Haemocytic encapsulation in the locust Schistocerca gregaria (Orthoptera) and in the cockroach Periplaneta americana (Dictyoptera)

Summary The numbers and types of haemocytes in adult male Schistocerca gregaria and Periplaneta americana have been studied in an attempt to explain the differences in thickness of haemocytic capsules formed around abiotic particles in the 2 species. Total and differential haemocyte counts and measurements of blood volume using ³H-inulin indicate that there are 3–4 times more plasmatocytes in the cockroach than in the locust. Although the three main haemocyte types are easily recognised by phase-contrast microscopy, there are few distinguishing ultrastructural characteristics and thus defining the cell types that make up the capsule is difficult. In early capsules in the locust, but not in the cockroach, signs of coagulocyte lysis are apparent, and in both species the bulk of the capsule appears to be made up of granular plasmatocyte-like cells. The relatively thinner capsules formed in the locust might be due to the slow, and limited, recruitment of plasmatocytes to the developing capsule. The material coating completed capsules appears ultrastructurally similar to the subepidermal basement membrane, and both these layers stain with Alcian blue. Once the coating material has formed, the capsule appears to be treated as self by the immunorecognition system.     

Neuropeptide-degrading endopeptidase activity of locust (Schistocerca gregaria) synaptic membranes.

Locust adipokinetic hormone (AKH, pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2) was used as the substrate to measure neuropeptide-degrading endopeptidase activity in neutral membranes from ganglia of the locust Schistocerca gregaria. Initial hydrolysis of AKH at neural pH by peptidases of washed neural membranes generated pGlu-Leu-Asn and Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 as primary metabolites, demonstrating that degradation was initiated by cleavage of the Asn-Phe bond. Amastatin protected the C-terminal fragment from further metabolism by aminopeptidase activity without inhibiting AKH degradation. The same fragments were generated on incubation of AKH with purified pig kidney endopeptidase 24.11, and enzyme known to cleave peptide bonds that involve the amino group of hydrophobic amino acids. Phosphoramidon (10 microM), a selective inhibitor of mammalian endopeptidase 24.11, partially inhibited the endopeptidase activity of locust neural membranes. This phosphoramidon-sensitive activity was shown to enriched in a synaptic membrane preparation with around 80% of the activity being inhibited by 10 microM-phosphoramidon (IC50 = 0.2 microM). The synaptic endopeptidase was also inhibited by 1 mM-EDTA, 1 mM-1,10-phenanthroline and 1 microM-thiorphan, and the activity was maximal between pH 7.3 and 8.0. Localization of the phosphoramidon-sensitive enzyme in synaptic membranes is consistent with a physiological role for this endopeptidase in the metabolism of insect peptides at the synapse.     

Hybridization studies in the Schistocerca americana complex: II. The Peruvian locust

A culture of swarming locusts from Peru was hybridized with locusts from Argentina and Mexico. The sex ratio and fertility of the hybrids was measured and male meiosis observed. It is concluded that the Peruvian locust is a subspecies of the Central American locust and a separate species from the Argentinian.     

Pharmacology of GABA receptors on skeletal muscle fibres of the locust (Schistocerca gregaria)

GABA and the trans isomer of 4-aminocrotonic acid are equally potent at inducing increases in Cl- conductance when applied to distal extensor tibia muscle fibres of the locust (Schistocerca gregaria). beta-Alanine, norvaline, glycine and norleucine induced conductance increases of less than 5% of GABA responses. C9 and meso-di-GABA did not alter input conductance in a manner consistent with actions on a GABA receptor Cl- channel complex. Picrotoxin and anisatin were equally potent GABA antagonists, however bicuculline and penicillin G did not reduce GABA-induced changes in input conductance. Pentobarbitone, in addition to inducing an increase in K+ conductance, potentiated GABA-induced increases in Cl- permeability.     

Lactate content and lactate dehydrogenase activity inPalaemon serratus abdominal muscle during temperature changes

Summary Lactate concentration was measured in the abdominal muscle of the shrimpPalaemon serratus. Rapid and seasonal temperature changes result in an increase of the lactate content of approximately 3–4 fold.Lactate dehydrogenase from the abdominal muscle exhibits a temperature dependent pyruvate inhibition with pyruvate as substrate.The kinetic parameters of lactate dehydrogenase fromPalaemon serratus are found to vary during rapid temperature changes: V_max increases with temperature from 0.06 mol min^–1 (mg protein)^–1 at 10°C to 0.28 mol min^–1 (mg protein)^–1 at 30°C with lactate as substrate, and from 5.5 mol min^–1 (mg protein)^–1 at 10°C to 26.2 mol min^–1 (mg protein)^–1 at 30°C, with pyruvate (Table 1). The Hill coefficientn _H, decreases with temperature from 2.2 to 1.2 when the pyruvate reduction is examined, but remains near 1.2 when the activity is measured with lactate as substrate (Table 1). The S_0.5 values for lactate show a tendency to increase below 30 °C (18.9 mM l^–1 at 20 °C) whereas the S_0.5 for pyruvate is found to increase greatly with temperature (0.004 mM l^–1 at 10 °C and 0.06 mM l^–1 at 20 °C).Long term temperature changes involve variations of lactate dehydrogenase activity leading to inverse thermal compensation (Table 2).Activation energy (about 56 kJ both with pyruvate and lactate) does not vary during the year, suggesting that temperature adaptation does not induce important catalytic changes (Table 3).Abbreviation LDH lactate dehydrogenase     

The action of philanthotoxin-343 and photolabile analogues on locust (Schistocerca gregaria) muscle

The effects of philanthotoxin-343 (PhTX-343; tyrosyl-butanoyl-spermine) and photolabile analogues of this synthetic toxin on locust (Schistocerca gregaria) skeletal muscle have been investigated using whole muscle preparations (twitch contractions), single muscle fibres (excitatory postsynaptic currents (EPSCs)) and muscle membrane patches containing single quisqualate-sensitive glutamate receptors (qGluR). Analogues containing an azido group attached to either the butanoyl side-chain of PhTX-343 or as a substitute for the hydroxyl moiety of the tyrosyl residue were about 6 fold more potent antagonists than PhTX-343; those with an azido group located at the distal end of the toxin molecule were generally 2–3 fold less potent than PhTX-343. When these compounds were tested in subdued light, they were reversible antagonists of the muscle twitch, EPSC and qGluR. When a muscle was irradiated with U.V. during application of photolabile toxin combined with either neural stimulation of the muscle orl-glutamate application, antagonism of the twitch, EPSC and qGluR was complete and irreversible.     

Ontogeny of tracheal system structure: a light and electron-microscopy study of the metathoracic femur of the American locust, Schistocerca americana

Does oxygen delivery become more challenging for insects as they increase in size? To partially test this hypothesis, we used quantitative light and electron microscopy to estimate the oxygen delivery capacity for two steps of tracheal oxygen delivery within the metathoracic femur (jumping leg) for 2nd instar (about 47 mg) and adult (about 1.7 g) locusts, Schistocerca americana. The fractional cross‐sectional areas of the major tracheae running longitudinally along the leg were similar in adults and 2nd instars; however, since the legs of adults are longer, the mass‐specific diffusive conductances of these tracheae were 4‐fold greater in 2nd instars. Diffusive gas exchange longitudinally along the leg is easily possible for 2nd instars but not adults, who have many air sacs within the femur. Mitochondrial content fell proximally to distally within the femur in 2nd instars but not adults, supporting the hypothesis that diffusion was more important for the former. Lateral diffusing capacities of the tracheal walls were 12‐fold greater in adults than 2nd instars. This was primarily due to differences in the smallest tracheal class (tracheoles), which had thinner epidermal and cuticular layers, greater surface to volume ratios, and greater mass‐specific surface areas in adults. Adults also had greater mitochondrial contents, larger cell sizes and more intracellular tracheae. Thus, larger insects do not necessarily face greater problems with oxygen delivery; adult grasshoppers have superior oxygen delivery systems and greater mass‐specific aerobic capacities in their legs than smaller/younger insects. J. Morphol. 262:800–812, 2004. © 2004 Wiley‐Liss, Inc.     

Inhibiton and gamma-aminobutyric acid-induced conductance on locust (Schistocerca gregaria) extensor tibiae muscle fibres

• 1.1. Increases in membrane conductance (g_m) were induced by GABA in distal bundles 32, 33 and 34 of extensor tibiae muscles of the locust (Schistocerca gregaria).
• 2.2. Bath application of GABA (10⁻⁵−5 × 10⁻³ M) induced reductions in muscle fibre space constant (λ).
• 3.3. GABA (5 × 10⁻³ M) induced additional membrane conductance of 2.21 ± 0.03 × 10⁻⁶ S/mm, 0.38 ± 0.03 × 10⁻⁶ S/mm and 0.29 ± 0.06 × 10⁻⁶ S/mm on muscle bundles 34, 33 and 32 respectively. The greater sensitivity of muscle fibres in bundle 34 to GABA is due at least in part to a larger number of GABA receptors on bundle 34 muscle fibres.
• 4.4. The decrement of electrotonic potentials in the presence of GABA were measured over distances of both half fibre length and whole fibre length. Good agreement was obtained between changes in space constant produced by GABA using half fibre length and whole fibre length data.
• 5.5. By taking into account changes in space constant induced by GABA it was possible to demonstrate that presynaptic GABA receptors were involved in the inhibition of slow excitatory postsynaptic potentials by GABA.
• 6.6. “Slow” excitatory postsynaptic potentials recorded under current clamp were inhibited in a dose-dependent manner by GABA. This inhibition was not dependent on muscle-fibre GABA sensitivity and could not be completely accounted for by GABA-induced changes in the cable properties of the muscle fibres.

     

Flight-correlated activity changes in neurons of the lateral accessory lobes in the brain of the locust Schistocerca gregaria

The study investigates activity changes in neurons of the lateral accessory lobes in the brain of the locust Schistocerca gregaria during wind-elicited tethered flight. Neurons with ascending projections from the ventral nerve cord to the lateral accessory lobes showed flight-associated excitations which were modulated in the flight motor rhythm. Descending neurons with ramifications in the lateral accessory lobes were tonically excited corresponding to flight duration. The onset of wind-elicited responses in the descending neurons preceded the onset of flight motor activity by 22–60 milliseconds. Neurons connecting the lateral accessory lobes with the central body, the anterior optic tubercles, or other brain areas showed a variety of responses including activity changes during flight initiation and flight termination. Activity in many of these neurons was less tightly coupled to the flight situation and often returned to background levels before flight was terminated. Most of the recorded neurons responded, in addition, to stationary visual stimuli. The results suggest that the lateral accessory lobes in the locust brain are integrative links between the central body, visual pathways, and the ventral nerve cord. The possible involvement of these brain areas in flight control is discussed.     

Intermolt development reduces oxygen delivery capacity and jumping performance in the American locust (<Emphasis Type="Italic">Schistocerca americana</Emphasis>)

Among animals, insects have the highest mass-specific metabolic rates; yet, during intermolt development the tracheal respiratory system cannot meet the increased oxygen demand of older stage insects. Using locomotory performance indices, whole body respirometry, and X-ray imaging to visualize the respiratory system, we tested the hypothesis that due to the rigid exoskeleton, an increase in body mass during the intermolt period compresses the air-filled tracheal system, thereby, reducing oxygen delivery capacity in late stage insects. Specifically, we measured air sac ventilation frequency, size, and compressibility in both the abdomen and femur of early, middle, and late stage sixth instar Schistocerca americana grasshoppers. Our results show that late stage grasshoppers have a reduced air sac ventilation frequency in the femur and decreased convective capacities in the abdomen and femur. We also used X-ray images of the abdomen and femur to calculate the total proportion of tissue dedicated to respiratory structure during the intermolt period. We found that late stage grasshoppers had a lower proportion of their body dedicated to respiratory structures, especially air sacs, which convectively ventilate the tracheal system. These intermolt changes make oxygen delivery more challenging to the tissues, especially critical ones such as the jumping muscle. Indeed, late stage grasshoppers showed reduced jump frequencies compared to early stage grasshoppers, as well as decreased mass-specific CO₂ emission rates at 3 kPa PO₂. Our findings provide a mechanism to explain how body mass changes during the intermolt period reduce oxygen delivery capacity and alter an insect’s life history.     

Partial purification and some properties of pyruvate carboxylase from the flight muscle of the locust (Schistocerca gregaria)

A procedure is described for the partial purification of pyruvate carboxylase (pyruvate:CO2 ligase (ADP-forming), EC 6.4.1.1) from the flight muscle of the locust (Schistocerca gregaria). Characterisation of the kinetic properties of this enzyme indicates that it is activated by acetyl-CoA, is insensitive to inhibition by di- and tricarboxylic acids and exhibits an apparent Km for HCO3-(16 mM) which differs by an order of magnitude from that observed for other pyruvate carboxylases. It is suggested that activation of this locust flight muscle pyruvate carboxylase during the rest leads to flight transition may result from increases in the concentrations of pyruvate and HCO3- under these conditions.     

FMRFamide-like immunoreactivity in the metathoracic ganglion of the locust (Schistocerca gregaria)

Summary The distribution of FMRFamide-like immunoreactivity in the metathoracic ganglion of the locust, Schistocerca gregaria, has been investigated in serial semithin transverse sections with the use of the peroxidase-antiperoxidase (PAP) technique. The topographical distribution of approximately 120 immunopositive neurons was established. Antiserum against bovine pancreatic polypeptide (BPP) stains the same ganglionic cells as FMRFamide-antiserum, yet this staining is largely blocked after preabsorption to FMRFamide. A comparison of these results with those from other studies suggests that there may be more than one type of endogenous RFamide-like peptide.     

Changes in lactate dehydrogenase and glucose-6-phosphate dehydrogenase activity in the course of muscle alteration]

Changes of outflux, extractability and activity of lactate dehydrogenase (LDG) and glucose-6-phosphate dehydrogenase (G-6PhDG) of muscles under the action of heating (at 32--44 degrees for 15 min) and urea (1 M during 10 min, 30 min, 2 hr. and 9 hr.) on the skeletal muscles of R. temporaria L. were studied. Under the thermal action not accompanied by contracture and fall of the excitability (32--36 degrees), the increase of outflux of LDG out of muscles into surrounding solutions is observed. G-6-PhDG in the external medium under any heating action was not revealed. Extractibility of LDG and G-6-PhDG did not change. Under the thermal action accompanied by the fall of excitability and by the contracture, along with the prolong increase of outflux of LDG, a decrease of extractability of LDG takes place. The decrease of G-6-PhDG is set at 42 degrees. Under the alteration of muscles by urea in the period of the temporary fall of excitability and contracture (10 and 30 min) an increase of the outflux of LDG out of muscles is observed. G-6PhDG in the surrounding medium was not revealed up to 9 hr. of incubation of muscle. In the period of the recovery of the excitability and relaxation of muscles (2hr.) the outflux of LDG approaches the control level. During the temporary loss and recovery of excitability, the extractability of LDG and G-6-PhDG does not change. In the period of irreversible contracture and loss of the excitability (6--10 hr.) a sharp increase of outflux of LDG out of muscles takes place. The extractability of the examined enzymes, especially of LDG, decreases.     

Intracellular distribution of NADP-linked isocitrate dehydrogenase,fumarase and citrate synthase in bovine heart muscle

By fractional extraction of minced bovine heart muscle with iso-osmotic sucrose and phosphate buffer solutions, it is shown that less than 4% of the total citrate synthase in the tissue is in the cytosol. Using citrate synthase as a marker for broken mitochondria, two methods of fractionation of 750 × g supernatants from homogenates of bovine heart muscle show that 10% of the total fumarase and NADP-linked isocitrate dehydrogenase activities are present in the cytoplasm. Homogenates prepared by sonication and osmotic shock and by sand-grinding gave closely similar results as regards enzyme distributions and extent of mitochondrial breakage. The results are compared with those reported for other tissues.