For the Insect Pathogen Photorhabdus luminescens, Which End of a Nematode Is Out?

The nematode Heterorhabditis bacteriophora is the vector for transmitting the entomopathogenic bacterium Photorhabdus luminescens between insect larvae. The dauer juvenile (DJ) stage nematode selectively retains P. luminescens in its intestine until it releases the bacteria into the hemocoel of an insect host. We report the results of studying the transmission of the bacteria by its nematode vector. Cells of P. luminescens labeled with green fluorescent protein preferentially colonized a region of the DJ intestine immediately behind the basal bulb, extending for various distances toward the anus. Incubation of DJ nematodes in vitro in insect hemolymph induced regurgitation of the bacteria. Following a 30-min lag, the bacteria migrated in a gradual and staggered movement toward and ultimately exited the mouth. This regurgitation reaction was induced by a low-molecular-weight, heat- and protease-stable, anionic component present in arthropod hemolymph and in supernatants from insect cell cultures. Nematodes anesthetized with levamisole or treated with the antihelmenthic agent ivermectin did not release their bacteria into hemolymph. The ability to visualize P. luminescens in the DJ nematode intestine provides the first clues to the mechanism of release of the bacteria during infection of insect larvae. This and the partial characterization of a component of hemolymph triggering release of the bacteria render this fascinating example of both a mutualistic symbiosis and disease transmission amenable to future genetic and molecular study.     

Opsonic involvement in phagocytosis by mollusk hemocytes.

Macrophages from the gastrophod mollusk Otala lactea are capable of in vitro recognition and phagocytosis of foreign particles such as yeast, mammalian erythrocytes, and bacteria. The degree of intensity of the phagocytic response, in certain instances, is governed by the surface characteristics of the particle in question as well as by the presence of opsonic factors.Hemagglutinins have been implicated as opsonins in certain invertebrates, including mollusks. Otala lacks serum lectins; however, its hemolymph stimulates phagocytosis of formalized yeast but not erythrocytes and bacteria. Hemagglutinin-containing extracts of Otala albumin gland were shown to opsonize formalized red cells. The rate of ingestion of the bacteria used in this study by Otala hemocytes was variable and was not influenced by the presence of hemolymph in the medium.     

Porphyromonas gingivalis Peptidoglycans Induce Excessive Activation of the Innate Immune System in Silkworm Larvae

Porphyromonas gingivalis, a pathogen that causes inflammation in human periodontal tissue, killed silkworm (Bombyx mori, Lepidoptera) larvae when injected into the blood (hemolymph). Silkworm lethality was not rescued by antibiotic treatment, and heat-killed bacteria were also lethal. Heat-killed bacteria of mutant P. gingivalis strains lacking virulence factors also killed silkworms. Silkworms died after injection of peptidoglycans purified from P. gingivalis (pPG), and pPG toxicity was blocked by treatment with mutanolysin, a peptidoglycan-degrading enzyme. pPG induced silkworm hemolymph melanization at the same dose as that required to kill the animal. pPG injection increased caspase activity in silkworm tissues. pPG-induced silkworm death was delayed by injecting melanization-inhibiting reagents (a serine protease inhibitor and 1-phenyl-2-thiourea), antioxidants (N-acetyl-l-cysteine, glutathione, and catalase), and a caspase inhibitor (Ac-DEVD-CHO). Thus, pPG induces excessive activation of the innate immune response, which leads to the generation of reactive oxygen species and apoptotic cell death in the host tissue.     

Proteomic analysis of nucleopolyhedrovirus infection resistance in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Silkworm hemolymph is an important defense tissue to resist bacteria and virus infections. To study the response of silkworm hemolymph in the resistance of Bombyx mori L. nucleopolyhedrovirus (BmNPV), we constructed a near-isogenic silkworm line with BmNPV resistance using highly resistant and highly susceptible parental strains. In this paper, two-dimensional gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization (MALDI)-mass spectrometry were employed to investigate the differences of protein patterns in the hemolymph of the highly resistant, highly susceptible and near-isogenic silkworm strains after BmNPV was administrated to the larvae. A comparison between the proteomes of these three silkworm strains led us to identify two differentially expressed proteins, beta-N-acetylglucosaminidase 2 and aminoacylase. The expression levels of these proteins were higher in the BmNPV resistant strains.     

Galleria mellonella infected with Bacillus thuringiensis involves Hsp90

Insects are good models for studying the innate immune response. We report that Galleria mellonella larvae infected with entomopathogenic bacteria Bacillus thuringiensis kurstaki show changes in the level of Hsp90. Our experimental approach was to pre-treat larvae with the Hsp90-binding compound, 17-DMAG, before infection with B. thuringiensis. We show that pre-treated animals display a higher level of immune response. This was mainly manifested by enhanced action of their hemolymph directed toward living bacteria as well as lysozyme activity digesting bacterial peptidoglycan. The observed phenomenon was due to the higher activity of antimicrobial peptides which, in contrast to healthy animals, was detected in the hemolymph of the immunestimulated larvae. Finally, the physiological significance of our observation was highlighted by the fact that G. mellonella pre-treated with 17-DMAG showed a prolonged survival rate after infection with B. thuringiensis than the control animals. Our report points to a role for Hsp90 in the immune response of G. mellonella after infection with B. thuringiensis at the optimal growth temperature.     

Spätzle1 is a constituent of the extracellular signaling pathway that promotes nodule formation,a cell-mediated immune response,in caterpillar hemolymph

The existence of an extracellular signaling pathway that mediates nodule formation, a cell-mediated immune response, has been reported in Bombyx mori larvae. In this pathway, C-type lectins and the hemolymph serine proteinase BmHP-8 function in pathogen associated molecular pattern (PAMPs) recognition and signaling transduction. However, which molecule elicits the cellular response at the end of the pathway is unknown. In this study, the Toll ligand Bombyx mori Spätzel1 was shown to be involved in the pathway by applying anit-Spätzel1 antiserum in an in vitro nodule-like aggregate formation assay and an in vivo nodule formation assay.     

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species^1-5, as well as the wealth of information available regarding the insect''s immune system^6-8, and the pending genome sequence⁹ make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter¹⁰. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking¹¹ and oral toxicity assays¹². Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides⁷; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR¹³. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes⁶. To analyze these responses, injected insects can be dissected and visualized by microscopy^{13, 14}.     

Proteomics of larval hemolymph in Bombyx mori reveals various nutrient-storage and immunity-related proteins

The silkworm, Bombyx mori, is an important economic insect for its production of silk. The larvae of many lepidopteran insects are major agricultural pests and often silkworm is explored as a model organism for other lepidopteran pest species. The hemolymph of caterpillars contains a lot of nutrient and immune components. In this study, we applied liquid chromatography–tandem mass spectrometry to gain a better understanding of the larval hemolymph proteomics in B. mori. We identified 752 proteins in hemolymph collected from day-4 fourth instar and day-7 fifth instar. Nearly half the identified proteins (49 %) were predicted to function as binding proteins and 46 % were predicted to have catalytic activities. Apolipophorins, storage proteins, and 30K proteins constituted the most abundant groups of nutrient-storage proteins. Of them, 30K proteins showed large differences between fourth instar larvae and fifth instar larvae. Besides nutrient-storage proteins, protease inhibitors are also expressed very highly in hemolymph. The analysis also revealed lots of immunity-related proteins, including recognition, signaling, effectors and other proteins, comprising multiple immunity pathways in hemolymph. Our data provide an exhaustive research of nutrient-storage proteins and immunity-related proteins in larval hemolymph, and will pave the way for future physiological and pathological studies of caterpillars.     

Insect Cytokine Paralytic Peptide (PP) Induces Cellular and Humoral Immune Responses in the Silkworm Bombyx mori

In the blood (hemolymph) of the silkworm Bombyx mori, the insect cytokine paralytic peptide (PP) is converted from an inactive precursor to an active form in response to the cell wall components of microorganisms and contributes to silkworm resistance to infection. To investigate the molecular mechanism underlying the up-regulation of host resistance induced by PP, we performed an oligonucleotide microarray analysis on RNA of blood cells (hemocytes) and fat body tissues of silkworm larvae injected with active PP. Expression levels of a large number of immune-related genes increased rapidly within 3 h after injecting active PP, including phagocytosis-related genes such as tetraspanin E, actin A1, and ced-6 in hemocytes, and antimicrobial peptide genes cecropin A and moricin in the fat body. Active PP promoted in vitro and in vivo phagocytosis of Staphyloccocus aureus by the hemocytes. Moreover, active PP induced in vivo phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) in the fat body. Pretreatment of silkworm larvae with ML3403, a pharmacologic p38 MAPK inhibitor, suppressed the PP-dependent induction of cecropin A and moricin genes in the fat body. Injection of active PP delayed the killing of silkworm larvae by S. aureus, whereas its effect was abolished by preinjection of the p38 MAPK inhibitor, suggesting that p38 MAPK activation is required for PP-dependent defensive responses. These findings suggest that PP acts on multiple tissues in silkworm larvae and acutely activates cellular and humoral immune responses, leading to host protection against infection.     

A Biochemical Evidence of the Inhibitory Effect of Diflubenzuron on the Metamorphosis of the Silkworm,Bombyx mori

Diflubenzuron (DFB) has been known to prevent metamorphosis of silkworm, Bombyx mori, from larval to pupal stage at low dose exposure. To explain this inhibitory action of DFB, a hypothesis was raised that DFB acts like juvenile hormone (JH) or DFB inhibits JH esterase to increase endogenous JH titer. A JH bioassay using isolated abdomen clearly indicates that DFB does not act as JH analog because DFB did not induce vitellogenesis in the isolated female abdomen, while endogenous JHs did significantly. General esterase activities in hemolymph were lower in DFB-treated fifth instar larvae than in the control larvae, but there was no difference between fat body esterase activities in both groups. Two hemolymph esterases (‘E1’ and ‘E2’) of the fifth instar larvae were separated and visualized by α-and β-naphthyl acetate. From in vitro incubation experiment, the cathodal esterase (‘E1’) was sensitive to DFB at its nanomolar range. Considering the fact that early fifth instar larvae have high level of JH esterase in the hemolymph, these results suggest that DFB inhibit larval to pupal metamorphosis by blocking JH degradation, which increases endogenous JH titer especially at the critical period when the larvae determine metamorphic development at the following molt.     

Pathogen Entrapment by Transglutaminase—A Conserved Early Innate Immune Mechanism

Clotting systems are required in almost all animals to prevent loss of body fluids after injury. Here, we show that despite the risks associated with its systemic activation, clotting is a hitherto little appreciated branch of the immune system. We compared clotting of human blood and insect hemolymph to study the best-conserved component of clotting systems, namely the Drosophila enzyme transglutaminase and its vertebrate homologue Factor XIIIa. Using labelled artificial substrates we observe that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Using both a human and a natural insect pathogen we provide functional proof for an immune function for transglutaminase (TG). Drosophila larvae with reduced TG levels show increased mortality after septic injury. The same larvae are also more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria while neither phagocytosis, phenoloxidase or—as previously shown—the Toll or imd pathway contribute to immunity. These results firmly establish the hemolymph/blood clot as an important effector of early innate immunity, which helps to prevent septic infections. These findings will help to guide further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions.     

Phagocytosis and nodule formation by hemocytes of Manduca sexta larvae following injection of Pseudomonas aeruginosa

The time course of clearance of an injected dose of 10⁶ CFU ml^?1 hemolymph of Pseudomonas aeruginosa ATCC 9027 in larvae of the tobacco hornworm, Manduca sexta, has been examined in detail. The clearance process has been subdivided into three stages during which the rates of reduction in concentration of circulating viable bacteria were clearly different. Contributions of hemocyte reactions to bacterial clearance were examined during stages I and II. During stage I (0–2 hr postinoculation (PI), nodule formation produced a dramatic reduction in circulating bacteria by entrapping over 90% of the injected dose in the first 30 min. Phagocytosis of bacteria by circulating hemocytes and subsequent intracellular digestion contributed significantly to reductions in circulating bacteria during stage II (2–8 hr PI). Viable cells of the virulent P. aeruginosa P11-1 were trapped in nodules as efficiently as the less virulent 9027 during the first 30 min after injection into M. sexta. Bacteria of strain P11-1 were also phagocytosed by hemocytes during stage II, however, phagocytosed bacteria were observed less frequently in P11-1-treated insects and intracellular digestion of these bacteria was only rarely observed. The increased virulence of P11-1 in larvae of M. sexta may be due to less efficient phagocytosis by circulating hemocytes and to insensitivity of this strain to killing reactions in nodules and following phagocytosis.     

Bombyx mori cathepsin D expression is induced by high temperature and H2O2 exposure

Cathepsin D is involved in the metamorphosis of the silkworm, Bombyx mori. Here, we show the expression profile of B. mori cathepsin D (BmCatD) in the fat body during exposure to stressors, such as high temperature and H₂O₂. Exposure of larvae in the fifth instar stage to high temperature (28 °C) led to accelerated metamorphosis and shortened larval stage compared to control larvae grown at 23 °C. Concomitantly, the expression level of BmCatD mRNA was greatly increased during exposure to high temperature. We also detected significantly elevated H₂O₂ levels in the hemolymph of larvae treated with high temperature. To confirm that oxidative stress induces BmCatD expression, B. mori larvae were injected with H₂O₂. As predicted, we observed increased expression of BmCatD following H₂O₂ exposure. Based on these results, we conclude that BmCatD expression is induced by high temperature and H₂O₂ exposure and that this stress-induced BmCatD expression leads to early metamorphosis.     

Eicosanoids up-regulate production of reactive oxygen species by NADPH-dependent oxidase in Spodoptera exigua phagocytic hemocytes

Eicosanoids mediate cellular immune responses in insects, including phagocytosis of invading microbes. Phagocytosis entails two major steps, the internalization of microbes and the subsequent killing of them via formation of reactive oxygen species (ROS). Here, we posed the hypothesis that eicosanoids mediate ROS production by activating NADPH-dependent oxidase (NOX) and tested the idea in the model insect, Spodoptera exigua. A NOX gene (we named SeNOX4) was identified and cloned, yielding a full open reading frame encoding 547 amino acid residues with a predicted molecular weight of 63,410 Da and an isoelectric point at 9.28. A transmembrane domain and a large intracellular domain containing NADPH and FAD-binding sites were predicted. Phylogenetic analysis indicated SeNOX4 clusters with other NOX4 genes. SeNOX4 was expressed in all life stages except eggs, and exclusively in hemocytes. Bacterial challenge and, separately, arachidonic acid (AA, a precursor of eicosanoid biosynthesis) injection increased its expression. The internalization step was assessed by counting hemocytes engulfing fluorescence-labeled bacteria. The phagocytic behavior was inhibited by dsRNA suppression of SeNOX4 expression and, separately by dexamethasone (DEX, a specific inhibitor of eicosanoid biosynthesis) treatments. However, injecting AA to dsSeNOX4-treated larvae did not rescue the phagocytic activity. Hemocytic ROS production increased following bacterial challenge, which was sharply reduced in dsSeNOX4-treated, and separately, in DEX-treated larvae. AA partially reversed the suppressed ROS production in dsSeNOX4-treated larvae. Treating larvae with either the ROS-suppressing dsSeNOX4 construct or DEX rendered experimental larvae unable to inhibit bacterial proliferation in their hemocoels. We infer that eicosanoids mediate ROS production during phagocytosis by inducing expression of SeNOX4.     

Cloning and Characterization of Bombyx mori PP-BP a Gene Induced by Viral Infection

The ENF peptide family, so termed after the consensus sequence in their amino termini (Glu-Asn-Phe-), is assumed to play multiple important roles in defense reactions, growth regulation, and homeostasis of Lepidopteran insects. The paralytic peptide of Bombyx mori (BmPP) is one such peptide that is involved in the paralytic and plasmatocyte-spreading activities in the hemocyte immune reaction. The growth-blocking peptide of Pseudaletia separata (PsGBP), which is also a member of the ENF peptide family, has similar functions that can reportedly be attenuated by the growth-blocking peptide-binding protein (GBP-BP). Using the fluorescent differential display (FDD) technique, the differential expression pattern of genes in highly susceptible silkworm strain 306 were analyzed, following infection with B. mori nuclear polyhedrosis virus (BmNPV), and a differential band (G12₇₈₂) was obtained from the hemolymph RNA pools. Using 5′-RACE with a specially designed primer based on the FDD study, a 1 401 bp cDNA clone was obtained containing a 1 311 bp open reading frame (ORF, GenBank accession number DQ306881). The deduced protein was highly homologous in primary structure to GBP-BP and was termed B. mori paralytic peptide-binding protein (PP-BP). The B. mori PP-BP gene is organized into two exons and only one intron, using bioinformatics searches.Using RT-PCR analysis, it was found that the B. mori PP-BP gene was expressed almost exclusively in the hemolymph. Real-time quantitative PCR analysis indicated that the B. mori PP-BP mRNA level in B. mori strain 306 exposed to BmNPV was much higher than that in B. mori strain without the virus infection. This result implies that the B. mori PP-BP is related to the cellular immune response after BmNPV invades the hemolymph.     

Is decreased generalized immunity a cost of Bt resistance in cabbage loopers Trichoplusia ni?

We studied the immune response to Bacillus thuringiensis kurstaki (Btk) in susceptible (Bt-RS) and resistant (Bt-R) Trichoplusia ni after exposure to low doses of Btk and injection with Escherichia coli. We measured the levels of resistance, the expression profiles of hemolymph proteins, the phenoloxidase (PO) activity, and the differential number of circulating hemocytes in resistant and susceptible individuals. Individuals from the Bt-RS line became more resistant following a previous exposure to sub lethal concentrations of Btk, but the resistance to Btk of the Bt-R line did not change significantly. Similarly the Bt-R strain showed no significant changes in any of the potential immune responses, hemolymph protein levels or PO activity. The number of circulating hemocytes was significantly lower in the Bt-R strain than in the Bt-RS strain. Exposure to Btk decreased the hemocyte counts and reduced PO activity of Bt-RS larvae. Hemolymph protein concentrations also declined significantly in the susceptible larvae continually exposed to Btk. Seven peptides with antibacterial activity were identified in the hemolymph of Bt-RS larvae after exposure to Btk and five were found in the Bt-R larvae. When exposed to a low level Bt challenge the susceptible strain increases in tolerance and there are concomitant reductions in hemolymph protein concentrations, PO activity and the number of circulating hemocytes.     

The delta-endotoxin of Bacillus thuringiensis. V. On the occurrence of endotoxin fragments in hemolymph

Leucine-³H labeled crystals of Bacillus thuringiensis δ-endotoxin were fed to last-instar larvae of spruce budworm, Choristoneura fumiferana, eastern forest tent caterpillar, Malacosoma disstria, and silkworm, Bombyx mori. Radioactivity was detected in hemolymph 1 min after feeding in the first two species, but not until 3–5 min after feeding in silkworm larvae. Most of the radioactivity from hemolymph of all three species eluted from gel filtration columns at the same elution volume indicating similar moleculear weights (<1800 daltons).