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Hemolymph
Kinds of Hemolymph Selected AbstractsLysozyme as Pathogen-Recognition Protein in the Hemolymph of Galleria mellonellaENTOMOLOGICAL RESEARCH, Issue 3 2003In Hee LEE ABSTRACT Recognition of invading micro-organisms into hemolymph is a pivotal event for triggering diverse immune mechanisms in insects. It has been known that this recognition was mediated by the binding of hemolymph proteins to pattern-molecules on the cell surface of microbes. Recently, I found that the lysozyme in the G. mellonella hemolymph has binding affinity to cell-walls of Gram (-), (±) bacteria and fungus (Candida albicans). After the hemolymph was incubated with heat-killed microbes and treated with acidic buffer containing high concentration of NaCl, several plasma proteins detached from microbes were detected by reverse phase HPLC and SDS-PAGE analyses. Of binding proteins, it was assumed that the major one might be a lysozyme, which was previously characterized in the G. mellonella hemolymph. Furthermore immunoblot analysis performed with antiserum to G. mellonella lysozyme revealed that it was a lysozyme. [source] In Vivo Transfection of Adult Eastern Oysters Crassostrea virginicaJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 3 2001John T. Buchanan The eastern oyster Crassostrea virginica provides a commercially valuable industry along the eastern and Gulf coasts of the United States. Recently this industry has been damaged by disease problems, creating an interest in the use of gene transfer (transfection) to improve disease resistance. We transfected adult oysters with two genes, red-shifted green fluorescent protein (rsGFP), commonly used as a reporter gene, and the lytic peptide cecropin B (cepB), known to have antimicrobial properties. Oysters were transfected by injecting DNA mixed with SuperFectŌ reagent (Qiagen Inc.) into the adductor muscle sinus. Oysters were assigned to three groups of 15: the first was injected with rsGFP complexed with transfecting reagent: the second was injected with cepB complexed with transfectlng reagent; and the third was injected with saline (control group). Hemolymph was collected at 4 and 10 d after injection. DNA was extracted for analysis by polymerase chain reaction (PCR), and hemocytes were examined by flow cytometry and fluorescence microscopy for detection of green fluorescence due to rsGFP expression. The rsGFP gene was detected by PCR in hemocytes from 14 of 15 oysters at day 4, and in 15 of 15 oysters at day 10. The cepB gene was detected by PCR in 12 of 15 oysters at day 4 and in 14 of 15 oysters at day 10. No oysters from the control group were positive for either gene at days 4 or 10. Green fluorescence was detected by flow cytometry at significantly higher levels (P < 0.05) in oysters injected with rsGFP than in other oysters at day 4, but not at day 10. This report indicates the ability to introduce DNA into adult eastern oysters with subsequent gene expression. Future work will involve developing these techniques for enhanced disease resistance in oysters. [source] Juvenile hormone III produced in male accessory glands of the longhorned beetle, Apriona germari, is transferred to female ovaries during copulationARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2010Ling Tian Abstract We report on juvenile hormone (JH) biosynthesis in vitro by male accessory glands (MAGs) in the longhorned beetle, Aprionona germari, accompanied by the transfer of JH from males to females during copulation. JH was extracted from the MAGs and separated by reversed-phase high-performance liquid chromatography. JH III was identified as the major JH by gas chromatography,mass spectrometry. A radiochemical assay and a non-radioactive method were used to measure the in vitro rate of JH biosynthesis by the MAGs. After 4,h of incubation with 3H-methionine in the medium, the radioactivity in the MAGs substantially increased. In a separate assay, incubation of the MAGs with non-radioactive methionine for 4,h resulted in a 39% increase in JH III. Seven-day-old males were injected with medium 199 containing 3H,methionine and 24,h later they were mated with virgin females. Hemolymph and the MAGs were collected from the mated males and hemolymph, ovaries and eggs were collected from the mated females for assaying radioactive JH. The radioactivity incorporated into JH in the MAGs was transferred to the females during copulation and later transferred into their eggs. Assayed 1,h after copulation, JH III level in the MAGs decreased 42% and the content of JH III in the male hemolymph did not change, whereas the content of JH III in the female hemolymph and ovaries both increased. © 2010 Wiley Periodicals, Inc. [source] Inhibition of Human Cell Apoptosis by Silkworm HemolymphBIOTECHNOLOGY PROGRESS, Issue 4 2002Shin Sik Choi Many studies on preventing apoptosis have been carried out from the viewpoint of anti-apoptotic cloned-gene expressions inside cells, whereas in this study, we investigated the inhibition of apoptosis by the addition of silkworm hemolymph, a natural compound, from outside of the cells. In a previous study, we reported the inhibition effect of silkworm hemolymph on the baculovirus-induced insect cell apoptosis. Using the vaccinia virus-HeLa cell system as a model system in this study, we found that silkworm hemolymph, the insect serum, inhibits apoptosis not only in the insect cell system but also in the human cell system. The vaccinia virus-induced HeLa cell apoptosis was analyzed using DNA electrophoresis, TUNEL, and flow cytometry, and the resulting data confirmed that silkworm hemolymph inhibits human cell apoptosis. The inhibition of apoptosis due to silkworm hemolymph was not caused by an inhibition of virus binding and internalization steps, nor did silkworm hemolymph interfere with the virus production. The inhibition of apoptosis by silkworm hemolymph decreased the cell detachment from an adhering surface. With these characteristics, silkworm hemolymph can be effectively used to minimize cell death in commercial animal cell culture. [source] Pigment-dispersing factor in the locust abdominal ganglia may have roles as circulating neurohormone and central neuromodulatorDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2001Magnus G. S. Persson Abstract Pigment-dispersing factor (PDF) is a neuropeptide that has been indicated as a likely output signal from the circadian clock neurons in the brain of Drosophila. In addition to these brain neurons, there are PDF-immunoreactive (PDFI) neurons in the abdominal ganglia of Drosophila and other insects; the function of these neurons is not known. We have analyzed PDFI neurons in the abdominal ganglia of the locust Locusta migratoria. These PDFI neurons can first be detected at about 45% embryonic development and have an adult appearance at about 80%. In each of the abdominal ganglia (A3,A7) there is one pair of lateral PDFI neurons and in each of the A5,A7 ganglia there is additionally a pair of median neurons. The lateral neurons supply varicose branches to neurohemal areas of the lateral heart nerves and perisympathetic organs, whereas the median cells form processes in the terminal abdominal ganglion and supply terminals on the hindgut. Because PDF does not influence hindgut contractility, it is possible that also these median neurons release PDF into the circulation. Release from one or both the PDFI neuron types was confirmed by measurements of PDF-immunoreactivity in hemolymph by enzyme immunoassay. PDF applied to the terminal abdominal ganglion triggers firing of action potentials in motoneurons with axons in the genital nerves of males and the 8th ventral nerve of females. Because this action is blocked in calcium-free saline, it is likely that PDF acts via interneurons. Thus, PDF seems to have a modulatory role in central neuronal circuits of the terminal abdominal ganglion that control muscles of genital organs. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 19,41, 2001 [source] Purification and cDNA Cloning of Lysozyme II from Cabbage Butterfly, Artogeia rapae LarvaeENTOMOLOGICAL RESEARCH, Issue 4 2005BANG In Seok ABSTRACT Last instar larvae of cabbage butterfly Artogeia rapae respond to injection of bacteria with a set of inducible antibacterial peptides/proteins. The inducible peptides/proteins are related to the known hinnavins (I and II) and lysozymes (I and II). The lysozyme II has been isolated by heat treatment, cation exchange, and reversed-phase chromatography from immunized hemolymph of last instar larvae. The lysozyme II gene of A. rapae was isolated and its nucleotide sequence was determined by the RACE-PCR from immunized fat body with E. coli. It has an open reading frame of 414 bp nucleotide corresponding to 138 amino acids including an 18 amino acid signal sequence. The molecular weight and the isoelectric point of Artogeia lysozyme II without a signal peptide were 13,649.38 Da and 9.11, respectively. It is great similarity with Manduca lysozyme among other lepidopteran. [source] Purification and Characterization of Acid Phosphatase from the Egg of the Lady Beetle, Harmonia axyridis (Coccinellidae: Coleoptera)ENTOMOLOGICAL RESEARCH, Issue 1 2004Jun Hyuk LEE ABSTRACT Acid phosphatase (AP) in the egg of the lady beetle, Harmonia axyridis, was purified and characterized. Ammonium sulfate precipitation, CM column and isoelectrofocusing (IEF) were applied to purify an estimated molecular weight of 66 kDa AP. The purity was checked by SDS PAGE, native PAGE and Western blot. AP was detected in the hemolymph of the female and the egg, but not in the male on the blotting. Km of AP for a substrate, p -nitrophenyl phosphate (p -NPP), was 1.64 x 10 -4 M. AP had the optimum enzymatic activity at pH 3.5. In inhibition tests performed with various chemicals, ammonium molybdate suppressed 99% of the enzyme activity of AP even at the concentration of 5 x 10 -4 mM. AP was stable up to 50°C. [source] Postendocytic Provitellin Processing in the Growing Oocyte of the Short Horned Grasshopper, Oxyajaponicajaponica (Orthoptera: Acrididae)ENTOMOLOGICAL RESEARCH, Issue 1 2004Sae Youll CHO ABSTRACT Polyclonal antibodies made against 86 kDa (86 k), 80 kDa (80 k) and 54 kDa (54 k) vitellins of Oxya japonica japonica are used for Western blotting. Anti-80k vitellin antibody is cross-reacted with a 95 kDa (95 k) vitellin. While 95 k vitellin is present both in the female hemolymph and in the oocyte, 80 k vitellin is detected only in the oocyte and the laid egg. In the growing oocytes, as 95 k vitellin is faded out gradually, 80 k vitellin is accumulated increasingly, indicating postendocytic processing of 95 k vitellin brings 80 k vitellin. Further conforming the hypothesis, partial digestion of 95 k vitellin with pepsin and ,-chymotrypsin makes several protein bands of molecular weight around 80 kDa. Thus, the 95k vitellin may have a cleavage site (s) to produce 80 k vitellin which forms fairly stable tertiary structure. In the reduced condition (20 mM glutathion), both 95 k and 80 k vitellins were digested throughly by endogenous proteinase at pH 4. Both 86 k and 54 k vitellins, respectively, show no apparent molecular weight changes in the growing oocyte and in the hemolymph. [source] Lysozyme as Pathogen-Recognition Protein in the Hemolymph of Galleria mellonellaENTOMOLOGICAL RESEARCH, Issue 3 2003In Hee LEE ABSTRACT Recognition of invading micro-organisms into hemolymph is a pivotal event for triggering diverse immune mechanisms in insects. It has been known that this recognition was mediated by the binding of hemolymph proteins to pattern-molecules on the cell surface of microbes. Recently, I found that the lysozyme in the G. mellonella hemolymph has binding affinity to cell-walls of Gram (-), (±) bacteria and fungus (Candida albicans). After the hemolymph was incubated with heat-killed microbes and treated with acidic buffer containing high concentration of NaCl, several plasma proteins detached from microbes were detected by reverse phase HPLC and SDS-PAGE analyses. Of binding proteins, it was assumed that the major one might be a lysozyme, which was previously characterized in the G. mellonella hemolymph. Furthermore immunoblot analysis performed with antiserum to G. mellonella lysozyme revealed that it was a lysozyme. [source] Immunocompetence of bivalve hemocytes as evaluated by a miniaturized phagocytosis assayENVIRONMENTAL TOXICOLOGY, Issue 3 2002C. Blaise Abstract Immune function in bivalves can be adversely affected by long-term exposure to environmental contaminants. Investigating alterations in immunity can therefore yield relevant information about the relationship between exposure to environmental contaminants and susceptibility to infectious diseases. We have developed a rapid, cost-effective, and miniaturized immunocompetence assay to evaluate the phagocytic activity, viability, and concentration of hemocytes in freshwater and marine bivalves. Preliminary experiments were performed to optimize various aspects of the assay including 1) the time required for adherence of hemocytes to polystyrene microplate wells, 2) the time required for internalization of fluorescent bacteria, 3) the ratio of hemocytes to fluorescent bacteria in relation to phagocytosis, 4) hemolymph plasma requirements, and 5) the elimination of fluorescence from (noninternalized) bacteria adhering to the external surface of hemocytes. The results of these experiments showed the optimal adherence time for hemocytes in microplate wells to be 1 h, that phagocytosis required at least 2 h of contact with fluorescently labeled E. coli cells, that the number of fluorescent E. coli cells had a positive effect on phagocytic activity, that at least 2.5 million cells/mL were required to measure a significant intake, and that a linear increase in uptake of bacteria (R = 0.91; p < 0.01) could be obtained with concentrations of up to 1.3 × 106 hemocytes/mL. Afterward, the assay was used in two field studies to identify sites having the potential to affect the immunocompetence of bivalves. The first study was conducted on Mya arenaria clams collected at selected contaminated sites in the Saguenay River (Quebec, Canada), and the second examined Elliptio complanata freshwater bivalves that had been exposed to a municipal effluent plume in the St. Lawrence River (Quebec, Canada). In the Saguenay River field study a significant increase in phagocytosis was observed at sites closest to polluted areas. Phagocytotic activity varied over time and was highest during the warmest months (June, July, and August), closely paralleling the spawning period of Mya arenaria clams. In contrast, a drop in phagocytic activity was observed in Elliptio complanata mussels exposed to surface water 4 km downstream of a major municipal effluent plume, with a concomitant increase in the number of hemocytes in the hemolymph. It appears that both immunosuppressive and immunostimulative effects are likely to occur in the field and that responses will be influenced by the type and intensity of contaminants at play. © 2002 Wiley Periodicals, Inc. Environ Toxicol 17: 160,169, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.10047 [source] Molluscan shellfish biomarker study of the Quebec, Canada, Saguenay Fjord with the soft-shell clam, Mya arenariaENVIRONMENTAL TOXICOLOGY, Issue 3 2002C. Blaise Abstract A spatial and temporal survey of six sites in the Saguenay Fjord and of one adjacent site in the St. Lawrence River estuary (Quebec, Canada) was undertaken to study the possible effects of anthropogenic contaminant input on soft-shell clam (Mya arenaria) populations. Bivalve sampling sites were selected because they reflected a range of areas representative of either no known (or apparent) pollution sources or of areas potentially influenced by different gradients and types of contamination sources. The most upstream site selected in the Saguenay Fjord, nearest to a highly populated and industrialized sector, and the most downstream site, near its mouth with the St. Lawrence River estuary, spanned a distance of some 70 km and encompassed the entire intertidal area suitable for Mya arenaria habitat. To measure effects in collected animals, we used a comprehensive battery of biomarkers composed of metallothionein-like proteins (MT), 7-ethoxyresorufin O-deethylase activity (EROD), DNA damage (DD), lipid peroxidation (LPO), vitellinlike proteins (Vn), phagocytosis (PHAG), nonspecific esterase (NspE) activity, and condition factor (weight-to-length ratio of clams). Vn, PHAG, DD, and NspE biomarkers were assayed in hemolymph (or hemocytes), whereas others (MT, EROD, LPO) were determined in the digestive gland. Whole-tissue metal content was also quantified in clams collected in the spatial survey. The spatial survey conducted in June 1997 showed significant effects at all sites, and principal component analysis indicated in addition that the more important responses were linked to the MT, LPO, and NspE biomarkers. Clams collected from sites closest to the upstream reaches of the fjord generally displayed higher levels of tissue metals (cadmium, manganese), as well as greater responses of NspE activity, MT, LPO, and PHAG. Animals collected from sites influenced by municipal wastewaters had higher levels of Vn, suggesting the presence of environmental estrogens. The results of the temporal survey (six monthly samplings of clams at three sites from May through October, 1997) showed that the bivalve reproductive cycle (vitellogenesis and spawning) can modulate the expression of several biomarkers. Vn levels, for example, were positively correlated with DD and EROD and negatively correlated with MT, suggesting that reproduction can influence the susceptibility of clams to some contaminants. Discrimination analysis over the 6 months of sampling revealed that the mean value of the discriminant function changed significantly over time, suggesting important changes in the relative contribution of each biomarker. In short, this study has provided evidence that clam populations in the Saguenay Fjord are impacted by multiple sources of contamination whose effects can be modulated by reproduction. © 2002 Wiley Periodicals, Inc. Environ Toxicol 17: 170,186, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.10048 [source] NUPTIAL GIFTS AND THE EVOLUTION OF MALE BODY SIZEEVOLUTION, Issue 3 2002Kenneth M. Fedorka Abstract In many insect systems, males donate nuptial gifts to insure an effective copulation or as a form of paternal investment. However, if gift magnitude is both body size-limited and positively related to fitness, then the opportunity exists for the gift to promote the evolution of large male size. In the striped ground cricket, Allonemobius socius, males transfer a body size-limited, somatic nuptial gift that is comprised primarily of hemolymph. To address the implications of this gift on male size evolution, we quantified the intensity and direction of natural (fecundity) and sexual (mating success) selection over multiple generations. We found that male size was under strong positive sexual selection throughout the breeding season. This pattern of selection was similar in successive generations spanning multiple years. Male size was also under strong natural selection, with the largest males siring the most offspring. However, multivariate selection gradients indicated that gift size, and not male size, was the best predictor of female fecundity. In other words, direct fecundity selection for larger gifts placed indirect positive selection on male body size, supporting the hypothesis that nuptial gifts can influence the evolution of male body size in this system. Although female size was also under strong selection due to a size related fecundity advantage, it did not exceed selection on male size. The implications of these results with regard to the maintenance of the female-biased size dimorphic system are discussed. [source] Increased bacterial load in shrimp hemolymph in the absence of prophenoloxidaseFEBS JOURNAL, Issue 18 2009Fernand F. Fagutao Invertebrates rely on their innate immune responses to protect themselves from pathogens, one of which is melanization of bacteria mediated by the activation of phenoloxidase (PO). Furthermore, invertebrate hemolymph, even that of healthy individuals, has been shown to contain bacterial species. The mechanisms that prevent these bacteria from proliferating and becoming deleterious to the host are, however, poorly understood. Here, we show that knocking down the activity of the inactive precursor of PO [prophenoloxidase (proPO)] by RNA interference resulted in a significant increase in the bacterial load of kuruma shrimp, Marsupenaeus japonicus, even in the absence of a bacterial or viral challenge. Silencing of proPO also led to a sharp increase in shrimp mortality. In addition, the hemolymph of proPO-depleted shrimp had significantly lower hemocyte counts and PO activity than control samples. Microarray analysis after proPO silencing also showed a decrease in the expression of a few antimicrobial peptides, but no effect on the expression of the genes involved in the clotting system. Treatment with antibiotics prior to and after proPO dsRNA injection, to counteract the loss of proPO, resulted in a significant increase in shrimp survival. Our results therefore show that the absence of proPO renders the shrimp incapable of controlling bacteria present in the hemolymph, and that proPO is therefore essential for its survival. [source] An ecdysteroid-inducible insulin-like growth factor-like peptide regulates adult development of the silkmoth Bombyx moriFEBS JOURNAL, Issue 5 2009Naoki Okamoto Insulin-like growth factors (IGFs) play essential roles in fetal and postnatal growth and development of mammals. They are secreted by a wide variety of tissues, with the liver being the major source of circulating IGFs, and regulate cell growth, differentiation and survival. IGFs share some biological activities with insulin but are secreted in distinct physiological and developmental contexts, having specific functions. Although recent analyses of invertebrate genomes have revealed the presence of multiple insulin family peptide genes in each genome, little is known about functional diversification of the gene products. Here we show that a novel insulin family peptide of the silkmoth Bombyx mori, which was purified and sequenced from the hemolymph, is more like IGFs than like insulin, in contrast to bombyxins, which are previously identified insulin-like peptides in B. mori. Expression analysis reveals that this IGF-like peptide is predominantly produced by the fat body, a functional equivalent of the vertebrate liver and adipocytes, and is massively released during pupa,adult development. Studies using in vitro tissue culture systems show that secretion of the peptide is stimulated by ecdysteroid and that the secreted peptide promotes the growth of adult-specific tissues. These observations suggest that this peptide is a Bombyx counterpart of vertebrate IGFs and that functionally IGF-like peptides may be more ubiquitous in the animal kingdom than previously thought. Our results also suggest that the known effects of ecdysteroid on insect adult development may be in part mediated by IGF-like peptides. [source] Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonellaFEBS JOURNAL, Issue 7 2000Andreas C. Fröbius Three inducible serine protease inhibitors (ISPI-1, 2, 3) have been purified from larval hemolymph of greater wax moth larvae, Galleria mellonella, and characterized at a molecular level. These inhibitors were synthesized after larvae were injected with a yeast polysaccharide, zymosan preparation. ISPI-1,2,3 were active against various serine proteases including trypsin and toxic proteases released by the entomopathogenic fungus Metarhizium anisopliae. Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitors from cell-free hemolymph samples. The molecular masses of purified proteins were determined by MS to be 9.2 kDa (ISPI-1), 6.3 kDa (ISPI-2) and 8.2 kDa (ISPI-3) with isoelectric points ranging between 7.2 and 8.3. The N-terminal amino-acid sequences of ISPI-1 and ISPI-3 are not similar to other known proteins, whereas that of ISPI-2 exhibits extensive similarity to known Kunitz-type protease inhibitors. [source] Physiological functions of hemocytes newly emerged from the cultured hematopoietic organs in the silkworm, Bombyx moriINSECT SCIENCE, Issue 1 2010Cheng-Long Wang Abstract, Cellular immunity is a very important part of insect innate immunity. It is not clear if hemocytes entering the hemolymph require a maturation process to become competent. The establishment of a tissue culture system for the insect hematopoietic organs would enable physiological function assays with hemocytes newly emerged from hematopoietic organs. To this end, we established a hematopoietic organ culture system for the purebred silkworm pnd pS and then studied the physiological functions of the newly emerged hemocytes. We found that Grace's medium supplemented with 10% heated silkworm larval plasma was better for culturing the hematopoietic organs of pnd pS. Newly emerged hemocytes phagocytosed propidium iodide-labeled bacteria and encapsulated the Iml-2 coated nickel beads as well as pupal tissue debris. This culture system is therefore capable of generating physiologically functional hemocytes. These hemocytes can be used to study the mechanisms of the hemocyte immune response among others. [source] ANALYSIS OF THE SOLUBLE PROTEINS IN THREE SPECIES OF PARASITOIDS AND MOLECULAR CHARACTERISTICS OF YOLK PROTEIN IN PTEROMALUS PUPARUM,INSECT SCIENCE, Issue 4 2001SUN Meng Abstract The results both from PAGE and capillary electrophoresis indicated that there was a female specific protein i.e. vitellogenin (Vg) or vitellin (Vt) in the female wasp of Pteromalus puparum (Hymenoptera: Pteromalidae). While there was no difference in the electrophoresis graph between soluble proteins of the female whole body and those of the male one both in two bracoids (Hymenoptera: Braconidae), i.e. Cotesia plutellae and Macrocentrus linears. According to the graph of the gradient SDS-PAGE, it was clear that the Vg or Vt of P. puparum consisted of two subunits with approximate molecular weights, and their molecular weights were 74.4 and 52.8 KDa, respectively. Both immunological reactions between some main different tissues of the female wasps and the male whole body and the polyantibody against the Vt of this parasitoid, and the graph of the gradient SDS-PAGE including soluble proteins sampled separately from hemolymph, fat body and ovary of the female and the whole body of the male demonstrated that Vg existed both in female fat body and hemolymph, and Vt deposited in the ovary, not in the male, as well as the Vg was synthesized in the female fat body. [source] Spatial organization and isotubulin composition of microtubules in epidermal tendon cells of Artemia franciscanaJOURNAL OF MORPHOLOGY, Issue 2 2005Godelieve R.J. Criel Abstract Epidermally derived tendon cells attach the exoskeleton (cuticle) of the Branchiopod crustacean, Artemia franciscana, to underlying muscle in the hindgut, while the structurally similar transalar tendon (epithelial) cells, which also arise from the epidermis and are polarized, connect dorsal and ventral exopodite surfaces. To establish these latter attachments the transalar tendon cells interact with cuticles on opposite sides of the exopodite by way of their apical surfaces and with one another via basal regions, or the cuticle attachments may be mediated through linkages with phagocytic storage cells found in the hemolymph. In some cases, phyllopod tendon cells attach directly to muscle cells. Tendon cells in the hindgut of Artemia possess microtubule bundles, as do the transalar cells, and they extend from the basal myotendinal junction to the apical domain located near the cuticle. The bundled microtubules intermingle with thin filaments reminiscent of microfilaments, but intermediate filament-like structures are absent. Microtubule bundles converging at apical cell surfaces contact structures termed apical invaginations, composed of cytoplasmic membrane infoldings associated with electron-dense material. Intracuticular rods protrude from apical invaginations, either into the cuticle during intermolt or the molting fluid in premolt. Confocal microscopy of immunofluorescently stained samples revealed tyrosinated, detyrosinated, and acetylated tubulins, the first time posttranslationally modified isoforms of this protein have been demonstrated in crustacean tendon cells. Microfilaments, as shown by staining with phalloidin, coincided spatially with microtubule bundles. Artemia tendon cells clearly represent an interesting system for study of cytoskeleton organization within the context of cytoplasmic polarity and the results in this article indicate functional cooperation of microtubules and microfilaments. These cytoskeletal elements, either acting independently or in concert, may transmit tension from muscle to cuticle in the hindgut and resist compression when connecting exopodite cuticular surfaces. © 2004 Wiley-Liss, Inc. [source] Effects of Salinity on Biogenic Amines, Hemolymph Osmotic Pressure, and Activity of Gill's Na+/K+ -ATPase in Charybdis japonica (Crustacea, Decapoda)JOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 6 2008Liu Hong-Yu The effects of salinity on hemolymph osmotic pressure, gill Na+/K+ -ATPase activity and dopamine (DA), norepinephrine (NE), serotonin (5-HT) in the gills, and hemolymph of the adult Charybdis japonica were studied. DA levels increased significantly (P < 0.05), while the NE and 5-HT revealed contrary change in hemolymph and gills. The iso-osmotic point of C. japonica (911.4 mOsm/kg) was at salinity of 27.87 ppt. The Na+/K+ -ATPase activity of gill showed negative correlation with salinity in the hypotonic environment (<27.87 ppt). The results of this experiment indicated that C. japonica had great capability to acclimate to low salinity. [source] Nitrite Toxicity to Litopenaeus vannamei in Water Containing Low Concentrations of Sea Salt or Mixed SaltsJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 4 2004Anthony Sowers The uptake, depuration and toxicity of environmental nitrite was characterized in Litopenaeus vannamei exposed in water containing low concentrations of artificial sea salt or mixed salts. In 2 g/L artificial sea salts, nitrite was concentrated in the hemolymph in a dose-dependent and rapid manner (steady-state in about 2 d). When exposed to nitrite in 2 g/L artificial sea salts for 4 d and then moved to a similar environment without added nitrite, complete depuration occurred within a day. Increasing salinity up to 10 g/L decreased uptake of environmental nitrite. Nitrite uptake in environments containing 2 g/L mixed salts (combination of sodium, potassium, calcium and magnesium chlorides) was similar to or lower than rates in 2 g/L artificial sea salt. Toxicity was inversely related to total dissolved salt and chloride concentrations and was highest in 2 g/L artificial sea salt (96-h medial lethal concentration = 8.4 mg/L nitrite-N). Animals that molted during the experiments did not appear to be more susceptible to nitrite than animals that did not molt. The shallow slope of the curve describing the relationship between toxicity and salinity suggests that management of nitrite toxicity in low-salinity shrimp ponds by addition of more salts may not be practical. [source] A Profound Effect of Hyperthermia on Survival of Litopenaeus vannamei Juveniles Infected with White Spot Syndrome VirusJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 4 2001Oscar M. Vidal This study was conducted to examine the effect of increasing seawater temperature on White Spot Syndrome Virus (WSSV) infection in juvenile Pacific White shrimp (Litopenaeus vannamei). Infection by WSSV was achieved using two methods, intramuscular injection and per os (oral) administration. Forty injected and 20 per os infected animals were kept in heated tanks at 32.3 ± 0.8 C, and the same number of WSSV infected animals were maintained in tanks at ambient temperature (25.8 ± 0.7 C). Despite the route of exposure, there were no survivors among the animals kept at ambient temperature; whereas, in heated tanks the survival of the WSSV infected juvenile shrimp was always above 80%, suggesting the existence of a beneficial effect from hyperthermia that mitigated the progression of WSSV disease. Moreover, this beneficial effect was not attributable to viral inactivation. Infected animals kept at 32 C had histologically detectable lymphoid organ spheroids suggestive of a chronic viral infection but were PCR negative (hemolymph) for WSSV. These findings might be related to low viral replication in WSSV-infected shrimp held at the higher environmental temperature. When the WSSV-infected shrimp were transferred from 32 C to ambient temperature, the mortality from WSSV ensued and was always 100%. Although the mechanism related to the beneficial effect of heating was not determined, our results indicate that increasing the water temperature modifies dramatically the natural history of the WSSV disease and the survival curves of WSSV-infected juvenile Pacific White shrimp. [source] Development of the Insect Pathogenic Alga HelicosporidiumTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 3 2006VERENA-ULRIKE BLÄSKE-LIETZE ABSTRACT. This study examined the morphogenesis and replication dynamics of the different life stages (cysts, filamentous cells, vegetative cells) of Helicosporidium sp., a non-photosynthetic, entomopathogenic alga. The isolate (SjHe) used originated from an infected black fly larva. Filamentous cell transformation into vegetative cells and autosporulation during vegetative cell replication were observed under controlled in vitro conditions. The transformation process was initiated by a partial swelling of the filamentous cell along with the reorganization of the nuclear material. Two subsequent nuclear and cell divisions resulted in the release of 4 rod-shaped daughter cells, which divided into oval to spherical vegetative cells. These underwent several cycles of autosporogenic cell division. Multiple-passaged vegetative cell cultures formed non-motile, adherent cell clusters (palmelloid colonies). Vegetative replication dynamics were also observed in 2 experimental noctuid hosts, Spodoptera exigua and Helicoverpa zea. The average density of helicosporidial cells produced per microliter hemolymph exceeded cell concentrations obtained in vitro by 15- and 46-fold in S. exigua and H. zea, respectively. Cyst morphogenesis was only observed in the hemolymph, whereas no cysts differentiated at various in vitro conditions. [source] Juvenile hormone III produced in male accessory glands of the longhorned beetle, Apriona germari, is transferred to female ovaries during copulationARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2010Ling Tian Abstract We report on juvenile hormone (JH) biosynthesis in vitro by male accessory glands (MAGs) in the longhorned beetle, Aprionona germari, accompanied by the transfer of JH from males to females during copulation. JH was extracted from the MAGs and separated by reversed-phase high-performance liquid chromatography. JH III was identified as the major JH by gas chromatography,mass spectrometry. A radiochemical assay and a non-radioactive method were used to measure the in vitro rate of JH biosynthesis by the MAGs. After 4,h of incubation with 3H-methionine in the medium, the radioactivity in the MAGs substantially increased. In a separate assay, incubation of the MAGs with non-radioactive methionine for 4,h resulted in a 39% increase in JH III. Seven-day-old males were injected with medium 199 containing 3H,methionine and 24,h later they were mated with virgin females. Hemolymph and the MAGs were collected from the mated males and hemolymph, ovaries and eggs were collected from the mated females for assaying radioactive JH. The radioactivity incorporated into JH in the MAGs was transferred to the females during copulation and later transferred into their eggs. Assayed 1,h after copulation, JH III level in the MAGs decreased 42% and the content of JH III in the male hemolymph did not change, whereas the content of JH III in the female hemolymph and ovaries both increased. © 2010 Wiley Periodicals, Inc. [source] Factors affecting proliferation and differentiation of lepidopteran midgut stem cells,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2010Marcia J. Loeb Midgut stem cells of last instar larvae and pupae of Heliothis virescens, Lymantria dispar and several other Lepidopteran species have been cultured in vitro and have been induced to proliferate using low titers of ecdysteroids and the 77-Kda peptide fragment, ,-arylphorin, isolated and identified from pupal fat body tissue. The insulin-related hormone, Bombyxin, also induced mitosis in cultured midgut stem cells; it appeared to be fast-acting and quickly inactivated, while ,-arylphorin was slower to act and had a longer lasting effect in vitro, indicating different functions for these proliferation agents. Changes in Calcium ion concentration within or outside the cells discretely affected stem cell differentiation, indicating a role for second messenger participation in peptide regulation of this process. Four different peptides (MDFs 1,4) that induced midgut stem cells to differentiate to mature midgut cell types in vitro were isolated and characterized from conditioned media and hemolymph of H. virescens and L. dispar. However, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and all-trans retinoic acid (RA) from vertebrate sources induced differentiation to non-midgut cell types as well. MDF1 was located in basal areas of columnar cells of midgut epithelium, although MDF2 was observed in all of the cytoplasm of columnar cells and in droplets of antibody positive material in the midgut lumen, suggesting a digestive function as well for this peptide. Anti-MDF-3 stained the central areas of cultured midgut columnar cells and the bases of columnar cells of midgut epithelium in vivo. Midgut secretory cells stained with anti-MDF-4; streams of MFD-4-positive material were observed extending from secretory cells facing the epithelial lumen, and as a layer on the hemolymph-facing side, suggesting an endocrine or paracrine function for this or an immunologically similar peptide. Published 2010 Wiley Periodicals, Inc. [source] Plant-insect interactions: what can we learn from plant lectins?ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2010Katrien Michiels Abstract Many plant lectins have high anti-insect potential. Although the effects of most lectins are only moderately influencing development or population growth of the insect, some lectins have strong insecticidal properties. In addition, some studies report a deterrent activity towards feeding and oviposition behavior. Transmission of plant lectins to the next trophic level has been investigated for several tritrophic interactions. Effects of lectins with different sugar specificities can vary substantially with the insect species under investigation and with the experimental setup. Lectin binding in the insect is an essential step in exerting a toxic effect. Attempts have been made to study the interactions of lectins in several insect tissues and to identify lectin-binding receptors. Ingested lectins generally bind to parts of the insect gut. Furthermore, some lectins such as the Galanthus nivalus agglutinin (GNA) cross the gut epithelium into the hemolymph and other tissues. Recently, several candidate lectin-binding receptors have been isolated from midgut extracts. To date little is known about the exact mechanism for insecticidal activity of plant lectins. However, insect glycobiology is an emerging research field and the recent technological advances in the analysis of lectin carbohydrate specificities and insect glycobiology will certainly lead to new insights in the interactions between plant lectins and insects, and to a better understanding of the molecular mechanisms involved. © 2010 Wiley Periodicals, Inc. [source] Hemocytes of the cochineal insect: ultrastructureARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2010Sandra Caselķn-Castro Abstract Using transmission electron microscopy, light microscopy (Giemsa May-Grumwald), and the Periodic Acid-Schif (PAS) and Sudan Black B staining techniques, hemocytes in the hemolymph of adult female Dactylopius coccus were characterized. The following, in order of abundance, were found: granulocytes, plasmatocytes, prohemocytes, and oenocytoids. Granulocytes varied in size with granulations in the cytoplasm, a large quantity of mitochondria, rugose endoplasmatic reticulum, ribosomes and vesicles, central or exocentric, spherical and occasionally lobulate nucleus. Plasmatocytes were polymorphic with irregularities in the plasma membrane; cytoplasm contained mitochondria, rugose endoplasmatic reticulum and vesicles, and exocentric, spherical, or irregular nucleus. In both types of hemocytes, scant polysaccharides and lipids were found. Prohemocytes were small and spherical with homogeneous cytoplasm and large exocentric nuclei. Oenocytoids were oval or irregular with dense homogeneous cytoplasm and elongated exocentric nuclei. The percentages of granulocytes on different days (d 1 and 10) during the life of the adult female were significantly different, as were those of plasmatocytes on d 30 and 50 and prohemocytes on d 1 and 50. © 2010 Wiley Periodicals, Inc. [source] Hemolymph ecdysteroids during the last three molt cycles of the blue crab, Callinectes sapidus: quantitative and qualitative analyses and regulationARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2010J. Sook Chung Abstract The profiles of circulating ecdysteroids during the three molt cycles prior to adulthood were monitored from the juvenile blue crab, Callinectes sapidus. Ecdysteroid patterns are remarkably similar in terms of peak concentrations ranging between 210,330,ng/ml hemolymph. Analysis of hemolymph at late premolt stage revealed six different types of ecdysteroids with ponasterone A (PoA) and 20-OH ecdysone (20-OH E) as the major forms. This ecdysteroid profile was consistent in all three molt cycles. Bilateral eyestalk ablation (EA) is a procedure that removes inhibitory neurohormones including crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) and often results in precocious molting in crustaceans. However, the inhibitory roles of these neuropeptides in vivo have not yet been tested in C. sapidus. We determined the regulatory roles of CHH and MIH in the circulating ecdysteroid from ablated animals through daily injection. A daily administration of purified native CHH and MIH at physiological concentration maintained intermolt levels of ecdysteroids in the EA animals. This suggests that Y organs (YO) require a brief exposure to CHH and MIH in order to maintain the low level of ecdysteroids. Compared to intact animals, the EA crabs did not exhibit the level of peak ecdysteroids, and the major ecdysteroid turned out to be 20-OH E, not PoA. These results further underscore the important actions of MIH and CHH in ecdysteroidogenesis, as they not only inhibit, but also control the composition of output of the YO activity. © 2009 Wiley Periodicals, Inc. [source] Inhibitory effect of molt-inhibiting hormone on phantom expression in the Y-organ of the kuruma prawn, Marsupenaeus japonicusARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2009Hideaki Asazuma Abstract Molting in crustaceans is induced by ecdysteroids as in insects. The ecdysteroid titre in hemolymph is negatively regulated by molt-inhibiting hormone (MIH) that inhibits the secretion of ecdysteroids from the Y-organ, an ecdysteroid-producing gland of crustaceans, whereas little is known about the molecular mechanism of inhibition by MIH. Recently, the Halloween genes encoding cytochrome P450 monooxygenases were characterized as the steroidogenic enzymes in insects. To elucidate whether the ecdysteroidogenesis in the Y-organ is regulated by molt-inhibiting hormone (MIH), we analyzed the expression level of an orthologue of a member of the Halloween genes, phantom (Cyp306a1, phm), in the Y-organ of a decapod crustacean, Marsupenaeus japonicus. A cDNA encoding phm (Mj-phm) was cloned by degenerate PCR and 5,- and 3,-RACEs. The deduced amino acid sequence of Mj-phm showed about 40% identity to those of insect phm. The six motif sequences and the four substrate recognition sites were well conserved between Mj-PHM and other PHM. RT-PCR showed the specific expression of Mj-phm mRNA in the Y-organ. In addition, quantitative real-time PCR verified that the expression level of Mj-phm was significantly increased at the pre-molt stage and decreased after ecdysis. Furthermore, exposure of the Y-organ to MIH significantly decreased the Mj-phm expression level in vitro. These results indicate that the transcription of Mj-phm in the Y-organ may be regulated by the inhibitory mechanism of MIH of M. japonicus, which involves the consequent negative regulation of ecdysteroidogenesis at the transcriptional level. © 2009 Wiley Periodicals, Inc. [source] Synthesis and mobilization of glycogen and trehalose in adult male Rhodnius prolixusARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2009Ana C. Mariano Abstract The vector of Chagas' disease, Rhodnius prolixus, feeds exclusively on blood. The blood meals are slowly digested, and these insects wait some weeks before the next meal. During the life of an insect, energy-requiring processes such as moulting, adult gonadal and reproductive growth, vitellogenesis, muscular activity, and fasting, lead to increased metabolism. Carbohydrates are a major source of energy and their mobilization is important. We determined the amounts of glycogen, trehalose, and glucose present in the fat body and/or hemolymph of adult males of R. prolixus and recorded the processes of accumulation and mobilization of these carbohydrates. We also tested our hypothesis that these processes are under endocrine control. The amount of glycogen in the fat body progressively increased until the fourth day after feeding (from 9.3±2.2 to 77. 3±7.5,µg/fat body), then declined to values around 36.3±4.9,µg/fat body on the fifteenth day after the blood meal. Glycogen synthesis was eliminated in decapitated insects and head-transplanted insects synthesized glycogen. The amount of trehalose in the fat body increased until the sixth day after feeding (from 16. 6±1.7 to 40. 6±5.3,nmol/fat body), decreased abruptly, and stabilized between days 7 and 15 at values ranging around 15,19,nmol/fat body. Decapitated insects did not synthesize trehalose after feeding, and this effect was reversed in head-transplanted insects. The concentration of trehalose in the hemolymph increased after the blood meal until the third day (from 0.07±0.01 to 0.75±0.05,mM) and at the fourth day it decreased until the ninth day (0.21±0.01,mM), when it increased again until the fourteenth day (0.79±0.06,mM) after the blood meal, and then declined again. In decapitated insects, trehalose concentrations did not increase soon after the blood meal and at the third day it was very low, but on the fourteenth day it was close to the control values. The concentration of glucose in the hemolymph of untreated insects remained low and constant (0.18±0.01,mM) during the 15 days after feeding, but in decapitated insects it progressively increased until the fifteenth day (2.00±0.10,mM). We recorded the highest trehalase activity in midgut, which was maximal at the eighth day after feeding (2,830±320,nmol of glucose/organ/h). We infer that in Rhodnius prolixus, the metabolism of glycogen, glucose, and trehalose are controlled by factors from the brain, according to physiological demands at different days after the blood meal. © 2009 Wiley Periodicals, Inc. [source] Role of allatostatin-like factors from the brain of Tenebrio molitor females,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2009O. Wasielewski Abstract The effect of brain extract from females of freshly emerged Tenebrio molitor on ovary, oocyte development, total protein content of hemolymph, and ovary was studied in 4-day-old adult mealworm females. Injections of extracts of 2-brain equivalents into intact (unligatured) Tenebrio females did not affect ovarian and oocyte development. Injections of ligated females, however, with 2-brain equivalents on day 1 and 2 after adult emergence strongly inhibited ovarian growth and oocyte development. At day 4, ligated and injected females did not develop their ovaries and pre-vitellogenic oocytes were not found. The changes in ovarian development correlated with an increase in the concentration of soluble proteins in the hemolymph as compared with the saline-injected controls. Additionally, a strong reduction of total protein content in ovarian tissue was observed. Reverse phase HPLC separation of a methanolic brain extract of T. molitor females showed that fraction 5 has a similar retention time to synthetic cockroach allatostatin. Fraction 5 was eluted at 12.88,min, which was closest to the internal standard Dippu-AST I, which eluted at 12.77,min. An ELISA of fraction 5 from the methanolic brain extract using antibodies against allatostatins Grybi-AST A1 and Grybi-AST B1 from cricket Gryllus bimaculatus showed that fraction 5 cross-reacted with Grybi-AST A1 antibodies. The cross-reactivity was similar to the synthetic allatostatin from D. punctata, which was used as a positive control. These observations demonstrate a possible role for allatostatin-like brain factor(s) in regulating the reproductive cycle of Tenebrio molitor. © 2009 Wiley Periodicals, Inc. [source] | ||||||||||||||||