Antimicrobial Peptide Genes Research Articles

Evidence that the loss of colonic anti-microbial peptides may promote dysbiotic Gram-negative inflammaging-associated bacteria in aging mice.

Introduction: Aging studies in humans and mice have played a key role in understanding the intestinal microbiome and an increased abundance of "inflammaging" Gram-negative (Gn) bacteria. The mechanisms underlying this inflammatory profile in the aging microbiome are unknown. We tested the hypothesis that an aging-related decrease in colonic crypt epithelial cell anti-microbial peptide (AMP) gene expression could promote colonic microbiome inflammatory Gn dysbiosis and inflammaging. Methods: As a model of aging, C57BL/6J mice fecal (colonic) microbiota (16S) and isolated colonic crypt epithelial cell gene expression (RNA-seq) were assessed at 2 months (mth) (human: 18years old; yo), 15mth (human: 50yo), and 25mth (human: 84yo). Informatics examined aging-related microbial compositions, differential colonic crypt epithelial cell gene expressions, and correlations between colonic bacteria and colonic crypt epithelial cell gene expressions. Results: Fecal microbiota exhibited significantly increased relative abundances of pro-inflammatory Gn bacteria with aging. Colonic crypt epithelial cell gene expression analysis showed significant age-related downregulation of key AMP genes that repress the growth of Gn bacteria. The aging-related decrease in AMP gene expressions is significantly correlated with an increased abundance in Gn bacteria (dysbiosis), loss of colonic barrier gene expression, and senescence- and inflammation-related gene expression. Conclusion: This study supports the proposed model that aging-related loss of colonic crypt epithelial cell AMP gene expression promotes increased relative abundances of Gn inflammaging-associated bacteria and gene expression markers of colonic inflammaging. These data may support new targets for aging-related therapies based on intestinal genes and microbiomes.

The life-history trait trade-offs mediated by reproduction and immunity in the brown planthopper, Nilaparvata lugens Stål

Reproduction and immune defense are costly functions, and they are expected to tradeoff with each other to drive evolution. The brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera, Delphacidae), is a global superpest that mostly damages rice crops. Yeast-like symbionts (YLS) exist in the abdominal fat body tissue and are tightly associated with the development, growth, and reproduction of BPH. Our previous research demonstrated that mating behavior promotes the release of YLS from the fat body into the hemolymph in the BPH, thereby triggering an immune response. Additionally, the fitness costs related to life-history traits of BPH (such as survival rate) have a strong dependence on the relative abundance of YLS. However, the possible relationship between reproduction and the immune response in BPH has not been identified. In this study, an omics-based approach was used to analyze the transcriptome of fat body tissues in mated and unmated BPH at 72 h post-eclosion, from which two antimicrobial peptide genes, NlDefensin A (NlDfA) and NlDefensin B (NlDfB), were selected since they were highly expressed in mated BPH. Subsequently, the full-length cDNA sequences of the NlDfA and NlDfB genes were cloned and analyzed. qPCR results showed up-regulation of the NlDfA and NlDfB genes in mated BPH when compared to unmated BPH. Spatial-temporal expression analysis indicated that the NlDfA and NlDfB genes were expressed in all tissues and developmental stages, and they were most highly expressed in the fat body at 24 h post-eclosion. Moreover, the symbionts in BPH were significantly inhibited by the in vitro expression of the NlDfA and NlDfB proteins. Furthermore, RNA interference (RNAi)-mediated suppression of NlDfA and NlDfB dramatically increased the relative abundance of YLS in the fat body, while YLS in the hemolymph decreased significantly. These BPHs also displayed some fitness disadvantages in survival, fecundity, hatchability, and possibly the vertical transmission of YLS from hemolymph to egg. Our results indicated that mating could heighten the immunity of BPH by up-regulating the expression of the NlDfA and NlDfB genes, which protect the host from pathogen challenges during reproduction. However, the reduced content of YLS may act as a fitness disadvantage in dictating the life-history traits of BPH. This work has significant theoretical and practical implications for the precise green control technology that involves crucial gene targeting, as well as for the “endosymbionts for pest control” strategy in insects.

Enhancement of an entomopathogenic fungal virulence against the seedcorn maggot, Delia platura, by suppressing immune responses with a bacterial culture broth of Photorhabdus temperata subsp. temperata.

In Korea, there are two maggot species in the Delia genus that commonly infest the roots and stems of the Welsh onion, thus causing serious economic damage on the crop at the seedling stage. In this study, the seedcorn maggot (Delia platura) was detected in onion fields in two different localities in Korea. After overwintering, maggot infestations occurred throughout the entire growing seasons from transplantation to harvest, but their specific patterns of occurrence varied in the two localities examined. Entomopathogenic fungi induced significant virulence against the maggot larvae, in which a strain of Beauveria bassiana was effective, though it exhibited limited mortality in its insecticidal activity. To enhance this insecticidal activity, a culture broth from an entomopathogenic bacterium, Photorhabdus temperata temperata (Ptt), was added to B. bassiana treatment. The addition of Ptt broth significantly increased the insecticidal activity of B. bassiana in a dose-dependent manner. To elucidate this enhancement in insecticidal activity, the immunosuppressive activity of Ptt broth was assessed by identifying the immune responses of the seedcorn maggots. The seedcorn maggots possessed at least three different hemocytes with plasmatocytes, crystal cells, and lamellocytes. These hemocytes exhibited nodule formation in response to the fungal infection. In addition to the cellular immunity, the maggots exhibited inducible expressions of antimicrobial peptide (AMP) genes such as cecropin and defensin. The addition of Ptt broth suppressed the nodule formation and the AMP expressions in response to the fungal infection. Altogether, this study demonstrated the innate immune responses in a non-model insect, D. platura, along with the application of immunosuppression to develop a highly efficient biological control by enhancing the virulence of B. bassiana.

BMP signaling to pharyngeal muscle in the C. elegans response to a bacterial pathogen regulates anti-microbial peptide expression and pharyngeal pumping.

Host response to pathogens recruits multiple tissues in part through conserved cell signaling pathways. In Caenorhabditis elegans, the bone morphogenetic protein (BMP) like DBL-1 signaling pathway has a role in the response to infection in addition to other roles in development and postdevelopmental functions. In the regulation of body size, the DBL-1 pathway acts through cell autonomous signal activation in the epidermis (hypodermis). We have now elucidated the tissues that respond to DBL-1 signaling upon exposure to two bacterial pathogens. The receptors and Smad signal transducers for DBL-1 are expressed in pharyngeal muscle, intestine, and epidermis. We demonstrate that expression of receptor-regulated Smad (R-Smad) gene sma-3 in the pharynx is sufficient to improve the impaired survival phenotype of sma-3 mutants and that expression of sma-3 in the intestine has no effect when exposing worms to bacterial infection of the intestine. We also show that two antimicrobial peptide genes - abf-2 and cnc-2 - are regulated by DBL-1 signaling through R-Smad SMA-3 activity in the pharynx. Finally, we show that pharyngeal pumping activity is reduced in sma-3 mutants and that other pharynx-defective mutants also have reduced survival on a bacterial pathogen. Our results identify the pharynx as a tissue that responds to BMP signaling to coordinate a systemic response to bacterial pathogens.

The interaction between lipid and vitamin D3 impacts lipid metabolism and innate immunity in Chinese mitten crabs Eriocheir sinensis

As a fat-soluble vitamin, vitamin D3 relies on fat to perform its biological function, affecting lipid metabolism and innate immunity. This study used different percentages of lipid and vitamin D3 diets to evaluate the synergistic effects on the growth, lipid metabolism and immunity of juvenile Eriocheir sinensis (5.83 ± 0.01 g) for 56 days, including low lipid (LL, 1.5%) and normal lipid (NL, 7.5%) and three levels of vitamin D3: low (LVD, 0 IU/kg), medium (MVD, 9000 IU/kg) and high (HVD, 27,000, IU/kg). The synergistic effect of lipid and vitamin D3 was not significant on growth but significant on ash content, total protein, hepatopancreas lipid content, hemolymph 1α,25-hydroxy vitamin D3 [1α,25(OH)2D3] content, hepatopancreas lipolysis and synthesis genes. Crabs fed normal lipid (7.5%) and medium vitamin D3 (9000 IU/kg) had the highest hepatopancreas index, hemolymph 1α,25(OH)2D3 content, antibacterial ability, immune-related genes and hepatopancreatic lipid synthesis genes expression, but down-regulated the lipolysis genes expression. In contrast, crabs fed diets with low lipid percentage (1.5%) had low growth performance, hemolymph 1α,25(OH)2D3, mRNA levels of lipid synthesis genes, antibacterial ability and immune-related gene expression. At the 1.5% lipid level, excessive or insufficient vitamin D3 supplementation led to the obstruction of ash and protein deposition, reduced growth and molting, aggravated the reduction in antioxidant capacity, hindered antimicrobial peptide gene expression and reduced innate immunity, and resulted in abnormal lipid accumulation and the risk of oxidative stress. This study suggests that diets' lipid and vitamin D3 percentage can enhance antioxidant capacity, lipid metabolism and innate immunity in E. sinensis. A low lipid diet can cause growth retardation, reduce antioxidant capacity and innate immunity, and enhance lipid metabolism disorder.

Humoral immune response of Galleria mellonella after mono- and co-injection with Hypericum perforatum extract and Candida albicans.

Galleria mellonella is used as a model organism to study the innate immune response of insects. In this study, the humoral immune response was assessed by examining phenoloxidase activity, fungal burden, and the expression of phenoloxidase and antimicrobial peptide genes at different time point following separate and combined injections of Hypericum perforatum extract and a nonlethal dose of Candida albicans. The administration of a plant extract at low doses increased phenoloxidase activity, while higher doses had no effect. Similarly, co-injection of a low dose of the extract with the pathogen allowed half of the yeast cells to survive after 24 h. Co-injection of plant extract with the pathogen decreased the phenoloxidase activity at the end of 4 h compared to C. albicans mono-injection. The phenoloxidase gene expressions was reduced in all experimental conditions with respect to the control. When plant extracts and the pathogen were administered together, gallerimycin and hemolin gene expressions were considerably higher compared to mono-injections of plant extracts and the pathogen. The results of this study reveal that gene activation and regulatory mechanisms may change for each immune gene, and that recognition and signaling pathways may differ depending on the involved immunoregulator.

BmPGPR-L4 is a negative regulator of the humoral immune response in the silkworm Bombyx mori.

Toll, immune deficiency and prophenoloxidase cascade represent vital immune signaling pathways in insects. Peptidoglycan recognition proteins (PGRPs) are innate immune receptors that activate and regulate the immune signaling pathways. Previously, we reported that BmPGPR-L4 was induced in the silkworm Bombyx mori larvae by bacteria and peptidoglycan challenges. Here, we focused on the function of BmPGRP-L4 in regulating the expression of antimicrobial peptides (AMPs). The hemolymph from BmPGRP-L4-silenced larvae exhibited an enhanced inhibitory effect on the growth of Escherichia coli, either by growth curve or inhibitory zone experiments. Coincidentally, most of the AMP genes were upregulated after RNAi of BmPGRP-L4. Oral administration of heat-inactivated E. coli and Staphylococcus aureus after RNAi of BmPGRP-L4 resulted in the increased expression of BmPGRP-L4 in different tissues of the silkworm larvae, revealing an auto-regulatory mechanism. By contrast, the expression of most AMP genes was downregulated by oral bacterial administration after RNAi of BmPGRP-L4. The above results demonstrate that BmPGRP-L4 recognizes bacterial pathogen-associated molecular patterns and negatively regulates AMP expression to achieve immunological homeostasis. As a negative regulator, BmPGPR-L4 is proposed to be involved in the feedback regulation of the immune signaling pathways of the silkworm to prevent excessive activation of the immune response.

Differential expression of antimicrobial peptide encoding genes of honey bee (Apis mellifera) after Varroa mite (Varroa destructor) infestation

Varroa destructor mite is a serious threat to the world apiculture industry, and severely hampered beekeeping practices. Innate immune systems are key defenses of animals and are particularly important in species that lack the sophisticated adaptive immune systems found in vertebrates. In honey bees, antimicrobial immune peptide genes play a significant immune response against pathogens. The present study analyzed the detrimental effects of Varroa infestation on Apis mellifera at all developmental stages in both Varroa challenged and controlled hives. Physical attributes were observed in Varroa hives at different time intervals (0, 30, and 45 days respectively). After 45 days of Varroa exposure, the samples were taken, and the AMPs immune response of bees parasitized by Varroa was detected by studying the expression levels of antibacterial peptides (defensin, apidaecin, abaecin, and hymenoptaecin). Significant alterations in the physical parameters viz. body length, body weight, wing shape, and mean numbers of brood cells were observed compared to the control. Colony density was also decreased with marked negative behavioural changes. Ct values of Real-Time PCR results showed that AMP-specific immune gene transcription level in Varroa-challenged bees was downregulated, except for abaecin and defensin (adult stage). There was a significant (p < 0.05) increase in abaecin gene transcription folds i.e., 1.064 larvae, 1.732 pupae, and 1.264 adults bees. As a whole, all these findings can be taken as critical biomarkers in the advancement of control techniques against Varroa and other pathogen infections as a part of wide-ranging programs of honey bee health and Varroa control.

Transcriptomic profiling of different developmental stages reveals parasitic strategies of Wohlfahrtia magnifica, a myiasis-causing flesh fly

BackgroundWohlfahrtia magnifica is an obligatory parasite that causes myiasis in several warm-blooded vertebrates. Adult females deposit the first-stage larvae directly onto wounds or natural body orifices (e.g., genitalia) of the host, from where they quickly colonize the host tissue and feed on it for development. The infestation of W. magnifica can lead to health issues, welfare concerns, and substantial economic losses. To date, little is known about the molecular mechanisms of the W. magnifica-causing myiasis.ResultsIn this study, we collected parasitic-stage larvae of W. magnifica from wounds of naturally infested Bactrian camels, as well as pupae and adult flies reared in vitro from the wound-collected larvae, for investigating the gene expression profiles of the different developmental stages of W. magnifica, with a particular focus on examining gene families closely related to the parasitism of the wound-collected larvae. As key proteins related to the parasite-host interaction, 2049 excretory/secretory (ES) proteins were identified in W. magnifica through the integration of multiple bioinformatics approaches. Functional analysis indicates that these ES proteins are primarily involved in cuticle development, peptidase activity, immune response, and metabolic processes. The global investigation of gene expression at different developmental stages using pairwise comparisons and weighted correlation network analysis (WGCNA) showed that the upregulated genes during second-stage larvae were related to cuticle development, peptidase activity, and RNA transcription and translation; during third-stage larvae to peptidase inhibitor activity and nutrient reservoir activity; during pupae to cell and tissue morphogenesis and cell and tissue development; and during adult flies to signal perception, many of them involved in light perception, and adult behavior, e.g., feeding, mating, and locomotion. Specifically, the expression level analysis of the likely parasitism-related genes in parasitic wound-collected larvae revealed a significant upregulation of 88 peptidase genes (including 47 serine peptidase genes), 110 cuticle protein genes, and 21 heat shock protein (hsp) genes. Interestingly, the expression of 2 antimicrobial peptide (AMP) genes, including 1 defensin and 1 diptericin, was also upregulated in the parasitic larvae.ConclusionsWe identified ES proteins in W. magnifica and investigated their functional distribution. In addition, gene expression profiles at different developmental stages of W. magnifica were examined. Specifically, we focused on gene families closely related to parasitism of wound-collected larvae. These findings shed light on the molecular mechanisms underlying the life cycle of the myiasis-causing fly, especially during the parasitic larval stages, and provide guidance for the development of control measures against W. magnifica.

The Toll/IMD pathways mediate host protection against dipteran parasitoids

Parasitoids have utilized a variety of strategies to counteract host defense. They are in different taxonomic status and exhibit phenotypic and genetic diversity, and thus are thought to evolve distinct anti-defense mechanisms. In this study, we investigated the performance of two closely related parasitoids, Exorista japonica and Exorista sorbillans (Diptera: Tachinidae) that are biological control agents in agriculture and major insect pests in sericulture, on the host Bombyx mori. We show that the host is more susceptible to E. sorbillans infection while relatively resistant to E. japonica infection. Moreover, the expression levels of host antimicrobial peptides (AMPs) genes are repressed at early infection and induced at late infection of E. japonica, while AMPs are over-expressed at early infection and return to normal levels at late infection of E. sorbillans. In parallel, Toll and IMD pathway genes are generally induced at late infection of E. japonica, whereas these genes are up-regulated at early infection and down-regulated at late infection of E. sorbillans. Activating of host Toll/IMD pathways and AMPs expression by lipopolysaccharide (LPS) represses the larval growth of E. sorbillans. Conversely, inhibiting host Toll/IMD pathways by RNA interference significantly promotes E. japonica development. Therefore, the Toll/IMD pathways are required in the host for defense against infection of dipteran parasitoids. Overall, our study provides the new insight into the diversified host-parasitoid interactions, and offers a theoretical basis for further studies of the adaptive mechanism of dipteran parasitoids.

Intercalated cell function, kidney innate immunity, and urinary tract infections.

Intercalated cells (ICs) in the kidney collecting duct have a versatile role in acid-base and electrolyte regulation along with the host immune defense. Located in the terminal kidney tubule segment, ICs are among the first kidney cells to encounter bacteria when bacteria ascend from the bladder into the kidney. ICs have developed several mechanisms to combat bacterial infections of the kidneys. For example, ICs produce antimicrobial peptides (AMPs), which have direct bactericidal activity, and in many cases are upregulated in response to infections. Some AMP genes with IC-specific kidney expression are multiallelic, and having more copies of the gene confers increased resistance to bacterial infections of the kidney and urinary tract. Similarly, studies in human children demonstrate that those with history of UTIs are more likely to have single-nucleotide polymorphisms in IC-expressed AMP genes that impair the AMP's bactericidal activity. In murine models, depleted or impaired ICs result in decreased clearance of bacterial load following transurethral challenge with uropathogenic E. coli. A 2021 study demonstrated that ICs even act as phagocytes and acidify bacteria within phagolysosomes. Several immune signaling pathways have been identified in ICs which may represent future therapeutic targets in managing kidney infections or inflammation. This review's objective is to highlight IC structure and function with an emphasis on current knowledge of IC's diverse innate immune capabilities.

LDLa containing C-type lectin mediates phagocytosis of V.anguillarum and regulates immune effector genes in shrimp

C-type lectins (CTLs) function as pattern recognition receptors (PRRs) by recognizing invading microorganisms, thereby triggering downstream immune events against infected pathogens. In this study, a novel CTL containing a low-density lipoprotein receptor class A (LDLa) domain was obtained from Litopenaeus vannamei, designed as LvLDLalec. Stimulation by the bacterial pathogen Vibrio anguillarum (V. anguillarum) resulted in remarkable up-regulation of LvLDLalec, as well as release of LvLDLalec into hemolymph. The rLvLDLalec protein possessed broad-spectrum bacterial binding and agglutinating activities, as well as hemocyte attachment ability. Importantly, LvLDLalec facilitated the bacterial clearance in shrimp hemolymph and protected shrimp from bacterial infection. Further studies revealed that LvLDLalec promoted hemocytes phagocytosis against V. anguillarum and lysosomes were involved in the process. Meanwhile, LvLDLalec participated in humoral immunity through activating and inducing nuclear translocation of Dorsal to regulate phagocytosis-related genes and antimicrobial peptides (AMPs) genes, thereby accelerated the removal of invading pathogens in vivo and improved the survival rate of L. vannamei. These results unveil that LvLDLalec serves as a PRR participate in cellular and humoral immunity exerting opsonin activity to play vital roles in the immune regulatory system of L. vannamei.

A parasitoid regulates 20E synthesis and antibacterial activity of the host for development by inducing host nitric oxide production.

Parasitoids are important components of the natural enemy guild in the biological control of insect pests. They depend on host resources to complete the development of a specific stage or whole life cycle and thus have evolved towards optimal host exploitation strategies. In the present study, we report a specific survival strategy of a fly parasitoid Exorista sorbillans (Diptera: Tachinidae), which is a potential biological control agent for agricultural pests and a pest in sericulture. We found that the expression levels of nitric oxide synthase (NOS) and nitric oxide (NO) production in host Bombyx mori (Lepidoptera: Bombycidae) were increased after E. sorbillans infection. Reducing NOS expression and NO production with an NOS inhibitor (NG-nitro-L-arginine methyl ester hydrochloride) in infected B. mori significantly impeded the growth of E. sorbillans larvae. Moreover, the biosynthesis of 20-hydroxyecdysone (20E) in infected hosts was elevated with increasing NO production, and inhibiting NOS expression lowered 20E biosynthesis. More importantly, induced NO synthesis was required to eliminate intracellular bacterial pathogens that presumably competed for shared host resources. Inhibiting NOS expression down-regulated the transcription of antimicrobial peptide genes and increased the number of bacteria in parasitized hosts. Collectively, this study revealed a new perspective on the role of NO in host-parasitoid interactions and a novel mechanism for parasitoid regulation of host physiology to support its development.

Immune signaling pathways in Rhodnius prolixus in the context of Trypanosoma rangeli infection: cellular and humoral immune responses and microbiota modulation.

Rhodnius prolixus is a hematophagous insect and one of the main vectors for Trypanosoma cruzi and Trypanosoma rangeli parasites in Latin America. Gut microbiota and insect immune responses affect T. cruzi and T. rangeli infection within triatomines. Particularly the Toll and IMD signaling pathways activations and how they orchestrate the antimicrobial peptides (AMPs) expressions in R. prolixus, especially when infected by T. rangeli. Examine how T. rangeli infection modulates R. prolixus cellular and humoral immunity and its impacts on insect microbiota. R. prolixus was fed on blood containing epimastigotes of T. rangeli, and infection was quantified in insect tissues. The gene expression of dorsal, cactus, relish, PGRP, and AMPs was examined in the midgut, fat body, and salivary glands by quantitative real-time PCR. Microbiota composition was analyzed using RT-q PCR targeting specific bacterial species. Hemocyte numbers and phenoloxidase activity were quantified to assess cellular immune responses. T. rangeli infection modulated triatomine immunity in midgut and hemocoel, activating the expression of the NF-kB gene dorsal, associated with the Toll pathway; increasing expression of the gene encoding PGRP receptor, a component involved in the IMD pathway, both in the intestine and fat body; repressing the expression of the relish transcription factor, mainly in salivary glands. Among the R. prolixus AMPs studied, T. rangeli infection repressed all AMP gene expression, other than defensin C which increased mRNA levels. The PO activity was enhanced in the hemolymph of infected insects. T. rangeli infection did not induce hemocyte number alterations compared to control insects. However, an increase in hemocyte microaggregation was detected in infected insects. R. prolixus recognizes T. rangeli infection and triggers humoral and cellular immune responses involving Toll pathway activation, defensin C synthesis, increased phenoloxidase activity, and enhanced hemocyte aggregation. On the other hand, T. rangeli infection suppressed some IMD pathway components, suggesting that, in R. prolixus, this pathway is involved in defensins A and B gene regulation. Importantly, these immune responses altered the bacterial microbiota composition, potentially favoring T. rangeli establishment in the insect vector.

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.

Diversity of MrTolls and their regulation of antimicrobial peptides expression during Enterobacter cloacae infection in Macrobrachium rosenbergii

Toll/Toll-like receptor (TLR) is an important pattern recognition receptor that plays an important role in the immunity of animals. Six Toll genes were identified in Macrobrachium rosenbergii, namely, MrToll, MrToll1, MrToll2, MrToll3, MrToll4, and MrToll5. SMART analysis showed that all six Tolls have a transmembrane domain, a TIR domain, and different number of LRR domains. The phylogenetic tree showed that six Tolls were located in six different branches. Among these six Tolls, only MrToll4 contains the QHR motif, which is similar to insect Toll9. MrToll4 belongs to V-type/scc Toll with only one LRRCT domain. MrToll1 and MrToll5 are classical P-type/mcc Toll with two LRRCT domains and an LRRNT. MrTolls were distributed in the hemocytes, heart, hepatopancreas, gills, stomach, and intestine. During the infection of Enterobacter cloacae, the expression level of MrToll and MrToll1-4 was upregulated in the intestine of M. rosenbergii. RNA interference experiments showed that the expression of most antimicrobial peptide (AMP) genes was negatively regulated by MrTolls during E. cloacae infection. On the contrary, crustin (Cru) 3 and Cru4 were inhibited after the knockdown of MrToll, and Cru1 and Cru4 were significantly downregulated with the knockdown of MrToll4 during E. cloacae challenge. These results suggest that MrTolls may be involved in the regulation of AMP expression in the intestine during E. cloacae infection.

Effects of the combination of Lactobacillus helveticus and isomalto-oligosaccharide on survival, gut microbiota, and immune function in Apis cerana worker bees.

The adult worker bees were fed sucrose syrup or sucrose syrup supplemented with Lactobacillus helveticus KM7, prebiotic isomalto-oligosaccharide (IMO), or L. helveticus KM7 combined with IMO. Survival rate, gut microbiota, and gene expression of gut antimicrobial peptides in worker honey bees were determined. Administration of L. helveticus KM7 and IMO significantly increased the survival rate in worker bees relative to bees fed sucrose only. Then, higher concentration of both lactic acid bacteria and Bifidobacterium in the gut and lower counts of gut fungi, Enterococcus, and Bacteroides-Porphyromonas-Prevotella were observed in bees fed the combination of KM7 and IMO compared with control bees. The combination of L. helveticus KM7 with IMO showed a greater or comparable modulating effect on those bacteria relative to either KM7 or IMO alone. Furthermore, the combination treatment of L. helveticus KM7 and IMO enhanced mRNA expression of antimicrobial peptide genes, including Abaecin, Defensin, and the gene encoding prophenoloxidase (PPO) in the gut compared with both control bees and those either L. helveticus KM7 or IMO alone. These results suggest that the combination of L. helveticus KM7 and IMO synergistically modifies the gut microbiota and immunity and consequently improves the survival rate of Apis cerana adult workers.

Biological Diversity of Genes Encoding Wheat Defensin Homologues

Antimicrobial peptides (AMPs) are major components of innate immunity in plants and animals. AMP genes have significant intra- and interspecific polymorphism, the role of which is poorly understood. Previously, by high throughput transcriptome sequencing of wheat plants, we identified defensin genes up-regulated upon infection with the pathogenic fungus Fusarium oxysporum and/or treatment with resistance inducers. In the present work, a bioinformatic search in NCBI databases for peptide homologues of these defensins was carried out using the sequences of their γ-cores, the sites of the molecules responsible for antimicrobial activity. DEFL1-16 homologues were identified in 95 species of angiosperms belonging to 48 families and 30 orders of monocotyledonous and dicotyledonous plants. The ubiquitous distribution of this defensin in angiosperms suggests its involvement not only in defense, but also in other processes in flowering plants. Homologues of other defensins induced by infection were found only in plants of the Poaceae family, which suggests the existence of a Poaceae-specific defense mechanism associated with the expression of these defensins. Among the γ-core variants of wild plant defensins identified in the study, the peptides with better antimicrobial activity compared to wheat, might be present, which are of considerable interest for the development of new antibiotics for medicine and agriculture.

Identification of antimicrobial peptide genes from transcriptomes in Mandarin fish (Siniperca chuatsi) and their response to infection with Aeromonas hydrophila

Mandarin fish (Siniperca chuatsi) is a valuable freshwater fish species widely cultured in China. Its aquaculture production is challenged by bacterial septicaemia, which is one of the most common bacterial diseases. Antimicrobial peptides (AMPs) play a critical role in the innate immune system of fish, exhibiting defensive and inhibitory effects against a wide range of pathogens. This study aimed to identify the antimicrobial peptide genes in mandarin fish using transcriptomes data obtained from 17 tissue in our laboratory. Through nucleotide sequence alignment and protein structural domain analysis, 15 antimicrobial peptide genes (moronecidin, pleurocidin, lysozyme g, thymosin β12, hepcidin, leap 2, β-defensin, galectin 8, galectin 9, apoB, apoD, apoE, apoF, apoM, and nk-lysin) were identified, of which 9 antimicrobial peptide genes were identified for the first time. In addition, 15 AMPs were subjected to sequence characterization and protein structure analysis. After injection with Aeromonas hydrophila, the number of red blood cells, hemoglobin concentration, and platelet counts in mandarin fish showed a decreasing trend, indicating partial hemolysis. The expression change patterns of 15 AMP genes in the intestine after A. hydrophila infection were examined by using qRT-PCR. The results revealed, marked up-regulation (approximately 116.04) of the hepcidin gene, down-regulation of the piscidin family genes expression. Moreover, most AMP genes were responded in the early stages after A. hydrophila challenge. This study provides fundamental information for investigating the role of the different antimicrobial peptide genes in mandarin fish in defense against A. hydrophila infection.

Identification and immune analysis of antimicrobial peptides from the cigarette beetle (Lasioderma serricorne).

Antimicrobial peptides (AMPs) in insects are endogenous peptides that are effector components of the innate defense system of the insect. AMPs may serve as antimicrobial agents because of their small molecular weight and broad-spectrum antimicrobial activity. In this study, we performed transcriptome analysis of cigarette beetle (Lasioderma serricorne) larvae, parasitized by the ectoparasitic wasp, Anisopteromalus calandrae. Several AMP genes were significantly upregulated following A.calandrae parasitism, postulating the hypothesis that the parasitization enhanced the host's resistance against pathogenic microorganisms through the regulation of host AMP genes. Specifically, 3 AMP genes (LsDef1, LsDef2, and LsCole) were significantly upregulated and we studied their immune function in L.serricorne. Immune challenge and functional analysis showed that LsCole was responsible for the immune response against Gram-negative and Gram-positive bacteria, while LsDef1 and LsDef2 were involved in insect defense against Gram-positive bacteria. Purified recombinant LsCole exhibited antimicrobial activities against the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. LsDef2 showed an antibacterial effect against S.aureus. LsCole and LsDef2 exhibited antibiofilm activity against S.aureus. The 2 AMPs disrupted cell membranes and caused leakage of S.aureus cell contents. The results indicated that the 3 AMPs in L.serricorne are involved in the innate immunity of this pest insect. These AMPs may have potential as antimicrobial agents for bacterial infection chemotherapy. Hence, data are discussed in relation to new control strategies with greater biosafety against pest insects with use of microbial biocontrol agents in combination with RNA interference against the insect's defensive AMP genes.