Larval and pupal induction and N-terminal amino acid sequence of lysozyme from Heliothis virescens

Fifth instar larvae and prepupae of Heliothis virescens (tobacco budworm) were injected with live Enterobacter cloacae and bled at different times after vaccination. Immune pupal hemolymph showed a 54 times increase in lysozyme activity when compared with normal larval hemolymph, and an 11 times increase of lysozyme activity when compared with immune larval hemolymph. Lysozyme activity of the normal pupal hemolymph increased as greatly as did lysozyme activity of the immune larval hemolymph after metamorphosis. The pupal immune response with regard to lysozyme was much greater than the larval immune response in H. virescens. Lysozyme was purified by heat treatment at 100 degrees C and a chromatography series that included reverse-phase HPLC. The molecular mass of H. virescens lysozyme was approximately 16 kDa by SDS-PAGE which is greater than other insect lysozymes and chicken lysozyme. Amino acid sequence of the N-terminus showed that H. virescens lysozyme is 82% homologous with lysozyme of Manduca sexta and Galleria mellonella. CNBr cleavage of H. virescens lysozyme produced 11 and 6 kDa peptide fragments indicating that one methionine was present, which was also supported by amino acid analysis. However, methionine was located at the carboxyl terminal side rather than the N-terminal side as judged by the N-terminal sequences of each peptide fragment. The residue 22 in most lepidopteran lysozymes is methionine, whereas H. virescens lysozyme had a leucine at residue 22. There was an amino acid deletion near the carboxyl terminal side of H. virescens lysozyme as also found in Trichoplusia ni.     

Isolation of mannose-binding C-type lectin from Heliothis virescens pupae

A mannose-binding C-type lectin (MBL) was isolated by affinity chromatography from Heliothis virescens immune pupal hemolymph. The immune pupal hemolymph was obtained after bacterial injection of live Enterobacter cloacae bacteria. MBL in mammals acts as an opsonin for phagocytosis and activates the lectin complement pathway of the innate immune response, which leads to killing of gram-negative bacteria and enveloped viruses. The affinity-purified and reduced pupal MBL showed a single band of 36 kDa by SDS-PAGE (12% gel). A dot-immunoblot ELISA (using guinea pig anti-MBL IgG as primary antibody) demonstrated specificity of the antibody for the affinity-purified pupal MBL. The immune pupal hemolymph contained 21 microg of MBL per ml of hemolymph. The amino acid composition of the purified pupal MBL was determined with high amounts of arginine and histidine detected. The presence of MBL in insect pupae has not before been reported and could be important in pupal innate immunity to bacterial infection.     

温度对黄粉虫幼虫生长发育及体液免疫防御能力的影响

【目的】探究饲养温度对黄粉虫Tenebrio molitor幼虫生长发育和体液免疫防御的影响。【方法】测定了不同温度（18, 22, 26和30℃）下饲养的黄粉虫幼虫的发育历期、蛹重、化蛹率;采用抑制区分析法测定了不同温度下饲养的免疫（用生理盐水将大肠杆菌Escherichia coli配制成1×104个菌体/μL悬浮液,用微量注射器将其注入虫体腹部的背面,每头幼虫注射1 μL）和非免疫(注射生理盐水)黄粉虫幼虫血淋巴的抑菌和溶菌酶活性,通过分光光度法测定了其酚氧化酶活性。【结果】结果显示,黄粉虫幼虫发育历期随饲养温度的上升而明显缩短（P<0.0001）,而不同温度下蛹重（P=0.067）与化蛹率（P=0.869）差异不显著。免疫组黄粉虫幼虫血淋巴的抑菌、酚氧化酶和溶菌酶活性随饲养温度上升而降低：抑菌和酚氧化酶活性随温度变化差异极显著（P<0.0001）,溶菌酶活性差异显著（P=0.013）。【结论】本研究结果表明,温度对黄粉虫的生长发育和免疫防御具有较大的影响,低温下黄粉虫幼虫的发育历期延长,但其体液免疫防御能力明显增强。     

Synergistic action of Galleria mellonella anionic peptide 2 and lysozyme against Gram-negative bacteria

Lysozyme and antimicrobial peptides are key factors of the humoral immune response in insects. In the present work lysozyme and anionic defense peptide (GMAP2) were isolated from the hemolymph of the greater wax moth Galleria mellonella and their antibacterial activity was investigated. Adsorption of G. mellonella lysozyme on the cell surface of Gram-positive and Gram-negative bacteria was demonstrated using immunoblotting with anti-G. mellonella lysozyme antibodies. Lysozyme effectively inhibited the growth of selected Gram-positive bacteria, which was accompanied by serious alterations of the cell surface, as revealed by atomic force microscopy (AFM) imaging. G. mellonella lysozyme used in concentrations found in the hemolymph of naive and immunized larvae, perforated also the Escherichia coli cell membrane and the level of such perforation was considerably increased by GMAP2. GMAP2 used alone did not perforate E. coli cells nor influence lysozyme muramidase activity. However, the peptide induced a decrease in the turgor pressure of the bacterial cell. Moreover, in the samples of bacteria treated with a mixture of lysozyme and GMAP2 the sodium chloride crystals were found, suggesting disturbance of ion transport across the membrane leading to cell disruption. These results clearly indicated the synergistic action of G. mellonella lysozyme and anionic peptide 2 against Gram-negative bacteria. The reported results suggested that, thanks to immune factors constitutively present in hemolymph, G. mellonella larvae are to some extent protected against infection caused by Gram-negative bacteria.     

Insect immunity. Isolation from the lepidopteran Heliothis virescens of a novel insect defensin with potent antifungal activity

Lepidoptera have been reported to produce several antibacterial peptides in response to septic injury. However, in marked contrast to other insect groups, no inducible antifungal molecules had been described so far in this insect order. Surprisingly, also cysteine-rich antimicrobial peptides, which predominate in the antimicrobial defense of other insects, had not been discovered in Lepidoptera. Here we report the isolation from the hemolymph of immune induced larvae of the lepidopteran Heliothis virescens of a cysteine-rich molecule with exclusive antifungal activity. We have fully characterized this antifungal molecule, which has significant homology with the insect defensins, a large family of antibacterial peptides directed against Gram-positive strains. Interestingly, the novel peptide shows also similarities with the antifungal peptide drosomycin from Drosophila. Thus, Lepidoptera appear to have built their humoral immune response against bacteria on cecropins and attacins. In addition, we report that Lepidoptera have conferred antifungal properties to the well conserved structure of antibacterial insect defensins through amino acid replacements.     

Involvement of apolipophorin III in antibacterial defense of Galleria mellonella larvae

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. We investigated involvement of apoLp-III in the antibacterial response in Galleria mellonella larvae. Immune challenge with Gram-negative (Escherichia coli, Klebsiella pneumoniae) and Gram-positive (Micrococcus luteus) bacteria led to an increase in the level of apoLp-III in G. mellonella hemolymph, 0.5-2h and 8h after treatment, respectively. ApoLp-III purified from larval hemolymph as well as that present in hemolymph extracts adsorbed on the surface of different bacteria. The adsorption capacity of apoLp-III on bacterial cells prompted us to investigate the effect of this phenomenon on bacterial growth. Our results demonstrate antibacterial activity of apoLp-III against selected Gram-positive and Gram-negative bacteria in vitro. Among bacteria tested, Salmonella typhimurium and K. pneumoniae were the most sensitive to apoLp-III. LIVE/DEAD staining of bacteria incubated with purified apoLp-III revealed their growth inhibition; however, neither morphological changes in the cell shape nor formation of cell aggregates was noticed. The results suggest that apoLp-III is a multifunctional protein in G. mellonella hemolymph.     

Changes in Trace Metals in Hemolymph of Baculovirus-Infected Noctuid Larvae

We studied how biologically relevant trace metals (i.e., micronutrients) in the hemolymph of larval Heliothis virescens and Helicoverpa zea (Lepidoptera: Noctuidae) changed in response to per os baculovirus infection, larval development, and injection of heat-killed bacteria. Concentrations of hemolymph Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, and Zn were measured using inductively coupled plasma-mass spectrometry. H. virescens larvae exhibited greater fluctuations in hemolymph trace metal levels in response to baculovirus infection and development than did H. zea larvae. H. zea single nucleopolyhedrosis virus infection significantly altered the levels of Cu, Fe, Mg, Mn, Mo, and Zn in fourth instar H. virescens larvae. Conversely, in fifth instar H. virescens and both H. zea instar infections, no metal levels were significantly different between infected and uninfected larvae. In fourth instar H. virescens hemolymph, Cu, Fe, Mo, and Zn increased during development. Cu, Fe, Mg, Mn, Mo, and Zn levels changed significantly during development in fifth instar H. virescens as well as both H. zea instars. Based on this analysis, metals were identified whose levels changed during development in both species and during the immune response of H. virescens larvae.     

Stem cells from midguts of Lepidopteran larvae: clues to the regulation of stem cell fate

Previously, we showed that isolated stem cells from midguts of Heliothis virescens can be induced to multiply in response to a multiplication protein (MP) isolated from pupal fat body, or to differentiate to larval types of mature midgut cells in response to either of 4 differentiation factors (MDFs) isolated from larval midgut cell-conditioned medium or pupal hemolymph. In this work, we show that the responses to MDF-2 and MP in H. virescens stem cells decayed at different time intervals, implying that the receptors or response cascades for stem cell differentiation and multiplication may be different. However, the processes appeared to be linked, since conditioned medium and MDF-2 prevented the action of MP on stem cells; MP by itself appeared to repress stem cell differentiation. Epidermal growth factor, retinoic acid, and platelet-derived growth factor induced isolated midgut stem cells of H. virescens and Lymantria dispar to multiply and to differentiate to mature midgut cells characteristic of prepupal, pupal, and adult lepidopteran midgut epithelium, and to squamous-like cells and scales not characteristic of midgut tissue instead of the larval types of mature midgut epithelium induced by the MDFs. Midgut stem cells appear to be multipotent and their various differentiated fates can be influenced by several growth factors.     

Antibacterial hemolymph proteins of Manduca sexta

Exclusion column fractionated immune hemolymph of the M. sexta larva contains five peaks of anti-E. coli activity with molecular weights of greater than 140 kD and approximately 91, 54, 14 and 4 kD, plus one peak of lysozyme activity with a molecular weight of 17 kD. Purification of the 54 kD peak showed that this peak consists of the previously described M18 proteins which have monomeric weights of approximately 20 kD and had antibacterial activity against certain gram negative bacteria. Approximately 80% of the total hemolymph antibacterial activity was detected in the 14 and 4 kD peaks. These proteins, which kill both gram negative and gram positive bacteria, appeared to be directly analogous to the cecropins of H. cecropia. The greater than 140 and 91 kD peaks constituted only a minor part of the total antibacterial activity.     

Immune depression in Rhodnius prolixus by seco-steroids, physalins

A comparative study of the effects of physalins, seco-steroidal substances of Physalis angulata (Solanaceae), on the immune reactions of R. prolixus was carried out. Ecdysis and mortality were not affected by treatment with physalins B, D, F or G (1-10 microg/ml of blood meal). R. prolixus larvae fed with blood containing physalins and inoculated with 1 microl of Enterobacter cloacae beta12 (5 x 10(3)/insect) exhibited mortality rates three times higher than controls. The insects treated with physalin B, and F (1 microg/ml) and inoculated with E. cloacae beta12 showed significant differences on lysozyme activity in the hemolymph compared to untreated insects. Furthermore, physalin D (1 microg/ml) significantly reduced the antibacterial activity. Concerning cellular immune reactions, all insects treated with physalins (1 microg/ml), exhibited drastic reductions in the quantity of yeast cell-hemocyte binding and subsequent internalization. Insects inoculated with bacteria and treated with physalins B, F and G showed reductions of microaggregate formation but physalin D did not. Physalins B and F also reduced total hemocyte count in the hemolymph. These results suggest that, in different ways, probably due to their different chemical structures, physalin B, D, F and G are immunomodulatory substances for the bloodsucking insect, R. prolixus.     

Correlation of hemocyte counts with different developmental parameters during the last larval instar of the tobacco hornworm, Manduca sexta

We determined the changes in hemocyte titer and in the abundance of hemocyte types of the tobacco hornworm Manduca sexta during the fourth and fifth larval stadium and the beginning of the pupal stadium. As we analyzed the samples of individual insects at daily intervals, we were able to correlate phenotypical features, body weight, as well as total protein content and lysozyme activity in the hemolymph with the observations on hemocytes. In the course of the fifth larval stadium, the hemocyte titer decreased slightly and declined further after pupation. Using calculated values for total hemocyte numbers, females had about five times and males three times more hemocytes in the circulating population at the beginning of the wandering stage (in the middle of the fifth larval stadium) than immediately after the last larval--larval molt (from the fourth to the fifth larval stadium). This sexual difference was mainly due to an increase in the number of plasmatocytes, which was more prominent in females than in males. Granular cells were dominant in early fifth larval stadium while plasmatocytes were the most abundant cells in pupae. Oenocytoids and spherule cells disappeared during the wandering stage. Lysozyme activity in the hemolymph rose to a maximum during the wandering stage, with females having lysozyme values twice as high as those for males. These changes in lysozyme activity, however, did not correlate with the increase of total hemolymph protein titer which occurred already at the beginning of the wandering stage. We postulate that changes in hemocyte titers are under direct hormonal control, which has to be proven in future experiments.     

Antibacterial activity in the hemolymph of myriapods (Arthropoda)

The hemolymphs of two diplopod (Chicobolus sp. and Rhapidostreptus virgator) and two chilopod species (Lithobius forficatus and Scolopendra cingulata) were tested for the presence of antibacterial substances using Petri dish tests. The native hemolymph of all species had substances acting on living Micrococcus luteus, whereas only Rhapidostreptus, Scolopendra, and Lithobius were effective against lyophilized Micrococcus. The antibacterial activity against living Micrococcus increased after inoculation with bacteria (Enterobacter cloacae beta-12) in Chicobolus and Rhapidostreptus and also against lyophilized Micrococcus in the latter. Thus, these effects appear to be inducible. None of the myriapods tested had any bacteriostatic effect on Escherichia coli D-31 whereas the growth of gram-negative E. cloacae was inhibited. The antibacterial substances in the diplopod species were unstable when heated but were resistant to freezing. At least two antibacterial substances (a lysozyme-like one and another substance) are considered to occur in Myriapoda.     

Characterization and cDNA cloning of hinnavin II, a cecropin family antibacterial peptide from the cabbage butterfly, Artogeia rapae

Hinnavins, together with lysozymes, are the main types of antibacterial peptides/proteins previously isolated from the larval haemolymph of the cabbage butterfly, Artogeia rapae as part of the humoral immune response to a bacterial invasion. One of these antibacterial peptides, named hinnavin II, was purified and characterized after cDNA cloning. The purified hinnavin II was more active against Gram negative than against Gram positive bacteria. Hinnavin II also showed a powerful synergistic effect on the inhibition of bacterial growth with purified lysozyme. The cDNA has a total length of 186 bp with a 114 coding region. The deduced protein sequence contains 38 amino acids with a coding capacity of 4142.8 Da. The result of a multiple sequence alignment and phylogenetic analysis with Clustal W indicated that mature hinnavin II showed an approximately 78.9% amino acid sequence identity with cecropin A and originated from a group containing mostly lepidopteran cecropins.     

Effect of Pseudomonas aeruginosa crude proteolytic fraction on antibacterial activity of Galleria mellonella haemolymph

The antibacterial activity of immune haemolymph Galleria mellonella directed against Escherichia coli D31 was destroyed by Pseudomonas aeruginosa crude proteolytic fraction. This was demonstrated by diffusion well assay and acid gel electrophoresis and subsequent bioautography. On the contrary, lysozyme activity appeared to be insensitive to extracellular proteases of P. aeruginosa when activity was determined using the bioautography method. In addition, no change in lysozyme protein level was observed by immunoblotting with specific antibodies directed against G. mellonella lysozyme, which confirmed that lysozyme was not degraded by the crude proteolytic fraction of P. aeruginosa. However, a significant decrease of lysozyme activity in naive and immune haemolymph exposed to the action of P. aeruginosa proteins determined by using diffusion well assay was observed. Mechanisms of the observed inhibition require further studies.     

The involvement of protein kinase A in the immune response of Galleria mellonella larvae to bacteria

The role of protein kinase A (PKA) in the humoral immune response of the greater wax moth Galleria mellonella larvae to live gram-positive bacteria Micrococcus lysodeikticus and gram-negative bacteria Escherichia coli was investigated. The immune challenge of larvae with both kinds of bacteria caused an increase in fat body PKA activity depending on the injected bacteria. Gram-positive M. lysodeikticus was a much better inducer of the enzyme activity than gram-negative E. coli. The PKA activity was increased about 2.5-fold and 1.5-fold, after M. lysodeikticus and E. coli injection, respectively. The in vivo inhibition of the enzyme activity by a cell permeable selective PKA inhibitor, Rp-8-Br-cAMPS, was correlated with considerable changes of fat body lysozyme content and hemolymph antimicrobial activity in bacteria-challenged insects. The kinetics of changes were different and dependent on the bacteria used for the immune challenge of G. mellonella larvae.     

蝶蛹金小蜂寄生对菜粉蝶体液免疫反应的影响(英文)

以蝶蛹金小蜂及其寄主菜粉蝶为研究对象 ,研究了内寄生蜂对寄主体液免疫反应的影响。当寄主蛹被寄生后 1 2h或第 4和 5d时 ,血淋巴中酚氧化酶活性明显增高。寄生蛹血淋巴中血细胞凝集素活性始终高于针刺和未寄生蛹 ;同样 ,寄生蛹血淋巴的抗菌活性也明显增强 ,而后两者处理蛹的活性则很微弱。由此可知 ,该蜂寄生能引起寄主体液免疫因子活性的不同程度的变化     

Dirofilaria immitis: effect on hemolymph polypeptide synthesis in Aedes aegypti during melanotic encapsulation reactions against microfilariae

[35S]Methionine-labeled hemolymph polypeptides from adult, female Aedes aegypti Liverpool strain mosquitoes inoculated with the microfilariae of the filarial nematode Dirofilaria immitis were compared with those from saline-inoculated and uninoculated controls by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. SDS-PAGE analysis of cell-free hemolymph collected via perfusion at 6, 12, 24, 48, 72, and 96 hr postinoculation (PI) detected the enhanced expression of an 84-kDa polypeptide. This polypeptide, expressed constitutively in the hemolymph of all three groups of mosquitoes, increased considerably in inoculated mosquitoes as time progressed as compared with uninoculated controls. Moreover, the 84-kDa polypeptide was expressed at higher levels in D. immitis-inoculated mosquitoes than in saline-inoculated controls. This stimulation of de novo biosynthesis of the 84-kDa polypeptide in inoculated mosquitoes may play a role in the immune response of mosquitoes. Since it is likely that the wound healing response in insects involves many of the same chemical processes as occur in melanotic encapsulation reactions of mosquitoes against filarial worms, the preferential expression of the 84-kDa polypeptide in saline-inoculated mosquitoes seen in this study may reflect a wound healing response. The greater increase in synthesis of this protein in D. immitis-inoculated mosquitoes may reflect production of melanotic material required for parasite destruction as well as for wound healing.     

革兰氏阳性菌苏云金芽孢杆菌和革兰氏阴性菌大肠杆菌诱导的柞蚕蛹生理指标变化

【目的】明确柞蚕Antheraea pernyi对外源微生物防御性生理变化规律,为柞蚕的病害防治和合理饲养提供理论依据。【方法】本研究选用革兰氏阳性菌苏云金芽孢杆菌Bacillus thuringeinsis(Bt)和革兰氏阴性菌大肠杆菌Escherichia coli(Ec)为外源诱导微生物,调整至10~6～10~8cfu/m L菌液,灭活后处理柞蚕蛹,诱导24,48,72和96 h后不同时间测定血淋巴蛋白含量、PO活性、CAT活性、抗菌活性和溶菌酶活性等生理指标。【结果】Ec和Bt诱导柞蚕蛹导致各生理指标出现显著变化,但两种菌株诱导生理指标变化规律差异明显,Ec高浓度诱导72 h会增加血淋巴蛋白含量,而Bt各浓度诱导会在24,48和96 h增加血淋巴蛋白含量。免疫防御关键酶系PO和CAT活性变化规律在不同菌株诱导后差异更明显,Ec诱导后,PO活性随着时间增加表现为先升高后降低的趋势,CAT活性呈现"升高-降低-升高"的规律;而Bt诱导后PO活性表现为"升高-降低-升高"的规律,CAT活性随诱导时间增加变化规律不明显,但有随菌液浓度增加而降低的趋势。对抗菌活性测定表明,Ec和Bt诱导都会显著增加蛹粗酶液抗菌活性,溶菌酶活性也会极显著增加,但2个指标高峰值出现的时间会有明显差别。【结论】本研究结果表明Ec和Bt不同处理均可诱导柞蚕蛹产生明显防御反应,但柞蚕蛹生理指标变化规律与不同种类微生物及处理时间和浓度有关,推测革兰氏阳性菌和革兰氏阴性菌具有不同的诱导防御反应机制。研究结果可以为外源微生物侵染柞蚕后的免疫防御反应规律提供理论指导。     

Purification and Characterization of Apolipohorin-III in Cabbage Butterfly, Artogeia rapae

ABSTRACT The apoLp-III in the adult hemolymph of Artogeia rapae can associate reversibly with lipophorin. The apoLp-III was purified from the adult and larval hemolymph by KBr density gradient ultracentrifugation, gel permeation chromatography anion exchange chromatography and preparative electrophoresis (Prep Cell). ApoLp-I, ApoLp-II and apoLp-III have the molecular weights of 212 kDa, 80 kDa respectively. N-terminal sequence of apoLp-III were determined. The N-terminal amino acid sequence of apoLp-III shows 50-57% identity with those of other lepidopteran insects. apoLp-III has the antibacterial activity. Injection of bacteria increase the concentration of apoLp-III in the hemolymph, indicating that apoLp-III plays a role in insect immunity. Immunological analysis was also investigated with the anti-apoLp-III.     

Purification of lectin induced in the hemolymph of Sarcophaga peregrina larvae on injury

A lectin was purified from the hemolymph of Sarcophaga peregrina larvae, obtained after injury of their body wall. This lectin agglutinated sheep red blood cells markedly and the hemagglutinating activity was inhibited by galactose and lactose. The active lectin was found to have a molecular weight of 190,000 and to consist of four alpha subunits and two beta subunits, with molecular weights of 32,000 and 30,000, respectively. During the early pupal stage, similar hemagglutinating activity in the hemolymph increased to several times than in larval hemolymph. This activity was completely inhibited by the antibody prepared against the lectin purified from the hemolymph of injured larvae. Thus, the same protein having lectin activity is apparently induced under two different physiological conditions: injury of the body wall of larvae and during pupation. The biological significance of this lectin is discussed.