Toll Pathway Research Articles

Toll receptor ligand Spätzle 4 responses to the highly pathogenic Enterococcus faecalis from Varroa mites in honeybees.

Honeybees play a major role in crop pollination, which supports the agricultural economy and international food supply. The colony health of honeybees is threatened by the parasitic mite Varroa destructor, which inflicts physical injury on the hosts and serves as the vector for variable viruses. Recently, it shows that V. destructor may also transmit bacteria through the feeding wound, yet it remains unclear whether the invading bacteria can exhibit pathogenicity to the honeybees. Here, we incidentally isolate Enterococcus faecalis, one of the most abundant bacteria in Varroa mites, from dead bees during our routine generation of microbiota-free bees in the lab. In vivo tests show that E. faecalis is only pathogenic in Apis mellifera but not in Apis cerana. The expression of antimicrobial peptide genes is elevated following infection in A. cerana. The gene-based molecular evolution analysis identifies positive selection of genes encoding Späetzle 4 (Spz4) in A. cerana, a signaling protein in the Toll pathway. The amino acid sites under positive selection are related to structural changes in Spz4 protein, suggesting improvement of immunity in A. cerana. The knock-down of Spz4 in A. cerana significantly reduces the survival rates under E. faecalis challenge and the expression of antimicrobial peptide genes. Our results indicate that bacteria associated with Varroa mites are pathogenic to adult bees, and the positively selected gene Spz4 in A. cerana is crucial in response to this mite-related pathogen.

Serpin-4 Negatively Regulates Prophenoloxidase Activation and Antimicrobial Peptide Synthesis in the Silkworm, Bombyx mori.

The prophenoloxidase (PPO) activation and Toll antimicrobial peptide synthesis pathways are two critical immune responses in the insect immune system. The activation of these pathways is mediated by the cascade of serine proteases, which is negatively regulated by serpins. In this study, we identified a typical serpin, BmSerpin-4, in silkworms, whose expression was dramatically up-regulated in the fat body and hemocytes after bacterial infections. The pre-injection of recombinant BmSerpin-4 remarkably decreased the antibacterial activity of the hemolymph and the expression of the antimicrobial peptides (AMPs) gloverin-3, cecropin-D, cecropin-E, and moricin in the fat body under Micrococcus luteus and Yersinia pseudotuberculosis serotype O: 3 (YP III) infection. Meanwhile, the inhibition of systemic melanization, PO activity, and PPO activation by BmSerpin-4 was also observed. Hemolymph proteinase 1 (HP1), serine protease 2 (SP2), HP6, and SP21 were predicted as the candidate target serine proteases for BmSerpin-4 through the analysis of residues adjacent to the scissile bond and comparisons of orthologous genes in Manduca sexta. This suggests that HP1, SP2, HP6, and SP21 might be essential in the activation of the serine protease cascade in both the Toll and PPO pathways in silkworms. Our study provided a comprehensive characterization of BmSerpin-4 and clues for the further dissection of silkworm PPO and Toll activation signaling.

An evolutionarily conserved serine protease network mediates melanization and Toll activation in Drosophila.

Melanization and Toll pathway activation are essential innate immune mechanisms in insects, which result in the generation of reactive compounds and antimicrobial peptides, respectively, to kill pathogens. These two processes are mediated by phenoloxidase (PO) and Spätzle (Spz) through an extracellular network of serine proteases. While some proteases have been identified in Drosophila melanogaster in genetic studies, the exact order of proteolytic activation events remains controversial. Here, we reconstituted the serine protease framework in Drosophila by biochemical methods. This system comprises 10 proteases, i.e., ModSP, cSP48, Grass, Psh, Hayan-PA, Hayan-PB, Sp7, MP1, SPE and Ser7, which form cascade pathways that recognize microbial molecular patterns and virulence factors, and generate PO1, PO2, and Spz from their precursors. Furthermore, the serpin Necrotic negatively regulates the immune response progression by inhibiting ModSP and Grass. The biochemical approach, when combined with genetic analysis, is crucial for addressing problems that long stand in this important research field.

Ubiquitin signalling in Drosophila innate immune responses.

Cells respond to invading pathogens and danger signals from the environment by adapting gene expression to meet the need for protective effector molecules. While this innate immune response is required for the cell and the organism to recover, excess immune activation may lead to loss of homeostasis, thereby promoting chronic inflammation and cancer progression. The molecular basis of innate immune defence is comprised of factors promoting survival and proliferation, such as cytokines, antimicrobial peptides and anti-apoptotic proteins. As the molecular mechanisms regulating innate immune responses are conserved through evolution, the fruit fly Drosophila melanogaster serves as a convenient, affordable and ethical model organism to enhance understanding of immune signalling. Fly immunity against bacterial infection is built up by both cellular and humoral responses, where the latter is regulated by the Imd and Toll pathways activating NF-κB transcription factors Relish, Dorsal and Dif, as well as JNK activation and JAK/STAT signalling. As in mammals, the Drosophila innate immune signalling pathways are characterised by ubiquitination of signalling molecules followed by ubiquitin receptors binding to the ubiquitin chains, as well as by rapid changes in protein levels by ubiquitin-mediated targeted proteasomal and lysosomal degradation. In this review, we summarise the molecular signalling pathways regulating immune responses to pathogen infection in Drosophila, with a focus on ubiquitin-dependent control of innate immunity and inflammatory signalling.

Comparative Transcriptome Analysis of the Response to Vibrio parahaemolyticus and Low-Salinity Stress in the Swimming Crab Portunus trituberculatus.

Vibrio parahaemolyticus is one of the main pathogenic bacteria of Portunus trituberculatus and causes mass mortality of P. trituberculatus in aquaculture. In addition, low-salinity stimulation makes P. trituberculatus more susceptible to V. parahaemolyticus infections. In order to elucidate the molecular mechanism of resistance to V. parahaemolyticus in P. trituberculatus, comparative transcriptomic analysis of blood cells stimulated by low salinity and V. parahaemolyticus was carried out in this study. Transcriptome sequencing of low-salinity stress and pathogen infection at different time points was completed using Illumina sequencing technology. A total of 5827, 6432, 5362 and 1784 differentially expressed genes (DEGs) involved in pathways related to ion transport and immunoregulation were found under low-salinity stress at 12, 24, 48 and 72 h compared with the control at 0 h. In contrast, 4854, 4814, 5535 and 6051 DEGs, which were significantly enriched in Toll and IMD signaling pathways, were found at 12, 24, 48 and 72 h compared with the control at 0 h under V. parahaemolyticus infection. Among them, 952 DEGs were shared in the two treatment groups, which were mainly involved in apoptosis and Hippo signaling pathway. Cluster analysis screened 103 genes that were differentially expressed in two factors that were negatively correlated, including immunoglobulin, leukocyte receptor cluster family, scavenger receptor, macroglobulin and other innate-immune-related genes. These results provide data support for the analysis of the mechanisms of immunity to V. parahaemolyticus under low-salinity stress in P. trituberculatus and help to elucidate the molecular mechanisms by which environmental factors affect immunity.

Gut dysbacteriosis induces expression differences in the adult head transcriptome of Spodoptera frugiperda in a sex-specific manner

Mounting evidence indicates that the gut microbiota influences the neurodevelopment and behavior of insects through the gut-brain axis. However, it is currently unclear whether the gut microbiota affect the head profiles and immune pathway in pests. Here, we find that gut bacteria is essential for the immune and neural development of adult Spodoptera frugiperda, which is an extremely destructive agricultural pest worldwide. 16 S rRNA sequencing analysis showed that antibiotics exposure significantly disturbed the composition and diversity of gut bacteria. Further transcriptomic analysis revealed that the adult head transcripts were greatly affected by gut dysbacteriosis, and differently expression genes critical for brain and neural development including A4galt, Tret1, nsun4, Galt, Mitofilin, SLC2A3, snk, GABRB3, Oamb and SLC6A1 were substantially repressed. Interestingly, the dysbacteriosis caused sex-specific differences in immune response. The mRNA levels of pll (serine/threonine protein kinase Pelle), PGRP (peptidoglycan-sensing receptor), CECA (cecropin A) and CECB (cecropin B) involved in Toll and Imd signaling pathway were drastically decreased in treated male adults’ heads but not in female adults; however, genes of HIVEP2, ZNF131, inducible zinc finger protein 1-like and zinc finger protein 99-like encoding zinc-finger antiviral protein (ZAP) involved in the interferon (IFNα/β) pathway were significantly inhibited in treated female adults’ heads. Collectively, these results demonstrate that gut microbiota may regulate head transcription and impact the S. frugiperda adults’ heads through the immune pathway in a sex-specific manner. Our finding highlights the relationship between the gut microbiota and head immune systems of S. frugiperda adults, which is an astonishing similarity with the discoveries of other animals. Therefore, this is the basis for further research to understand the interactions between hosts and microorganisms via the gut-brain axis in S. frugiperda and other insects.

Altered Gene Expression of the Parasitoid Pteromalus puparum after Entomopathogenic Fungus Beauveria bassiana Infection

Both parasitoids and entomopathogenic fungi are becoming increasingly crucial for managing pest populations. Therefore, it is essential to carefully consider the potential impact of entomopathogenic fungi on parasitoids due to their widespread pathogenicity and the possible overlap between these biological control tools during field applications. However, despite their importance, little research has been conducted on the pathogenicity of entomopathogenic fungi on parasitoids. In our study, we aimed to address this knowledge gap by investigating the interaction between the well-known entomopathogenic fungus Beauveria bassiana, and the pupal endoparasitoid Pteromalus puparum. Our results demonstrated that the presence of B. bassiana significantly affected the survival rates of P. puparum under laboratory conditions. The pathogenicity of B. bassiana on P. puparum was dose- and time-dependent, as determined via through surface spraying or oral ingestion. RNA-Seq analysis revealed that the immune system plays a primary and crucial role in defending against B. bassiana. Notably, several upregulated differentially expressed genes (DEGs) involved in the Toll and IMD pathways, which are key components of the insect immune system, and antimicrobial peptides were rapidly induced during both the early and late stages of infection. In contrast, a majority of genes involved in the activation of prophenoloxidase and antioxidant mechanisms were downregulated. Additionally, we identified downregulated DEGs related to cuticle formation, olfactory mechanisms, and detoxification processes. In summary, our study provides valuable insights into the interactions between P. puparum and B. bassiana, shedding light on the changes in gene expression during fungal infection. These findings have significant implications for the development of more effective and sustainable strategies for pest management in agriculture.

Larval diet and temperature alter mosquito immunity and development: using body size and developmental traits to track carry-over effects on longevity

BackgroundEstimating arbovirus transmission potential requires a mechanistic understanding of how environmental factors influence the expression of adult mosquito traits. While preimaginal exposure to environmental factors can have profound effects on adult traits, tracking and predicting these effects remains challenging.MethodsUsing Aedes albopictus and a structural equation modeling approach, we explored how larval nutrition and temperature jointly affect development rate and success, female body size, and whether these metrics capture carry-over effects on adult female longevity. Additionally, we investigated how larval diet and temperature affect the baseline expression of 10 immune genes.ResultsWe found that larval development success was primarily determined by diet, while temperature and diet both affected development rate and female body size. Under a low larval diet, pupal wet weight and wing length both declined with increasing temperature. In contrast, responses of the two morphometric measures to rearing temperature diverged when females were provided higher larval nutrition, with pupal wet weight increasing and wing length decreasing at higher temperatures. Our analyses also revealed opposing relationships between adult female lifespan and the two morphometric measures, with wing length having a positive association with longevity and pupal weight a negative association. Larval diet indirectly affected adult longevity, and the time to pupation was negatively correlated with longevity. The expression of eight immune genes from the toll, JAK-STAT and Imd pathways was enhanced in mosquitoes with higher nutrition.ConclusionsOur results highlight deficiencies from using a single body size measure to capture carry-over effects on adult traits. Further studies of larval development rate under varying environmental conditions and its potential for tracking carry-over effects on vectorial capacity are warranted.Graphical

Gcm counteracts Toll-induced inflammation and impacts hemocyte number through cholinergic signaling.

Hemocytes, the myeloid-like immune cells of Drosophila, fulfill a variety of functions that are not completely understood, ranging from phagocytosis to transduction of inflammatory signals. We here show that downregulating the hemocyte-specific Glial cell deficient/Glial cell missing (Glide/Gcm) transcription factor enhances the inflammatory response to the constitutive activation of the Toll pathway. This correlates with lower levels of glutathione S-transferase, suggesting an implication of Glide/Gcm in reactive oxygen species (ROS) signaling and calling for a widespread anti-inflammatory potential of Glide/Gcm. In addition, our data reveal the expression of acetylcholine receptors in hemocytes and that Toll activation affects their expressions, disclosing a novel aspect of the inflammatory response mediated by neurotransmitters. Finally, we provide evidence for acetylcholine receptor nicotinic acetylcholine receptor alpha 6 (nAchRalpha6) regulating hemocyte proliferation in a cell autonomous fashion and for non-cell autonomous cholinergic signaling regulating the number of hemocytes. Altogether, this study provides new insights on the molecular pathways involved in the inflammatory response.

DsFoxO knockout affects development and fecundity of Drosophila suzukii

Forkhead box O (FoxO), a key transcription factor in many species, participates in numerous physiological and pathological processes of organisms through a variety of signaling pathways. In the present study, we established DsFoxO knockout (DsFoxO-KO) strain using CRISPR/Cas9, and the influence on development and fecundity of mutant strain were evaluated. To clarify the corresponding mechanism, a transcriptome analysis was conducted subsequently. The results showed that the survival rates of the DsFoxO-KO strain in larval, pupal, and adult stages were all significantly lower than those of control. The duration of the pupal stage was similar between the two strains; however, durations of egg, larva, adult preoviposition period (APOP), and total APOP (TPOP) in the DsFoxO-KO strain were all significantly longer compared to those of the control strain. The fecundity of the DsFoxO-KO strain was 20.31 eggs/female, which was significantly lower than that of the control strain (430.47 eggs/female). With the transcriptome analysis, 612 differentially expressed genes (DEGs) were identified. Following COG and GO analyses, we found that most of the DEGs were associated with the metabolic process. According to the KEGG database, the mTOR signaling, MAPK signaling, Wnt signaling, and Toll and Imd signaling pathways; insect hormone biosynthesis; autophagy; and apoptosis were altered in the DsFoxO-KO strain. These results demonstrated that knockout of DsFoxO in D. suzukii significantly influenced its development and fecundity, while transcriptome analysis provided insights to explore the corresponding molecular mechanism. These findings highlighted the critical role of FoxO in D. suzukii and might contribute to the development of novel management strategies for these flies in the future.

Nutritional stress compromises mosquito fitness and antiviral immunity, while enhancing dengue virus infection susceptibility

Diet-induced nutritional stress can influence pathogen transmission potential in mosquitoes by impacting life history traits, infection susceptibility, and immunity. To investigate these effects, we manipulate mosquito diets at larval and adult stages, creating two nutritional levels (low and normal), and expose adults to dengue virus (DENV). We observe that egg number is reduced by nutritional stress at both stages and viral exposure separately and jointly, while the likelihood of laying eggs is exclusively influenced by adult nutritional stress. Adult nutritional stress alone shortens survival, while any pairwise combination between both-stage stress and viral exposure have a synergistic effect. Additionally, adult nutritional stress increases susceptibility to DENV infection, while larval nutritional stress likely has a similar effect operating via smaller body size. Furthermore, adult nutritional stress negatively impacts viral titers in infected mosquitoes; however, some survive and show increased titers over time. The immune response to DENV infection is overall suppressed by larval and adult nutritional stress, with specific genes related to Toll, JAK-STAT, and Imd immune signaling pathways, and antimicrobial peptides being downregulated. Our findings underscore the importance of nutritional stress in shaping mosquito traits, infection outcomes, and immune responses, all of which impact the vectorial capacity for DENV transmission.

Evidence of a conserved mammalian immunosuppression mechanism in Lutzomyia longipalpis upon infection with Leishmania.

Sand flies (Diptera: Phlebotominae) belonging to the Lutzomyia genus transmit Leishmania infantum parasites. To understand the complex interaction between the vector and the parasite, we have been investigating the sand fly immune responses during the Leishmania infection. Our previous studies showed that genes involved in the IMD, Toll, and Jak-STAT immunity pathways are regulated upon Leishmania and bacterial challenges. Nevertheless, the parasite can thrive in the vectors' gut, indicating the existence of mechanisms capable of modulating the vector defenses, as was already seen in mammalian Leishmania infections. In this study, we investigated the expression of Lutzomyia longipalpis genes involved in regulating the Toll pathway under parasitic infection. Leishmania infantum infection upregulated the expression of two L. longipalpis genes coding for the putative repressors cactus and protein tyrosine phosphatase SHP. These findings suggest that the parasite can modulate the vectors' immune response. In mammalian infections, the Leishmania surface glycoprotein GP63 is one of the inducers of host immune depression, and one of the known effectors is SHP. In L. longipalpis we found a similar effect: a genetically modified strain of Leishmania amazonensis over-expressing the metalloprotease GP63 induced a higher expression of the sand fly SHP indicating that the L. longipalpis SHP and parasite GP63 increased expressions are connected. Immuno-stained microscopy of L. longipalpis LL5 embryonic cells cultured with Leishmania strains or parasite conditioned medium showed cells internalization of parasite GP63. A similar internalization of GP63 was observed in the sand fly gut tissue after feeding on parasites, parasite exosomes, or parasite conditioned medium, indicating that GP63 can travel through cells in vitro or in vivo. When the sand fly SHP gene was silenced by RNAi and females infected by L. infantum, parasite loads decreased in the early phase of infection as expected, although no significant differences were seen in late infections of the stomodeal valve. Our findings show the possible role of a pathway repressor involved in regulating the L. longipalpis immune response during Leishmania infections inside the insect. In addition, they point out a conserved immunosuppressive effect of GP63 between mammals and sand flies in the early stage of parasite infection.

Insight into the structural hierarchy of the protease cascade that regulates the mosquito melanization response

Serine protease cascades regulate important insect immune responses, including melanization and Toll pathway activation. In the context of melanization, central components of these cascades are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and their non-catalytic homologs (cSPHs). Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family, central players in melanization, and characterize their relative contributions to bacterial melanization and to mosquito susceptibility to bacterial infections. Using in vivo genetic analysis we show that the protease cascade branches downstream of the cSPs CLIPB4 and CLIPB17 into two branches one converging on CLIPB10 and the second on CLIPB8. We also show that the contribution of key cSPHs to melanization in vivo in response to diverse microbial challenges is more significant than any of the individual cSPs, possibly due to partial functional redundancy among the latter. Interestingly, we show that the key cSPH CLIPA8 which is essential for the efficient activation cleavage of CLIPBs in vivo is efficiently cleaved itself by several CLIPBs in vitro, suggesting that cSPs and cSPHs regulate signal amplification and propagation in melanization cascades by providing positive reinforcement upstream and downstream of each other.

Imidacloprid and acetamiprid synergistically downregulate spaetzle and myD88 of the Toll pathway in haemocytes of the European honeybee (Apis mellifera)

Pollinator health has been of critical concern over the last few decades. The prevalence of the honeybee Colony Collapse Disorder (CCD), changing climate, and the rise of vector-borne honeybee diseases by Varroa destructor, have played a major role in the rapid decline of global honeybee populations. Honeybees are environmentally and economically significant actors in biodiversity. The impact of agricultural practices, such as pesticide use, has exacerbated the negative effects on honeybees. We demonstrate the synergistic effect of cocktails of the neonicotinoids imidacloprid and acetamiprid on honeybee haemocytes. Two genes responsible for critical immune responses, spaetzle and myD88, are consistently dysregulated following exposure to either neonicotinoid alone or as a mixture with or without an immune challenge. The 2018 ban of neonicotinoids in Europe, followed by the 2020 reauthorisation of imidacloprid in France and the current consideration to reinstate acetamiprid underscores the need to evaluate their cumulative impact on honeybee health.

Serpin-1a and serpin-6 regulate the Toll pathway immune homeostasis by synergistically inhibiting the Spätzle-processing enzyme CLIP2 in silkworm, Bombyx mori.

The Toll receptor signaling pathway is an important innate immune response of insects to pathogen infection; its extracellular signal transduction involves serine protease cascade activation. However, excessive or constitutive activation of the Toll pathway can be detrimental. Hence, the balance between activation and inhibition of the extracellular protease cascade must be tightly regulated to achieve favorable outcomes. Previous studies have shown that serpins-serine protease inhibitors-negatively regulate insect innate immunity by inhibiting extracellular protease cascade signaling. Although the roles of serpins in insect innate immunity are well described, the physiological mechanisms underlying their synergistic effects remain poorly understand. Here, we characterize the molecular mechanism by which serpin-1a and serpin-6 synergistically maintain immune homeostasis of the silkworm Toll pathway under physiological and pathological conditions. Through in vitro biochemical assays and in vivo bioassays, we demonstrate that clip-domain serine protease 2 (CLIP2), as the Toll cascade-activating terminal protease, is responsible for processing proSpätzle1 to induce the expression of antimicrobial peptides. Further biochemical and genetic analyses indicate that constitutively expressed serpin-1a and inducible serpin-6 synergistically target CLIP2 to maintain homeostasis of the silkworm Toll pathway under physiological and pathological conditions. Taken together, this study provides new insights into the precise regulation of Toll cascade activation signals in insect innate immune responses and highlights the importance and complexity of insect immune homeostasis regulation.

Transcriptomic landscape of the interaction between the entomopathogenic fungus Beauveria bassiana and its tolerant host Tribolium castaneum revealed by dual RNA-seq

Entomopathogenic fungi such as Beauveria bassiana are the only insect pathogens able to start the infection process by penetrating through the host cuticle. However, some insects try to avoid fungal infection by embedding their cuticle with antifungal compounds. This is the case of the red flour beetle Tribolium castaneum, which generates economical loss of great significance in stored product environments worldwide. In this study, T. castaneum adults were fed during different time periods (from 3 to 72 h) on B. bassiana conidia-covered corn kernels. The progression of fungal infection was monitored using the dual RNA-seq technique to reconstruct the temporal transcriptomic profile and to perform gene enrichment analyses in both interacting organisms. After mapping the total reads with the B. bassiana genome, 904 genes were identified during this process. The more expressed fungal genes were related to carbon catabolite repression, cation binding, peptidase inhibition, redox processes, and stress response. Several immune-related genes from Toll, IMD, and JNK pathways, as well as genes related to chitin modification, were found to be differentially expressed in fungus-exposed T. castaneum. This study represents the first dual transcriptomic approach to help understand the interaction between the entomopathogenic fungus B. bassiana and its tolerant host T. castaneum.

Matrix metalloproteinase 2 contributes to adult eclosion and immune response in the small hive beetle, Aethina tumida.

During the pupal-adult eclosion process of holometabolous insects, the old cuticle is shed and replaced by a completely different new cuticle that requires tanning and expansion, along with extensive extracellular matrix (ECM) remodeling. In vertebrates, matrix metalloproteinases (MMPs), a class of zinc-dependent endopeptidases, play key roles in regulating the ECM that surrounds cells. However, little is known about these extracellular proteinases available in insects. The small hive beetle (SHB), Aethina tumida, is a widespread invasive parasite of honey bees. In this study, 6 MMP homologs were identified in the SHB genome. RNA interference experiments showed that all 6 AtMmps are not required for the larval-pupal transition, only AtMmp2 was essential for pupal-adult eclosion in SHB. Knockdown of AtMmp2 resulted in eclosion defects and wing expansion failure, as well as mortality within 3d of adult eclosion. Transcriptomic analysis revealed that knockdown of AtMmp2 significantly increased expression of the Toll and Imd pathways, chitin metabolism, and cross-linking (such as the pro-phenoloxidase activating cascade pathway and the tyrosine-mediated cuticle sclerotization and pigmentation pathway). These data revealed evolutionarily conserved functions of Mmp2 in controlling adult eclosion and wing expansion, also provided a preliminary exploration of the novel function of regulating Toll and Imd pathways, as well as new insights into how MMPs regulate insect development and defense barriers.

Pathogenicity and transcriptomic exploration of Vibrio fortis in Penaeus monodon

In this study, a strain (recorded as Y6) was isolated from the biofloc pool, its DNA was extracted for 16S rDNA sequencing and compared in the NCBI database, and it was identified as Vibrio fortis. The V. fortis was activated, cultured, and artificially injected into Penaeus monodon to observe the symptoms and calculate the semi-lethal concentration (LC50). It was found that the symptoms of the red leg, an empty stomach, and enlarged hepatopancreas of P. monodon after infection with V. fortis. The LC50 was 4.00 × 107, 2.24 × 107, 1.82 × 107, 1.41 × 107, 7.52 × 106 and 3.31 × 106 CFU/mL at 16, 24, 32, 48, 128, and 144 hpi, respectively. The K–B disk method was used to detect the sensitivity of V. fortis to various antibiotic drugs. V. fortis resisted Ampicillin, Piperacillin, Cefazolin, Cephalothin and Cefoxitin. Highly sensitive to Polymyxin B, Tobramycin, Gentamicin, Cefepime, Cefoperazone and Streptomycin. To explore the molecular response mechanism of V. fortis infection in P. monodon, the hepatopancreas of P. monodon infected with V. fortis at 24 and 48 hpi by transcriptome sequencing, and a total of 347 DEGs were obtained (214 up-regulated DEGs and 133 down-regulated DEGs). In the KEGG pathway enrichment analysis of DEGs, significant changes were found in genes and signaling pathways related to immune system and substance metabolism, including NOD-like receptor signaling pathways, Toll and Imd signaling pathways, C-type lectin receptor signaling pathways and pyruvate metabolism. This study initially revealed the immune response of P. monodon to V. fortis infection from the molecular level and provided a reference for further understanding of the study and control of the vibriosis of shrimp.

Drosophila Gut Immune Pathway Suppresses Host Development-Promoting Effects of Acetic Acid Bacteria.

The physiology of most organisms, including Drosophila, is heavily influenced by their interactions with certain types of commensal bacteria. Acetobacter and Lactobacillus, two of the most representative Drosophila commensal bacteria, have stimulatory effects on host larval development and growth. However, how these effects are related to host immune activity remains largely unknown. Here, we show that the Drosophila development-promoting effects of commensal bacteria are suppressed by host immune activity. Mono-association of germ-free Drosophila larvae with Acetobacter pomorum stimulated larval development, which was accelerated when host immune deficiency (IMD) pathway genes were mutated. This phenomenon was not observed in the case of mono-association with Lactobacillus plantarum. Moreover, the mutation of Toll pathway, which constitutes the other branch of the Drosophila immune pathway, did not accelerate A. pomorum-stimulated larval development. The mechanism of action of the IMD pathway-dependent effects of A. pomorum did not appear to involve previously known host mechanisms and bacterial metabolites such as gut peptidase expression, acetic acid, and thiamine, but appeared to involve larval serum proteins. These findings may shed light on the interaction between the beneficial effects of commensal bacteria and host immune activity.

Spätzle processing enzyme is required to activate dorsal switch protein 1 induced Toll immune signalling pathway in Tenebrio molitor.

Dorsal switch protein 1 (DSP1) acts as a damage-associated molecular pattern (DAMP) molecule to activate immune responses in Tenebrio molitor. From a previous study in Spodoptera exigua, we found that DSP1 activates Toll immune signalling pathway to induce immune responses by melanisation, PLA2 activity and AMP synthesis. However, the target site of DSP1 in this pathway remains unknown. The objective of this study was to determine the role of spätzle processing enzyme in the DSP1 induced toll immune signalling pathway. To address this, we analyzed spätzle processing enzyme (Tm-SPE) of the three-step serine protease cascade of T. molitor Toll pathway. Tm-SPE expressed in all developmental stages and larval tissues. Upon immune challenge, its expression levels were upregulated but significantly reduced after RNA interference (RNAi). In addition, the induction of immune responses upon immune challenge or recombinant DSP1 injection was significantly increased. Loss of function using RNA interference revealed that the Tm-SPE is involved in connecting DSP1 induced immune responses like hemocyte nodule formation, phenoloxidase (PO) activity, phospholipase A2 (PLA2) activity and antimicrobial peptide (AMP) synthesis. These suggest that Tm-SPE controls the DSP1 induced activation of Toll immune signalling pathway required for both cellular and humoral immune responses. However, to confirm the target molecule of DSP1 in three-step proteolytic cascade, we have to check other upstream serine proteases like Spatzle activating enzyme (SAE) or modular serine protease (MSP).