R.H.C. Strang

Physiological and metabolic responses of the shore crab Carcinus maenas (L.) during environmental anoxia and subsequent recovery

The degree of hypoxia experienced by Carcinus maenas (L.) in littoral rock pools varies throughout the year. In summer, conditions may become extremely hypoxic or even anoxic at night. Crabs which remain in the pools utilize anaerobic metabolism for energy production as indicated by an increase in tissue lactate. In the laboratory, l-lactate was found to be the major end-product of anaerobic metabolism for although fumarate and alanine accumulated during anoxia, the amounts accumulated indicate that alternative metabolic pathways are of only very limited importance in C. maenas. Survival for long periods under anoxic conditions necessitates a considerable reduction in metabolic rate. Calorimetric measurements indicated that, during anoxia, the metabolic rate of C. maenas is reduced to <20% of the normoxic rate. Exposure to anoxia resulted in significant acid-base disturbances. Although a marked metabolic acidosis developed, there was no indication that exoskeletal carbonates were important in buffering these changes. Recovery from anoxia resulted in a pronounced increase in the rates of O2 consumption and heart rate. A transient but significant increase in tissue lactate concentration was observed during the initial stages of recovery suggesting that anaerobic glycolysis may also be important in meeting the high energy demands of the crabs at this time.

The antimitotic effect of the neem terpenoid azadirachtin on cultured insect cells

When cultured insect cells (Sf9) were grown in the presence of 5 x 10(-6) M azadirachtin, there was a rapid increase in the mitotic index, with the appearance of many aberrant mitotic figures. Flow cytometry established that cells accumulated in the G2/M phase of the cell cycle, and that the effect was concentration-dependent. At 10(-8) M a period of 20 h was necessary to raise the proportion in G2/M to 42% above the control values, but at 5 x 10(-6) M more than 90% of the cells were in this phase. Azadirachtin had the same effect on C6/36 mosquito cells, but failed to affect L929 murine fibroblast cells even at a concentration of 10(-4) M over 72 h. Experiments with colchcine and taxol showed similarities of action between azadirachtin and colchicine, and azadirachtin was apparently able to displace colchicine-fluorescein from binding-sites in living insect cells. Another similarity between azdirachtin and colchicine was that both phytochemicals prevented the polymerisatrion in vitro of mammalian tubulin, although the azadirachtin was much less effective.

Effect of aerial exposure on concentrations of selected metabolites in blood of the Norwegian lobsterNephrops norvegicus (Crustacea: Nephropidae)

The Norwegian lobsterNephrops norvegicus (L.) collected from Firth of Clyde, Scotland between December 1987 and March 1988, was unable to survive longer than 18 h experimental emersion at 10°C. During this time the partial pressure of oxygen (PνO2) in the venous blood decreased rapidly and the lobster supplemented cellular energy requirements by anaerobic metabolism. This was indicated by the rapid accumulation ofL-lactate in the blood. Although the survival rate increased (to ca 36 to 48 h) if lobsters were kept on ice, the accumulation ofL-lactate in the blood was not significantly different from lobsters at 10°C, despite the temperature difference. There was no indication thatN. norvegicus was able to further metabolize circulatingL-lactate during emersion. On emersion there was also a marked hyperglycemia in the blood due to the stress of handling and asphyxiation. There was fairly good agreement between results obtained during laboratory studies and simulated fishing activity in the Firth of Clyde. Both sets of results are discussed in the context of adaptation to air breathing within the Crustacea and an assesment of post-harvest treatment of lobsters.

The distribution of taurine in the tissues of some species of insects

Abstract Taurine concentration was measured in the tissues of Schistocerca americana gregaria, Blatella orientalis and Tenebrio molitor and was found to be present in all those examined. In the locust Schistocerca gregaria it was found in particularly high concentration in active flight muscle (26 μmol/g) to a lesser extent in the eye (7 μmol/g). The thoracic concentration of taurine in developing locusts showed a strong correlation with the development of flight muscle, increasing from 4.4 to 11.3 μmol/g in the thorax during the first 24 days of adult life. Analysis of the thoracic content of taurine in adults of the three species examined confirmed that high taurine concentrations are associated with fully functional flight muscle. The concentration in the thorax of the flightless flour beetle T. molitor was only 1.79 μmol/g compared to 11.33 μmol/g for the locust. Stress due to flying and picrotoxin poisoning caused the release of taurine from the muscles into the haemolymph, causing the concentration to rise from 1.1 to 2.2 and 5.76 μmol/g respectively. Analysis of the distribution of arginine kinase showed that this release was not due to breakdown of the muscle tissue.

Radioisotope studies of the energy metabolism of the shore crab Carcinus maenas (L.) during environmental anoxia and recovery

Under both normoxic and anoxic conditions, 14C from d-[U14C]-glucose injected into the haemolymph of Carcinus maenas (L.) was rapidly incorporated into various metabolic fractions. Under both conditions, the greatest proportion of label was found in the amino acid fraction (>70%). During normoxia, the steady accumulation of acid labile, volatile 14C in the water indicated the complete oxidation of the glucose to CO2. Under both normoxia and anoxia, a considerable part of the label appeared in glycogen. The main differences between aerobic and anaerobic conditions were (a) the slower disappearance of label from the neutral sugar fraction and (b) the accumulation of label in glycolyctic phosphate material under anaerobic conditions. The proportion of label in lactate doubled during the 1st h of recovery period under normoxic condition, increasing to a maximum of 23% of the non-amino acid radioactivity before decreasing during the rest of the recovery period. Label from l-[U14C]-lactate injected into crabs immediately before they were returned to normoxic conditions following exposure to anoxia was partly oxidased, appearing as acid labile material in the medium. Most of the injected label, however, was retained within the animal and appeared as glycogen and subsequently in the amino acid fraction. There was no evidence for the excretion of lactate.

Effect of Taurine on Neurotransmitter Release from Insect Synaptosomes

Abstract: The effect of taurine on the release of [3H]acetylcholine ([3H]ACh) and [3H]‐γ‐aminobutyric acid ([3H]GABA) from preloaded locust synaptosomes has been studied. Veratridine (100 μM) and K+ (100 mM) both evoked [3H]ACh release and this was reduced in a concentration‐dependent manner by taurine (5, 10, and 20 mM). In contrast to this, veratridine induced no observable release of [3H]GABA, and the response to K+ was slight. In the presence of taurine, however, a concentration‐dependent enhancement of [3H]GABA release was observed. Since nipecotic acid (1 mM), an inhibitor of neuronal GABA uptake, also revealed [3H]GABA release induced by veratridine, it is suggested that both this effect and that of taurine are due to prevention of GABA reuptake. These results suggest that taurine may act as a neuromodulator in insects.

A COMPARISON OF SOME ASPECTS OF THE PHYSIOLOGY AND METABOLISM OF THE NERVOUS SYSTEM OF THE LOCUST SCHISTOCERCA GREGARIA IN VITRO WITH THOSE IN VIVO

Abstract— Studies were made on isolated ganglia of the locust in a modified O2 electrode. The basic requirements of a suitable incubation medium were determined and found to include saturation of the medium with 100% O2, and 10mm‐glucose as energy source. The effects of incubation in vitro were determined for a number of biochemical characteristics including O2 uptake and the concentrations of Arg P, ATP, ADP, glycogen, glucose, lactate and pyruvate. Action potentials were recorded from a major nerve both in vivo and in vitro. On the basis of these biochemical and physiological studies, it was concluded that the tissue functions well in vitro. Some comparisons were made between the metabolic characteristics found in the locust nervous system, and those of mammalian tissue. The locust ganglia were found to have a rate of O2 uptake much higher than comparable mammalian tissue, and to contain a considerable reserve of glycogen. The low ratio of lactate/pyruvate suggests a more aerobic metabolism than is the case in mammalian brain.

Effect of Taurine on Calcium Accumulation in Resting and Depolarised Insect Synaptosomes

Abstract: The effect of taurine (2‐aminoethanesulphonic acid) on 45Ca2+ accumulation in resting and depolarised synaptosomes obtained from the locust Schistocerca americana gregaria was studied. Taurine reduced 45Ca2+ accumulation in resting synaptosomes, and this effect was more pronounced when synaptosomes were depolarised with either high [K+] or veratridine. Veratridine‐induced 45Ca2+ accumulation was not affected by either γ‐aminobutyric acid or leucine, but was reduced by both verapamil and tetrodotoxin.

Electrophysiological responses of isolated locust (Schistocerca gregaria) somata to taurine and GABA application

Abstract The comparative effects of applied taurine and GABA on isolated locust neural somata has been studied. Iontophoretically applied taurine and GABA both hyperpolarized and decreased membrane resistance in the preparation. GABA was markedly the more potent of the two amino acids in this respect. Both amino acids showed similar reversal potentials (around −75 mV) when tested on somata held at a series of membrane potentials. The actions of both amino acids were antagonized by picrotoxin and the proposed specific taurine antagonist 6-aminomethyl-3-methyl-4 H -1,2,4-benzthiadiazine-1,1-dioxide hydrochloride. Responses to both amino acids were augmented by the benzodiazepine flunitrazepam. The data suggest that taurine and GABA act at a common site in locust somata.

Biosynthesis of taurine in tissues of the locust (Schistocerca americana gregaria) and the effect of physiological and toxicological stresses on biosynthetic rate of this amino acid

Abstract The biosynthesis of the amino acid taurine has been studied in the locust Schistocerca americana gregaria . Tissue concentrations of putative precursors of taurine, and taurine itself were estimated in haemolymph, nervous tissue and muscle. Following this locusts were injected with 20 μCi of [ 35 S]cysteine and tissue samples were taken at times thereafter, and taurine and its precursors were extracted and separated by thin-layer chromatography (TLC). Taurine, cysteine sulphinic acid (CSA) were quantified by scintillation counting. Cysteic acid and hypotaurine were qualitatively detected by autoradiography of TLC plates. Results indicate that in haemolymph and muscle the biosynthetic pathway for taurine is cysteine then CSA, cysteic acid and thereafter taurine. In nervous tissue both cysteic acid and hypotaurine were detected suggesting two possible pathways for taurine since both are immediate precursors of this amino acid. Taurine biosynthesis was markedly greater in juvenile compared with mature adult locusts. It was also observed that picrotoxin treatment and prolonged flying, which have both previously been found to cause a redistribution of taurine in the locust, increased the biosynthesis of this amino acid.