Flight capability and flight motor pattern in a sedentary South African grasshopper, Phymateus morbillosus– a comparison with migratory species

Abstract Among the Acridoidea, not all species are strong fliers. We have examined the possible causes for loss of flight in a species with a reduced flight system, the South African grasshopper, Phymateus morbillosus (L.). This is a sedentary species that, in the field, displays only marginal flight (in males) or no flight (in females). In a wind tunnel, however, this species can be stimulated to perform flight movements for a short time. In the present study, several morphometric parameters and aspects of the flight motor output have been examined. The data are compared with those of the migratory locust species ( Locusta migratoria L. and Schistocerca gregaria Forsk.). Phymateus morbillosus can exhibit the typical flight motor pattern for short periods of up to 1 min. Morphometric data and wing-beat frequency in P. morbillosus are similar to that of other members of this insect group. However, female specimens of P. morbillosus are too heavy to lift themselves for active flight. We assume that females of this species invest in the augmentation of reproduction rather than investing in the flight system.     

A third active AKH is present in the pyrgomorphid grasshoppers Phymateus morbillosus and Dictyophorus spumans

The corpora cardiaca of the African pyrgomorphid grasshoppers Phymateus morbillosus and Dictyophorus spumans contain three adipokinetic hormones (AKHs): besides two already known AKHs, Phm-AKH-I and Scg-AKH-II (G?de et al., 1996 [G?de, G., Kellner, R., Rinehart, K.L., 1996. Pyrgomorphid grasshoppers of the genus Phymateus contain species-specific decapeptides of the AKH/RPCH family regulating lipid-mobilisation during flight. Physiol. Entomol. 21, 193-202]), a new AKH-III, denoted Phm-AKH-III, pGlu-Ile-Asn-Phe-Thr-Pro-Trp-Trp-NH(2), has been characterised. This is only the second AKH-III identified so far, thus, only three insect species - all of them grasshoppers - contain three active AKHs. Phm-AKH-III differs from Lom-AKH-III from the migratory locust, Locusta migratoria, only in position 2: isoleucine is present instead of leucine. The structure of the Phm-AKH-III was confirmed by synthesis, subsequent mass determination and reversed-phase high-performance liquid chromatography. The synthetic peptide also induced hyperlipaemia in D. spumans and L. migratoria.     

Isolation and identification of AKH/RPCH family peptides in blister beetles (Meloidae)

Abstract. Two peptides were isolated from methanolic extracts of corpora cardiaca of the blister beetle, Decupotoma lunata , by a single-step purification procedure, utilizing C-18 reversed-phase high-performance liquid chromatography (RP-HPLC) for separation, and the increase of haemolymph lipids in Locusta migratoria for bioassay. The native peptides were analysed by matrix-assisted laser-desorption ionization mass spectrometry revealing main ions at m/z 1180 and 1009 respectively which were attributed to the [M + Na]⁺ form of the respective peptides. After deblocking of the N-terminal pyroglutamate residue of each peptide, the structures of the deblocked peptides were determined by pulsed-liquid phase sequencing employing Edman chemistry. The sequences of the two peptides, (1) pGlu-Leu-Asn-Phe-Ser-Pro-Am-Trp-Gly-AsnNH₂ and (2) pGlu-Leu-Asn-Phe-Ser-Pro-Asn-TrpNH₂, characterize them as deca- and octapeptide members of the AKH/RPCH family. Whereas the decapeptide is a novel member of this family and is given the acronym Del-CC ( Decupotoma lunata corpus cardiacum peptide), the octapeptide has previously been found in tenebrionid beetles and has the acronym Tem-HrTH. The corpora cardiaca of two other species of blister beetles ( Cyaneolytta pectoralis and Mylabris coeca ) contain the same two peptides as D. lunata , as judged by RP-HPLC and biological activity. Neither a corpus cardiacum extract of Decupotoma lunata nor the synthetic peptides Del-CC and Tem-HrTH were active in mobilizing carbohydrates or lipids in the blister beetle.     

Effects of rapid changes in oxygen concentration on the respiration of carrot roots

Rates of CO₂ production and O₂ consumption from aged disks of carrot ( Daucus carota L.) root tissues were measured for 4 h after they were transferred from 21% to 0, 1, 2, 4 or 8% O₂ in gas mixtures. A transient peak in the rate of CO₂ production started 5 to 7 min after transfer to 2% or lower O₂ mixtures and peaked at 50 min. After the peaks in CO₂ production from the 0, 1 and 2% O₂ treatments and after the stable production from the 4 and 8% O₂ treatments, the rate of CO₂ production from all low O₂ treatments started to decline at 50 min, reaching stable rates by 160 to 240 min. Concentrations of lactate and ethanol that were significantly higher than the 21% O₂ controls had started to accumulate in disks between 10 and 50 min after exposure to atmospheres containing 2% or less O₂. Production of CO₂ started to increase 5 to 7 min after transfer to 0, 1 and 2% O₂, while the initial decline and then rise in pH and the accumulation of ethanol did not occur until 30 min after the change in atmosphere. Ethanol accumulation paralleled the increase in pH; first at 0.4 μmol g⁻¹ h⁻¹ from 30 to 60 min as the pH shifted from 5.97 to 6.11, and then at 0.08 μmol g⁻¹ h⁻¹ from 60 to 100 min as the pH stablized around 6.12. The peak at 50 min in CO₂ production roughly coincided with the shift from the rapid to the slow change in pH and ethanol accumulation.     

Evidence for the direct 2beta- and 3beta-hydroxylation of [2H2]GA20-13-O-[6'-2H2]glucoside in seedlings of Phaseolus coccineus

[17-²H₂]GA₂₀-13-O-[6'-²H₂]glucoside was synthesized and applied to seedlings of Phaseolus coccineus L. After incubation for 72 h the conjugate metabolites were purified and shown by LC-ESI-tandem-MS and GC-MS to be [17-²H₂]GA₁-13-O-[6'-²H₂]glucoside and [17-²H₂]GA₂₉-13-O-[6'-²H₂]glucoside. This is the first evidence for the conversion of intact GA-O-glucosides, and represents an additional metabolic pathway of the gibberellin metabolism in P. coccineus L. The results indicate that intact GA-O-glucosides are accepted by 2- and 3-oxidases in the plant.     

Ozone-induced ultrastructural changes in the plasma membrane of Chlorella sorokiniana

Abstract. Modifications in plasma membrane structure and permeability were observed in Chlorella sorokiniana following exposure to 0.2 gm⁻³(140 p.p.m.) O₃ for 30 min. Sixty-eight per cent of the cells were plasmolysed after 15 min O₃ exposure with disruption of organelles similar to that previously described in higher plants. Freeze-fracture exposed large areas of plasma membrane in 90% of the control cells and those exposed to O₃for short periods. After 20 min O₃ 90% of the cells cross-fracture, which indicates a change in molecular interactions in the membrane exposed to O₃ The earliest observed ultraslructural alteration is an aggregation of particles on the plasma membrane P face, statistically significant after 10 min O₃ Changes in ⁸⁶Rb influx occur during a similar time. After more extended exposure to O³ the plasma membrane P face shows regions of lipid phase transition to the crystalline state.     

Parthenogenesis in Urechis caupo (Echiura) II. Role of intracellular pH in parthenogenesis induction

A peptide (P23) isolated from sperm acrosomal protein initiates development in eggs of the marine worm Urechis caupo . We have shown previously that eggs exposed to P23 for ≥3min complete meiosis but fail to cleave. However, a brief (1.5–2 min) exposure to P23 at pH 8, followed by either acidification of the seawater to pH 7 or dilution of P23 at pH 8 causes germinal vesicle breakdown (GVBD), but eggs fail to complete meiosis and many then later advance to mitosis. In the present study we investigated the hypothesis that partial activation leading to parthenogenesis occurs when there is a partial intracellular alkalinization. Measurements with the fluorescent pH indicator bis(carboxyethyl)-carboxyfluorescein (BCECF) showed that P23 induces a pH, increase similar to that occurring during fertilization and that parthenogenesis-inducing treatments interupt this rise in pH₁ In eggs exposed to P23 for >3 min the pH₁ increase was 0.31–0.49 units, slightly higher than in fertilized eggs. In partially activated eggs exposed to P23 for 1.5–2 min at pH 8, pH₁ began to rise but then returned to control values or remained only partially elevated (< 0.2 pH units average increase). Electrophysiological measurements revealed that removal of P23 during the first few minutes of exposure caused the activation potential to terminate and experiments with [¹⁴C]-P23 confirmed that dilution results in a rapid unbinding of P23 from eggs. If proton export is driven by membrane potential as well as the pH gradient, these results explain why dilution of P23 at pH 8 also interrupts the pH_i increase.     

Gas exchange measurements required to predict the newly fixed carbon pool size in a source leaf of corn (Zea mays L.)

Abstract. Steady-state photosynthesis (P_n), post-illumination CO₂ release rates (R), sucrose-phosphate synthase (SPS) activities, and levels of starch, sucrose and hexoses were measured in the source leaf of corn ( Zea mays L.) during a 16-h photoperiod at 800 μmol m ² s ¹. P_n and SPS activity remained constant. Carbohydrate pools increased at a linear rate, except the first and last hour of the photoperiod. Both the CO₂ evolution rate at the moment of light removal (R_max) and SPS activity decreased by one half after the onset of darkness (0 60 min). Sucrose diminished during this period by 40%, whereas the starch remained constant. Thereafter, starch mobilization began, followed by a gradual decline in leaf respiration. The average dark export rate was calculated to be 60% less than that during the day. Maintenance respiration (R_m) of an attached leaf after 48 h darkness was determined. Plants were illuminated for different intervals (e.g. 5, 10 or 20 min), each followed by dark periods sufficient for respiration to decline to R_m. The ratio of C assimilated in light to that released in dark was 6:1. After the 48-h dark period, the minimal period of illumination (T_min) required to restore P_n and R_max to the original level was determined. A mathematical analysis of the kinetics involved in the recovery of P_n and R_max provided an estimate of turnover time (0.22h) and pool size 9.15 mmol m ²) for the newly fixed carbon.     

On the diel rhythms in metabolism and activity of post-hatching lesser spotted dogfish, Scyliorhinus canicula

The diel rhythms in metabolic rate ( M_R ) and activity level ( A_L ) were measured for single post-hatching dogfish (weight range, 2.76–10.61 g) at 15° C by the indirect calorimetric method of rate of oxygen consumption ( V O₂) and by video-observation respectively, over a period of 72 b. The mean VO ₂ increased from 62.0 (s.e. 2.9) mg O₂ kg⁻¹ h⁻¹ in the daylight hours to 85.5 (s.e. 3.1) mg O₂ kg⁻¹ h⁻¹ during the dark (light regíme, 12 h L: 12 h D). The simultaneous measurement of A _L also showed mean night elevation from 0.6 (s.e. 0.2) min h⁻¹ in the light phase to 14.5 (s.e. 1.6) min h⁻¹ during the darkness. Bimodal nocturnal activity (BNA) was exhibited by the post-hatching dogfish within the 12 h dark period, with V O₂ increasing from 71.4 (s.e. 2.8) mg O₂ kg⁻¹ h⁻¹ before 01.00 hours to 99.5 (s.e. 4.2) mg O₂ kg⁻¹ h⁻¹ after 01.00 hours. Similarly, A _L also increased from 8.9 (s.e. I.7)min h⁻¹ before 01.00 hours to 21.1 (s.e. 2.8) min h⁻¹ after 01.00 hours. The importance of the results presented to the natural behavioural ecology of the hatching dogfish are discussed.     

Metabolic Precursors and Compartmentation of Cerebral GABA in Vigabatrin-Treated Rats

Abstract: The metabolic precursors and cerebral compartmentation of the augmented GABA pool induced by vigabatrin, an irreversible inhibitor of GABA transaminase, have been investigated by ¹³C NMR. Adult rats receiving rat chow ad libitum were given either drinking water only or drinking water containing 2.5 g/L vigabatrin for 7 days. Both groups of animals were infused either with [1,2-¹³C₂]acetate (15 µmol/min/100 g body weight), an exclusive precursor of GABA formation through the glial glutamine pathway, or with [1,2-¹³C₂]glucose (15 µmol/min/100 g body weight), a substrate that can produce GABA through the glial glutamine pathway or by direct metabolism in the neurons. The brains were frozen in situ, extracted with perchloric acid, and analyzed by ¹³C NMR. In vigabatrin-treated animals [¹³C]glutamine, a common intermediate for [¹³C]GABA synthesis from glucose or acetate, was accumulated to similar amounts during infusions with [1,2-¹³C₂]glucose or [1,2-¹³C₂]acetate. However, [¹³C]GABA accumulation was sevenfold higher during [1,2-¹³C₂]glucose infusions or twofold higher during [1,2-¹³C₂]acetate infusions. These results show that the direct pathway of GABA formation by neuronal metabolism of glucose predominates over the alternative pathway through glial glutamine. Near-equilibrium relationships of the aminotransferases of GABA and aspartate imply that the observed [¹³C]GABA accumulation occurs initially in the neuronal compartment.     

Rod visual pigment changes in the elver of the eel Anguilla anguilla L. measured by microspectrophotometry

The visual pigments in the retinal rods of elvers ( Anguilla anguilla L.) were examined by microspectrophotometry and high performance liquid chromatography. The glass elver stage had a mixture of rhodopsin P501₁ and porphyropsin P523₂ with the former predominating (P501₁:P523₂= c. 60:40). More mature pigmented elvers had a predominance of the porphyropsin P523₂ (P501₁:P523₂= c. 10:90). The shift between the proportions of each pigment was monitored over a 7-month period and was shown to occur in individual retinal rods. This shift is a reverse of the pigment shift that occurs in adult eels during their downstream migration.     

Non-stomatal limitation of CO2 assimilation in three tree species during natural drought conditions

The effect of drought on CO₂ assimilation and leaf conductance was studied in three northern hardwood species: Quercus rubra L., Acer rubrum L. and Populus grandidentata Michx. Leaf gas exchange characteristics at two CO₂ levels (320 and 620 μl I⁻¹) and temperatures from 20 to 35°C were measured at the end of a dry period and shortly after 10 cm of rainfall. The effects of drought varied with species, temperature and CO₂ level. Calculated values of internal CO₂ concentration showed little or no decline during drought. Differences in assimilation, before vs after the rains, were most apparent at the higher CO₂ level. These latter two observations indicate nonstomatal disruption of CO₂ assimilation during the dry period. In P. grandidentata there was a substantial interaction between drought and temperature, with a resultant shift in the temperature for maximum assimilation to lower temperatures during drought. During drought, internal CO₂ concentrations increased sharply in all three species under the combined conditions of high temperatures and the higher CO₂ level.     

Mhp493 (P216) is a proteolytically processed, cilium and heparin binding protein of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae induces respiratory disease in swine by colonizing cilia causing ciliostasis, cilial loss and epithelial cell death. Heparin binds to M. hyopneumoniae cells in a dose-dependent manner and blocks its ability to adhere to porcine cilia. We show here that Mhp493 (P216), a paralogue of the cilium adhesin P97 (Mhp183), is cleaved between amino acids 1040 and 1089 generating surface-accessible, heparin-binding proteins P120 and P85. Antiphosphoserine antibodies recognized P85 in 2-D immunoblotting studies and TiO₂ chromatography of trypsin digests of P85 isolated a single peptide with an m/z of 917.3. A phosphoserine residue in the tryptic peptide ⁹⁰VSELpSFR⁹⁶ (position 94 in P85) was identified by MALDI-MS/MS. Polyhistidine fusion proteins (F1_P216, F2_P216, F3_P216) spanning Mhp493 bound heparin with biologically significant Kd values, and heparin, fucoidan and mucin inhibited this interaction. Latex beads coated with F1_P216, F2_P216 and F3_P216 adhered to and entered porcine kidney epithelial-like (PK15) cell monolayers. Microtitre plate-based assays showed that sequences within P120 and P85 bind to porcine cilia and are recognized by serum antibodies elicited during infection by M. hyopneumoniae . Mhp493 contributes significantly to the surface architecture of M. hyopneumoniae and is the first cilium adhesin to be described that lacks an R1 cilium-binding domain.     

Foraging energetics of Grey-headed Albatrosses Diotnedea chrysostoma at Bird Island, South Georgia

At-sea metabolism (CO₂ production) and water turnover of six breeding Grey-headed Albatrosses Diomedea chrysostoma were measured, using the doubly labelled water method, at Bird Island, South Georgia, Mean food consumption (estimated from a water influx rate of 1.01 1 d^-1 and data on dietary composition) was 1200gd^-1 or 50.4 W. At-sea metabolism (derived from a rate of CO₂ production of 3.98 1 h^-1) was 27.7 W, 2.5 times the estimated basal metabolic rate (BMR). On average the birds ingested nearly twice as much food energy as they expended to obtain it. The metabolic rate during flight (estimated from at-sea metabolism and activity budget data) was 36.3 W (range 34.7–39.0 W) or 3.2 (range 3.0–3.4) times the predicted BMR. This is the lowest cost of flight yet measured, but consistent with the highly developed adaptations for economic flight shown by albatrosses. These results are briefly compared with data for other polar vertebrates (penguins, fur seals) exploiting similar prey.     

Influence of dietary acetylated peptides on fermentation and peptidase activities in the sheep rumen

The predominant mechanism of peptide breakdown by rumen micro-organisms is aminopeptidase. Thus acetylation of the N-terminus of peptides inhibits their degradation by rumen micro-organisms in short-term incubations with rumen fluid in vitro . An experiment was undertaken to determine if adaptation of the rumen microbial population would take place when acetylated peptides were fed for a prolonged period, which would enable the microbial population to break down the protected peptides and thus decrease their nutritive value. Three adult sheep, fitted with permanent rumen cannulae, received a maintenance hay/concentrate diet to which was added, at each meal, 20 g of casein enzymic hydrolysate ('peptides') or 20 g of peptides previously treated with acetic anhydride. The diets were fed for 28 d in a 3 × 3 latin square and samples were taken during the last 7 d. Fermentation products and NH₃ concentrations indicated that acetylated peptides remained less degradable than untreated peptides. There was a trend towards increased proteolytic activity with acetylated peptides, and dipeptidase activity increased by 18% and 28%, respectively, compared with untreated peptides and control treatments. Activity against N-acetyl-Ala₂ also increased when acetylated peptides were fed, but it remained only 13% of the rate of Ala₂ hydrolysis. No increase was found in the rate of ammonia production from acetylated peptides in animals receiving acetylated peptides–this rate was 26% of that found with untreated peptides–and acetylated peptides continued to persist for longer in the rumen than untreated peptides after feeding. Thus it was concluded that the rumen microbial population did not adapt to utilize acetylated peptides.     

Lactate uptake contributes to the NAD(P)H biphasic response and tissue oxygen response during synaptic stimulation in area CA1 of rat hippocampal slices

Synaptic train stimulation (10 Hz × 25 s) in hippocampal slices results in a biphasic response of NAD(P)H fluorescence indicating a transient oxidation followed by a prolonged reduction. The response is accompanied by a transient tissue PO₂ decrease indicating enhanced oxygen utilization. The activation of mitochondrial metabolism and/or glycolysis may contribute to the secondary NAD(P)H peak. We investigated whether extracellular lactate uptake via monocarboxylate transporters (MCTs) contributes to the generation of the NAD(P)H response during neuronal activation. We measured the effect of lactate uptake inhibition [using the MCT inhibitor α-cyano-4-hydroxycinnamate (4-CIN)] on the NAD(P)H biphasic response, tissue PO₂ response, and field excitatory post-synaptic potential in hippocampal slices during synaptic stimulation in area CA1 (stratum radiatum). The application of 4-CIN (150–250 μmol/L) significantly decreased the reduction phase of the NAD(P)H response. When slices were supplemented with 20 mmol/L lactate in 150–250 μmol/L 4-CIN, the secondary NAD(P)H peak was restored; whereas 20 mmol/L pyruvate supplementation did not produce a recovery. Similarly, the tissue PO₂ response was decreased by MCT inhibition; 20 mmol/L lactate restored this response to control levels at all 4-CIN concentrations. These results indicate that lactate uptake via MCTs contributes significantly to energy metabolism in brain tissue and to the generation of the delayed NAD(P)H peak after synaptic stimulation.     

31P NMR Relaxation Does Not Affect the Quantitation of Changes in Phosphocreatine, Inorganic Phosphate, and ATP Measured In Vivo During Complete Ischemia in Swine Brain

Abstract: Ischemia-induced changes in ³¹P NMR relaxation were examined in 16 piglets. NMR spectra were acquired under control conditions and during complete cerebral ischemia induced via cardiac arrest. Changes in T ₁ were assessed directly in six animals during control conditions and after 30–45 min of complete ischemia when changes in brain P₁ levels had reached a plateau. The T ₁ for P₁ did not change, i.e., 2.3 ± 0.5 s during control conditions versus 2.4 ± 1.0 s during ischemia. To evaluate phosphocreatine and ATP, two types of spectra, with a long (25-s) or short (1-s) interpulse delay time, were collected during the first 10 min of ischemia (n = 10). Both types of spectra showed the same time course of changes in phosphocreatine and ATP levels, implying that the T ₁ relaxation times do not change during ischemia. There were no changes in the linewidths of phosphocreatine, ATP, or P₁ during ischemia, implying that the T *₂ values remain constant. Our results suggest that the ³¹P T ₁ and T *₂ for phosphocreatine, P_i, and ATP do not change during ischemia, and therefore changes in ³¹P NMR peak intensity accurately reflect changes in metabolite concentrations.     

The Effect of Oxygen on the Germination and Outgrowth of Clostridium butyricum Spores and Changes in the Oxidation-Reduction Potential of Cultures

S ummary . The E_h fall observed during incubation of Clostridium butyricum spores occurred during germination and emergence, not during the log phase; it is attributed to the H₂ tension resulting from metabolism. When the O₂ tension in the medium was increased, the E_h fell only after a few spores outgrew and replicated; germination of remaining spores then followed. It is suggested that the few cells able initially to metabolize can (a) elaborate NADH etc. which reduce the O₂ tension to a level non-inhibitory for the remaining spores, and (b) produce the H₂ tension recorded by the Pt electrode.     

Carbon dioxide anaesthesia in rainbow trout: effects of hypercapnic level and stress on induction and recovery from anaesthetic treatment

The physiological and anaesthetic effects of three different levels of air-saturated and buffered CO₂ anaesthesia, P _w co ₂=31, 78, or 125 mmHg, were examined in cannulated rainbow trout Oncorhynchus mykiss . Complete anaesthesia (no opercular movements) was not achieved by these hypercapnic levels after 20 min of CO₂ exposure. Although increasing P _w co ₂ reduced the induction times to the early stages of anaesthesia, it also resulted in increasing hyperventilatory, hypoxaemic, and acid-base disturbances. After a 10-min recovery period, while the respiratory acidosis component of the acid-base disturbance was corrected, there was a significant metabolic acidosis. Recovery time was longest in the high P_w co₂ treatment where 33% of the fish died. Two additional groups ( P _w co ₂=37 and 78 mmHg) were exposed to an acute stress prior to the anaesthetic treatment. Stress reduced the hypoventilatory effects of the low P _w co₂ treatment, increased the recruitment of anaerobic metabolism, and prolonged recovery time. Although the increase in plasma catecholamines elicited by the stress was small relative to the response obtained with the anaesthetic, stress prior to CO₂ anaesthesia impaired the efficiency of the treatment. Overall, our results suggest that P _w co₂ levels above 37 mmHg and/or stress prior to the anaesthesia impair the efficiency of air-saturated and buffered CO₂ anaesthesia by exacerbating the hypoxaemic effects of the hypercapnic treatment.     

Tris stimulates ecdysteroid secretion via Ca2+ messenger system in the prothoracic glands of Locusta migratoria

Abstract.  Secretion of ecdysteroids by the prothoracic glands of the migratory locust, Locusta migratoria L., is enhanced in vitro by tris(hydroxymethyl)aminomethane (tris) in a dose-dependent manner. Glands from larvae on the second day of the penultimate stadium are most sensitive. This action of tris depends on the uptake of calcium; increased production of ecdysteroid does not occur in the presence of cadmium, verapamil or TMB-8, or in calcium-free media. The concentration of unbound Ca²⁺, [Ca²⁺]_i, in the cytoplasm is measured with the aid of FURA 2/AM. Tris causes a rise of [Ca²⁺]_i that is fully suppressed by lanthanum and partially by nitrendipine. Two antagonists of IP₃ receptors elicit mutually opposite effects: heparin blocks, whereas 2-APB accelerates, the rise in [Ca²⁺]_i. Ryanodine exerts only a slight effect. It is proposed that tris activates locust prothoracic glands in a Ca²⁺-dependent manner that exhibits many similarities to the transduction pathway of prothoracicotropic hormone.