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PubMed1986

Multiple forms of juvenile hormone esterase active sites in the hemolymph of larvae of Trichoplusia ni.

Kinetic analysis was performed on the juvenile hormone (JH) esterase activity in the hemolymph of feeding, last instar larvae of Trichoplusia ni (Lepidoptera: Noctuidae). When the results were analyzed by several different graphical and regression procedures, all approaches yielded the same conclusion that at least two forms of JH esterase active sites exist in the hemolymph. The apparent Km for one site for JH I, II and III was 8.5 X 10(-8) M, and 6.6 X 10(-8) M, respectively. The Km for the other site for JH I, II and III was 6.6 X 10(-7) M, 7.6 X 10(-7) M, 40 X 10(-7) M, respectively. When hemolymph JHE activity was subjected to high resolution isoelectric focusing (IEF), two distinct large peaks of JHE activity were observed, with pIs of 5.3 and 5.5, as well as a small peak at pI 5.1. Separate kinetic analysis of the JHE activity in each peak showed that only the higher Km active site for each substrate was present (in the 10(-7) M range). These data necessitate a change in the current model for JHE in T. ni, and some other insects, which states that a single active site is responsible for most or all of the JH esterase activity in vivo. The data also explain the different estimates of the Km of JHE in T. ni obtained by different laboratories. Studies on the purification of, and the development of inhibitors for, JHE esterase must consider the role of both JHE forms and sites in regulation of T. ni metamorphosis.

Animals↗
PubMed1991

Methyl farnesoate in the prawn Macrobrachium rosenbergii: synthesis by the mandibular organ in vitro, and titers in the hemolymph.

1. The mandibular organ of Macrobrachium rosenbergii was identified and cultured in the presence of [methyl-3H]-methionine. 2. Radiolabelled methyl farnesoate was extracted and quantitated from cultured glands and culture media of both males and females: variation in synthesis rates was observed (1558-52,652 DPM/hr per gland) between individual prawns. 3. No significant incorporation of isotope into hexane-extractable material was observed in any other tissue cultured. 4. The concentration of methyl farnesoate in the hemolymph of M. rosenbergii was determined using normal phase HPLC with cis-trans (non-biological isomer) as an internal standard. 5. Hemolymph from males contained 17.3 +/- 9.5 ng/ml methyl farnesoate. 6. Female hemolymph contained 24 +/- 13.1 ng/ml methyl farnesoate.

Animals↗
PubMed1993

The occurrence of inducible anti-Escherichia coli activity in hemolymph from the migratory grasshopper, Melanoplus sanguinipes.

1. Inducible antibacterial activity against an Escherichia coli strain has been shown to occur in the hemolymph of Melanoplus sanguinipes. 2. This anti-E. coli activity was induced by the infection with Serratia marcescens or E. coli K12 but not Enterobacter cloacae. 3. The greatest anti-E. coli activity was observed in hemolymph extracted from grasshoppers 6 hr after the injection of any of the two species of bacteria. 4. Anti-E. coli activity disappeared in hemolymph extracted from grasshoppers 8 hr after injection or longer.

Animals↗
PubMed2005

Putative antiviral activity in hemolymph from adult Pacific oysters, Crassostrea gigas.

Innate, non-specific resistance mechanisms are important to pathogens, particularly for delaying virus replication at the onset of infection. Innate immunity constitutes the first line of defense in vertebrates and is the only one in invertebrates. Little is known about possible antiviral substances in invertebrates. The present work concerns a study of antiviral substances in hemolymph from adult Crassostrea gigas oysters. Despite the detection of cytotoxicity in fresh filtered hemolymph for both mammalian (CC50: 750 microg/ml) and fish cells (CC50: > 2000 microg/ml for EPC cells and 345 microg/ml for RTG-2 cells), an antiviral substance was detected. Fresh filtered hemolymph was capable of inhibiting the replication of herpes simplex virus type 1 in vitro at an EC50 of 425 microg/ml (total proteins) and the replication of infectious pancreatic necrosis virus in EPC and RTG-2 cells at 217 and 156 microg/ml (total proteins), respectively.

Animals↗
PubMed2004

Isolation and characterization of hemolymph clotting factors in Drosophila melanogaster by a pullout method.

Clotting is critical in limiting loss of hemolymph and initiating wound healing in insects as well as in vertebrates. Clotting is also an important immune defense, quickly forming a secondary barrier to infection, thereby immobilizing, and possibly killing bacteria directly. Here, we describe methods to assess clotting and to extract the clot from Drosophila larval hemolymph by using aggregation of paramagnetic beads. The validity of the assay was demonstrated by characterization of mutants. We show that clotting occurs in the absence of phenoloxidase and that the Drosophila clot binds bacteria. We also describe a pullout assay to purify the clot as a whole, free from entrapped hemocytes and cellular debris. Proteins subsequently identified by mass spectrometry include both predicted and novel clot proteins. Immune induction has been shown for three of the latter, namely Tiggrin and two unknown proteins (GC15825 and CG15293) that we now propose function in hemolymph clotting. The most abundant clot protein is Hemolectin, and we confirm that hemolectin mutant larvae show clotting defects.

Animals↗
PubMed2005

Effects of two hemolymph proteins on humoral defense reactions in the wax moth, Galleria mellonella.

Two hemolymph proteins were isolated from the wax moth, Galleria mellonella, larvae by a two-step procedure consisting of acid extraction and reversed phase (RP)-HPLC. One was an apolipophorin III (apoLp-III) previously characterized as a lipopolysaccharide (LPS) binding protein in the hemolymph of G. mellonella. The other was confirmed to be a new protein with a molecular mass of 23,768.69 Da, referred to as Gm protein-24. The full-length cDNA of Gm protein-24 was cloned from the fat body. The cDNA structure showed that it is a 219-residues protein derived from the precursor of 236 amino acids. The effects of apoLp-III and Gm protein-24 have been tested on the insect humoral immunity. ApoLp-III enhanced the activity of antibacterial peptide such as cecropin but Gm protein-24 had no effect on cecropin activity. On the other hand, Gm protein-24 and apoLp-III were both involved in the activation of prophenoloxidase (PPO) cascade, which has been regarded as a critical immune reaction in insect hemolymph. Of note, the Gm protein-24 was a significantly stronger activator of PPO cascade than apoLp-III.

Amino Acid Sequence↗
PubMed2005

Effects of juvenile hormone III on Reticulitermes flavipes: changes in hemolymph protein composition and gene expression.

Termites express polyphenism during caste differentiation that is mostly undefined at the molecular level. Using the eastern subterranean termite, Reticulitermes flavipes Kollar, we wanted (1) to test juvenile hormone (JH) model assays for their ability to induce detectable molecular changes in worker termites and (2) to investigate hemolymph proteins and their corresponding genes during JH-induced soldier caste differentiation. Our results illustrate pronounced changes in two hemolymph proteins after JH treatment, as well as differences among several caste phenotypes. Significant increases in the expression of four genes encoding hemolymph proteins, including two vitellogenins and two hexamerins, were observed after JH exposure. These findings are the first to demonstrate such protein and gene expression changes during termite caste differentiation. These results also validate the utility of JH model assays for inducing detectable molecular changes in worker termites that have begun presoldier differentiation.

Animals↗
PubMed2005

Molecular identification of a bevy of serine proteinases in Manduca sexta hemolymph.

Extracellular serine proteinase pathways control immune and homeostatic processes in insects. Our current knowledge of their components is limited-prophenoloxidase-activating proteinases (PAPs) are among the few hemolymph proteinases (HPs) with known functions. To identify components of proteinase systems in the hemolymph of Manduca sexta, we amplified cDNAs from larval fat body or hemocytes using degenerate primers coding for two conserved regions in S1 family serine proteinases. PCR yielded fragments encoding seven known (HP1-HP4, PAP-1, PAP-2 and PAP-3) and 18 unknown (HP5-HP22) serine proteinases. We screened cDNA libraries and isolated clones for 17 of the newly discovered HPs (HP5-HP22 except for HP11) and prepared antibodies to 14 recombinant proteins (HP6, HP8-HP10, HP12, HP14-HP19, HP21 and HP22). Fourteen of the HPs contain regulatory clip domain(s) at their amino-terminus--HP1, HP2, HP6, HP8, HP13, HP17, HP18, HP21, HP22 and PAP-1 have one, whereas HP12, HP15, PAP-2 and PAP-3 have two clip domains. Multiple sequence alignment of catalytic domains in these and other arthropod serine proteinases provided useful clues for future functional analysis. Northern blot and reverse transcription PCR (RT-PCR) analyses showed increases in HP2, HP7, HP9, HP10, HP12-HP22 mRNA levels at 24h after a bacterial challenge, and immunoblot analysis confirmed elevated concentrations of HP12, HP14-HP19, HP21 and HP22 proteins in plasma in response to injected bacteria. Hemocytes express HP13 and HP18; fat body produces HP12, HP20-HP22; both tissues synthesize the other HPs. These results collectively indicate the existence of a complex serine proteinase network in M. sexta hemolymph, predicted to mediate rapid defense responses upon wounding and/or microbial infection.

Amino Acid Sequence↗
PubMed2005

Membrane-bound sorbitol 6-phosphatase in fat body cells controls the dynamics of sorbitol 6-phosphate, a major hemolymph sugar in the silkworm.

Sorbitol 6-phosphate (S6P) is one of two major sugars (another is trehalose) in the larval hemolymph of Bombyx mori, and its amount dramatically decreases concomitantly with the onset of prepupal period. In the last (fifth) instar larvae, the amount of S6P is approximately 30 micromol/larva at its maximum and decreases to less than 1 micromol at the wandering stage. Incubation of fat bodies of wandering larvae with S6P generates sorbitol in the medium, while S6P in the medium decreases, indicating that fat body possesses sorbitol 6-phosphatase (S6Pase) activity. S6Pase activity in the fat body remains low during the feeding period, abruptly increases at the wandering and decreases to a low level after gut purge. 20-Hydroxyecdysone (20E) increases S6Pase activity in the fat body of feeding larvae, and the activation is dose-dependent. Cell fractionation studies show that S6Pase is mainly associated with the membrane and the optimal pH for membrane-bound S6Pase is 5.5, which is different from that for soluble acid phosphatase (pH 4). Present findings indicate that the S6Pase responsible for a decrease in hemolymph S6P is membrane-bound, and its activity is controlled by a rise of hemolymph ecdysteroid titer at the onset of the wandering stage.

Animals↗
PubMed2004

The basis for colorless hemolymph and cocoons in the Y-gene recessive Bombyx mori mutants: a defect in the cellular uptake of carotenoids.

Bombyx mori is an excellent model for the study of carotenoid-binding proteins (CBP). In previous papers, we identified and molecularly characterized a CBP from the Y-gene dominant mutants. In the present study, we attempted to correlate and establish lipid metabolism and distribution in these mutants. When [3H]-triolein was fed to the mutants, typical patterns of uptake of labeled fatty acids from midgut to hemolymph and subsequent delivery to fat body and silk glands were obtained in all mutants. Further analysis of lipid and carotenoid profiles revealed that the yellow coloration in the hemolymph associated with lipophorin is not attributed to a difference in lipophorin concentrations among the mutants, nor to its lipid composition, but rather to its carotenoid content. Lipophorin of the Y+I mutant exhibited the highest concentration of total carotenoids of 55.8 microg/mg lipophorin compared to 3.1 microg/mg in the +Y+I mutant, 1.2 microg/mg in the YI mutant and 0.5 microg/mg in the +YI mutant. Characteristic retention time in HPLC of the different classes of carotenoids of lipophorin identified the presence of lutein as the major chromophore (62-77%), followed by beta-carotenes (22-38%). Although lutein and beta-carotene content of mutants' lipophorin differed significantly, the ratio of lutein to beta-carotene of 3:1 was not different among mutants. Similarly, lipid compositions of mutant silk glands were not significantly different, but carotenoid contents were. The significantly high concentration of lutein in the Y+I mutant silk gland represented more than 160-fold increase compared to +Y+I mutant (p<0.001). In this report, we conclude that lipid metabolism in the mutants is not defected and that the molecular basis for colorless hemolymph and cocoons is a defect in the cellular uptake of lutein associated with the Y-gene recessive mutants.

Animals↗
PubMed2005

Reproductive division of labor, dominance, and ecdysteroid levels in hemolymph and ovary of the bumble bee Bombus terrestris.

To determine whether ecdysteroids are associated with reproductive division of labor in Bombus terrestris, we measured their levels in hemolymph and ovaries of queens and workers. Queens heading colonies had large active ovaries with high ecdysteroid content, whereas virgin gynes and mated queens before and after diapause had undeveloped ovaries with low ecdysteroid content. The hemolymph ecdysteroid titer was rather variable, but in a pooled analysis of mated queens before and after diapause versus colony-heading queens, ecdysteroid titers were higher in the latter group. In workers, agonistic behavior, ovarian activity, ovarian ecdysteroid content, and hemolymph ecdysteroid titers were positively correlated, and were lowest when a queen was present. In queenless workers, ecdysteroid levels were elevated in dominant workers, and were also influenced by the presence of brood and by group demography; hormone levels were higher in bees kept in larger groups. These findings are consistent with the premise that in B. terrestris the ovary is the primary site of ecdysteroid synthesis, and they show that ecdysteroids levels vary with the social environment.

Animals↗
PubMed2004

Detection of white spot syndrome virus (WSSV) from hemolymph of Penaeid shrimps Penaeus japonicus by reverse passive latex agglutination assay using high-density latex particles.

A simple reverse passive latex agglutination (RPLA) method for detecting white spot syndrome virus (WSSV) in the hemolymph of infected Kuruma shrimp (Penaeus japonicus) was developed. It was confirmed that WSSV could be detected from the shrimp hemolymph when the latex particles blocked with a casein protein were used as detection reagent. It became clear from the result of the infection trial that viruses are detectable by RPLA before the appearance of overt symptoms of this disease. In addition, an amplification product of 982 bp (s) derived from WSSV by PCR was detected in all the samples in which WSSV was detected by RPLA. This newly developed RPLA assay can examine many samples in a simple manner since hemolymph can be extracted more easily than any other organs. This assay can be used conveniently for virus detection in the culture pond of shrimps or in the field.

Animals↗
PubMed2005

Nitric oxide production in blowfly hemolymph after yeast inoculation.

Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses that include both cellular and humoral components. Cellular responses are mediated by hemocytes and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In this work, we determined NO production in Chrysomya megacephala hemolymph and hemocytes after yeast inoculation. Assays were performed with non-infected controls (NIL), saline-injected larvae (SIL) or larvae injected with Saccharomyces cerevisiae (YIL). The hemolymph of injected groups was collected 0.5, 1, 2, 4, 12, 24 or 48h post-injection. NO levels in SIL were comparable to those measured in NIL until 12h, which might be considered the basal production, increasing at 24 and 48h post-injection, probably in response to the increased larval fragility after cuticle rupture. YIL exhibited significantly higher levels of NO than were found in other groups, peaking at 24h. l-NAME and EDTA caused a significant reduction of NO production in YIL at this time, suggesting the activity of a Ca(2+)-dependent NOS. Plasmatocytes and granular cells phagocytosed the yeasts. Plasmatocytes initiated the nodule formation and granular cells were the only hemocyte type to produce NO. These results permit us to conclude that yeasts induced augmented NO production in C. megacephala hemolymph and granular cells are the hemocyte type involved with the generation of this molecule.

Animals↗
PubMed2006

In vitro transport of an allatostatin across the foregut of Manduca sexta larvae and metabolism by the gut and hemolymph.

The degradation of synthetic cydiastatin 4 by enzymes of the foregut and hemolymph, and transport across the foregut of larvae of the tobacco hawkmoth moth, Manduca sexta, were investigated using reversed-phase high performance liquid chromatography (RP-HPLC) together with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In the hemolymph in vitro, cydiastatin 4 had a half-life of ca. 30 min. Two degradation products were identified; cydiastatin 4(1-6), due to cleavage of the C-terminal di-peptide GL-amide, and cydiastatin 4(2-8), due to cleavage of the N-terminal A residue. This hydrolysis could be inhibited by up to 93% by 1,10-phenanthroline. Other protease inhibitors had lesser effects (<21% inhibition of degradation) including the aminopeptidase inhibitors amastatin and bestatin, and the chelator EDTA. When incubated with foregut extract in vitro, cydiastatin 4 had a half-life of 23 min, and the hydrolysis products detected were also cydiastatin 4(1-6) and cydiastatin 4(2-8). Similarly, 1-10 phenanthroline inhibited foregut enzyme degradation of cydiastatin 4 by ca. 80%, whereas amastatin, bestatin, and EDTA had very little effect (<10% inhibition). Cydiastatin 4 was transported, intact, from the lumen to the hemolymph side of foregut tissues that were mounted as flat sheets in modified Ussing chambers. This trans-epithelial flux of peptide was dose and time-dependent, but was <3% of the amount of cydiastatin 4 present in the lumen bathing saline. In contrast, no trans-epithelial transport of peptide was apparent across everted foregut sac preparations.

Animals↗
PubMed2003

Inhibition of apoptosis by recombinant 30K protein originating from silkworm hemolymph.

In a previous study, we reported that silkworm hemolymph inhibits apoptosis and that the anti-apoptotic component in silkworm hemolymph is a 30K protein. In this study, the 30K protein encoded by 30Kc6 was expressed in Escherichia coli. The recombinant 30K protein was expressed as an inclusion body, and the inclusion body was separated and refolded by affinity column chromatography using a 6xHis tag. We demonstrated that apoptosis is inhibited by supplementing the culture medium with this purified recombinant 30K protein. The recombinant 30K protein inhibited the virus- or chemical-induced apoptosis in human cells as well as insect cells. Apoptosis-inhibitory activity of recombinant 30K protein was comparable to that of whole silkworm hemolymph. The recombinant 30K protein can be effectively used to minimize cell death and consequently increase the productivity by extending the production time of host cells in commercial animal cell culture.

Amino Acid Sequence↗
PubMed2002

Effect of ice fraction and dilution factor on the antifreeze activity in the hemolymph of the cerambycid beetle Rhagium inquisitor.

The freezing-melting hysteresis in a given volume of hemolymph from the cerambycid beetle Rhagium inquisitor was linearly and negatively related to the logarithm of the mass fraction of ice in the sample. When the ice fraction dropped by a factor of 10, the hysteresis activity increased by about 2 degrees C. When the hemolymph was diluted, the hysteresis activity was linearly and negatively related to the logarithm of the dilution factor. Dilution of the hemolymph by a factor of 2 led to a 1 degree C reduction in hysteresis activity. In the diluted samples, the ice growth took place along the a-axes, implying that the antifreeze peptides of insects block ice growth along the c-axis, in addition to the a-axis.

Animals↗
PubMed2002

Basic pattern of fluctuation in hemolymph PTTH titers during larval-pupal and pupal-adult development of the silkworm, Bombyx mori.

General features of the changes in hemolymph PTTH titers during larval-pupal and pupal-adult development of the silkworm Bombyx mori were analyzed by comparing the patterns of the titer changes between different races and between silkworms reared under different environmental conditions. In common to all types of the silkworms tested, we observed low PTTH titers during the phagoperiod of the final instar, a small rise in PTTH titer on the day before wandering, two middle-sized peaks of the titer at the wandering and prepupal stages, high PTTH titers during early pupal-adult development, and a gradual titer increase shortly before adult eclosion. Increases in hemolymph PTTH titer were closely correlated with increases in ecdysteroid titers and with subsequent occurrences of morphological and behavioral changes characteristic of the initiation or progression of metamorphosis. The timing of the increase in hemolymph PTTH titer on the day of wandering was photoperiodically controlled, but that timing at the later stages seemed not to be influenced by the light-dark cycle.

Animals↗
PubMed1988

Liquid chromatographic procedures for the analysis of compounds in the serotonergic and octopamine pathways of lobster hemolymph.

High-performance liquid chromatography, with serial electrochemical and ultraviolet detectors, was used with a reduced activity catecholamine C18 column to separate and quantify compounds important in the serotonergic and octopamine pathways in lobster hemolymph. The chromatographic mobile phase was composed of potassium dihydrogenphosphate buffer, trichloroacetic acid, sodium dodecyl sulfate, the sodium salt of ethylenedinitrilotetraacetic acid and the organic solvents, acetonitrile and methanol. The compounds serotonin, 5-hydroxyindoleacetic acid, tryptophan, 5-hydroxytryptophan, tryptamine, melatonin, octopamine and tyrosine were well resolved within 13 min. Good electrode maintenance, the use of a silica gel precolumn and careful sample preparation were necessary to give a stable baseline, high resolution of these compounds and reproducibility of retention times and peak heights. The electrochemical detector extended the range of detection to the picogram level. Because of the instability of the solutes and of the chromatographic baseline, sample preparation procedures were investigated. Deproteinization with ammonium sulfate gave the best recovery of the compounds of interest and the most stable baseline with the electrochemical detector. Peaks in the hemolymph were characterized by addition of standards, dual detection (electrochemical and ultraviolet) and the enzyme peak shift technique. With this methodology, important endogenous neurohormones in the hemolymph of lobsters can be quantitatively determined with respect to the molt cycle.

Animals↗