Free amino acids of the haemolymph of the southwestern corn borer and the European corn borer in relation to their diapause

The titres of free amino acids present in the haemolymph of diapausing larvae of the southwestern corn borer, Diatraea grandiosella and the European corn borer, Ostrinia nubilalis, were examined. High titres of serine were found in the haemolymph of both species. Serine may serve as a storage form of compounds that are required for the synthesis of uric acid and other purines. The high titres of proline found in the haemolymph of O. nubilalis during the fall and winter may contribute to the freezing tolerance of this species. Alanine accumulated in the haemolymph of both species during the winter.     

Distribution of nutrient reserves during spinning in tissues of the larva of the fly, Rhynchosciara americana

The amount of protein, carbohydrate, lipids, and free amino acids were examined in the spinning stage in the fat body, haemolymph, skeletal muscle, and gut of Rhynchosciara americana. Protein and lipids increase in the fat body soon after the animal stopped feeding, probably at the expense of the digestion of the gut contents and of the reserves of the gut wall. Afterwards there is a fall in protein and lipids in the fat body. Haemolymph protein rises a little at the beginning of spinning and then decreases steadily during cocoon production. Carbohydrate and free amino acids decrease from the beginning of spinning in all tissues studied. Quantitatively, the most important decrease of carbohydrate during spinning occurs in the fat body whereas that of free amino acids occurs in the haemolymph. Lipid increases during spinning in the skeletal muscle, probably due to enlargement of the lateral fat body which occurs as a contaminant in the skeletal muscle preparation. The Malpighian tubules contain a large amount of calcium carbonate, which is eliminated during spinning. A correlation of our chemical data with histochemical data recently published is presented and the physiological implications of our findings are discussed in comparison to other insects.     

Amino acid and protein changes in the haemolymph of developing fourth instar Chironomus tentans

The concentration of free amino acids, total soluble protein, and haemoglobin in the haemolymph of fourth instar Chironomus tentans was investigated.The concentration of the free amino acid pool increases between the early (15.7 mM/l) and mid-(33.9 mM/l) fourth larval stages followed by a decline during the late (16.9 mM/l) fourth larval period. Alanine, serine, and the amides of aspartic acid and glutamic acid are the predominant free amino acids at all stages. Physiological fluid analysis of late fourth instar haemolymph detected 32 ninhydrin positive components including 18 common amino acids plus homoarginine, ornithine, citrulline, β-alanine, α-aminoadipic acid, α-aminoisobutyric acid, and sarcosine.The concentration of total soluble protein steadily increases during fourth instar larval development to a maximum of $\text{9.3}\text{g}\text{100}\text{ml}$ followed by a decline during the pharate pupal period. A similar pattern of variation occurs in haemoglobin content which comprises from 51 to 66% of Chironomus tentans haemolymph protein.The mM percentage of individual amino acids of total haemolymph protein varies little during the fourth instar. At all stages alanine and aspartic acid are the predominant amino acids.     

Chemical composition and rate of synthesis of the larval salivary secretion of the fly Rhynchosciara americana

The salivary secretion of Rhynchosciara americana was chemically analysed. The secretion shows a yellow colour, with a pH of 7·5 and protein as its major component (94·5 per cent of the secretion dry weight). Carbohydrates are minor components of the secretion which amount to 3·4 per cent of the secretion dry weight, of which 2·3 per cent are neutral carbohydrates and 1·1 per cent are galactosamine. The major amino acids present in the secretion proteins are aspartic acid, glycine, serine, and glutamic acid. The salivary secretion proteins can be separated into eleven protein fractions by urea-acrylamide gel electrophoresis from which nine fractions are PAS positive. The salivary pigment moves together with the protein fraction No. 8, which is quantitatively the most important one, and has spectral characteristics identical to a haemolymph pigment. The higher rate of gland protein labelling by ¹⁴C-phenylalanine determined in vivo and in vitro occurs around the middle of the spinning stage at the same time as the appearance of the large chromosomal puffs. The rôle of the salivary secretion in cocoon production is discussed.     

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.     

Effect of various kinds of dietary protein and supplementation with limiting amino acids on growth,haemolymph components and uric acid excretion in the silkworm, Bombyx mori

Effects of various kinds of dietary protein on growth of the silkworm, Bombyx mori, were determined using semi-synthetic diets. Also, the ingestion, digestion and utilization of dry matter and of nitrogen were measured. Nutritive effects of dietary proteins and supplementation of limiting amino acids on haemolymph protein and amino acids pattern were also investigated. Larval growth was largely dependent on the dietary proteins. When the larvae were reared on a diet containing weakly nutritive proteins such as gluten and zein, haemolymph protein was decreased and uric acid excretion was markedly accelerated. The free amino acid composition of the haemolymph manifested characteristic patterns according to the kinds of dietary protein.The supplementation of gluten and zein with their limiting amino acids resulted in a rise of haemolymph protein and a drop in uric acid excretion. The amino acid patterns in the haemolymph were greatly changed according to supplementation.     

Some features of the regulation of the free amino acids in adult Calliphora erythrocephala

The amounts of nearly every amino acid in Calliphora remain unchanged inspite of stress. Thus the free amino acid pool is regulated. The amounts of free amino acid in the haemolymph account for only a small part of the total free amino acid in a fly and therefore most free amino acid is located and regulated intracellularly. A comparison of the rates of conversion and turnover of glycine-C¹⁴ in flies fed protein and sugar and in flies fed sugar alone indicates that in the absence of a dietary source of amino acid, not only is the rate of total turnover appreciably lower but the rates of conversion to other amino acids also change. With a dietary source of amino acid, the rates of incorporation into protein account for only a part of the total turnover of each amino acid. With a sufficiency of amino acid, substantial amounts are probably used as an energy source.     

Identification of hornet silk gene with a characteristic repetitive sequence in Vespa simillima xanthoptera

Vssilk 5 is a gene encoding a component protein of the silk produced by the larvae of the yellow hornet (Vespa simillima, Vespinae, Vespidae). In this study, we deduced the complete cDNA sequence of Vssilk 5. It was found that 2 silk proteins, Vssilk 5 N and Vssilk 5 C, in the cocoon of the yellow hornet are both encoded by the Vssilk 5 gene. Vssilk 5 N and 5 C are the N- and C-terminal regions, respectively, of the Vssilk 5 pro-protein (Vssilk 5p). The complete amino acid sequences of Vssilk 5 N and Vssilk 5 C were deduced. Although a non-repetitive amino acid sequence and coiled-coil structure are properties common to the major components of silk proteins produced by the larvae of the social superfamilies Apoidea and Vespoidea of the Apocrita, nearly the entire sequence of Vssilk 5 C consisted of a repeated sequence of amino acids, and the calculated coiled-coil probability for this protein was low. Vssilk 5 N is a protein without a repetitive amino acid sequence and has a low coiled-coil probability. Moreover, we found a water soluble protein, Vssilk 5S that is likely segmented from Vssilk 5 C and contains an N-terminal sequence identical to that of Vssilk 5 C.     

Structure and expression of the silk adhesive protein Ser2 in Bombyx mori

Sericins are soluble silk components encoded in Bombyx mori by three genes, of which Ser1 and Ser3 have been characterized. The Ser1 and Ser3 proteins were shown to appear later in the last larval instar as the major sericins of cocoon silk. These proteins are, however, virtually absent in the highly adhesive silk spun prior to cocoon spinning, when the larvae construct a loose scaffold for cocoon attachment. We show here that the silk-gland lumen of the feeding last instar larvae contains two abundant adhesive proteins of 230 kDa and 120 kDa that were identified as products of the Ser2 gene. We also describe the sequence, exon–intron structure, alternative splicing and deduced translation products of this gene in the Daizo p50 strain of B. mori. Two mRNAs of 5.7 and 3.1 kb are generated by alternative splicing of the largest exon. The predicted mature proteins contain 1740 and 882 amino acid residues. The repetitive amino acid sequence encoded by exons 9a and 9b is apparently responsible for the adhesiveness of Ser2 products. It has a similar periodic arrangement of motifs containing lysine and proline as a highly adhesive protein of the mussel Mytilus edulis.     

Changes of haemocyanin,protein and free amino acid levels in the haemolymph of Penaeus japonicus exposed to ambient ammonia

Haemolymph ammonia-N of Penaeus japonicus (15.6 ± 2.17 g) increased with increased ambient ammonia-N, as they were exposed individually in 30 ppt seawater to 0.003 (control), 0.367, 0.731, 1.439 and 3.665 mmol/1 ammonia-N after 24 hr. Ammonia-N excretion was inhibited and net ammonia-N uptake occurred, as shrimps were exposed to 0.367 mmol/1 ammonia-N or greater. Haemocyanin and protein levels in the haemolymph of shrimp decreased, whereas free amino acid levels increased with increased ambient ammonia-N in the range of 0.003–1.439 mmol/1 ammonia-N. Exposure of shrimp to ambient ammonia-N at 1.439 mmol/1 caused accumulation of haemolymph ammonia and urea, and caused catabolism of haemocyanin and protein to free amino acids. Urea, taurine and glutamine are the major organic constituents in the haemolymph of shrimp under ammonia stress.     

The larval silk of Hypera spp. (Coleoptera: Curculionidae). A new example of the cross-β protein conformation in an insect silk

X-ray diffraction studies on the larval cocoon silk of the weevils Hypera postica and H. rumicis (Coleoptera: Curculionidae) indicate that it is a new example of the cross-β conformation in proteins. This silk is secreted by Malpighian tubules and stored in the rectum. Chemically it is more complex, with a greater range of constituent amino acids, than Chrysopa (Neuroptera) silk (the first well-documented example of a naturally occurring cross-β protein). There is a relatively high proline content, an unusual feature for an arthropod silk. X-ray data, and direct measurement of dispersed ribbons, suggest that the micellar width is about 3.0 compared to 2.5 nm in Chrysopa silk. This latter silk is the ‘type structure’ for naturally occurring cross-β protein conformations. Hypera silk can be considered the ‘next one up’ in a possible hierarchy of cross-β structures.     

Free amino acids in some sponges

Most invertebrates, particularly those of marine origin, have relatively high concentrations of free amino acids which are considered an important constituent of their osmoregulatory mechanisms [1]. Very little information is available on the free amino acid distribution in Porifera [2,3]. Common amino acids in some sponges were recognised by paper chromatography by Inskip and Cassidy [4] and Ackermann et al. [5,6] included a few sponges in their survey of the occurence of nitrogen compounds in marine invertebrates. More recently Bergquist and Hartman [7] surveyed semiquantitatively the distribution of free amino acids in several sponges. In the present paper we report on the amino acid composition of 12 species of sponges belonging to the class Demospongiae as a part of a study on the metabolites of Porifera [8]. Fresh sponges were extracted with aqueous ethanol. The organic solvent was removed and the aqueous solution, after removal of the ether soluble compounds, was separated into cationic, anionic and neutral fractions by ion-exchange chromatography. The cation fraction was analysed for amino acids using an automatic amino acid analyser. The results, which are presented in Table 1, show that all species of sponges examined have a similar composition in common amino acids. Glycine almost always appears as the dominant protein amino acid, followed by high concentrations of alanine and glutamic acid, whereas relatively lower concentrations of basic amino acids are present. In Axinella cannabina, Chondrosia reniformis, Chondrilla nucula, Cliona viridis and Hymeniacidon sanguinea, glycine represents more than 77% of the total amino acids. The high percentage of free glycine (90.4%) in Chondrosia reniformis is noteworthy. The anionic and the neutral fractions were examined for sulfur-containing amino acids using PC. Taurine (Table 2) was detected in all the Porifera examined; this is in agreement with previous observations [5–7]. N-Methyltaurine was identified in some of the species examined, whereas neither N,N-dimethyltaurine nor N,N,N-trimethyltaurine were found.     

Physiological pattern and further characterization of the haemolymph violet carotenoprotein of the fly Rhynchosciara americana

• 1.1. An electrophoretic purification procedure for the haemolymph violet carotenoprotein of R. americana was described. The purified protein was used for obtaining a specific antiserum.
• 2.2. This carotenoprotein contains: (1) a high weight percentage of glutamic acid, threonine and proline and a low weight percentage of histidine; (2) mannose and/or glucose as suggested by the interaction with concanavalin A; (3) phosphoryl groups.
• 3.3. The concentration of the violet carotenoprotein in the haemolymph is approximately constant during all the life cycle of R. americana.
• 4.4. The haemolymph of four species of Rhynchosciara genus shows the presence of proteins immunologically related with the R. americana violet carotenoprotein.

     

Influence d'une microsporidiose sur les acides aminés libres de Carcinus mediterraneus Czerniavsky, 1884 soumis à diverses salinités et à des valeurs extrêmes de température

The influence of a microsporidian, Thelohania maenadis Pérez, 1904 (Protozoa: Microspora), on the free amino-acid content of the haemolymph and muscle tissue of Carcinus mediterraneus Czerniavsky, 1884 (Crustacea: Decapoda: Brachyura) is studied.Analysis of free amino acids reveals the presence in affected crabs, whose muscle tissue is 70–80% destroyed, of four additional, non-identified compounds in the haemolymp and eleven in the muscle. Parasitization does not provoke a substantial variation in the total free amino acids of the haemolymph and the muscle. In the latter, methionine sulphoxide, methionine and serine levels increase considerably; glycine, proline and arginine levels fall while alanine and taurine levels rise.The desalination at moderate temperatures of healthy and parasitized crabs alike results in a reduction of the total free amino acids of the haemolymph, mainly due to the lowering of proline, glycine and alanine concentrations. Supersalination leads to only a slight reduction of the total free amino acids in healthy specimens, and has no effect on parasitized crabs. The sharp increase in proline and alanine is offset by a reduction in the levels of other amino acids - arginine, glutamic acid, tryptophan and taurine in healthy crabs; and arginine, tryptophan, taurine and serine in parasitized specimes.The influence of the combined factors of salinity and temperature was studied in conditions comparable to those pertaining in winter and summer in the lagoon which is the crab's natural habitat. At low and high extremes of temperature, the effect of salinity on healthy crabs is reversed: desalination results in increased free amino-acid levels. Parasitization reduces this effect of temperature extremes on the influence exercised by salinity, and may annul it completely at high temperatures.     

Variation in protein intake induces variation in spider silk expression

Background

It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved.

Methodology/Principal Findings

We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species'' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake.

Conclusions

Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics.     

Effect of sound and Lenzites-decayed wood on the amino acid composition of Reticulitermes flavipes

In hydrolysates of the eastern subterranean termite, Reticulitermes flavipes, the most abundant protein amino acids (μmoles) were glycine, alanine, and glutamic acid; the least abundant were methionine and histidine. Sawdust from both sound and Lenzites trabea-decayed sapwood blocks of sugar maple, loblolly pine, and slash pine was force-fed to termites. A diet of decayed rather than sound wood had little effect on protein amino acid composition of the termites; glycine content varied the most. In contrast, diet affected the free amino acid composition. Except for glutamic acid, the major protein amino acids of the termites were not the predominant free amino acids. Tyrosine and histidine were relatively more abundant as free than as protein amino acids. Greatest differences in protein amino acid compositions of sound and decayed wood were in contents of glycine, leucine, lysine, and arginine.     

Characterization of the peptidases of Calliphora: Many features allow the utilization of small peptides as an amino acid reservoir

Electrophoretic separation of whole flies and of haemolymph indicates the presence of four peptidases, named dipeptidase A, B and C (Dip A, B and C) and leucine amino peptidase (LAP) after enzymes of similar substrate specificities and electrophoretic mobilities found in Drosophila (Laurie-Ahlberg, Biochem. Genet.20, 407–424, 1982; Walker et al., Insect Biochem.10, 535–541, 1980). Prominent in both tissues and haemolymph, dipeptidase A and B together hydrolyse a variety of dipeptides in vitro and probably most of the fly's small peptide component in vivo. Though Dip A and Dip B hydrolyse many of the same substrates, their activities differ in at least several respects. Dip A's K_ms are higher than Dip B's K_ms and hence in vivo the two enzymes together are likely to provide peptide hydrolysis through a wide range of substrate concentration. Dip A's unique hydrolyses are of peptides with biosynthesized amino acids in the N-terminal position and Dip B's unique hydrolyses are of peptides with essential amino acids in the N-terminal position. Dip B, but not Dip A, is inhibited by free amino acid. It is inhibited non-competitively and most strongly by essential amino acids. In cell-free haemolymph Dip B's activity is more stable than Dip A's. The accumulation and maintenance of small peptides in times of dietary sufficiency and the utilization of the small peptides as a source of amino acid in times of dietary scarcity (Collett, Insect Biochem.6, 179–185, 1976a; J. Insect Physiol.22, 1433–1440, 1976b) may be attributed to these features.     

Tyrosine and phenylalanine concentrations in haemolymph and tissues of the American cockroach, Periplaneta americana, during metamorphosis

Phenylalanine and tyrosine concentrations were measured in the haemolymph, fat body, and abdominal integument of the American cockroach, Periplaneta americana, during the pre- and post-ecdysial periods of cuticle formation and sclerotization.Gas-liquid chromatography of trimethylsilyl derivatives of phenylalanine, tyrosine, and their metabolites provided a very sensitive and rapid method for determining those amino acids in small haemolymph and tissue samples.Haemolymph tyrosine increased in two stages: initially near apolysis and 16 to 25 hr pre-ecdysis, reaching its highest concentration at ecdysis (3·5 μg tyrosine/mg haemolymph). During that time, total haemolymph tyrosine increased by approximately 700 μg/insect. Fat body and abdominal integument began to accumulate tyrosine near apolysis. Fat body tyrosine peaked between ecdysis and 3·3 hr post-ecdysis whereas abdominal integument tyrosine peaked at ecdysis. Maximum concentrations were 6·0 μg and 4·1 μg tyrosine/mg wet wt. of tissue, respectively. Between ecdysis and 24 hr post-ecdysis, the period of maximum sclerotization, total tyrosine in haemolymph and fat body decreased by approximately 600 μg and 420 μg/insect, respectively. Phenylalanine concentrations did not change significantly in the haemolymph, fat body, or abdominal integument during the pre- and post-ecdysial periods.The cockroach apparently does not store free phenylalanine or tyrosine in the fat body during larval development as compared to tyrosine storage in some Diptera. The rapid increase of haemolymph, fat body, and integument tyrosine just prior to ecdysis suggests another form of storage for this important amino acid.     

Salivary amino acids in Lygus species (Heteroptera:Miridae)

Free and protein-bound amino acids were investigated in the phytophagous bug Lygus rugulipennis and its salivary gland. Over 38 substances were separated. The total content of amino compounds in the insects was about 1400 μmol/g fr. wt (16% by weight), of which 97% was amino acid residues in proteins.The salivary glands, which comprise about 1.5% of the live weight of the insects, contain 3.5% of the total free amino acids and 1% of the whote insect. Free and protein-bound amino acids comprise, respectively, about 1.4 and 11.6% of the fresh weight of the gland. The total concentration of free amino acids in the saliva was estimated to range from 0.5 to 2.2% by weight (ca. 0.1 M).The composition of free amino acids in the salivary gland of Lugus varies markedly. In four studied species (L. rugulipennis, L. gemellatus, L. pratensis, L. punctatus), the most abundant compounds were proline, arginine, lysine, leucine, glutamic acid, methionine sulphoxide and glycerophosphoethanolamine. In whole specimens of L. rugulipennis the predominant free amino acids were proline, alanine, taurine, glutamic acid, glutamine and methionine sulphoxide. The most abundant amino acids in proteins were glutamic and aspartic acid, glycine, alanine and leucine. The results indicate that the amino acid composition in the salivary glands of Lygus species does not differ markedly from that of the whole insect. The functions of salivary amino acids are discussed.     

Amino acid and protein metabolism in the nervous system of the praying mantid

An analysis of four ganglia of the mantid Stagmatopera biocellata (‘brain’, B; prothoracic ganglion, P; mesothoracic ganglion, M; and metathoracic ganglion, T) is presented. The four ganglia are alike in total protein contents, total free amino acids, concentration of free leucine, and concentrations of every one of 17 protein-bound amino acids. Electrophoretic separation of the proteins show almost identical curves for the four ganglia with 18 peaks at the same points of relative mobility. Thus, according to these analysis, B, P, M, and T appear to be similar masses of nervous tissue. On the other hand, they behave in different ways when the dynamic state of free leucine is studied. Both, the flux leucine from the haemolymph into the ganglia and the turnover rate of protein bound leucine differ between ganglia. Results indicate a metabolic gradient M→T→P→B of protein synthesis. A correlation between the metabolic gradient and differences in the control of motor activity and sensorial input in the ganglia is suggested. Data on total free amino acids and total protein in haemolymph are given. The probable meaning of the noticeable contrast between the constancy in values of aminoacidemia and the high variability in values of proteinemia is discussed.