Lipase activity in the hemolymph of Biomphalaria glabrata (Mollusca) challenged with bacterial lipids

The levels of lipase activity in both the cellular and serum constituents of the hemolymph of Biomphalaria glabrata that had been challenged in vitro to beat-killed and sonicated Bacillus megaterium as well as samples challenged with live B. megaterium were ascertained. There were no significant alterations in the levels of enzyme activity in both cells and serum of the samples that had been challenged with sonicated bacteria; however, there was a signficant elevation in the enzyme activity associated with both the cells and serum of hemolymph that had been challenged with live bacteria. It has been concluded that live B. megaterium can stimulate hypersynthesis of lipase, a lysosomal enzyme, in phagocytes of B. glabrata and that this enzyme subsequently is released into serum. Consequently, the hydrolysis of lipid constituents of bacteria could theoretically occur in serum as well as within phagocytes.     

Developmental and sex-dependent regulation of storage protein synthesis in the silkworm,Bombyx mori

The mechanism of sex-dependent expression of a major plasma protein, referred to as storage protein 1 (SP-1) was studied during development of the silkworm, Bombyx mori. SP-1 occurred in the hemolymph of the female as well as in the male larvae until the end of the fourth larval instar. In the last instar larvae, the amount of SP-1 in the hemolymph greatly increased in females, but markedly declined in males. The level of fat body mRNA for SP-1 reflected the developmental and sex-dependent changes in the hemolymph concentration of SP-1. The developmental patterns of hemolymph proteins in the third and the fourth instar larvae of sex-mosaic individuals were quite analogous to those observed in normal larvae at the same developmental stages. The hemolymph concentration of SP-1 at the last larval instar of the sex mosaics varied among individuals irrespective of the gonad compositions. In vitro culture of the fat body cells dissected from several locations of a sex-mosaic larva provided evidence that each fat body cell in a common hemolymph milieu synthesizes a high (female type) or a low (male type) level of SP-1 depending on the sex chromosome composition. The amount of vitellogenin in the hemolymph of the sex-mosaic pupae was in proportion to that of SP-1 at the last larval instar. From these results, it is suggested that the sex-dependent expression of SP-1 and vitellogenin in B. mori is genetically determined and developmentally regulated without participation of the reproductive organs or any sex-specific humoral factors.     

A trehalose 6-phosphate synthase gene of the hemocytes of the blue crab, Callinectes sapidus: cloning, the expression, its enzyme activity and relationship to hemolymph trehalose levels

Trehalose in ectoderms functions in energy metabolism and protection in extreme environmental conditions. We structurally characterized trehalose 6-phosphate synthase (TPS) from hemocytes of the blue crab, Callinectes sapidus. C. sapidus Hemo TPS (CasHemoTPS), like insect TPS, encodes both TPS and trehalose phosphate phosphatase domains. Trehalose seems to be a major sugar, as it shows higher levels than does glucose in hemocytes and hemolymph. Increases in HemoTPS expression, TPS enzyme activity in hemocytes, and hemolymph trehalose levels were determined 24 h after lipopolysaccharide challenge, suggesting that both TPS and TPP domains of CasHemoTPS are active and functional. The TPS gene has a wide tissue distribution in C. sapidus, suggesting multiple biosynthetic sites. A correlation between TPS activity in hemocytes and hemolymph trehalose levels was found during the molt cycle. The current study provides the first evidence of presence of trehalose in hemocytes and TPS in tissues of C. sapidus and implicates its functional role in energy metabolism and physiological adaptation.     

Homologous Expression of the Lipase and ABC Transporter Gene Cluster, tliDEFA, Enhances Lipase Secretion in Pseudomonas spp.

The ABC transporter TliDEF was found to be an efficient secretory apparatus for extracellular lipase TliA in Pseudomonas fluorescens. For the enhanced secretion of the lipase, we tried to coexpress tliA and tliDEF in various Pseudomonas species. Whereas the coexpression of tliA and tliDEF was required for the lipase secretion in P. fragi, the expression of tliA was sufficient for the lipase secretion in P. fluorescens, P. syringae, and P. putida, indicating the existence of compatible ABC transporter in these species. However, P. fluorescens harboring tliDEFA secreted much more lipase than P. fluorescens harboring only tliA, but the tliDEF was functional only at temperatures below 30°C. The recombinant P. fluorescens overexpressing tliDEFA showed the highest secretion level, 217 U/ml · OD (optical density) (28 μg/ml · OD) of lipase in Luria-Bertani medium under microaerated conditions. With the increase of aeration, the lipase production was decreased and the lipase seemed to be degraded as the cells entered the cell death phase. These results demonstrate that P. fluorescens can be used as a host system for the secretory production of the lipase using the ABC transporter, thus producing lipase in over 14% of the total protein.     

Hemolymph pH of the larvae of three species of mosquitoes,and the effect of Romanomermis culicivorax parasitism on the blood pH of Culex pipiens

The hemolymph pHs of late fourth-instar Culex pipiens, Aedes taeniorhynchus, and Toxorhynchites amboinensis were measured with pH-sensitive glass microelectrodes and were slightly alkaline; pH 7.51, 7.62, and 7.37, respectively. The hemolymph pH remained relatively constant during the development of C. pipiens larvae through the third and early and late fourth instars. The hemolymph pH in C. pipiens larvae parasitized with the mermithid nematode Romanomermis culicivorax was unaltered. These measurements provide an approximate pH (ca 7.4) which is normal for the hemolymph of larval mosquitoes, and should be useful for further development of a culture medium for R. culicivorax.     

Experimentally induced elevation of aminopeptidase activity in hemolymph cells of the American oyster,Crassostrea virginica

The levels of aminopeptidase activity in the serum and hemolymph cells of the American oyster, Crassostrea virginica, have been determined to be 2.89 ± 2.25 and 0.62 ± 0.39 Sigma units/ml, respectively. Low levels of aminopeptidase activity have been demonstrated cytochemically within cytoplasmic granules, i.e., secondary phagosomes, of circulating hemolymph cells. Exposure of whole hemolymph to heat-killed Bacillus megaterium or to sterile sea water results in a significant increase in cellular aminopeptidase activity, with the level of activity being the highest in cells that had been exposed to bacteria. The level of aminopeptidase activity in serum is unaltered in similarly challenged whole hemolymph. It is concluded that aminopeptidase synthesized in cells during phagocytosis or as a result of stimulation by exposure to sea water is not released into the serum but is retained intracellularly. Our studies suggest that it is the intracellular and not the serum aminopeptidases whith are of primary importance in the degradation of B. megaterium.     

Uptake of the yolk protein, lipovitellin, by developing crustacean oocytes

A variety of cytochemical techniques were used to demonstrate how crustacean lipovitellin accumulates within the egg. It was found that a protein serologically identical to the lipovitellin of yolk spheres was present in the hemolymph of vitellogenic crustaceans, but was absent from the hemolymph of males and immature females.In the three crustacean species studied (Uca pugilator, Cambarus clarkii, and Libinia emarginata), pinocytosis of fluorescein-conjugated lipovitellin and trypan blue occurred only during those periods when oocytes were accumulating yolk.It may be concluded from the present studies that yolk spheres develop in crustacean eggs primarily through micropinocytotic uptake of lipovitellin from the hemolymph, although other oocyte proteins appear to be made in the oocyte.     

Inducement of miracidia-immobilizing substance in the hemolymph of Biomphalaria glabrata

Lie K. J., Jeong K. H. and Heyneman D. 1980. Inducement of miracidia-immobilizing substance in the hemolymph of Biomphalaria glabrata. Intemational Journal for Parasitology10: 183–188. More than 85% of echinostome-infected albino B. glabrata laboratory strain snails develop miracidia-immobilizing substance(s) (MIS) in the hemolymph, while less than 5% of control uninfected snails show this ability. Snails infected with Echinostoma lindoense show a strong miracidial immobilizing test (MIT) when homologous miracidia are exposed to the hemolymph and a moderate response when E. liei and Paryphostomum segregatum miracidia are used. Infection with E. paraensei results in a high level of hemolymph MIS with E. lindoense miracidia, a moderate one with P. segregatum miracidia, and a weak one when hemolymph is tested against E. liei as well as the homologous E. paraensei miracidia. Infection with E. liei induces a strong MIT with E. lindoense miracidia whereas only a moderate one was observed when using homologous or P. segregatum miracidia. Infection with P. segregatum gives a moderate MIT reaction to miracidia of the homologous species, as well as to E. lindoense and E. liei, and only a weak response to E. paraensei miracidia. Infection with S. mansoni fails to induce hemolymph that shows MIS to any of the parasites tested. Production of hemolymph MIS is temporary. It begins one day postexposure, reaches its maximum 10–14 days postexposure, and declines to the preinfection level several weeks later. Infection of snails with irradiated parasites also results in a temporary production of hemolymph MIS.Uninfected snails show a tissue-extract MIS, which is especially strong when digestive gland extracts are used. However, these snails give little or no evidence of a hemolymph MIS.     

Stages of Infection during the Tripartite Interaction between Xenorhabdus nematophila, Its Nematode Vector, and Insect Hosts

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.     

The synthesis of hemolymph proteins by the larval midgut of an insect Calpodes ethlius (Lepidoptera:Hesperiidae)

Ligated tubes of Calpodes ethlius (Lepidoptera:Hesperiidae) larval midgut with normal (i.e. apical secretions into the lumen and basal into the hemocel or medium) or inverted orientation (i.e. apical secretions into the hemocoel or medium and basal into the lumen) were incubated in artificial hemolymph in the presence of [³⁵S]methionine to investigate protein synthesis and vectorial secretion. The midgut synthesizes and secretes at least eight polypeptides basally and seven apically. The tissue also synthesizes many other polypeptides that are not released at either surface. Two dimensional analysis of proteins labeled in vitro and in vivo showed that (a) proteins synthesized in vitro are the same as those synthesized in vivo, (b) different proteins are secreted on apical and basal surfaces, (c) in vitro apical secretions are the same as in vivo luminal proteins, (d) at least two of the basal secretions can be demonstrated in the hemolymph labeled in vivo. Almost all basal secretions showed immunological similarity with hemolymph proteins as observed by immunoprecipitation and fluorography. Arylphorin is a main hemolymph protein synthesized by the midgut. Midgut arylphorin has been identified by its precipitation by antibodies to hemolymph arylphorin. We conclude that insect midgut has bi-directional secretion. Luminal proteins (presumably digestive enzymes and perhaps goblet cell luminal contents) are carried to the apical face. A different set of proteins are carried basally to the hemolymph.     

New Insights into the Colonization and Release Processes of Xenorhabdus nematophila and the Morphology and Ultrastructure of the Bacterial Receptacle of Its Nematode Host, Steinernema carpocapsae

We present results from epifluorescence, differential interference contrast, and transmission electron microscopy showing that Xenorhabdus nematophila colonizes a receptacle in the anterior intestine of the infective juvenile (IJ) stage of Steinernema carpocapsae. This region is connected to the esophagus at the esophagointestinal junction. The process by which X. nematophila leaves this bacterial receptacle had not been analyzed previously. In this study we monitored the movement of green fluorescent protein-labeled bacteria during the release process. Our observations revealed that Xenorhabdus colonizes the distal region of the receptacle and that exposure to insect hemolymph stimulated forward movement of the bacteria to the esophagointestinal junction. Continued exposure to hemolymph caused a narrow passage in the distal receptacle to widen, allowing movement of Xenorhabdus down the intestine and out the anus. Efficient release of both the wild type and a nonmotile strain was evident in most of the IJs incubated in hemolymph, whereas only a few IJs incubated in nutrient-rich broth released bacterial cells. Incubation of IJs in hemolymph treated with agents that induce nematode paralysis dramatically inhibited the release process. These results suggest that bacterial motility is not required for movement out of the distal region of the receptacle and that hemolymph-induced esophageal pumping provides a force for the release of X. nematophila out of the receptacle and into the intestinal lumen.     

Changes observed in hemolymph proteins of the lawn armyworm,Spodoptera mauritia acronyctoides,during growth,development, and exposures to a nuclear polyhedrosis virus

Vertical electrophoresis with acrylamide gel was used to study the effects of a nuclear polyhedrosis virus (NPV) on the hemolymph proteins of Spodoptera mauritia acronyctoides. An electrophoretic pattern consisting of 20 basic bands of proteins was separated in hemolymph of normal larvae which were older than 17 days. These hemolymph proteins increased quantitatively during growth. All 20 proteins could not be detected in hemolymph of younger larvae by the techniques utilized. Additional proteins were separated with metamorphosis. Lethal doses of NPV resulted in a general reduction of hemolymph proteins (hypoproteinemia) in infected larvae. Sublethal doses of NPV elicited an increase in certain hemolymph proteins. Similar increases in proteins were also observed in larvae surviving ostensibly lethal levels of NPV, in larvae subjected to physical stress, and in larvae reared axenically without formaldehyde in their diets. These same proteins, however, were present in approximately the same quantities in mature larvae. Physiopathology of NPV in S. mauritia appears to involve stress factors, host reactions, and the host endocrine system.     

Analysis of the hemagglutinin and general protein element of the hemolymph of the West Indian leaf cockroach, Blaberus craniifer

Only one protein component of Blaberus craniifer hemolymph migrates toward the anode in an electrophoretic field, within a pH range of 6.5 to 9.0. This sample was separated out of the whole hemolymph using ethanol fractionation, gel filtration, and ultracentrifugation. Results indicate it to be a glycoprotein with a maximum Svedberg value of 5S. This protein, Group I, is not involved in the naturally occuring hemagglutination reaction of the hemolymph. All other hemolymph proteins, including any with agglutinating activity, exceed a molecular size of 21.4S.     

The synthesis of hemolymph proteins by the larval epidermis of an insect Calpodes ethlius (Lepidoptera: Hesperiidae)

Sheets of the dorsal abdominal integument from fifth instar larvae of Calpodes ethlius (Lepidoptera: Hesperiidae) were incubated in artificial hemolymph in the presence of [³⁵S]methionine to investigate protein synthesis and vectorial secretion. The epidermis synthesizes and secretes at least 13 polypeptides basally and 15 apically. Two dimensional analysis of proteins labeled in vitro and in vivo showed that (a) most of the polypeptides secreted on apical and basal surfaces are different, (b) in vitro apical secretions are the same as in vivo cuticular proteins, (c) at least four of the basal secretions can be demonstrated in hemolymph labeled in vivo.Antibodies made against whole hemolymph recognized five basally secreted polypeptides and one apically secreted polypeptide both on fluorograms of immunoprecipitates and immunoblots. Arylphorin is secreted from both surfaces. Arylphorin synthesized in vitro has been identified through its precipitation by antibodies to hemolymph arylphorin in epidermis, cuticle and medium. We conclude that insect epidermis has bi-directional secretion. Cuticular proteins are carried to the apical face. A different set of proteins are carried basally to the hemolymph.     

Effect of salinity on the osmotic,chloride, total protein and calcium concentrations in the hemolymph of the prawn Peneaus monodon (fabricius)

• 1.1. Osmolality and chloride concentrations in the hemolymph of Penaeus monodon became stable 1 day after molting in 32 ppt, while total protein and calcium concentrations remained stable throughout the molting cycle. When intermolt (≥ 36 hr postmolt) animals were transferred from control (32 ppt) to experimental (8–40 ppt) salinities, osmolality, chloride and total protein, but not calcium, concentrations in the hemolymph achieved steady state values 24–48 hr after transfer.
• 2.2. The hemolymph osmolality was a linear function (slope = 0.28) of medium osmolality at salinities between 8 and 40 ppt. It was isosmotic to seawater at 698 mOsm (10 g prawns) and 752 mOsm (30 g), and was hyperosmotic to the medium below isosmotic concentrations, and hypoosmotic to those above.
• 3.3. Hemolymph chloride concentration was isoionic to seawater at 334 mM, and was hyperregulated below isoionic concentrations, and hyporegulated to those above.
• 4.4. P. monodon maintained its hemolymph calcium concentration between 6.4 and 10 mM when medium salinities increased from 8 to 40 ppt.
• 5.5. Total protein concentration in the hemolymph was independent of medium salinity (8–40 ppt) and hemolymph osmolality (540–850 mOsm).

     

Alkenyl sex pheromone analogs in the hemolymph of an arctiid Eilema japonica and several non-arctiid moths

The majority of moth species utilize compounds derived from de novo synthesized fatty acids as their sex pheromones (type I). In contrast, species belonging to two recently diverged moth families, Arctiidae and Geometridae, utilize alkenes and their epoxides, which are derived from dietary essential fatty acids (EFAs), as their sex pheromones (type II). In the latter species, EFAs are considered to be converted into alkenes, often after chain elongation, in specialized cells called oenocytes. These alkenes are transported through the hemolymph to the pheromone gland, from which they are secreted with or without further modifications. We confirmed that the appearance of EFA-derived alkenes in the hemolymph was closely associated with the completion of pheromone gland formation in an arctiid moth Eilema japonica. Analyses of the hemolymph of several moth species utilizing type-I sex pheromones demonstrated the occurrence of (Z,Z,Z)-3,6,9-tricosatriene (T23), a typical type-II component, in the hemolymph of a noctuid Mamestra brassicae and two crambids Ostrinia furnacalis and Ostrinia scapulalis. Our results demonstrated that moths utilizing type-I pheromones have the ability to synthesize type-II sex pheromones, and suggested that recently diverged groups of moths may have secondarily exploited EFA-derived alkenes as sex pheromones.     

Metabolism of amino acids during hyposmotic adaptation in the whiteleg shrimp, Litopenaeus vannamei

The penaeid prawn, Litopenaeus vannamei, was employed to investigate intracellular isosmotic regulation in situations where invertebrates encounter hyposmosis. Hemolymph osmolality was first analyzed to confirm osmoregulatory conditions in the experimental animals, followed by analysis of amino acids in muscle and hemolymph using high-performance liquid chromatography. Total muscle amino acid levels decreased when hemolymph osmolality was extremely low, whereas glycine and l-serine levels increased in the hemolymph. These results suggest that tissue amino acids were released into the hemolymph to lower the osmolality of the tissues for purposes of low-salinity adaptation. Next, oxygen consumption and ammonia excretion rates were examined, and the O/N ratio was determined. Oxygen consumption levels and ammonia excretion rates increased, and the O/N ratio decreased when the animals were exposed to low salinity. These results suggest that amino acids were abundantly consumed as an energy source when animals were exposed to low salinity. To confirm the consumption of particular amino acids, the specific activity of l-serine ammonia lyase was also examined. Specific activity was highest when l-serine levels in the hemolymph were highest. Thus, it appears that l-serine levels increased under hyposmotic conditions due to the consumption of l-serine as an energy source. It was concluded that particular amino acids as osmolytes are likely metabolized as energy sources and consumed for purposes of hyposmotic adaptation.     

Improved replication of the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) in vitro using proteins from Lonomia obliqua hemolymph

The baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV), a member of the family Baculoviridae, has been widely applied as a biopesticide for the control of the velvetbean caterpillar, a pest of soybean crop field. Baculoviruses are considered safe and efficient agents for this purpose, because they do not infect vertebrates, being safe for the health of humans and animals, as well as to the environment. The objective of this work was to identify proteins obtained from Lonomia obliqua hemolymph with potential application in the optimization of baculovirus AgMNPV replication in Sf9 insect cell culture. In this work the improvement of the cell culture and viral replication of the AgMNPV baculovirus was observed when Grace medium was supplemented with 10 % (v/v) Fetal Bovine Serum (FBS), 1 % (v/v) hemolymph extract, or 3 % (v/v) of hemolymph fractions or hemolymph sub-fractions obtained by purifying hemolymph through High Performance Liquid Chromatography. Hemolymph presented a positive effect on the synthesis of polyhedra and enhanced baculovirus replication in Spodoptera frugiperda (Sf9) cells (TCID₅₀/mL), and led to Sf9 cell culture improvement. Grace medium supplemented with 10 % (v/v) FBS and 1 % (v/v) hemolymph provided an increase of baculovirus replication, when the cells were infected with multiplicity of infection of 1. In this case, the baculovirus replication was 6,443.91 times greater than that obtained with the control: Grace medium supplemented with 10 % (v/v) FBS. In addition, this work suggests that hemolymph from L. obliqua could have an interesting application in biotechnology, due to an increase in the viability of the cells and virus replication.