Antimicrobial Peptide Genes Research Articles

Transcription factor STAT enhanced antimicrobial activities in Bombyx mori

STAT, a transcription factor in the JAK/STAT signaling pathway, regulates immune response to pathogens. In the silkworm (Bombyx mori), STAT exists as two split-forms, STAT-S and STAT-L. However, the role of STAT in silkworm immunity remains unclear. Our purpose was to investigate the effect of STAT on the expression of antimicrobial peptide (AMP) genes and resistance against pathogens. The expression levels of STAT-S and STAT-L were significantly up-regulated after induction by pathogenic microorganisms. In BmE cells, lipopolysaccharide (LPS), peptidoglycan (PGN) and β-glucan stimulated STAT-S and STAT-L to transfer from the cytoplasm to the nucleus. We found that overexpression of STAT-S and STAT-L in cells could promote the expression of AMPs. We generated transgenic silkworm lines overexpressing STAT-L or STAT-S (OE-STAT-S; OE-STAT-L) or interfering with STAT (A4-dsSTAT). Overexpression of STAT-S and STAT-L upregulated the expression of AMP genes in the OE-STAT-S and OE-STAT-L, increased the survival rates of the OE-STAT-S silkworms and lowered the mortality of OE-STAT-L silkworms infected with S. aureus or Beauveria bassiana. By contrast, the death rate of A4-dsSTAT silkworms was higher after infection with these pathogenic microorganisms. These findings may provide insights into the role of STAT in the antimicrobial immune response of silkworms.

The first identification and functional analysis of two drosophila mothers against decapentaplegic protein genes (SpSmad1 and SpSmad2/3) and their involvement in the innate immune response in Scylla paramamosain

Smad，a member of the TGF-β superfamily，controls cell proliferation，growth and guiding cell differentiation, thus playing a crucial role in diseases. However, the presence as well as specific function of Smad in crabs is still unknown. In this study, two Smads (Smad1 and Smad2/3) were identified for the first time from the mud crab Scylla paramamosain. The complete open reading frames of SpSmad1 and SpSmad2/3 were 1,497bp and 1,338bp, encoding deduced proteins of 498 and 445 amino acids respectively. Moreover, under the administration of Vibrio alginolyticus and WSSV, the relative expression levels of SpSmad1 and SpSmad2/3 were significantly increased, indicating their involvement in the innate immune response of mud crabs. Knockdown of SpSmad1 and SpSmad2/3 in vivo not only led to the increasement of the expressions of NF-κB signaling genes and antimicrobial peptides genes, but also significantly affected the bacterial clearance process of mud crabs. Additionally, overexpression of SpSmad1 and SpSmad2/3 in HEK293T cells could markedly activate NF-κB signaling. These results indicated that Smad1 and Smad2/3 participated in the innate immunity of Scylla paramamosain, and might provide a better understanding of the presence and immune regulatory functions of Smad1 and Smad2/3 in crabs and even invertebrates.

Functional Characterization of A Deformed Epidermal Autoregulatory Factor 1 Gene in Litopenaeus vannamei

Innate immunity is crucial for invertebrate defense against pathogenic infections. Numerous studies have indicated that the Toll–NF–κB pathway plays an important role in this process, particularly in anti-bacterial and anti-fungal immunity. Although the function of this pathway has been studied extensively, there are still uncertainties regarding its role in shrimp. In this study, we investigated the functions of Deformed Epidermal Autoregulatory Factor 1 (LvDEAF1) in Litopenaeus vannamei, a member of the Toll–NF–κB pathway. Our findings revealed that LvDEAF1 interacts with L. vannamei Pellino1 (LvPellino1). LvDEAF1 enhances the promoter activity of certain antimicrobial peptide genes, such as Metchnikowin and Drosomycin, in Drosophila Schneider 2 (S2) cells by binding to the NF-κB binding site. LvDEAF1 and LvPellino1 exhibit positive and synergistic effects. Additionally, the expression of LvDEAF1 is induced by Vibrio parahaemolyticus infection and lipopolysaccharides or zymosan treatment. Knockdown LvDEAF1 expression resulted in a decrease in Penaeidins 4 expression and an increase in the cumulative mortality of shrimp infected with V. parahaemolyticus. These findings indicate that LvDEAF1 plays an important role in the Toll–NF–κB pathway of L. vannamei and is essential for its immune response against pathogens.

Characterization of Domeless receptors and the role of BdDomeless3 in anti-symbiont-like virus defense in Bactrocera dorsalis

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway play a pivotal role in innate immunity. Among invertebrates, Domeless receptors serve as the key upstream regulators of this pathway. In our study on Bactrocera dorsalis, we identified three cytokine receptors: BdDomeless1, BdDomeless2, and BdDomeless3. Each receptor encompasses five fibronectin-type-III-like (FN III) extracellular domains and a transmembrane domain. Furthermore, these receptors exhibit the increased responsiveness to diverse pathogenic challenges. Notably, only BdDomeless3 is upregulated during symbiont-like viral infections. Moreover, silencing BdDomeless3 enhanced the infectivity of Bactrocera dorsalis cripavirus (BdCV) and B. dorsalis picorna-like virus (BdPLV), underscoring BdDomeless3’s crucial role in antiviral defense of B. dorsalis. Following the suppression of Domeless3 expression, six antimicrobial peptide genes displayed decreased expression, potentially correlating with the rise in viral infectivity. To our knowledge, this is the first study identifying cytokine receptors associated with the JAK/STAT pathway in tephritid flies, shedding light on the immune mechanisms of B. dorsalis.

Basigin binds bacteria and activates Dorsal signaling to promote antibacterial defense in Penaeus vannamei

The NF-κB pathway plays an important role in immune regulation. Basigin, an immunoglobulin superfamily membrane protein, is involved in the activation of NF-κB. However, its role in NF-κB signaling in response to pathogen infection remains unclear. In this study, we identified the Basigin gene from Pacific white shrimp, Penaeus vannamei, a representative species for studying the innate immune system of invertebrates. Basigin promoted the degradation of the IκB homolog Cactus, facilitated the nuclear translocation of the NF-κB family member Dorsal, and positively regulated the expression of Dorsal pathway downstream antimicrobial peptide genes. Interestingly, recombinant Basigin protein could bind a variety of Gram-positive and Gram-negative bacteria. Silencing of Basigin inhibited the Dorsal signaling activated by V. parahaemolyticus infection and significantly decreased the survival rate of V. parahaemolyticus-infected shrimp. The expression levels of the antimicrobial peptides ALF1 and ALF2 were downregulated, and the phagocytosis of hemocytes was attenuated in Basigin-silenced shrimp. Similar results were observed in shrimp treated with a recombinant extracellular region of the Basigin protein that was able to compete with endogenous Basigin. Therefore, to the best of our knowledge, this study is the first to demonstrate the function of Basigin as a pathogen recognition receptor that activates NF-κB signaling for antibacterial immunity in shrimp.

Differential responses of Ceratitis capitata to infection by the entomopathogenic fungus Purpureocillium lilacinum.

The medfly Ceratitis capitata is one of the most damaging fruit pests with quarantine significance due to its extremely wide host range. The use of entomopathogenic fungi constitutes a promising approach with potential applications in integrated pest management. Furthermore, developing insect control methods can involve the use of fungal machinery to cause metabolic disruption, which may increase its effectiveness by impairing insect development. Insect species, including C. capitata, relies on reproduction potential, nutrient reserves, metabolic activities, and immune response for survival. Accordingly, the purpose of this study was to investigate the impacts of the entomopathogenic fungus Purpureocillium lilacinum on C. capitata pre-mortality. The medfly V8 strain was subjected to laboratory bioassays, which consisted on determining the virulence of P. lilacinum on the medfly. Purpureocillium lilacinum was applied on abdominal topical of 5-day-old males and females. Following the fungal inoculation, we have confirmed (i) a significant increase in tissue sugar content, (ii) a significant decrease in carbohydrase activities, digestive glycosyl hydrolase, and proteinase activities in whole midguts of treated flies, (iii) the antimicrobial peptides (AMPs) genes expression profile was significantly influenced by fly gender, fly status (virgin, mature, and mated), and time after infection, but infection itself had no discernible impact on the AMPs for the genes that were examined. This study provides the first insight into how P. lilacinum could affect C. capitata physiological mechanisms and provides the foundation for considering P. lilacinum as a novel, promising biocontrol agent.

Iron metabolism in autism spectrum disorder; inference through single nucleotide polymorphisms in key iron metabolism genes

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental problems with various genetic and environmental components. The ASD diagnosis is based on symptom expression without reliance on any biomarkers. The genetic contributions in ASD remain elusive. Various studies have linked ASD with iron. Since iron plays a crucial role in brain development, neurotransmitter synthesis, neuronal myelination and mitochondrial function, we hypothesized that iron dysregulation in the brain could play a role and contribute to the pathogenesis of ASD. In this study, we investigated single nucleotide polymorphisms in ASD in various iron metabolism genes, including the Transferrin Receptor (TFRC) gene (rs11915082), the Solute Carrier Family 11 Member 2 (SLC11A2) gene (rs1048230 and rs224589), the Solute Carrier Family 40 Member 1 (SLC40A1) gene (rs1439816), and hepcidin antimicrobial peptide (HAMP) gene (rs10421768). We recruited 48 patients with ASD and 88 matched non-ASD controls. Our results revealed a significant difference between ASD and controls in the G allele of the TFRC gene rs11915082, and in the C allele of the SLC40A1 gene rs1439816. In silico analysis demonstrated potential positive role of the indicated genetic variations in ASD development and pathogenesis. These results suggest that specific genetic variations in iron metabolism genes may represent part of early genetic markers for early diagnosis of ASD. A significant effect of SNPs, groups (ASD/control) as well as interaction between SNPs and groups was revealed. Follow-up post hoc tests showed a significant difference between the ASD and control groups in rs11915082 (TFRC gene) and rs1439816 (SLC40A1 gene). Backward conditional logistic regression using both the genotype and allele data showed similar ability in detecting ASD using allel model (Nagelkerke R2 = 0.350 p = 0.967; Variables: rs1439816, rs11915082) compared to genotype model (Nagelkerke R2 = 0.347, p = 0.430; Variables: rs1439816 G, rs1439816 C, rs10421768 A). ROC curve showed 54% sensitivity in detecting ASD compared to 47% for the genotype model. Both models differentiated controls with high accuracy; the allele model had a specificity of 91% compared to 92% for the genotype model.In conclusion, our findings suggest that specific genetic variations in iron metabolism may represent early biomarkers for a diagnosis of ASD. Further research is needed to correlate these markers with specific blood iron indicators and their contribution to brain development and behavior.

Genome-Wide Identification of Trachinotus ovatus Antimicrobial Peptides and Their Immune Response against Two Pathogen Challenges.

Golden pompano, Trachinotus ovatus, as a highly nutritious commercially valuable marine fish, has become one of the preferred species for many fish farmers due to its rapid growth, wide adaptability, and ease of feeding and management. However, with the expansion of aquaculture scale, bacterial and parasitic diseases have also become major threats to the golden pompano industry. This study, based on comparative genomics, shows the possibility of preferential evolution of freshwater fish over marine fish by analyzing the phylogenetic relationships and divergence times of 14 marine fish and freshwater fish. Furthermore, we identified antimicrobial peptide genes from 14 species at the genomic level and found that the number of putative antimicrobial peptides may be related to species evolution. Subsequently, we classified the 341 identified AMPs from golden pompano into 38 categories based on the classification provided by the APD3. Among them, TCP represented the highest proportion, accounting for 23.2% of the total, followed by scolopendin, lectin, chemokine, BPTI, and histone-derived peptides. At the same time, the distribution of AMPs in chromosomes varied with type, and covariance analysis showed the frequency of its repeat events. Enrichment analysis and PPI indicated that AMP was mainly concentrated in pathways associated with disease immunity. In addition, our transcriptomic data measured the expression of putative AMPs of golden pompano in 12 normal tissues, as well as in the liver, spleen, and kidney infected with Streptococcus agalactiae and skin infected with Cryptocaryon irritans. As the infection with S. agalactiae and C. irritans progressed, we observed tissue specificity in the number and types of responsive AMPs. Positive selection of AMP genes may participate in the immune response through the MAPK signaling pathway. The genome-wide identification of antimicrobial peptides in the golden pompano provided a complete database of potential AMPs that can contribute to further understanding the immune mechanisms in pathogens. AMPs were expected to replace traditional antibiotics and be developed into targeted drugs against specific bacterial and parasitic pathogens for more precise and effective treatment to improve aquaculture production.

Tension as a key factor in skin responses to pollution

Being the more apparent organ exposed to the outdoor stressors, the effect of pollution on the skin has been widely studied in the last few decades. Although UV light is known as the most aggressive stressor to which our cutaneous tissue is daily exposed, other components of the tropospheric pollution have also shown to affect skin health and functionality. Among them, ozone has been proven to be one of the most toxic due to its high reactivity with the epidermal lipids. Studying the cutaneous effect of pollution in a laboratory setting presents challenges, therefore it becomes critical to employ appropriate and tailored models that aim to answer specific questions. Several skin models are available nowadays: in vitro models (2D cell lines and 3D cutaneous tissues), ex vivo skin explants and in vivo approaches (animals and humans). Although in the last 20 years researchers developed skin models that closely resemble human skin (3D cutaneous tissues), ex vivo skin explants still remain one of the best models to study cutaneous responses. Unfortunately, one important cutaneous property that is not present in the traditional ex vivo human skin explants is the physiological tension, which has been shown to be a cardinal player in skin structure, homeostasis, functional properties and responses to external stimuli. For this reason, in this study, to confirm and further comprehend the harmful mechanism of ozone exposure on the integumentary system, we have performed experiments using the state of art in cutaneous models: the innovative TenSkin™ model in which ex vivo human skin explants are cultured under physiologically relevant tension during the whole experimental procedure. Specifically, we were interested in corroborating previous findings showing that ozone exposure modulates the expression of cutaneous antimicrobial peptides (AMPs). The present work demonstrates that cutaneous exposure to ozone induces AMPs gene and protein levels (CAMP/LL-37, hBD2, hBD3) and that the presence of tension can further modulate their expression. In addition, different responses between tension and non-tension cultured skin were also observed during the evaluation of OxInflammatory markers [cyclooxygenase-2 (COX2), aryl hydrocarbon receptor (AhR), matrix-metallo-proteinase 9 (MMP9) and 4-hydroxy-nonenal (4HNE)]. This current study supports our previous findings confirming the ability of pollution to induce the cutaneous expression of AMPs via redox signaling and corroborates the principle that skin explants are a good and reliable model to study skin responses even though it underlines the need to holistically consider the role of skin tension before extrapolating the data to real life.

Transcriptome and Metabolome Analyses of Thitarodes xiaojinensis in Response to Ophiocordyceps sinensis Infection.

Ophiocordyceps sinensis exhibits more than 5 months of vegetative growth in Thitarodes xiaojinensis hemocoel. The peculiar development process of O. sinensis has been elucidated through morphological observation and omics technology; however, little information has been reported regarding the changes that occur in the host T. xiaojinensis. The RNA sequencing data showed that when O. sinensis blastospores were in the proliferative stage, the greatest change in the infected larval fat body was the selectively upregulated immune recognition and antimicrobial peptide genes. When O. sinensis blastospores were in the stationary stage, the immune pathways of T. xiaojinensis reverted to normal levels, which coincides with the successful settlement of O. sinensis. Pathway enrichment analysis showed a higher expression of genes involved in energy metabolism pathway in this stage. Metabolomic analyses revealed a reduction of amino acids and lipids in hemolymph, but an upregulation of lipids in the fat body of the host larvae after O. sinensis infection. We present the first transcriptome integrated with the metabolome study of T. xiaojinensis infected by O. sinensis. It will improve our understanding of the interaction mechanisms between the host and entomopathogenic fungi, and facilitate future functional studies of genes and pathways involved in these interactions.

Maturation of the medaka immune system depends on reciprocal interactions between the microbiota and the intestinal tract.

The interactions between the host immune system and intestinal microorganisms have been studied in many animals, including fish. However, a detailed analysis has not been performed in medaka, an established fish model for biological studies. Here, we investigated the effect of immunodeficiency on the microbiota composition and the effect of gut bacteria on intestinal epithelial development and immune responses in medaka. Chronological analysis of the intestinal microbiota of interleukin 2 receptor subunit gamma (il2rg) mutant medaka showed a gradual decrease in the evenness of operational taxonomic units, mainly caused by the increased abundance of the Aeromonadaceae family. Exposure of wild-type medaka to high doses of an intestine-derived opportunistic bacterium of the Aeromonadaceae family induced an inflammatory response, suggesting a harmful effect on adult il2rg mutants. In addition, we established germ-free conditions in larval medaka and observed large absorptive vacuoles in intestinal epithelial cells, indicating a block in epithelial maturation. Transcriptome analysis revealed a decrease in the expression of genes involved in the defense response, including the antimicrobial peptide gene hepcidin, whose expression is induced by lipopolysaccharide stimulation in normal larvae. These results show that reciprocal interactions between the microbiome and the intestinal tract are required for the maturation of the medaka immune system.

Mannose-binding C-type lectin from Procambarus clarkii exhibited antimicrobial activity to mediate crayfish innate immunity

Mannose-binding lectin (MBL), which is a member of the C-type lectin (CTL) family, plays crucial roles in non-self recognition and the clearance of invading microorganisms. In this study, an MBL (designated as PcMBL) was cloned and characterized from the crayfish Procambarus clarkii. PcMBL cDNA consisted of 816 bp encoding a protein with 271 amino acid residues. The deduced PcMBL contained a signal peptide, three low-complexity regions, and a carbohydrate-recognition domain, as found in most CTLs. The mRNA encoding PcMBL was detected in all the examined tissues. PcMBL expression in hemocytes and intestines was significantly upregulated after stimulation with Vibrio parahaemolyticus and Staphylococcus aureus. RNA interference studies revealed that PcMBL silencing remarkably downregulated the transcription of five antimicrobial peptide genes, namely, Anti-lipopolysaccharide factor 1 (ALF-1), ALF-4, ALF-10, Crustin 1 (Crus-1), and Crus-4. Recombinant PcMBL protein was used for the functional analysis. The results showed that rPcMBL had the capacity to bind to all seven tested microorganisms and three carbohydrates. rPcMBL facilitated the clearance of V. parahaemolyticus and S. aureus and inhibited the formation of biofilms and growth of the two bacteria. In addition, rPcMBL could reduce the mortality of crayfish infected with V. parahaemolyticus or S. aureus. All these results suggest that PcMBL may be involved in immune recognition and pattern elimination in the innate immunity of P. clarkii.

Kruppel homolog 1 modulates ROS production and antimicrobial peptides expression in shrimp hemocytes during infection by the Vibrio parahaemolyticus strain that causes AHPND.

Shrimp aquaculture has been seriously affected by acute hepatopancreatic necrosis disease (AHPND), caused by a strain of Vibrio parahaemolyticus that carries the Pir toxin plasmids (V. parahaemolyticus (AHPND)). In this study, the transcription factor, Kruppel homolog 1-like of Peneaus vannamei (PvKr-h1), was significantly induced in shrimp hemocytes after V. parahaemolyticus (AHPND) challenge, suggesting that PvKr-h1 is involved in shrimp immune response. Knockdown of PvKr-h1 followed by V. parahaemolyticus (AHPND) challenge increased bacterial abundance in shrimp hemolymph coupled with high shrimp mortality. Moreover, transcriptome and immunofluorescence analyses revealed that PvKr-h1 silencing followed by V. parahaemolyticus (AHPND) challenge dysregulated the expression of several antioxidant-related enzyme genes, such as Cu-Zu SOD, GPX, and GST, and antimicrobial peptide genes, i.e., CRUs and PENs, and reduced ROS activity and nuclear translocation of Relish. These data reveal that PvKr-h1 regulates shrimps' immune response to V. parahaemolyticus (AHPND) infection by suppressing antioxidant-related enzymes, enhancing ROS production and promoting nuclei import of PvRelish to stimulate antimicrobial peptide genes expression.

Identification of two galectin-4 proteins (PcGal4-L and PcGal4-L-CRD) and their function in AMP expression in Procambarus clarkii

Galectins, a family of lectins that bind to β-galactoside, possess conserved carbohydrate recognition domains (CRDs) and play a crucial role in recognizing and eliminating pathogens in invertebrates. Two galectin-4 genes (PcGal4) isoforms, named PcGal4-L and PcGal4-L-CRD, were cloned from the cDNA library of Procambarus clarkia in our study. PcGal4-L contains an open reading frame (ORF, 1089 bp), which encodes a protein consisting of 362 amino acids including a single CRD and six low complexity regions. The full-length cDNA of PcGal4-L-CRD contains a 483 bp ORF that encodes a protein of 160 amino acids, with a single CRD and a low-complexity region. The difference between the two PcGal4 isoforms is that PcGal4-L has 202 additional amino acids after the CRD compared to the PcGal4-L-CRD. These two isoforms are grouped together with other galectins from crustaceans through phylogenetic analysis. Further study revealed that total PcGal4 (including PcGal4-L and PcGal4-L-CRD) was primarily expressed in the muscle, gills and intestine. The mRNA levels of total PcGal4 in gills and hemocytes were significantly induced after challenge with Aeromonas hydrophila. Both recombinant PcGal4-L and its spliced isoform, PcGal4-L-CRD, could directly bind to lipopolysaccharides, peptidoglycan and five tested microorganisms, inducing a wide spectrum of microbial agglutination. The spliced isoform PcGal4-L-CRD showed a stronger binding ability than PcGal4-L. In addition, when the PcGal4 was knockdown, transcriptions of seven antimicrobial peptides (AMPs) genes (ALF5, ALF6, ALF8, CRU1, CRU2, CRU3 and CRU4) in gills and seven AMPs genes (ALF5, ALF6, ALF8, ALF9, CRU1, CRU3 and CRU4) in hemocytes were significantly decreased. Meanwhile, the survival rate of P. clarkii decreased in the PcGal4-dsRNA group. In summary, these results indicate that PcGal4 can mediate the innate immunity in P. clarkii by bacterial recognition and agglutination, as well as regulating AMP expression, thus recognition and understanding of the functions of galectin in crustaceans in immune resistance.

Effects of different diets on the growth and development of young silkworms

To investigate the impact of different diets on the growth and development of the silkworm Bombyx mori (L.; Lepidoptera: Bombycidae), we conducted an analysis of differentially expressed genes (DEGs) between larvae fed an artificial diet (referred to as the AD group) and those fed mulberry leaves (referred to as the ML group) during the first, second, and third instars using RNA sequencing (RNA-Seq) technology. Our results revealed that the DEGs primarily belonged to pathways associated with lipid metabolism, carbohydrate metabolism, amino acid metabolism, the endocrine system, the nervous system, the digestive system, the immune system, and oxidation–reduction processes. Notably, in the AD group, there was a decrease in transcript levels of genes related to amino acid metabolism synthases, while the transcript levels of antimicrobial peptide genes were up-regulated. Furthermore, genes associated with cytochrome P450 (CYP) enzymes exhibited down-regulation in the AD group. These observed changes likely contributed to the delayed growth and compromised robustness observed in the AD group. Overall, these findings provided valuable insights into the mechanisms underlying the differences in growth, development, disease resistance, and adversity resistance between silkworms fed an artificial diet and those fed mulberry leaves.

Effect of aminergic signaling on the humoral innate immunity response of Drosophila.

Biogenic amines are crucial signaling molecules that modulate various physiological life functions both in vertebrates and invertebrates. In humans, these neurotransmitters influence the innate and adaptive immunity systems. In this work, we analyzed whether the aminergic neurotransmission of dopamine, serotonin, and octopamine could have an impact on the humoral innate immune response of Drosophila melanogaster. This is a powerful model system widely used to uncover the insect innate immunity mechanisms which are also conserved in mammals. We found that the neurotransmission of all these amines positively modulates the Toll-responsive antimicrobial peptide (AMP) drosomycin (drs) gene in adult flies infected with the Micrococcus luteus bacterium. Indeed, we showed that either blocking the neurotransmission in their specific aminergic neurons by expressing shibirets (Shits) or silencing the vesicular monoamine transporter gene (dVMAT) by RNAi caused a significantly reduced expression of the Toll-responsive drs gene. However, upon M. luteus infection, the block of aminergic transmission did not alter the expression of AMP attacin genes responding to the immune deficiency (Imd) and Toll pathways. Overall, our results not only reveal a neuroimmune function for biogenic amines in humoral immunity but also further highlight the complexity of the network controlling AMP gene regulation.

The effect of alginate oligosaccharides on intestine barrier function and Vibrio parahaemolyticus infections in the white shrimp Litopenaeus vannamei

The intestine is a host-pathogen interaction site and improved intestinal barrier function help to prevent disease in shrimp. Alginate oligosaccharides (AOS) are derived from resourceful brown algae. The intestine protection properties of AOS were widely recognized, and their benefits in fish have been reported. Nevertheless, there are no reports on AOS in shrimp and other crustaceans. In the present work, we measured the effects of AOS on growth performance and disease resistance in the white shrimp Litopenaeus vannamei and investigated their effects on intestinal health. Shrimps with an initial weight of about 2 g were fed with diets supplemented with 0 (control), 0.07%, 0.2%, 0.6%, or 1.2% of AOS for 56 days and were sampled and challenged with Vibrio parahaemolyticus. Dietary AOS did not significantly influence weight gain or feed utilization (P > 0.05). However, AOS considerably decreased the seven-day cumulative mortality after the challenge at any dose (P < 0.05). Dietary AOS improved the intestinal structure, significantly boosted the intestinal villus height at 0.6% and 1.2% levels, and increased intestinal wall thickness by 0.2%, 0.6%, and 1.2%. The alkaline phosphatase and maltase activities were also increased, suggesting that AOS improved the intestinal condition. Redox homeostasis in intestinal was improved by AOS, as expressed by the enhanced total antioxidant capacity and decreased malonaldehyde content, partly due to the increased superoxide dismutase and catalase activities. Compared with the antioxidant system, AOS's stimulating effects on immunity were more significant. At any level, AOS significantly activated lysozyme activity, the expression of propo and two antimicrobial peptide genes (pen-3 and crusin). However, the lowest concentration of AOS did not stimulate the gene expression of all three assayed pattern recognition receptors (LGBP, Toll, and IMD), and only the highest concentration of AOS increased the expression of imd. These findings suggest that AOS are highly efficient immunostimulants, and various immune pathways in shrimp are differentially sensitive to AOS. Finally, our findings suggest that AOS significantly alter the gut microbiota and their relative abundance at the phylum, family, and genus levels. In conclusion, AOS significantly enhances disease resistance in L. vannamei, possibly attributed to improved intestinal development, increased intestinal immunity and altered microbiota. These findings could provide a basis for future studies on the practical use of AOS and its mechanisms of action.

Protective Effects of Bacteriocin-Producing Lactiplantibacillus plantarum on Intestinal Barrier of Mice.

Bacteriocins are crucial metabolites of probiotics that display beneficial functions. The intestinal barrier is an important target on which probiotics exert their intestinal health activity. However, the impacts of bacteriocin-producing probiotics on the intestinal barrier are unclear. In this study, the effects of bacteriocin-producing Lactiplantibacillus plantarum Q7 and L. plantarum F3-2 on the intestinal barrier of mice were explored. It was shown that L. plantarum Q7 promoted the expression of mucin MUC2 to enhance the protection provided by the intestinal mucus layer. L. plantarum Q7 up-regulated the gene expression of intestinal tight junction proteins ZO-1 and JAM-1 significantly, and L. plantarum F3-2 up-regulated ZO-1 and Claudin-1 markedly, which exhibited tight junction intestinal barrier function. The two strains promoted the release of IgA and IgG at varying degrees. The antimicrobial peptide gene RegIIIγ was up-regulated markedly, and the gene expression of inflammatory cytokines appeared to exhibit an upward trend with L. plantarum Q7 treatment, so as to enhance intestinal immune regulation function. Furthermore, L. plantarum Q7 and L. plantarum F3-2 increased the abundance of the beneficial bacteria Muribaculaceae, inhibited the growth of the harmful bacteria Parabacteroides, and facilitated the synthesis of total short-chain fatty acids (SCFAs), which seemed to favor the prevention of metabolic diseases. Our results suggested that L. plantarum Q7 and L. plantarum F3-2 showed strain specificity in their protective effects on the intestinal chemical, physical, immunological and biological barriers of mice, which provided theoretical support for the selective utilization of bacteriocin-producing strains to regulate host health.

The plant toxin 4-methylsulfinylbutyl isothiocyanate decreases herbivore performance and modulates cellular and humoral immunity.

Insect herbivores frequently encounter plant defense molecules, but the physiological and ecological consequences for their immune systems are not fully understood. The majority of studies attempting to relate levels of plant defensive chemistry to herbivore immune responses have used natural population or species-level variation in plant defensive chemistry. Yet, this potentially confounds the effects of plant defense chemistry with other potential plant trait differences that may affect the expression of herbivore immunity. We used an artificial diet containing known quantities of a plant toxin (4-methylsulfinylbutyl isothiocyanate; 4MSOB-ITC or ITC, a breakdown product of the glucosinolate glucoraphanin upon herbivory) to explicitly explore the effects of a plant toxin on the cellular and humoral immune responses of the generalist herbivore Trichoplusia ni (Lepidoptera: Noctuidae) that frequently feeds on glucosinolate-containing plants. Caterpillars feeding on diets with high concentrations of ITC experienced reduced survivorship and growth rates. High concentrations of ITC suppressed the appearance of several types of hemocytes and melanization activity, which are critical defenses against parasitic Hymenoptera and microbial pathogens. In terms of T. ni humoral immunity, only the antimicrobial peptide (AMP) genes lebocin and gallerimycin were significantly upregulated in caterpillars fed on diets containing high levels of ITC relative to caterpillars that were provided with ITC-free diet. Surprisingly, challenging caterpillars with a non-pathogenic strain of Escherichia coli resulted in the upregulation of the AMP gene cecropin. Feeding on high concentrations of plant toxins hindered caterpillar development, decreased cellular immunity, but conferred mixed effects on humoral immunity. Our findings provide novel insights into the effects of herbivore diet composition on insect performance demonstrating the role of specific plant defense toxins that shape herbivore immunity and trophic interactions.

A novel perlucin-like protein (PLP) protects Litopenaeus vannamei against Vibrio harveyi infection.

C-type lectins (CTLs), as pattern recognition receptors (PRRs), play an important role in the innate immunity of Litopenaeus vannamei. In this study, a novel CTL, named perlucin-like protein (PLP), was identified from L. vannamei, which shared homology sequences of PLP from Penaeus monodon. PLP from L. vannamei was expressed in the hepatopancreas, eyestalk, muscle and brain and could be activated in the tissues (hepatopancreas, muscle, gill and intestine) after infection with the pathogen Vibrio harveyi. Bacteria (Vibrio alginolyticus, V. parahaemolyticus, V. harveyi, Streptococcus agalactiae and Bacillus subtilis) could be bound and agglutinated by the PLP recombinant protein in a Ca2+-dependent manner. Moreover, PLP could stabilise the expression of the immune-related genes (ALF, SOD, HSP70, Toll4 and IMD) and apoptosis gene (Caspase2). The RNAi of PLP could remarkably affect the expression of antioxidant gene, antimicrobial peptide genes, other CTLs, apoptosis genes, Toll signaling pathways, and IMD signaling pathways. Moreover, PLP reduced the bacterial load in the hepatopancreas. These results suggested that PLP was involved in the innate immune response against V. harveyi infection by recognising bacterial pathogens and activating the expression of immune-related and apoptosis genes.