Hemolymph Serum Research Articles

Biochemical and proteomic characterisation of haemolymph serum reveals the origin of the alkali-labile phosphate (ALP) in mussel (Mytilus galloprovincialis)

Mollusc haemolymph proteins are known to play several important physiological roles in the immune system, heavy metal transport and the tissue distribution of lipophilic compounds.In this study, we analysed acetone-extracted proteins from mussel haemolymph by one- and two-dimensional gel electrophoresis. The proteins were identified by comparing mass spectrometry data with the invertebrate EST database, allowing us to establish the mussel haemolymph serum proteome. Extrapallial protein (EP) precursor represents the most abundant serum protein; astacin and CuZn superoxide dismutase were also detected. Slight contamination from muscle proteins, due to the sampling method, was also found. No differences were observed in the profiles obtained for male and female serum proteins. One aspect of interest was the previously reported finding that alkali-labile phosphate (ALP) from haemolymph serum may be representative of vitellogenin (vtg)-like protein content in the circulatory fluid of molluscs. In our analysis of mussel haemolymph serum, vitellogenin-like proteins were never found. To confirm these data, a typical methyl-tert-butyl-ether (MTBE) extraction, which is specific for vtg-like proteins, was performed, and the results of the electrophoretic analyses were compared with those obtained by acetonic precipitation. The results showed that the electrophoretic profiles are similar and that vtg-like proteins cannot be identified. Moreover, the main phosphoprotein present in female and male extracts is EP protein precursor. In addition, agarose gel electrophoresis demonstrates that high-molecular-weight forms of vtg-like proteins are not detectable.

Vibrio cholerae interactions with Mytilus galloprovincialis hemocytes mediated by serum components

Edible bivalves (e.g., mussels, oysters) can accumulate large amount of bacteria in their tissues and act as passive carriers of pathogens to humans. Bacterial persistence inside bivalves depends, at least in part, on hemolymph anti-bacterial activity that is exerted by both serum soluble factors and phagocytic cells (i.e., the hemocytes). It was previously shown that Mytilus galloprovincialis hemolymph serum contains opsonins that mediate D-mannose-sensitive interactions between hemocytes and Vibrio cholerae O1 El Tor bacteria that carry the mannose-sensitive hemagglutinin (MSHA). These opsonins enhance phagocytosis and killing of vibrios by facilitating their binding to hemocytes. Since V. cholerae strains not carrying the MSHA ligand (O1 classical, non-O1/O139) are present in coastal water and can be entrapped by mussels, we studied whether in mussel serum, in addition to opsonins directed toward MSHA, other components can mediate opsonization of these bacteria. By comparing interactions of O1 classical and non-O1/O139 strains with hemocytes in artificial sea water and serum, it was found that M. galloprovincialis serum contains components that increase by at approximately twofold their adhesion to, association with, and killing by hemocytes. Experiments conducted with high and low molecular mass fractions obtained by serum ultrafiltration indicated that these compounds have molecular mass higher than 5000 Da. Serum exposure to high temperature (80°C) abolished its opsonizing capability suggesting that the involved serum active components are of protein nature. Further studies are needed to define the chemical properties and specificity of both the involved bacterial ligands and hemolymph opsonins. This information will be central not only to better understand V. cholerae ecology, but also to improve current bivalve depuration practices and properly protect human health.

Hemolytic activity is mediated by the endogenous lectin in the mosquito hemolymph serum

Although cytolysis of invading organisms is an innate form of immunity used by invertebrates, so far the underlying mechanism remains less explored. The pupal hemolymph of the mosquito Armigeres subalbatus induces an activity that causes hemolysis of human red blood cells (HRBC). This hemolytic activity was inhibited by sialic acid (N-acetylneuraminic acid) and serine protease inhibitors. We purified the sialic acid-specific lectin(s) from the pupal hemolymph using formaldehyde-fixed HRBC and determined the sequence of the amino-terminal 19 amino acid residues. A polyclonal antibody produced against this N-terminal peptide clearly inhibited the hemolytic activity of the hemolymph in vitro, thus suggesting that the hemolysis of HRBC is caused by the lectin present in the mosquito hemolymph. We suggest that mosquitoes possess a cytolysis system.

Development of cell culture system from the giant freshwater prawn Macrobrachium rosenbergii (de Man)

A new cell culture system (MRH) was developed for the first time from 2 months old freshwater prawn, Macrobrachium rosenbergii. Primary cultures were developed from heart tissues by explant culture technique. Cell outgrowth was obtained from the heart explant after 14 days of explant culture. The culture medium used was Leibovitz-15 supplemented with 20% Fetal Bovine Serum along with 1% prawn hemolymph serum, 0.1% glucose, 0.5% NaCl and antibiotics (Penicillin 10,000 Units ml(-1), Streptomycin 10,000 microg ml(-1), Amphotericin B 500 mg ml(-1)) with a final osmomolality of 470-550 mmol kg(-1). The pH of the growth medium found suitable for the growth of the cells was 7.20. The viability of cells was found to be 60% when revived after a month of storage in liquid nitrogen.

Influence of cell density and phase variants of bacterial symbionts (Xenorhabdus spp.) on dauer juvenile recovery and development of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida)

The rhabditid nematodes Steinernema carpocapsae and Steinernema feltiae are used in biological control of insect pests. Mass production is done in liquid culture media pre-incubated with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, before nematode dauer juveniles (DJs) are inoculated. As a response to food signals produced by the bacterial symbionts, the DJs exit from the developmentally arrested dauer stage (they recover development) and grow to adults, which produce DJ offspring. Variable DJ recovery after inoculation often causes process failure due to non-synchronous population development and low numbers of adult nematodes. This contribution investigated the influence of the bacterial cell density on DJ recovery and development to adults. At higher density of 10(10) bacterial cells ml(-1), a higher percentage of DJ recovery was induced, and adults occurred earlier in both Steinernema spp. than at lower density of 10(9) and 10(8) cells ml(-1). Xenorhabdus symbionts produce phase variants. Recovery in bacteria-free supernatants was lower than in supernatants containing bacterial cells for both primary and secondary phase Xenorhabdus spp. and lower in secondary than in primary phase supernatants or cell suspensions. In general, recovery was lower for Steinernema feltiae and the time at which 50% of the population had recovered after exposure to the food signal was longer (RT(50) = 17.1 h) than for Steinernema carpocapsae (RT(50) = 6.6 h). Whereas >90% S. carpocapsae DJs recovered in hemolymph serum of the lepidopteran insect Galleria mellonella, recovery of S. feltiae only reached 31%. Penetration into a host insect prior to exposure to the insect's food signal did not enhance DJ recovery. Consequences for liquid culture mass production of the nematodes and differences between species of the genera Steinernema and Heterorhabditis are discussed.

Enhanced expression of a cloned and sequenced Ciona intestinalis TNFα-like (CiTNFα) gene during the LPS-induced inflammatory response

A tumor necrosis factor-alpha (TNFalpha)-like gene from Ciona intestinalis (CiTNF alpha-like) body wall challenged with bacterial lipopolysaccharide (LPS) was cloned and sequenced 4 h after LPS inoculation. An open reading frame of 936 bp encoding a propeptide of 312 amino acids (35.4 kDa) displaying a transmembrane domain from positions 7 to 29, a TACE cleavage site, and a mature peptide domain of 185 amino acids (20.9 kDa), was determined with a predicted isoelectric point of 9.4. The phylogenetic tree based on deduced amino acid sequences of invertebrate TNF-like protein and vertebrate TNFs supported the divergence between the ascidian and vertebrate TNF families, whereas D. melanogaster Eiger A and B TNF-like sequences were distinctly separated from the chordate TNFs. Thus, the ascidian TNFalpha-like cytokine was upregulated by in vivo LPS challenge supporting its pro-inflammatory role. In the pharynx, increased expression levels were found following analysis by real-time polymerase chain reaction, whereas in situ hybridization assay showed positive hemocytes both in the tissue and in circulating hemocytes. Finally, Western blot with monoclonal antibodies disclosed human TNFalpha epitopes in a 15-kDa protein component of the hemolymph serum and in a 43-kDa protein contained in the hemocyte lysate supernatant prepared in the presence of detergents. Both soluble and hemocyte-bound CiTNF alpha-like protein therefore appeared to be modulated by the LPS challenge.

Oceaniserpentilla haliotis gen. nov., sp. nov., a marine bacterium isolated from haemolymph serum of blacklip abalone

An aerobic, Gram-negative bacterial isolate, strain DSM 19503(T), was isolated from haemolymph serum of the blacklip abalone Haliotis rubra. Cells of strain DSM 19503(T) were vibrioid to spiral, motile and were able to pass through sterile filters with a pore size of 0.2 microm, indicating the small width of the bacterium. The isolate was psychrophilic, with the ability to grow at 2-8 degrees C. Oxidase activity was present, whereas catalase activity was absent. The nearly complete 16S rRNA gene sequence of strain DSM 19503(T) was obtained and phylogenetic sequence analysis showed that it formed a distinct genus in the family Oceanospirillaceae with highest sequence similarity of 92.9% to Oleispira antarctica RB-8(T). The cellular fatty acid composition was dependent on the growth medium used for cultivation. During growth on seawater agar, the fatty acid composition was most similar to that of Oleispira antarctica DSM 14852(T), with mainly C(16:0) (90.3%). In contrast, Columbia blood agar/NaCl-grown cells exhibited mainly C(10:0) 3-OH (11.8%), C(12:1)cis5 (8.2%), C(16:1)cis9 (29.6%), C(16:0) (19.3%) and C(18:1)cis9 (13.1%) fatty acids. On the basis of phenotypic, chemotaxonomic and genotypic data, strain DSM 19503(T) is considered to represent a novel species in a new genus for which the name Oceaniserpentilla haliotis gen. nov., sp. nov. is proposed. The type strain of Oceaniserpentilla haliotis is DSM 19503(T) (=LMG 24225(T)).

Effect of immunostimulants on the haemolymph haemagglutinins of tiger shrimpPenaeus monodon

The levels of haemagglutinins in Penaeus monodon, following administration of immunostimulants, β-glucans and/or vibrio bacterin either orally or by immersion, were studied. The freshly drawn haemolymph was incubated with microbial materials like β-glucans/vibrio bacterin and serum obtained after coagulation was administered to naïve animals. The immunostimulant treatments either via immersion, feeding or injection were found to cause an increase (P<0.006) in haemagglutination activity (HA) of the haemolymph against mouse erythrocytes. Injection of saline or heterologous haemolymph caused an increase in the HA, but injection of haemolymph serum obtained by clotting haemolymph in the presence of vibrio bacterin or glucan did not bring about an increase in HA. There was no change in the haemolymph protein profile of the groups receiving immunostimulants through immersion or feed. Two protein bands (27 and 30 kDa), which were present in the uninjected group, were found to be overexpressed in the haemolymph-injected groups. Three bands of 17, 21 and 23 kDa, which were absent in control or saline-injected groups, were present in all the haemolymph serum-injected groups. The study indicates that modulation of HA may partly account for the immunomodulatory activity of immunostimulants like β-glucan or vibrio bacterins.

Ecological and physiological effects of PaV1 infection on the Caribbean spiny lobster ( Panulirus argus Latreille)

Pathogens can profoundly impact the ecology of the organisms they infect through changes in host behavior that influence demographic processes. For example, juvenile Caribbean spiny lobsters ( Panulirus argus Latreille) infected with the PaV1 virus ( Panulirus argus Virus 1) are avoided by their normally social conspecifics, which alters local spatial distributions and presumably rates of disease transmission. PaV1 infections are nearly always lethal, but prior to succumbing to the disease, infection may impact other host dynamics (e.g., movement, growth, or survival) that effect transmission of the virus. We used mark-recapture surveys and laboratory studies to determine the impact of PaV1 infection on lobster movement and physiological condition, and in turn, the effect of condition on susceptibility to infection. Significantly more healthy lobsters were recaptured than heavily diseased lobsters in 5-d mark-recapture surveys, indicating either greater emigration or greater mortality of infected lobsters. Results of a laboratory bioassay suggest that lobsters with early-stage infections moved at an equivalent rate to healthy lobsters, however, as infection progressed, lobsters inoculated with PaV1 moved less than healthy lobsters ultimately remaining sedentary. Infected lobsters captured in nature also had significantly lower hemolymph (blood) serum protein values, indicating poor physiological condition. However, poor condition did not predispose lobsters to greater risk of infection because prevalence was not different between starved lobsters and healthy lobsters challenged with PaV1. Although lobsters infected with PaV1 eventually become sedentary and of poor nutritional condition, during the early stages of infection they remain active and are thus capable of dispersing the virus throughout the population.

Hemagglutinating activity and electrophoretic pattern of hemolymph serum proteins of Penaeus monodon and Macrobrachium rosenbergii to white spot syndrome virus injections

The giant fresh water prawn Macrobrachium rosenbergii is known to be highly tolerant to white spot syndrome virus (WSSV) infections when compared to the widely cultured marine tiger shrimp Penaeus monodon. At present, the exact mechanism of tolerance by M. rosenbergii to WSSV is not known. In this study, we attempt to study the effect of WSSV injections on the hemagglutination activity of the hemolymph serum of both P. monodon and M. rosenbergii and look for changes if any, on their hemolymph serum protein electrophoretic patterns. Our results show that M. rosenbergii had significantly ( p < 0.05) higher hemagglutinating activity against mouse erythrocytes when compared to P. monodon. As the infection progressed to 48 h there was a further increase ( p < 0.05) in the hemagglutination activity in M. rosenbergii, while it decreased in P. monodon. 12% SDS-PAGE analysis of the hemolymph serum of M. rosenbergii infected with WSSV did not show any new protein bands, whereas few bands with decreased intensity was observed in moribund P. monodon where the hemagglutinating activity was also observed to be decreased. The results indicate that hemolymph hemagglutinin levels are modulated in crustaceans as a response to viral infections.

TISSUE DISTRIBUTION AND HEMOLYMPH ACTIVITY OF SIX ENZYMES IN THE AMERICAN LOBSTER (HOMARUS AMERICANUS): POTENTIAL MARKERS OF TISSUE INJURY

Abstract Biochemistry panels are used to help identify tissue injury (e.g., because of inflammation, trauma or hypoxia) in human and veterinary medicine in part, by detecting increased enzyme activity in serum or plasma after release from damaged tissues. To determine if a similar approach can be used in Homarus americanus, activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamate dehydrogenase (GD), sorbitol dehydrogenase (SDH), amylase (AMY) and lipase (LIP) were measured in tissue homogenates of heart, hepatopancreas, abdominal muscle, proximal intestine, antennal gland, hemocyte lysate supernatant and hemolymph plasma and serum. Activities of ALT and AST were significantly higher in serum than plasma, which was attributed to release of enzymes from hemocytes during coagulation. Reference intervals calculated for plasma enzyme activity at ambient holding temperatures of 2°C to 4°C and 15°C were quite similar. Plasma enzyme activity was not a sensitive test for detecting ...

Effects of tumour necrosis factor α (TNFα) on Mytilus haemocytes: role of stress‐activated mitogen‐activated protein kinases (MAPKs)

Many studies indicate that innate immunity in invertebrates can be modulated by a cytokine network like in vertebrates. In molluscs, the immune response is carried out by circulating haemocytes and soluble haemolymph factors. In the present study, the effects of heterologous TNFalpha (tumour necrosis factor alpha) on cell signalling and function in the haemocytes of the bivalve Mytilus galloprovincialis Lam. were investigated. Addition of TNFalpha in the absence of haemolymph serum [in ASW (artificial sea water)] induced cellular stress, as indicated by lysosomal destabilization, and decreased phagocytosis; on the other hand, in the presence of serum, TNFalpha did not affect lysosomal stability and even stimulated phagocytosis. TNFalpha induced rapid phosphorylation of the stress-activated p38 and JNK (c-Jun N-terminal kinase) MAPKs (mitogen-activated protein kinases); both effects were persistent in ASW but transient in serum. Activation of p38 and JNKs in mediating the effects of TNFalpha was confirmed by the use of specific MAPK inhibitors. Moreover, flow cytometric analysis indicated that TNFalpha in the presence of serum induced transient phosphatidylserine exposure on the haemocyte surface, evaluated as annexin V binding; in ASW, the cytokine resulted in a stable increase in the percentage of both annexin- and propidium iodide-positive cells, indicating possible apoptotic/necrotic processes. The results indicate that TNFalpha can affect the function of bivalve haemocytes through conserved transduction pathways involving stress-activated MAPKs and suggest that the haemocyte response to the cytokine is influenced by soluble haemolymph components.

Interactions between Mytilus haemocytes and different strains of Escherichia coli and Vibrio cholerae O1 El Tor: role of kinase-mediated signalling

In the article by Canesi et al. (2005), Fig. 1 was published incorrectly. The correct figure is presented below. We apologize for this error. Changes in phosphorylation of MAPK-like proteins in mussel haemocytes incubated with wild-type and mutant E. coli and V. cholerae strains in the presence of haemolymph serum for different periods of time. Lanes: 1 = control; 2 = 5 min; 3 = 15 min; 4 = 30 min; 5 = 60 min. Haemocyte protein extracts were subjected to 12% SDS-PAGE followed by Western blotting using polyclonal phosphospecific antibodies to ERK1/2, p38 and SAPK/JKN MAPKs. W E.c., wild-type E. coli; M E.c., mutant E. coli; W V.c., wild-type V. cholerae; M V.c., mutant V. cholerae. A. Effect on ERK MAPK phosphorylation. B. Effect on p38 MAPK phosphorylation. C. Effect on SAPK/JNK MAPK phosphorylation. Results are representative of three independent experiments. Insets: densitometric analyses of blots from three independent experiments (mean ± SD). Relative changes in band optical densities (arbitrary units) were normalized for the control band in each series. D. Anti-actin blots are shown as loading controls. Bands were detected using enhanced chemioluminescence reagents (see Experimental procedures).

Induced resistance to infection of lobsters Homarus americanus by Aerococcus viridans (var.) homari, the bacterium causing gaffkemia

A vaccine composed of steam sterilized (autoclaved) cells of a virulent strain of Aerococcus viridans (var.) homari was effective in protecting lobsters Homarus americanus against gaffkemia. At 15 degrees C the heat-killed vaccines (HKV) at concentrations between 1 and 5 x 10(7) particles kg(-1) lobster body wt induced maximal protection in induction periods ranging from 7 to 11 d. Protection was substantial over the course of a 30 d post-induction trial period. Spring-caught lobsters (i.e. those more fully rehabilitated following ecdysis) gained more protection (LD50 = 1.9 x 10(4)) from the vaccination than did those caught in the late fall-early winter period (lobsters that were not yet fully recovered from ecdysis) (LD50 = 3.2 x 10(3)). The protection offered by the HK vaccine was comparable to that induced by a vaccine produced by incubating the pathogen with low concentrations (2 pg ml(-1)) of the antibiotic vancomycin. The bacterins produced by both methods exhibited similar new properties: (1) agglutination at low titres by lobster hemolymph serum, suggesting an impaired capsule layer, and (2) increased permeability to the large Alcian Blue molecule. With both vaccines, the protection may be a direct result of increased exposure to intact bacterial cell structures by the lobster defences, an exposure which otherwise would be prevented by an intact capsule.

Role for mannose-sensitive hemagglutinin in promoting interactions between Vibrio cholerae El Tor and mussel hemolymph.

The role of mannose-sensitive hemagglutinin (MSHA) in Vibrio cholerae O1 El Tor interactions with hemolymph of the mussel Mytilus galloprovincialis was studied. Bacterial adherence to and association with hemocytes were evaluated at 4 and 18 degrees C, respectively. In hemolymph serum, the wild-type strain N16961 adhered to and associated with hemocytes about twofold more efficiently than its mutant lacking MSHA. In artificial seawater (ASW), no significant differences between the two strains were observed. N16961 was also more sensitive to hemocyte bactericidal activity than its MSHA mutant; in fact, the percentages of killed bacteria after 120 min of incubation were 60 and 34%, respectively. The addition of D-mannose abolished the serum-mediated increase in adherence, association, and sensitivity to killing of the wild-type strain without affecting the interactions of the mutant. A similar increase in N16961 adherence to hemocytes was observed when serum was adsorbed with MSHA-deficient bacteria. In contrast, serum adsorbed with either wild-type V. cholerae El Tor or wild-type Escherichia coli carrying type 1 fimbriae was no longer able to increase adherence of N16961 to hemocytes. The results indicate that hemolymph-soluble factors are involved in interactions between hemocytes and mannose-sensitive adhesins.

Surface interactions between Escherichia coli and hemocytes of the Mediterranean mussel Mytilus galloprovincialis lam. leading to efficient bacterial clearance.

The role of type 1 fimbriae in the interactions between Escherichia coli and Mytilus galloprovincialis Lam. hemocytes was evaluated. The association of fimbriated strain MG155 with hemocyte monolayers at 18 degrees C was 1.5- and 3- to 4-fold greater than the association of unfimbriated mutant AAEC072 in artificial seawater and in hemolymph serum, respectively. Such differences were apparently due to different adhesive properties since MG155 adhered more efficiently than AAEC072 when hemocytes were incubated at 4 degrees C to inhibit the internalization process. Hemolymph serum increased both association and adherence of MG155 two- to threefold but did not affect association and adherence of AAEC072. MG155 was also 1.5- to 1.7-fold more sensitive to killing by hemocytes than AAEC072, as evaluated by the number of culturable bacteria after 60 and 120 min of incubation. The role of type 1 fimbriae in MG155 interactions with hemocytes was confirmed by the inhibitory effect of D-mannose. In in vivo experiments MG155 cells were cleared from circulating hemolymph more rapidly than AAEC072 cells were cleared. These results confirm that surface properties are crucial in influencing bacterial persistence and survival within mussel hemolymph.

Increases in tissue free amino acid levels in response to prolonged emersion in marine crabs: an ammonia-detoxifying process efficient in the intertidal Carcinus maenas but not in the subtidal Necora puber.

Carcinus maenas and Necora puber were exposed to air for 72 h and 18 h, respectively, at 18 °C. Changes in the free amino acid (FAA) content of their muscle, hepatopancreas and haemolymph were recorded during air-exposure and subsequent reimmersion. Muscle and hepatopancreas urate contents and haemolymph serum protein levels were also measured during emersion. In air-exposed C. maenas, the muscle FAA pool increased significantly within the first 24 h of emersion. This increase was due to an increase in the non-essential amino acid (NEAA) pool only; the essential amino acid (EAA) pool did not change. In haemolymph, the EAA pool decreased during the first 24 h of emersion, whereas the FAA and NEAA pools did not change. However, in this compartment, glutamine levels increased throughout the air-exposure period. No significant changes in FAA, NEAA and EAA contents of the hepatopancreas were observed during the 72 h emersion. In air-exposed N. puber, the FAA pools of muscle and hepatopancreas did not change, although changes in the levels of some amino acids were observed during the 18 h emersion period. In this species, large increases in both the NEAA and EAA pools in the haemolymph were recorded. High levels of urate were observed in the muscle and hepatopancreas of immersed N. puber, but no significant changes occurred during emersion. In contrast, immersed C. maenas exhibited low levels of urate in both compartments, and hepatopancreas urate levels increased slightly during emersion. Haemolymph protein content did not change in air-exposed N. puber, whereas it increased in the haemolymph of 72 h emersed C. maenas. The origin of newly formed NEAAs and their role in ammonia detoxification, particularly in C. maenas, which is able to regulate its internal ammonia levels during such a prolonged emersion, are discussed.

Evidence for the presence of a pheromonotropic factor in hemolymph and regulation of sex pheromone production in Helicoverpa zea

In intact animal assays, 2-day-old females of Helicoverpa zea injected with 2 pmol synthetic H. zea-pheromone biosynthesis activating neuropeptide (Hez-PBAN) exhibited significant stimulation of pheromone production. In contrast, octopamine did not stimulate pheromone production when injected 30 min before lights off. Additionally, females injected with PBAN and mianserin (an octopamine antagonist) or reserpine, produced significant amounts of pheromone. These results suggest that octopamine may not be involved in regulation of pheromone production in H. zea. In abdomen incubation assays, synthetic Hez-PBAN stimulated pheromone production at doses as low as 10 fmol. Additionally, one female equivalent of hemolymph or serum from scotophase females exhibited pheromonotropic activity that was comparable to activity elicited by 10 fmol synthetic Hez-PBAN. High pressure liquid Chromatographic (HPLC) fractionation of scotophase H. zea hemolymph serum filtrates, obtained after filtration through molecular weight filters, suggested the presence of a pheromonotropic fraction with molecular weight that appears to be less than 3000.

Phenoloxidase and its zymogen from the haemolymph of larvae of the lepidopteran Spodoptera littoralis (Lepidoptera: Noctuidae)

Haemolymph serum phenoloxidase from larvae of the noctuid moth Spodoptera littoralis is present as an inactive proenzyme, prophenoloxidase. Partially purified serum prophenoloxidase was activated by methanol, but not by laminarin, lipopolysaccharides, bovine trypsin or chymotrypsin. Phenoloxidase activity was optimal between pH 7.0 and 7.5 for the oxidation of l-DOPA, with an apparent K m of 1.35 mM for this substrate. Both Mg 2+ and Ca 2+ stimulated phenoloxidase activity compared with controls and maximal stimulation was observed at about 30 mM for both ions. EDTA had little effect on activity even at high concentrations. Phenoloxidase activity was inhibited by dithiothreitol (50% inhibition at 20 μM) and kojic acid (50% inhibition at 135 μM, inhibition constant of 69 μM).

Neuroendocrine factors affecting the glycogen metabolism of purified Mytilus edulis glycogen cells: Partial characterization of the putative glycogen mobilization hormone—demonstration of a factor that stimulates glycogen synthesis

A putative glycogen mobilizing hormone (GMH) from the marine mussel Mytilus edulis L. has been partially characterized. GMH activity is present in the cerebral ganglia and the hemolymph serum and promotes the mobilization of glycogen in isolated glycogen cells. The cerebral GMH is trypsin sensitive and partially heat labile and has an apparent molecular mass of >20 kDa. Following fractionation of cerebral extracts by molecular mass, a second factor, with a molecular mass of ca. 1.5 kDa, was discovered. This factor stimulates post-incubation incorporation of 14C into glycogen in isolated glycogen cells.