Water balance and osmoregulation in Stenocara gracilipes,a wax-blooming tenebrionid beetle from the Namib Desert

Dehydration (10 days at 27 degrees C) of the Namib tenebrionid Stenocara gracilipes resulted in a rapid weight loss (17.5%), and a substantial decline in haemolymph volume (72%). Although the lipid content decreased significantly, metabolic water production was insufficient to maintain total body water (TBW). Rehydration (no food) resulted in increases in haemolymph volume, body weight (sub-normal), and TBW to normality. Haemolymph osmolality, sodium, potassium, chloride, amino acids, and sugars (trehalose and glucose), were all subject to osmoregulatory control during both dehydration and rehydration. Major osmolar effectors in this species are sodium, chloride, and amino acids, with most of the contribution to regulation of haemolymph osmolality coming from changes in the levels of these constituents. Changes in amino acid levels are not the result of interchange with soluble protein during dehydration (the possibility exists during extended rehydration, however). Despite faecal losses of sodium being low (8.2% of that removed from the haemolymph during dehydration), sodium concentrations do not return to normal during rehydration. Chloride concentrations increase supra-normally when access to water is allowed, and remain elevated throughout the rehydration period. Although faecal loss of potassium greatly exceeded the amount removed from the haemolymph (by approximately 1.8 times), haemolymph potassium levels were strongly regulated during rehydration. S. gracilipes demonstrates an exquisite capacity to regulate haemolymph osmolality under conditions of both acute water-shortage and -abundance. Together with an efficient water economy (drinking when fog-water is available, and a superb water conservation mechanism in the form of wax-bloom production), this must serve to contribute to long-term survival of this species in an otherwise harsh abode.     

Composition of fluid from the notochordal canal of the coelacanth, Latimeria chalumnae.

Fluid from the notochordal canal of the coelacanth, Latimeria chalumnae, was analyzed for major inorganic and organic constituents and compared with blood serum from the same fish. Significantly or suggestively lower levels of sodium, magnesium, calcium, bicarbonate, sulfate, total carbohydrates, glucose, lactate, cholesterol, bound phosphate and total proteins were found in notochordal fluid than in serum, whereas potassium, chloride, urea, trimethylamine oxide, and total free amino acids were higher and inorganic phosphorus essentially identical. Osmolarity of notochordal fluid (1058 mOsm) exceeds that of serum (942 mOsm). A whitish precipitate in the fluid consisted of a matrix of fibers 100 A in diameter and of indefinite length. It resembled a sialoglycoprotein in composition and was stabilized by disulfide bonds. The fluid contained cellular debris.     

Haemolymph composition in the larva of the dragonfly,Uropetala carovei

Analysis of the haemolymph composition of the semi-terrestrial larva of the dragonfly, Uropetala carovei, yielded the following data. The osmotic pressure of the haemolymph is equivalent to 174.0 mM/1 solution of sodium chloride, the pH 8.2, sodium ion concentration 152.0 mM/1, potassium 4.6 mM/1, calcium 3.8 mM/1, magnesium 3.9 mM/1, bicarbonate 27.6 mM/1, and chloride 121.0 mM/1; total ninhydrin-positive substances 58.7 mM/1 (of which the major components are alanine, glycine, glutamic acid, and leucine); total carbohydrates 8.9 ± 0.4 μg/μl haemolymph (trehalose 7.88 μg/μl and glucose 0.85 μg/μl). Twelve protein bands were found, one of which showed alkaline phosphatase activity, three, acid phosphatase, and four, esterase activity. The significance of these results is discussed.     

The composition of the haemolymph of the New Zealand earwig, Anisolabis littorea

The haemolymph of the maritime earwig, Anisolabis littorea, has been analysed for inorganic and organic components. The inorganic fraction has a high sodium ion concentration and osmotic pressure but a relatively lower chloride ion concentration. The organic fraction has a high amino acid content—the principal ones being proline, alanine, glutamic acid, and glutamine, and tyrosine. In contrast to the high aminoacidemia there is a very low carbohydrate content and no detectable trehalose in the haemolymph. The evolution of the composition of insect blood is discussed.     

Osmoregulation in Onymacris rugatipennis, a free-ranging tenebrionid beetle from the Namib Desert.

Haemolymph levels of organic and inorganic constituents were investigated in the tenebrionid Onymacris rugatipennis during dehydration and rehydration. The major osmolar effectors are sodium (26%), chloride (24%), amino acids (18%), and sugars (11%); regulation of haemolymph osmotic pressure (OP) during dehydration is effected largely by a reduction in the haemolymph content of these constituents. Changes in amino acid levels are not the result of interchanges with soluble protein. During rehydration, the main contributors to osmoregulation are sodium (26%), chloride (24%), and an increase in haemolymph solute(s) not measured in this study (31%). Of the sodium removed from the haemolymph during dehydration, 21.2% was excreted. Faecal losses of potassium during dehydration far exceeded the amounts removed from the haemolymph; however, haemolymph potassium levels were strongly regulated during rehydration. Regulatory efficiency increases as desiccation proceeds, and is greatest only when this species is severely challenged.     

Haemolymph constituents and osmolality as functions of moult stage,body weight,and feeding status in marron,Cherax cainii () and yabbies,Cherax destructor (Clark, 1936)

The study investigates the change in osmolality and haemolymph constituents in marron Cherax cainii and yabbies Cherax destructor associated with moult stages, body weights and their feeding status. A total of 582 haemolymph samples from 5 moult stages (postmoult-AB, intermoult-C, and premoult stages – D₀, D₁, D₂), two body weight classes (2–15 g and 61–75 g) and nutritional status were used for analysis of osmolality, protein, glucose, and ionic concentrations of potassium and chloride following the standard biochemical procedures. The haemolymph protein, glucose, potassium and chloride levels were highest at intermoult and early premoult stages, and lowest at postmoult in both crayfish species. Except protein, no significant differences were seen in analyzed parameters between various weight classes and two species. Haemolymph osmolality, protein and glucose were significantly higher in fed crayfish, whereas no variations in haemolymph potassium and chloride concentrations were observed between the fed and unfed crayfish. Maximum osmolality was recorded at 7–8 h after feeding in both crayfish species. The results showed that the biochemical changes in the haemolymph of marron and yabbies are related to moult stages, body weight and feeding and thus can be used as tools for determining suitable diets.     

Influence of Medetomidine on Acid-base Balance and Urine Excretion in Goats

Seven goats were given medetomidine 5 μg/kg as an iv bolus injection. Venous blood samples were taken repeatedly and urine was collected continuously via a catheter up to 7h after the injection. Medetomidine caused deep clinical sedation. Base excess, pH and PCO2 in venous blood rose after medetomidine administration. There were no significant changes in plasma concentrations of sodium, calcium, magnesium, creatinine or osmolality, whereas potassium and bicarbonate concentrations increased, and phosphate and chloride decreased. Medetomidine increased plasma glucose concentration, and in 4 of 7 goats glucose could also be detected in urine. Medetomidine did not influence urine flow rate, free water clearance, bicarbonate and phosphate excretion or pH, but renal chloride, sodium, potassium, calcium, magnesium and creatinine excretion were reduced. The results suggest that the metabolic alkalosis recorded after medetomidine administration is not caused by increased renal acid excretion.     

The biochemical effects of potassium chloride on the silkworm, （ Bombyx mori L.）

The supplementation with 50, 100 and 150μg/mL potassium chloride to the fifth instar larvae of the silkworm Bombyx mori on fat body glycogen, protein, total lipids and haemolymph protein and trehalose were analyzed. The fat body glycogen and protein and haemolymph protein were increased significantly in all the treated groups; whereas fat body total lipids increased only in 100 and 150μg/mL and haemolymph trehalose increased only in 150μg/mL potassium chloride-treated groups when compared with those of the corresponding parameters of the carrier controls.     

Plasma chemistry of rockhopper (Eudyptes crestatus), magellanic (Spheniscus magellanicus) and gentoo (Pygoscelis papua) wild penguins in relation to moult

• 1.1. Both the post-moult, rockhopper (Eudyptes crestatus) and magellanic penguins (Spheniscus magellanicus) had significantly lower plasma total protein, albumin, urate, iron and potassium and higher alkaline phosphatase activity than pre-moult birds. In addition creatinine, conjugated bilirubin and inorganic phosphate in the magellanics; globulin, urate, calcium, alanine and aspartate transaminases in the rockhoppers were significantly decreased.
• 2.2. There were significant differences in plasma bicarbonate, inorganic phosphate, alkaline phosphatase and iron concentrations between non-moulting adult and post-moult gentoo penguin (Pygoscelis papua) chicks.
• 3.3. Absence or scarcity of the preferred nutrient requirement during the period preceding moulting could threaten the survival of any of the species, particularly of those of narrow dietary speciality.

     

Transport protein synthesis in non-growing yeast cells

Addition of a metabolizable substrate (glucose, ethanol and, to a degree, trehalose) to non-growing baker's yeast cells causes a boost of protein synthesis, reaching maximum rate 20 min after addition of glucose and 40–50 min after ethanol or trehalose addition. The synthesis involves that of transport proteins for various solutes which appear in the following sequence: H⁺, l-proline, sulfate, l-leucine, phosphate, α-methyl-d-glucoside, 2-aminoisobutyrate. With the exception of the phosphate transport system, the K_t of the synthesized systems is the same as before stimulation. Glucose is usually the best stimulant, but ethanol matches it in the case of sulfate and exceeds it in the case of proline. This may be connected with ethanol's stimulating the synthesis of transport proteins both in mitochondria and in the cytosol while glucose acts on cytosolic synthesis alone. The stimulation is often repressed by ammonium ions (leucine, proline, sulfate, H⁺), by antimycin (proline, trehalose, sulfate, H⁺), by iodoacetamide (all systems tested), and by anaerobic preincubation (leucine, proline, trehalose, sulfate). It is practically absent in a respiration-deficient petite mutant, only little depressed in the op₁ mutant lacking ADP/ATP exchange in mitochondria, but totally suppressed (with the exception of transport of phosphate) in a low-phosphorus strain. The addition of glucose causes a drop in intracellular inorganic monophosphate by 30%, diphosphate by 45%, ATP by 70%, in total amino acids by nearly 50%, in transmembrane potential (absolute value) by about 50%, an increase of high-molecular-weight polyphosphate by 65%, of total cAMP by more than 100%, in the endogenous respiration rate by more than 100%, and a change of intracellular pH from 6.80 to 7.05. Ethanol caused practically no change in ATP, total amino acids, endogenous respiration, intracellular pH or transmembrane potential; a slight decrease in inorganic monophosphate and diphosphate and a sizeable increase in high-molecular-weight polyphosphate. The synthesis of the various transport proteins thus appears to draw its energy from different sources and with different susceptibility to inhibitors. It is much more stimulated in facultatively aerobic species (Saccharomyces cerevisiae, Endomyces magnusii) than in strictly aerobic ones (Rhodotorula glutinis, Candida parapsilosis) where an inhibition of transport activity is often observed after preincubation with metabolizable substrates.     

13C and 31P NMR studies on sugar metabolism in Bombyx mori and Philosamia cynthia ricini larvae

Studies were made on ¹³C and ³¹P NMR in larvae of two species of silkworm, Bombyx mori and Philosamia cynthia ricini, in vivo as well as in vitro to determine the pathways of glucose utilization, especially those to amino acids as components of silk fibroin. Results showed that the ¹³C of [1-¹³C]glucose administered orally into 5th instar larvae of both species was incorporated into glucose-1-phosphate, glucose-6-phosphate and trehalose. Serine, glutamate, glutamine, citrate, malate, trehalose and sorbitol-6-phosphate were detected in the hemolymphs of these larvae as metabolites of [1-¹³C]glucose. Two days after [1-¹³C]glucose administration, labeled alanine, glycine, serine, urea, glycogen, trehalose and glycerol were clearly detected in Bombyx larvae. Starvation caused rapid consumption of administered [1-¹³C]glucose with very little accumulation of ¹³C in glycogen or trehalose. In the in vivo³¹P NMR spectra of Bombyx larvae, ATP, arginine phosphate, sorbitol-6-phosphate, uridine diphosphoglucose, phosphoenolpyruvate and inorganic phosphate were detected with some sugar phosphates, such as glucose-1-phosphate and glucose-6-phosphate. During starvation, the intensity of the signal of inorganic phosphate increased and those of sugar phosphate other than sorbitol-6-phosphate decreased, but these changes were reversed by oral administration of glucose.     

Water and ion regulation in cicadas in relation to xylem feeding

Cicadas feed on xylem fluid. This is hypotonic to the haemolymph and contains high concentrations of potassium, sodium, calcium, magnesium, chloride, and phosphate ions. The urine contains the same ions in the same proportions but in slightly lower concentrations. Amino acids and sucrose are present in xylem fluid and traces of amino acids are also found in urine.Water is rapidly shunted from foregut to hindgut via the filter chamber. Injection of xylem fluid into the oesophagus results in an immediate tenfold increase in flow rate in the ileum. The osmotic pressure of xylem fluid in the filter chamber rapidly rises whilst the osmotic pressure in the anterior part of the ileum rapidly falls.Absorption of nutrients and ions into the haemolymph probably occurs in the conical segment and anterior tubular midgut. Storage excretion of divalent ions occurs in the mid-midgut and ions may be transported from the haemolymph into the posterior tubular midgut.The Malpighian tubules secrete a fluid slightly hypertonic to blood containing K⁺ (42 mM/l.] and Na⁺ (14 mM/l.).The osmotic pressures within the internal Malpighian tubules and internal midgut in the filter chamber are considerably higher than the osmotic pressure of the xylem fluid when it first enters the filter chamber proper. Passive osmosis will occur and water will be shunted into the ileum.Reabsorption of K⁺ and Na⁺ occurs in the ileum.     

Comparative chemical composition of protoscolices and hydatid cyst fluid of Echinococcus granulosus (Cestoda)

Hydatid cysts were analysed for electrolytes, nucleic acids, proteins, enzymes, nitrogenous waste products, carbohydrates and lipids. Substantial amounts of cations were detected. Potassium, magnesium and calcium were concentrated in protoscolices as compared to cyst fluid, but sodium levels were similar in both. Of the anions, chloride constituted the major bulk in the fluid anions, and was not found in protoscolices. In contrast, phosphate and bicarbonate had more significant values in the protoscolices. RNA and DNA which were found in substantial amounts in the protoscolices were nearly absent in the fluid ; so was ammonia. The reverse pattern of distribution was observed in urea, uric acid, creatinine and bilirubin. The proteins of cyst fluid were mainly albumin and globulin, the latter having always double the concentration of the former. Albumins and globulins also formed J of protoscolex protein. Among enzymes, LDH, phosphatases, GOT and GPT exhibited high activities. Cholesterol, cholesterol esters, mono-, di- and triglycerides, fatty acids and phospholipids were detected mainly in the protoscolices with phospholipids constituting the major portion. The carbohydrates of the protoscolices were glycogen, trehalose, glucose and alkali stable carbohydrates. Of particular interest was the detection of sucrose in both protoscolices and fluid.     

Tissue specific glucose-trehalose variations prior to spinning in the eri-silkworm, Philosamia ricini.

A sharp rise in trehalose level of haemolymph is observed towards the end of 4th instar accompanied with sudden fall of the sugar in fat body during the same period, but after moulting blood trehalose abruptly decreases. Whereas, blood glucose level is maintained constant throughout life span of both the instars. Silkgland of 5th instar larvae presents a consistent gradual increase in glucose-trehalose levels reaching to the maximum prior to spinning with a higher value for trehalose than glucose which can be attributed to the decrease in blood trehalose level during the period.     

Use of benzyldimethyl-n-hexadecylammonium chloride (“16-BAC”), a cationic detergent,in an acidic polyacrylamide gel electrophoresis system to detect base labile protein methylation in intact cells

A discontinuous polyacrylamide gel system operating at pH 4.0–1.5 which resolves proteins bearing base labile groups extracted from intact cells is described. It uses potassium phosphate buffer in the running and stacking gel and glycine as the trailing ion component. Proteins are solubilized with urea and benzyldimethyl-n-hexadecylammonium chloride, a cationic detergent. The utility of the system is illustrated by fluorographs of the pattern of protein methylation in blood platelets and the HL60 promyelocyte cell line.     

Haemolymph amino acid and sugar levels in locusts fed nutritionally unbalanced diets

Aspects of pre- and post-ingestive compensation were investigated in locusts (Locusta migratoria) fed nutritionally unbalanced artificial diets containing 7% protein and 21% digestible carbohydrate (7:21) or 21% protein and 7% digestible carbohydrate (21:7). Feeding behaviour and haemolymph levels of amino acids and sugars were measured in locusts fed ad libitum on these diets. Locusts fed the high-protein diet had chronically elevated haemolymph levels of 15 out of 19 amino acids measured compared to locusts fed the low protein diet. However, haemolymph levels of lysine, alanine, aspartic acid and glutamic acid did not differ between diets, suggesting some specific regulatory mechanism for these amino acids. Haemolymph glucose and trehalose reflected levels of carbohydrate in the diets, being high in insects fed diet 7:21 relative to those given diet 21:7. These data are discussed in relation to the physiological and behavioural bases of nutritional homeostasis.Abbreviations AA amino acid(s) - PRO protein - CHO carbohydrate - PBS phosphate-buffered saline - MW molecular weight     

α-Glucosidase activity in haemolymph of the American cockroach, Periplaneta americana

α-Glucosidase activity of whole haemolymph has been investigated in adult males of the American cockroach, Periplaneta americana. Two electrophoretically distinguishable enzymes capable of hydrolysing α-glucosidic linkages are present in the serum component of the haemolymph, and one of these hydrolyses trehalose. Trehalase activity is also present in haemocytes, and the haemocyte enzyme shares an identical electrophoretic mobility and similar pH sensitivity with the serum trehalase. Furthermore, both enzymes are inhibited to the same extent by sodium ethylene diamine tetracetate (EDTA); thus it is suggested that the same enzyme may be responsible for trehalase activity in the two components. The K_m of EDTA-inhibited trehalase is 3·3 mM and this value is reduced to 1·8 mM upon activation of the enzyme by calcium ions. The properties of the trehalase are discussed in light of the possible rôle of the enzyme in regulating haemolymph trehalose and glucose concentrations.     

Trehalose turnover and the coexistence of trehalose and active trehalase in cockroach haemolymph

In the cockroaches Periplaneta americana, Periplaneta australasiae, Leucophaea maderae, and Nauphoeta cinerea, undiluted haemolymph, undiluted haemolymph to which 10% solid trehalose was added, and haemolymph diluted 100 or more times with 1% trehalose solution showed approximately equal trehalase activities (3 to 8 mg/ml per hr). No evidence for the presence of a trehalase inhibitor was found.Freshly drawn haemolymph of Periplaneta americana contained 14 to 16 mg trehalose/ml, which on standing was hydrolyzed to glucose at a rate of 4 to 8 mg/ml per hr. In this cockroach, the rate of haemolymph trehalose turnover was only 1.3 mg/ml per hr. This means that in vitro trehalose is hydrolyzed by undiluted haemolymph at several times the rate at which it is replaced in the haemolymph of the intact insect. The mechanism through which trehalose and trehalase can coexist in the haemolymph of the intact cockroach remains therefore unexplained.     

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.     

Fate of glucose in haemolymph of the American cockroach, Periplaneta americana

The rate of removal of high concentrations of glucose (10 μg/μl haemolymph) from haemolymph of adult male cockroaches, Periplaneta americana, was studied in normal and ligated insects. More than 50% of the injected glucose is removed from the haemolymph of normal insects within 20 min of injection. A period of rapid trehalose synthesis occurs during the initial 10 min following injection of glucose into the haemocoele, and this is succeeded by a period of glycogen synthesis. The results are discussed in terms of earlier observations on ‘stress-induced hypertrehalosemia’ and the possible involvement of a glycogenic agent.