Insect Muscle Research Articles

Denervation of insect muscle: A comparative study of the changes in L-Glutamate sensitivity on locust retractor unguis and extensor tibiae muscle

The sensitivity of the extrajunctional membrane of the locust retractor unguis (R.U.) muscle to microiontophoretic application of l-glutamate was compared with that of the extensor tibiae (E.T.) muscle. The distribution of extrajunctional D-receptors for l-glutamate on R.U. muscle was more uniform than on E.T. muscle. Chronic denervation (at 30°C) caused an increase in the mean extrajunctional D-sensitivity of both muscles, complete development of which occurred 12–14 days post-denervation for R.U. muscle and 10–12 days post-denervation for E.T. muscle. The maximum mean extrajunctional glutamate D-sensitivity of denervated R.U. fibres was about 20 times greater than the sensitivity of innervated control fibres, while the increase for denervated E.T. fibres was about 6-fold. For both muscles the increase in glutamate sensitivity was only partially accounted for by an increase in the input resistance of the fibres after denervation. The extrajunctional D-receptors on denervated R.U. muscle were uniformly distributed, but on E.T. fibres there was considerable variation in local glutamate sensitivity. Between 20–28 days post-denervation the extrajunctional sensitivity of both muscles declined at a time when muscle atrophy was evident. The reversal potential of extrajunctional glutamate responses of denervated muscle, determined using voltage-clamp, was +3.5 ± 2.6 mV (mean ± S.D., n = 8), similar to the reversal potential of junctional glutamate responses of both innervated and denervated fibres.

Permeation of sodium through calcium channels of an insect muscle membrane

The contribution of Na ions to the electrically excited response was studied in the muscle fibres of mealworm larvae, Tenebrio molitor, using microelectrode techniques. When Ca ions were omitted from the external solution, no action potential could be elicited. However, addition of Na ions to Ca-free medium rendered the fibre excitable again. The amplitude of these action potentials increased with a slope of about 40 mV for a 10-fold elevation of external Na concentrations. Tetrodotoxin had no effect on the initiation of the spike, and Co ions completely suppressed it. Therefore, it seems likely that a Ca-channel, which is utilized by both Na and Ca ions, is the sole factor responsible for the action potential in the mealworm larval muscle fibre membrane.

Polyphosphoinositides in insect muscle and sensory tissues

Polyphosphoinositides in insect muscle and sensory tissues

Programmed cell death. Electrophysiological and ultrastructural correlations in metamorphosing muscles of lepidopteran insects

Individual spontaneously degenerating fibers of moths have been examined successively by electrophysiological and ultrastructural techniques. The correlations observed by this method have revealed two phases of lysis. The first lytic phase begins at or before the ecdysis of the moth. It is characterized by a probable but occult respiratory lesion, normal movement of the ions across the plasma membrane, and limited degradation of myofilaments and autophagy of mitochondria. There are nevertheless early degenerative changes in the Z-band and a slight swelling of the T system. The second lytic phase is introduced, at approximately 15 hr after ecdysis, by collapse of the tracheal airways. The collapse of the tracheae is followed within 2 or 3 hr by a drastic reduction of input resistance, depolarization of the fiber, and loss of contractility. The rate of proteolysis increases, and the remaining myofilaments lose all structural identity by the twentieth hour. During this period of rapid change, the electron density of the reticular portion of the dyads decreases before a compactive process increases their opacity. Neural elements degenerate secondarily. The early lytic phases are apparently non-lysosomal for the myofilaments, although organelles such as mitochondria, ribosomes, and glycogen are destroyed in autophagic vacuoles. Elimination of oxygen and collapse of the resting potential, with the presumed equilibration of intracellular ionic content, provokes a rapid dissolution of the myofilament, with continued autophagic elimination of the organelles.

A study on the mode of action and composition of a toxin from the female abdomen and eggs of Arctia caja (L.) (Lep. Arctiidae): An electrophysiological, ultrastructural and biochemical analysis

Cajin, the toxic principle extracted from the female abdomen and eggs of the aposematic moth Arctia caja, was investigated by means of column chromatography, and its effects on tissues were studied by electrophysiology and electron microscopy. Partial purification suggests that one fraction is a polypeptide with a molecular weight (mol. wt) of about 1000. It is lethal to both mammals and insects if injected into the body, but inactive via the oral route. Crude extracts applied to insect muscles produce a slow contracture which lasts for over 1 1 2 hr and is irreversible. Fine structural analyses of insect tissues after 5 min toxin treatment reveal disruption of both internal membranous systems and mitochondria. Unlike many other toxins, cajin appears to act by stimulating the muscle directly. The results of experiments involving Ca 2+-free solutions plus cajin suggest that it may increase the permeability of the muscle membrane to Ca 2+, perhaps behaving as a calcium ionophore, or may act as an analogue of an insect neurotransmitter.

The inhibitory action of L-glutamic acid esters on the insect neuromuscular junction

1. γ-Methyl and γ-ethyl esters of L-glutamic acid in 10 mM concentration blocked the mealworm larval neuromuscular transmission while diesters of L-glutamic acid were less effective. 2. The amplitude of the intra- and extracellular EPSPs was reduced by the treatment of 10 mM γ-esters in a few minutes. 3. The result suggest that the glutamte receptor in insect muscles resembles to that of the crayfish muscle and differs to some degree from the glutamate receptor on mammalian central neurones.

Mechanical properties of succinylcholine activated muscle fibers in the inferior oblique muscle of the cat.

To determine the mechanical properties of the multi-innervated fibres in inferior oblique muscle of the cat, these fibers were selectively activated by means of an intravenous injection of succinylcholine chloride (Sch). For small sinusoidal length changes (+/- 1 mm) of low frequency, tension in the Sch activated muscle exhibited a component that was sensitive to the direction of stretch but was independent of the velocity. At higher frequencies of length change (16 Hz) a form of negative friction was observed. Negative friction had not been previously noted in mammalian muscles, although a similar friction was observed both in the muscles of insects and amphibians. These properties were expressed in the form of a simple mechanical model.

Intrinsic rhythm and basic tonus in insect skeletal muscle.

The jumping muscle of orthopterous insects contains fibres that possess an intrinsic rhythm (IR) of slow contraction. The contributing fibres are generally synchronized, but as many as three or four pacemakers are present. The frequency, amplitude and duration of IR contractions fluctuate erratically over a 24 h period. Metathoracic DUM neurone bursts suppress IR for a few minutes. Other, unidentified dorsal neurones enhance its amplitude. In addition to IR, the extensor tibiae shows intrinsic basic tonus (BT). BT is relaxed for several s by low-frequency burst output from unidentified metathoracic dorsal neurones. DUM neurone bursts may enhance extensor BT, relax it, or leave it unaffected. The effects on IR of various regimes of activity in the slow extensor tibiae (SETi) and the common inhibitor (CI) axons were examined. CI affects IR when stimulated at frequencies above 2 Hz. It causes amplitude depression and reduced duration of individual IR contractions as well as increased frequency. At 30 Hz and above, CI completely suppresses IR. An enhanced IR contraction starts within a few milliseconds of the termination of a CI train. At low frequencies (below 10 Hz) SETi causes increased frequency and decreased amplitude of IR, with a depressed IR contraction following cessation of the SETi burst. At frequencies above 15 Hz the SETi-evoked contraction dominates tension development, though IR summates with it during the rising phase. In quiescent preparations not showing IR, SETi stimulation at 10 Hz often started up IR. Single SETi or FETi impulses can initiate an IR contraction, and cause altered phasing, with up to a quintupling of frequency. After a critical period has elapsed following the onset of an IR contraction, a single single impulse in any one of the three axons will terminate it abruptly. The early termination is followed by a reduced interval which is proportional to the reduced IR contraction time. The rhythm of accumulated readiness to go into an IR contraction is independent of the pacemaker rhythm that initiates the contraction.

Identification et biologie du Gongylonème parasite du Macroscélide en Tunisie

Its larval form obtained from experimentally infected intermediate hosts, differs from those of other Gongylonematids. In the genus Gongylonema four types of larvae were recognized. They are characterized by the size and the cephalic and caudal structures of the larvae. The cellular reactions caused by the larvae of G. brevispiculum in the insect muscles are similar to those caused by infections with larvae of Acuarid nematodes.

Differential effect of anaerobiosis on phospholipid metabolism in insect muscle

An in vivo anaerobic exposure of 80 min duration induces little change in the rate of incorporation of 32P into flight muscle phospholipids of male American cockroaches taken 1–3 days after adult ecdysis. In mature animals (1–3 months after adult ecdysis) the incorporation rate is strongly depressed, phosphatidylcholine and phosphatidylethanolamine being distinctly more affected than phosphatidylinositol. During 60 min recovery from anaerobiosis only phosphatidylinositol reaches the specific radioactivity found in control muscles. In phosphatidylcholine and phosphatidylethanolamine not even the control rate of labelling is attained during this period. The results are discussed.

Initiation of Ca-spikes from an insect muscle fiber immersed in a low pH saline solution containing carboxylic anions

Skeletal muscle fibers of beetle larvae, Xylotrupes dichotomus L., which were inexcitable in the standard saline solution at pH 7.4 became capable of generating all-or-none spikes when a certain amount of acetate was added to a low pH saline solution. The optimum condition for the spike initiation was the presence of 20–30 mM acetate at pH 6. The spikes thus elicited were pure Ca-spikes as they were dependent on the external Ca ++ concentration but were not inhibited either by removal of the external Na + or by application of tetrodotoxin. This facilitation of the Ca-spike generation was probably due to a combined action of acetate and of the low pH upon the muscle membrane.

Ultrastructural study of the normal degeneration of the intersegmental muscles of Anthereae polyphemus and Manduca sexta (Insecta, Lepidoptera) with particular reference of cellular autophagy.

AbstractIn studying ultrastructural changes in metamorphosis‐related degeneration of intersegmental muscles in Antheraea polyphemus, particular attention was directed to the mechanisms and timing of degradation of organelles and myofilaments. At emergence, the muscles are typical slowly contracting insect muscles, with a few dense body lysosomes and occasional autophagic vacuoles containing mitochondria. During the early phases of degradation the number of autophagic vacuoles, dense bodies, and lamellar bodies increases rapidly, along with an expansion of the Golgi system and the T system. Free glycogen particles and glycogenosomes are demonstrated by the PATAg test.Between 7 and 20 hours after ecdysis the T system continues to expand, the fibers subdivide, and the contractile system is degraded. Myofibrils fragment; myofilaments are not enclosed in isolating membranes at the time of their dissolution. The destruction of individual filaments occurs rapidly, with few intermediate stages being seen, while thick filaments tend to disappear earlier than thin filaments and Z‐line material. The process is generalized and not confined to specific regions of the fiber. Autophagy destroys cell organelles in apparent synchrony with the first signs of nuclear pycnosis.By 20 to 30 hours after emergence, the fibers are reduced to lamellae of polynucleate sarcoplasm containing no organized contractile material. The sarcoplasm is filled with autophagic vacuoles containing mitochondria, dense lamellar or residual bodies, and ribosome‐rich sarcoplasm. The number of mitochondria is drastically reduced at this time.In the final phases of involution (40–49 hours after emergence) shedding of the residual sarcoplasm precedes the expulsion of the pycnotic nuclei into the hemocoele.These results indicate that autophagy is responsible for the selective destruction of mitochondria, glycogen particles, ribosomes, and other organized sarcoplasmic structures. The one exception is the dissolution of the myofilaments, a process which remains undefined but which appears to be independent of lysosomal activity.

L-Arginine kinase from tobacco hornworm, Manduca sexta (L.). Purification, properties, and interaction with L-canavanine.

Arginine kinase (adenosine 5'-triphosphate: L-arginine phosphotransferase, EC 2.7.3.3) was purified from the larvae of the tobacco hornworm, Manduca sexta (L). This enzyme catalyzes the production of L-phosphoarginine, which is the principal reserve of high energy phosphate compounds in insect muscle. The enzyme also phosphorylates L-canavanine, a guanidinooxy analogue of arginine which severely disrupts all developmental stages of this insect. Evaluations of certain kinetic and thermodynamic parameters of the reactions with arginine and canavanine suggest that reactions known to be much more sensitive to canavanine, such as protein synthesis or genome expression, rather than phosphagen formation and function account for the pronounced toxicity of canavanine in this insect. Sedimentation equilibrium and electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate indicate that this insect enzyme has a molecular weight of about 40,000. This value is consistent with molecular weights of arginine kinases of non-insect arthropods. Its amino acid composition is also very similar to that of other arthropod arginine kinases. Km values for the enzyme are: L-arginine, 0.5 mM; Mg-ATP, 2.5 mM; L-canavanine, 22 mM; L-phosphoarginine, 0.7 mM; Mg-ADP, 0.45 mM; and L-phosphocanavanine, 27 mM. Turnover numbers (expressed as moles of product per min per mol of enzyme) are: L-arginine, 8,320; L-canavanine, 1,635; L-phosphoarginine, 25,875; and L-phosphocanavanine, 3,040. The apparent equilibrium constants at 37 degrees for phosphagen formation are 0.44 with arginine and 0.1 with canavanine. A procedure for L-phosphocanavanine synthesis is also presented.

Permeation of manganese, cadmium, zinc, and beryllium through calcium channels of an insect muscle membrane.

Larval muscle fibers of a beetle, Xylotrupes dichotomus, produce calcium spikes that are maintained when the fibers are bathed in saline solutions containing manganese, cadmium, zinc, or beryllium instead of calcium. This indicates that these cations permeate the calcium channels of the muscle fiber. By contrast, cobalt, nickel, and magnesium are nonpermeating and behave as competitive inhibitors of the permeation of the other divalent cations. Some of the permeating cations suppress delayed rectification.

The electrical and mechanical properties of supercontracting body-wall muscles of the blowfly larva, Calliphoraerythrocephala (Meig.)

1. 1. The muscle membrane constants are calculated and fall within the range of values reported for other insect muscles. The membranes show a delayed rectification response to depolarizing current injected intracellularly, but no electrically-excited depolarizing electrogenesis. 2. 2. A large, fast, EPSP and a small, slow, EPSP are recorded intracellularly in response to nerve stimulation. The properties of these EPSP's are described and compared with other insect systems. 3. 3. A preparation is described for recording isometric tension from a single muscle fibre with intact innervation. The muscles appear to be tonic, with low twitch:tetanus ratios.

Metabolic Rate and Body Temperature in Singing Katydids

The oxygen uptake of the katydids Neoconocephalus robustus and Euconocephalus nasutus is about 0.03-1.5 ml O₂ h⁻¹ g⁻¹ at rest. During the warm-up that precedes stridulation and during stridulation this rate increases to 18.4 ml O₂ h⁻¹ g⁻¹ (maximum 26.4) in Euconocephalus and to 15.8 ml O₂ h⁻¹ g⁻¹ (maximum 19.5) in Neoconocephalus. Oxygen uptake during stridulation is 1.5-1.9 ml O₂ min⁻¹ per gram active muscle, a value similar to that of wing muscle of other insects in flight. The thorax temperature 'increases during warm-up and stridulation. Cooling that occurs when singing ceases is not a simple exponential function. Metabolic rates calculated from cooling curves are lower than measured values; we suggest they are lower because the rate of heat loss is about 23% greater during singing because heat is convected by wing movement. On the basis of measurements of abdominal temperatures we suggest that about 40% of the heat produced by the thorax is transferred to the abdomen and lost via this avenue.

Effects of cobalt and manganese on the calcium-action potentials in larval insect muscle fibres ( Ephestia kühniella)

The effects of cobalt and manganese ions on the calcium action potentials in larval insect muscle fibres were investigated. 1. 1. In either cobalt or manganese (5–25 mM) containing solutions, action potentials could still be elicited by direct stimulation. The maximal rates of rise and fall of these action potentials were however considerably reduced. 2. 2. The repolarization phase of the action potentials was delayed by cobalt and manganese. 3. 3. The membrane input resistance increased in the presence of cobalt and manganese (25 mM) by a factor of 2–4, while the membrane rectification was abolished 4. 4. It is suggested that cobalt and manganese ions reduced both the inward and the outward current of the muscle membrane. These effects of cobalt and manganese are compared with those described for other excitable cells.

Programmed cell death. Measurement of several ATP-digesting enzymes in degenerating insect muscles

Several phosphate-digesting enzymes were purified from muscles of pharate adults of Antheraea polyphemus and Manduca sexta. The various fractions resulting from differential centrifugation and centrifugation on sucrose gradients were analyzed by electron microscopy. Lysosomes were found in mitochondrial fractions prepared by both procedures. Myosin, mitochondrial, and membrane-associated ATPases could be differentiated according to pH optima, movement under gravitational fields, and sensitivity to divalent cations. The enzyme digesting glycerophosphate at acid pH proved to be soluble in preparations from Manduca muscles, but ATP was digested at acid pH by a Ca 2+-activated small particle enzyme which attacked p- nitrophenyl phosphate at a slower rate. This enzyme appears to be a lysosomal enzyme, but another, Mg 2+-stimulated enzyme may be that which has been reported to be a lysosomal ATPase.

Effects of monocarboxylates and external pH on some parameters of insect muscle fibres

The resting membrane potential and the conductance of flight muscle fibres of the butterfly Pieris brassicae were measured by means of two intracellular electrodes. The intracellular potassium, chloride, and hydrogen ion concentrations were estimated by measuring the concentrations in diluted muscle homogenates. Chloride in the bath was replaced in part by monocarboxylates and the pH was subsequently lowered. Replacement of chloride by propionate caused decrease in the internal chloride concentration, a marked hyperpolarization and a small increase in conductance. Lowering the pH in the propionate saline caused an additional decrease in the internal chloride concentration, an increase in the internal hydrogen ion concentration, a marked depolarization and a concurrent decrease in conductance. This was followed by an irreversible increase in conductance. With glycolate the effects on the membrane parameters were small and with pyruvate no effect was observed.

Nicotinamide-adenine dinucleotide-linked “malic” enzyme in flight muscle of the tse-tse fly (Glossina) and other insects

1. A high activity of NAD-linked "malic" enzyme was found in homogenates of flight muscle of different species of tse-tse fly (Glossina). The activity was the same as, or higher than, that of malate dehydrogenase and more than 20-fold that of NADP-linked "malic" enzyme. A similar enzyme was found in the flight muscle of all other insects investigated, but at much lower activities. 2. ACa2+-stimulated oxaloacetate decarboxylase activity was present in all insect flight-muscle preparations investigated, in constant proportion to the NAD-linked "malic" enzyme. 3. A partial purification of the NAD-linked "malic" enzyme from Glossina was effected by DEAE-cellulose chromatography, which separated the enzyme from malate dehydrogenase and NADP-linked "malic" enzyme, but not from oxaloacetate decarboxylase. 4. The intracellular localization of the NAD-linked "malic" enzyme was predominantly mitochondrial; latency studies suggested a localization in the mitochondrial matrix space. 5. Studies on the partially purified enzyme demonstrated that it had a pH optimum between 7.6 and 7.9. It required Mg2+ or Mn2+ for activity; Ca2+ was not effective. The maximum rate was the same with either cation, but the concentration of Mn2+ required was 100 times less than that of Mg2+. Acitivity with NADP was only 1-3% of that with NAD, unless very high (greater than 10mM) concentrations of Mn2+ were present. 6. It is suggested that the NAD-linked "malic" enzyme functions in the proline-oxidation pathway predominant in tse-tse fly flight muscle.