Impact of captivity and natural habitats on gut microbiome in Epinephelus akaara across seasons.

BACKGROUND: The gut microbiota significantly influences the health and growth of red-spotted grouper (Epinephelus akaara), a well-known commercial marine fish from Fujian Province in southern China. However, variations in survival strategies and seasons can impact the stability of gut microbiota data, rendering it inaccurate in reflecting the state of gut microbiota. Which impedes the effective enhancement of aquaculture health through a nuanced understanding of gut microbiota. Inspired by this, we conducted a comprehensive analysis of the gut microbiota of wild and captive E. akaara in four seasons. RESULTS: Seventy-two E. akaara samples were collected from wild and captive populations in Dongshan city, during four different seasons. Four sections of the gut were collected to obtain comprehensive information on the gut microbial composition and sequenced using 16S rRNA next-generation Illumina MiSeq. We observed the highest gut microbial diversity in both captive and wild E. akaara during the winter season, and identified strong correlations with water temperature using Mantel analysis. Compared to wild E. akaara, we found a more complex microbial network in captive E. akaara, as evidenced by increased abundance of Bacillaceae, Moraxellaceae and Enterobacteriaceae. In contrast, Vibrionaceae, Clostridiaceae, Flavobacteriaceae and Rhodobacteraceae were found to be more active in wild E. akaara. However, some core microorganisms, such as Firmicutes and Photobacterium, showed similar distribution patterns in both wild and captive groups. Moreover, we found the common community composition and distribution characteristics of top 10 core microbes from foregut to hindgut in E. akaara. CONCLUSIONS: Collectively, the study provides relatively more comprehensive description of the gut microbiota in E. akaara, taking into account survival strategies and temporal dimensions, which yields valuable insights into the gut microbiota of E. akaara and provides a valuable reference to its aquaculture.

Chronic exposure to environmental concentrations of benzo[a]pyrene causes multifaceted toxic effects of developmental compromise, redox imbalance, and modulated transcriptional profiles in the early life stages of marine medaka (Oryzias melastigma).

Polycyclic aromatic hydrocarbons (PAHs) accumulate and integrate into aquatic environments, raising concerns about the well-being and safety of aquatic ecosystems. Benzo[a]pyrene (BaP), a persistent PAH commonly detected in the environment, has been extensively studied. However, the broader multifaceted toxicity potential of BaP on the early life stages of marine fish during chronic exposure to environmentally relevant concentrations needs further exploration. To fill these knowledge gaps, this study assessed the in vivo biotoxicity of BaP (1, 4, and 8 µg/L) in marine medaka (Oryzias melastigma) during early development over a 30-day exposure period. The investigation included morphological, biochemical, and molecular-level analyses to capture the broader potential of BaP toxicity. Morphological analyses showed that exposure to BaP resulted in skeletal curvatures, heart anomalies, growth retardation, elevated mortality, delayed and reduced hatching rates. Biochemical analyses revealed that BaP exposure not only created oxidative stress but also disrupted the activities of antioxidant enzymes. This disturbance in redox balance was further explored by molecular level investigation. The transcriptional profiles revealed impaired oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle pathways, which potentially inhibited the oxidative respiratory chain in fish following exposure to BaP, and reduced the production of adenosine triphosphate (ATP) and succinate dehydrogenase (SDH). Furthermore, this investigation indicated a potential connection to apoptosis, as demonstrated by fluorescence microscopy and histological analyses, and supported by an increase in the expression levels of related genes via real-time quantitative PCR. This study enhances our understanding of the molecular-level impacts of BaP's multifaceted toxicity in the early life stages of marine medaka, and the associated risks.

A 14-amino acid cationic peptide Bolespleenin334-347 from the marine fish mudskipper Boleophthalmus pectinirostris exhibiting potent antimicrobial activity and therapeutic potential.

Antimicrobial peptides (AMPs) are an important component of innate immunity in both vertebrates and invertebrates, and some of the unique characteristics of AMPs are usually associated with their living environment. The marine fish, mudskipper Boleophthalmus pectinirostris, usually live amphibiously in intertidal environments that are quite different from other fish species, which would be an exceptional source of new AMPs. In the study, an AMP named Bolespleenin334-347 was identified, which was a truncated peptide derived from a new functional gene found in B. pectinirostris, that was up-regulated in response to bacterial challenge. Bolespleenin334-347 had only 14 amino acid residues, including five consecutive arginine residues. It was found that the peptide had broad-spectrum antibacterial activity, good thermal stability and sodium ion tolerance. Bolespleenin334-347 killed Acinetobacter baumannii and Staphylococcus aureus by disrupting the structural integrity of the bacterial membrane, leading to leakage of the cellular contents, and inducing accumulation of bacterial endogenous reactive oxygen species (ROS). In addition, Bolespleenin334-347 effectively inhibited biofilm formation of A. baumannii and S. aureus and long-term treatment did not lead to the development of resistance. Importantly, Bolespleenin334-347 maintained stable activity against clinically multi-drug resistant bacterial strains. In addition, it was noteworthy that Bolespleenin334-347 showed superior efficacy to LL-37 and vancomycin in a constructed mouse model of MRSA-induced superficial skin infections, as evidenced by a significant reduction in bacterial load and more favorable wound healing. This study provides an effective antimicrobial agent for topical skin infections with potential therapeutic efficacy for infections with drug-resistant bacteria, including MRSA.

A novel antimicrobial peptide Scyreptin1-30 from Scylla paramamosain exhibiting potential therapy of Pseudomonas aeruginosa early infection in a mouse burn wound model.

Antimicrobial resistance (AMR) constitutes a significant global threat to human health. In recent years, there has been a concerning surge in infections caused by multidrug-resistant bacteria, highlighting the pressing need to urgently explore novel and effective alternatives to conventional antibiotics. Antimicrobial peptides (AMPs) have emerged as a focal point of research, capturing significant attention as promising antimicrobial agents. In this study, we have identified a novel cationic antimicrobial peptide (AMP) named Scyreptin1-30, derived from the marine invertebrate Scylla paramamosain. The results showed that Scyreptin1-30 exhibits a broad-spectrum antimicrobial activity, demonstrating significant potency against both bacteria and fungi, and even against the clinically isolated multidrug-resistant bacteria Pseudomonas aeruginosa. Moreover, Scyreptin1-30 exhibited rapid bactericidal kinetic. The results of antibacterial mechanism showed that Scyreptin1-30 destroyed the integrity of bacterial membranes, leading to bacterial death and exhibited potent anti-biofilm activity against P. aeruginosa. The activity of Scyreptin1-30 against bacteria had a favorable thermal stability, displayed a certain ion tolerance, and showed no discernible cytotoxicity when assessed against both the mammalian cell line HEK293T and the fish cell lines ZF4. In an In vivo study, Scyreptin1-30 exhibited a remarkably reduction in the bacterial load caused by multidrug-resistant P. aeruginosa at the site of infection, and promoted wound healing in a mouse model of burn infection. This study indicated that Scyreptin1-30 holds promise as an effective antibacterial agent, potentially serving as a topical skin treatment against multidrug-resistant bacterial infections, including those caused by P. aeruginosa.

Upregulation of FAM50A promotes cancer development.

FAM50A encodes a nuclear protein involved in mRNA processing; however, its role in cancer development remains unclear. Herein, we conducted an integrative pan-cancer analysis using The Cancer Genome Atlas, Genotype-Tissue Expression, and the Clinical Proteomic Tumor Analysis Consortium databases. Based on the gene expression data from TCGA and GTEx databases, we compared FAM50A mRNA levels in 33 types of human cancer tissues to those in corresponding normal tissues and found that FAM50A mRNA level was upregulated in 20 of the 33 types of common cancer tissues. Then, we compared the DNA methylation status of the FAM50A promoter in tumor tissues to that in corresponding normal tissues. FAM50A upregulation was accompanied by promoter hypomethylation in 8 of the 20 types of tumor tissues, suggesting that promoter hypomethylation contributes to the upregulation of FAM50A in these cancer tissues. Elevated FAM50A expression in 10 types of cancer tissues was associated with poor prognosis in patients with cancer. FAM50A expression was positively correlated with CD4+ T-lymphocyte and dendritic cell infiltration in cancer tissues but was negatively correlated with CD8+ T-cell infiltration in cancer tissues. FAM50A knockdown caused DNA damage, induced interferon beta and interleukin-6 expression, and repressed the proliferation, invasion, and migration of cancer cells. Our findings indicate that FAM50A might be useful in cancer detection, reveal insights into its role in cancer development, and may contribute to the development of cancer diagnostics and treatments.

Effect of Cross-Linking Density on Non-Linear Viscoelasticity of Vulcanized SBR: A MD Simulation and Experimental Study.

In recent years, there has been a growing interest in changes in dynamic mechanical properties of mixed rubber during dynamic shear, yet the influence of vulcanized characteristics on the dynamic shear behavior of vulcanized rubber, particularly the effect of cross-linking density, has received little attention. This study focuses on styrene-butadiene rubber (SBR) and aims to investigate the impact of different cross-linking densities (Dc) on dynamic shear behavior using molecular dynamics (MD) simulations. The results reveal a remarkable Payne effect, where the storage modulus experiences a significant drop when the strain amplitude (γ0) exceeds 0.1, which can be attributed to the fracture of the polymer bond and the decrease in the molecular chain's flexibility. The influence of various Dc values mainly resides at the level of molecular aggregation in the system, where higher Dc values impede molecular chain motion and lead to an increase in the storage modulus of SBR. The MD simulation results are verified through comparisons with existing literature.

A new pathogenic isolate of Kocuria kristinae identified for the first time in the marine fish Larimichthys crocea.

In recent years, new emerging pathogenic microorganisms have frequently appeared in animals, including marine fish, possibly due to climate change, anthropogenic activities, and even cross-species transmission of pathogenic microorganisms among animals or between animals and humans, which poses a serious issue for preventive medicine. In this study, a bacterium was clearly characterized among 64 isolates from the gills of diseased large yellow croaker Larimichthys crocea that were raised in marine aquaculture. This strain was identified as K. kristinae by biochemical tests with a VITEK 2.0 analysis system and 16S rRNA sequencing and named K. kristinae_LC. The potential genes that might encode virulence-factors were widely screened through sequence analysis of the whole genome of K. kristinae_LC. Many genes involved in the two-component system and drug-resistance were also annotated. In addition, 104 unique genes in K. kristinae_LC were identified by pan genome analysis with the genomes of this strain from five different origins (woodpecker, medical resource, environment, and marine sponge reef) and the analysis results demonstrated that their predicted functions might be associated with adaptation to living conditions such as higher salinity, complex marine biomes, and low temperature. A significant difference in genomic organization was found among the K. kristinae strains that might be related to their hosts living in different environments. The animal regression test for this new bacterial isolate was carried out using L. crocea, and the results showed that this bacterium could cause the death of L. crocea and that the fish mortality was dose-dependent within 5 days post infection, indicating the pathogenicity of K. kristinae_LC to marine fish. Since K. kristinae has been reported as a pathogen for humans and bovines, in our study, we revealed a new isolate of K. kristinae_LC from marine fish for the first time, suggesting the potentiality of cross-species transmission among animals or from marine animals to humans, from which we would gain insight to help in future public prevention strategies for new emerging pathogens.

Newly Discovered Antimicrobial Peptide Scyampcin44-63 from Scylla paramamosain Exhibits a Multitargeted Candidacidal Mechanism In Vitro and Is Effective in a Murine Model of Vaginal Candidiasis.

The emergence of azole-resistant and biofilm-forming Candida spp. contributes to the constantly increasing incidence of vulvovaginal candidiasis. It is imperative to explore new antifungal drugs or potential substituents, such as antimicrobial peptides, to alleviate the serious crisis caused by resistant fungi. In this study, a novel antimicrobial peptide named Scyampcin44-63 was identified in the mud crab Scylla paramamosain. Scyampcin44-63 exhibited broad-spectrum antimicrobial activity against bacteria and fungi, was particularly effective against planktonic and biofilm cells of Candida albicans, and exhibited no cytotoxicity to mammalian cells (HaCaT and RAW264.7) or mouse erythrocytes. Transcriptomic analysis revealed four potential candidacidal modes of Scyampcin44-63, including promotion of apoptosis and autophagy and inhibition of ergosterol biosynthesis and the cell cycle. Further study showed that Scyampcin44-63 caused damage to the plasma membrane and induced apoptosis and cell cycle arrest at G2/M in C. albicans. Scanning and transmission electron microscopy demonstrated that Scyampcin44-63-treated C. albicans cells were deformed with vacuolar expansion and destruction of organelles. In addition, C. albicans cells pretreated with the autophagy inhibitor 3-methyladenine significantly delayed the candidacidal effect of Scyampcin44-63, suggesting that Scyampcin44-63 might contribute to autophagic cell death. In a murine model of vulvovaginal candidiasis, the fungal burden of vaginal lavage was significantly decreased after treatment with Scyampcin44-63.

A New Gene SCY3 Homologous to Scygonadin Showing Antibacterial Activity and a Potential Role in the Sperm Acrosome Reaction of Scylla paramamosain.

In the study, a new gene homologous to the known antimicrobial peptide Scygonadin was identified in mud crab Scylla paramamosain and named SCY3. The full-length sequences of cDNA and genomic DNA were determined. Similar to Scygonadin, SCY3 was dominantly expressed in the ejaculatory ducts of male crab and the spermatheca of post-mating females at mating. The mRNA expression was significantly up-regulated after stimulation by Vibrio alginolyticus, but not by Staphylococcus aureus. The recombinant protein rSCY3 had a killing effect on Micrococcus luteus and could improve the survival rate of mud crabs infected with V. alginolyticus. Further analysis showed that rSCY3 interacted with rSCY1 or rSCY2 using Surface Plasmon Resonance (SPR, a technology for detecting interactions between biomolecules using biosensor chips) and Mammalian Two-Hybrid (M2H, a way of detecting interactions between proteins in vivo). Moreover, the rSCY3 could significantly improve the sperm acrosome reaction (AR) of S. paramamosain and the results demonstrated that the binding of rSCY3, rSCY4, and rSCY5 to progesterone was a potential factor affecting the sperm AR by SCYs on. This study lays the foundation for further investigation on the molecular mechanism of SCYs involved in both immunity and physiological effects of S. paramamosain.

Growth-promoting effect of antimicrobial peptide Scy-hepc on mariculture large yellow croaker Larimichthys crocea and the underlying mechanism.

With the antibiotics prohibition in feedstuffs worldwide, antimicrobial peptides (AMPs) are considered a more promising substitute for antibiotics to be used as feed additives, and positive results have been reported in livestock feeding studies. However, whether dietary supplementation of AMPs could promote the growth of mariculture animals such as fish and the underlying mechanism has not been elucidated yet. In the study, a recombinant AMP product of Scy-hepc was used as a dietary supplement (10 mg/kg) to feed mariculture juvenile large yellow croaker (Larimichthys crocea) with an average initial body weight (BW) of 52.9 g for 150 days. During the feeding trial, the fish fed with Scy-hepc showed a significant growth-promoting performance. Especially at 60 days after feeding, fish fed with Scy-hepc weighed approximately 23% more than the control group. It was further confirmed that the growth-related signaling pathways such as the GH-Jak2-STAT5-IGF1 growth axis, the PI3K-Akt and Erk/MAPK pathways were all activated in the liver after Scy-hepc feeding. Furthermore, a second repeated feeding trial was scheduled for 30 days using much smaller juvenile L. crocea with an average initial BW of 6.3 g, and similar positive results were observed. Further investigation revealed that the downstream effectors of the PI3K-Akt pathway, such as p70S6K and 4EBP1, were significantly phosphorylated, suggesting that Scy-hepc feeding might promote translation initiation and protein synthesis processes in the liver. Taken together, as an effector of innate immunity, AMP Scy-hepc played a role in promoting the growth of L. crocea and the underlying mechanism was associated with the activation of the GH-Jak2-STAT5-IGF1 axis, as well as the PI3K-Akt and Erk/MAPK signaling pathways.

A Novel Antimicrobial Peptide Spampcin56-86 from Scylla paramamosain Exerting Rapid Bactericidal and Anti-Biofilm Activity In Vitro and Anti-Infection In Vivo.

The abuse of antibiotics leads to the increase of bacterial resistance, which seriously threatens human health. Therefore, there is an urgent need to find effective alternatives to antibiotics, and antimicrobial peptides (AMPs) are the most promising antibacterial agents and have received extensive attention. In this study, a novel potential AMP was identified from the marine invertebrate Scylla paramamosain and named Spampcin. After bioinformatics analysis and AMP database prediction, four truncated peptides (Spa31, Spa22, Spa20 and Spa14) derived from Spampcin were screened, all of which showed potent antimicrobial activity with different antibacterial spectrum. Among them, Spampcin56-86 (Spa31 for short) exhibited strong bactericidal activity against a variety of clinical pathogens and could rapidly kill the tested bacteria within minutes. Further analysis of the antibacterial mechanism revealed that Spa31 disrupted the integrity of the bacterial membrane (as confirmed by scanning electron microscopy observation, NPN, and PI staining assays), leading to bacterial rupture, leakage of cellular contents (such as elevated extracellular ATP), increased ROS production, and ultimately cell death. Furthermore, Spa31 was found to interact with LPS and effectively inhibit bacterial biofilms. The antibacterial activity of Spa31 had good thermal stability, certain ion tolerance, and no obvious cytotoxicity. It is worth noting that Spa31 could significantly improve the survival rate of zebrafish Danio rerio infected with Pseudomonas aeruginosa, indicating that Spa31 played an important role in anti-infection in vivo. This study will enrich the database of marine animal AMPs and provide theoretical reference and scientific basis for the application of marine AMPs in medical fields.

Multi-dimensional investigation and distribution characteristics analysis of gut microbiota of different marine fish in Fujian Province of China.

The gut microbiota plays an important role in animal health and behavior. In marine fish, the composition of the gut microbiota is affected by many complex factors, such as diet, species, and regional factors. Since more than one hundred fish species have been cultured in fish farms along with the 3,324 km coastline of Fujian Province in South China, we chose this region to study the gut microbiota composition of marine commercial fishes because sufficient different species, diets, and regional factors were observed. We investigated the distribution characteristics of the gut microbiota of seven cultured species (Epinephelus akaara, Epinephelus coioides, Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀, Siganus fuscescens, Pagrus major, Lateolabrax japonicus, and Acanthopagrus schlegelii) living in the same aquatic region and one species (E. akaara) living separately in five regions separated by latitude. The impacts of diet, region, and species factors on fish gut microbiota were also evaluated. Diversity and multivariate analyses showed that the patterns of the microbiota were significantly different in different fish species within the same habitat and E. akaara with five latitude regions. Mantel analysis showed that AN, SiO3 2-, DO, and NO2 - were the principal factors affecting the microbial community of E. akaara in the five habitats. Additionally, similar distribution characteristics occurred in different gut parts of different fishes, with an increasing trend of Proteobacteria and Vibrionaceae abundance and a decreasing trend of Firmicutes and Bacillaceae abundance from the foregut to the hindgut. Vibrionaceae was the most abundant family in the content. This study highlights that a persistent core microbiota was established in marine commercial fishes spanning multiple scales. The factors with the greatest effect on fish gut microbiota may be (i) host genetics and (ii) geographic factors rather than the microbiota in the diet and water environment. These core microbes regularly colonized from the foregut to the hindgut, which was driven by their underlying functions, and they were well adapted to the gut environment. Moreover, the microbiota in the content may have contributed more to the gut microbial communities than previously reported. This study could complement basic data on the composition of marine commercial fishes and facilitate relatively complete investigations, which would be beneficial for the healthy and sustainable development of aquaculture.

The Antimicrobial Peptide LJ-hep2 from Lateolabrax japonicus Exerting Activities against Multiple Pathogenic Bacteria and Immune Protection In Vivo.

Hepcidin is widely present in many kinds of fish and is an important innate immune factor. A variety of HAMP2-type hepcidins have strong antimicrobial activity and immunomodulatory functions and are expected to be developed as substitutes for antibiotics. In this study, the antimicrobial activity of Hepc2 from Japanese seabass (Lateolabrax japonicus) (designated as LJ-hep2) was investigated using its recombinant precursor protein (rLJ-hep2) expressed in Pichia pastoris and a chemically synthesized mature peptide (LJ-hep2(66-86)). The results showed that both rLJ-hep2 and synthetic LJ-hep2(66-86) displayed broad antimicrobial spectrum with potent activity against gram-negative and gram-positive bacteria, and fungi. Especially, LJ-hep2(66-86) had stronger antimicrobial activity and exhibited potent activity against several clinically isolated multidrug-resistant bacteria, including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterococcus faecium. Moreover, LJ-hep2(66-86) exerted rapid bactericidal kinetic (killed tested bacteria within 2 h), induced significant morphological changes and promoted agglutination of E. coli, P. aeruginosa and Aeromonas hydrophila. The activity of LJ-hep2(66-86) against E. coli, P. aeruginosa and A. hydrophila was stable and remained active when heated for 30 min. In addition, LJ-hep2(66-86) exhibited no cytotoxicity to the mammalian cell line HEK293T and fish cell lines (EPC and ZF4). In vivo study showed that LJ-hep2(66-86) could improve the survival rate of marine medaka (Oryzias melastigma) by about 40% under the challenge of A. hydrophila, indicating its immunoprotective function. Taken together, both rLJ-hep2 and LJ-hep2(66-86) have good prospects to be used as potential antimicrobial agents in aquaculture and medicine in the future.

A truncated peptide Spgillcin177-189 derived from mud crab Scylla paramamosain exerting multiple antibacterial activities.

Antimicrobial peptides (AMPs) may be the most promising substitute for antibiotics due to their effective bactericidal activity and multiple antimicrobial modes against pathogenic bacteria. In this study, a new functional gene named Spgillcin was identified in Scylla paramamosain, which encoded 216 amino acids of mature peptide. In vivo, Spgillcin was dominantly expressed in the gills of male and female crabs, offering the highest expression level among all tested organs or tissues. The expression pattern of Spgillcin was significantly altered when challenged by Staphylococcus aureus, indicating a positive immune response. In vitro, a functional truncated peptide Spgillcin177-189 derived from the amino acid sequence of Spgillcin was synthesized and showed a broad-spectrum and potent antibacterial activity against several bacterial strains, including the clinical isolates of multidrug-resistant (MDR) strains, with a range of minimum inhibitory concentrations from 1.5 to 48 µM. Spgillcin177-189 also showed rapid bactericidal kinetics for S. aureus and Pseudomonas aeruginosa but did not display any cytotoxicity to mammalian cells and maintained its antimicrobial activity in different conditions. Mechanistic studies indicated that Spgillcin177-189 was mainly involved in the disruption of cell membrane integrity where the membrane components lipoteichoic acid and lipopolysaccharide could significantly inhibit the antimicrobial activity in a dose-dependent manner. In addition, Spgillcin177-189 could change the membrane permeability and cause the accumulation of intracellular reactive oxygen species. No resistance was generated to Spgillcin177-189 when the clinical isolates of methicillin-resistant S. aureus and MDR P. aeruginosa were treated with Spgillcin177-189 and then subjected to a long term of continuous culturing for 50 days. In addition, Spgillcin177-189 exerted a strong anti-biofilm activity by inhibiting biofilm formation and was also effective at killing extracellular S. aureus in the cultural supernatant of RAW 264.7 cells. Taken together, Spgillcin177-189 has strong potential as a substitute for antibiotics in future aquaculture and medical applications.

Environmentally relevant concentrations of microplastics modulated the immune response and swimming activity, and impaired the development of marine medaka Oryzias melastigma larvae.

Microplastics (MPs), due to their impacts on the ecosystem and their integration into the food web either through trophic transfer or ingestion directly from the ambient environment, are an emerging class of environmental contaminants posing a great threat to marine organisms. Most reports on the toxic effects of MPs exclusively focus on bioaccumulation, oxidative stress, pathological damage, and metabolic disturbance in fish. However, the collected information on fish immunity in response to MPs is poorly defined. In particular, little is known regarding mucosal immunity and the role of mucins. In this study, marine medaka (Oryzias melastigma) larvae were exposed to 6.0 µm beads of polystyrene microplastics (PS-MPs) at three environmentally relevant concentrations (102 particles/L, 104 particles/L, and 106 particles/L) for 14 days. The experiment was carried out to explore the developmental and behavioural indices, the transcriptional profiles of mucins, pro-inflammatory, immune, metabolism and antioxidant responses related genes, as well as the accumulation of PS-MPs in larvae. The results revealed that PS-MPs were observed in the gastrointestinal tract, with a concentration- and exposure time-dependent manner. No significant difference in the larval mortality was found between the treatment groups and the control, whereas the body length of larvae demonstrated a significant reduction at 106 particles/L on 14 days post-hatching. The swimming behaviour of the larvae became hyperactive under exposure to 104 and 106 particles/L PS-MPs. In addition, PS-MP exposure significantly up-regulated the mucin gene transcriptional levels of muc7-like and muc13-like, however down-regulated the mucin gene expression levels of heg1, muc2, muc5AC-like and muc13. The immune- and inflammation and metabolism-relevant genes (jak, stat-3, il-6, il-1ß, tnf-а, ccl-11, nf-κb, and sod) were significantly induced by PS-MPs at 104 and 106 particles/L compared to the control. Taken together, this study suggests that PS-MPs induced inflammation response and might obstruct the immune functions and retarded the growth of the marine medaka larvae even at environmentally relevant concentrations.

A New Crustin Gene Homolog SpCrus8 Identified in Scylla paramamosain Exerting In Vivo Protection Through Opsonization and Immunomodulation.

Crustins are the most abundant class of antimicrobial peptides in crustaceans and are essential for protecting animals from infection. Among them, type II crustins usually exhibit potent antimicrobial activity. Interestingly, in this study, a newly identified type II crustin gene homolog (named SpCrus8) from mud crab Scylla paramamosain, the recombinant proteins of which (rSpCrus8 and rTrx-SpCrus8) showed no obvious antibacterial effects, but could significantly reduce the bacterial load in crab hemolymph and improve the survival rate of crabs infected with Vibrio alginolyticus. The immune-related function of SpCrus8 and the underlying mechanism deserve further investigation. It was found that the SpCrus8 gene was widely distributed in various tissues of adult crabs. In the hepatopancreas of crabs infected with V. alginolyticus or Staphylococcus aureus, transcripts of the SpCrus8 gene were remarkably induced, indicating that the SpCrus8 gene was involved in the immune response to bacterial infection in vivo. In addition, rSpCrus8 and rTrx-SpCrus8 had strong binding activity not only to microbial surface components (lipopolysaccharide, lipoteichoic acid, peptidoglycan, and glucan), but also to the tested bacteria (S. aureus, Pseudomonas aeruginosa and V. alginolyticus). Notably, rSpCrus8 and rTrx-SpCrus8 could significantly promote hemocyte phagocytosis. After rSpCrus8 and rTrx-SpCrus8 treatment, a large number of fluorescent microspheres were observed to aggregate into clusters and be phagocytosed by multiple hemocytes, while hemocytes in the control group phagocytosed only individual microspheres, indicating that SpCrus8 played an important role in opsonization. When the SpCrus8 gene was knocked down, the expression levels of the key phagocytosis-related genes SpRab5 and SpRab7 were significantly downregulated, as well as the IMD signaling pathway genes SpIKKß and SpRelish, and another crustin gene SpCrus5. Correspondingly, all the SpIKKß, SpRelish and SpCrus5 genes were significantly upregulated after rSpCrus8 treatment, suggesting that SpCrus8 might be involved in the immunomodulation of S. paramamosain. Taken together, this study revealed the immune-related functions of the SpCrus8 gene in opsonization and regulation, which will help us further understand the role of the crustin gene family in the immune system of mud crabs and provide new insights into the function of type II crutins.

The Anticancer Activity Conferred by the Mud Crab Antimicrobial Peptide Scyreprocin through Apoptosis and Membrane Disruption.

Scyreprocin is an antimicrobial peptide first identified in the mud crab Scylla paramamosain. Herein, we showed that its recombinant product (rScyreprocin) could significantly inhibit the growth of human lung cancer NCI-H460 cells (H460), but showed no cytotoxicity to human lung fibroblasts (HFL1). rScyreprocin was a membrane-active peptide that firstly induced the generation of reactive oxygen species (ROS) in H460, and led to endoplasmic reticulum stress and Ca2+ release, which resulted in mitochondrial dysfunction and subsequently activation of caspase-3 cascades, and ultimately led to apoptosis. The comprehensive results indicated that rScyreprocin exerted anticancer activity by disrupting cell membrane and inducing apoptosis. The in vivo efficacy test demonstrated that intratumoral injection of rScyreprocin significantly inhibited the growth of H460 xenografts, which was close to that of the cisplatin (inhibition rate: 69.94% vs. 80.76%). Therefore, rScyreprocin is expected to become a promising candidate for the treatment of lung cancer.

Two Male-Specific Antimicrobial Peptides SCY2 and Scyreprocin as Crucial Molecules Participated in the Sperm Acrosome Reaction of Mud Crab Scylla paramamosain.

Antimicrobial peptides (AMPs) identified in the reproductive system of animals have been widely studied for their antimicrobial activity, but only a few studies have focused on their physiological roles. Our previous studies have revealed the in vitro antimicrobial activity of two male gonadal AMPs, SCY2 and scyreprocin, from mud crab Scylla paramamosain. Their physiological functions, however, remain a mystery. In this study, the two AMPs were found co-localized on the sperm apical cap. Meanwhile, progesterone was confirmed to induce acrosome reaction (AR) of mud crab sperm in vitro, which intrigued us to explore the roles of the AMPs and progesterone in AR. Results showed that the specific antibody blockade of scyreprocin inhibited the progesterone-induced AR without affecting intracellular Ca2+ homeostasis, while the blockade of SCY2 hindered the influx of Ca2+. We further showed that SCY2 could directly bind to Ca2+. Moreover, progesterone failed to induce AR when either scyreprocin or SCY2 function was deprived. Taken together, scyreprocin and SCY2 played a dual role in reproductive immunity and sperm AR. To our knowledge, this is the first report on the direct involvement of AMPs in sperm AR, which would expand the current understanding of the roles of AMPs in reproduction.

A Novel Antimicrobial Peptide Sp-LECin with Broad-Spectrum Antimicrobial Activity and Anti-Pseudomonas aeruginosa Infection in Zebrafish.

New antimicrobial agents are urgently needed to address the increasing emergence and dissemination of multidrug-resistant bacteria. In the study, a chemically synthesized truncated peptide containing 22-amino acids derived from a C-type lectin homolog SpCTL6 of Scylla paramamosain was screened and found to exhibit broad-spectrum antimicrobial activity, indicating that it is an antimicrobial peptide (AMP), named Sp-LECin. Sp-LECin possessed the basic characteristics of most cationic AMPs, such as positive charge (+4) and a relatively high hydrophobicity (45%). After treatment with Sp-LECin, the disruption of microbial membrane integrity and even leakage of cellular contents was observed by scanning electron microscopy (SEM). In addition, Sp-LECin could bind lipopolysaccharide (LPS), increase the outer and inner membrane permeability and induce reactive oxygen species (ROS) production, ultimately leading to the death of Pseudomonas aeruginosa. Furthermore, Sp-LECin exhibited potent anti-biofilm activity against P. aeruginosa during both biofilm formation and maturation. Notably, Sp-LECin had no obvious cytotoxicity and could greatly improve the survival of P. aeruginosa-infected zebrafish, by approximately 40% over the control group after 72 h of treatment. This study indicated that Sp-LECin is a promising antibacterial agent with the potential to be used against devastating global pathogen infections such as P. aeruginosa.

A Novel Antimicrobial Peptide Sparamosin26-54 From the Mud Crab Scylla paramamosain Showing Potent Antifungal Activity Against Cryptococcus neoformans.

Due to the increasing prevalence of drug-resistant fungi and the limitations of current treatment strategies to fungal infections, exploration and development of new antifungal drugs or substituents are necessary. In the study, a novel antimicrobial peptide, named Sparamosin, was identified in the mud crab Scylla paramamosain, which contains a signal peptide of 22 amino acids and a mature peptide of 54 amino acids. The antimicrobial activity of its synthetic mature peptide and two truncated peptides (Sparamosin1-25 and Sparamosin26-54) were determined. The results showed that Sparamosin26-54 had the strongest activity against a variety of Gram-negative bacteria, Gram-positive bacteria and fungi, in particular had rapid fungicidal kinetics (killed 99% Cryptococcus neoformans within 10 min) and had potent anti-biofilm activity against C. neoformans, but had no cytotoxic effect on mammalian cells. The RNA-seq results showed that after Sparamosin26-54 treatment, the expression of genes involved in cell wall component biosynthesis, cell wall integrity signaling pathway, anti-oxidative stress, apoptosis and DNA repair were significantly up-regulated, indicating that Sparamosin26-54 might disrupt the cell wall of C. neoformans, causing oxidative stress, DNA damage and cell apoptosis. The underlying mechanism was further confirmed. Sparamosin26-54 could bind to several phospholipids in the cell membrane and effectively killed C. neoformans through disrupting the integrity of the cell wall and cell membrane observed by electron microscope and staining assay. In addition, it was found that the accumulation of reactive oxygen species (ROS) increased, the mitochondrial membrane potential (MMP) was disrupted, and DNA fragmentation was induced after Sparamosin26-54 treatment, which are all hallmarks of apoptosis. Taken together, Sparamosin26-54 has a good application prospect as an effective antimicrobial agent, especially for C. neoformans infections.