Eicosanoids mediate Galleria mellonella immune response to hemocoel injection of entomopathogenic nematode cuticles.

Entomopathogenic nematodes are symbiotically associated with bacteria and widely used in biological control of insect pests. The interference of symbiotic bacteria with insect host immune responses is fairly well documented. However, knowledge of mechanisms regulating parasite­host interactions still remains fragmentary. In this study, we used nematode (Steinernema carpocapsae and Heterorhabditis bacteriophora) cuticles and Galleria mellonella larvae as parasite­host model, focused on the changes of innate immune parameters of the host in the early phase of nematode cuticle infection and investigated the role of eicosanoid biosynthesis pathway in the process. The results showed that injection of either S. carpocapsae or H. bacteriophora cuticles into the larval hemocoel both resulted in significant decreases in the key innate immune parameters (e.g., hemocyte density, microaggregation, phagocytosis and encapsulation abilities of hemocyte, and phenoloxidase and antibacterial activities of the cell-free hemolymph). Our study indicated that the parasite cuticles could actively suppress the innate immune response of the G. mellonella host. We also found that treating G. mellonella larvae with dexamethasone and indomethacin induced similar depression in the key innate immune parameters to the nematode cuticles. However, these effects were reversed when dexamethasone, indomethacin, or nematode cuticles were injected together with arachidonic acid. Additionally, we found that palmitic acid did not reverse the influence of the dexamethasone, indomethacin, or nematode cuticles on the innate immune responses. Therefore, we inferred from our results that both S. carpocapsae and H. bacteriophora cuticles inhibited eicosanoid biosynthesis to induce host immunodepression.

The lipopolysaccharide (LPS) of Photorhabdus luminescens TT01 can elicit dose- and time-dependent immune priming in Galleria mellonella larvae.

In this work, we primed Galleria mellonella larvae by haemocoel injection of lipopolysaccharide (LPS) extracted from Photorhabdus luminescens TT01 to determine whether bacterial LPS can induce enhanced immune protection (recently called immune priming). We also analyzed the relationship between changes in the levels of innate immune elements and the degree of enhanced immune protection in the larvae at designated time points after priming. The larvae that received experimental doses (20.0, 10.0 and 5.0µg per larva) of LPS demonstrated increased resistance against lethal challenge with P. luminescens TT01; the degree and period of protection correlated positively with the priming dose. These results indicated that the P. luminescens TT01 LPS could induce typical immune priming in G. mellonella. Moreover, the levels of innate immune parameters (i.e. haemocyte density, phagocytosis, haemocyte encapsulation ability, and antibacterial activity of cell-free haemolymph) and endogenous enzyme activities (i.e. acid phosphatase, ACP; alkaline phosphatase, AKP; superoxide dismutase, SOD and lysozyme, LSZ) were significantly increased following priming of the larvae with LPS, whereas the activities of peroxidase (POD) and catalase (CAT) were significantly decreased. All of the parameters examined changed in a dose- and time-dependent manner. This study demonstrated that G. mellonella larvae could modulate their immune responses based on different doses of LPS used for priming, and that priming phenomenon in G. mellonella larvae elicited by LPS was mediated by the innate immune elements and enzyme activity.

Prior infection of Galleria mellonella with sublethal dose of Bt elicits immune priming responses but incurs metabolic changes.

Invertebrate immune priming has attracted wide attention of biologists in recent years because it challenges core notions about the disparate nature of acquired and innate immunity. However, the metabolic switch and energetic cost during eliciting immune priming are poorly investigated issues, which could widen and deepen our understanding of the physiological mechanism of immune priming. In this study, using sublethal dose of Bacillus thuringiensis (Bt) as an elicitor, we detected typical immune priming responses in Galleria mellonella. We found that the intensity of immune priming is positively correlated with the levels of antimicrobial peptides and phagocytosis ability of hemocytes. Subsequently, we employed LC-MS/MS-based untargeted metabolomics techniques to analyze the metabolic changes in the fat body of G. mellonella larvae during immune priming. The results showed that there were 74 and 56 significantly altered metabolites in positive and negative ion mode, respectively, after Bt priming. Most of the differential metabolites were enriched in the following metabolic pathways: amino acid biosynthesis, carbon metabolism, aminoacyl-tRNA biosynthesis and ABC transporters. The energetic cost of immune priming was depicted mainly in the slow growth of body mass and decreased levels of sucrose, lactose, D-ribulose 1,5-bisphosphate, Glycerate-3P and isocitric acid, which are enriched in carbon metabolism and involved in energy production. Meanwhile, correlation and interaction network analysis showed negative correlations between carbohydrates and metabolites involved in amino acid biosynthesis, suggesting that amino acids acted as the main energy source and helped the organisms synthesize immune effectors to participate in the immune priming response. Our results pave the way for uncovering the physiological mechanism of insect immune priming and discovering novel targets for Bt insecticide.

Transcriptome analysis reveals immune and metabolic regulation effects of Poria cocos polysaccharides on Bombyx mori larvae.

Poria cocos polysaccharides (PS) have been used as Chinese traditional medicine with various pharmacological effects, including antiviral, anti-oxidative, and immunomodulatory activities. Herein Bombyx mori silkworm was used as a model animal to evaluate the immunomodulatory effects of PS via detecting the changes of innate immune parameters and explore the underlying molecular mechanism of the immunoregulatory effect of PS using Illumina HiSeq Xten platform. The results presented here demonstrated that a hemocoel injection of PS significantly enhanced the cellular immunity of silkworm, including hemocyte phagocytosis, microaggregation, and spreading ability. A total of 335 differentially expressed genes (DEGs) were screened, including 214 upregulated genes and 121 downregulated genes by differential expression analysis. Gene annotation and enrichment analyses showed that many DEGs related to immune signal recognition, detoxification, proPO activation, carbohydrate metabolism, and lipid metabolism were significantly upregulated in the treatment group. The Kyoto Encyclopedia of Genes and Genomes-based Gene Set Enrichment Analysis also revealed that the more highly expressed gene sets in the PS treatment silkworm were mainly related to immune signal transduction pathways and energy metabolism. In addition, the activity of four enzymes related to immunity and energy metabolism-including phenoloxidase, glucose-6-phosphate dehydrogenase, hexokinase, and fatty acid synthetase-were all significantly increased in the larvae injected with PS. We performed qRT-PCR to examine the expression profile of immune and metabolic-related genes, which further verified the reliability of our transcriptome data and suggested that PS can regulate the immunity of silkworm by enhancing the cellular immunity and modulating the expression levels of genes related to immune responses and physiological metabolism. These findings will lay a scientific foundation for the use of PS as an immunomodulator in disease prevention in human beings or animals.

RNA-seq profiles of putative genes involved in specific immune priming in Bombyx mori haemocytes.

BACKGROUND: The immune system of many invertebrates, including insects, has been shown to comprise memory, or specific immune priming. However, knowledge of the molecular mechanisms especially the candidate immune-related genes mediated the specificity of the immune priming are still very scarce and fragmentary. We therefore used two closely related Gram-negative pathogenic bacteria (Photorhabdus luminescens TT01 and P. luminescens H06) as the priming agents and employed Illumina/Solexa platform to investigate the transcriptional changes of the haemocytes of Bombyx mori larvae after priming. RESULTS: In total, 23.0 Gbp of sequence data and 153,331,564 reads were generated, representing 10,496 genes. Approximately 89% of the genes or sequenced reads could be aligned to the silkworm reference genome. The differentially expressed genes (DEGs) of PBS-vs-TT01 (up-regulated expression of TT01 relative to PBS), PBS-vs-H06 (up-regulated expression of H06 relative to PBS) and TT01-vs-H06 (up-regulated expression of H06 relative to TT01) were 707, 159 and 461 respectively. In addition, expression patterns of 25 selected DEGs derived from quantitative real-time polymerase chain reaction (qRT-PCR) were consistent with their transcript abundance changes obtained by transcriptomic analyses. The DEGs are mainly related to pattern recognition receptors (PRRs), antimicrobial peptides (AMPs), signaling molecular, effector molecules, phagosome and spliceosome, indicating that they have participated in the regulation of the specific immune priming in the B. mori larvae. CONCLUSIONS: The transcriptome profiling data sets from this study will provide valuable resources to better understand the molecular and biological mechanisms regulating the specificity of invertebrates' immune priming. All these will shed light on controlling insect pests or preventing epidemic of infectious diseases in economic invertebrates.

Transcriptome analysis of differentially expressed genes involved in innate immunity following Bacillus thuringiensis challenge in Bombyx mori larvae.

In this study, we describe RNA-seq expression profiling of larval Bombyx mori response to hemocoel injection of Bacillus thuringiensis (Bt). Two transcriptomes were generated from the hemocytes of the PBS- and Bt-injected B. mori larvae. More than 49 million 100-bp paired-end reads, encompassing over 7.3 Gb of sequence data, were generated for each library. After filtering the raw reads and removing the rRNA mapped reads, more than 89% of the reads in each library could be mapped to the silkworm genome reference sequence. Comparison of gene expression levels revealed that a total of 133 unigenes were upregulated while 84 unigenes were downregulated in PBS vs Bt. To further investigate the biological functions of different expression genes (DEGs), gene ontology (GO) and functional enrichment analysis were performed to map all the DEGs to terms in the GO, euKaryotic Ortholog Groups of proteins (KOG) and Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) database. Among these DEGs, many genes involved in immunity against Bt challenge were identified. These included genes participated in pattern recognition, antimicrobial peptides, insecticide resistance or detoxification, immune melanization, cytoskeleton reorganization and many other immune effectors. To confirm the gene expression patterns identified by the RNA-seq data, the transcript levels of 10 immune related DEGs were examined by quantitative real-time PCR (qRT-PCR). The results showed that the DEGs obtained from the deep sequencing data were accurate and gene expression profiles from RNA-Seq data were reliable. Our studies provide insights into the to immune response of B. mori underling the stress of Bt, which is valuable to understand how Bt affects the innate immune system of silkworm and provide new approaches to control insect pests by using Bt as a biological insecticide.

Haemocoel injection of PirA1B1 to Galleria mellonella larvae leads to disruption of the haemocyte immune functions.

The bacterium Photorhabdus luminescens produces a number of insecticidal proteins to kill its larval prey. In this study, we cloned the gene coding for a binary toxin PirA1B1 and purified the recombinant protein using affinity chromatography combined with desalination technology. Furthermore, the cytotoxicity of the recombinant protein against the haemocytes of Galleria mellonella larvae was investigated. We found that the protein had haemocoel insecticidal activity against G. mellonella with an LD50 of 131.5 ng/larva. Intrahaemocoelic injection of PirA1B1 into G. mellonella resulted in significant decreases in haemocyte number and phagocytic ability. In in vitro experiments, PirA1B1 inhibited the spreading behaviour of the haemocytes of G. mellonella larvae and even caused haemocyte degeneration. Fluorescence microscope analysis and visualization of haemocyte F-actin stained with phalloidin-FITC showed that the PirA1B1 toxin disrupted the organization of the haemocyte cytoskeleton. Our results demonstrated that the PirA1B1 toxin disarmed the insect cellular immune system.

Galleria mellonella larvae are capable of sensing the extent of priming agent and mounting proportionatal cellular and humoral immune responses.

Larvae of Galleria mellonella are useful models for studying the innate immunity of invertebrates or for evaluating the virulence of microbial pathogens. In this work, we demonstrated that prior exposure of G. mellonella larvae to high doses (1×10(4), 1×10(5) or 1×10(6) cells/larva) of heat-killed Photorhabdus luminescens TT01 increases the resistance of larvae to a lethal dose (50 cells/larva) of viable P. luminescens TT01 infection administered 48h later. We also found that the changes in immune protection level were highly correlated to the changes in levels of cellular and humoral immune parameters when priming the larvae with different doses of heat-killed P. luminescens TT01. Priming the larvae with high doses of heat-killed P. luminescens TT01 resulted in significant increases in the hemocytes activities of phagocytosis and encapsulation. High doses of heat-killed P. luminescens TT01 also induced an increase in total hemocyte count and a reduction in bacterial density within the larval hemocoel. Quantitative real-time PCR analysis showed that genes coding for cecropin and gallerimycin and galiomycin increased in expression after priming G. mellonella with heat-killed P. luminescens TT01. All the immune parameters changed in a dose-dependent manner. These results indicate that the insect immune system is capable of sensing the extent of priming agent and mounting a proportionate immune response.

Ultrastructural and functional characterization of circulating hemocytes from Galleria mellonella larva: Cell types and their role in the innate immunity.

Galleria mellonella larvae have been widely used as a model to study the virulence of various human pathogens. Hemocytes play important roles in the innate immune response of G. mellonella. In this study, the hemocytes of G. mellonella larvae were analyzed by transmission electron microscope, light microscope, and cytochemistry. The cytological and morphological analyses revealed four types of hemocytes; Plasmatocytes, granular cells, spherule cells and oenocytoids. Differential hemocyte counts showed that under our conditions plasmatocytes and granular cells were the most abundant circulating cell types in the hemolymph. We also investigated the role of different types of hemocytes in the cellular and humoral immune defenses. The in-vivo experiment showed that plasmatocytes, granular cells and oenocytoids phagocytized FITC-labelled Escherichia coli bacteria in larvae of G. mellonella, whereas the granular cells exhibited the strongest phagocytic ability against these microbial cells. After incubation with L-DOPA, plasmatocytes, granular cells and oenocytoids are stained brown, indicating the presence of phenoloxidase activity. These results shed new light on our understanding of the immune function of G. mellonella hemocytes.

Effects of dietary heavy metals on the immune and antioxidant systems of Galleria mellonella larvae.

In this work, we analyzed the effects of chromium (Cr) and lead (Pb) on immune and antioxidant systems of Galleria mellonella. In particular, after exposure to diets containing environmentally relevant concentrations (5, 50 and 100 µg/g) of Cr or Pb for 7 d, alterations in innate immune parameters and the activity of endogenous enzymes were measured in larvae. The results showed that 1) compared with the control, the lowest doses (5 µg/g) of Cr and Pb significantly increased the levels of innate immune parameters (total hemocyte count, THC; phagocytic activity; extent of encapsulation) of the larvae and hemolymph immune enzyme activities (acid phosphatase, ACP; alkaline phosphatase, AKP; phenoloxidase, PO), whereas the highest doses (100 µg/g) of Cr and Pb inhibited them; 2) the activity of antioxidant enzymes (superoxide dismutase, SOD; peroxidase, POD; catalase, CAT) showed significant increases with increasing concentrations of dietary Cr and Pb, and were significantly higher than those of the control; and 3) feeding the larvae with experimental concentrations of either Cr or Pb resulted similar patterns of changes of all the parameters examined. The current study suggested that moderate amounts of Cr and Pb enhance the innate immunity of G. mellonella, but that large amounts led to the inhibition of larval immune function, and also indicated that the experimental concentrations of Cr and Pb used caused strong oxidative stresses in the larvae.

No evidence for priming response in Galleria mellonella larvae exposed to toxin protein PirA2B2 from Photorhabdus luminescens TT01: An association with the inhibition of the host cellular immunity.

There is accumulating evidence that many invertebrates including insects can acquire enhanced immune protection against subsequently pathogens infection through immune priming. However, whether the toxin protein from pathogenic bacteria can induce such priming response remains unclear. Here we cloned, expressed and purified the toxin Photorhabdus insect-related proteins A2B2 (PirA2B2) from Photorhabdus luminescens TT01. We primed Galleria mellonella with sublethal dose of PirA2B2 and then challenged the larvae with viable P. luminescens TT01 at 48 h after priming. We found no evidence for immune priming in G. mellonella larvae exposed to PirA2B2. Priming the larvae with PirA2B2 did not improve their resistance in a subsequent challenge with P. luminescens TT01. Whereas a robust priming response was observed when the larvae exposed to lipopolysaccharide (LPS) extracted from P. luminescens TT01. Because the larvae primed with LPS showed significant higher resistance against P. luminescens TT01 infection than those of the PBS and BSA controls. Furthermore, we investigated the changes of the cellular immune parameters, such as hemocyte counts, phagocytic activity and encapsulation ability of the hemocytes, after priming. We found that the toxin PirA2B2 significantly decreased the cellular immunity of the larvae, whereas the LPS significantly increased them. These results indicated that the degree of priming response in G. mellonella correlated positively to the levels of cellular immune parameters, and the underlying mechanism in regulating the immune priming of invertebrates was not homologous to that of the immunological memory of vertebrates.

The specificity of immune priming in silkworm, Bombyx mori, is mediated by the phagocytic ability of granular cells.

In the past decade, the phenomenon of immune priming was documented in many invertebrates in a large number of studies; however, in most of these studies, behavioral evidence was used to identify the immune priming. The underlying mechanism and the degree of specificity of the priming response remain unclear. We studied the mechanism of immune priming in the larvae of the silkworm, Bombyx mori, and analyzed the specificity of the priming response using two closely related Gram-negative pathogenic bacteria (Photorhabdus luminescens TT01 and P. luminescens H06) and one Gram-positive pathogenic bacterium (Bacillus thuringiensis HD-1). Primed with heat-killed bacteria, the B. mori larvae were more likely to survive subsequent homologous exposure (the identical bacteria used in the priming and in the subsequent challenge) than heterologous (different bacteria used in the priming and subsequent exposure) exposure to live bacteria. This result indicated that the B. mori larvae possessed a strong immune priming response and revealed a degree of specificity to TT01, H06 and HD-1 bacteria. The degree of enhanced immune protection was positively correlated with the level of phagocytic ability of the granular cells and the antibacterial activity of the cell-free hemolymph. Moreover, the granular cells of the immune-primed larvae increased the phagocytosis of a previously encountered bacterial strain compared with other bacteria. Thus, the enhanced immune protection of the B. mori larvae after priming was mediated by the phagocytic ability of the granular cells and the antibacterial activity of the hemolymph; the specificity of the priming response was primarily attributed to the phagocytosis of bacteria by the granular cells.