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PubMed1992

Storage proteins are present in the hemolymph from larvae and adults of the Colorado potato beetle.

The protein composition of larval and adult hemolymph from the Colorado potato beetle, Leptinotarsa decemlineata, was investigated and some abundant, high molecular weight proteins were identified and characterized. Diapause protein 1, which occurs in the hemolymph of last instar larvae and short-day adults, appeared to be a storage protein. This protein dissociated into two bands due to the high pH used in nondenaturing gels. Its quaternary structure was established by chemical crosslinking. It appeared to be a hexamer. Diapause protein 1 is composed of approximately 82,000 subunits. The amino acid composition and N-terminal sequence of this protein has been determined. Specific antibodies against diapause protein 1 have been developed. Topical application of 1 microgram pyriproxyfen, a juvenile hormone analog, to last instar larvae and short-day adults suppressed the appearance of this protein in the hemolymph. Pyriproxyfen prematurely induced vitellogenin, when applied to last instar larvae. A larval specific protein was also identified in the hemolymph. Its temporary appearance in the hemolymph of last instar larvae, its subunit composition (M(r) approximately 82,000) and its suppression by pyriproxyfen suggests that this protein is a storage protein as well.

Amino Acid Sequence↗
PubMed1992

Hemolymph concentrations of host ecdysteroids are strongly suppressed in precocious prepupae of Trichoplusia ni parasitized and pseudoparasitized by Chelonus near curvimaculatus.

Regulation of ecdysteroid production in lepidopteran prepupae was studied using a parasitic wasp (C. near curvimaculatus) which specifically suppresses host prepupal ecdysteroid production after the induction of precocious host metamorphosis. At the developmental stage at which the hemolymph of the unparasitized metamorphosing host has its maximum titer of prepupal ecdysteroids, the hemolymph of 4th instar "truly parasitized" hosts (hosts with a surviving endoparasite) had a strongly reduced ecdysteroid titer. However, during the photophase about 12 h later, just prior to emergence of the parasite larva, an ecdysteroid peak was observed in the host hemolymph. Fourth instar pseudoparasitized prepupal hosts (in which the endoparasite was not present or died early in development) exhibited a sustained suppression in the hemolymph ecdysteroid titer. Small 5th instar pseudoparasitized hosts, which normally would molt to a 6th instar prior to metamorphosis, but which precociously attained the prepupal stage, also had a strongly reduced ecdysteroid titer. The late increase observed in truly parasitized hosts could be completely prevented by surgical removal of the parasite 24 h earlier, resulting in a titer similar to that in pseudoparasitized hosts. HPLC analysis of ecdysteroids in normal, truly parasitized, and 4th or 5th instar pseudoparasitized prepupae showed that both ecdysone and 20-OH ecdysone* were suppressed in truly and pseudoparasitized prepupae, with ecdysteroid levels being lowest in pseudoparasitized hosts. These data, and those of Brown and Reed-Larsen (Biol Contr 1, 136 [1992]), showing endoparasite secretion of ecdysteroids just prior to its emergence from the host, strongly indicate that: (1) the prepupal peak in truly parasitized hosts originates from the endoparasite, and (2) the low level of ecdysteroids in pseudoparasitized hosts results from the host's intrinsic inability to express a normal level of prepupal ecdysteroid titer. While precocious 4th or 5th instar prepupae of similar size had similarly suppressed ecdysteroid titers, smaller 4th instar prepupae had a lower ecdysteroid titer than larger, precocious 5th instar prepupae. Rare 5th instar pseudoparasitized prepupae that were of nearly normal size showed a prepupal ecdysteroid titer distinctly greater than those of the usual smaller, precocious 5th instar prepupae. The data suggest that the competence of the host to express a normal hemolymph titer of prepupal ecdysteroids is more closely correlated with the size of the prepupae than with the instar attained.

Animals↗
PubMed1994

Properties and significance of a riboflavin-binding hexamerin in the hemolymph of Hyalophora cecropia.

A riboflavin-binding hexamerin isolated from pupal hemolymph of Hyalophora cecropia has a native M(r) of 510,000, subunit M(r) of 85,000, and a 5% carbohydrate content. An intrachain cross-link was confirmed in protease limit digests. Ellman titration confirmed the presence of a sulfhydryl group, which is needed for this linkage. Though Cu2+ is known to promote the linkage, heavy metals were not detected in the isolate. Heat denaturation released ligand with the absorbency, fluorescence spectra, and chromatographic behavior of riboflavin. Binding resulted in substantial quenching of the fluorescence of both the isoalloxazine in riboflavin and of aromatic groups in the apoprotein. Kinetic analysis indicated a KD of 2.5 x 10(-7) M for riboflavin, 1.3 x 10(-7) M for lumiflavin, and greater than 1 x 10(-6) M for FMN and FAD. Over four moles of flavin were bound per mole of hexamerin. The amount of riboflavin in pupal hemolymph is sufficient to occupy only 2-3 of these sites. Riboflavin is also associated with lipophorin and vitellogenin, but the molar ratios after protein isolation were low. On a standard laboratory diet, riboflavin is in great excess, but most of it is apparently excreted before the apoprotein first appears in the hemolymph, just before wandering. The concentration of riboflavin-binding hexamerin rises to 15-30 mg/ml in pupae; relative to other hexamerins, very little is stored in the fat body. All of the apoprotein and 75% of riboflavin disappear from the hemolymph during adult development. An amount of flavin at least equal to that stored in pupal hemolymph is transferred to the eggs formed during this period.

Animals↗
PubMed2003

Dopaminergic regulation of crustacean hyperglycemic hormone and glucose levels in the hemolymph of the crayfish Procambarus clarkii.

The effects of dopamine on crustacean hyperglycemic hormone (CHH) release and hemolymph glucose levels in the crayfish Procambarus clarkii were investigated. A quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using antibodies specific for Prc CHH was developed and characterized. The sensitivity of the ELISA was about 1 fmol/well. Specific measurement of CHH in hemolymph samples by the ELISA was demonstrated by the parallelism between CHH standard curve and sample (hemolymph) titration curve. Moreover, thermally stressed P. clarkii exhibited a characteristic change of hemolymph CHH levels as revealed by the ELISA. CHH and glucose levels increased significantly within 30 min of dopamine injection, peaked at 1 h, and returned to the basal levels at 4 h. Dose-dependent effects of dopamine on CHH and glucose levels were observed between 10(-8) to 10(-6) mol/animal. Dopamine-induced increases in CHH and glucose levels were absent in eyestalk-ablated animals. Finally, dopamine significantly stimulated the release of CHH from in vitro incubated eyestalk ganglia. These results suggest that dopamine enhances release of CHH into hemolymph that in turn evokes hyperglycemic responses and that the predominant site of dopamine-induced CHH release is the X-organ-sinus gland complex located within the eyestalk.

Animals↗
PubMed2000

Silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis.

The effect of silkworm hemolymph on baculovirus-induced insect cell apoptosis was investigated. The addition of silkworm hemolymph into the culture medium either before or during the baculovirus infection increased the host cell longevity; however, its addition after the infection was less effective. This can be explained by the higher transfer rate of silkworm hemolymph which is caused by endocytosis during the virus internalization step. The delayed cell death due to silkworm hemolymph was not caused by an inhibition of the virus attachment and internalization steps. The apoptosis was analyzed using DNA fragmentation and TUNEL assays, and the resulting data confirm that silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis.

Animals↗
PubMed1996

Ovarian and hemolymph ecdysteroids in the terrestrial isopod Armadillidium vulgare (Malacostracan Crustacea).

Ovarian and hemolymph ecdysteroids in Armadillidium vulgare were analyzed at four stages of ovarian maturation through the reproductive molt cycle using high-performance liquid chromatography and radioimmunoassay. The major ecdysteroids in the ovaries and hemolymph of A. vulgare were 20-hydroxyecdysone and ecdysone in free and conjugated forms. The concentration of ovarian ecdysteroids reached a maximal level in maturing ovaries during stage D (premolt period) of the molt cycle. At the end of stage D, a high level of ecdysteroids was detected in fully matured ovaries. On the other hand, hemolymph ecdysteroid titers in reproductive females showed a peak during stage D and declined rapidly to a low level at the end of stage D. No appreciable differences in the amounts of hemolymph ecdysteroids and in molecular species of them were apparent in females in reproductive and nonreproductive molt cycles. The amounts of hemolymph ecdysteroids were about fivefold higher in females than those in males. These ecdysteroids may have a role in controlling ovarian development in female A. vulgare.

Animals↗
PubMed1992

Effect of age on acetylcholinesterase and other hemolymph proteins in Aplysia.

Hemolymph was examined in young (ca. 86 days old), mature (ca. 163 days old), and old (ca. 294 days old) Aplysia for age-related changes in constituent proteins. In young, mature, and old animals protein concentrations were 1.6 +/- 0.27, 1.41 +/- 0.53, and 1.45 +/- 0.43 mg.ml-1, respectively. The copper-containing respiratory protein, hemocyanin, measured by determining the copper concentration, was found to increase significantly from young (0.98 +/- 0.51 microgram.ml-1) to mature (2.02 +/- 0.95 micrograms.ml-1) Aplysia, with little change between mature and old (1.92 +/- 0.43 micrograms.ml-1) animals. These findings were consistent with the results obtained when hemocyanin was directly measured by spectrophotometric absorption at 340 nm. Acetylcholinesterase (AChE) was present in the hemolymph of Aplysia. Its activity was highest in mature animals (3121 +/- 1627 units.mg-1) and least in old animals (1463 +/- 599 units.mg-1). Young animals had intermediate levels (2080 +/- 762 units.mg-1). SDS-PAGE revealed a distinct pattern of protein bands for hemolymph from each age group; hemolymph from the young group contained six prominent protein bands with molecular weights (MW) from 13 to 300 kDa. Hemolymph of mature and old animals exhibited four and three prominent protein bands, respectively, with MW between 45 and 300 kDa. A prominent band at 97 kDa was present in samples from the mature group, but was faint in samples from the old group and absent in samples from the young group.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1988

Quantitative description of the development of Babesia ovis in Rhipicephalus bursa (hemolymph, ovary, eggs).

The development and infection dynamics of Babesia ovis in the hemolymph, ovaries, and eggs of Rhipicephalus bursa are described quantitatively, based mainly on examination of Giemsa-stained smears. After alimentary infection of female ticks, their hemolymph became infected 5 days after repletion (p.repl.). The prevalence and mean intensity of infection increased during the course of infection studied, up to 17 days p.repl. After vertical infection of female ticks, their hemolymph was infected only during the first 3 days after the onset of infestation (p. infest.) and again after the onset of alimentary infection 5 days p.repl. There was a positive correlation between prevalence and mean intensity of infection in the hemolymph. The prevalence of infection decreased with aging of the unfed adult ticks. After alimentary infection, the ovaries became infected 6 days p.repl., and after vertical infection, 3 days p. infest; they remained infected until the death of the tick. Ticks selected for susceptibility during 18 and 19 vertically infected generations were more susceptible than ticks in their first to third vertically infected generations or alimentarily infected ticks. Eggs deposited on day 1 of oviposition were noninfected after alimentary infection of the female tick. After vertical infection of the tick, even such eggs became infected; the infection, then, was detectable in eggs produced throughout the oviposition period regardless of the infection mode. Intense hemolymph infections induced an increase of egg degeneration and a decrease of total as well as infected egg production. There was a positive correlation between the number of deposited and infected eggs as well as between prevalence and mean intensity of infection in eggs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

A study of steady state and kinetic regulation of chloride ion and osmotic pressure in hemolymph of oysters, Crassostrea virginica, exposed to tri-n-butyltin.

Two sets of experiments were made to determine if bis(tri-n-butyl)tin oxide (TBT) [IUPAC: hexabutyldistannoxane] influenced osmotic pressure or chloride ion concentration in hemolymph of adult American oysters, Crassostrea virginica. In the first set, oysters were acclimated to 5, 25 or 40%;, then exposed to bis(tri-n-butyl)tin oxide dissolved in seawater (0.5, 1.0 or 2.0 micrograms/L) for 11 days. Measurements of the total osmotic pressure and chloride ion concentration in hemolymph indicated that mean values of neither was significantly affected under steady state conditions. Oysters exposed to TBT exhibited a higher variation around the mean value for both total osmotic pressure and chloride ion concentration. There was significant mortality of oysters exposed to TBT in 25 and 40, but not in 5%. In a second experiment, adult oysters were acclimated to 25%, and simultaneously exposed to the TBT concentrations listed above for 10 days. Then, subgroups of oysters were abruptly moved to 5 or 40% and the time-course of adjustment of hemolymph osmotic pressure and chloride ion concentration was measured. During the first 25 hr, there was little adjustment to 5% in controls or TBT exposed oysters; apparently, they remained closed most or all of the time. In marked contrast, hemolymph of oysters moved to higher salinities rapidly readjusted. Exposure to TBT had a delaying effect on the schedule of the increase in hemolymph osmotic pressure and chloride ion concentration. Results of these experiments show that TBT has an indirect effect on osmotic pressure and chloride ion concentration adjustment in oysters; it does not appear to act strongly as an anionophore to influence anion transfer across epithelia.

Animals↗
PubMed2003

Synthesis of new uracil-5-sulfonamide derivatives and immunostimulatory effect of a chemically modified hemolymph of Biomphalaria alexandrina on Schistosoma mansoni infected mice.

Some N-p-substituted phenyl uracil-5-sulphonamide derivatives have been synthesized to be evaluated as molluscicides against Biomphalanaria alexandrina snails, the intermediate host of Schistosoma mansoni. Schistosoma mansoni infected mice were treated with hemolymph obtained from pre-treated Biomphalana alexandrina snails with the products 4a, 10a, 10b and 4b or obtained from non-treated snails. The selection of the concentration based on the predetermined dose which caused mortality of less than 50% of snails/24 h. LC50 of compounds 4a, 10a, 10b and 4b was 50, 100, 200 and 50 ppm respectively. The result showed that immunostimulatory effect of treated hemolymph with compounds 4a, 10a and 4b was related to significant protective effects (44.4, 34.6 and 50.4% reduction in worm burden respectively). In addition, mean total worm burdens were significantly reduced in non treated hemolymph by 33.8%. The effect of hemolymph obtained from treated or non treated snails on S. mansoni adult worms antigens was studied by indirect immunofluorescence technique using chronic mouse sera (CMS). The results indicated that there was a strong reaction with epitopes in gut epithelium, tubercles, teigument and subtegumental musculature of untreated and treated S. mansoni adult worms antigens. Therefore, treatment of hemolymph obtained from pre-treated snails with compounds 4a, 10a, and 4b can stimulate specific immune response and induce protective effects against S. mansoni infection.

Adjuvants, Immunologic↗
PubMed1996

The effects of parasitism by Glyptapanteles liparidis (Braconidae: Hymenoptera) on the hemolymph and total body composition of gypsy moth larvae (Lymantria dispar, Lymantriidae: Lepldoptera).

The hemolymph and total body tissue composition (carbohydrates, lipids, proteins, and free amino acids) of gypsy moth larvae (Lymantria dispar, Lymantriidae:Lepidoptera) were analyzed and compared with the composition of larvae infected by their main parasitoid, Glyptapanteles liparidis (Braconidae:Hymenoptera). In the body tissue the concentrations of total lipids and total proteins decreased, whereas the glycogen concentration in the total body tissue was significantly elevated and exceeded the concentration of the nonparasitized larvae by a factor of approximately 3. Trehalose, glucose, and sorbitol were found in the hemolymph of both parasitized and nonparasitized larvae, but levels of all three carbohydrates were significantly reduced in parasitized individuals. The total lipid concentration was not altered due to parasitism. In the hemolymph the concentrations of total proteins nearly doubled in parasitized larvae, whereas the total free amino acid concentration was reduced; however, the levels of some single amino acids were reduced and those of others were elevated in the parasitized larvae. The polypeptide profile of the host hemolymph provided one parasitism-specific polypeptide at approximately 80 kDa. The results demonstrate that parasitization of gypsy moths by G. liparidis leads to significant alterations in levels of nearly all substance classes within the host's hemolymph and tissue.

Animals↗
PubMed2002

Why does the larval integument of some sawfly species disrupt so easily? The harmful hemolymph hypothesis.

The larvae of several sawfly species belonging to the Tenthredinidae (Hymenoptera) have such a low mechanical resistance in the integument that slight mechanical damage to the integument is enough to provoke the release of hemolymph at a given spot. We quantified this phenomenon, which we call "easy bleeding", by measuring the pressure needed to pierce dissected sawfly integument. We also investigated the feeding deterrance of ethanolic extracts of the hemolymph by laboratory bioassays using Myrmica rubra ant workers. These traits, integument resistance and hemolymph deterrence, were inversely related, considering 22 tenthredinid species. A negative correlation was obtained by only taking into account the species of one tenthredinid tribe, namely the Phymatocerini (nine species studied). Our results support the "harmful hemolymph hypothesis" that we present here and that assumes a functional link between these morphological and chemical traits, jointly acting as a chemical defense strategy. We suspect hemolymph deterrence to be often due to sequestration of plant secondary metabolites. We discuss the role of easy bleeding, considering the fact that sawfly larvae are frequently the prey of invertebrate and vertebrate predators. It is suggested that invertebrates such as ants were more important than vertebrates in the evolution of easy bleeding.

Animals↗
PubMed1992

Molecular mechanism of hemolymph clotting system in Limulus.

Limulus (horseshoe crab) hemolymph is known to be very sensitive to bacterial endotoxin (LPS), which causes a rapid coagulation response. Hemolymph contains a single type of hemocyte that undergoes aggregation, adhesion, and degranulation in response to LPS. The granule contents are released into the hemolymph, where they form an insoluble gel. We have characterized four components involved in this coagulation response that comprise a cascade of three serine protease zymogens (factor C, factor B, and proclotting enzyme) and one clottable protein (coagulogen). Of these components, factor C sensitive to LPS is a protein composed of five complement-related domains ("Sushi" or SCR), an EGF-like domain, and a C-type lectinlike domain as well as a putative amino-terminal LPS-binding domain. This domain structure is very similar to that of selectin family of cell adhesion molecules, suggesting that it might also function as a cell adhesion molecule after the release into the hemolymph. Factor B and the proclotting enzyme share a common Cys-rich motif ("cliplike" domain) in the amino-terminal portions. This domain is also found in a putative serine protease zymogen ("easter") in Drosophila, which is essential for normal embryonic development. All four of the components of the cascade and an antibacterial protein (anti-LPS factor) are localized to a specific type of the hemocyte granule. Another antibacterial peptide (tachyplesins I and II) is localized in a distinct granule population. The contents of both granule populations are released into the hemolymph in response to LPS, where they cooperate in immobilization and killing of Gram-negative bacteria.

Amino Acid Sequence↗
PubMed1990

Hemolymph lectin of the pearl oyster, Pinctada fucata martensii: a possible non-self recognition system.

To elucidate a mutual correlation between the hemolymph lectin and hemocytes of the pearl oyster, Pinctada fucata martensii, we searched for common epitopes and ligands. Neither the hemocyte plasma membrane nor cytoplasm was immunoreactive to anti-hemolymph lectin antibody. The hemolymph lectin strongly bound to D-galactose and N-acetyl-D-galactosamine, and the plasma membrane of both granulocytes and agranulocytes had affinity only for D-mannose and N-acetyl-D-glucosamine-binding plant lectins. In the gonad, the hemolymph lectin selectively adsorbed injected horse red blood cells (HRBC), and its hemagglutinating activity probably prevented them from dispersing. Pinctada sp. may possess system of recognition of non-self by the hemolymph lectin.

Animals↗
PubMed2006

Hemolymph osmolality and cation concentrations in Litopenaeus vannamei during exposure to artificial sea salt or a mixed-ion solution: relationship to potassium flux.

Interest in culturing the Pacific white shrimp Litopenaeus vannamei in low-salinity and brackish-well waters has led to questions about the ability of this species to osmo- and ionoregulate in environments containing low concentrations of ions and in environments with ionic ratios that differ from those found in sea water. After seven days, hemolymph osmolality and potassium, sodium and calcium values were all significantly affected by salinity (as artificial sea salt) with values decreasing with decreasing salinity. These decreases were small, however, relative to decreases in salinity, indicating iono- and osmoregulation with adjustment for gradients. The hemolymph osmolality and sodium and calcium concentrations in shrimp exposed to either 2 g/L artificial sea salt or 2 g/L mixed-ion solution (a mixture of sodium, potassium, calcium, and magnesium chlorides that approximate the concentrations and ratios of these cations found in 2 g/L dilute seawater) did not differ significantly. However, hemolymph potassium levels were significantly lower in shrimp held in the mixed-ion environment. Potassium influx rates were similar in shrimp held in either artificial sea salt or mixed ions. The results of this study indicate that salinity affects hemolymph-cation concentrations and osmolality. Further, differential potassium-influx rates do not appear to be the basis for low hemolymph potassium levels observed in shrimp held in mixed-ion environments.

Animals↗
PubMed2005

Differential gene expression by Metarhizium anisopliae growing in root exudate and host (Manduca sexta) cuticle or hemolymph reveals mechanisms of physiological adaptation.

Like many other fungal pathogens Metarhizium anisopliae is a facultative saprophyte with both soil-dwelling and insect pathogenic life-stages. In addition, as M. anisopliae traverses the cuticle and enters the hemolymph it must adapt to several different host environments. In this study, we used expressed sequence tags and cDNA microarray analyses to demonstrate that physiological adaptation by M. anisopliae to insect cuticle, insect hemolymph, bean root exudate (a model for life in the soil), and nutrient rich Sabouraud dextrose broth (SDB) involves different subsets of genes. Overall, expression patterns in cuticle and hemolymph clustered separately from expression patterns in root exudates and SDB, indicative of critical differences in transcriptional control during pathogenic and saprophytic growth. However, there were differences in gene expression between hemolymph and cuticle and these mostly involved perception mechanisms, carbon metabolism, proteolysis, cell surface properties, and synthesis of toxic metabolites. These differences suggest previously unsuspected stratagems of fungal pathogenicity that can be tested experimentally. Examples include the switch-off of cuticle-degrading proteases and a dramatic cell wall reorganization during growth in hemolymph.

Adaptation, Physiological↗
PubMed2005

The hemolymph proteome of Anopheles gambiae.

We used two-dimensional SDS-PAGE and microsequencing or peptide mass fingerprinting to identify major proteins in the hemolymph of Anopheles gambiae. We found approximately 280 protein spots in hemolymph and identified 28 spots, representing 26 individual proteins. Most of these proteins have known or predicted functions in immunity, iron transport, or lipid biology. Many of the proteins have been found in hemolymph in other insects but one protein is novel: a new member of the ML family (involved in lipid recognition). Three of the identified proteins increased in spot intensity or appeared de novo following bacterial injection: a phenoloxidase, and two chitinase-like proteins. A subset of proteins decreased following bacterial injections: these included the light and heavy chains of ferritin. Several proteins appeared in hemolymph following any wound or injection. Most of these are metabolic enzymes lacking signal peptides that are likely to be released as a result of damage to muscles and other tissues by injury. The map will provide a useful tool for examining changes in hemolymph proteins following blood feeding and infection by parasites.

Animals↗
PubMed2005

Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori.

Mulberry leaves are the sole diet of the silkworm, Bombyx mori. The host urease is incorporated into the larval hemolymph and involved in nitrogen metabolism in the insect. To investigate the selective absorption of the host urease to the larvae, crude urease was prepared from mulberry leaves and roots. Root urease was identical to leaf urease on the basis of electrophoretic analyses: (1) the urease activity appeared in the same migration position in a native gel; (2) There was no difference in molecular mass of the subunit. The root urease was orally injected to the fifth instar larvae of the silkworm. Just before spinning, the larvae absorbed intact urease from the midgut lumen to the hemolymph without the loss of activity. The capacity to absorb urease occurred only at the specific stage. Localization of host urease in midgut tissue was observed using confocal laser scanning microscopy and transmission electron microscopy. Based on spatial distribution of immunofluorescent signals and immunogold particles, host urease specifically attached to the surfaces of microvilli existing in the apical side of columnar cells and appeared in the cytoplasm of the cells for transport to the hemolymph. The incorporation efficiency of root urease into the hemolymph was significantly higher than for ureases from jack bean seeds and Bacillus pasteurii. The urease that was transported to the hemolymph was electrophoretically altered, compared with the host urease extracted.

Aging↗