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PubMed1992

Separation of insect hemolymph proteins by cascade-mode multi-affinity chromatography.

The hemolymph of the adult female Manduca sexta was fractionated by cascade-mode multi-affinity chromatography (CASMAC) on a main-line tandem column chain containing Zn(2+)-TED, T-gel, Ni(2+)-DPA, and phenylsepharose and a side-line column containing Zn(2+)-DPA. The technique separated some of the previously described major hemolymph proteins, and yielded a number of fractions with simple composition. Some of these fractions contained only less abundant proteins of Manduca hemolymph. Thus, it appears that CASMAC would be a very useful fractionation technique for purification and characterization of the minor proteins of insect hemolymph.

Animals↗

PubMed1994

Purification and characterization of a flavin-binding storage protein from the hemolymph of Galleria mellonella.

The 85K storage protein that accumulates in the hemolymph of Galleria mellonella during the final larval instar was isolated and purified from newly molted pupae. The separation of fresh hemolymph proteins from larvae or pupae by different chromatographic and electrophoretic procedures indicated the native protein had a M(r) of 170,000 and consisted of two identical 85K subunits. Crosslinking experiments using fresh hemolymph followed by Western blotting also indicated a dimeric structure for the native protein. Analyses of the dimer purified from pupal hemolymph indicated that 85K was a glycoprotein, containing approximately 6.5% neutral sugar and about 1.9% amino sugar. Like other insect flavin-binding proteins, 85K has a relatively high histidine content but an uncharacteristically high arginine content. The purified 85K dimer did not bind riboflavin, suggesting that the integrity of the molecule had been altered during purification. However, 85K purified in low yield by Affi-Gel Blue chromatography, did bind riboflavin, indicating that under certain, undefined conditions the functional integrity of the protein could be retained during purification.

Amino Acid Sequence↗

PubMed1994

Isolation and characterization of a hemocyte aggregation inhibitor from hemolymph of Manduca sexta larvae.

A protein that inhibits hemocyte aggregation has been isolated from hemolymph of Manduca sexta larvae and named hemocyte aggregation inhibitor protein (HAIP). HAIP has a M(r) = 50,000, pI = 8.5, and contains 7% carbohydrate. It is present at 230 +/- 20 micrograms/ml in hemolymph of day 3 fifth instar larvae. Antibodies to HAIP do not cross-react with M. sexta hemolin, which is similar in size and charge and also inhibits hemocyte aggregation. HAIP and hemolin have some similarity in amino acid composition and NH2-terminal sequence, but are different in overall secondary structure, as determined by CD spectroscopy. The concentration of HAIP in hemolymph is not affected by injection of larvae with bacteria. A protein of approximately 50,000 daltons that reacts with antibody to M. sexta HAIP is present in hemolymph of Bombyx mori, Heliothis zea, and Galleria mellonella. Although the function of HAIP in vivo is not yet clear, it may have a role in modulating adhesion of hemocytes during defensive responses.

Amino Acid Sequence↗

PubMed2004

Hemolymph-dependent and -independent responses in Drosophila immune tissue.

Insects possess an antimicrobial defense response that is similar to the mammalian innate immune response. The innate immune system is designed to recognize conserved components of microorganisms called pathogen-associated molecular patterns (PAMPs). How host receptors detect PAMPs and transmit the signals to mount the immune response is being elucidated. Using GFP-Dorsal, -Dif, and -Relish reporter proteins in ex vivo assays, we demonstrate that Drosophila fat bodies, a major immune tissue, have both hemolymph-dependent and -independent responses. Microbial preparations such as lipoteichoic acid (LTA) and peptidoglycan (PGN) can stimulate some responses from dissected and rinsed larval fat bodies. Therefore, at least some aspects of recognition can occur on fat body cell surfaces, bypassing the requirement of hemolymph. Our results also show that supernatants from bacterial cultures can stimulate the nuclear translocation of Dorsal in dissected fat bodies, but this stimulation is strictly hemolymph-dependent. Various biochemical assays suggest that the factors from bacterial supernatants that stimulate the hemolymph-dependent nuclear translocation are likely made up of proteins. We further show that Dorsal mutant larvae have much lower phenoloxidase activity, consistent with a more important role of Dorsal in innate immunity than previously shown.

Animals↗

PubMed1999

EPR detection of reactive oxygen species in hemolymph of Galleria mellonella and Dendrolimus superans sibiricus (Lepidoptera) larvae.

The formation of reactive oxygen species (ROS) in hemolymph and hemocytes of Galleria mellonella and Dendrolimus superans sibiricus larvae was studied by ESR spectroscopy using spin-trap 1-hydroxy-3-carboxy-pyrrolidine (CP-H). The background level of ROS formation was detected in the intact hemolymph. The addition of dihydroxyphenylalanine (DOPA) into free cells of the hemolymph increased CP-H oxidation about two times for G. mellonella and about four times for D. superans sibiricus. This increase was completely inhibited by a specific inhibitor of phenoloxidase, phenylthiourea. The presence of exogenous superoxide dismutase (SOD) did not change CP-H oxidation in the hemolymph. The data obtained in hemocytes showed only a DOPA-induced increase in CP-H oxidation. Phagocytosis activators did not affect ROS formation in hemocytes of both insect species. SOD decreased DOPA-induced CP-H oxidation 20-30% in suspension of hemocytes of D. superans sibiricus only. Our results are in agreement with the contribution of superoxide radical and DOPA-derived quinones/semiquinones in the immune response of insects.

Animals↗

PubMed1999

A newly established ELISA showing the effect of the androgenic gland on secondary-vitellogenic-specific protein in the hemolymph of the crayfish Cherax quadricarinatus.

A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to monitor the onset of secondary vitellogenesis in Cherax quadricarinatus females and in intersex individuals (having both male and female reproductive systems) after removal of the androgenic gland (AG). As a prerequisite for the assay, the 106-kDa polypeptide was separated from newly laid C. quadricarinatus eggs by SDS-PAGE, and anti-106-kDa antibody was raised in rabbit. The specificity of the anti-106-kDa polypeptide for proteins specific for the hemolymph of secondary-vitellogenic females was confirmed by double immunodiffusion and immunoblot cross-reactivity tests. A characteristic standard ELISA curve, using egg high-density lipoprotein (HDL), showed linearity between 16 and 500 ng (r = 0. 953) and was sensitive for amounts as low as 8 ng. The inter- and intraassay coefficients of variance were 14.8 and 7.2%, respectively. Only traces of egg HDL equivalents were detected in the hemolymph of male and primary-vitellogenic females (11 to 110 microg/ml), confirming the specificity of the assay, whereas high levels of such a protein (8-35 mg/ml) were detected in the hemolymph of secondary-vitellogenic females. Removal of the AG from intersex individuals leads to a significant increase in the concentration of vitellogenic-specific protein in the hemolymph (up to 2 mg/ml). Moreover, a significantly lower concentration was found in females subjected to AG transplant (79.3 microg/ml). The ELISA thus provided an accurate and sensitive tool to investigate the influence of the AG on the expression of a vitellogenic-specific protein in female and intersex C. quadricarinatus, confirming the central role of this gland in tuning sexual plasticity in this species.

Animals↗

PubMed2000

Enzymatic activities in European flat oyster, Ostrea edulis, and pacific oyster, Crassostrea gigas, hemolymph.

Enzymatic activities in the hemolymph of healthy and Bonamia-infected Ostrea edulis and Crassostrea gigas were studied with a commercial kit for the detection of 19 enzymes: 15 and 16 enzymes, respectively, were detected in the hemolymph of O. edulis and C. gigas and 10 of them showed relatively high activity levels. Most of them existed in both the cell-free fraction of the hemolymph and in the hemocytes. The cell-free hemolymph fraction of Bonamia ostreae-infected European flat oysters showed an elevated enzymatic activity level compared with that of healthy individuals. C. gigas hemocytes possessed higher enzymatic activity levels than O. edulis hemocytes. Differences in enzymatic activities existed in granulocytes and hyalinocytes in both oyster species. The enzyme release from oyster hemocytes seemed to be selective. The infection by B. ostreae induced enzymatic activity variations in European flat oysters. Higher enzyme levels within hemocytes may contribute partly to the natural resistance of C. gigas to the infection by B. ostreae.

Animals↗

PubMed1980

Carbohydrate reserves and hemolymph sugars of the African giant snail, Achatina fulica in relation to parasitic infection and starvation.

In the snail, Achatina fulica, parasitized by the rat lungworm Angiostrongylus cantonensis, levels of carbohydrate in hemolymph and digestive glands were determined. The normal level of hemolymph glucose of 11.7 mg% dropped to 4.25 mg% in the infected snails, a significant difference after only one week of infection. The level of total reducing sugar in the hemolymph also decreased significantly (12.3 mg% to 3.6 mg%) in this period. Later on, the snails were capable of adapting themselves to the parasitic infection and the concentration of hemolymph sugars returned to the normal range. Starvation caused a decrease in the carbohydrate reserves of the digestive gland only when the starved snails had been previously infected.

Angiostrongylus↗

PubMed1995

Degradation of pheromone biosynthesis-activating neuropeptide (PBAN) by hemolymph enzymes of the tobacco hornworm, Manduca sexta, and the corn earworm, Helicoverpa zea.

The tritium-labeled bis-norleucine analog of Helicoverpa zea pheromone biosynthesis-activating neuropeptide ([3H]NLPBAN) was incubated in vitro with hemolymph from Manduca sexta or H. zea adult females. The incubations resulted in the formation of several tritium-labeled degradation products. At a [3H]NLPBAN concentration of 0.9 microM the degradation proceeded at a very slow but physiologically plausible rate (2-10 fmol/min/microliters hemolymph). The primary [3H]NLPBAN degradation reaction in M. sexta hemolymph was not inhibited by 20 microM leupeptin, 0.1 mM amastatin, 1 mM EDTA, 1 mM EGTA, 1 mM 1,10-phenanthroline, or 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride; but secondary reactions may have been affected, as some of the inhibitors changed the radio-HPLC profile of the degradation products. It is concluded that hemolymph of M. sexta and H. zea contains peptidase(s) capable of inactivating circulating PBAN.

Amino Acid Sequence↗

PubMed1996

Aplysia hemolymph promotes neurite outgrowth and synaptogenesis of identified Helix neurons in cell culture.

Hemolymph of adult Aplysia californica significantly affects neurite outgrowth of identified neurons of the land snail Helix pomatia. The metacerebral giant cell (MGC) and the motoneuron C3 from the cerebral ganglion and the neuron B2 from the buccal ganglion of H. pomatia were isolated by enzymatic and mechanical dissociation and plated onto poly-L-lysine-coated dishes either containing culture medium conditioned by Helix ganglia, or pre-treated with Aplysia hemolymph. To determine the extent of neuronal growth we measured the neurite elongation and the neuritic field of cultured neurons at different time points. Aplysia hemolymph enhances the extent and rate of linear outgrowth and the branching domain of Helix neurons. With the hemolymph treatment the MGC neuron more consistently forms specific chemical synapses with its follower cell B2, and these connections are more effective than those established in the presence of the conditioned medium.

Animals↗

PubMed1985

Hemagglutinin activity in the hemolymph of Anticarsia gemmatalis larvae infected with the fungus Nomuraea rileyi.

Hemolymph samples from larvae of 3 lepidopteran species (Anticarsia gemmatalis, Trichoplusia ni and Spodoptera frugiperda) were tested for hemagglutination activity. Samples from A. gemmatalis larvae which had been injected 12-24 hrs previously with hyphal bodies of the entomopathogenic fungus Nomuraea rileyi showed agglutination activity against human 0, rabbit and sheep erythrocytes. Little or no activity was detected in samples from the other 2 larval species. Low titers (approximately 1:2) were observed when rabbit and sheep erythrocytes were tested with hemolymph from non-injected or water-injected A. gemmatalis control larvae. Higher titers (1:256-1:1024) were obtained when human erythrocytes were incubated with control hemolymph, but values were greater in the hyphal body-injected samples (1:2048-1: greater than 32,000). These results indicate a direct correlation between agglutinin production and the presence of fungal cells in the larval hemolymph. Injection with heat-killed or homogenated hyphal bodies did not cause increased activity. Decreases in titer values after these injections and for 1-12 hrs after injection with viable hyphal bodies suggest that the agglutinin(s) may function in immune surveillance. Agglutination of rabbit erythrocytes was inhibited by lactose, galactose and L-fucose. N-acetylneuraminic acid inhibited agglutination of human erythrocytes.

Animals↗

PubMed1991

Isolation of a phosphoryl choline-binding protein from the hemolymph of the snail, Achatina fulica.

A phosphorylcholine-binding protein from the hemolymph of the snail Achatina fulica was purified to near homogeneity using a Sepharose phenylphosphorylcholine affinity column. The protein bound to the affinity column was eluted with 5 mM phosphorylcholine as a single symmetrical peak. The purified protein (400 Kda) contained 35-40% carbohydrate. On SDS-PAGE the protein separated into two bands of 20 and 24 Kda, and had a pI of 5.9. On immunodiffusion, antiserum to the snail phosphorylcholine binding protein did not cross-react against other phosphorylcholine binding proteins, like rat serum phosphorylcholine-binding protein (PCBP), limulus C-reactive protein (CRP), or human CRP. On pretreatment of the snail hemolymph with this antiserum, the hemagglutination titer of the hemolymph was markedly decreased. The purified snail phosphorylcholine binding protein agglutinated rabbit erythrocytes in the absence of divalent cation (Ca+2) but trace amount of Ca+2 increased its binding. The strongest inhibitor of the agglutination reaction was lactose, followed by melibiose and 2-deoxygalactose. The relationships of the snail phosphorylcholine binding protein to other hemolymph agglutinins and to CRPs are discussed in light of common phylogeny.

Animals↗

PubMed1996

Functional bovine immunoglobulins in Boophilus microplus hemolymph.

The aim of the present work was to quantify the passage of bovine immunoglobulins into the hemolymph of the tick Boophilus microplus during the feeding process and to determine their antibody activity. The knowledge is of paramount importance when vector control or blocking of disease transmission is attempted by vaccination of cattle. Approximately 2% of bovine immunoglobulin present in the serum as determined by competitive ELISA was demonstrated in hemolymph of B. microplus and antibody activity against an antigen of B. microplus in the hemolymph of ticks fed on bovine immunized with the antigen purified from tick eggs was detected by Western blot assay. The antibody reactivity detected against the B. microplus antigen showed that functional antibodies are present in the hemolymph of fully engorged ticks for at least 48 h after completing the parasitic life cycle.

Animals↗

PubMed1979

Selected enzyme activities in Mya arenaria hemolymph.

1. The activities of lysozyme, acid and alkaline phosphatases, beta-glucuronidase, amylase, lipase, glutamate-oxalacetate transaminase, and glutamate-pyruvate transaminase in the whole hemolymph and 4000 g pellets and supernatants of Mya arenaria were determined. 2. All of these enzymes, except for amylase, occurred in whole hemolymph as well as in the 4000 g pellet and supernatant. 3. Based on earlier observations, these enzymes are believed to be of cellular origin within hemolymph cells. 4. In the case of amylase, it only occurred in the whole hemolymph and/or serum and is believed to have originated from the crystalline style.

Acid Phosphatase↗

PubMedDate not supplied

Antiviral melanization reaction of Heliothis virescens hemolymph against DNA and RNA viruses in vitro.

1. Antiviral activity of Heliothis virescens larval hemolymph was determined using a cytotoxicity/virus inhibition test (TClD50) done in Vero cell tissue cultures. Excellent antiviral activity was found especially against herpes simplex viruses-1 and -2 and also against vesicular stomatitis, parainfluenza-3, coxsackie B3 and sindbis viruses. 2. Prolonged incubation of herpes simplex virus-1 and vesicular stomatitis virus with hemolymph was virucidal and greatly reduced infectivity of the two viruses in tissue culture. 3. Antiviral activity was produced by both normal and immune (vaccinated larvae) cell-free hemolymphs. 4. Antiviral activity against herpes simplex virus-1 could be generated in vitro with hemolymph phenoloxidase or mushroom tyrosinase using four different substrates including tyrosine. 5. Activation of the insect melanization reaction by phenoloxidase was necessary for antiviral activity to occur.

Animals↗

PubMed2005

The effect of seawater composition and osmolality on hemolymph levels of methyl farnesoate in the green crab Carcinus maenas.

Green crabs, Carcinus maenas, exposed to dilute seawater (e.g., 5 ppt salinity, approximately 150 mOsm/kg) have hemolymph levels of methyl farnesoate (MF) that are up to 10-fold higher than animals in isosmotic seawater (27 ppt, approximately 800 mOsm/kg). In this paper, we examine aspects of osmotic and ionic stress to identify factors involved in elevating MF levels. MF levels did not rise after exposure to concentrated seawater, so only hypoosmotic stress elevates MF. MF levels rose in animals exposed to dilute seawater containing mannitol to make it isosmotic, indicating that the hypoosmotic rise in MF is due to decreased ion concentrations. Individual ions were investigated by exposing crabs either to isosmotic seawater with low concentrations of an ion or to dilute seawater with high concentrations of an ion. Ca(2+) and Mg(2+) in combination affected MF levels. Finally, we found that the increase in MF levels was accelerated when hemolymph osmolality was precociously lowered by partially replacing hemolymph with deionized water prior to transferring animals to dilute seawater. Thus, the 6-8 h delay between exposing crabs to dilute sea water and observing an increase in MF appears to reflect the time needed for specific hemolymph ions to decrease below a threshold concentration.

Acclimatization↗

PubMed2005

Isolation and characterization of hemolymph antifreeze proteins from larvae of the longhorn beetle Rhagium inquisitor (L.).

We describe a simple and effective procedure to isolate antifreeze proteins (AFPs) from the hemolymph of larvae of the longhorn beetle Rhagium inquisitor, and present some characteristics of their structures. Several AFPs were isolated from the hemolymph of this species by heat and acid extraction followed by cation exchange. The hemolymph contains at least six AFPs ranging in size from 12.5 to 12.8 kDa. Of these, three were separated to purity by the ion exchange step, as indicated by mass spectrometry. The remaining three forms were further separated by size exclusion chromatography, but could not be isolated to purity. All AFPs in the hemolymph of this species appears to have isoelectric points above 8.00. The dominant form, RiAFP(H4), was purified by the ion exchange step. Its amino acid composition reveals a lower level of cysteine and a higher level of threonine, arginine, alanine and glycine than seen in other insect AFPs. Its trypsin fingerprint does not match that of any known protein. It interacts with ice both in the anionic and cationic state.

Alanine↗

PubMed2004

The hemolymph JH titer exhibits a large-amplitude, morph-dependent, diurnal cycle in the wing-polymorphic cricket, Gryllus firmus.

The hemolymph juvenile hormone (JH) titer was measured in over 500 flight-capable and flightless, adult female Gryllus firmus at 3-6 h intervals during each of days 2-8 of adulthood. The flight-capable morph exhibited a large-amplitude daily cycle in the hemolymph JH titer, while the flightless morph exhibited a barely perceptible cycle. The JH titer cycle was observed on all days in the flight-capable morph, but the large amplitude cycle (>15-20 fold increase in mean titer; >100-fold increase in some individuals), began on day 5. For both the large and small amplitude cycles, the JH titer peaked near the end of the photophase-beginning of the scotophase. The hemolymph ecdysteroid titer did not exhibit a corresponding large amplitude daily cycle, although a low amplitude cycle (1-3-fold change) was seen in both morphs. The large magnitude rise in the JH titer in the flight-capable morph during the photophase was not due to decreased hemolymph volume or JH degradation. Daily cycles in the JH titer may be common, but may have gone unnoticed in other insect species due to restricted temporal sampling. Failure to identify these cycles can result in substantial errors in inferring biological roles for JH. Because JH regulates flight behaviors, morph-specific daily cycles in the JH titer may be especially common in dispersal-polymorphic insects, in which flight is restricted to one morph during a limited period of the day or night. However, because JH regulates numerous biological traits, analogous cycles may be common in insects exhibiting other types of complex (e.g. caste or phase) polymorphism, in which morphs differ in a biological characteristic that is restricted to a specific period of the photophase or scotophase.

Analysis of Variance↗