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PubMed1991

Quantification of methyl farnesoate levels in hemolymph by high-performance liquid chromatography.

Methyl farnesoate (MF) is an acyclic sesquiterpenoid that has been detected in hemolymph and other tissues of crustaceans and insects. This paper describes a rapid and sensitive method for measuring MF in crustacean hemolymph. Extracts of hemolymph samples were separated by normal-phase high-performance liquid chromatography (5-microns silica, 250 x 4.6 mm I.D., 1.3% diethyl ether in hexane) and detected by UV (220 nm). The limit of detection with this method was less than 250 pg/ml. This method should be useful for studying the physiological functions of MF in crustaceans and other arthropods.

Animals↗

PubMed1997

Degradation of Dip-allatostatins by hemolymph from the cockroach, Diploptera punctata.

Incubation of Dip-AST 7 (APSGAQRLYGFGLa) or Dip-AST 9 (GDGRLYAFGLa) (5 microM) with hemolymph for 30 min results in cleavage by a putative endopeptidase, yielding the C-terminal hexapeptide. This metabolic product is subsequently cleaved by an amastatin-sensitive aminopeptidase to yield the the C-terminal pentapeptide, as treatment with the competitive aminoexopeptidase inhibitor, amastatin, results in a significant accumulation of the C-terminal hexapeptide. Interestingly, Dip-AST 5 (DRLYSFGLa) (6 microM), which in common with Dip-AST 7 and 9 possesses Arg-Leu-Tyr, is not rapidly cleaved. However, [3H-Tyr]Dip-AST 5 at physiological concentrations (4 nM), appears to be cleaved by the same enzymes that cleave Dip-AST 7 and 9, albeit at a reduced rate. Incubation of other members of the Dip-allatostatin family with hemolymph also results in cleavage of the peptides, suggesting that there are a variety of endo- and/or exopeptidases present in the hemolymph of D. punctata.

Animals↗

PubMed2003

Allelic variation in the hemolymph juvenile hormone binding protein gene of Manduca sexta.

Peripheral distribution of the insect juvenile hormones (JHs) requires hemolymph transport proteins. A comparison of three strains of the tobacco hornworm Manduca sexta indicates that hemolymph JH binding protein (hJHBP) levels in the Madison wild-type (Mwt) strain are significantly higher than in the black larval mutant (bl) and Seattle wild-type (Swt) strains. To correlate differences in hJHBP levels between strain phenotypes with the hJHBP locus, we sequenced 8.4 kb of the hJHBP gene locus from each strain. Snb, an allele found in the Swt and bl strains, contains a 408 bp repetitive nuclear element flanked by 15 bp direct repeats. Mating studies coupled with molecular genotyping demonstrate that the presence of Snb correlates with a twofold lower hJHBP level relative to the allele found in the Mwt strain. Despite the lower hJHBP levels in individuals carrying the Snb gene, hemolymph levels of JH do not appear to be significantly affected.

Alleles↗

PubMed1998

Isolation and partial characterization of chromoprotein from the larval hemolymph of the Japanese oak silkworm (Antheraea yamamai).

A total of two different hemolymph proteins (designated P-I and P-II) of the Japanese oak silkworm, Antheraea yamamai, were purified from the hemolymph of the fifth instar larvae using four chromatographic steps: (a) hydrophobic interaction chromatography; (b) ion exchange chromatography; (c) gel-filtration; and (d) reverse-phase high performance liquid chromatography (HPLC). These two proteins were separated by TSKgel Phenyl-5PW RP column chromatography. P-I has an apparent molecular weight of 31,000 or 35,000, as determined by gel-filtration and SDS-PAGE, respectively. P-II shows a molecular weight of 22,000 or 25,000, by gel-filtration and SDS-PAGE, respectively. The molecular weight of P-I and P-II were determined to be 31,076 and 21,500 by MALDI-TOF MS, respectively. These results suggest that both P-I and P-II are monomers. The N-terminal sequence analysis suggests that P-I is closely related to the ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta, with 40% identity in the first 30 residues, while P-II is similar to the biliproteins (BPs) from other lepidopteran insects (50% identity). Spectroscopic analysis shows that the blue chromophore of A. yamamai BP is not biliverdin IX, which is present in the biliproteins of most insects.

Amino Acid Sequence↗

PubMed1998

Sialic acid in hemolymph and affinity purified lectins from two marine bivalves.

Sialic acids have been implicated in a variety of complex biological regulatory and signalling events and their functional importance is reflected by their presence in a wide variety of phyla. Potentially they may inhibit intermolecular and intercellular interactions. Lectins that exhibit specificity for sialic acid or sialoglycoconjugates are ubiquitous in the body fluids of invertebrates and this has supported the assumption that these lectins are involved in defense against microbes that express sialic acids on their surfaces. This biological function has also been inferred from the absence of sialic acids in lower invertebrates. However, most invertebrate lectins are heterogeneous and may also bind other ligands. The biological significance of the different carbohydrate specificities are not yet known. We have demonstrated the presence of sialic acids in hemolymph from two marine bivalves, the Pacific oyster Crassostrea gigas (approximately 15 micrograms ml-1) and the horse mussel Modiolus modiolus (48-100 micrograms ml-1) by several different assays. The sialic acid was mostly in free form. Affinity purified lectins from the horse mussel also contained bound sialic acids (2-5 mumol g-1). Oyster hemolymph stimulated the in vitro phagocytosis of bacteria by oyster hemocytes. The stimulation by hemolymph is facilitated by a dialyzable component, that apparently is active irrespective of the binding to sialic acid (BSM). Addition of sialic acid had no significant effect on the in vitro phagocytosis of bacteria by oyster hemocytes.

Agglutination↗

PubMed1999

Catechol conjugation with hemolymph proteins and their incorporation into the cuticle of the American cockroach, Periplaneta americana.

Newly ecdysed American cockroaches, Periplaneta americana (sixth to last instar) were injected with radioactive dopamine (DA) and hemolymph was collected at 10-60 min post-ecdysis. Size-exclusion chromatography established the presence of at least three proteins that serve as catecholamine carriers. Reinjection of the smaller radiolabeled phenol-bound proteins into newly ecdysed animals results in in vivo aggregation, with the radiolabel bound to large MW proteins (30- > 200 kDa). In addition, the reinjection of radiolabeled protein of any size resulted in the incorporation of the label into the newly sclerotized cuticle. Hemolymph proteins were synthesized in vivo using [14C]leucine and subsequently double labeled in vivo with [3H]dopamine. After sclerotization (7 h post-ecdysis) the cuticle was extirpated, hydrolyzed and counted. An identical ratio of 14C to 3H was found in cuticle extracts as in the double-labeled hemolymph proteins, suggesting that the phenol-bound protein was incorporated in the cuticle unchanged. It appears that the catechol bound to the proteins exists as a beta-glucoside.

Animals↗

PubMed2000

Isolation and characterization of juvenile hormone esterase from hemolymph of Lymantria dispar by affinity- and by anion-exchange chromatography.

Juvenile hormone esterase (JHE), which catalyzes the hydrolysis of juvenile hormone, was isolated from the hemolymph of 5(th) instars of Lymantria dispar by two different procedures. One procedure was based on affinity chromatography and the other on anion-exchange chromatography. The material from both purifications showed bands of approximately 50 kDa when analyzed by SDS-PAGE. Isoelectric focusing (IEF) gels in combination with enzyme activity assays indicated two isoelectric forms with the same pI values (pH 5.1. and 5.3) from affinity purification and from anion-exchange chromatography. Amino acid sequencing of several internal peptides from the 50 kDa band following affinity purification and alignment of these sequences with JHEs from previously purified lepidopteran species (Heliothis virescens, Manduca sexta) showed high homology of these enzymes. The isolated JHE, at least in the stage of insect used, was different from the enzyme reported earlier [Valaitis, A.P., 1991. Characterization of hemolymph juvenile hormone esterase from Lymantria dispar. Insect Biochemistry 21, 583-595] to hydrolyze JH in the hemolymph of gypsy moth, based on molecular weight and amino acid sequence.

Amino Acid Sequence↗

PubMed2001

Identification of a defensin from the hemolymph of the American dog tick, Dermacentor variabilis.

Hemolymph from partially fed virgin Dermacentor variabilis females was collected following Borrelia burgdorferi challenge and assayed for antimicrobial activity against Bacillus subtilis and B. burgdorferi. A small inducible cationic peptide was identified by SDS-PAGE in the hemolymph of these ticks as early as 1h post challenge. Following purification by a three-step procedure involving sequential SepPak elution, reversed phase high performance liquid chromatography (RP-HPLC) and gel electrophoresis, the yield of the active peptide was approximately 0.1% of the total protein in the hemolymph plasma. The molecular weight, 4.2kDa, was determined by MALDI-TOF mass spectrometry. N-terminal sequencing by the Edman degradation method gave a sequence for the first 30 amino acids as: G-F-G-C-P-L-N-Q-G-A-C-H-N-H-C-R-S-I-(R)-(R)-(R)-G-G-Y-C-S-Q-I-I-K. A computer search of databases showed that the peptide had 83% similarity to a defensin found in a scorpion. This is the first report of a defensin from a tick. The peptide was stable at least up to 70 degrees C. Although the tick defensin alone was not immediately effective against B. burgdorferi, tick defensin plus lysozyme killed more than 65% of cultured B. burgdorferi within 1h.

Amino Acid Sequence↗

PubMed2002

A new member of the PBAN family in Spodoptera littoralis: molecular cloning and immunovisualisation in scotophase hemolymph.

In this article, we report evidence suggesting that the immunoreactive factor previously detected in Spodoptera littoralis scotophase hemolymph is PBAN, which supports a humoral route of the hormone to the pheromone gland. Western blot after native-PAGE of prepurified scotophase hemolymph extracts yielded an immunoreactive band with the same mobility as S. littoralis Br-SOG factor and the expected mobility for a noctuid PBAN. This band was not detected in photophase hemolymph extract. The identity of S. littoralis Br-SOG factor as PBAN was obtained from cDNA cloning using RT-PCR strategy. This allowed us to deduce the amino acid sequence of Spl-PBAN, which is highly homologous to other known PBANs. Moreover, we found that the PBAN encoding cDNA also encoded four other putative amidated peptides (Spl-DH homologue, Spl-alpha-NP, Spl-beta-NP and Spl-gamma-NP) that are identical or highly conserved among noctuids, and two non amidated peptides of unknown function. This cDNA organization is common to all known cDNAs encoding PBANs, leading to the release of different peptides after putative enzymatic cleavage of the preprohormone.

Amino Acid Sequence↗

PubMed2000

Purification and characterization of epidermis-origin hemolymph protein in Galleria mellonella.

Epidermis-origin hemolymph protein (EOHP) was identified and purified from the last instar larval hemolymph of Galleria mellonella by anion exchange chromatography, chromatofocusing chromatography, and Sephadex G-100. The EOHP has a native molecular mass of 47 kDa and is composed of one subunit. The isoelectric point of the EOHP was determined to be 5.3. The amino acid composition of the EOHP was rich in aspartic acid, glutamic acid and lysine, but poor in tyrosine, methionine, and tryptophan. EOHP is present in hemolymph over the period from the 4th instar larvae to the adult stage examined. Concentration of EOHP is high during the larval stage but gradually decreased during the developmental stage from pupal to adult stage. EOHP is present in the cuticle, fat bodies and trachea but not in hemocytes, fore gut, mid gut and hind gut.

Amino Acids↗

PubMed2002

Vitellogenin levels in mussel hemolymph--a suitable biomarker for the exposure to estrogens?

Increased vitellogenin (vtg) levels in the blood of male fish are frequently used as an indicator of estrogenic exposure. Similar responses are expected for mussels, where the concentration of vtg-like proteins has been reported to depend on estrogens. To verify the role of hemolymph during vitellogenesis of mussels, the saltwater mussel Mytilus edulis and the freshwater mussel Anodonta cygnea were exposed to 17beta-estradiol (E2) and wastewater treatment plant effluents, known for their estrogenic potential. Gel electrophoresis did not reveal any significant induction (or repression) of plasma proteins compared to control plasma. Our results do not support the hypothesis that mussel hemolymph is a carrier of estrogen-dependent major egg-yolk precursors (vtg-like proteins). However, additional information on a 35+/-2-kDa hemolymph protein, previously reported to bind heavy metals, was obtained by high-resolution two-dimensional electrophoresis. It was resolved in a cluster of single proteins with properties that match the characteristics of a previously reported histidine-rich glycoprotein.

Animals↗

PubMed1987

Purification and properties of a very high density lipoprotein from the hemolymph of the honeybee Apis mellifera.

A larval-specific very high density lipoprotein (VHDL) has been isolated from the hemolymph of the honeybee Apis mellifera. VHDL was isolated by a combination of density gradient ultracentrifugation and gel filtration. The purified protein is a dimer of Mr 160,000 apoproteins as shown by chemical cross-linking with dimethyl suberimidate. N-Terminal sequence analysis indicates that the two polypeptide chains are identical. The holoprotein contains 10% lipid by weight and 2.6% covalently bound carbohydrate. A native Mr 330,000 species was obtained by gel permeation chromatography. Antiserum directed against VHDL was used to show that VHDL is distinct from other hemolymph proteins and appears to constitute a novel lipoprotein of unknown function. However, the lipoprotein is present in high amounts in hemolymph only at the end of larval life, suggesting a potential role in lipid transport and/or storage protein metabolism during metamorphosis.

Amino Acid Sequence↗

PubMed2001

Sequestration of host plant glucosinolates in the defensive hemolymph of the sawfly Athalia rosae.

Interactions between insects and glucosinolate-containing plant species have been investigated for a long time. Although the glucosinolate-myrosinase system is believed to act as a defense mechanism against generalist herbivores and fungi, several specialist insects use these secondary metabolites for host plant finding and acceptance and can handle them physiologically. However, sequestration of glucosinolates in specialist herbivores has been less well studied. Larvae of the tumip sawfly Athalia rosae feed on several glucosinolate-containing plant species. When larvae are disturbed by antagonists, they release one or more small droplets of hemolymph from their integument. This "reflex bleeding" is used as a defense mechanism. Specific glucosinolate analysis, by conversion to desulfoglucosinolates and analysis of these by high-performance liquid chromatography coupled to diode array UV spectroscopy and mass spectrometry, revealed that larvae incorporate and concentrate the plant's characteristic glucosinolates from their hosts. Extracts of larvae that were reared on Sinapis alba contained sinalbin, even when the larvae were first starved for 22 hr and, thus, had empty guts. Hemolymph was analyzed from larvae that were reared on either S. alba, Brassica nigra, or Barbarea stricta. Leaves were analyzed from the same plants the larvae had fed on. Sinalbin (from S. alba), sinigrin (B. nigra), or glucobarbarin and glucobrassicin (B. stricta) were present in leaves in concentrations less than 1 micromol/g fresh weight, while the same glucosinolates could be detected in the larvae's hemolymph in concentrations between 10 and 31 micromol/g fresh weight, except that glucobrassicin was present only as a trace. In larval feces, only trace amounts of glucosinolates (sinalbin and sinigrin) could be detected. The glucosinolates were likewise found in freshly emerged adults, showing that the sequestered phytochemicals were transferred through the pupal stage.

Adaptation, Physiological↗

PubMed1999

A novel lipoprotein from the hemolymph of the cochineal insect, Dactylopius confusus.

A new type of insect lipoprotein was isolated from the hemolymph of the female cochineal insect Dactylopius confusus. The lipoprotein from the cochineal insect hemolymph was found to have a relative molecular mass of 450 000. It contains 48% lipid, mostly diacylglycerol, phospholipids and hydrocarbons. The protein moiety of the lipoprotein consists of two apoproteins of approximately 25 and 22 kDa, both of which are glycosylated. Both apolipoproteins are also found free in the hemolymph, unassociated with any lipid. Purified cochineal apolipoproteins can combine with Manduca sexta lipophorin, if injected together with adipokinetic hormone into M. sexta. This could indicate that the cochineal lipoprotein can function as a lipid shuttle similar to lipophorins of other insects, and that the cochineal insect apolipoproteins have an overall structure similar to insect apolipophorin-III.

Amino Acid Sequence↗

PubMed2004

A proteomic approach for the analysis of instantly released wound and immune proteins in Drosophila melanogaster hemolymph.

Insects respond to microbial infection by the rapid and transient expression of several genes encoding antibacterial peptides. In this paper we describe a powerful technique, two-dimensional difference gel electrophoresis, that, when combined with mass spectrometry, can be used to study the immune response of Drosophila melanogaster at the protein level. By comparatively analyzing the hemolymph proteome of 2,000 third-instar Drosophila larvae, we identified 10 differential proteins that appear in the fruit fly hemolymph very early after an immune-challenge with lipopolysaccharides. These proteins can be assigned to the immune response, because they are not induced after sterile injury. Reduction of integral variability or quantification problems related to conventional two-dimensional electrophoresis and improvement of image analysis were achieved by the use of two fluorescent dyes to label the two different protein samples. Some of the immune-induced proteins, such as thioester-containing protein 2, can be assigned to specific aspects of the immune response; others were already reported as being involved in stress response. An immune-induced protein (CG18594) is homologous to a mammalian serine protease inhibitor that mediates the mitogen-activated protein kinase and the NF-kappa B signaling pathways. In addition, a number of proteins that had not been associated with the immune response before were isolated and identified, and some of these were still present in the hemolymph 4 h after injury. Determining the function of all of these immune-induced proteins represents an exciting challenge for increasing our knowledge of insect immunity.

Animals↗

PubMed2006

Interaction of beta-1,3-glucan with its recognition protein activates hemolymph proteinase 14, an initiation enzyme of the prophenoloxidase activation system in Manduca sexta.

A serine proteinase pathway in insect hemolymph leads to prophenoloxidase activation, an innate immune response against pathogen infection. In the tobacco hornworm Manduca sexta, recombinant hemolymph proteinase 14 precursor (pro-HP14) interacts with peptidoglycan, autoactivates, and initiates the proteinase cascade (Ji, C., Wang, Y., Guo, X., Hartson, S., and Jiang, H. (2004) J. Biol. Chem. 279, 34101-34106). Here, we report the purification and characterization of pro-HP14 from the hemolymph of bacteria-injected M. sexta larvae. The zymogen, consisting of a single polypeptide with a molecular mass of 68.5 kDa, is truncated at the amino terminus. It is converted to a two-chain active form in the presence of beta-1,3-glucan (a fungal cell wall component) and beta-1,3-glucan recognition protein-2. The 45-kDa heavy chain contains four low-density lipoprotein receptor A repeats, one Sushi domain, and one unique cysteine-rich region, whereas the 30-kDa light chain contains a serine proteinase domain, which was labeled by [(3)H]diisopropyl fluorophosphate. Pro-HP14 in the plasma strongly binds curdlan, zymosan, and yeast and interacts with peptidoglycan and Micrococcus luteus. Addition of autoactivated HP14 elevated phenoloxidase activity level in the larval plasma. Recombinant M. sexta serpin-1I reduced prophenoloxidase activation by inhibiting HP14. These data are consistent with the current model on initiation and regulation of the prophenoloxidase activation cascade upon recognition of pathogen-associated molecular patterns by specific pattern recognition proteins.

Animals↗

PubMedDate not supplied

Maximum titers of vitellogenin and total hemolymph protein occur during the canalized phase of grasshopper egg production.

Many organisms exhibit developmental plasticity only in sensitive phases and cannot respond to environmental perturbations at other times. However, we know little about the physiological events that define plastic and canalized phases. During egg production in insects, vitellogenin (Vg) accumulates first in the hemolymph and then in the eggs. In addition, storage proteins may be important resources for egg production. Therefore, we tested hypotheses on the relationships of Vg and TP (total hemolymph protein minus Vg) titers to the transition from flexible to inflexible development during egg production. In lubber grasshoppers, approximately 70% of TP is contained in three proteins that range from 68 to 83 kDa. We maintained females on food treatments that produced defined plastic and canalized periods, collected hemolymph every approximately 4 d, and determined the ages at which oviposition and the maximum Vg and TP titers occurred. Both Vg(max) titer and especially TP(max) titer were predictors of the number of eggs produced. The time from eclosion to Vg(max) was significantly affected by diet, but the time from Vg(max) to oviposition was not. Similarly, the time from eclosion to TP(max) was significantly affected by diet, while the time from TP(max) to oviposition was not. Hence, Vg(max) and TP(max) are physiological landmarks that occur during the canalized phase of egg production.

Adaptation, Physiological↗

PubMed1991

Determination of vitellogenin titer in the hemolymph of Dermacentor variabilis (Acarina: Ixodidae) using an indirect enzyme-linked immunosorbent assay.

Using polyclonal antibodies against Dermacentor variabilis (Say) egg vitellin, we developed an indirect, competitive enzyme-linked immunosorbent assay (ELISA). Vitellogenin in the hemolymph of females was quantitatively monitored through feeding (slow feeding and rapid engorgement feeding periods) and postfeeding (preoviposition and oviposition) until death. Vitellogenin concentrations in hemolymph ranged from 1 microgram/ml on the first day of feeding to a maximum of 430 micrograms/ml on the third day of oviposition. The temporal pattern of vitellogenin production in this tick shows a gradual increase in the hemolymph vitellogenin titer throughout the slow feeding period followed by a rapid and substantial rise during the rapid engorgement feeding period and a continuation of this rise in titer through the most productive egg laying part of oviposition. The vitellogenin titer rapidly falls toward the end of oviposition as the tick nears death. There was a 170-fold increase in vitellogenin concentration as measured from the onset of rapid engorgement to the maximum vitellogenin titer. The rapid engorgement feeding period signaled a sharp increase in vitellogenin production.

Animals↗