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PubMed2006

WEB 2086, a platelet-activating factor antagonist, inhibits prophenoloxidase-activating system and hemocyte microaggregation reactions induced by Trypanosoma rangeli infection in Rhodnius prolixus hemolymph.

The effects of the triazolodiazepine WEB 2086, a platelet-activating factor (PAF) antagonist, on hemocyte microaggregation and prophenoloxidase (proPO)-activating system in the hemolymph, hemocoelic infection and mortality in fifth-instar larvae of Rhodnius prolixus inoculated with Trypanosoma rangeli were investigated. Hemocoelic injection of short T. rangeli epimastigotes (1x10(4) parasites/insect) in R. prolixus that were previously fed with blood containing 1muM of WEB 2086 resulted in (i) reduced hemocyte microaggregations as well as an attenuated proPO system in the hemolymph and (ii) greater parasitemia and mortality among the insects. In vitro assays using hemolymph from insects previously fed with blood containing WEB 2086 exhibited attenuated hemocyte microaggregations when T. rangeli was employed as the inducer of the reaction, and this effect was not counteracted by PAF treatment. In vitro assays using hemolymph from insects previously fed with blood, regardless of WEB 2086 presence increased the PO activity when incubated with the parasites. However, the PO activity was drastically inhibited when hemolymph from insects fed with blood, whether or not it contained WEB 2086, was incubated with fat body homogenates from insects fed with blood containing WEB 2086. The addition of PAF did not enhance the PO activity. These analyses did not reveal any PAF influence on WEB 2086 effects in the two defense reactions.

Animals↗
PubMed2005

A simple wax-embedding method for isolation of aphid hemolymph for detection of luteoviruses in the hemocoel.

A protocol for isolating hemolymph from viruliferous aphids has been developed. This method uses warm melted wax to immobilize the aphid. Following removal of a hind leg, the hemolymph can be collected readily. Flushing with RNase-free water allows for collection of sufficient hemolymph for RNA extraction from individual aphids. The extracted RNA was successfully used for detection of barley yellow dwarf virus (BYDV) and pea enation mosaic virus (PEMV) from individual viruliferous Rhopalosiphum padi and Acyrthosiphon pisum aphids, respectively. A TaqMan real-time RT-PCR protocol for quantitation of PEMV in the hemolymph of individual aphids was developed. The wax-embedding hemolymph collection technique provides a useful tool for studying molecular interactions between persistent and circulative plant viruses and their insect vectors.

Animals↗
PubMed1988

Hemostasis in larvae of Manduca sexta: formation of a fibrous coagulum by hemolymph proteins.

Little is known of the participation of insect hemolymph proteins in wound healing and clot formation. We describe the assembly of purified hemolymph protein from the tobacco hornworm into an extended fibrous coagulum in the absence of hemocytes. This coagulum resembles the clot formed from bovine fibrinogen and thrombin. Structural components of the coagulum are present in hemolymph, however, spontaneous assembly occurs only in hemolymph collected through a wound. The fibrous coagulum assembles from purified structural protein(s) following addition of a non-protein factor from hemolymph, which is also present in Grace's insect cell culture medium.

Animals↗
PubMed1982

Effects of hemolymph from immune and non-immune larvae of Galleria mellonella on the ultra-structure of Pseudomonas aeruginosa.

The ultrastructure of Pseudomonas aeruginosa, a pathogen of Galleria mellonella is rapidly altered after in vitro exposure to the hemolymph of vaccinated larvae. The bacteria were treated with normal and immune hemolymph for periods of time ranging from 7 to 28 min at 28 degrees C. In contrast to the apparent non-damaging effects of normal hemolymph, the immune hemolymph caused progressive damage to the cells within 7 min. The initial attack was directed towards the cell wall. Complete degradation was observed after 14 to 28 min exposure to the immune hemolymph.

Animals↗
PubMed2000

Isolation of lectins from hemolymph of decapod crustaceans by adsorption on formalinized erythrocytes.

Hemolymph of decapod crustaceans contains lectins of important specificity. An isolation procedure, based on adsorption of hemolymph lectins on red blood cells (RBC) fixed with formaldehyde, is described. Hemolymph is let to clot for 3 h at 22-28 degrees C (RT) and for 24 h at 5 degrees C; centrifuged at 13000 g for 30 min; filtered through 5-microm filters; diluted with an equal volume of 50 mM NaCl, 100 mM CaCl(2); supplemented with protease as well as phenoloxidase inhibitors; centrifuged at 13000 g for 20 min. Formalinized RBC (FRBC) are mixed with diluted hemolymph to a suspension of about 20% v/v FRBC. After incubation for 30 min at RT, FRBC are washed five times with 150 mM NaCl, 10 mM CaCl(2). The lectins adsorbed on FRBC are desorbed using either 100-500 mM of carbohydrate solutions in 0.9% NaCl or 50 mM Tris-HCl buffer, pH 8.0 containing 100 mM NaCl and 20 mM entylenediaminetetraacetate (EDTA). The procedure is efficient in isolating the hemolymph lectins of the decapods Liocarcinus depurator and Potamon potamios.

Adsorption↗
PubMed2002

Search for peptidic molecular markers in hemolymph of crowd-(gregarious) and isolated-reared (solitary) desert locusts, Schistocerca gregaria.

An HPLC analysis of hemolymph extracts was undertaken to uncover differences between desert locusts, Schistocerca gregaria, reared under either crowded or isolated conditions. Some differences in the chromatographic pattern could be detected. One of the major peaks in the hemolymph of crowd-reared adults was found to be a minor one in isolated-reared individuals, whereas other peaks increased after solitarization. The differences became even more pronounced after several generations of isolated rearing. The dominant chromatographic peak in hemolymph extracts of the crowd-reared animals was identified as a novel peptide with a molecular mass of 6080Da. Edman degradation in combination with enzymatic fragmentation and quadrupole-time of flight (Q-Tof) mass spectrometry revealed the full sequence: DNADEDTICVAADNKFYLYANSLKLYTCYNQLPKVYVVKPKSQCRSSLSDCPTS. This 54 aa-peptide is very abundant in hemolymph of crowd-reared adults. Its concentration in hemolymph amounts to 0.1mM. To uncover the function, its effects were investigated in several bioassays, so far without positive results. One of the other peaks differentially expressed in the individuals of the two phases was identified as SGPI-2 (MW=3794Da), which is a serine protease inhibitor in locusts.

Amino Acid Sequence↗
PubMed1997

The concentrations of vitellogenin (vitellin) and protein in hemolymph, ovary and hepatopancreas in different ovarian stages of the freshwater prawn, Macrobrachium rosenbergii.

The objectives were to measure the concentrations of vitellogenin (vitellin) and protein in hemolymph, ovary, and hepatopancreas of the freshwater prawn, Macrobrachium rosenbergii, in different stages of ovarian development. The ovarian development of M. rosenbergii was classified into five developmental stages (Stages I-V). Vitellogenin concentrations increased in the hemolymph of prawns in the early stages of ovarian development (Stage I or II) and were maintained at high levels until Stage V. There was no close association between ovarian development and the concentrations of circulating vitellogenin. Concentrations of protein in hemolymph and hepatopancreas remained constant during various stages of ovarian development. The ovarian stages closely correlated with the gonadosomatic index, the concentrations of ovarian vitellin and protein, respectively. Vitellogenin levels in hepatopancreas remained very low in different stages of the prawn although the highest levels were observed in Stage IV. No close association between hemolymph vitellogenin and ovarian vitellin was observed. The increase of vitellogenin concentration in hemolymph occurred earlier than vitellin content in ovary.

Animals↗
PubMed1998

Purification and characterization of two insecticyanin-type proteins from the larval hemolymph of the Eri-silkworm, Samia cynthia ricini.

Two different biliverdin-binding proteins, designated BBP-I and BBP-II, were purified from the larval hemolymph of the Eri-silkworm, Samia cynthia ricini. These proteins were readily isolated from the hemolymph of fifth instar larvae using two chromatographic steps, hydrophobic interaction chromatography and ion exchange chromatography. Both BBPs were easily separated by Q-Sepharose HP column chromatography. BBP-I has an apparent molecular weight of 24 kDa, as determined by gel-filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Native BBP-II had a molecular weight of 48 kDa estimated by gel-filtration. SDS-PAGE revealed a single band with a molecular weight of 26 kDa. Moreover, the molecular weights of BBP-I and BBP-II were determined to be 20,468 and 22,708 by MALDI-TOF/MS (matrix-assisted laser desorption ionization-time of flight/mass spectrometry), respectively. On this basis, BBP-I and BBP-II molecules are assumed to be a monomer and a dimer, respectively. The blue color of BBPs collected from the hemolymph is attributed to the presence of biliverdin IX gamma, which is non-covalently and stoichiometrically bound to the apoprotein, based on absorbance maxima at 359 and 695 nm in methanol:HCl (95:5, v/v). One molecule of BBP-I contains one molecule of biliverdin IX gamma, whereas BBP-II contains two molecules of biliverdin IX gamma. The amino acid compositions of BBP-I and BBP-II are different, although the N-terminal sequences of both BBPs have a 48% identity. These BBPs were found in the hemolymph of fourth and fifth instar larvae. The newly molted fifth instar larvae had the highest concentration of BBP-I in the hemolymph. This gradually decreased during larval development. In contrast to BBP-I, the level of BBP-II was low, and increased slightly at the same developmental stage in S. cynthia ricini larvae.

Amino Acid Sequence↗
PubMed2000

Binding sites for human interferon-gamma in protocerebrum and hemolymph of tobacco hornworm (Manduca sexta) larvae differ in sensitivity to polycationic peptides.

We have recently characterized specific binding sites for human interferon-gamma on particulates prepared from the protocerebrum and hemolymph of tobacco hornworm larvae, Manduca sexta ¿(Parker, M.S., Ourth, D.D., 1999. Comp. Biochem. Physiol. B 122, 155-163). The sensitivity to sulfated polysaccharides indicated an involvement of oligobasic epitopes of hIFN-gamma in the binding. In the present study, we found that polycationic peptides inhibited the binding of [125I]hIFN-gamma to particulates from either the hemolymph or the protocerebrum of Manduca sexta larvae. With amino acid homopolymers, the rank order of potency was poly-L-lysine > poly-L-arginine >> poly-L-ornithine, while the acidic side chain polymer poly-L-aspartate was not inhibitory. However, the potency of all polycationic peptides was at least three-fold greater at the hemolymph particulates. Also, acidic polysaccharides such as heparin were much more efficacious in the inhibition of hIFN-gamma binding to hemolymph relative to protocerebral particulates. The peptide polycations inhibited the binding of [125I](Leu31,Pro34)human peptide YY, a ligand selective for the Y1 subtype of the neuropeptide Y receptor, to rabbit kidney or to parietal cortex particulates with the expected rank order of poly-L-arginine > poly-L-lysine >> poly-L-ornithine, and with little cross-tissue difference in affinity. The selectivity observed with M. sexta particulates indicates a preferential involvement of oligobasic lysine-rich C-terminal sequences of IFN-gamma, while large insect tissue-related affinity differences point to involvement of diverse oligoacidic sequences in binding to protocerebrum and hemolymph sites. This study provides evidence for the presence of molecules in lepidopteran larvae that are similar in structure to vertebrate co-receptors of IFN-gamma, and adds to the characterization of these binding sites.

Amino Acids↗
PubMed2001

In vitro degradation of the Neb-Trypsin modulating oostatic factor (Neb-TMOF) in gut luminal content and hemolymph of the grey fleshfly, Neobellieria bullata.

The unblocked hexapeptidic Trypsin Modulating Oostatic Factor of the fleshfly, an inhibitor of both trypsin and ecdysone biosynthesis, resists very well proteolytic breakdown by enzymes present in the lumen of the gut of previtellogenic fleshflies. However, when incubated in hemolymph of adult flies, females and males, its half-life time is a mere 0.5 min. In hemolymph of last instar larvae, this value increases to about 1.5 min. Whereas PMSF, a potent inhibitor of serine proteases has no effect, captopril and lisinopril, both known to be specific inhibitors of mammalian angiotensin I converting enzyme (ACE), effectively inhibit TMOF breakdown in fly hemolymph. Digestion of Neb-TMOF by recombinant Drosophila AnCE on itself results in identical degradation products as with total hemolymph. In both cases ESI-Qq-oa-Tof mass spectrometry demonstrated the appearance of peptide fragments with the sequences NPTN, LH and NP. These observations not only confirm the reported presence of circulating ACE-like activity in flies but also strongly suggest that in flies this hemolymph ACE-like activity might be involved in the regulation of the oostatic activity as exerted by Neb-TMOF.

Animals↗
PubMed2002

Hemolymph of reproductives of Solenopsis invicta (Hymenoptera: Formicidae)-amino acids, proteins and sugars.

Free amino acid composition and carbohydrate and protein concentration of the hemolymph of the pupal and adult stages of reproductives of Solenopsis invicta are presented. The physico-chemical properties of the hemolymph differ between both sexes of fire ants during development. Male alates (459 mmol/kg) have a higher osmolality than pupae (388 mmol/kg). Osmolality during the pupal stage was 428 mmol/kg, decreasing to 354 mmol/kg soon after de-alation and increasing again to 463 mmol/kg in active queens. The hemolymph of newly eclosed adults was more basic than the pupa and older adults. The pH of the hemolymph ranged from 6.40 to 6.71 for males and 6.47 to 7.01 for females. An increase in the carbohydrate and protein content was found for both sexes after emergence. Newly de-alated females had a decrease in their carbohydrate titers, although no changes were found in the protein concentration. An increase in the total free amino acid concentration was recorded for males after emergence, while changes in females were noted only after de-alation. However, females had a 2.7-fold increase in the free amino acid pool after they de-alated, with proline, glutamine and taurine showing the highest increase. Changes in the organic composition and in the physico-chemical properties of the hemolymph of S. invicta are discussed in regard to the physiological processes involved in the maturation of the reproductives of this insect.

Amino Acids↗
PubMed2005

Plant phenolics behave as radical scavengers in the context of insect (Manduca sexta) hemolymph and midgut fluid.

To evaluate the prooxidant versus antioxidant properties of plant phenolics toward leaf-feeding caterpillars, quenching of the stable ABTS radical by five phenolics was measured in two physiological contexts: hemolymph and midgut fluid. Addition of tannic acid, chlorogenic acid, quercetin, or catechin to Manduca sexta (L.) gut fluid increased its total antioxidant capacity by 12-45%, with tannic acid and quercetin being the most powerful in this regard. The antioxidant contribution of the phenolics increased with longer (30-60 min) incubation time in gut fluid. Chlorogenic acid and caffeic acid exhibited the weakest antioxidant activity in gut fluid. The total antioxidant capacity of hemolymph is considerably less than that of gut fluid, and in hemolymph chlorogenic and caffeic acids sometimes acted as mild prooxidants, particularly after longer incubation periods (30-60 min), although this trend was not statistically significant. Tannic acid, catechin, and quercetin behaved as antioxidants in hemolymph. These results suggest that many phenolics have radical scavenging activity in the digestive tract, but some may have more detrimental effects after absorption into the hemolymph compartment.

Animals↗
PubMed2000

Viresin. A novel antibacterial protein from immune hemolymph of Heliothis virescens pupae.

Immune hemolymph was collected from fifth instar larvae and 1-day-old pupae of Heliothis virescens after injection of prepupae with live Enterobacter cloacae. Induction of antibacterial activity against Escherichia coli K12 D31 was 7.5 times greater in pupal than in larval immune hemolymph. Lysozyme activity of immune pupal hemolymph against Micrococcus lysodeikticus was 11 times greater when compared with lysozyme activity of immune larval hemolymph. Early pupal immune response with regard to antibacterial activity was much greater than larval immune response in H. virescens. Normal pupal hemolymph showed an increase in antibacterial activity and lysozyme that was induced during metamorphosis. Antibacterial protein was isolated together with lysozyme by gel filtration chromatography and then separated from lysozyme by sequential electrophoresis with a native acid gel and SDS gel. Molecular mass of antibacterial protein was estimated to be 12 kDa. The N-terminal amino acid sequence of 12-kDa protein was different from those of antibacterial molecules found in other insects and has not been identified before. A sample containing 12-kDa protein was negative for immunoblotting with anti-synthetic cecropin B antibody. We have named the novel 12-kDa antibacterial protein viresin. Viresin showed antibacterial activity against several Gram-negative bacteria including E. cloacae but not against Gram-positive bacteria.

Amino Acid Sequence↗
PubMed2000

Cuticular pro-phenoloxidase of the silkworm, Bombyx mori. Purification and demonstration of its transport from hemolymph.

Pro-phenoloxidase (proPO) in insects is implicated in the defense against microbes and wounding. The presence of proPO in the cuticle was suggested more than 30 years ago, but it has not been purified. The extract of cuticles of the silkworm, Bombyx mori, was shown to contain two proPO isoforms (F-type and S-type proPOs, which have slightly different mobilities in polyacrylamide gel electrophoresis under nondenaturing conditions). The two isoforms were purified to homogeneity. From hemolymph of the same insect, two types of proPO with the same electrophoretic mobilities as those of cuticular isoforms were separated and were shown to be different at five amino acid residues in one of their subunits. The isoforms in the hemolymph and cuticle were activated by a specific activating enzyme. The resulting active phenoloxidases exhibited almost the same substrate specificities and specific activities toward o-diphenols. The substrate specificities and the susceptibilities to inhibitors, including carbon monoxide, indicated that the purified proPO isoforms were not zymogens of laccase-type phenoloxidase. The proPO in hemolymph was shown to be transported to the cuticle. This demonstration was corroborated by the failure to detect proPO transcripts by Northern analysis of total RNA from epidermal cells. In reversed-phase column chromatography, cuticular and hemolymph proPOs gave distinct elution profiles, indicating that some yet to be identified modification occurs in hemolymph proPO and results in the formation of cuticular proPO. There was little transportation of cuticular proPO to the cuticle when it was injected into the hemocoel. The nature of the modification is described in the accompanying paper (Asano, T., and Ashida, M. (2001) J. Biol. Chem. 276, 11113-11125).

Amino Acid Sequence↗
PubMed2004

Proteomic analysis of the Drosophila larval hemolymph clot.

Components of the insect clot, an extremely rapid forming and critical part of insect immunity, are just beginning to be identified (1). Here we present a proteomic comparison of larval hemolymph before and after clotting to learn more about this process. This approach was supplemented by the identification of substrates for the enzyme transglutaminase, which plays a role in both vertebrate blood clotting (as factor XIIIa) and hemolymph coagulation in arthropods. Hemolymph proteins present in lower amounts after clotting include CG8502 (a protein with a mucin-type domain and a domain with similarity to cuticular components), CG11313 (a protein with similarity to prophenoloxidase-activating proteases), and two phenoloxidases, lipophorin, a secreted gelsolin, and CG15825, which had previously been isolated from clots (2). Proteins whose levels increase after clotting include a ferritin-subunit and two members of the immunoglobulin family with a high similarity to the small immunoglobulin-like molecules involved in mammalian innate immunity. Our results correlate with findings from another study of coagulation (2) that involved a different experimental approach. Proteomics allows the isolation of novel candidate clotting factors, leading to a more complete picture of clotting. In addition, our two-dimensional protein map of cell-free Drosophila hemolymph includes many additional proteins that were not found in studies performed on whole hemolymph.

Amino Acid Sequence↗
PubMed1998

Quantitation of cat immunoglobulins in the hemolymph of cat fleas (Siphonaptera: Pulicidae) after feeding on blood.

Passage of ingested cat immunoglobulin G (IgG) into the hemocoel of cat fleas, Ctenocephalides felis (Bouché), was examined using antibody capture enzyme-linked immunosorbent assays (ELISA) and Western blotting. Fleas were fed heparinized cat blood via membrane feeders. Cat IgG was present in the hemolymph of engorged female fleas 1 h after ingestion at an estimated quantity of 35 +/- 14 micrograms/ml. The prevalence of fleas with demonstrable cat IgG in their hemolymph 1 h after feeding was 100% for both female and male fleas. Following a single blood meal, cat IgG was present in the hemolymph of all 15 fleas tested 1 h after ingestion but dissipated below detectable levels in 10 of 20 fleas examined 3 h after ingestion, and was detectable in only 1 of 10 fleas examined 18 h after ingestion. However, when fleas were provided with continual access to blood over a 72-h period, IgG content in hemolymph, as measured in excised, triturated legs of individual fleas, remained fairly constant (3-16 pg IgG per sample). Flea feeding studies using specific antisera indicated that IgG in flea hemolymph retained its binding activity, and that at least a portion of the IgG was intact. Passage of ingested host antibody from gut into hemocoel is a prerequisite for the possible development of antiflea vaccines that target antigens outside of the flea midgut lumen (e.g., key components of the flea endocrine system controlling oogenesis).

Animals↗
PubMed1974

Insect immunity. I. Characteristics of an inducible cell-free antibacterial reaction in hemolymph of Samia cynthia pupae.

Pupae of the silk moth, Samia cynthia, were found to contain an inducible antibacterial activity in their hemolymph. This immunity response was provoked by primary infections with either Escherichia coli K-12 or Enterobacter cloacae. In both cases the antibacterial activity was directed chiefly towards E. coli. During standard conditions, 1% of hemolymph could kill 10(3) to 10(4) viable E. coli, strain D31, within 5 min. A lower level of antibacterial activity was induced by injections of a sterile salt solution. The killing of strain D31 followed single-hit kinetics, and increasing rate constants were obtained for increasing amounts of hemolymph. The reaction was sensitive to pretreatment with trypsin and it was protected by reducing agents. The activity was inhibited by microgram quantities of lipopolysaccharide (LPS) prepared from certain LPS mutants of E. coli K-12. A comparison of the susceptibility showed that "heptose-less" LPS mutants were more sensitive to killing than other strains. During standard conditions hemolymph will lyse both E. coli and Micrococcus lysodeikticus. Lysis of E. coli followed a multi-hit kinetics and it was inhibited by LPS, whereas lysis of M. lysodeikticus was unaffected by LPS. Hemolymph was fractionated on Sephadex G-200, and the lytic activities were recovered in partly overlapping peaks. Reconstitution with pooled fractions gave synergistic effects with the killing assay.

Animals↗
PubMed1999

Respecified larval proleg and body wall muscles circulate hemolymph in developing wings of Manduca sexta pupae.

Most larval external muscles in Manduca sexta degenerate at pupation, with the exception of the accessory planta retractor muscles (APRMs) in proleg-bearing abdominal segment 3 and their homologs in non-proleg-bearing abdominal segment 2. In pupae, these APRMs exhibit a rhythmic 'pupal motor pattern' in which all four muscles contract synchronously at approximately 4 s intervals for long bouts, without externally visible movements. On the basis of indirect evidence, it was proposed previously that APRM contractions during the pupal motor pattern circulate hemolymph in the developing wings and legs. This hypothesis was tested in the present study by making simultaneous electromyographic recordings of APRM activity and contact thermographic recordings of hemolymph flow in pupal wings. APRM contractions and hemolymph flow were strictly correlated during the pupal motor pattern. The proposed circulatory mechanism was further supported by the findings that unilateral ablation of APRMs or mechanical uncoupling of the wings from the abdomen essentially abolished wing hemolymph flow on the manipulated side of the body. Rhythmic contractions of intersegmental muscles, which sometimes accompany the pupal motor pattern, had a negligible effect on hemolymph flow. The conversion of larval proleg and body wall muscles to a circulatory function in pupae represents a particularly dramatic example of functional respecification during metamorphosis.

Animals↗