Diurnal and temporal changes in the chemical profile of xylem exudate from Vitis rotundifolia

Positive root pressure in Vitis rotundifolia Michx. cv. Noble was employed to quantify diurnal and temporal changes in the chemical profile of xylem exudate. Xylem fluid osmolarity (7.2 to 16.8 m M ), water flux (8.2 to 18.5 ml h⁻¹) and solute flux (0.7 to 2.2 mmol h⁻¹) from a cut spur exhibited a diurnal pattern with maxima during midday and minima at night. Total osmolarity was similar to the sum of all organic and inorganic entitites quantified, indicating that the major solutes have been identified. Total amino acid and organic acid concentration were about equal (2 to 7 m M ), and sugars accounted for a minor fraction of the total profile (<0.2 m M ). Glutamine represented ca 80% of the organic N and 70% of the total N transported in the xylem fluid. A circadian rhythm in water flux and net flux of most organic and inorganic entities was observed with maxima during midday and minima at night. The increase in xylem fluid osmolarity occurring during midday was primarily a consequence of increased organic acid (oxalic, citric, tartaric, malic and succinic acids) and ion (NH₄⁺, No₃⁻, P and Ca) concentration. A diurnal cycle in amino acid concentration was less clear. The concentration of individual organic and inorganic entities varied asynchronously with time. Xylem solute was comprised of 80% organic and 20% inorganic components when collected 5 min to 2 h after the commencement of bleeding, but the ratio of organic to inorganic components fell to about 50% after 7 days.     

Biochemical aspects of Balanus hameri haemolymph,together with some comparative haemolymph data for Balanus balanus and Lepas anatifera

• 1.1. The concentration of protein in the haemolymph of Balanus hameri ranged from 2.0 to 17.3 mg/ml, and the lipid from 1.4 to 7.7 mg/ml; the haemolymph protein and lipid levels increased significantly prior to cross-fertilization.
• 2.2. The protein and lipid concentrations in Balanus balanus haemolymph were 8.1 and 1.7 mg/ml respectively.
• 3.3. The lipid concentration of Lepas anatifera haemolymph was 1.2 mg/ml.
• 4.4. The neutral lipid and phospholipid components of B. hameri and L. anatifera haemolymph were the same, with the major components of the phospholipid fraction being phosphatidyl ethanolamine and phosphatidyl choline.
• 5.5. The osmolarity (970.4 mOsm), chloride ion concentration (501.3 m-eq/l) and pH (7.29) of B. hameri haemolymph were also determined.

     

Freeze-tolerance adaptations,including haemolymph protein and lipoprotein nucleators,in the larvae of the cranefly Tipula trivittata

The terrestrial overwintering larvae of the cranefly Tipula trivittata were freeze tolerant (able to survive the freezing of their extracellular body fluids) throughout the winter and spring of 1982–1983 until they pupated in mid-May. The larvae were most cold tolerant (24 h lower lethal temperatures of ?25 to ?30°C) in late January and early February. Sorbitol, at a maximal concentration of ～0.4 M, was the only polyol determined to be present at high levels and sorbitol accounted for most of the seasonal fluctuation in osmotic concentration. Haemolymph inorganic ion (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?) concentrations did not vary seasonally.The supercooling points of the larvae remained constant at ?6 to ?7°C over the study period because of the presence of haemolymph ice nucleating factors. These ice nucleating factors consist not only of haemolymph proteins, as had been demonstrated previously in other insect species, but also lipoproteins.     

The composition of larval-pupal moulting fluid in the tobacco hornworm, Manduca sexta

The concentrations of sodium, potassium, chloride, bicarbonate, total phosphate, labile phosphate, inorganic phosphate, protein, polypeptides, amino acids, trehalose, and glucose, as well as pH and osmotic pressure of larval-pupal moulting fluid and haemolymph were measured in the tobacco hornworm, Manduca sexta, during the secretion and resorption of moulting fluid. Moulting fluid is a mildly alkaline (pH 7.2), iso-osmotic (330 mOsm) potassium bicarbonate salt solution containing within it the sol form of moulting gel. Bicarbonate is the principal anion in moulting fluid. It is only a minor component of haemolymph. Significant differences in the concentrations of potassium, chloride, bicarbonate, non-labile phosphate, labile phosphate, inorganic phosphate, protein, polypeptide, amino acids, trehalose, and glucose indicate that the integumentary epithelium does not act as a semi-permeable membrane, and that free diffusion between moulting fluid and haemolymph can account for only a small fraction of the molecular species movement between these two fluids.     

Ionic and osmotic regulation of the haemolymph of the dragonfly, Libellula quadrimaculata (Odonata : Libellulidae)

Larvae of the widespread dragonfly, Libellula quadrimaculata, were adapted to a series of salt solutions, and the osmotic pressure, and sodium, potassium and chloride concentrations in the haemolymph measured. The regulation of potassium is extremely efficient over the range 0–50 m-mole/l. external concentration. Above this, larvae die. Sodium and chloride are regulated to a lesser extent, the larvae being able to withstand considerable changes in the concentration of these ions in the haemolymph. However, at higher external concentrations, the haemolymph concentration of these ions is maintained below that of the external medium. The osmotic pressure is regulated in parallel with sodium concentration over most of the range tested. However, in higher salinities, the osmotic pressure of the haemolymph does not fall below that of the external medium. This is seen as a strategy to limit the amount of drinking in saline media. Overall, the osmoregulatory system of L. quadrimaculata resembles that of brackish-water insects, rather than that of the more strictly freshwater dragonflies that have been studied.     

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.     

Changes taking place in the electrophoretic haemolymph protein pattern during metamorphosis of Polytela gloriosae (Lepidoptera)

The haemolymph proteins of the larva, pupa and adult of Polytela gloriosae have been fractioned by Polyacrylamide gel disc electrophoresis. In the haemolymph of the fifth instar larval stage a total of ten protein fractions have been detected. The concentration of the protein fractions 2, 3, 4, 9 and 10 shows oscillations in their concentration in the early fifth instar, middle fifth instar and late fifth instar larval stage. In all 11 protein fractionswere detected in the haemolymph of different stages of the pupa. The protein bands 1, 7 and 10 of the pupa appear newly in the haemolymph as these bands were not found in the haemolymph of the larvae. The protein fraction 9 of larva was not found in the pupa. In the haemolymph of adult insect sexual difference was observed in the haemolymph protein pattern. In the haemolymph of adult female a total of 10 protein fractions were detected while from the male haemolymph a total of 8 protein fractions were detected. The pupal band 7 was not found in the adults of both the sexes. In the haemolymph of larva and adult one pigmented protein fraction was observed. No pigmented protein fraction was found in the haemolymph of pupa. Iron - containing protein fraction and the acid mucopolysaccharides were not found in the haemolymph. The protein fractions 3, 4, 5, 6 and 7 of adult haemolymph were darkly stained by the Schiff reagent and, thus, they are the fractions of glycoprotein. One protein fraction of lipoprotein was also found in the haemolymph.     

Binding of juvenile hormone,methoprene and hydroprene to haemolymph proteins of larvae of the southwestern corn borer,Diatraea grandiosella

The binding of tritiated juvenile hormone I, and the juvenile hormone mimics, methoprene and hydroprene (alkyl-3,7,11-trimethyl-2,4-dodecadienoates), to proteins of the haemolymph of larvae of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. The techniques of analytical isoelectric focusing, disc electrophoresis and gel filtration were employed. Juvenile hormone was shown to bind to a high affinity low molecular weight protein in the haemolymph of diapausing larvae. This protein has an apparent dissociation constant of about $\text{3.1 × 10}{}^{\mathrm{-7}}$ M at 4°C, an isoelectric point of about 4.85, and a molecular weight of about $\text{3 × 10}{}^4$. Juvenile hormone was also bound to at least one low affinity high molecular weight protein fraction. High juvenile hormone binding activity was found in the haemolymph of newly diapaused larvae. Methoprene and hydroprene bound selectively to a different protein fraction present in the haemolymph of pre-diapausing larvae. This protein fraction had a low relative mobility when subjected to electrophoresis at pH 8.9.A preliminary study of the binding of tritiated juvenile hormone I to proteins of the haemolymph of pre-diapausing larvae of the southern cornstalk borer, Diatraea crambidoides, was also conducted in vitro. A low molecular weight binding protein with identical electrophoretic properties to that of D. grandiosella was present. The high molecular weight binding protein fraction of the two species, however, had different electrophoretic properties.     

Haemolymph amino acids and related compounds during cocoon production by the larvae of the fly, Rhynchosciara americana

A qualitative and quantitative analysis of free amino acids and related compounds in the haemolymph of Rhynchosciara americana was carried out for different periods of the fourth larval instar. Threonine, serine, proline, and glutamic acid make up 50 per cent of the total free amino acids in R. americana haemolymph just before the larvae start spinning the communal cocoon; after this the titre of most of the amino acids declines continuously. There are few peptides but these are present in high titres; they consist of two to three amino acid residues, of which the most important are histidine and aspartic acid. The fall in the haemolymph amino acid and peptide titres is insufficient to account for the silk protein which accumulates on the communal cocoon during the same period. The results are consistent with a silk protein origin from haemolymph proteins and haemolymph free amino acids. The origin and metabolic rôle of some haemolymph ninhydrin-positive compounds are discussed.     

Ion regulatory capacity and the biogeography of Crustacea at high southern latitudes

Brachyuran and anomuran decapod crabs do not occur in the extremely cold waters of the Antarctic continental shelf whereas caridean and other shrimp-like decapods, amphipods and isopods are highly abundant. Differing capacities for extracellular ion regulation, especially concerning magnesium, have been hypothesised to determine cold tolerance and by that the biogeography of Antarctic crustaceans. Magnesium is known to have a paralysing effect, which is even more distinct in the cold. As only few or no data exist on haemolymph ionic composition of Sub-Antarctic and Antarctic crustaceans, haemolymph samples of 12 species from these regions were analysed for the concentrations of major inorganic ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl⁻, SO₄ ²⁻) by ion chromatography. Cation relationships guaranteed neuromuscular excitability in all species. Sulphate and potassium correlated positively with magnesium concentration. The Antarctic caridean decapod as well as the amphipods maintained low (6–20% of ambient sea water magnesium concentration), Sub-Antarctic brachyuran and anomuran crabs as well as the Antarctic isopods high (54–96% of ambient sea water magnesium concentration) haemolymph magnesium levels. In conclusion, magnesium regulation may explain the biogeography of decapods, but not that of the peracarids.     

Haemolymphal activation of protyrosinase and the site of synthesis of haemolymph protyrosinase in larvae of the fleshfly, Sarcophaga barbata

When whole blood from 5 day third instar larvae of the fleshfly, Sarcophaga barbata was incubated under nitrogen at 25°C for 16 hr in the presence of salivary glands there was an increase in its protyrosinase content, which amounted to 53% of that which occurs in vivo over the same period. The protyrosinase in ammonium sulphate fractions of haemolymph that were allowed to stand at 4°C for 24 hr following the incubation at 25°C was found to have autoactivated. Analysis of all these fractions revealed the presence of a protyrosinase activator in the 30% saturated ammonium sulphate fraction. When proenzyme and haemolymph activator were mixed there followed a lag period before the rapid phase of activation, the duration of the lag being dependent upon the concentration of both proenzyme and activator. The final activity attained was dependent upon the concentration of proenzyme, but was independent of the activator concentration and was comparable to that obtained using the cuticle activator. The level of activator in the haemolymph increased as larvae aged from 4 to 7 days.The effect of several compounds on the catecholase activity of the activated haemolymph protyrosinase and on the cuticle enzyme is reported and the significance of haemolymphal activation of protyrosinase is discussed.     

Effects of methoprene,a juvenile hormone analogue,on the larval and pupal stages of the yellow fever mosquito, Aedes aegypti

Exposure of early fourth-instar larvae of Aedes aegypti to the juvenile hormone analogue Altosid ZR15^® (methoprene) significantly increased the concentration of carbohydrates in the haemolymph of late fourth-instar larvae and reduced the haemolymph carbohydrate concentration of 24-h-old pupae relative to controls. Such treatment also effected a decline in haemolymph amino nitrogen levels of the pupal stage and a depletion of haemolymph proteins in late fourth-instar larvae as well as pupae. Two of nine protein fractions in the haemolymph of larvae were significantly depleted following methoprene treatment. Fourteen soluble protein fractions were present in the haemolymph of control pupae; two of these were missing from the pupae which were treated as larvae with methoprene. A further protein fraction, common to the haemolymph of both treated and control pupae, was significantly reduced in concentration as a consequence of exposure to methoprene. The juvenile hormone analogue impaired the capacity of the fat bodies of late fourth-instar larvae and pupae to synthesise proteins, resulting in a lowered concentration of fat body proteins. Glycogen levels in the fat bodies of treated larvae were significantly lower than in controls and glycogenolysis was suppressed due to an overall depletion of glycogen phosphorylase and, in pupae, a lowered ratio of active: inactive enzyme. The data are consistent with the proposition that the juvenile hormone analogue elicits neuroendocrinological changes in the target insect.     

Adipokinetic hormone-induced lipid mobilization and lipophorin interconversions in fifth larval instar locusts

The response of fifth larval instar locusts to injected adipokinetic hormone (AKH) is only poor, as is reflected in both a very moderate elevation of the haemolymph lipid concentration and the slight occurrence of the haemolymph lipophorin interconversions characteristic for adult locusts, resulting in formation of only small quantities of the low density lipophorin (A⁺). However, an additional lipophorin fraction (A′) is induced, which is intermediate in density and size between high and low density lipophorin and which is not identified in adult haemolymph. As in adults, larval A⁺ formation includes association of the resting high density lipophorin with a non-lipid containing protein (C₂), the haemolymph concentration of which is only one-fifth relative to adults. However, the larval haemolymph protein composition is not the primary cause of the incomplete adipokinetic response, as elevation of the concentration of protein C₂ by injection of isolated adult C₂, whether or not in combination with adult high density lipophorin, did not increase lipophorin conversions nor haemolymph lipid elevation.In vitro incubation of larval fat bodies in adult haemolymph showed that competency to both the AKH-induced lipid release and the haemolymph lipophorin conversions of the larval fat body are reduced compared to equal amounts of adult tissue. Reciprocal incubation of adult fat body in larval haemolymph resulted in only a very moderate adipokinetic response, demonstrating that larval haemolymph protein composition is restrictive for full development of hormone action.Both immunoblotting experiments and enzyme-linked immunosorbent assays (ELISA), using monoclonal antibodies specific for the adult lipophorin apoproteins, indicated that the larval lipophorins closely resemble the adult forms. Apparently the structure of locust lipophorins is remarkably constant throughout development despite changes in metabolic functions.     

Diuresis in the cabbage white butterfly, Pieris brassicae: Water and ion regulation and the rôle of the hindgut

The significance of diuresis in the water and ion balance of newly emerged Pieris was examined by comparing the composition of haemolymph and urine during diuresis. The high potassium content of the urine results in a marked increase in the Na/K ratio of the haemolymph. The haemolymph osmolarity is well regulated, in spite of the very hypo-osmotic urine. By means of an isolated preparation of the ileum, it was shown that rapid resorption of potassium ions by this part of the hindgut is responsible for the low osmolarity of the urine.     

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.     

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.

     

Côntrole neuroendocrine des protéines sanguines vitellogenes d'Anacridium aegyptium sain et parasite

The changing patterns of haemolymph proteins were followed in male and female adults of normal and parasitized Anacridium aegyptium during diapause (autumn, winter) or during activity (spring) of their endocrine system without or with electrostimulations of the pars intercerebralis (PI).The haemolymph protein concentration is high in winter and decreases in spring. It is comparatively depleted in locusts infected by the fly Metacemyia calloti. However, the depletion is significant only in ‘castrated’ females.Fifteen protein fractions were resolved by polyacrylamide disk gel electrophoresis in haemolymph of normal and infected locusts during diapause and activity. Some fractions decrease in quantity during activity in males, normal females, and parasitized females with complete ovarian development. One fraction disappears in females with mature eggs and seems correlated with formation of the eggshell. Eight others protein fractions exhibit electrophoretic mobility identical to the 7 protein fractions of homogenates of eggs. There is little doubt that these haemolymph protein fractions are involved in yolk synthesis and are thus ‘vitellogenic’. One of these ‘vitellogenic’ fractions (band 6) is larger in yolk than in blood.Five protein fractions were demonstrated by electrophoresis of homogenates of parasites. Their electrophoretic mobilities are similar to those of 5 of the 8 haemolymph ‘vitellogenic’ fractions of the host. There is little doubt that these 5 haemolymph protein fractions (one of them is the band 6) are involved in the nutritional requirements of the parasite.Electrostimulation of the PI, during diapause and activity, increase the haemolymph protein concentration and chiefly the protein concentration of the blood band 6. Thus, the median neurosecretory cells of the brain (M-NSC) regulate protein synthesis and chiefly the synthesis of ‘vitellogenic’ proteins.In parasitized females, the increase of the haemolymph protein concentration after electrostimulations of the PI is associated with an enhancement of ovarian development. The depletion of the haemolymph protein concentration in ‘castrated’ females is thus involved in the inability of the oöcytes to sequester available proteins from the haemolymph. The haemolymph protein deficiency may be attributed to (1) an impairment of protein synthesis, attendant upon the hypoactivity of the M-NSC, and (2) the nutritional requirements of the parasite.     

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.     

Oxidative effects of inorganic and organic contaminants on haemolymph of mussels

We applied a newly-established method in haemolymph of mussels, Mytilus galloprovincialis, exposed to different concentrations of heavy metals, such as zinc and cadmium and organic pollutants, such as PAHs and lindane, for the detection of total antioxidant capacity (TAC). The susceptibility of exposed mussels was increased in relation to oxidative stress induced by contaminants tested. Oxidative modifications of proteins were estimated by measuring protein carbonyl content (PCC) and malondialdehyde levels (MDA). For PCC measurement, a highly sensitive and accurate ELISA method, which requires only 5 µg of protein, was used. The significant increase of PCC and MDA in haemolymph of exposed mussels reinforces its role as biomarkers of oxidative stress. Significant correlation of TAC assay, PCC and MDA was conducted in order to evaluate the utility of PCC and TAC assay, used in the present study, as tools for determining oxidative effects of pollutants in mussels. The results reinforce the application of PCC method as useful tool for the determination of PCC alterations in haemolymph of mussels exposed to different levels of contaminants. In addition, the TAC method gives encouraging results, concerning its ability to predict antioxidant efficiency in haemolymph of mussels exposed to inorganic and organic contaminants.     

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.