Friedrich Buchholz

Ontogeny of osmoregulatory structures and functions in the green crab Carcinus maenas (Crustacea, Decapoda)

SUMMARY The ontogeny of osmoregulation, the development of branchial transporting epithelia and the expression of the enzyme Na+/K+-ATPase were studied in Carcinus maenas (L.) obtained from the North Sea, Germany. Laboratory-reared zoea larvae, megalopae and young crabs were exposed to a wide range of salinities, and hemolymph osmolality was measured after 24 h exposure time (72 h in juveniles). Zoea I larvae slightly hyper-regulated in dilute media (10.2‰ and 17.0‰) and osmoconformed at> 17‰. All later zoeal stages (II-IV) osmoconformed in salinities from 10.2‰ to 44.3‰. The megalopa hyper-regulated at salinities from 10.2 to 25.5‰. Young crabs hyperregulated at salinities from 5.3‰ to 25.5‰, showing an increase in their osmoregulatory capacity. The development of transporting epithelia and the expression of Na+/K+-ATPase were investigated by means of transmission electron microscopy and immunofluorescence microscopy. In the zoea IV, only a very light fluorescence staining was observed in gill buds. Epithelial cells were rather undifferentiated, without showing any features of ionocytes. Gills were present in the megalopa, where Na+/K+-ATPase was located in basal filaments of the posterior gills. In crab I juveniles and adults, Na+/K+-ATPase was noted in the three most posterior pairs of gills, but lacking in anterior gills. Ionocytes could first be recognized in filaments of megalopal posterior gills, persisting through subsequent stages at the same location. Thus, the development of the gills and the expression of Na+/K+-ATPase are closely correlated with the ontogeny of osmoregulatory abilities. The morphological two-step metamorphosis of C. maenas can also be regarded as an osmo-physiological metamorphosis, (i) from the osmoconforming zoeal stages to the weakly regulating megalopa, and (ii) to the effectively hyper-regulating juvenile and adult crabs.

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

SummaryChitinolytic activity was quantified in euphausiid integuments in relation to moulting. In Euphausia superba, shortly before moult the activity increased in chitinase and N-acetyl-β-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.

Meganyctiphanes norvegica in the Kattegat

From 1979 to 1983 a population ofMeganyctiphanes norvegica was regularly found in a confined location in the northern Kattegat. The development of this pelagic population was followed over one complete year-cycle, and is discussed in light of published data on landlocked and oceanic populations. In the Kattegat,M. norvegica mature at the age of 9 to 12 months. Copulation starts in january, but fully developed spermatophores are present in males for at least 9 months. Spawning starts in April and continues until October with a marked peak in July. As only 10 to 15% of the gravid females belong to the II-group, it is concluded that recruitment is due to the I-group. Linear growth is fastest in spring and early summer, rises to a second maximum in October and stagnates during winter. The weight development shows some discontinuties caused by spawning or the accumulation of reserves towards winter. Weight decreases during winter. Growth in length and in weight runs largely parallel in both sexes, and can be closely correlated with the prevailing food supply. Diurnal vertical migration could be demonstrated. The results indicate a continuous development, which suggests the existence of an autonomous palagic population, a well suited subject for long-term investigation.

Drach's molt staging system adapted for euphausiids

A method is introduced to determine intermolt stages in Antarctic and boreal euphausiids. The system is based on the classical scheme elaborated by Drach and Tchernigovtzeff. In addition to the definitions of these authors several features unique to euphausiids are described and combined to establish a system of at least 12 easily distinguishable stages. The method can be used to investiate specific cyclical physiological changes as well as answer questions regarding general growth physiology.

Fluoride in tissues of Krill Euphausia superba Dana and Meganyctiphanes norvegica M. Sars in relation to the moult cycle

SummaryThe fluoride content of whole animals and different tissues of the euphausiid species Euphausia superba and Meganyctiphanes norvegica was analyzed by two different and improved methods of isolation and determination. In contrast to other authors our findings show that the internal organs (muscle, hepatopancreas and hemolymph) contain less than 6 ppm d.w. fluoride this being the same order of magnitude as for vertebrates. The high concentrations reported by other authors must be mainly due to contamination of the soft tissue during storage (post-mortem migration of fluoride from shell) and/or contamination caused by minute fractions of cuticle during dissection. Over 99% of the total fluoride content is located in the cuticle (i.e. integument) of the euphausiids (2600 ppm/d.w. in E. superba and 3300 ppm/d.w. in M. norvegica in pleon cuticle). Analysis of F- levels in relation to the moulting cycle showed that the uptake in both euphausiids occurs at a comparable and fast rate during the same physiological phase shortly after moult, parallel to the general construction of the cuticle. The internal organs show homeostasis in respect to fluoride. Accordingly, no internal deposition takes place, and F- is reaccumulated from the external medium at each moult.

Diel vertical migrations ofMeganyctiphanes norvegica in the Kattegat: Comparison of net catches and measurements with Acoustic Doppler Current Profilers

Diel vertical migration of a stable and well-defined population of Nordic krill,Meganyctiphanes norvegica (Crustacea, Euphausiacea) was investigated during eight days in August 1989, in the Läsö-Deep, East of the Danish island Läsö. Net catches with a multi-net (MOCNESS) and measurements with a moored and a shipboard Acoustic Doppler Current Profiler (ADCP) were compared. Backscattered energy as a measure for biomass gave good correlations to the dry weight ofM. norvegica and smaller zooplankton from net catches. Diel migratory patterns matched well, as determined, parallel with both methods. Migratory vertical velocity was determined with ADCP at 2–3 cm sec−1. The potential for the use of ADCPs for biological investigation is discussed. Vertical migration was dependent on environmental parameters. The krill did not cross a temperature barrier of 14°C, although rich food sources were situated beyond it. Differences in salinity did not play a role. Currents were involved in plankton distribution. Light was an important Zeitgeber (synchronizer) and determined the density of the krill aggregations. Feeding behaviour did not interfere with the light-induced migratory pattern of Nordic krill at the Läsö-Deep.

Citrate synthase and pyruvate kinase activities during early life stages of the shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae): effects of development and temperature.

Energy metabolism in early life stages of the shrimp Farfantepenaeus paulensis subjected to temperature reduction (26 and 20 degrees C) was determined using the activities of citrate synthase (CS) and pyruvate kinase (PK). At both temperatures, weight-specific activity of CS decreased throughout the ontogenetic development from protozoea II (PZ II) to postlarva XII-XIV (PL XII-XIV). PK activity reached a pronounced peak in PL V-VI, followed by a further decrease in PL XII-XIV. Temperature reduction produced variation in oxygen consumption rates (QO(2)), ammonia-N excretion and in enzyme activities. Ammonia-N excretion was higher at 20 degrees C in mysis III (M III), PL V-VI and PL XII-XIV, resulting in substantially lower O:N ratios in these stages. QO(2) was increased in protozoea II (PZ II) and mysis I (M I) at 26 degrees C, while no difference in QO(2) was detected in the subsequent stages at either temperature. This fact coincided with higher CS and PK activities in M III, PL V-VI and PL XII-XIV at 20 degrees C compared with 26 degrees C. Regressions between individual enzyme activities and dry weight exhibited slope values of 0.85-0.92 for CS and 1.1-1.2 for PK and temperature reduction was reflected by higher slope values at 20 than at 26 degrees C for both enzymes. Weight-specific CS activity was positively correlated with QO(2) at 20 and 26 degrees C, and may thus be used as an indicator of aerobic metabolic rate throughout the early stages of F. paulensis. The variation in enzyme activities is discussed in relation to possible metabolic adaptations during specific ontogenetic events of the F. paulensis life cycle. Here, the catalytic efficiency of energy-metabolism enzymes was reflected in ontogenetic shifts in behaviour such as larval settlement and the adoption of a benthic existence in early postlarvae. In most cases, enhanced enzyme activities appeared to counteract negative effects of reduced temperature.

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

SummaryChitinase and N-acetyl-β-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-β-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.

Ultrastructure of the integument of a pelagic Crustacean: moult cycle related studies on the Antarctic krill, Euphausia superba

The ultrastructure of euphausiid integument was examined in relation to the moult cycle and supplemented by investigations of chitinase activity in the integument and content of N-acetyl-β-D-glucosamine in the hemolymph. The Antarctic krill, Euphausia superba was collected in 1983 in Admiralty Bay, King George Island, Antarctica. Some specimens of the Northern krill, Meganyctiphanes norvegica, from the Danish Kattegat served for comparison. As a major aim of the study, the moult staging system developed for living tissue could be verified by ultrastructural findings. Under experimental high production conditions of the Antarctic summer, no period of rest or “intermoult” between post- and premoult was observed in subadult E. superba. Neither was a resting phase seen at the cellular level, the epidermis remained active. The epidermal gland cells did not show any cyclical changes, and the organelles of protein synthesis were generally well developed in all moult stages. In order to follow the physiological course of events, structural and biochemical methods were combined and showed as a result that the last moult stage before ecdysis is characterized by massive cuticular resorption. The epicuticle remained ultrastructurally unchanged before and after ecdysis, even though its permeability should alter at ecdysis. The existence of muscle insertions which connect the old and the new cuticle across the exuvial space suggests an answer to the question why E. superba is hardly impaired in swimming almost up to the time of ecdysis.

Isoforms of an N-acetyl-beta-D-glucosaminidase from the Antarctic krill, Euphausia superba: purification and antibody production.

Two forms of the chitinolytic enzyme N-acetyl-beta-D-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.