Characterization of a novel protein with homology to C-type lectins expressed by the Cotesia rubecula bracovirus in larvae of the lepidopteran host,Pieris rapae

Polydnaviruses are essential for the survival of many Ichneumonoid endoparasitoids, providing active immune suppression of the host in which parasitoid larvae develop. The Cotesia rubecula bracovirus is unique among polydnaviruses in that only four major genes are detected in parasitized host (Pieris rapae) tissues, and gene expression is transient. Here we describe a novel C. rubecula bracovirus gene (CrV3) encoding a lectin monomer composed of 159 amino acids, which has conserved residues consistent with invertebrate and mammalian C-type lectins. Bacterially expressed CrV3 agglutinated sheep red blood cells in a divalent ion-dependent but Ca2+-independent manner. Agglutination was inhibited by EDTA but not by biological concentrations of any saccharides tested. Two monomers of approximately 14 and approximately 17 kDa in size were identified on SDS-PAGE in parasitized P. rapae larvae. The 17-kDa monomer was found to be an N-glyscosylated form of the 14-kDa monomer. CrV3 is produced in infected hemocytes and fat body cells and subsequently secreted into hemolymph. We propose that CrV3 is a novel lectin, the first characterized from an invertebrate virus. CrV3 shows over 60% homology with hypothetical proteins isolated from polydnaviruses in two other Cotesia wasps, indicating that these proteins may also be C-type lectins and that a novel polydnavirus lectin family exists in Cotesia-associated bracoviruses. CrV3 is probably interacting with components in host hemolymph, resulting in suppression of the Pieris immune response. The high similarity of CrV3 with invertebrate lectins, as opposed to those from viruses, may indicate that some bracovirus functions were acquired from their hosts.     

Juvenile hormone esterase activity and ecdysteroid titer in Heliothis virescens larvae injected with Microplitis croceipes teratocytes

Juvenile hormone esterase (JHE) activity in the hemolymph of 5th-instar Heliothis virescens larvae injected with Microplitis croceipes teratocytes was inversely related to the number of teratocytes injected. JHE activity in the hemolymph of larvae injected with 750 3-day-old teratocytes (the approximate number from one parasitoid embryo) was depressed to less than 5% of those levels found in control larvae. During the latter portion of the digging stage and in the burrowing-digging (BD) stage JHE activity in larvae treated with 350 teratocytes was approximately 40% of control values. However, injection of 180 teratocytes did not significantly affect JHE titers. Two-day-old teratocytes caused the greatest reduction in JHE titer with decreasing effects observed with injections of 3- to 6-day-old teratocytes. Nevertheless, because 2-day-old teratocytes were difficult to separate from host hemocytes, 3-day-old teratocytes were used in most of these studies. Injections of nonparasitized H. virescens hemolymph plasma, Micrococcus luteus bacterial cell walls, washed M. croceipes eggs, or teratocytes from Cotesia congregata did not depress JHE titers. Teratocyte injections also significantly reduced growth of host fat body. Ecdysteroid titers in cell formation, day 2 (CF2) larvae injected as new 5th instars with 350 3-day-old teratocytes failed to increase, as compared to noninjected and saline-injected controls. An injection of 1 μg/larva of 20-hydroxyecdysone at the BD stage permitted normal pupation in 50% of the teratocyte-treated larvae as compared to 0% pupation for teratocyte-treated control larvae not treated with 20-hydroxyecdysone. Teratocytes seem to be responsible for the inhibition of JHE release and thus indirectly impact on ecdysteroid titers. © 1992 Wiley-Liss, Inc.     

Apoferritin in the vacuolar system of insect hemocytes

Electron microscopy showed no holoferritin in either the cytosol or the vacuolar system of hemocytes (granulocytes) from normal Calpodes ethlius larvae. This does not mean that ferritin is normally absent from hemocytes, since apoferritin lacks contrast and would not be observed. In vitro iron in glycerol treatment of hemocytes from normal larvae caused holoferritin cores to be visible in the rough endoplasmic reticulum, suggesting that hemocytes from normal larvae contain apoferritin. Hemocytes are therefore like the fat body, and could also be a source of hemolymph ferritin. After loading the hemolymph with iron in vivo, many holoferritin cores were resolvable in the vacuolar system of some hemocytes. Ferritin synthesis can therefore be induced by elevated hemolymph iron levels. Iron loading of epidermis and heart showed similar ferritin cores but more rarely. In all tissues they occurred in the secretory pathway and not in the cytosol.     

Wound Responses in Insects

For over a century, the study of specific antipathogenic strategiesin insects has been confounded by non-specific responses tointegumental invasion. Experimental injury to diapausing Hyalophoracecropia silkmoth pupae elucidated some of the events inherentin this response—increased oxygen consumption and DNAand RNA synthesis leading to de novo synthesis of proteins,some of which are constituents of the adult protein cohort aswell as some injury-specific ones. The mechanism which enforcesdiapause is apparently released by integumental injury as wellas by normal developmental stimuli. Recent work has concentratedon purification of antipathogenic and injury-specific proteins,the possible involvement of lectins in the immune response,and localization of synthesis of these proteins in hemocytesand fat body cells. At least ten different hemolymph proteinswhich are synthesized by fat body cells in response to inoculationof lepidopteran species with bacteria currently are being isolated.The hemolymph of H. cecropia contains lectins which are synthesizedby hemocytes. Analysis of in vitro incorporation of ['H]leucineby hemocytes into proteins reveals that these lectins apparentlyare not constituents of the secreted injury response proteincomplex in fifth instar caterpillars or diapausing pupae, norare hemolymph lectin titers significantly different in healthyversus diseased or injured animals. However, intracellular lectinconcentrations may increase upon injury. Increased lectin titerand induction of bactericidal activity coincide in another holometabolousspecies, the fleshfly Sarcophaga peregrina. Pursuit of thesestudies may elaborate our knowledge of insect cellular immunity.     

Esterification of cholesterol and cholestanol in the whole body,tissues, and frass of Heliothis zea

The quantity of free and esterified sterols in the whole body, intestine, hemolymph, fat body, and frass of 6th-instar larvae of H. zea, fed cholesterol or cholestanol, was measured in order to determine if there was a difference in the utilization of these two molecules. The principal sterol in the tissues of the larvae was cholestanol or cholesterol, when they were fed diet containing these two molecules, respectively; there was little, if any, metabolism of dietary cholestanol to cholesterol. There was little or no difference in the amount of total sterol in the whole body, tissues, or frass of larvae fed the two different diets, indicating that the absence of a Δ⁵-bond in cholestanol does not prevent the uptake or distribution of this sterol to various tissues. However, the relative percentage of steryl ester was significantly higher in prepupae reared on a diet containing cholestanol instead of cholesterol (6–7-, 4-, 13-, 4-, and 2-fold increase, for the whole body, intestine, hemolymph, fat body, and frass, respectively). The average percentage of total sterol that was esterified in the tissues was greater in the fat body (10.8 ± 15.4 and 44.2 ± 12.3%, respectively, for larvae fed cholesterol and cholestanol) than in the hemolymph (0.5 ± 0.1 and 6.3 ± 0.8%) and intestine (1.2 ± 0.1 and 4.7 ± 1.1%). The percentage of sterol that was esterified in the frass of larvae was large (26.9 ± 3.7 and 48.2 ± 0.5%, respectively, for larvae fed cholesterol and cholestanol). Therefore, the fact that larvae of H. zea fed cholestanol, instead of cholesterol, contain this saturated molecule as their principal tissue sterol and preferentially esterify it may explain, at least in part, why their rate of growth on cholestanol is slower than on cholesterol.     

Humoral factor activating the Sarcophaga lectin gene in cultured fat body

On culture of the fat body of Sarcophaga peregrina (flesh fly) larvae in modified Grace's medium, the Sarcophaga lectin gene was activated only when the medium was supplemented with larval hemolymph. The expressions of the genes for the storage protein and the sarcocystatin A were not affected by supplementing the medium with the hemolymph. Thus the hemolymph contained a factor that specifically activated the Sarcophaga lectin gene. This factor seemed to be a heat-stable, low molecular weight compound that was probably not a peptide, and to activate genes in the fat body for defense proteins that are known to be expressed in response to injury of Sarcophaga larvae.     

Purification and characterization of a male-specific protein in the hemolymph of the wax moth,Galleria mellonella L.

A male-specific protein (MSP) present only in males was identified from the hemolymph of the wax moth, Galleria mellonella L., by polyacrylamide gel electrophoresis (PAGE) and purified by anion-exchange chromatography. MSP has a native molecular mass of 55 kDa and consists of two 27-kDa subunits. An isoelectric point of MSP was measured to be approximately 5.8. MSP is a glycoprotein that contains 1.7% carbohydrate. The compositional analysis of carbohydrate component indicated a predominance of fructose and glucose. MSP also contains large amounts of asparagine, aspartic acid, glutamine, glutamic acid, and lysine but small amounts of tyrosine, methionine, and tryptophan. Western blot analysis of the hemolymph of each developmental stage indicated that MSP is present in the hemolymph of 8-day-old pupa and adult. Also, results from Western blotting indicated that MSP is not present in the tissues of larvae and of female adults but appears in the fat body of male pupae and adult and testis of adult. The fat body and testis of male pupae and adult were cultured in vitro to trace the place and time of MSP synthesis. The fat body began to synthesize MSP in late pupae and showed active synthesis during the adult stage. The distribution of MSP in the testis was observed by electron microscopic immunogold labeling, using the antibody against MSP. MSP is present between the germinal cysts and is taken up through the basal surface of the seminiferous tubular epithelium. Arch. Insect Biochem. Physiol. 37:257–268, 1998. © 1998 Wiley-Liss, Inc.     

Isolation of cDNA clones coding for humoral lectin of silkworm, Bombyx mori, larvae.

In the hemolymph of the silkworm, Bombyx mori, lectin with hemagglutinating activity against sheep red blood cells increases at larval-larval ecdysis and at spinning stage (Suzuki and Natori, 1983) and is induced by infection with cytoplasmic polyhedrosis virus. A Bombyx lectin polypeptide with molecular weight approx 280K is responsible for hemagglutinating activity, since antiserum raised against this polypeptide inhibited hemagglutinating activity. The site of synthesis of Bombyx lectin was determined by primary tissue cultures of fat body and hemocytes. A hemagglutinating activity assay demonstrated that hemocyte is responsible for the release of hemagglutinin into the culture medium. Isolation of cDNA clones coding for Bombyx lectin was carried out on the cDNA library prepared in an expression vector lambda gt11 starting with poly(A)+ RNA from spinning larval hemocytes. As a result of immunoscreening, several positive clones were obtained, and the cDNA clones were characterized.     

Fat body and hemocyte contribution to the antimicrobial peptide synthesis in <Emphasis Type="Italic">Calliphora vicina</Emphasis> R.-D. (Diptera: Calliphoridae) larvae

Antimicrobial peptides accumulated in the hemolymph in response to infection are a key element of insect innate immunity. The involvement of the fat body and hemocytes in the antimicrobial peptide synthesis is widely acknowledged, although release of the peptides present in the hemolymph from the immune cells was not directly verified so far. Here, we studied the presence of antimicrobial peptides in the culture medium of fat body cells and hemocytes isolated from the blue blowfly Calliphora vicina using complex of liquid chromatography, mass spectrometry, and antimicrobial activity assays. Both fat body and hemocytes are shown to synthesize and release to culture medium defensin, cecropin, diptericins, and proline-rich peptides. The spectra of peptide antibiotics released by the fat body and hemocytes partially overlap. Thus, the results suggest that insect fat body and blood cells are capable of releasing mature antimicrobial peptides to the hemolymph. It is notable that the data obtained demonstrate dramatic difference in the functioning of insect antimicrobial peptides and their mammalian counterparts localized into blood cells’ phagosomes where they exert their antibacterial activity.     

Venom of Euplectrus separatae causes hyperlipidemia by lysis of host fat body cells

Although the lepidopteran larva Pseudaletia separata is attacked by the gregarious ectoparasitoid Euplectrus separatae, it continues to feed and grow. Lipid concentration in the hemolymph of the parasitized host was higher than that of the nonparasitized host from 3 to 8 days after parasitization. Artificial injection of parasitoid venom also elevated lipid concentration in the host hemolymph. One day after venom injection the host's fat body contained many lipid particles, but most of the lipid particles disappeared 7 days later. Light microscopy and transmission electron microscopy showed the lipid particles leaving the fat body cells as a result of the lysis of the fat body cells. These results suggest that the venom elevated the lipid concentration in the host hemolymph by provoking the release of lipid particles from the fat body. Though most of the lipid particles were freely floating in the host hemolymph, a portion of the released lipid particles were phagocytized by hemocytes. The amount of lipid that was loaded to lipophorin in the hemolymph of the venom-injected host was measured, but it was not sufficient to explain the high lipid titer in the hemolymph of parasitized and venom-injected host larvae. The fact that parasitoid larva consumed many hemocytes as evidenced by their presence in the midgut supported the hypothesis that the parasitoid larvae fed on the host hemolymph containing the free lipid particles, the hemocytes phagocytizing the lipid particles, and the lipid-loaded lipophorin. The possibility of the venom contribution to the disruption of the intercellular matrix was examined. The venom showed high activity of matrix metalloproteinase (MMP), especially when it was mixed with the hemolymph of non-parasitized 5th instar larvae. We suggest that the MMP in the venom was activated by some components of the host hemolymph. On the other hand, the venom mixed with hemolymph could not decompose gelatin on zymography, suggesting that the venom-MMP is a different type from gelatinase. Activity of phospholipases A(2), B, C and hyaluronidase were measured with agar plates. High activities of phospholipase B and hyaluronidase were detected. These results suggest that the venom-MMP initially attacked the specific site of the intercellular-matrix of the fat body, and then the hyaluronidase and the phospholipase B cause lysis of the fat body cell, allowing lipid particles to be released into the host hemolymph.     

In vivo development of the entomogeneous hyphomycetePaecilomyces farinosus in hostSpodoptera exigua (beet armyworm) larvae

The in vivo development of the entomogenous hyphomycetePaecilomyces farinosus inSpodoptera exigua (beet armyworm) larvae was examined using light and electron microscopic techniques. Blastospores injected into larval hemocoels (500 blastospores/larva) were immediately ingested by phagocytic hemocytes, and no fungal cells were detected in the hemolymph until 36 h post-injection. As indicated by immunocytochemical methods, the in vivo-produced blastosopres, in contrast to in vitro blastospores, lacked a galacto-mannan surface layer required for opsonization by aS. exigua humoral lectin. Therefore, these in vivo cells were not recognized by phagocytic granulocytes and were freely-circulating in the hemolymph. Hyphae differentiating from the blastospores were recognized by the hemocytes and induced formation of multicellular hemocytic nodules. By 72 h post-injection, mycelia were observed emerging from the nodules and by 96 h, larvae had become mummified due to extensive proliferation of the fungus throughout host tissues. Neither phagocytosis of the initially injected in vitro-produced blastospores nor nodule formation around hyphal cells later in the infection process was effective in stopping fungal growth. The in vivo development ofP. farinosus was similar to that of another hyphomycete,Beauveria bassiana except that in the latter case, extensive nodule formation was inhibited by the production of fungal metabolites.     

Different course of proteolytic inhibitory activity and proteolytic activity in Galleria mellonella larvae infected by Nosema algerae and Vairimorpha heterosporum

The activity of protease inhibitors and proteases was studied in the hemolymph, gut, and fat body of 7th-instar larvae of Galleria mellonella infected by two microsporidia, Nosema algerae and Vairimorpha heterosporum. The increase in inhibitory activity in the hemolymph was substantial, and coincided with the development of the disease. The increase in inhibitory activity in the gut was almost doubled by N. algerae as compared with V. heterosporum, whereas the increase in inhibitory activity in fat body was found only in V. heterosporum-infected larvae. The course of proteolytic activity followed an inverse pattern to the elevated activity of inhibitors in the gut and the fat body, and rose only in moribund larvae at the end of the course of V. heterosporum infection. The differences in the pattern of proteases and inhibitors reflect the organ specificity of each of the microsporidia.     

Anti-tumor factor in insects' hemolymph

We have purified a lectin from the hemolymph of Sarcophaga peregrina larvae, obtained after injury of their body wall. This lectin recognizes galactose residues and suggested to be involved in the defense mechanism of this insect. We also demonstrated that this lectin induced cytotoxic effects on tumor cells in the presence of murine macrophages. It was found that the murine macrophages had Sarcophaga lectin binding proteins. Using pupal hemocytes and fatbody of this insect, we established an in vitro system that mimics dissociation of the fatbody in vivo. New membrane protein, induced on the surface of the hemocytes at pupation, suggested to participate in the recognition of the fatbody.     

Regulation of hemoglobin synthesis by ecdysterone and juvenile hormone during development of Chironomus thummi (Diptera)

Chironomus thummi contains nine soluble hemoglobins (Hbs) in the larval hemolymph which can be resolved by 12.7% acrylamide gel electrophoresis (pH 8.65). Hemoglobins 2 and 3 are stage specific for the 4th instar and are first detected by day 4 of this stage in vivo, being absent in the 3rd instar. Fat-body cultures in the presence of 3H-delta-aminolevulinic acid and 14C-amino acids synthesize and secrete labelled Hbs, as was assayed by acrylamide gel electrophoresis and immunoprecipitation of Hbs recovered from the culture medium. During development from 3rd instar to pupa, Chironomus fat body undergoes functional changes, being actively involved in Hb synthesis in intermolt periods and inactive with respect to Hb production during molting. The repression of Hb synthesis is reversed following the molt from the 3rd instar to the 4th instar. Metamorphosis is related to a gradual and irreversible loss of Hb synthesis and secretion by the fat body. The treatment of fat body in vitro with ecdysterone inhibits Hb synthesis in tissue from intermolt animals, even in the presence of excess methoprene, a potent juvenile hormone analogue. In contrast, immunoprecipitation of the translation products from a wheat-germ cell-free system, using mRNA from ecdysterone-treated 4th-instar fat body as a template, shows significant synthesis of globins, suggesting that ecdysterone does not affect the amount or template activity of globin messages. Methoprene induces the precocious in vitro synthesis of Hbs 2 and 3 in day-2 4th-instar fat body and enhances all Hb synthesis in the absence of ecdysterone. In vitro treatment with methoprene activates newly molted fat body to synthesize Hbs 2 and 3 in vitro. The process of Hb induction by this analogue is completely inhibited by actinomycin D or ecdysterone. Fat body from animals already exposed to high endogeneous ecdysterone titer are insensitive to treatment with this juvenile hormone analogue. Intermolt larvae normally possess stable Hb mRNA molecules, because actinomycin-D administration in vitro does not affect Hb synthesis for as long as 30 h, whereas it effectively inhibits all RNA synthesis in the fat body. Immunoprecipitation of globin translated in vitro from mRNA from 2-day-old 4th-instar larvae treated in vivo with methoprene shows enhanced synthesis of globins 2 and 3, as compared to controls with no treatment. It is suggested that both juvenile hormone and ecdysterone regulate Hb synthesis in Chironomus; juvenile hormone affecting the activity of Hb genes, and ecdysterone modulating the level of Hb gene expression.     

Changes in <Emphasis Type="Italic">Galleria mellonella</Emphasis> lysozyme level and activity during <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> infection

The level of lysozyme in fat body, hemocytes and cell-free hemolymph from Galleria mellonella larvae infected with Pseudomonas aeruginosa was determined and evaluated. In the samples of fat body and hemocytes, an increase in lysozyme content was detected 1 d after infection and then a significant decrease was observed after a prolonged infection time. In the case of cell-free hemolymph, an increase in the lysozyme level was noticeable during the first 30 h post injection and stayed at a similar level for 42 h. The smaller decrease of the lysozyme level after 42 h might be associated with the development of bacteremia of P. aeruginosa in insects. In addition, the gradual increase in the content of lysozyme correlated with the increase of its activity in the hemolymph of the infected larvae as a response to injection with P. aeruginosa. The G. mellonella lysozyme appeared to be insensitive to extracellular proteinases produced in vivo by P. aeruginosa.     

In vivo and in vitro effects of epofenonane on juvenile hormone esterase activity in tissues of last stadium larvae of Trichoplusia ni

Topical application of the juvenoid, epofenonane, to last stadium postwandering larvae of Trichoplusia ni caused a precocious elevation of juvenile hormone esterase (JHE) activity that was tissue speific and time dependent. This increase in enzyme activity over controls was most dramatic in the hemolymph, whereas increases in the fat body were lower. Antibodies raised against JHE reacted on Western blots with a fat body and hemolymph protein present in epofenonane treated and untreated last stadium day 3 larvae. The abundance of this protein, which comigrated with JHE, closely coincided with the temporal increases in JHE catalytic activity that occurred in response to treatment in vivo with epofenonane.The presence of epofenonane (5–10,000 nM) in the medium at the start of fat body incubations failed to shift the temporal appearance of JHE activity or boost activity levels significantly over those of controls. If larvae were treated in vivo with epofenonane before fat body tissue was removed, only a small, but significant increase in JHE activity was found in vitro. The rate of enzyme secretion was insufficient to account for the rapid increases in enzyme activity that occur in the hemolymph in response to epofenonane, even though tissue held in vitro was deemed viable by monitoring lactate dehydrogenase activity in the medium, fat body intracellular ATP, and the incorporation of [³⁵S]methionine into fat body protein. Fat body tissue removed from various aged last stadium larvae released enzyme at different rates in vitro.     

Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.     

In vitro release of lipophorin from the fat body of nondiapause and diapause larvae of the Southwestern corn borer,Diatraea grandiosella

The release of lipophorin and total protein was examined from the fat body of nondiapause and diapause larvae of the southwestern corn borer, Diatraea grandiosella, incubated in vitro in Grace's medium. The characteristics of the released lipophorin were compared to those of the high-density lipophorin present in the hemolymph of nondiapause and diapause larvae. Over a 4 h incubation period, the fat body of nondiapause larvae released about 1.5 times more total protein and 2 times more lipophorin per mg dry weight than did that of diapause larvae. Lipophorin isolated from the medium in which fat bodies of nondiapause and diapause larvae had been incubated and from the plasma of nondiapause and diapause larvae had similar mean densities of 1.115, 1.112, 1.117 and 1.119 g/ml, respectively. Although the lipid classes detected in lipophorin isolated from the fat body incubation medium and hemolymph were identical, more polar lipids and less diacylglycerol were associated with lipophorin isolated from fat body incubation medium then were associated with lipophorin isolated from the hemolymph. Sterols accounted for about 11% of the total lipids of lipophorin isolated from the fat body incubation medium, whereas they accounted for about 20% of the total lipids of lipophorin from hemolymph. We conclude that the fat body of feeding nondiapause larvae and nonfeeding diapause larvae releases high-density lipophorin.