Haemolymph protein patterns during the spinning stage and metamorphosis of Rhynchosciara americana

An acrylamide gel electrophoretical analysis of the haemolymph proteins of R. americana was carried out at different stages of development. In mature larvae there are about 14 haemolymph protein fractions from which one stains heavily and two others faintly for lipoprotein, while three fractions stain for glycoprotein. The haemolymph protein fraction with Rm 0·25 decreases remarkably in mass during spinning, while the others decrease to a lesser extent. The protein fractions could be used in cocoon spinning since previous work suggests that haemolymph proteins are a major pool of cocoon protein precursors. The finding of a protein fraction in the salivary glands with an electrophoretical mobility similar to that of the haemolymph fraction with Rm 0·25 reinforces our hypothesis.     

SOURCES OF LARVAL SALIVARY GLAND SECRETION IN THE DIPTERAN CHIRONOMUS TENTANS

The soluble proteins in the hemolymph, the salivary gland, and the salivary secretion of fourth instar Chironomus tentans were examined by disc electrophoresis in acrylamide gels. Of the 11 protein fractions detected in buffered saline extracts of the gland, 10 are present also in the hemolymph. Amino acid isotope incorporation experiments indicate that the protein fractions shared by the salivary gland and the hemolymph are not synthesized in the gland but are synthesized in other larval tissues. Immunochemical studies show that most of these proteins eventually are secreted from the gland. The salivary gland in vivo and in vitro is active in de novo protein synthesis. The protein synthesized tends to form large molecular weight aggregates. As demonstrated by radioautography, at least 80% of this protein is secreted from the 30 large cells forming most of the gland. The proteins synthesized in the salivary gland cannot be detected in the hemolymph. The results of this investigation are consistent with a mechanism of secretion formation involving both de novo synthesis of some secretion proteins and the selective uptake, transport, and secretion of hemal proteins by the salivary gland.     

Relationship between protein and proteolytic activity in the midgut of mosquitoes.

In female mosquitoes anal injections (enemas) of nutrient solutions were administered in measured amounts to allow direct comparison of protease activities.The amount of protein ingested had a pronounced effect upon the rate of protein digestion, but had little influence upon the rate of protease secretion. Maximal protease activity increased only slightly with increasing meal size and always coincided with the digestion of about 80 per cent of the protein ingested.The use of the enema technique provided an experimental means to reject clearly a neural stimulus for protease secretion. Proof is given for a secretagogue stimulus: the presence of globular proteins with a minimal molecular weight is required for protease secretion.Despite antitryptic factors which are known to occur in different titres in vertebrate bloods, no inhibition was observed in vivo when proteases were recorded after enemas of blood or plasma from several hosts into Aedes aegypti, Anopheles quadrimaculatus, and Culex pipiens quinquefasciatus.Mosquito trypsin was shown to account for about 75 per cent of the proteolytic activity in midgut homogenates. Chymotrypsin was present although with very low activity. Calcium did not stimulate mosquito trypsin as it does mammalian trypsin. Between 22 and 32°C a Q₁₀ of 2·0 was observed for proteases as well as for protein digestion.     

Migration of the monarch butterfly, Danaus plexippus: energy sources,

The origin and nature of the metabolic reserves of the monarch butterfly during its autumn migration through Missouri were examined. Information was obtained about the butterfly's protein, glycogen, lipid, and lipid-soluble pigment composition. The principal results showed that lipids, which made up ca. 45 per cent of the insect's dry weight, were the major reserve. Analyses of the lipid classes showed that triglycerides were the dominant class in each sex. Separate analyses of the thorax and abdomen showed that >90 per cent of the lipids were present in the massive fat body which is restricted to the abdomen. The adult female rapidly incorporated U-¹⁴C-d-glucose into abdominal glycerides. The rôle of larval reserves and nectar consumption in migration and ovarial maturation was discussed.     

Tissue specific gene activities and proteins in the Chironomus salivary gland

Summary The secretory proteins of the larval salivary gland of some Chironomus species were analysed according to a method developed by Grossbach (1969). After reduction of the disulphide bonds and alkylation the secretion of Chironomus thummi was separated by disc electrophoresis at acrylamide concentrations of 4.8 to 9.5% into seven main and some minor fractions. By increasing the acrylamide concentration up to 20% nine main fractions could be resolved. In addition to these high molecular weight structural proteins a number of proteins soluble in simple buffer solutions and separable at pH 8.8 in a 15% acrylamide gel are present in the secretion but only to a very small amount.The electrophoretic pattern of the secretory proteins of Chironomus strenzkei, Ch. luridus and Ch. obtusidens are qualitatively the same as the Ch. thummi pattern. Some quantitative differences may exist. In the secretion of Chironomus plumosus at least eleven main fractions were detected in 20% acrylamide gels. A comparison between the number of Balbiani rings of the salivary gland chromosomes and the number of main protein fractions in the salivary gland secretion yielded no direct correlation.The results are discussed in respect to the question wether the major puffs of the gland, the Balbiani rings, encode the major products of the gland, the secretory proteins.Abbreviations used BR Balbiani ring - bis N,N-Methylenebisacrylamide     

Disk electrophoresis fractionation of hemolymph proteins during the metamorphosis of different castes and sexes of Formica pratensis

Using polyacrylamide electrophoresis the proteins of the haemolymph of the different developmental stages can be separated into eight strong and nine weak coloured fractions during the cocoon period of Formica pratensis. The proteins were stained with aniline black and measured quantitatively by a Chromoscan densitometer. The values were compared with those maintained with bovine serum albumin.The total protein content of the haemolymph was calculated as the sum of the different fractions; at maximum it amounts to 2·1 per cent (w/v). The maximum is reached during the pharate pupal stage and during the pigmentation of the eyes; the minimum can be observed at the end of pupal ecdysis. At the beginning of body pigmentation in all the forms the protein content of the haemolymph was very much reduced, especially in workers and females.All fractions change independently resulting in a different composition of the haemolymph proteins in pharate pupae, eclosed pupae, and pharate adults. The slow-running fractions f1, f3, f5, and f6 and the mean bands f8 and f11 are reduced weakly until body pigmentation, and from the eleventh day strongly in both castes. All fractions are reduced during the cocoon period, but mostly the slow-running ones. Only the front band f14 increases to nearly twice that of the protein content. The importance of the changes in the protein fractions for development of different organs and for the synthesis of the haemolymph proteins and the influence of hormones are discussed.     

Cultivation of Lemna gibba under desert conditions. I: Twelve months of continuous cultivation in open ponds

Duckweed, Lemna gibba, was grown in 12 m² shallow ponds in the Negev desert, during 12 months of continuous cultivation, beginning April 1984. Average monthly growth rates varied with the season of the year. The lowest daily yield, 2·6±0·4 g dry weight m⁻² day⁻¹, was obtained during January. Highest daily yields, 7·9±2·6 g dry weight m⁻² day⁻¹ and 7·0±1·2 g dry weight m⁻² day⁻¹, were obtained during September and May. A 35% decline of the yield was seen during midsummer (July), 4·8±1·2 g dry weight m⁻² day⁻¹. The average rate for the year was 5·15±1·7 g dry weight m⁻² day⁻¹. The protein content of the plants ranged from 30 to 38% per unit dry weight.Growth performance is discussed in relation to the prevailing climatic conditions.     

Molecular weight hetergeneity of proteins of the ribosomes and ribosomal subunits from dry pea seeds]

The molecular weight distribution of the total protein of ribosomes and ribosomal subunits isolated from dry pea seeds was studied by electrophoresis in polyacrylamide gel, containing sodium dodecyl sulfate. It was demonstrated that overall protein of 80 S ribosomes is separated into a number of fractions with molecular weights of 10000-64000. Treatment of ribosomes with 0.5 per cent tritone, 0.5 per cent and 1 per cent deoxycholate does not change the general pattern of the molecular weight distribution of ribosomal proteins. The large subunit reveals 19 protein zones (14 major and 5 minor zones), their molecular weights are varying from 10000 to 54000. The majority of proteins of the large subunit have molecular weights of 14000--32000. The molecular weights of 17 protein zones of the small subunit (7 major and 10 minor zones) vary from 10000 to 64000. The majority of proteins of both large and small subunits have molecular weights of 14000--32000. Electrophoretic separation of proteins in the split gel confirmed the fact that the proteins of large subunit differ in molecular weights from those of the small subunit. Thus, ribosomal proteins of pea seeds are shown to produce a typical (for 80S ribosomes) pattern of molecular weight distribution under polyacrylamide gel electrophoresis in the presence of sodium dodecul sulphate.     

Self-selection of an optimum diet from a mixture of wheat fractions by the larvae of Tribolium confusum

Tribolium confusum was reared from the beginning of the first instar to the pupa on wheat bran, endosperm or germ or on a 1 : 1 : 1 mixture of these three fractions. The food intake of the larvae reared on the mixture included more or less similar proportions of the three wheat fractions, averaging 1·5 per cent bran, 17·1 per cent endosperm and 81·4 per cent germ. Past work on the nutrition of stored-product insects has involved the assumption that the insects cannot feed selectively from a finely powdered diet. Our findings show that this assumption may not be warranted. The mixed diet supported growth better than any one of the pure fractions or any wheat diet previously tested by us. It was superior to germ, the best of the pure fractions, because it was more digestible and because the digested portion was more efficiently utilized for growth. Endosperm, the poorest diet of the fractions, was highly digestible but was not efficiently utilized. Bran, better than endosperm but inferior to germ, was relatively indigestible although the digested portion was efficiently utilized. Germ was both digestible and efficiently utilized.     

Patterns of nitrogen utilization between the ambrosia beetle Xyleborus dispar and its symbiotic fungus

Some parameters of nitrogen utilization between the ambrosia beetle Xyleborus dispar in mutualistic association with its symbiotic fungus Ambrosiella hartigii, were examined. Qualitative and quantitative analyses of the major nitrogenous excretory products were made on the various life stages of X. dispar. The main nitrogenous product found in excreta and hindguts of beetles, larvae, and pupae, was uric acid (range 7·6–14·8 μg uric acid/beetle). No ninhydrin-positive compounds were located in excreta of the beetles. The concentration of ammonia-nitrogen in the various life stages averaged between 0·70 and 1·13 μg NH₃-N/beetle.Total nitrogen determinations were made on sapwood samples of Malus sylvestris (0·34 ± 0·005% N by dry weight), attacked wood, ‘pre-brood’ (0·31 ± 0·005% N by dry weight), and attacked wood ‘post-brood’ (0·17 ± 0·02% N). Similar determinations of the artificial medium (l-asparagine medium) indicated that a nitrogen requirement of about 0·08–0·1% N by dry weight was necessary before oviposition could occur.Fixation of atmospheric nitrogen by individual X. dispar beetles in vitro was not indicated using the acetylene ethylene reductase method. Proteolytic enzyme activity was not found on examination of diapause beetles, their excreta, larval and pupal excreta, and the ambrosial and mycelial forms of A. hartigii.Comparative concentrations of soluble proteins and free amino acids suggested that fungus in the mycangia was built up from free amino acids of the insects. At the period of emergence, flight, and attack of new hosts, the females were found to have a concentration of soluble proteins more than double that found in the beetles during the remainder of the year. The free amino acids were the lowest values recorded during this period (March–October).     

Dietary cholesterol requirements of housefly, Musca domestica, larvae.

Quantitative analyses have been made of the dietary cholesterol requirement for the growth of the larvae of Musca domestica. The larvae will not grow on diets to which no cholesterol is added, a few pupae and adults are obtained when the concentration of cholesterol is 0·05 μmol/g of diet, but the concentration has to be raised to 0·36 μmol/g of diet before the maximum numbers of pupae and adults are obtained. Further addition of cholesterol above 0·36 μmol/g diet did not have any significant effect on the weight and growth of the larvae. However, the ratios of the cholesterol to phospholipid fractions recovered from the larvae increased rapidly when the concentration of cholesterol in the diet was raised from 0·05 to 0·56 μmol/g of diet. Above this concentration only a slight increase in the ratios was observed. Larvae reared on diets containing 0·05 μmol cholesterol/g of diet contain only 25 per cent of the cholesterol content of the larvae reared on the diets containing more than 0·28 μmol of cholesterol/g of diet, the cholesterol content being expressed relative to the weight of the larvae,The absence of cholesterol synthesis has been demonstrated in the larvae by feeding [4-¹⁴C] cholesterol. The specific activity of the cholesterol recovered from the larvae is the same as that of cholesterol added to the diet. Irrespective of the cholesterol concentration of the larval diet, approximately 97 per cent of the radioactivity recovered from the larvae behaved as free cholesterol, less than 1 per cent as cholesterol esters and the rest as unidentified ‘polar sterols’. The results are compared with those from similar studies on other insects.     

ISOLATION OF AN INSULIN SECRETION GRANULE FRACTION

Goosefish islets were homogenized in 0.25 M sucrose and separated into nuclear, mitochondrial + secretion granule, microsomal, and supernatant fractions. Eighty per cent of the cytochrome oxidase activity and 75 per cent of the bioassayed insulin activity were found in the mitochondrial + secretion granule fraction (6000 g for 10 minutes). The mitochondrial + secretion granule fraction was further subfractionated by centrifugation (2 hours at 100,000 g and 0°C) using a continuous linear density gradient 1.0–2.0 M sucrose). Eighteen to 20 subfractions were collected by piercing the bottom of the tube and collecting drops. The total protein was distributed into a bimodal curve consisting of a high density component, which contained 90 per cent of the insulin (secretion granules), and a lower density component, which contained the cytochrome oxidase activity (mitochondria).     

Differential net food utilization by larvae of Sitophilus oryzae and Sitophilus granarius

The net utilization of a meridic diet by Sitophilus oryzae from the newly hatched larva to the pupa was more efficient compared with that of S. granarius. The artificial diet was highly digestible to both species, but S. oryzae converted 24·1 per cent of the ingested food into body substance compared with 12·4 per cent converted by S. granarius. The mean pupal weight of S. granarius (1·402 mg) was greater than that of S. oryzae (1·012 mg); however, larvae of S. granarius consumed 2·6 times as much food and excreted 8·1 times as much faecal material to obtain that weight. Even though S. granarius consumed the diet at a greater rate, the relative growth rate of S. oryzae was faster. The intracellular symbiotes present in S. oryzae may have a rôle in the species' faster development and more efficient utilization of its food.     

Effect of particle size on the anthelmintic efficacy of mebendazole against Nippostrongylus brasiliensis in the rat.

The effect of reduction in particle size on the anthelmintic efficacy of mebendazole (methyl 5 (6)- benzoyle 1–2 benzimidazole carbamate) was studied in rats undergoing a primary infection with N. brasiliensis. A single oral treatment with fine ground mebendazole (particle size spectrum—54·95 per cent of particles less than 10·62 μ dia.; 86·06 per cent less than 21·27 μ) removed more than 98 per cent of adult worms from the intestine at a dose rate of 12·5 mg/kg body wt. On the other hand the best result achieved with coarse ground mebendazole (18·47 per cent of particles less than 10·62 μ dia; 42·26 per cent less than 21·27 μ dia) was 58 per cent of adult worms removed at a dose rate of 100 mg/kg body wt. It was also shown that fine ground mebendazole adversely affected migrating third stage larvae of N. brasiliensis.     

Characterization of a glycoprotein in the cyst fluid of Cysticercus tenuicollis from the goat

Dixon S. N., Gibbons R., Parker Janice and Sellwood R. 1973. Characterization of a glycoprotein in the cyst fluid of Cysticercus tenuicollis from the goat. International Journal for Parasitology3:419–424. In the fluids of two tapeworm cysts from goats a prominent periodic acid- Schiff reagent staining protein was found on gel electrophoretograms. In one case serum proteins from the host were also observed. The glycoprotein has been isolated and found to contain about 7·7 per cent heterosaccharide consisting of glucose, galactose, mannose, fucose, neuraminic acid, glucosamine and an unidentified component. The amino acid portion is extremely rich in glutamic acid, aspartic acid, lysine and arginine. Its molecular weight is approximately 90,000. A small degree of heterogeneity was demonstrated by ultra-centrifugal analysis; this may be due to the presence of about 3 per cent of a glycosaminoglycan. In view of the high proportion of charged amino acids it is suggested that this glycoprotein, which appears to be derived from the parasite, functions as the osmotically active macromolecule in the cyst fluid maintaining the turgidity of the cyst.     

The chemical basis of honeybee, Apis mellifera, caste formation. Partial purification of queen bee determinator from royal jelly

On royal jelly, 1- to 2-day-old honeybee worker larvae have been reared in vitro to adults in a yield of 67±18 per cent. Up to 100 per cent and, on an average, 60 per cent of them were queens and intercaster. The preparation of a basic food from royal jelly by extensive alcohol extraction is described. With this control food, a survival rate of 47±18 per cent was achieved; 15 per cent of the adults were determined, 4·3 per cent of them were queens. Rearing of 1- to 2-day-old worker larvae on a basic food, to which unknown fractions may be added, was used as a biological test for the partial purification of queen bee determinator from royal jelly. By chromatography of the ethanol extract, previously treated with charcoal, on the cation exchanger Dowex 50 WX4 and rechromatography on silica gel, a 10⁵-fold purification of determinator was achieved. Chemical properties of the highly hydrophilic, low molecular active fraction are described.     

Brain cardiolipin: isolation and fatty acid positions

Abstract— Cardiolipin (diphosphatidyl glycerol) was isolated from bovine brain grey matter in pure form by column chromatography. A combination of a DEAE cellulose column, an acid-silicic acid column and a bicarbonate-treated silicic acid column was used for the isolation. Analytical data on cardiolipin (and lyso-cardiolipin), including phosphorus and fatty ester values, infrared and ultraviolet spectra, and chromatographic behaviour of intact cardiolipin, lyso-cardiolipin and their water-soluble hydrolysis products gave results which were consistent with the diphosphatidylglycerol structure proposed by MacFarlane. Cardiolipin constituted 1·2 per cent of the total lipids from grey matter, or 0·43 per cent of the dry weight. No aldehydes were detected in purified cardiolipin. The major fatty acids were 16:0, 16:1, 18:1, 18:2 and 20:4. Cardiolipin contained much higher concentrations of 18:2 (17·8 per cent) than any of the other major grey matter glycerophosphatides. Cardiolipin was hydrolysed by incubation with snake venom (Trimeresurus flavoviridis) phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) to determine the distribution of fatty acids on the carbons of its diglyceride moieties. The fatty acids were not distributed randomly; 16:0, 18:0 and 18:2 were predominantly localized in the α and α positions, while 18:1 and 20:4 were predominantly localized in the β and β positions.     

Effect of Feeding Millet (Sorghum vulgarie) Protein on Growth,Nucleic Acids and Proteins of Liver and Plasma of Rats

Effect of feeding millet (Sorghum vulgarie) at 5, 10 and 15 per cent protein levels respectively for a period of six weeks to rats on their liver DNA, RNA and proteins of liver, its subcellular fractions and plasma has been studied, and results compared with rats fed casein at 10 per cent level. Both liver DNA and RNA of rats fed millet at 5 per cent protein level were significantly increased. Liver proteins (mg/l00 g body weight) of rats fed millet at 5 and 10 per cent protein level were significantly increased and plasma proteins decreased. Incorporation of leucine-I-¹⁴C into both liver and plasma proteins of rats fed millet was significantly higher than the control.     

Utilization of fatty acids by Pieris brassicae reared on artificial and natural diets

The fatty acid composition of Pieris brassicae was measured from larvae reared on four different diets. Pieris can alter the composition of fatty acids in the diet through selective incorporation and synthesis. Oleate is preferentially accumulated on artificial diets (15·9 per cent in diet, 43·8 per cent in neutral lipid (NL) of fifth instar larvae), but not equally on natural diets (18·1 per cent in Brassica napus, 25·6 per cent in the NL of fifth instar larvae). Incorporation of linolenate appears to depend on the concentration of both linolenate and linoleate in the diet. With dietary levels of 35·7% linolenate and 32·2% linoleate, fifth instar larvae contain 12·2 and 16·0 per cent, respectively, of these acids. With 45·8% linolenate and 12·5% linoleate in the diet, fifth instar larvae contain 44·1 and 11·6 per cent of these acids, respectively, in the NL. Palmitoleate is actively synthetized on the artificial diets; with trace amounts of dietary palmitoleate, fifth instar larvae have 9·3 per cent of this acid in the NL. Pieris regulates the uptake of linoleate from the diet at the intestinal wall as was shown by linoleic acid-1-¹⁴C, and is unable to convert dietary linoleate to any of the 18-carbon analogues. The female apparently accumulates linolenate into egg phospholipids on the artificial diet, but in general the fatty acid composition of the eggs resembles that of the fat body.     

The antiserotonin and antihistamine activities of salivary secretion of Rhodnius prolixus

The antiserotonin and antihistamine activities of Rhodnius prolixus salivary secretion were studied using the rat uterus and the guinea pig ileum preparations. Serotonin antagonism was not competitive and the salivary secretion dose which abolished a half-maximal uterine contraction was 13 ± 2 (S.E.) μg protein/ml (n = 9). No inhibition was seen for the acetylcholine and bradikinin contractures. On the other hand, the observed antihistamine activity was typically competitive. The mean concentration of Rhodnius salivary secretion of 14 ± 2 (S.E.) μg protein/ml (n = 9) reduced the effect of histamine to that of a half dose. Specificity was demonstrated when testing salivary secretion in the acetylcholine and BaCl₂-induced guinea pig ileum contractures. In both antagonisms, no preincubation was needed for the full expression of the inhibitory effect, which did not persist after washing the preparation. The presence of enzymes that could be degrading the amines was excluded.Susceptibility to trypsin and heat treatments as well as the behaviour of the principles on gel filtration and preparative agarose electrophoresis experiments, suggest that the activities reside in the same peptide molecule with an apparent molecular weight of 39,000 daltons.