Induction kinetics of immune antibacterial proteins in pupae of Galleria mellonella and Pieris brassicae

• 1.1. Pupae of Galleria mellonella and Pieris brassicae given an injection with live, non-pathogenic Enterobacter cloacae or abiotic foreign molecules induce an acquired immunity that corresponds with the synthesis of haemolymph proteins of antibacterial activity.
• 2.2. This humoral defensive response which persists for several days, differs quantitatively between insect species and between the inducers used, although very different foreign bodies induced the same immune proteins in both lepidopteran insects.
• 3.3. A stronger and longer lasting response was consistently noticed in pupae immunized with non-pathogenic bacterium than after sterile nutrient broth injections.
• 4.4. A demonstrably elevated activity of haemolymph lysozyme and trace activity of cecropins found in pupae of Galleria treated with saline W, a salt solution physiological to moths, disappear soon after 36 hr from injection.
• 5.5. In P. brassicae, however, sterile insect Ringer can give a varying, if present at all, immune response.
• 6.6. A mechanical injury (sterile wounding of insect body) can occasionally induce a similar but much weaker response.
• 7.7. The antibacterial activity was drastically reduced in Pieris or completely depressed in most pupae of Galleria when actinomycin D or cycloheximide was given at an early time post-immunization with E. cloacae.
• 8.8. It is concluded that the de novo synthesis of ribonucleic acid and immune proteins is required for expression of antibacterial activity in pupal haemolymphs.
• 9.9. The synthesis of an immune mRNA was completed about 7 hr after the injection of the immunizing bacteria.

     

Phenoloxidase activity of acridid grasshoppers from the subfamilies melanoplinae and oedipodinae

• 1.1. Phenoloxidase activity and wound melanization was studied in five species of grasshoppers representing the subfamilies Melanoplinae and Oedipodinae.
• 2.2. Most of the phenoloxidase activity was detected in the plasma fraction of grasshopper whole-body homogenates and supernatant fractions of the hemolymph. The species representing the Oedipodinae had 20–50% higher percentage of the total phenoloxidase activity associated with particulate matter from a whole-body homogenate when compared to the Melanoplinae.
• 3.3. Phenoloxidase activity could not be detected in sclerotized cuticle of adult grasshoppers.
• 4.4. The phenoloxidase existed as a zymogen which could be activated by chymotrypsin and inhibited by KCN and NaCN while EDTA showed no effect. It had optimum activity at 37°C and pH 7.3.
• 5.5. These findings are discussed in relation to wound repair and immune responses to infection in grasshopper species.

     

Effect of salinity on the osmotic,chloride, total protein and calcium concentrations in the hemolymph of the prawn Peneaus monodon (fabricius)

• 1.1. Osmolality and chloride concentrations in the hemolymph of Penaeus monodon became stable 1 day after molting in 32 ppt, while total protein and calcium concentrations remained stable throughout the molting cycle. When intermolt (≥ 36 hr postmolt) animals were transferred from control (32 ppt) to experimental (8–40 ppt) salinities, osmolality, chloride and total protein, but not calcium, concentrations in the hemolymph achieved steady state values 24–48 hr after transfer.
• 2.2. The hemolymph osmolality was a linear function (slope = 0.28) of medium osmolality at salinities between 8 and 40 ppt. It was isosmotic to seawater at 698 mOsm (10 g prawns) and 752 mOsm (30 g), and was hyperosmotic to the medium below isosmotic concentrations, and hypoosmotic to those above.
• 3.3. Hemolymph chloride concentration was isoionic to seawater at 334 mM, and was hyperregulated below isoionic concentrations, and hyporegulated to those above.
• 4.4. P. monodon maintained its hemolymph calcium concentration between 6.4 and 10 mM when medium salinities increased from 8 to 40 ppt.
• 5.5. Total protein concentration in the hemolymph was independent of medium salinity (8–40 ppt) and hemolymph osmolality (540–850 mOsm).

     

Characteristics of Crassostrea virginica crystalline style chitin digestion

• 1.1. Fundamental chitin digestion characteristics of Crassostrea virginica crystalline style were investigated.
• 2.2. Optimum temperature and pH were 34°C and 4.8. respectively.
• 3.3. The colloidal regenerated chitin (0.56mol/0.5 ml: GlcNAc equivalents) was saturating under all enzyme levels encountered.
• 4.4. There was no evidence of end product inhibition, even after 100 hr incubation.
• 5.5. Calculated K_m for the chitinase complex was 1.19mM when determined using a 30 min assay, but was only 0.70 mM when determined using a 4.6 hr assay.
• 6.6. Both K_m values are lower than reported for similar assays in other molluscs and for most bacteria.
• 7.7. Effect of substrate preparation on the kinetics are discussed.
• 8.8. Eight peaks of chitinase activity were resolved by DEAE-Fractogel ion exchange chromatography.

     

The qualitative and quantitative analysis of insect hemolymph sugars by high performance thin-layer chromatography

• 1.1. A procedure is described for the analysis of sugars in insect hemolymph by high performance thin-layer chromatography.
• 2.2. Densitometric scanning of spots after plate developments shows linear responses with most sugars in an analytical range of 125ng-2.0 /gmg; detection limits range from 30/2–60 ng.
• 3.3. Quantitative measurements of trehalose, glucose and fructose can be made on hemolymph sample volumes of less than one microliter, allowing blood sugar analysis of individual insects.
• 4.4. Hemolymph sugar determinations on six species of insects agree well with values reported in the literature, but mean values for a species often showed higher variability because samples were taken from individuals.

     

Alterations in proteases,protease inhibitors and ecdysone levels: a profile of stress in insects

• 1.1. A comparison of proteolytic and protease inhibitory activity, and ecdysteroid levels in body fluids was made between normal larvae of the flesh fly, Sarcophaga bullata, and those that had been water-stressed for two days.
• 2.2. The course of proteolytic activity in water stressed flies decreases 6 hr after beginning the experiment and remains low in comparison with control flies.
• 3.3. The course of protease inhibitors exhibits a mirror image pattern to proteases.
• 4.4. Ecdysteroid pattern shows two peaks in control animals: minor at 24 hr and major at pupariation, in experimental animals: at 1 hr, at 6 hr and at white pupal stage.

     

Effect of inorganic phosphate on synthesis of 5-phosphoribosyl-1-pyrophosphate in cultured plant cells

• 1.1. Inorganic phosphate (Pi) was absorbed rapidly by suspension-cultured cells of Catharanthus roseus which had previously been cultured in Pi-free Murashige Skoog medium.
• 2.2. The intracellular levels of ATP, ADP and 5-phosphoribosyl-l-pyrophosphate (PRPP) increased markedly during the 24 hr which followed the addition of Pi (1.25mM).
• 3.3. Availability of PRPP in vivo, estimated by the measurement of nucleotide synthesis from [8-¹⁴C]adenine, was also increased by addition of Pi.
• 4.4. Only a 20% increase in the maximum catalytic activity of PRPP synthetase was observed in extracts of cells, prepared 24 hr after addition of Pi.
• 5.5. In contrast to results for mammalian PRPP synthetase, the activity of PRPP synthetase, partially purified from Catharanthus roseus, was inhibited by concentration of Pi greater than 5mM.
• 6.6. The mechanisms involved in the increased availability of PRPP and the synthesis of adenine nucleotides in the plant cells cultured in Pi-containing medium are discussed.

     

Light-induced transient increase of the activity of topoisomerase I in plasmodia of Physarum polycephalum

• 1.1. Activity of topoisomerase I and incorporation of [³H]uridine and [¹⁴C]thymidine were monitored during light-induced sporulation of the slime mold Physarum polycephalun.
• 2.2. A 4-fold transient increase of topoisomerase I activity but not of [³H]uridine or [¹⁴C]thymidine incorporation was observed after 42 hr of illumination with 6 hr impulses.
• 3.3. The activity of topoisomerase I did not increase in the absence of light impulses. However, ca 5-fold increase of the activity was observed in dark when 100 μ M dibutyryl-cAMP was administered 12 hr before harvesting of plasmodia.
• 4.4. Fluorodeoxyuridine and cycloheximide administered 36 hr after starting of the illumination cancelled the increase of the activity of topoisomerase I.
• 5.5. After 7 days of the illumination, when fruiting bodies appeared, the activity of topoisomerase I dropped to about 15% of the initial value.

     

Increased muscle glucose uptake in response to chronic glyburide treatment is not related to changes in glucose transporter (GLUT4) protein

• 1.1. The mechanism of action of glyburide (a sulfonylurea) on muscle has been investigated by measuring glucose uptake and glucose transporter (GLUT4) protein levels after chronic glyburide treatment.
• 2.2. A dietary induced insulin resistant rat model (4 wk of high-fat, high-sucrose feeding) was given glyburide (2mg/kg/day) for 10 days and glucose uptake was measured in a perfused hindquarter preparation.
• 3.3. Protein levels of the GLUT4 glucose transporter were determined by Western analysis.
• 4.4. After 7 days of treatment, rats fed glyburide had lower blood glucose concentrations 2 hr (72 ± 5 vs 103 ± 12 mg/dl) and 24 hr (97 ± 7 vs 123 ± 7 mg/dl) after glyburide administration with no difference in serum insulin levels compared to vehicle treated animals.
• 5.5. Glucose uptake was approx doubled in basal state (0 insulin) in response to glyburide (2.8 + 0.4 vs 1.7 ± 0.2μ mol/g per hr).
• 6.6. Maximal insulin (100 nM) stimulated glucose uptake tended to be higher in the glyburide treated group, but did not reach statistical significance (8.0 ± 0.7 vs 7.0 ± 0.6 μmol/g per hr).
• 7.7. Western analysis revealed no significant effect of glyburide on the GLUT4 protein level in skeletal muscle.
• 8.8. These results suggest that glyburide alters glucose uptake through some mechanism other than alterations in the level of the GLUT4 glucose transporter protein.

     

Hemolymph osmo-ionic concentrations in terrestrial crabs following desiccation

• 1.1. The hemolymph osmotic, Na⁺ K⁺, Ca⁺⁺ and Mg⁺⁺ concentrations were determined for both sexes in crabs from mangrove Ucides cordatus and Goniopsis cruentata and supralittoral Ocypode quadrala after aerial desiccation, for 10 hr.
• 2.2. There was no difference between sexes in the two mangrove crabs, but in O. quadrata the females were the most significantly affected (P < 0.01).
• 3.3. Hemolymph osmotic Na⁺, Ca⁺⁺ and Mg⁺⁺ concentrations increased significantly in desiccated ghost crabs, while K ⁺ concentration was not significantly changed. In the two mangrove crabs, only hemolymph Ca⁺⁺ concentration increased significantly, after desiccation.
• 4.4. The short time desiccation is suggested to be a device to study the steps of osmo-ionic regulation in terrestrial crabs.

     

Identification and characterization of vitellin in a hermophrodite shrimp,Pandalus kessleri

• 1.1. A female specific protein (FSP, vitellogenin) in hemolymph and its related ovarian protein (vitellin) of Pandalus kessleri were studied by means of electrophoretical and immunological procedures.
• 2.2. The vitellin was purified from vitellogenic ovaries using hydroxylapatite, DEAE cellulose and Sepharose 6B columns, consecutively.
• 3.3. The vitellin had a molecular weight of approximately 560 kD and was composed of two subunits, 81 and 110 kD, respectively.
• 4.4. The vitellogenin concentrations in the hemolymph increased as vitellogenesis in the ovarian oocytes advanced and dropped markedly after the release of mature eggs.

     

Haemolytic activity in the brown shrimp (Penaeus californiensis holmes) haemolymph

• 1.1. Natural haemolytic activity in brown shrimp (Penaeus californiensis) haemolymph was detected using mouse erythrocytes as target cells. This activity is unrelated to agglutinating and phenoloxidase activity, but it is another probable component of the shrimp defence system.
• 2.2. The haemolytic reaction is time and dose dependent, and a serine-protease is involved.
• 3.3. The haemolytic factor is thermolabile and has an apparent molecular weight of 23.5 kDa.

     

Time-dependent cadmium-neurotoxicity in rat brain synaptosomal plasma membranes

• 1.1. The modulation of lipid dynamics and lipid protein interactions were studied in rat brain synaptosomal plasma membranes (SPM) up to 24 hr after exposure to cadmium (Cd).
• 2.2. The activity of acetylcholinesterase and adenylate cyclase showed a considerable decrease after 6 hr of Cd exposure, followed by a progressive increase up to 24 hr.
• 3.3. SPM chemiluminescence showed a maximum decrease at 12 hr, demonstrating a considerable increase in lipid peroxidation.
• 4.4. SPM of Cd-exposed animals showed a statistical significant increase in fluorescence anisotropy parameter [(r₀/r) — 1]⁻¹ at 18 and 24 hr compared to SPM of the control, indicating a decrease of membrane fluidity.

     

Correlation of water translocation,cuticle dehydration and post-ecdysial sclerotization by the american cockroach

• 1.1. Haemolymph volume decreases during the initial 16 hr post-ecdysial period, increases after water ingestion and subsequently drops until the inter-ecdysial level is reached.
• 2.2. Total body water follows a similar pattern, but the changes are not as pronounced.
• 3.3. Tissue water is inversely proportional to the total body water.
• 4.4. Soluble cuticle protein declines throughout the initial 16 hr period while both β-glucosidase and alkaline phosphatase activity is lost within 6 hr after ecdysis.
• 5.5. Dehydration of the cuticle also occurs during the immediate 6 hr post-ecdysial period.
• 6.6. These data suggest that the formation of the protein-insoluble matrix is linked with water loss.
• 7.7. Water removal may decrease the distance between molecules allowing specific reactions to take place.

     

A comparison of aspartate transcarbamylase activity in juvenile pacific oysters (Crassostrea gigas) when fed various diets

• 1.1. The optimum pH for measurement of aspartate transcarbamylase activity in oyster tissue was determined to be 9.35 while the optimum temperature was 39.5°C.
• 2.2. Aspartate transcarbamylase activity varied significantly over short periods of time (hr) possibly due to fluctuations in the amount of food digested.
• 3.3. The composition of the oyster's diet also affected the levels of aspartate transcarbamylase activity in oyster tissues.
• 4.4. Those oysters fed an egg yolk-starch diet contained significantly lower aspartate transcarbamylase activity than oysters fed an egg yolk-starch-salmon oil diet or a casein-starch-salmon oil diet.
• 5.5. The aspartate transcarbamylase activities in oysters fed Phacedactylum tricornutum or a starch diet were not significantly different from the activities in oysters fed the egg yolk-starch diet.

     

Regulatory role of δ-aminolevulic acid dehydratase in the dimorphic fungus Mucor rouxii

• 1.1. With the aim of finding a possible relationship between the known dimorphism phenomenon existing in the fungus Mucor rouxii and the biosynthesis of respiratory pigments, the activity of aminolevulic acid synthetase (ALA-S) and ALA-dehydratase (ALA-D) was studied in crude extracts and in 15,000 g supernatants of both mycelium and yeast-like cells.
• 2.2. The activity of ALA-S was unusually high (3 nmol ALA/hr/mg protein) compared with that reported for other tissues and did not vary with the fungus morphology.
• 3.3. Instead, ALA-D specific activity was found to be 16.5 nmol PBG/hr/mg protein in mycelium extracts, that is 7-fold greater than that measured in the yeast-like morphology (2.6 nmol PBG/hr/mg protein).
• 4.4. It was of importance to determine the activity levels of ALA-D along with the morphogenic transition from yeast to mycelium. It was observed that the greatest change and enhancing of specific activity occurred 2 hr before the emergence of the germ tubes and was held constant up to the complete development of mycelium.
• 5.5. Both hyphae formation and enhancement of ALA-D activity were diminished when cAMP was added to the culture shifted from the anaerobic atmosphere to air.
• 6.6. These findings and preliminary studies on the characterization of M. rouxii ALA-D indicate that this enzyme plays a regulatory role in porphyrin biosynthesis in this fungus as well as a key function in the characteristic morphogenic transition.

     

Relationship between brain trehalase activity and trehalosema during direct and diapausing development in Pieris brassicae

• 1.1. Brain trehalase specific activity and trehalosemia were measured during the end of the developmental life cycle in non-diapausing and diapausing insects.
• 2.2. During non-diapausing development, trehalosemia reached maximum values at the beginning of pupal life. Then a constant decrease was observed up to the end of adult life.
• 3.3. The specific activity of brain trehalase was maximum when the insects were in active feeding periods, minimum activity appearing during moulting phases.
• 4.4. During diapausing development, trehalosemia was very high at the beginning of pupal life, particularly when insects were exposed to wintering conditions.
• 5.5. When diapause was broken, trehalosemia fell, announcing adult emergence.
• 6.6. Brain trehalase activity showed the same qualitative variations as in non-diapausing larvae, but with rather lower values.
• 7.7. During pupal life, brain trehalase activity decreased markedly during the long period necessary to obtain diapause breakdown.
• 8.8. Wintering conditions allow a progressive increase of brain trehalase activity, which preceded the fall of trehalosemia.

     

Blood volume recovery in hemorrhaged japanese quail

• 1.1. Blood volume and plasma biochemical changes and feed and water consumption in response to a hemorrhage by phlebotomy of 30% of the calculated total blood volume with and without replacement of blood volume with physiological saline were determined in juvenile male Coturnix coturnix japonica.
• 2.2. Plasma protein and osmolality decreased rapidly posthemorrhage and did not recover by 72 hr posthemorrhage.
• 3.3. Plasma glucose, Na⁺ and K⁺ increased within Ihr postphlebotomy. Plasma Na⁺ returned to nonphlebotomized levels within 6 hr postphlebotomy.
• 4.4. Saline replacement of blood volume resulted in hypervolemia within 3–5 min postphlebotomy.
• 5.5. Phlebotomized quail receiving no saline recovered blood volume to 0 hr (nonphlebotomized) levels within l hr postphlebotomy.

     

Osmolality and electrolyte concentration of hemolymph and the problem of ion and volume regulation of cells in higher insects

• 1.1. The study was carried out on 22 species of insects from 5 orders. The osmolality of their hemolymph varied from 319 to 421 mOsm/kg H₂O, concentration of Na⁺ 4.6 to 118 mM/l, K⁺ 6.3 to 73mM/l, Ca²⁺ 3.6 to 12.9 mM/l, Mg²⁺ 2.3 to 76 mM/l. The most abundant cation in the hemolymph of insects from higher orders is either K⁺ or Mg²⁺.
• 2.2. In the muscles of lower and higher insects K⁺ is usually within 80–120 mM/kg wet wt.
• 3.3. Most Ca²⁺ and Mg²⁺ in hemolymph is bound with protein and low molecular anions, concentration of free Ca²⁺ is 0.9-2.1mM/l Mg²⁺ 3.7–8.0 mM/l.
• 4.4. It is concluded that, in insects, potassium hemolymph, cell volume regulation and accumulation of ions in the cell, are ensured by an increased osmolality of hemolymph due to a high percentage contribution of low molecular organic substances which are retained in the hemolymph due to the absence of filtration apparatus in the Malpighian tubules.

     

The effects of temperature and moisture on survival capacity,cuticular permeability,hemolymph osmoregulation and metabolism in the scorpion,Centruroides hentzi (banks) (scorpiones,buthidae)

• 1.1. The temperature and water relations of Centruroides hentzi females were investigated. At 12 and 72% relative humidity (RH), the lower and upper Lt₅₀ were -4.5 and 43.7°C, and -4.7 and 45.1°C, respectively. When exposed to high temperature stress, survivorship was significantly greater under mesic conditions.
• 2.2. Cuticular water loss was higher under xeric conditions (12% RH), ranging from 0.061 mg/cm²/hr at 30°C to 0.211 at 41°C.
• 3.3. Exposure to dry air (0–5% RH) resulted in a significant increase in hemolymph osmolality: from 441 to 688 mOsm over a 5 day period.
• 4.4. Mean oxygen consumption rates increased from 161.7 mm³/g/hr at 34°C to 541.6 at 44°C. ATPase activity was significantly higher in animals acclimated and tested at 35°C.