Identification of proteolytic isozymes from Antarctic krill (Euphausia superba) in an enzymatic debrider

• 1.1. A partially purified krill extract (enzymatic debrider) intended for clinical use was electrophoretically characterized by polyacrylamide gel electrophoresis (PAGE) and by crossed immunoelectrophoresis (CIE) using polyclonal rabbit antibodies.
• 2.2. Three main types of proteolytic enzymes (serine proteinase, carboxypeptidase A and B) with mol. wts of 33,000, 28,000 and 35,000, respectively, could be separated by SDS—g-PAGE under reducing conditions.
• 3.3. Routine CIE analysis of krill samples revealed four protease-active immunoprecipitates. Two of these precipitates were associated with the proteinase activity, one with carboxypeptidase A and one with carboxypeptidase B.
• 4.4. Improving resolution of CIE by extending electrophoresis in the first dimension permitted separation of three serine proteinases of which two were isozymes (II and III) and the third one was unique (I).
• 5.5. Furthermore carboxypeptidase A could also be separated into two isozymes (AI and AII) while carboxypeptidase B still exhibited one single component.
• 6.6. Six individual immunoprecipitates were thus identified and proved to be related to the protease activity. Highly purified enzymes were used as references in CIE and tandem-CIE to establish identification of each enzyme in the krill mixture.

     

Classification of Proteases in Antarctic Krill

The species of endogenous proteases in Antarctic krill, Euphausia superba, were investigated using the homogenate of krill and the active fractions after gel filtration of the homogenate as to the following criteria: Substrate specificity (benzyloxycarbonyl (Z)-Phe-Ala, Z-Glu-Tyr, hippuryl-Arg, hippuryl-Phe, benzoyl Arg-p-nitroanilide, Leu-p-nitroanilide, ¹⁴C-hemoglobin), sensitivity to protease inhibitors (diisopropyl fluorophosphate, iodoacetamide, EDTA, soybean trypsin inhibitor and pepstatin), molecular weight and isoelectric point.

From the experimental results, we found that the carboxypeptidase A and B, aminopeptidase, trypsin and cathepsin A types of proteases were present in krill.     

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.     

Distribution,size frequencies and maturity stages of krill,Euphausia superba,in relation to sea-ice in the Northern Weddell Sea

Summary The spring (November) and early summer (December) distribution pattern and demography of Euphausia superba in the seasonally ice covered areas of the north-western Weddell Sea are described. Sampling with a Rectangular Midwater Trawl (RMT) and during SCUBA diving were undertaken in the pack-ice during the EPOS expedition 1988/1989 of RV Polarstern. These data were compared with former cruises during late winter (October) and summer (February). The biomass values of krill (1–27 g/m²) in the water column within the ice covered areas were of the same order of magnitude as earlier acoustic estimates in the ice-edge region of the same area. No significant correlation between percentage ice cover and krill abundance in the water column was found. Cluster analyses of size frequency distributions and maturity stage composition revealed the demography of the animals sampled. Samples obtained by SCUBA and RMT are compatible and results show a geographic and demographic separation of the krill. It is proposed that krill in the northern zone are largely emigrants from the Bellingshausen Sea, while the krill in the southern zone are of eastern Weddell Sea origin. Further studies concentrating on the under-ice distribution of krill are recommended.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation. Publication no 62 of the Alfred-Wegener-Institut für Polar- und Meeresforschung     

Isoforms of an N-acetyl-β-d-glucosaminidase from the Antarctic krill, Euphausia superba: purification and antibody production

Two forms of the chitinolytic enzyme N-acetyl-β- -glucosaminidase (NAGase, EC 3.2.1.52) have been isolated from the Antarctic krill, Euphausia superba, in order to study their potential role in temperature adaptation processes. A chromatographic protocol was developed that allowed complete separation of the two enzyme forms, named NAGase B and NAGase C. The latter was purified to homogeneity with 600-fold enrichment and a yield of 17%. The molecular mass was 150 kDa. NAGase B showed characteristics of a glycoprotein due to affinity towards concanavalin A sepharose, while NAGase C did not. Highly specific polyclonal antibodies to NAGase C [anti-(E. superba-NAGase C)-IgG] showed only negligible cross-reactivity with NAGase B isoforms. A comparison with the Northern krill, Meganyctiphanes norvegica, revealed a corresponding chromatographic pattern with two main activity peaks, for differentiation named NAGase II and NAGase III. Application of the antibody on M. norvegica revealed a high specificity toward NAGase III and a low cross-reactivity with NAGase II. First indication is given that the two forms are no isoenzymes in a strict sense but instead may have different functions in the metabolism of krill.     

Identifying patterns in the diet of mackerel icefish (<Emphasis Type="Italic">Champsocephalus gunnari)</Emphasis> at South Georgia using bootstrapped confidence intervals of a dietary index

Ontogenetic, inter-annual and regional variations in diet were investigated for mackerel icefish, Champsocephalus gunnari, in three successive summer seasons around South Georgia. Stomach contents from 2239 C. gunnari (130–560 mm total length) were examined. A bootstrapping technique was used to calculate confidence intervals for an index of relative importance of prey categories (% IRI_DC). Diet varied significantly between years and age classes but there was little regional difference in diet. In general, diet was dominated by krill, Euphausia superba and by the amphipod Themisto gaudichaudii. Smaller (younger) fish tended to prey on a higher proportion of T. gaudichaudii and small euphausiids such as Thysanoessa sp. and took smaller quantities of E. superba. In a season of poor krill availability (summer of 2003–2004) the proportion of krill in the diet, stomach fullness and fish condition (indicated by length–weight relationships) were significantly lower than in the other summer seasons. A large reduction (>80%) in the estimated annual (2005) biomass of the C. gunnari stock directly followed the season of poor krill availability. This decline was largely because of mortality of 2+ and 3+ fish, which were more krill dependent than 1+ fish. Younger fish appear to have survived, leading to an increase in the estimated population biomass in 2006.     

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     

Diet and reproductive success of Adélie and chinstrap penguins: linking response of predators to prey population dynamics

The diet and reproductive performance of two sympatric penguin species were studied at Signy Island, South Orkney Islands between 1997 and 2001. Each year, Adélie (Pygoscelis adeliae) and chinstrap (P. antarctica) penguins fed almost exclusively (>99% by mass) on Antarctic krill; however, there was considerable inter-annual variation in reproductive output. In 1998, chinstrap penguins were adversely affected by extensive sea-ice in the vicinity of the colony, whereas Adélie penguins were unaffected by this. However, in 2000, both species suffered reduced reproductive output. Detailed analysis of the population-size structure of krill in the diet indicated a lack of recruitment of small krill into the population since 1996. A simple model of krill growth and mortality indicated that the biomass represented by the last recruiting cohort would decline dramatically between 1999 and 2000. Thus, despite the lack of a change in the proportion of krill in the diet, the population demographics of the krill population suggested that the abundance of krill may have fallen below the level required to support normal breeding success of penguins sometime before or during the 2000 breeding season. The role of marine predators as indicator species is greatly enhanced when studies provide data reflecting not only the consequences of changes in the ecosystem but also those data that elucidate the causes of such changes.     

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.     

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.     

Interannual variation in the diet of non-breeding male Antarctic fur seals, <Emphasis Type="Italic">Arctocephalus gazella</Emphasis>, at Isla 25 de Mayo/King George Island

The diet of non-breeding male Antarctic fur seals, Arctocephalus gazella, was investigated at Stranger Point, King George Island, through the analysis of scats during three consecutive summer seasons (1996, 1997, 1998). Overall, fish and krill were the most frequent prey occurring, respectively, in an average of 82.9% and 78.8% of samples (n = 131), followed by penguins (22.8%) and cephalopods (17.8%). Myctophids constituted almost 90% of the fish predated, with Electrona antarctica and Gymnoscopelus nicholsi being the most abundant and frequent species consumed. All fish taxa identified were krill feeding species suggesting that seals foraged mainly on a krill and a fish community associated with krill aggregations. However, a seasonal change was observed in the relative proportions of the different prey taxa, with a progressive decrease with time in the occurrence of krill and a concomitant increase of fish, penguins and squid. Possible influence of the strong 1997/98 ENSO event is discussed.     

A comparison of Antarctic petrel (Thalassoica antarctica) diets with net samples of Antarctic krill (Euphausia superba) taken from the Prydz Bay region

Samples of the stomach contents of Antarctic petrels (Thalassoica antarctica) were obtained on board ship in the Prydz Bay region of Antarctica from birds which had spontaneously regurgitated. The weight of food and the species composition of the stomach contents were measured. Antarctic krill,Euphausia superba, the sole prey item taken, were compared to krill obtained by nets in the same region as part of a large-scale krill survey. Krill from petrel stomach samples were larger in mean size than those sampled by nets. This may be attributed to selection of the larger sized krill by the petrels, it may be caused by the nets sampling different populations of krill or it may be due to net avoidance by the larger krill.     

The diet of Antarctic fur seals, Arctocephalus gazella, at King George Island, during the summer–autumn period

The diet of non-breeding male Antarctic fur seals, Arctocephalus gazella, was investigated at Stranger Point, King George Island, by scat analysis from February to April 1996. Overall, krill and fish were the most frequent prey, occurring in an average of 97% and 69% of samples (n=128), followed by cephalopods (12%). Myctophids constituted almost 90% of the fish predated, with Electrona antarctica and Gymnoscopelus nicholsi being the most abundant and frequent species consumed. All fish taxa identified were krill-feeding species suggesting that seals foraged primarily on krill and opportunistically on fish species associated with krill swarms. A seasonal change observed in the relative proportions of the different fish prey taxa indicates that fur seals spent more time foraging over the shelf in summer and off the shelf in autumn. During the study period, commercial fishing in the area was not based upon any of the fish identified in this study.     

Eugregarine infection within the digestive tract of larval Antarctic krill, Euphausia superba

The infection rate of eugregarine parasites, Cephaloidophora pacifica, within the digestive tract of larval Antarctic krill, Euphausia superba, was examined using samples collected from the Indian sector of the Southern Ocean. Immature and mature eugregarine gamont stages were found at all larval stages older than Calyptopis I stage. Eugregarine infection in 14.0% (N = 108) of the first feeding stage (Calyptopis I) suggested that krill larvae are at risk from being infected during physiological transition from non-feeding to feeding stages. Eugregarine prevalence and intensity of infection at the three calyptopis stages increased with stage/krill length. Statistical analysis showed that the intensity of C. pacifica infection also increased with host density. Thus, krill density is probably a key determinant of the intensity of infection. We found gamont stage eugregarines in the host hind-gut, blocking the passage of food. Eugregarine infections in larval krill may have a negative impact on digestion and absorption in the host digestive tract.     

Genetic variation and population structure of krill (Euphausia superba dana) from the Prydz Bay region of Antarctic waters

Summary Samples of Antarctic krill E. superba from six locations near Prydz Bay were analysed electrophoretically to detect genetically-based protein variation. Analyses of allele distributions at four polymorphic loci indicate no evidence of significant heterogeneity, a result consistent with the hypothesis that all samples were derived from a single breeding population of krill. The results of this study agree closely with genetic data from other studies on krill in the Atlantic sector of the Southern Ocean, suggesting that krill stocks over at least 6000 km of Antarctic waters are derived from a single interbreeding population.     

Melatonin and its possible role in mediating seasonal metabolic changes of Antarctic krill, Euphausia superba

Melatonin, the chief secretory product of the vertebrate pineal gland is suspected to be a ubiquitous molecule principally involved in the transduction of photoperiodic information. Besides vertebrates, melatonin has been detected throughout phylogeny in numerous non-vertebrate taxa. In the present study, the occurrence of melatonin in Antarctic krill Euphausia superba and its possible role in mediating seasonal metabolic changes was evaluated. Melatonin was quantified by enzyme linked immunosorbent assay (ELISA) in high performance liquid chromatography (HPLC) purified extracts of eyestalks and hemolymph of krill sampled in the Lazarev Sea during the Antarctic winter and summer. In addition, oxygen uptake rates and the activities of the metabolic enzyme malate dehydrogenase (MDH) were recorded to assess the metabolic status of krill. Validation of melatonin measurements was carried out on the basis of three different extraction methods with parallel determination of melatonin by ELISA in crude extracts and in HPLC purified extracts, and after derivatization of melatonin under alkaline conditions in the presence of hydrogen peroxide. A significantly higher respiration rate and MDH activity was found in summer krill than in winter krill indicating that krill was in a state of reduced metabolic activity during winter. However, neither during winter nor during summer there were detectable melatonin concentrations in the visual system or hemolymph of krill. Based on these results, we question a mediating role of melatonin in the control of seasonal metabolic changes in Antarctic krill.     

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.     

The diet of Antarctic fur sealsArctocephalus gazella during the breeding season at South Georgia

 The diet of lactating female Antarctic fur seals Arctocephalus gazella at South Georgia was investigated during the pup-rearing period (January/March) of 1991–1994. Antarctic krill Euphausia superba was the main prey item, occurring in 88% of all scats (n=497), whereas fish occurred in 47% and squid in 5% of all scats. There was considerable intra- and inter-annual variation in the characteristics of krill taken by fur seals. The distribution of krill sizes taken suggests that fur seals are not actively selecting particular sizes of krill and, therefore, that the krill in the diet reflects the krill available around South Georgia. The absence of group 3 krill (44–48 mm in length) in the South Georgia area, as indicated by their absence in the diet of seals, is suggested as a possible reason for low availability of krill and the subsequent reproductive failure among krill predators. The frequency of occurrence of fish was much higher than in previous studies; the pattern of fish consumption showed a consistent seasonal pattern in 3 of the 4 years studied. Of the total number of the myctophid Protomyctophum choriodon, the most numerous fish taxon, 98% were taken between early February and the middle of March. Champsocephalus gunnari and Lepidonotothen larseni agg., which both feed on krill, dominated the fish component of the diet outside this period and together constituted 94% of the total estimated biomass of fish consumed. The intra- and inter-annual variability in the diet of Antarctic fur seals emphasise the need for diet studies to be conducted during the entire pup-rearing periods of several years. Received: 15 March 1995/Accepted: 13 May 1995     

Essential krill species habitat resolved by seasonal upwelling and ocean circulation models within the large marine ecosystem of the California Current System

Due to their global distribution, high biomass and energy content, euphausiids (krill) are important prey for many mid and upper trophic level marine organisms. Understanding drivers of krill habitat is essential for forecasting range shifts, and to better understand the response of krill predators to climate change. We hypothesized that the distribution and abundance of krill species derived from ecosystem surveys in spring/summer relates to geomorphic features, coastal upwelling during the preceding winter, and spring mesoscale oceanographic conditions. To test this hypothesis, we used boosted regression trees with environmental data and ocean model output to quantify the habitat associations of two primary krill species (Euphausia pacifica and Thysanoessa spinifera) in the central California Current Ecosystem from 2002 to 2018. Models confirmed the neritic distribution of T. spinifera and pelagic, outer slope association of E. pacifica (deviance explained ~35%). Distribution of these species were influenced by depth and bottom rugosity; chlorophyll-a concentrations and increased winter upwelling conditions; and spring surface currents and wind stress. Thysanoessa spinifera and E. pacifica abundance responded negatively (positively) to warm (cold) climate events, confirming known relationships. As an independent evaluation of krill models, observations of krill predator (seabirds, marine mammals) distribution indicated they were present within habitats of predicted high krill species abundance. Our framework indicates species-specific habitat relationships for these foundational forage species and their negative response to large-scale climate variations, such as El Niño and marine heatwave conditions. The approach can be easily transferred to other ecosystems or krill species that respond to similar ocean and climate forcing.     

Inter-sexual differences in Antarctic fur seal pup growth rates: evidence of environmental regulation?

We investigated the variation of Antarctic fur seal (Arctocephalus gazella) pup growth rates in response to sex, breeding season and duration of both maternal foraging trip and attendance bouts. Data were collected during five consecutive rearing seasons at Cape Shirreff, the most important breeding colony in the South Shetland Islands, Antarctica. Our results showed significant interannual and sexual variations in pup growth rates. Male pups grew significantly faster than female pups during 2000, 2001, 2002 and 2004 seasons, whilst during 2003 no difference was found. The interannual variation in pup growth rates was correlated with the interannual fluctuation in maternal foraging trip and attendance bouts. There was a significant effect of pup sex and maternal foraging trip duration on pup growth rates, which varied between years having foraging trip duration a major effect during 2003, when females spent more time at sea and interestingly on that year there were no sexual differences in pup growth rates. The effect of attendance bout on pup growth rates was not significant. Diet analysis showed that Antarctic krill (Euphausia superba) was the most frequent prey item during the study period. Analysis of krill size distribution showed a significant difference in krill length, during 2003, when A. gazella preyed upon the smallest sizes of krill. In this study, sex was the most important factor on pup growth rates, but when prey availability seemed more limited, there are longer foraging trips and shorter attendance bouts the sex factor became less significant.