were collected near the west coast of the Antarctic Peninsula rather than in the Bellingshausen Sea proper. In this paper we describe the first biochemical genetic data obtained on krill from the central Bellingshausen Sea and from the Ross Sea. Analyses of eight polymorphic loci in samples from these two areas have failed to provide any evidence of population structuring within the Pacific sector of the Southern Ocean, and have indicated that Pacific sector krill cannot be genetically discriminated from Atlantic sector krill or Indian Ocean sector krill. These findings further support the hypothesis of a single circumpolar breeding population of Antarctic krill.     

Antarctic krill (Euphausia superba) acquire a UV-absorbing mycosporine-like amino acid from dietary algae

We hypothesised that Antarctic krill acquire UV-absorbing mycosporine-like amino acids (MAAs) from dietary algae, which produce MAAs in response to ultraviolet (UV) irradiation. To test this hypothesis, we grew cultures of Phaeocystis antarctica that had been grown under either photosynthetically active radiation (PAR, 400-750 nm) plus UV irradiation (UVR, 280-400 nm), or else PAR-only. Algae grown under PAR-only produced high concentrations of porphyra-334, whereas additional UVR caused formation of high concentrations of mycosporine-glycine:valine and lower concentrations of porphyra-334. Krill were fed with either of these two cultures on eight occasions over 63 days. A third group was starved for the duration of the experiment. Animals were analysed after 36 and 63 days for MAA content. Remaining animals from all treatments were starved for a further 35 days and analysed to examine MAA retention characteristics. Our findings are that krill acquired different MAAs from dietary algae depending on the light conditions under which the algae were grown. Specifically, krill fed algae grown under PAR-only had higher concentrations of porphyra-334 than starved krill. Conversely, krill fed algae grown under PAR with additional UVR had high body concentrations of mycosporine-glycine:valine. MAA concentrations in starved krill remained static throughout the experiment. However, long term starvation (35 days) caused levels of certain acquired MAAs to decline. From this we can infer that MAA concentrations in krill are dependent on the MAA content of phytoplankton, and therefore the algae's response to UV exposure. This has implications for transfer of MAAs through marine trophic webs.     

Purification and partial characterization of a novel hyaluronic acid-degrading enzyme from Antarctic krill (Euphausia superba)

Summary A novel enzyme degrading hyaluronic acid has been isolated, purified and characterized from Antarctic krill (Euphausia superba). A combination of affinity chromatography (Con A-Sepharose), gel filtration (Superose 6) and fast protein liquid chromatography (Mono Q) was used for the purification. The hyaluronidase activity was determined by a radial diffusion method based on hyaluronic acid incorporated into an agarose gel. Moreover, the beta-glucuronidase and endo-(1,3)-beta-D-glucanase activities were also followed through the process using phenolphtalein mono beta-glucuronic acid and laminarin as substrates. After the final purification step on Mono Q column, the chromatogram showed three main peaks designated A, B and C. Peak C contained high hyaluronidase activity undetectable in peak A and B. The betaglucuronidase activity was associated with peak A, while the endo-(1,3)-beta-D-glucanase activity was found in peak B and slight in peak C. The hyaluronidase was purified about 85-fold. It had a pH optimum of 5.3, a temperature optimum of 37°C and a molecular weight of 80 000 Daltons. On polyacrylamide gradient gel electrophoresis the enzyme fraction showed one major band associated with hyaluronic acid decomposition, slightly contaminated with a few other components. Isoelectric focusing in combination with a hyaluronic acid zymogram demonstrated one major band at pH 6.7 with high enzyme activity. Preliminary data on enzyme specificity suggest that krill hyaluronidase is a new endo-beta-glucuronidase and support the concept of krill enzymes as a remarkable and unusually effective digestive system adapted to the Antarctic marine ecosystem.