Phospholipase A2 activity is required for immune defense of European (Apis mellifera) and Asian (Apis cerana) honeybees against American foulbrood pathogen, Paenibacillus larvae.

Honeybees require an efficient immune system to defend against microbial pathogens. The American foulbrood pathogen, Paenibacillus larvae, is lethal to honeybees and one of the main causes of colony collapse. This study investigated the immune responses of Apis mellifera and Apis cerana honeybees against the bacterial pathogen P. larvae. Both species of honeybee larvae exhibited significant mortality even at 102 103 cfu/mL of P. larvae by diet-feeding, although A. mellifera appeared to be more tolerant to the bacterial pathogen than A. cerana. Upon bacterial infection, the two honeybee species expressed both cellular and humoral immune responses. Hemocytes of both species exhibited characteristic spreading behaviors, accompanied by cytoskeletal extension along with F-actin growth, and formed nodules. Larvae of both species also expressed an antimicrobial peptide called apolipophorin III (ApoLpIII) in response to bacterial infection. However, these immune responses were significantly suppressed by a specific inhibitor to phospholipase A2 (PLA2). Each honeybee genome encodes four PLA2 genes (PLA2A ~ PLA2D), representing four orthologous combinations between the two species. In response to P. larvae infection, both species significantly up-regulated PLA2 enzyme activities and the expression of all four PLA2 genes. To determine the roles of the four PLA2s in the immune responses, RNA interference (RNAi) was performed by injecting gene-specific double stranded RNAs (dsRNAs). All four RNAi treatments significantly suppressed the immune responses, and specific inhibition of the two secretory PLA2s (PLA2A and PLA2B) potently suppressed nodule formation and ApoLpIII expression. These results demonstrate the cellular and humoral immune responses of A. mellifera and A. cerana against P. larvae. This study suggests that eicosanoids play a crucial role in mediating common immune responses in two closely related honeybees.

Identification of four secretory phospholipase A2s in a lepidopteran insect, Acrolepiopsis sapporensis, and their functional association with cellular immune responses.

Background: Eicosanoids are a group of the oxygenated C20 polyunsaturated fatty acids and play crucial roles in mediating various insect physiological processes. Catalytic activity of phospholipase A2 (PLA2) provides an initial substrate, arachidonic acid (AA), for subsequent eicosanoid biosynthesis. Results: This study identified four different secretory PLA2 (As-PLA2A-As-PLA2D) genes encoded in the Asian onion moth, Acrolepiopsis sapporensis. A phylogenetic analysis indicated that As-PLA2A and As-PLA2D are clustered with Group III PLA2s while As-PLA2B and As-PLA2C are clustered with Group XII and Group X PLA2s, respectively. Expression levels of these PLA2 genes increased along with larval development, especially in the fat body. A bacterial immune challenge upregulated the basal expression levels of the four PLA2 genes, which resulted in significant increases of the PLA2 enzyme activity. The enzyme activity was susceptible to a calcium chelator or reducing agent, suggesting Ca2+ dependency and disulfide linkage required for the catalytic activities of the secretory type of PLA2s. In addition, the PLA2 activity was also susceptible to bromophenacyl bromide (BPB), a specific inhibitor to sPLA2, but not to intracellular PLA2 inhibitors. An addition of BPB to the immune challenge significantly prevented hemocyte-spreading behavior of A. sapporensis. BPB treatment also suppressed a cellular immune response measured by hemocyte nodule formation. However, the immunosuppression was significantly rescued by the AA addition. To determine the PLA2(s) responsible for the immunity, individual RNA interference (RNAi) treatments specific to each of the four PLA2s were performed. Injection of gene-specific double-stranded RNAs caused significant reductions in the transcript level in all four PLA2s. In all four PLA2s, the RNAi treatments prevented the cellular immune response even after the immune challenge. Conclusion: This study reports four secretory PLA2s encoded in A. sapporensis and their function in mediating cellular immunity.

Immune responses of the Asian onion moth, Acrolepiopsis sapporensis, and their genetic factors from RNA-Seq analysis.

A nonmodel insect, Acrolepiopsis sapporensis, has been analyzed in immune responses. The total hemocytes in the fifth instar larvae were 2.33 × 106 cells/mL. These hemocytes comprised at least five different types and different relative ratios: 47% granulocytes, 26% plasmatocytes, 11% oenocytoid, 8% prohemocytes, and 5% spherulocytes. Upon bacterial challenge, some of the hemocytes exhibited typical hemocyte-spreading behaviors, such as focal adhesion, and filopodial and lamellipodial cytoplasmic extensions. The hemocyte behaviors induced cellular immune responses demonstrated by nodule formation. In addition, the plasma collected from the immune-challenged larvae exhibited humoral immune responses by bacterial growth inhibition along with enhanced phenoloxidase enzyme activity. These cellular and humoral immune responses were further analyzed by determining the immune-associated genes from a transcriptome generated by RNA-Seq. A total of about 12 Gb sequences led to about 218,116 contigs, which were predicted to encode about 46,808 genes. Comparative expression analysis showed 8392 uniquely expressed genes in the immune-challenged larvae. Differentially expressed gene (DEG) analysis among the commonly expressed genes indicated that 782 genes were upregulated and 548 genes were downregulated in the expressions after bacterial challenge. These immune-associated genes included pattern recognition receptors, immune mediation/signaling genes, and various immune effectors. Specifically, the genetic components of the Toll, IMD, and JAK/STAT immune signaling pathways were included in the DEG database. These results demonstrate the immune responses of A. sapporensis larvae and suggest the genes associated with the immune responses in this nonmodel insect.

Damage signal induced by Bacillus thuringiensis infection triggers immune responses via a DAMP molecule in lepidopteran insect, Spodoptera exigua.

Insect immunity defends the infection of an insect pathogenic bacterium, Bacillus thuringiensis (Bt). However, it was not clear on the recognition of Bt infection by the insect immune system. This study tested a physiological function of dorsal switch protein 1 (DSP1) in the Bt infection. DSP1 is classified into HMGB1-like damage-associated molecular pattern (DAMP) in insects. Upon Bt infection in a lepidopteran Spodoptera exigua, DSP1 was released from the nuclei of the midgut epithelium and activated immune responses. For this DSP1 release, a functional binding between Bt and its receptors on the midgut epithelium was required because any RNA interference (RNAi) treatments of Bt receptor (cadherin or ABCC) prevented the DSP1 release and became susceptible to the bacterial infection. The DSP1 release was required for the gene induction of Repat33, which is a member of response to pathogen gene family and its gene product mediated cellular and humoral immune responses against pathogen infection in S. exigua. The released DSP1 activated phospholipase A2 (PLA2) to produce eicosanoids, which induced the Repat33 expression because a hemocoelic injection of a recombinant DSP1 induced the Repat33 expression without Bt infection. However, any inhibition of PLA2 activity impaired the DAMP signaling between DSP1 and Repat33. DSP1 also up-regulated two other immune mediators, nitric oxide (NO) and a cytokine called plasmatocyte-spreading peptide (PSP). Either NO or PSP activated PLA2 to up-regulate Repat33 expression. These results suggest that Bt infection of the insect midgut generates a DAMP signal via DSP1 release, which turns on NO or the cytokine-PLA2-Repat33 immune signaling pathway.

Manipulation of GameXPeptide synthetase gene expression by a promoter exchange alters the virulence of an entomopathogenic bacterium, Photorhabdus temperata temperata, by modulating insect immune responses.

An entomopathogenic bacterium, Photorhabdus temperata subsp. temperata, is mutualistic to its host nematode, Heterorhabditis megidis. The infective juvenile nematodes enter target insects through natural openings and release the symbiotic bacteria into the insect hemocoel. The released bacteria suppress the insect immune responses and cause septicemia through their secondary metabolites. GameXPeptide (GXP) is one of the common secondary metabolites of most Photorhabdus species and is produced by the catalytic activity of a specific non-ribosomal peptide synthetase called GxpS encoded by the gxpS gene. This study confirmed gxpS to be encoded in the P. temperata temperata genome and analyzed its expression during bacterial growth. LC-MS/MS analysis of the bacterial culture broth contained at least four different GXPs (GXP-A to GXP-D), in which GXP-A was the most abundant. To investigate GXP synthesis following gxpS expression, the gxpS promoter of P. temperata temperata was replaced with an inducible arabinose promoter by homologous recombination. The gxpS transcript levels in the mutant were altered by the addition of l-arabinose. Without the inducer, the gxpS transcript level was significantly lower compared to the wild type and produced significantly lower amounts of the four GXPs. The addition of the inducer to the mutant significantly increased gxpS expression and produced significantly higher levels of the four GXPs compared to the wild type. The metabolite extracts obtained from wild-type and mutant bacteria showed differential immunosuppressive activities according to their GXP contents against the cellular and humoral immune responses of a lepidopteran insect, Spodoptera exigua. Interestingly, the gxpS-mutant bacteria showed less insecticidal activity compared to the wild type, whereas the addition of GXP to the mutant significantly restored insecticidal activity. These results suggest that the gxpS gene encoded in P. temperata temperata is responsible for the production of at least four different GXPs, which play crucial roles in bacterial virulence.

Integrated Biological Control Using a Mixture of Two Entomopathogenic Bacteria, Bacillus thuringiensis and Xenorhabdus hominickii, against Spodoptera exigua and Other Congeners.

Insect immunity defends against the virulence of various entomopathogens, including Bacillus thuringiensis (Bt). This study tested a hypothesis that any suppression of immune responses enhances Bt virulence. In a previous study, the entomopathogenic bacterium, Xenorhabdus hominickii (Xh), was shown to produce secondary metabolites to suppress insect immune responses. Indeed, the addition of Xh culture broth (XhE) significantly enhanced the insecticidal activity of Bt against S. exigua. To analyze the virulence enhanced by the addition of Xh metabolites, four bacterial secondary metabolites were individually added to the Bt treatment. Each metabolite significantly enhanced the Bt insecticidal activity, along with significant suppression of the induced immune responses. A bacterial mixture was prepared by adding freeze-dried XhE to Bt spores, and the optimal mixture ratio to kill the insects was determined. The formulated bacterial mixture was applied to S. exigua larvae infesting Welsh onions in a greenhouse and showed enhanced control efficacy compared to Bt alone. The bacterial mixture was also effective in controlling other Spodopteran species such as S. litura and S. frugiperda but not other insect genera or orders. This suggests that Bt+XhE can effectively control Spodoptera-associated pests by suppressing the immune defenses.

Repat33 Acts as a Downstream Component of Eicosanoid Signaling Pathway Mediating Immune Responses of Spodoptera exigua, a Lepidopteran Insect.

Repat (=response to pathogen) is proposed for an immune-associated gene family from Spodoptera exigua, a lepidopteran insect. In this gene family, 46 members (Repat1-Repat46) have been identified. They show marked variations in their inducible expression patterns in response to infections by different microbial pathogens. However, their physiological functions in specific immune responses and their interactions with other immune signaling pathways remain unclear. Repat33 is a gene highly inducible by bacterial infections. The objective of this study was to analyze the physiological functions of Repat33 in mediating cellular and humoral immune responses. Results showed that Repat33 was expressed in all developmental stages and induced in immune-associated tissues such as hemocytes and the fat body. RNA interference (RNAi) of Repat33 expression inhibited the hemocyte-spreading behavior which impaired nodule formation of hemocytes against bacterial infections. Such RNAi treatment also down-regulated expression levels of some antimicrobial genes. Interestingly, Repat33 expression was controlled by eicosanoids. Inhibition of eicosanoid biosynthesis by RNAi against a phospholipase A2 (PLA2) gene suppressed Repat33 expression while an addition of arachidonic acid (a catalytic product of PLA2) to RNAi treatment recovered such suppression of Repat33 expression. These results suggest that Repat33 is a downstream component of eicosanoids in mediating immune responses of S. exigua.