Physiological evidence of integrin-antibody reactive proteins influencing the innate cellular immune responses of larval Galleria mellonella hemocytes.

Larval Galleria mellonella (L.) hemocytes form microaggregates in response to stimulation by Gram-positive bacteria. Hemocyte adhesion to foreign materials is mediated by the cAMP/ protein kinase A pathway and the ß-subunit of cholera toxin using a cAMP-independent mechanism. Cholera toxin-induced microaggregation was inhibited by the integrin inhibitory RGDS peptide, implying integrins may be part of the mechanism. Based on the types of mammalian integrin-antibody reactive proteins affecting hemocyte adhesion and bacterial-induced responses α5 , αv , ß1 , and ß3 subunits occurred on both granular cell and plasmatocyte hemocyte subtypes. A fluorescent band representing the binding of rabbit α5 -integrin subunit antibodies occurred between adhering heterotypic hemocytes. The frequency of the bands was increased by cholera toxin. The α5 and ß1 rabbit integrin subunit antibodies inhibited removal of Bacillus subtilis (Cohn) from the hemolymph in vivo. A α5 ß1 -specific synthetic peptide blocker similarly diminished hemocyte function whereas the αv ß3 -specific inhibitory peptide and the corresponding integrin subunit antibodies did not influence nonself hemocyte activities. Western blots revealed several proteins reacting with a given integrin-antibody subtype. Thus integrin-antibody reactive proteins (which may include integrins) with possible α5 and ß1 epitopes modulate immediate hemocyte function. Confocal microscopy established plasmatocyte adhesion to and rosetting over substrata followed by granular cell microaggregate adhesion to plasmatocytes during early stage nodulation.

Functional Characterization of Outer Membrane Proteins (OMPs) in Xenorhabdus nematophila and Photorhabdus luminescens through Insect Immune Defense Reactions.

Xenorhabdus nematophila and Photorhabdus luminescens are entomopathogenic bacterial symbionts that produce toxic proteins that can interfere with the immune system of insects. Herein, we show that outer membrane proteins (OMPs) could be involved as bacterial virulence factors. Purified totals OMPs of both bacterial species were injected into fifth instar larvae of Spodoptera exigua Hübner. Larvae were surveyed for cellular defenses fluctuations in total haemocyte counts (THC) and granulocyte percentage and for the humoral defenses protease, phospholipase A2 (PLA2), and phenoloxidase (PO) activities at specific time intervals. Changes in the expression of the three inducible antimicrobial peptides (AMPs), cecropin, attacin, and spodoptericin, were also measured. Larvae treated with OMPs of both bacterial species had more haemocytes than did the negative controls. OMPs of X. nematophila caused more haemocyte destruction than did the OMPs of P. luminescens. The OMPs of both bacterial species initially activated insect defensive enzymes post-injection, the degree of activation varying with enzyme type. The AMPs, attacin, cecropin, and spodoptericin were up-regulated by OMP injections compared with the normal larvae. The expression of these three AMPs was maximal at four hours post injection (hpi) with P. luminescens OMPs treatment. Expression of the three AMPs in X. nematophila treated insects was irregular and lower than in the P. luminescens OMPs treatment. These findings provide insights into the role of OMPs of entomopathogenic nematode bacterial symbionts in countering the physiological defenses of insects.

Use of Greater Wax Moth Larvae (Galleria mellonella) as an Alternative Animal Infection Model for Analysis of Bacterial Pathogenesis.

Alternative infection models of bacterial pathogenesis are useful because they reproduce some of the disease characteristics observed in higher animals. Insect models are especially useful for modeling bacterial infections, as they are inexpensive, generally less labor-intensive, and more ethically acceptable than experimentation on higher organisms. Similar to animals, insects have been shown to possess innate immune systems that respond to pathogenic bacteria.

The role of pilin protein of Xenorhabdus nematophila against immune defense reactions of insects.

Xenorhabdus nematophila is a symbiotic bacterium of the entomopathogenic nematode Steinernema carpocapsae (Weiser). It produces several toxic proteins which interfere with the immune system of insects. The current study shows that purified pilin protein could be a virulence trait of X. nematophila. The fifth instar larvae of Spodoptera exigua (Hübner) was injected with purified pilin. Changes in the cellular defenses in terms of total haemocyte counts and granulocyte percentage and humoral factors including total protease, phospholipase A2, and phenoloxidase activities (humoral defense) as well as the expression of the three main antimicrobial peptides attacin, cecropin, and spodoptericin were measured at specific times. The level of THC and granulocytes in larvae with different concentrations of pilin protein were less than the negative control. Also agglutination of haemocytes was observed 8-16h post-injection. The pilin protein activated phenoloxidase in the initial hour post-injection, by 2hpi, activity was stable. The activities of phospholipase A2 and protease activities reached maximum levels at 12 and 4hpi, respectively, and then decreased. The expressions of attacin, cecropin, and spodoptericin in larvae treated with pilin protein were up-regulated above that of the normal sample. The overexpression of cecropin was greater than the other antimicrobial protein mRNA transcripts. The spodoptericin expression had an irregular trend while expressions of attacin and cecropin reached maximum levels at 4hpi and then decreased. Generally, after the injection of pilin protein, the cellular and humoral immune system of S. exigua is activated but this toxin was able to inhibit them. This is the first report of the role of pilin protein when the bacterial symbiont of S. carpocapsae encounters the humoral defense of an insect.

Hemocyte-hemocyte adhesion and nodulation reactions of the greater wax moth, Galleria mellonella are influenced by cholera toxin and its B-subunit.

Nodulation, the lepidopteran insect immune response to large numbers of microbes in the blood (hemolymph) consists of the coordination of the blood cell (hemocyte) types the granular cells and plasmatocytes in terms of granular cell-bacteria adhesion and hemocyte-hemocyte adhesion (microaggregation). Hemocyte-microbe adhesion is influenced by the secondary messenger, cAMP, and cAMP-dependent protein kinase A. In the present study, cholera toxin, an AB5 protein known to indirectly stimulate adenylate cyclase, is used to examine the hemocyte responses to glass, bacteria and hemocyte-hemocyte microaggregates. In vitro, this toxin induces a bimodal hemocyte adhesion response that varies with the holotoxin concentration in terms of the individual and aggregated hemocyte adhesion responses: the lower CTX concentration (1.2 nM) increases microaggregate adhesion and decreases individual hemocyte binding to glass, as does higher concentrations (6-120 nM), however microaggregates induced by lower concentrations do not adhere to glass. Cholera toxin-induced microaggregation is inhibited by RGDS, suggestive of integrin involvement. In vivo, cholera toxin (1.2-120 nM) injected into larvae induces also a bimodal hemocytic response: low levels (1.2-6 nM) cause reduced hemocyte adhesion, while high levels (12-120 nM) increase hemocyte release or mobilization of adhesive hemocyte counts in the hemolymph. Increasing levels of cholera toxin concomitantly injected with the non-pathogenic bacterium, Bacillus subtilis produces a bimodal pattern in bacterial removal from the hemolymph which correlates with nodule frequency in larvae injected with cholera toxin only. The effects of higher concentrations of cholera toxin in vitro (6-120 nM) and in vivo (12-120 nM) are mediated by the B-subunit, whereas the isolated A-subunit has no effect on hemocyte activity. Cholera toxin and its individual subunits did not detectably alter levels of intracellular cAMP in the hemocytes, suggesting a cAMP-independent mechanism stimulating the nodulation response.

Cell lines, Md108 and Md66, from the hemocytes of Malacosoma disstria (Lepidoptera) display aspects of plasma-free innate non-self activities.

The innate non-self response systems of the deciduous tree pest, the forest tent caterpillar, Malacosoma disstria has been documented by us in terms of in vitro and in vivo reactions towards the Gram-positive nonpathogenic bacterium, Bacillus subtilis and Gram-negative pathogenic microbe, Xenorhabdus nematophila and their respective surface antigens, lipopoteichoic acids (LTA) and lipopolysaccharides (LPS). These studies, often conducted in whole and diluted hemolymph, preclude examination of plasma-free cellular (hemocyte) responses. Plasma-free hemocytes as primary cultures are difficult to obtain. The floating cell line Md66 and attached cell line Md108 from M. disstria hemocytes were examined as a model for plasma-free M. disstria hemocyte non-self responses. Herein, it was established that although both lines differed from each other and from the primary hemocyte cultures of M. disstria in growth parameters, cell composition and sizes both cell lines displayed granular cell-like (GL) cells and plasmatocyte-like (PL) cells according to morphological criteria and to some extent antigenic similarities based on labeling with anti-Chrysodeixis includens hemocyte monoclonal antibodies. Hemocyte-specific neuroglian-like protein was detected on cells of both cell lines and in the primary hemocyte cultures albeit with staining patterns differing according to culture and cell types, confluency levels and cell-cell adhesion. Both cell lines bound B. subtilis and X. nematophila, the reaction extent varying with the cell line and its cell types. LPS damaged both cell types in the two cell lines whereas LTA enhanced the adhesion of Md66 GL cells to flask surfaces followed by PL cell adhesion. PL cells of both lines, like the primary cultures, phagocytosed FITC-labeled B. subtilis; only Md108 GL cells phagocytosed B. subtilis. In either case phagocytosis was always less in frequency and intensity than the primary cultures. Proteins released from the cell lines differed in pattern and magnitude but contained bacterial binding proteins that enhanced differential bacterial adhesion to both cell types in both cell lines: the GL cells both cultures, and those of granular cells in primary cultures, were more involved than the primary plasmatocytes and PL cells. Only Md66 cells possessed lysozyme and both cell types of both lines contained phenoloxidase. Neither enzyme type was released during early phase reaction with the bacteria. LPS inhibited phenoloxidase activity. The similarities and differences between the lines and primary cultures make Md66 and Md108 useful for the systematic examination of plasma-free cellular non-self reactions.

Innate hemocyte responses of Malacosoma disstria larvae (C. Insecta) to antigens are modulated by intracellular cyclic AMP.

Invertebrate intracellular hemocyte signaling pathways affecting cellular-antigen responses, although defined for molluscs and some arthropods including dipteran insects, is less known for lepidopterans. Hemocytic-antigen responses of the arboreal pest lepidopteran Malacosoma disstria are linked to cAMP-dependent protein kinase A implicating cAMP in cellular hemocyte immune responses. The purpose in the present study was to determine intracellular cAMP effects on larval M. disstria hemocytes adhering to slides and bacteria. Altering adenylate cyclase and phosphodiesterase activities as well as cAMP levels in vitro and in vivo changed hemocyte responses to antigens. Quiescent hemocytes had high cAMP levels due to adenylate cyclase activity and possibly low phosphodiesterase (type 4) activity. Antigen contact diminished hemocytic cAMP levels. Inhibiting adenylate cyclase increased hemocyte-antigen and hemocyte-hemocyte adhesion, the latter producing nodules in vivo without bacterial antigens. Inhibiting phosphodiesterase type 4 produced the reverse effects. Pharmacologically increasing intracellular cAMP in attached hemocytes caused many of the cells to detach. Diminished intracellular cAMP changed hemograms in vivo in bacteria-free larvae comparable to changes induced by the bacterium, Bacillus subtilis, by producing nodules. Lowering cAMP enhanced also the removal of Xenorhabdus nematophila and B. subtilisin vivo.

Steinernema carpocapsae DD136: metabolites limit the non-self adhesion responses of haemocytes of two lepidopteran larvae, Galleria mellonella (F. Pyralidae) and Malacosoma disstria (F. Lasiocampidae).

Live adult and juvenile entomopathogenic Steinernema carpocapsae DD136 (P. Nematoda) were not subjected to adhesion by haemocytes of lepidopteran insect larvae of Galleria mellonella or Malacosoma disstriain vitro or in vivo. In vitro freeze-killed nematodes exhibited haemocyte attachment, the intensity increasing with time. Accumulation of haemocytes on the dead nematodes was associated with host phenoloxidase activity; live nematodes and their exudates did not activate the enzyme whereas dead nematodes but not their exudate did activate phenoloxidase. Live-nematode exudate inhibited granular cell and some plasmatocyte adhesion to slides, increased granular cell but not plasmatocyte dissociation from preformed haemocyte monolayers and in vivo elevated total haemocyte counts and changed the floating haemocyte types while impairing bacterial removal from the haemolymph. Dead-nematode exudate did not affect these parameters thus immunosuppressant activity by live nematodes may represent the release of inhibitors not associated with their cuticle. The third stage juveniles released the inhibitors.

Surface antigens of Xenorhabdus nematophila (F. Enterobacteriaceae) and Bacillus subtilis (F. Bacillaceae) react with antibacterial factors of Malacosoma disstria (C. Insecta: O. Lepidoptera) hemolymph.

Previous research established different interactions of the insect pathogen, Xenorhabdus nematophila and nonpathogen, Bacillus subtilis, with antimicrobial hemocytes and humoral factors of larval Malacosoma disstria [Giannoulis, P., Brooks, C.L., Dunphy, G.B., Mandato, C.A., Niven, D.F., Zakarian, R.J., 2007. Interaction of the bacteria Xenorhabdus nematophila (Enterobacteriaceae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasicocampidae). J. Invertebr. Pathol. 94, 20-30]. The antimicrobial systems were inhibited by X. nematophila and stimulated by B. subtilis. The bacterial surface antigens participating in these reactions were unknown. Thus, herein the effects of lipopolysaccharide (endotoxin) from X. nematophila and lipoteichoic acid from B. subtilis on the larval M. disstria immune factors, the hemocytes and phenoloxidase, were determined. Endotoxin elevated the level of damaged hemocytes limiting the removal of X. nematophila from the hemolymph and enhancing the rapid release of bacteria trapped by nodulation. Similar effects were observed with the lipid A moiety of the endotoxin. The effects of lipopolysaccharide and lipid A on the hemocyte activities were abrogated by polymyxin B (an antibiotic that binds to lipid A) confirming lipopolysaccharide as the hemocytotoxin by virtue of the lipid A moiety. Lipoteichoic acid elicited nodulation and enhanced phenoloxidase activation and/or activity. Although lipoidal endotoxin and lipid A inhibited phenoloxidase activation they enhanced the activity of the enzyme. Apolipophorin-III precluded the effects of lipopolysaccharide, lipid A, and lipoteichoic acid on the hemocytes and prophenoloxidase until the antigens exceeded a critical threshold.

The antioxidants dimethylsulfoxide and dimethylthiourea affect the immediate adhesion responses of larval haemocytes from 3 lepidopteran insect species.

Antioxidants, dimethylsulfoxide (DMSO) and dimethylthiourea (DMTU), at concentrations not affecting the viability of blood cells (haemocytes) from the larval stage of 3 lepidopteran insects - Galleria mellonella, Lymantria dispar, and Malacosoma disstria - differed in their influence on the innate binding of haemocytes to glass, bacteria to haemocytes, and on humoral responses to alien materials. In vitro DMSO had little effect, whereas DMTU substantially impaired the adhesion of the haemocyte types, the plasmatocytes and granular cells, to slides as well as the attachment of Bacillus subtilis to these haemocytes. Although both antioxidants increased lysozyme and phenoloxidase activities, there was no correlation of enzyme activity and haemocyte adhesion responses, possibly reflecting sequestered radicals. Nitric oxide and hydroxyl radicals offset the DMTU effect. In the absence of antioxidants, inactivate protein kinases A (PKA) and C (PKC) enhanced haemocyte aggregation. In general, DMSO, as opposed to DMTU, did not alter the effects of PKA and PKC activators and inhibitors on haemocyte aggregation or of PKC and PKA activities. High concentrations of DMSO and all levels of DMTU, although inhibiting PKA and PKC, inhibited haemocyte adhesion to slides. Comparable results occurred for DMTU-treated haemocytes incubated with B. subtilis. In vivo DMSO, unlike DMTU, did not impair plasmatocyte or granular cell responses to foreign materials, including bacterial removal from the haemolymph and nodulation.

Interaction of the bacteria Xenorhabdus nematophila (Enterobactericeae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasiocampidae).

Malacosoma disstria larvae are a pest of deciduous trees. Little is known on the interaction of bacteria with the immediate hemocytic antimicrobial responses of these insects. Incubating dead Xenorhabdus nematophila and Bacillus subtilis with a mixture of serum-free granular cells and plasmatocytes in vitro revealed differential bacterial-hemocyte adhesion and differential discharge of lysozyme and phenoloxidase but not total protein. Although active phenoloxidase adhered equally to both bacterial species, X. nematophila limited enzyme activation whereas B. subtilis enhanced activation. Serum with active phenoloxidase (as opposed to tropolone-inhibited phenoloxidase) and purified insect lysozyme increased bacterial-hemocyte adhesion of both bacterial species. An apolipophorin-III-like protein when incubated with hemocytes, limited their responses to glass slides and bacterial adhesion. However, initial binding of the protein to both bacteria increased granular cell levels with bacteria while lowering the plasmatocyte levels with adhering procaryotes. The protein also increased lysozyme and phenoloxidase activities. Although B. subtilis in vivo elicited a nodulation-based decline in total hemocyte counts and did not affect hemocyte viability, dead X. nematophila elevated hemocyte counts and damaged the hemocytes as lipopolysaccharide levels increased and X. nematophila emerged into the hemolymph. Apolipophorin-III-like protein once bound to the bacteria slowed their removal from the hemolymph.

Cyclic AMP affects the haemocyte responses of larval Galleria mellonella to selected antigens.

Signal transduction of the innate immediate responses of insect haemocytes to foreign matter is rarely considered. Herein using a combination of adenylate cyclase inhibitors and activators and phosphodiesterase inhibitors we determined that cyclic adenosine monophosphate (cAMP) at high levels normally impairs non-self response. Haemocyte contact with glass and bacteria lowered cAMP in vitro. Inactive phosphodiesterases, including type 4, impaired haemocyte reactions in vitro. Using the drugs in vivo to modulate adenylate cyclase and phosphodiesterases altered the total and types of haemocytes. Adenylate cyclase inhibitors and etazolate (a type 4 phosphodiesterase inhibitor) alone produced changes in the haemograms similar to those caused by Bacillus subtilis. Sequential injections of an enzyme modulator followed by B. subtilis impaired bacterial removal due (1) in the case of enzyme inhibitors, to the removal of haemocytes prior to bacterial challenge and (2) in the case of forskolin and IBMX to the shut-down of the haemocytes. Activating adenylate cyclase or inhibiting phosphodiesterase impaired bacterial removal when co-injecting the compounds and bacteria.

Protein kinase A affects Galleria mellonella (Insecta: Lepidoptera) larval haemocyte non-self responses.

We used the protein kinase A (PKA) specific activator Sp-8-Br-cAMPS and type I inhibitor Rp-8-Br-cAMPS alone and in combination to define the role of PKA in the non-self responses of larval Galleria mellonella haemocytes in vitro and in vivo. Active PKA depressed haemocyte responses whereas PKA inhibition enhanced activities, including bacterial phagocytosis, the number of haemocytes with adherent bacteria, bacterial-induced haemocytic protein release and haemocyte adhesion to slides in vitro, as well as in vivo bacterial removal from the haemolymph. Non-attached haemocytes had more PKA activity than attached haemocytes; therefore, active PKA limited haemocyte response to foreign materials. We found that (i) PKA inhibitor alone induced non-self responses, including haemocyte protein discharge and lowered haemocyte counts in vivo, and induced nodulation; (ii) the enzyme activator produced effects opposite to those of the inhibitor; and (iii) together, the modulators offset each others' effects and influenced haemocyte lysate PKA activity. These findings establish PKA as a mediator of haemocytic non-self responses.

Bacterial formyl peptides affect the innate cellular antimicrobial responses of larval Galleria mellonella (Insecta: Lepidoptera).

The non-self cellular (hemocytic) responses of Galleria mellonella larvae, including the attachment to slides and the removal of the bacteria Xenorhabdus nematophila and Bacillus subtilis from the hemolymph, were affected by N-formyl peptides. Both N-formyl methionyl-leucyl-phenylalanine (fMLF) and the ester derivative decreased hemocyte adhesion in vitro, and both elevated hemocyte counts and suppressed the removal of both X. nematophila and B. subtilis from the hemolymph in vivo. The amide derivative and the antagonist tertiary-butoxy-carbonyl-methionyl-leucyl-phenylalanine (tBOC) increased hemocyte attachment to glass. The fMLF suppressed protein discharge from monolayers of granular cells with and without bacterial stimulation, while tBOC stimulated protein discharge. The peptide tBOC offset the effects of fMLF in vitro and in vivo. This is the first report implying the existence of formyl peptide receptors on insect hemocytes in which the compounds fMLF and tBOC inhibited and activated hemocyte activity, respectively.

Virulence of Candida albicans mutants toward larval Galleria mellonella (Insecta, Lepidoptera, Galleridae).

Culture medium affected the virulence of a strain of Candida albicans toward Galleria mellonella larvae, but the yeast growth rates in yeast extract - peptone - dextrose broth and synthetic Galleria serum were not correlated with yeast virulence. Virulent C. albicans grew rapidly in larval serum, whereas, it limited nodulation and continued development in vivo, producing toxins that damaged the hemocytes and fat body. Nonpathogenic yeast-phase cells grew slowly in larval serum but induced extensively melanized nodules in vivo and developed no further. There was no discernible relationship in 14 exo-enzymes between the virulent and avirulent yeast strains and virulence. The avirulent myosin-I-defective yeast cells were rapidly removed from the hemolymph in vivo because of lysozyme-mediated yeast agglutination and the possible binding of the yeast cells by lysozyme and apolipophorin-III. Both lysozyme and apolipophorin-III are proteins that bind beta-1,3-glucan. Finally, insects with nonpathogenic C. albicans exhibited induced immunity and were more resistant to candidiasis from the wild-type yeast cells than were noninduced insects.

Kinases, intracellular calcium, and apolipophorin-III influence the adhesion of larval hemocytes of the lepidopterous insect, Galleria mellonella.

Based on the results from the use of selective inhibitors and activators, active protein kinase A, protein tyrosine kinase, and protein kinase C (PKC) isoforms decreased the adhesion of larval Galleria mellonella hemocytes to glass slides. The protein kinase A inhibitor at all concentrations increased granular cell adhesion only whereas protein tyrosine kinase elevated both granular and plasmatocyte attachment at the lowest concentration. Active, Ca(2+)- and lipid-dependent PKC isoforms limited plasmatocyte and granular cell adhesion whereas PKC that was inhibited by selected compounds (with differed modes of PKC inhibition) enhanced hemocyte attachment. The granular cells were more sensitive to the PKC inhibitors than were plasmatocytes. Phospholipase C and its diacylglyceride product were necessary to reduce hemocyte adhesion and maintain PKC activity. Extracellular Ca(2+), possibly transported through L-channels, was required for plasmatocyte attachment. In contrast, lowering the levels of cytosolic Ca(2+) was associated with decreased PKC activity and was required for hemocyte adhesion.

Growth of an ovarian cell line of Galleria mellonella and its response to immune-inducing factors.

Antibacterial proteins are produced in the reproductive tracts of some insect species. The advent of a pupal ovarian cell line of the lepidopteran Galleria mellonella offered an opportunity for exploring the use of ovarian tissue culture to induce antimicrobial proteins in lieu of the larvae. The ovarian cell growth rates and cell yields were maximized by adjusting Grace's medium to pH 6.5, adding 15% (v/v) qualified heat-inactivated fetal calf serum, and lowering the sucrose concentration to 9.3 g/L. Five cell forms and biochemical profiles of the collective cell types were analyzed throughout the culture growth cycle. The final modified culture medium did not affect morphogenesis, whereas it increased the culture growth rate by 50% and the final cell yield threefold. The molting and immunoprotein-inducing hormone, 20-hydroxyecdysone, increased culture growth rate and altered the levels of cell types A and D. Neither 20-hydroxyecdysone nor the larval immunizing agents, apolipophorin-III or Bacillus subtilis, in combination or alone, induced antibacterial activity. The bacterium did induce immunity in both larval and adult stages.

Iron contributes to the antibacterial functions of the haemolymph of Galleria mellonella.

Studies with Galleria mellonella larvae and the iron chelating agent EDDA showed that iron was essential for the removal of dead Xenorhabdas nematophila and Bacillus subtilis from the haemolymph. The delay in removal of the bacteria from the iron-restricted haemolymph was attributed to reduced adhesiveness of the haemocytes and prophenoloxidase activity. Iron augmentation returned these activities to control levels. Whereas dead B. subtilis had no effect on the concentration of ferrozine-detectable iron (henceforth iron) in the haemolymph, dead X. nematophila was associated with substantially lower levels of iron as the number of damaged haemocytes increased. Haemocyte lysate lowered the concentrations of iron in both FeCl(3) solutions and deproteinized larval serum independent of serum lipids. Haemocyte lysate added to tryptic soybroth lowered the level of iron and limited the growth of X. nematophila. X. nematophila limited iron availability in the plasma by releasing lipopolysaccharides; such a mechanism may be a means of impairing the antimicrobial defences of the insects.

Apolipophorin-III affects the activity of the haemocytes of Galleria mellonella larvae.

Apolipophorin-III (apoLp-III) impaired the adhesion of plasmatocytes and a granular cell-subpopulation of larval Galleria mellonella to glass slides. The protein bound to haemocytes, limited the responses of the plasmatocytes to Bacillus subtilis and increased the percentage of a subgroup of granular cells with adhering bacteria. The total number of bacteria adhering to all the haemocytes on the slides declined. Injections of apoLp-III slowed bacterial removal from the haemolymph without affecting total haemocyte counts and impaired haemocyte attachment to glass slides. Purified apoLp-III bound to B. subtilis. ApoLp-III in serum bound to bacteria within 5 min, peaked at 15 min and was either shed or dissociated by 60 min. ApoLp-III bound to B. subtilis lowered the adhesion of the bacteria to the haemocytes and slowed the removal of the bacteria from the haemolymph.

Generation and properties of a luminescent insect pathogen Xenorhabdus nematophilus (Enterobacteriaceae).

Studies on the interaction of the insect pathogenic bacterium, Xenorhabdus nematophilus (Enterobacteriaceae), with its nematode and insect hosts would be greatly assisted if a luminescent phenotype were generated that would allow the detection of viable bacteria in vivo without the necessity for disruption of the cellular interactions. The plasmid, pMGM221, containing the luminescence gene (luxCDABE) of Vibrio harveyi was introduced into different strains (DD136 and 19061) and phases (one and two) of X. nematophilus by triparental mating. For reproducible and efficient conjugation, it was necessary to use older cultures (96-160 h) in the stationary phase of X. nematophilus for mating with relatively small differences (<2-fold) in transconjugant yield for the different strains and phases of X. nematophilus. All transconjugants emitted high levels of light with optimum bioluminescence at 27 degrees C in Luria broth at pH 8.0 containing 20 g/L NaCl; pH, osmolarity, and temperature conditions were similar to those encountered by the bacteria in the hemolymph of the larvae of Galleria mellonella. Plasmids were detected in the transconjugants after 6 months of subculturing the bacteria without antibiotic selection. Aside from light emission, luminescent transconjugants had the same physiological properties as the nonluminescent parental strains, including identical rates of growth, production of exoenzymes, removal from and subsequent emergence into the insect's hemolymph, bacterial-induced hemocyte damage, suppression of prophenoloxidase activation, and the ability to kill G. mellonella larvae. Light-emitting larvae could readily be detected by eye in a dark room, and all bacteria reisolated from dead larvae were luminescent. These properties validate the use of luminescent X. nematophilus not only as a means of following bacterial host interactions, but also as a potential agent to follow the infection and death of the insect population.