Hemolymph Research Articles

Caterpillars have evolved lungs for hemocyte gas exchange

Since insect blood usually lacks oxygen-carrying pigments it has always been assumed that respiratory needs are met by diffusion in the gas-filled lumen of their tracheal systems. Outside air enters the tracheal system through segmentally arranged spiracles, diffuses along tubes of cuticle secreted by tracheal epithelia and then to tissues through tracheoles, thin walled cuticle tubes that penetrate between cells. The only recognized exceptions have been blood cells (hemocytes), which are not tracheated because they float in the hemolymph. In caterpillars, anoxia has an effect on the structure of the hemocytes and causes them to be released from tissues and to accumulate on thin walled tracheal tufts near the 8th (last) pair of abdominal spiracles. Residence in the tufts restores normal structure. Hemocytes also adhere to thin-walled tracheae in the tokus compartment at the tip of the abdomen. The specialized tracheal system of the 8th segment and tokus may therefore be a lung for hemocytes, a novel concept in insect physiology. Thus, although as a rule insect tracheae go to tissues, this work shows that hemocytes go to tracheae.

Allatostatin in hemocytes of the cockroach Diploptera punctata.

Allatostatins are neuropeptides that inhibit the production, by the corpora allata, of a major insect hormone, juvenile hormone. These peptides are produced by cells of the brain and ganglia as well as by midgut endocrine cells. Transport from these sites may contribute to the allatostatin content in the hemolymph (insect blood). Using a monoclonal antibody against Diploptera punctata allatostatin I (A-P-S-G-A-Q-R-L-Y-G-F-G-L-NH2) and in situ hybridization with a digoxigenin-labeled cRNA probe generated from a portion of the allatostatin gene, it is demonstrated that allatostatin is present in and synthesized by granular hemocytes of D. punctata. About 5% of the hemocytes react with anti-allatostatin antibody and a similar number hybridize with a cRNA probe that detects allatostatin-specific mRNA. Electron micrographs showed that allatostatin-immunoreactive material occurs in membrane-bound, uniformly dense granules that frequently fill fusiform-shaped cells. Allatostatin in cell and plasma fractions of hemolymph quantified by enzyme-linked immunosorbent assay and by bioassay for inhibition of juvenile hormone synthesis in vitro indicated that about equal quantities (0.1-0.2 fmol/microl) are present in cell and plasma fractions. The production of allatostatin by hemocytes suggests that allatostatins may function as regulatory peptides in hemolymph activities in addition to their other known functions.

Isolation and Identification of a Plasmatocyte-spreading Peptide from the Hemolymph of the Lepidopteran InsectPseudoplusia includens

Insect blood cells (hemocytes) play an essential role in defense against parasites and other pathogenic organisms that infect insects. A key class of hemocytes involved in insect cellular immunity is plasmatocytes. Here we describe the isolation and identification of a peptide from the moth Pseudoplusia includens that mediates the spreading of plasmatocytes to foreign surfaces. This peptide, designated plasmatocyte-spreading peptide (PSP1), contains 23 amino acid residues in the following sequence: H-ENFNGGCLAGYMRTADGRCKPTF-OH. In vitro assays using the synthetic peptide at concentrations >/=2 nM induced plasmatocytes from P. includens to spread on the surface of culture dishes. Injection of this peptide into P. includens larvae caused a transient depletion of plasmatocytes from circulation. Labeling studies indicated that this peptide induced 75% of plasmatocytes that were double-labeled by the monoclonal antibodies 49G3A3 and 43E9A8 to spread, whereas plasma induced significantly more plasmatocytes to spread. This suggests that only a certain subpopulation of plasmatocytes responds to the peptide and that other peptidyl factors mediate plasmatocyte adhesion responses.

Metabolism of Stored Reserves in Insect Fat Body: Hormonal Signal Transduction Implicated in Glycogen Mobilization and Biosynthesis of the Lipophorin System*

The mobilization of carbohydrate and lipid reserves from the insect fat body as fuels for migratory flight activity is controlled by adipokinetic hormone (AKH), of which in Locusta migratoria three different forms occur: AKH-I, -II and -III. In fat body in vitro, each AKH is capable of activating glycogen phosphorylase and of stimulating cAMP production, but only in the presence of extracellular Ca2+. The hormones stimulate both the influx and the efflux of Ca2+, the higher influx probably causing an increase in intracellular [Ca2+]. AKH enhances the production of inositol phosphates among which inositol 1,4,5-triphosphate may mediate the mobilization of Ca2+ from intracellular stores. Evidence is presented in favor of the occurrence of a capacitative calcium entry mechanism. Results suggest that transduction of the AKH signal occurs through stimulatory G protein-coupled receptor(s). A tentative model is presented for the interactions between the AKH signaling pathways in the locust fat body cell. AKH-induced lipid mobilization during flight requires the presence in the insect blood of high-density lipophorin (HDLp) particles and apolipophorin III (apoLp-III). Both protein components are synthesized in the fat body. In the locust, the two integral, nonexchangeable HDLp apolipophorins (apoLp-I and -II) were shown to originate from a common precursor; an mRNA of 10.3 kb seems to code for this precursor protein. The models proposed for lipophorin assembly and secretion in a number of insects are not in agreement. The exchangeable apoLp-III may occur in two or more isoforms; locust apoLp-III is secreted from the fat body as one of the two isoforms and in the hemolymph converted into the truncated second one. The rationale for this process is as yet unknown.

VITELLOGENESIS IN THE MUD CRAB SCYLLA SERRATA-AN IN VIVO ISOTOPE STUDY

Vitellogenesis in the mud crab Scylla serrata was investigated in vivo using the radiolabeled amino acid ['4C]phenylalanine to trace the synthetic site(s) of the major yolk protein lipovitellin. The incorporation of [14C]phenylalanine into the synthesized proteins of different tissues, such as ovary (OV), hepatopancreas (HP), subepidermal adipose tissue (SAT), and hemolymph (HL) of vitellogenic phases I and II, provided the first in vivo demonstration of possible extraovarian synthesis of lipovitellin precursor, vitellogenin, in S. serrata. The results indicated that among different tissues of vitellogenic phase I, SAT and HP actively take part in synthesizing the yolk proteins which are immediately released into the HL. This is evidenced by a high level of ['4C]phenylalanine incorporation into the protein fraction of the HL. The incorporation of ['4C]phenylalanine into the synthesized proteins of the OV indicates the fact that OV is also capable of synthesizing the precursors to some extent. However, a dramatic increase of ['4C]phenylalanine incorporation into the synthesized ovarian proteins of vitellogenic phase II, concurrent with a decline in the level of ['4C]phenylalanine incorporation into the HL proteins, suggests the probable sequestration of the synthesized proteins from an extraovarian site. The data also suggest a dual role for the HP, in-asmuch as it synthesizes and stores the vitellogenin prior to its release into the OV for sequestration during intense vitellogenesis.

Growth effects of Phaenicia sericata larval extracts on fibroblasts: mechanism for wound healing by maggot therapy.

The potential growth stimulating effects of the blow fly, Phaenicia sericata, on mammalian tissue were assessed by exposing human fibroblast tissue culture to maggot extracts. The growth effects of these extracts were compared to those of epidermal growth factor (EGF), recombinant interleukin 6 (IL6), and the insect hormone 20-hydroxyecdysone (EC). Results of dose-response experiments revealed that EGF had a maximum fibroblast stimulation at 66078 +/- 1979 counts per minute (cpm), with peak counts on day 6 of culture, as measured by [3H]-thymidine incorporation. P. sericata hemolymph (HL) and alimentary secretions (AS) and EC were also demonstrated to stimulate resting fibroblast tissue cultures, but the maximal stimulations only achieved 12% of EGF. Their growth rates plateaued between days 4 and 6. Addition of both HL and AS, as well as EC, significantly increased the growth rate of EGF-stimulated fibroblasts; AS increased the maximal stimulation of IL6-stimulated fibroblasts. These studies suggest the existence of intrinsic factors within the maggot which may be responsible for the growth-stimulating effects seen in maggot-infested wounds.

Expression of biologically active human granulocyte-macrophage colony-stimulating factor in the silkworm (Bombyx mori).

Using virus derived from Bombyx mori (silkworm) nuclear polyhedrosis virus (BmNPV), we constructed an infectious recombinant virus carrying the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) cDNA placed downstream from the polyhedrin promoter. The BmN cells infected with the recombinant baculovirus BmNPV-GM-CSF expressed recombinant (r)hGM-CSF up to 3.9 x 10(5) colony-formation units/ml in the medium. The maximum activity of rhGM-CSF was 4.3 x 10(6) colony-formation units/ml in the haemolymph of silkworm (Bombyx mori) larvae infected with BmNPV-GM-CSF. Three distinct species of GM-CSF of molecular masses 15, 18 and 20 kDa were detected by immunoblotting. The specific activity of rhGM-CSF purified from the haemolymph was up to 2.92 x 10(7) colony-formation units/mg. The isoelectric point of the purified rhGM-CSF was 5.5-5.7.

Vitellogenins and vitellins in the bean bug,Riptortus clavatus (hemiptera: alydidae): Purification, immunological identification, and induction by juvenile hormone

Vitellogenins (Vgs) and vitellins (Vns) were purified from reproductive female adult hemolymph (HL) and newly laid eggs of the bean bug, Riptortus clavatus. They were separated into two (Vg-1 and 2) and three (Vn-1′, Vn-1, and Vn-2) sub-components, respectively, by ion exchange chromatography. Fused rocket immunoelectrophoresis using anti-(Vn-1) serum showed that they are distinguished clearly into two immunologically distinct groups, i.e., Vg-1/Vn-1/Vn-1′ and Vg-2/Vn-2. SDS-PAGE analysis showed Vn-1 (Vn-1′) and Vn-2 have the same and/or smaller subunit component polypeptides as Vg-1 and Vg-2, respectively. Vn-1′ has some lower molecular weight peptides than Vn-1. Quantitative rocket immunoelectrophoretic analysis showed that Vg-1 and Vn-1 (plus V-1′) were detected first on day 3 in the HL and day 4 in the ovary, respectively, after adult emergence, and increased by day 10 in non-diapause female adults. Vg-1 synthesis is induced in diapause female adults in 1 day after the treatment with juvenile hormone (JH) and JH analog (JHA) or in a few days after transfer from short day to long day conditions. © 1996 Wiley-Liss, Inc.

Yeast-prepro-alpha-factor-leader-region-directed synthesis and secretion of truncated human macrophage colony-stimulating factor in the silkworm Bombyx mori.

Human macrophage colony-stimulating factor (hM-CSF) cDNA joined to the leader region of the precursor of the yeast mating pheromone alpha-factor (MF alpha L) was expressed at high levels in BmN cells and in silkworm (Bombyx mori) larvae, using recombinant Bombyx mori nuclear polyhedrosis virus, as a vector. The biological activity of rhM-CSF detected in the haemolymph was 1 x 10(6) colony-formation units/ml, approximately half of the expression level directed by the native signal peptide of hM-CSF in silkworm larvae. The secreted rhM-CSF was purified to homogeneity. N-terminal analysis showed that the signal peptide had been removed, indicating that insect cells possess the enzymic activity necessary to cleave the pro-alpha-factor leader region from the fusion protein at the carboxy side of Lys-Arg dibasic residues, which is the cleavage site recognized by KEX2 endopeptidase in yeast cells.

Hemolymph Profiles of Juvenoid Substances in the Giant Freshwater Prawn <i>Macrobrachium rosenbergii</i> in Relation to Reproduction and Molting

Hemolymph Profiles of Juvenoid Substances in the Giant Freshwater Prawn <i>Macrobrachium rosenbergii</i> in Relation to Reproduction and Molting

Changes in Haemolymph Ecdysteroid Levels and Cns Contents of Crustacean Cardioactive Peptide-Immunoreactivity During the Moult Cycle of the Isopod Oniscus Asellus

By use of newly developed enzyme immunoassays for ecdysteroids (ECD) and crustacean cardioactive peptide (CCAP) it was found (1) that in the isopod Oniscus asellus a peak occurs of hemolymph (HL)-ECD contents in prcmoult stages followed by a second but smaller peak 1 h after posterior ecdysis, and (2) that concomitantly with a drop in the first ECD-peak, CCAP-contcnts of the ventral nerve cord rise tenfold basal level around posterior ecdysis. This suggests that CCAP plays a role in isopod moulting processes.

Acid phosphatase activity in hemolymph of the migratory grasshopper, Melanoplus sanguinipes, during Beauveria bassiana infection

AbstractThe distribution and abundance of acid phosphatase (AP) in hemolymph (HL), plasma (PM) and hemocyte lysate supernatant (HLS) of the migratory grasshopper, Melanoplus sanguinipes infected with Beauveria bassiana (strain GK 2016) has been examined. AP activity was determined at intervals from 30 min to 60 h postinjection of 2 μl of 1 times 108conidia/ml per grasshopper. The enzyme was detected with the substrate β‐glycerophosphate in sodium acetate acetic acid buffer form hemocytes (HC) and with p‐nitrophenol phosphate sodium salt for HL, PM and HLS. In results of experiment 1 proportion of HC showing AP activity increased 1–2 h, then returned to normal after 4 h. However, in B. bassiana‐injected grasshoppers, a second increase was noted 24 h later which was not seen in the Tween‐80‐injected insects. Uninjected controls showed no change with time in the proportion of HC with AP activity.Studies were also made of the distribution of AP activity in the HL, PM, and HLS. AP activity in HL appeared to vary with the sex of the grasshoppers. Females showed increase in AP activity in HL 18–24 h after injection with B. bassiana, whereas males only showed an increase 1 h after injection. Assay of HLS showed that the level of AP activity did not change significantly throughout the experiment. Changes in AP activity in PM, in both B. bassiana – and Tween‐80‐injected insects (both sexes) paralleled those of the HL, indicating that the enzyme is released from the HC. The observations are discussed in terms of the possible role of AP in the immune response of M. sanguinipes.

沿岸甲殻類の血リンパ中の細菌相

沿岸甲殻類の血リンパ中の細菌相

Long-Term GrowthIn Vitroof Isolated, Fully Differentiated Neurones from the Central Nervous System of an Adult Insect

A method is described for the isolation and growth in vitro of fully differentiated neurones from the thoracic ganglia of adult cockroaches. The presence of insect blood in the culture system is shown to promote growth. The morphology of the growing neurones and the plasticity of the branching processes are described and growth rates are measured. Using a fluorescent Ca2+ indicator dye, changes of intracellular calcium levels in the growing neurones in response to K+ depolarization have been measured. The results, indicating the presence of voltage-dependent Ca2+ channels on neuronal processes in vitro, show that neurones can be maintained in a functional state for several weeks by this technique. Such preparations could prove useful for studying a variety of physiological and pharmacological properties of neurones, including the mechanisms controlling growth, synapse formation and neuronal interactions with other cell types.

Naturally occurring agglutinin in the hemolymph of Japanese coastal Crustacea.

Naturally occurring agglutinin in the hemolymph of Japanese coastal Crustacea.

Regional Variation in the Current Flow Across an Insect Blood–Brain Barrier

One notable characteristic of the insect and vertebrate central nervous system is the presence of a clearly defined blood-brain barrier. In the insect this barrier is made up of the perineurial glial cells, and shows a heterogeneity in structure and possibly function between the connectives and ganglia. In this paper we have used a two-dimensional vibrating probe to investigate the net flow of electrical current across the barrier at various locations in the abdominal nervous system. The results show clear differences between different areas. There is a strong and consistent outward current flow (3.16 microA cm-2) perpendicular to the ganglion surface over the equatorial plane. Current returns through the peripheral nerves and the connectives. A detailed study of the latter shows that the net inward flow is principally over the initial section, immediately adjacent to the ganglia (2.11 microA cm-2). The different current polarities can be correlated with structural differences in the underlying glial cells.

Trehalose, the insect blood sugar, inhibits loading of diacylglycerol by lipophorin from the fat body in locusts

Trehalose, the insect blood sugar, was found to inhibit diacylglycerol uptake by lipophorin from the fat body in vitro . Trehalose inhibited diacylglycerol uptake by about 40%–50% at various physiological concentrations. This suggests that trehalose may play a dual role in the hemolymph, i.e. serving as the insect's fuel and as a regulator in lipid transport.

Agglutinin mediated immune recognition in Locusta migratoria (Insecta)

The haemagglutinating activity in the haemolymph of Locusta migratoria fifth-instar larvae was examined using several vertebrate erythrocytes. No agglutination was observed using red blood cells from chicken, sheep, rat, guinea pig or human A, B, O. The highest titres were observed with rabbit erythrocytes (up to 2 12). When tested with one kind of red blood cell, the agglutinin titres were similar in the plasma and in the serum of locusts. The agglutination was heat labile (65°C for 30 min). Its titre showed a natural development in the course of the fifth larval instar. Inhibition studies with a range of carbohydrates revealed a high specificity of the agglutinin molecule for the d-galactosides with α 1–6 linkage. In vitro and in vivo experiments were conducted comparing the rabbit red blood cells which are strongly agglutinated, with the sheep red blood cells which are not. After injection, the clearance rate of the rabbit cells was much higher than that of the sheep cells. In vitro, the rabbit red blood cells readily attached to haemocytes and “rosette” formations were numerous whereas the sheep cells only weakly adhered to the insect blood cells and never exhibited “rosette” formations. The haemagglutinin demonstrated in the haemolymph of L. migratoria acts as opsonin for the particles which possess receptors for this molecule.

Characterization of the Molt Stages inPenaeus vannamei: Setogenesis and Hemolymph Levels of Total Protein, Ecdysteroids, and Glucose

The molting cycle of Penaeus vannamei ju veniles was characterized by distinct and predictable changes in the setae of pleopods. The molt pattern was diecdysic with a relatively short intermolt period (40%) and a long proecdysial period (>53%). The levels of both total protein and ecdysteroids increased in the hemo lymph during proecdysis, whereas the level of hemo lymph glucose was low at metecdysis and proecdysis and maximal during anecdysis. As revealed by SDS-PAGE, the relative concentrations of two polypeptides (32 kD; 175 kD) changed during the molting cycle.

Distribution of tetracycline in haemolymph and tissue of all stages of corn earworm ( Heliothis zea boddie)

1. 1. Insects reared on tetracycline containing diets had lower mortality, but required more time to reach the pupal stage. 2. 2. Tetracycline diffuses and dissipates readily into and out of insect blood and tissues. 3. 3. It reduces biosynthesis of high molecular weight proteins in blood. 4. 4. Apportionment of tetracycline in the pupal tissue is uneven.