Cyclic AMP-dependent protein kinase: role in anoxia and freezing tolerance of the marine periwinkle Littorina littorea

A key regulatory mechanism underlying the switch between aerobic and anaerobic metabolism amongst anoxia-tolerant marine molluscs is reversible protein phosphorylation. To assess the role of cAMP-dependent protein kinase (PKA) in aerobic–anaerobic transitions, the effects of anoxia on the activity and subcellular distribution of PKA were assessed in foot and hepatopancreas of the marine periwinkle, Littorina littorea. Exposure to N₂ gas at 5 °C caused a rapid decline in the percentage of total enzyme present as the free catalytic subunit (PKAc) in both tissues; the percentage of PKAc fell from ∼30% in controls to 3% after 1 h anoxia and remained low over 72 h. Total PKA also fell by 30% after 72 h anoxia in hepatopancreas but rebounded during aerobic recovery. Freezing at −8 °C elicited parallel results for both percentage of PKAc and total PKA, suggesting that PKA responses to freezing were stimulated by the ischemia that develops when hemolymph freezes. Anoxia also led to a shift in PKA subcellular distribution in hepatopancreas (but not in foot), the percentage of total PKA activity associated with the nuclear fraction dropping from 25% in controls to 8% in 12 h anoxic snails with opposite changes in the cytosolic fraction. The catalytic subunit (PKAc) of foot PKA was purified to a final specific activity of 63.5 nmol phosphate transferred per minute per milligram protein. Enzyme properties included a molecular weight of 33 to 35 kDa, an activation energy from Arrhenius plots of 65.1 ± 4.8 kJ mol⁻¹, and substrate affinity constants of 151 ± 6 μM for the phosphate acceptor, Kemptide, and 72 ± 9 μM for Mg.ATP. Activity was strongly reduced by mammalian PKA inhibitors (H-89, PKA-I), by neutral chloride salts (I₅₀ values 165 to 210 mM) and by NaF (I₅₀ 62 mM). Reduced PKA activity under anoxic or freezing conditions would facilitate the observed suppression of the activities of numerous enzymes that are typically PKA-activated and thereby contribute to the overall anoxia-induced metabolic rate depression. Received: 19 November 1997 / Accepted: 30 September 1998     

Movements of tiger sharks (Galeocerdo cuvier) in coastal Hawaiian waters

The giant clam Tridacna crocea harbors in the mantle tissue symbiotic microalgae commonly called zooxanthellae. Isolated zooxanthellae release glycerol into the medium in the presence of mantle tissue homogenate (MH), but it is not clear whether the cells do so in situ. In order to determine the photosynthetic products released by zooxanthellae in the mantle of the giant clam we traced photosynthetic fixation products from ¹³C- and ¹⁴C-bicarbonate both in the clam and in isolated zooxanthellae (IZ) in the presence or absence of MH. After 15 min incubation in the absence of MH the IZ released less than 0.6% of the fixed labeled carbon, mainly as glucose. The major intracellular photosynthates were neutral lipids, which constituted 20 to 40% of the total extractable ¹⁴C. In the presence of MH, the IZ released up to 5.6% of the total fixed ¹⁴C, mostly as glycerol, and the major intracellular photosynthate was glucose. In an intact clam incubated in sea water containing ¹⁴C-bicarbonate, 46 to 80% of the fixed ¹⁴C was translocated from the zooxanthellae to the host tissues. Most of the ¹⁴C in the hemolymph, in the isolated zooxanthellae and in intact mantle tissue (containing zooxanthellae) was recovered as glucose. No ¹⁴C-glycerol was detected in the mantle after 1 to 30 min incubation, and, even after 60 min, far less ¹⁴C-glycerol was synthesized than by IZ in the presence of MH. The possibility that in clam tissue glycerol is converted to glucose was examined by tracing the labeled carbon from ¹⁴C-glycerol injected into the adductor muscle. After 5 min incubation, no labeled glucose was found in the hemolymph, but after 60 min, some 20% was found as glucose. Thin slices containing zooxanthellae, cut from the surface of the mantle, fixed inorganic carbon supplied as NaH¹⁴CO₃ in the medium and mainly released ¹⁴C-glucose. The addition of MH to the surrounding medium did not affect the release rate or form of release product. When the slices were cut into smaller pieces, however, the ratio of glycerol to glucose in the release product increased. These results indicate that in the presence of MH the metabolism of isolated zooxan- thellae was different from that of zooxanthellae in the mantle. In the presence of MH, isolated zooxanthellae release mostly glycerol, whereas in the mantle they release glucose. Received: 18 February 1998 / Accepted: 4 December 1998     

Primary cell-culture of the digestive gland of the marine mussel Mytilus edulis: a time-course study of antioxidant- and biotransformation-enzyme activity and ultrastructural changes

The extent to which the American lobster, Homarus americanus (H. Milne-Edwards), utilizes estuarine habitats is poorly understood. From 1989 to 1991 we examined lobster movements in and around the Great Bay estuary, New Hampshire using tag/recapture and ultrasonic telemetry. A total of 1212 lobsters were tagged and recaptured at sites ranging from the middle of Great Bay, 23.0 km from the coast, to Isles of Shoals, 11.2 km offshore. Twenty-six lobsters equipped with ultrasonic transmitters were tracked for periods ranging from 2 weeks to >1 year. Most lobsters moved <5 km toward the coast, with those furthest inland moving the greatest distance. Lobsters with transmitters moved in a sporadic fashion, with residency in one area for 2 to 4 weeks alternating with rapid movement to a new location (mean velocity = 0.3 km d⁻¹, 1.8 km d⁻¹ max.). Site of release influenced distance moved, but there was no significant relationship between lobster size and distance traveled, days at large, or rate of movement. Most movement into the estuary occurred in the spring, while during the remainder of the year there was a strong tendency to move downriver, toward the coast. These seasonal migrations of estuarine lobsters may enhance their growth and survival by enabling them to avoid low salinity events in the spring and fall, and to accelerate their growth in warmer estuarine waters during the summer. Received: 26 January 1996 / Accepted: 22 January 1999     

Simple molecular method to distinguish the identity of Calanus species (Copepoda: Calanoida) at any developmental stage

Diagnostic morphological characteristics of copepods of the genus Calanus are restricted largely to minor variations in secondary sex characteristics. This presents a persistent problem in the identification of individuals to species level, especially for immature stages. We have developed a simple molecular technique to distinguish between the North Atlantic Calanus species (C. helgolandicus, C. finmarchicus, C. glacialis and C. hyperboreus) at any life stage. Using the polymerase chain-reaction (PCR), the mitochodrial large subunit (16S) ribosomal RNA (rRNA) gene was amplified from individual copepods preserved in ethanol. Subsequent digestion of the amplified products with the restriction enzymes DdeI and VspI, followed by electrophoretic separation in 2% agarose (Metaphor, FMC Ltd), produced a characteristic pattern for each species. The versatility of the method is demonstrated by the unambiguous identification to species of any life stage, from egg to adult, and of individual body parts. Received: 11 May 1998 / Accepted: 5 August 1998     

Production of glycine betaine and dimethylsulfoniopropionate in marine phytoplankton. II. N-limited chemostat cultures

The influence of salinity on the time elapsed between two successive molts and the size reached after each molt were studied at 30, 21, 12 and 3‰S in juveniles of two co-occurring grapsid species, Cyrtograpsus angulatus and C. altimanus, cultured under identical conditions of temperature, photoperiod and food. Juvenile growth patterns were compared between these species (which differ in size-at-maturity and maximum size). C. angulatus grew faster than C. altimanus, reflecting a higher increment per molt and a shorter intermolt period. A significant difference existed between the number of instars preceding the size of maturity in both species: >11 in C. angulatus, 6 in C. altimanus. There was evidence of a differential effect of low salinity on growth. By the end of the experiment, individuals of both species were smaller at the lowest salinity (3‰) tested; the largest crabs developed at 21‰ (C. angulatus) and 30‰ (C. altimanus). The size difference between the “optimal” and the less suitable salinities in the sixth crab instar was 12.4% in C. angulatus and 35% in C. altimanus. During early juvenile development (Crab Instars 1 to 4), there were slight differences in intermolt period among salinities in C. angulatus, but large differences in C. altimanus. The longest intermolt period of C. altimanus was at 3‰S and the shortest at 30‰S. In the following instars (5 to 10 in C. angulatus and 5 to 6 in C. altimanus), the longest intermolt period occurred at 21‰S, the shortest at 3‰S, in both species. Interspecific differences in response to low salinities may explain why C. angulatus occurs throughout a whole temperate coastal lagoon, whereas C. altimanus is restricted to its mouth. Received: 12 July 1998 / Accepted: 24 February 1999     

Photoperiodic control of growth and segment proliferation by Nereis (Neanthes) virens in relation to state of maturity and season

The effects of two fixed photoperiodic regimes on the rate of somatic growth and segment proliferation in the semelparous polychaete Nereis (Neanthes) virens Sars has been investigated. The two photoperiods (16 h light:8 h dark and 8 h light:16 h dark, hereafter LD 16:8 and LD 8:16) were close to the extremes experienced naturally by N. (N.) virens in the course of a year. The experiments were carried out during the ambient winter and all worms (including four-setiger larvae raised out of season) showed elevated feeding, growth and enhanced segment-proliferation rates when reared under LD 16:8, compared to worms reared under LD 8:16. The rate of replacement of lost segments after caudal ablation was also higher under LD 16:8. The number of segments present when the segment proliferation rate had fallen to zero was higher in individuals grown from birth under LD 16:8 compared to those grown under LD 8:16. Feeding activity showed a degree of spontaneous recovery under LD 8:16 from January onwards, but remained lower than under LD 16:8. This difference continued into the following spring, and was not attributable to differences in sexual maturity. We conclude that the rate of feeding in N. (N.) virens and other indicators of somatic growth rate such as rates of caudal regeneration and segment proliferation are directly influenced by the photoperiod, but that the seasonal cycle of growth also includes a circa-annual component modifying the response to static photoperiods according to the time of year. Received: 20 October 1998 / Accepted: 4 February 1999     

Early life history and recruitment of the tropical eel Anguilla bicolor pacifica, as revealed by otolith microstructure and microchemistry

Otolith microstructure and microchemistry of the tropical eel Anguilla bicolor pacifica Schmidt were examined in glass eels collected at the mouth of the Dumoga River, North Sulawesi Island, Indonesia. Ages of the glass eels examined (age at recruitment) ranged from 124 to 202 d (167 ± 19.3 d; mean ± SD), hatching being estimated as having occurred between November 1995 and March 1996. Otolith increment widths markedly increased from age 101 to 172 d (135 ± 18.2 d; mean ± SD), coincident with a drastic decrease in otolith Sr:Ca ratios, suggesting that metamorphosis began during that period. The duration of metamorphosis was estimated as 20 to 40 d, on the basis of otolith microstructural characteristics. The fluctuation patterns in otolith increment widths and Sr:Ca ratios were similar to those of the temperate Japanese eel A. japonica. Received: 20 May 1998 / Accepted: 7 October 1998     

Estimation of Normal Variance Based on Balanced and Unbalanced Ranked Set Samples

In this paper we address the problem of estimation of the variance of a normal population based on a balanced as well as an unbalanced ranked set sample (RSS), which is a modification of the original RSS of McIntyre (1952).We have proposed several methods of estimation of variance by combining different unbiased between and within estimators, and compared their performances     

A study of the genetic relationships within and among wolf packs using DNA fingerprinting and mitochondrial DNA

Summary DNA fingerprinting and mitochondrial DNA analyses have not been used in combination to study relatedness in natural populations. We present an approach that involves defining the mean fingerprint similarities among individuals thought to be unrelated because they have different mtDNA genotypes. Two classes of related individuals are identified by their distance in standard errors above this mean value. The number of standard errors is determined by analysis of the association between fingerprint similarity and relatedness in a population with a known genealogy. We apply this approach to gray wolf packs from Minnesota, Alaska, and the Northwest Territories. Our results show that: (1) wolf packs consist primarily of individuals that are closely related genetically, but some packs contain unrelated, non-reproducing individuals; (2) dispersal among packs within the same area is common; and (3) short-range dispersal appears more common for female than male wolves. The first two of these genetically-based observations are consistent with behavioral data on pack structure and dispersal in wolves, while the apparent sex bias in dispersal was not expected.USA Offprint requests to: N. Lehman at the present address     

Studies on feeding repellents for fishes and shellfishes

The present study on chemotaxis activity was conducted between May 1989 and March 1990. In order to establish a chemotaxis index, with special reference to a repellence index of food for black abalone Haliotis discus, a logistic curve y=g/{1+ exp [-r(j-a)]}, where g, r and a are constants, was applied to time-courses of abalone responses to fresh and spoiled algal extracts. The time-courses were respresented by the number of individuals (y _i) displaying three behaviors (entering. leaving and remaining in a test compartment with algal sample) over time (j=1 to 10, 2-min intervals). Repellence and attraction specimens were prepared from a homogenate of green laver Ulva pertusa allowed to spoil for 2, 4 and 6 d at 40°C, and from fresh homogenate, respectively. Of 32 time-courses (estimated according to a method whereby the observed number was weighted by j for each behavior, involving 30 individuals), 32 entered, 19 left and 19 remained time-courses fitted the curve at the 0.10 level, as determined by a chi-square test. A general evaluation of repellence and attraction, determined by observable behavior, agreed well with the difference in a between the dummy (control) and specimen curves, and with the ratio of a in the dummy curve to a in the specimen curve, for logistic curves fitted to the entred time-course. The difference was >0 and the ratio was >1 in the repellence index, and <0 or <1 respectively in the attraction index. The validity of the indexes was ascertained using food specimens prepared from three kinds of algae.