Anti-lipopolysaccharide Factor Research Articles

HSP40 mediated TLR-Dorsal-AMPs pathway in Portunus trituberculatus

Heat shock protein 40 (HSP40) is a kind of molecular chaperone involved in various immune responses. However, the exact roles of HSP40 in immune defense against bacteria remain largely unclear. In this study, the activation function of a type Ⅰ HSP40 from Portunus trituberculatus (PtHSP40-Ⅰ) in the TLR pathway was investigated. The results showed that PtHSP40-Ⅰ can bind to lipopolysaccharide (LPS) and peptidoglycan (PGN). The PtHSP40-Ⅰ also exhibited binding activity toward the extracellular leucine-rich repeat (LRR) domain of Toll-like receptor (TLR). Moreover, the PtHSP40-Ⅰ could promote the transcription factor Dorsal translocated from cytoplasm into the nucleus in hemocytes and participated in regulating the expression of anti-lipopolysaccharide factor (ALF) and crustin. These findings provided insights into the activation mechanisms of TLR pathway mediated by HSP40 and offered basic theory of disease control in P. trituberculatus aquaculture.

Effects of Dietary Eucommia ulmoides Leaf Extract on Growth, Muscle Composition, Hepatopancreas Histology, Immune Responses and Microcystin-LR Resistance of Juvenile Red Claw Crayfish (Cherax quadricarinatus)

The objective of the present study was to investigate the effects of different doses of dietary Eucommia ulmoides leaf extract (ELE) on juvenile red claw crayfish (Cherax quadricarinatus). A total number of 720 red claw crayfish (initial body weight of 0.24 ± 0.01 g) were randomly assigned to six groups and fed diets containing 0 (Diet 1), 0.5 (Diet 2), 1 (Diet 3), 2 (Diet 4), 4 (Diet 5) and 10 (Diet 6) g dry weight (dw) ELE kg (dw)−1 diets for eight weeks and challenged with microcystin-LR stress. The results indicated that dietary supplementation with 1–2 g dw ELE kg (dw)−1 diet could significantly improve the weight gain rate (WGR) and specific growth rate (SGR) of crayfish. Muscle crude protein contents of crayfish fed Diet 2, Diet 3, and Diet 4 were significantly higher than those of the control group. Compared with the control group, dietary ELE could increase total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GPx), acid phosphatase (ACP), alkaline phosphatase (AKP), and phenoloxidase (PO) activities and decrease malondialdehyde (MDA) level of crayfish. Dietary ELE significantly increased the relative expression levels of SOD, thioredoxin 1 (TRX1), GPx, selenium-dependent glutathione peroxidase (Se-GPx), cytochrome P450 (CYP450), anti-lipopolysaccharide factor (ALF) and C-type lysozyme (C-LZM) mRNA of crayfish compared with the control group during the feeding experiment. When subjected to MC-LR stress for 48 h, the mRNA expression levels of SOD, GPx, Se-GPx, glutathione-s-transferase 1 (GST1), ALF, hemocyanin (HEM), and C-LZM in the hepatopancreas could be improved to varying degrees compared with the Diet 1. Supplementation of 1–2 g dw ELE kg (dw)−1 diet could improve the survival rate (SR) of crayfish under MC-LR stress. These results indicated that dietary ELE (1–2 g dw ELE kg (dw)−1 diet) could improve the growth performance, muscle protein, and non-specific immune response and increase the SR of crayfish under MC-LR stress by regulating the mRNA expression levels of the immune- and antioxidant-related genes.

Stress and Disease Resistance in Crayfish (Procambarus clarkii) Breed “Huachizhen-1”

Stress and disease are critical factors hindering the industrial development of red swamp crayfish (Procambarus clarkii). Breeding crayfish with stress- and disease-resistant characteristics can overcome these limitations and promote their industrial development. In this study, the crayfish breed F3, which exhibits rapid growth and a favoured haplotype combination of the immune genes R, ALF, and crustin2, encoding the Toll-like receptor, anti-lipopolysaccharide factor, and antimicrobial peptide, respectively, were selected as parents to breed offspring (the selected group (SG)). The genotype, stress resistance, and disease resistance of crayfish in the SG and unselected group (USG) were compared. The results showed that the ratio of the favoured haplotype was higher in the SG crayfish than in the USG crayfish, leading to stronger stress and disease resistance. Compared to that of the USG crayfish, the mortality of the SG crayfish subjected to stress during eight days of transport and challenged with bacteria (Aeromonas hydrophila or Vibrio parahaemolyticus) or white spot syndrome virus were significantly reduced by 60% and 20%, respectively (p < 0.05). Based on these results, the stress- and disease-resistant SG crayfish were named the “Huachizhen-1” breed. Additionally, the ratio of the unfavoured homozygous genotypes of R, ALF, and crustin2 sharply decreased, whereas those of the heterozygous genotypes increased together with stress and disease resistance during crayfish maturation under natural conditions, indicating that the heterozygotes of these genes also exhibit strong stress and disease resistance. All of this taken together, the crayfish breed “Huachizhen-1” may be applicable for improving stress and disease resistance and the production of crayfish.

Effect of Rice Protein Meal Replacement of Fish Meal on Growth, Anti-Oxidation Capacity, and Non-Specific Immunity for Juvenile Shrimp Litopenaeus vannamei.

This study assessed the effect of rice protein meal replacement for fish meal on the growth, nonspecific immunity, and disease resistance on juvenile shrimp Litopenaeus vannamei. Six groups of iso-nitrogenous and iso-lipid feeds named FM, R10, R20, R40, R60, and R80 were prepared by replacing 0%, 10%, 20%, 40%, 60%, and 80% in FM protein with RPM, respectively, and then fed to the shrimps (0.54 ± 0.01 g). An amount of 720 healthy and evenly sized shrimps were allocated to six groups (three replicates per group) and fed four times a day (7:00, 11:00, 17:00 and 21:00) for eight weeks. Results revealed no significant differences in WG, FCR, and SGR of shrimps after replacing FM with 10% RPM (p > 0.05). In the R10 and R20 groups, SOD and T-AOC activities were significantly higher than those in the FM group, whereas the opposite was observed for MDA content (p < 0.05). CAT, ACP, and LZM were all significantly higher in the R10, R20, and R40 groups than in the FM group (p < 0.05). GSH-Px activity in the R10 group was significantly higher than the activity in the FM group (p < 0.05). AKP, PO, TYS, GPT, and GOT activities were significantly higher in the R10 group than in the FM group (p < 0.05). Compared to the FM group, the eukaryotic translation initiation factor 3K (eif3k) gene was significantly up-regulated in the R10 group, whereas the penaiedin 3a (pen 3a) and anti-lipopolysaccharide factor (alf) genes were significantly up-regulated in the R10 and R20 groups (p < 0.05). The crustin a (cru a), immune deficiency (imd), and lysozyme (lzm) mRNA levels were significantly higher in the R10, R20, and R40 groups than in the other groups (p < 0.05). The prophenoloxidase (PO) mRNA levels in the R20 group were significantly higher than those in the FM group (p < 0.05). The replacement of 10-40% of FM with RPM improved the gut flora composition of shrimps, increasing beneficial bacteria (Bacteroidetes) abundance and reducing harmful bacteria (Aspergillus and Vibrio) abundance. After the challenge test of Vibrio parahaemolyticus (7 days), the cumulative mortality in the R10 group significantly decreased (p < 0.05). In conclusion, replacement of 10% FM by RPM significantly improved digestibility, protein synthesis, antioxidant capacity, and disease resistance in L. vannamei.

Fucosyltransferase 2 is involved in immune-related functions in Penaeus vannamei by modulating antimicrobial peptides’ expression

In mammals fucosyltransferase 2 (FUT2) plays an important regulatory role in inflammation, bacterial or viral infection, and tumor metastasis. However, the specific role of FUT2 in invertebrate immunity has not been reported. Here, the FUT2 homolog of Penaeus vannamei (designated as PvFUT2) was cloned and found to have a full-length cDNA of 1104 bp with an open reading frame (ORF) encoding 316 amino acids. PvFUT2 is constitutively expressed in all shrimp tissues tested with the highest found in intestines. Moreover, PvFUT2 was induced in the main immune organs (hemocytes and hepatopancreas) of shrimp by Gram-positive (Vibrio parahaemolyticus), Gram-negative (Streptococcus iniae) bacteria and virus (White Spot Syndrome Virus), indicating the involvement of PvFUT2 in shrimp antimicrobial response. Intriguingly, PvFUT2 knockdown with or without pathogen challenge reduced the expression of Pvβ-catenin and antimicrobial peptides genes, particularly anti lipopolysaccharide factor and lysozyme. Further analysis revealed that the knockdown of PvFUT2 increased Vibrio abundance in hemolymph and resulted in an increase in shrimp cumulative mortality rate. Thus, during pathogen challenge, the expression of PvFUT2 is induced to regulate β-catenin and subsequently antimicrobial peptides expression to augment shrimp antimicrobial immune response.

Cloning and expression of two anti-lipopolysaccharide factors in Eriocheir hepuensis under Vibrio alginolyticus-induced stress.

Anti-lipopolysaccharide factors (ALFs) are small basic proteins that exhibit broad-spectrum antiviral properties and antibacterial activity. In this research, we cloned and studied two Eriocheir hepuensis ALFs, EhALF2 and EhALF3. The results showed that the open reading frame lengths of EhALF2 and EhALF3 were 363 and 372 bp, encoding 120 and 123 amino acids, respectively. Their sequences both contained an Lipopolysaccharide-binding (LPS) domain and were highly similarity to other crab ALFs. qRT-PCR showed that EhALF2 and EhALF3 were detected in nine examined tissues and were expressed the highest in the haemocytes. After challenge with Vibrio alginolyticus, in the hepatopancreas, the expression levels of EhALF2 and EhALF3 reached the highest levels at 48 and 3h, respectively. In the heart, the expression levels of the two genes were highest at 12 h. These results indicate that EhALF2 and EhALF3 could participate in the resistance of E. hepuensis to V. alginolyticus stress within a short time. They have potential applications in the study of environmental stress markers and disease-resistance factors in E. hepuensis.

Identification and functional analysis of two drosophila mothers against decapentaplegic protein(Smad)genes and their involvement in immune responses in the red swamp crayfish Procambarus clarkii

Drosophila mothers against decapentaplegic proteins (Smads), the crucial signal transducers in activating downstream gene transcription through transforming growth factor beta (TGF-β) receptors, are the pleiotropic factors with important role in mediating cell proliferation, homeostasis, differentiation, apoptosis and immune response. However, whether Smads are involved in immune response in crustaceans remains unexplored. In the present study, the Smad3 and Smad4 were firstly identified and functionally characterized from the Red Swamp Crayfish Procambarus clarkii. The full-length cDNAs of pcSmad3 and pcSmad4 were 1, 670 bp and 3, 060 bp with 1, 326 bp and 1, 875 bp open reading frame (ORF), respectively. Real-time PCR analysis of the expression profiles demonstrated that pcSmad3 and pcSmad4 were predominantly expressed at in stomach, heart, and hemocytes. Notably, the expression levels of pcSmad3 and pcSmad4 both Aeromonas hydrophila and WSSV challenges were significantly altered, suggesting the involvement of pcSmad3 and pcSmad4 in innate immune responses. Knockdown of pcSmad3 and pcSmad4 in vivo dramatically activated the transcriptions of NF-κB signaling genes and anti-lipopolysaccharide factor genes. The overexpression of pcSmad3 and pcSmad4 could significantly activate NF-κB signaling in HEK293T cells. Meanwhile, the clearance of bacteria was significantly reduced with knockdown of pcSmad3 and pcSmad4 in vivo. Results indicated that pcSmad3 and pcSmad4 played an immune-regulatory role in crayfish's innate immunity, which might pave the for a better understanding of the TGF-β superfamily members in crustacean.

ERK is involved in the regulation of CpG ODN 2395 on the expression levels of anti-lipopolysaccharide factors in Chinese mitten crab, Eriocheir sinensis

CpG oligodeoxynucleotides (ODN), as an effective adjuvant or immunopotentiator, activate the immune system and induce various immune responses. Recently, it has also been reported that high dose of CpG ODN can lead to immunosuppression. However, the underlying mechanism of CpG ODN-mediated immune response remains largely unknown in invertebrates. In the present study, the role of ERK in regulating expression levels of anti-lipopolysaccharide factors (ALFs) induced by different doses of CpG ODN 2395 was analyzed in Chinese mitten crab, Eriocheir sinensis. The mRNA expression levels of EsALFs (EsALF1, EsALF2 and EsALF3) and EsERK in haemocytes were observed to increase from 6 h to 48 h post low doses of CpG ODN 2395 (0.5 μg and 2.5 μg) stimulation, while they were suppressed after high dose of CpG ODN 2395 (12.5 μg) injection. Meanwhile, the phosphorylation levels of ERK in haemocytes were significantly promoted after low doses of CpG ODN 2395 injection, and a reduce level of ERK phosphorylation was observed after high dose of CpG ODN 2395 injection. Further investigation showed that the expression levels of EsALFs induced by CpG ODN 2395 were markedly down-regulated after knocking down the expression of EsERK. Similarly, the EsALFs mRNA expression were also inhibited post different doses of CpG ODN 2395 stimulation in PD98059 (ERK inhibitor) injection crabs. These results collectively suggest that ERK is involved in regulating the expression level of EsALFs induced by different dose of CpG ODN 2395 in Chinese mitten crab, which contribute to the understanding of the regulation of CpG ODN involving in immune response in crustaceans.

Garlic Powder Supplementation Improves Growth, Nonspecific Immunity, Antioxidant Capacity, and Intestinal Flora of Chinese Mitten Crabs (Eriocheir sinensis).

This study was conducted to survey the effects of garlic powder on growth performance, nonspecific immunity, antioxidant capacity, and intestinal flora structure of Chinese mitten crabs. Altogether, 216 crabs which originally weigh 20.71 ± 0.13 g were randomly allocated into three treatment groups with 6 replicates of 12 crabs per replicate. The control group (CN) was fed a basal diet, while the other two groups were fed the basal diet supplemented with 1000 mg/kg (GP1000) and 2000 mg/kg (GP2000) garlic powder, respectively. This trial lasted 8 weeks. The results showed that the supplementation of garlic powder improved the final body weight, weight gain rate, and specific growth rate of the crabs (P < 0.05). Meanwhile, in serum, better nonspecific immune was confirmed by the enhancement of phenoloxidase and lysozyme levels, with the improvement of phosphatase activities in GP1000 and GP2000 (P < 0.05). On the other hand, the levels of total antioxidant capacity, glutathione peroxidases, and total superoxide dismutase in serum and hepatopancreas were increased (P < 0.05) while malondialdehyde content declined (P < 0.05) as the garlic powder was added to the basal diet. And, catalase in serum also shows an increase (P < 0.05). In both GP1000 and GP2000, genes related to antioxidant and immunity, for instance, Toll-like receptor 1, glutathione peroxidase, catalase, myeloid differentiation factor 88, TuBe, Dif, relish, crustins, antilipopolysaccharide factor, lysozyme, and prophenoloxidase mRNA expression levels, were increased (P < 0.05). The abundance of Rhizobium and Rhodobacter was reduced by adding garlic powder (P < 0.05). This study indicated that dietary addition of garlic powder promoted growth, enhanced nonspecific immunity and antioxidant capacity, activated Toll pathway, IMD pathway, and proPO system, increased antimicrobial peptide expression, while simultaneously improving the intestinal flora of Chinese mitten crabs.

Biomolecules of the Horseshoe Crab’s Hemolymph: Components of an Ancient Defensive Mechanism and Its Impact on the Pharmaceutical and Biomedical Industry

Without adaptive immunity, invertebrates have evolved innate immune systems that react to antigens on the surfaces of pathogens. These defense mechanisms are included in horseshoe crab hemocytes’ cellular responses to pathogens. Secretory granules, large (L) and small (S), are found on hemocytes. Once the invasion of pathogens is present, these granules release their contents through exocytosis. Recent data in biochemistry and immunology on the granular constituents of granule-specific proteins are stored in large and small granules which are involved in the cell-mediated immune response. L-granules contain most clotting proteins, which are necessary for hemolymph coagulation. They also include tachylectins; protease inhibitors, such as cystatin and serpins; and anti-lipopolysaccharide (LPS) factors, which bind to LPS and agglutinate bacteria. Big defensin, tachycitin, tachystatin, and tachyplesins are some of the essential cysteine-rich proteins in S-granules. These granules also contain tachycitin and tachystatins, which can agglutinate bacteria. These proteins in granules and hemolymph act synergistically to fight infections. These biomolecules are antimicrobial and antibacterial, enabling them to be drug resistant. This review is aimed at explaining the biomolecules identified in the horseshoe crab’s hemolymph and their application scopes in the pharmaceutical and biotechnology sectors.

Effect of dietary replacement of fish meal by poultry by-product meal on the growth performance, immunity, and intestinal health of juvenile red swamp crayfish, procambarus clarkia

The present study was conducted to investigate the dietary replacement of fish meal with poultry by-product meal (PBM) on the growth performance, immunity, antioxidant properties, and intestinal health of red swamp crayfish (Procambarus clarkia). A diet containing 20% fish meal (FM) and complex plant ingredients as the main protein resources was set as the FM group (crude protein 32%, crude lipid 6%). Four diets replacing 25%, 50%, 75%, and 100% fish meal of the FM diet with PBM were set as the PBM25, PBM50, PBM75, and PBM100 groups, respectively. Compared to the FM group, the PBM100 diet significantly decreased growth performance and feed utilization of crayfish, while markedly increasing the activity of serum aspartate aminotransferase. The immune response was depressed in crayfish fed the PBM100 diet as the activities of serum lysozyme and phenoloxidase, gene expression of anti-lipopolysaccharide factors (alf), cyclophilin A (cypa), crustin, and hemocyanin-1 (hep-1) in hepatopancreas were remarkably decreased. The activities of antioxidases and expression of antioxidant-relevant genes in the hepatopancreas were not influenced by PBM inclusion. Crayfish fed different diets exhibited no obvious symptoms of enteritis, but the PBM100 diet destructed intestinal morphology by significantly decreasing the average length of longitudinal ridges. The α-diversity and overall community structure were not significantly influenced but variations were found in the relative abundance of some genera by PBM inclusion. In summary, CAP could successfully replace 75% dietary FM in a basal diet containing 20% fish meal, while higher CAP level compromised growth performance, immunity, and intestinal histology of crayfish.

Comparative transcriptome analysis of Portunus trituberculatus in response to Vibrio parahaemolyticus and low salinity

Vibrio parahaemolyticus is a common pathogen of marine crustaceans and is responsible for large losses in aquaculture. Salinity is an important environmental factor, and abrupt changes in salinity can affect the immunity of crustaceans. In this study, we carried out a transcriptomic analysis under pathogenic infection and low salt stress, and conducted a comparative analysis of the differentially expressed genes (DEGs) after stimulation by the two factors. Illumina sequencing technology was used for the transcriptome sequencing of 27 hepatopancreas samples, and 178.77 G sequencing data and an average of 44,142,119 clean reads per sample were obtained. A total of 3,047 and 3,710 DEGs were found after V. parahaemolyticus infection and low salt stress, which included a number of innate immunity genes, such as Toll-like receptor (TLR), anti-lipopolysaccharide factor (ALF), lectin, and hemocyanin. In addition, 2,016 common DEGs were found, accounting for 42.52% of the total DEGs. Among these, 169 DEGs were up-regulated after pathogenic infection and down-regulated after low salt stress, and were mainly enriched in starch and sucrose metabolism, nitrogen metabolism, amino sugar and nucleotide sugar processes, and other pathways. Collectively, these results provide data support for the analysis of the immune mechanism of crabs against V. parahaemolyticus and will also help to clarify the molecular mechanism by which salinity affects immunity.

Comparative study of five anti-lipopolysaccharide factor genes in Litopenaeus vannamei

Anti-lipopolysaccharide factors (ALFs) are a family of common innate immune effectors in crustaceans, and they exhibit broad spectrum antimicrobial activity. In this study, we identified and characterized five novel ALF genes (designated as LvALF1–5) from the Pacific white shrimp (Litopenaeus vannamei) to investigate their potential immune functions. The amino acid sequence alignments showed that LvALFs contained two conserved cysteine residues, a hydrophobic N-terminal region, and the conserved signature sequence W(T/K)CPG(S)WT(A). They all shared high similarity with previously reported ALFs and were clearly novel members of the ALF family. The mRNA transcripts of LvALFs were most highly expressed in hemocytes and the hepatopancreas. After shrimp were stimulated with Vibrio parahaemolyticus or white spot syndrome virus, expression of the LvALFs was significantly induced in hemocytes and the hepatopancreas with various expression profiles. Recombinant proteins of LvALFs exhibited potent bacteriostatic activity in vitro. Together, these results suggest that LvALF1–5 participate in the immune response of Pacific white shrimp against invading pathogens.

Effect of feeding different types of β-glucans derived from two marine diatoms (Chaetoceros muelleri and Thalassiosira weissflogii) on growth performance and immunity of banana shrimp (Penaeus merguiensis)

β-glucans are produced by many organisms and could be used as supplementary feed to enhance immunity and growth in some aquatic animals. This study aimed to compare the effectiveness of β-glucans derived from two marine diatoms (Chaetoceros muelleri and Thalassiosira weissflogii) as growth promoters and immunity enhancers in banana shrimp (Penaeus merguiensis). Shrimp were divided into 3 groups: the control group was fed without β-glucan; the second and the third group were fed with 2 g kg−1 of β-glucan derived from C. muelleri and T. weissflogii, respectively. Shrimp were fed over a 30-day period to determine growth performance (final weight, weight gain, average daily gain (ADG), and feed conversion ratio (FCR)) at day 15 and day 30, respectively. The immune parameters determined were total hemocyte count (THC), phenoloxidase activity (PO) and immune gene expression. Survival rates were measured after 14 days of the feeding trial and Vibrio parahaemolyticus infection (6, 24, 48 h post infection). There was no significant difference (P > 0.05) for growth stimulation of shrimps between the two types of β-glucans (C. muelleri or T. weissflogii). Notably, shrimps fed with β-glucans had a higher final weight, weight gain, and ADG (P < 0.05) than shrimps fed with the control diet, while FCR of shrimps fed with both β-glucans was lower when compared to the control diet. Immune parameters, THC, PO, and gene expression of anti-lipopolysaccharide factor (ALF) and crustin were significantly higher (P < 0.05) in shrimps fed with β-glucans, especially with β-glucans from C. muelleri than the control group both before and after V. parahaemolyticus infection. Expression of penaeidin 3 and peroxiredoxin genes was significantly higher in shrimps fed with β-glucans after bacterial infection. Histopathology of hepatopancreas revealed an increase in blasenzellen hepatopancreatic epithelial cells (B cells) after 14 days of feeding which remained higher following infection with V. parahaemolyticus. The survival rate of shrimps fed with the diet containing β-glucan derived from either C. muelleri (82.2%) or T. weissflogii (77.8%) after V. parahaemolyticus infection was significantly higher than for the control group (51.1%) (P < 0.05). In conclusion, we propose that feeding banana shrimps with β-glucans derived from marine diatoms either C. muelleri or T. weissflogii at a 2 g kg−1 diet can significantly improve their growth performance and immunity.

Effects of recombinant anti-lipopolysaccharide factor expressed by Pichia pastoris on the growth performance, immune response and disease resistance of Litopenaeusvannamei

The codon-optimized anti-lipopolysaccharide factor (ALF) sequence was introduced into pPICZαA vector and transformed into Pichia pastoris GS115. The recombinant ALF yeast supernatant (rALF-mix) was freeze-dried and evaluated as a feed additive for Litopenaeus vannamei. It was found by antibacterial activity test in vitro that the rALF-mix had antibacterial activity under different pH and temperature conditions. The 0, 0.00375%, 0.0075%, 0.015%, 0.03% and 0.06% of rALF-mix were added respectively to make the six experimental diets. After a 10-week feeding trial with shrimps (2.36 ± 0.02 g), it was found that the weight gain rate (WGR) and protein efficiency ratio (PER) of shrimp in the groups with 0.0075%, 0.015% and 0.03% of dietary rALF-mix supplementation were significantly higher than those in the control group (P < 0.05). Dietary rALF-mix supplementation significantly increased the total haemocyte count, respiratory burst, phagocytic activity, total anti-oxidative capacity (T-AOC), phenol oxidase activity, nitric oxide synthase activity, lysozyme (LYZ) activity, serum antibacterial capacity in the hemolymph and the T-AOC, LYZ in the hepatopancreas of shrimps (P < 0.05). The malondialdehyde contents in hemolymph and hepatopancreas were significantly decreased (P < 0.05). Meanwhile, the expression levels of toll, immune deficiency, heat shock protein 70, crustin and lipopolysaccharide-β-glucan binding protein in the gill of shrimps were significantly increased (P < 0.05). After the challenge test, it was showed that dietary rALF-mix supplementation significantly improved the resistance of L. vannamei to Vibrio parahaemolyticus (P < 0.05). In conclusion, the rALF-mix can be used as a functional feed additive to improve the growth, immunity and disease resistance of shrimp. Based on the quadratic regression analysis for WGR, the optimal supplemental level of rALF-mix in diet for shrimp was estimated to be 0.02813%.

SpRab11a-Regulated Exosomes Inhibit Bacterial Infection through the Activation of Antilipopolysaccharide Factors in Crustaceans.

Exosomes, secreted by most cells, are critical antimicrobial immune factors in animals. Recent studies of certain key regulators of vesicular transport, the Rab GTPases, have linked Rab dysfunction to regulation of innate immune signaling. However, the relationship between exosomes and Rab GTPases, resulting in antimicrobial activity in vertebrates and invertebrates during pathogenic infection, has not been addressed. In this study, SpRab11a was reported to have a protective effect on the survival rate of mud crabs Scylla paramamosain after Vibrio parahaemolyticus challenge through the stimulation of exosome secretion and modulation of anti-LPS factor (ALF) expression. Furthermore, Sp14-3-3 was confirmed to be densely packaged in exosomes after V. parahaemolyticus infection, which could recruit the MyD88 and TLR by binding the Toll/IL-1R domain to the plasma membrane, promoting the translocation of Dorsal from the cytoplasm into the nucleus, and thereby regulating ALFs expression in the hemocytes of mud crab in response to the bacterial infection. The findings therefore provide, to our knowledge, a novel mechanism that underlies the cross-talk between SpRab11a-regulated exosome formation and ALFs expression in innate immune response in invertebrates, with a crustacean species, mud crab S. paramamosain, as a model study.

Effects of dietary yeast culture on health status in digestive tract of juvenile Pacific white shrimp Litopenaeus Vannamei

A feeding trial was conducted to investigate the effects of dietary yeast culture (YC) on health status in digestive tract of juvenile Pacific white shrimp Litopenaeus Vannamei. Shrimps (initial weight: 3.33 ± 0.06 g) were fed with graded levels of dietary YC (control, 0.3%, 0.5% and 1.0%). Results of the present study showed that villus height and the ratio between villus height and crypt depth in the digestive tract of juvenile shrimp was significantly increased by dietary 0.5% and 1.0%YC (P < 0.05). Besides, dietary 0.5% and 1.0%YC significantly activities of phenoloxidase (PO), lysozyme (LZ), acid phosphatase (ACP) and alkaline phosphatase (AKP) (P < 0.05), significantly up-regulated mRNA levels of prophenoloxidase (propo), lysozyme (lz), anti-lipopolysaccharide factor (alf), crustin and penaienadin (P < 0.05) and down-regulated mRNA levels of caspase-1, nuclear factor κB p65 (nf-κbp65) myeloid differentiation primary response protein (myd88) and toll like receptor (tlr) in the digestive tract of juvenile shrimp (P < 0.05). Compared with the control, dietary 0.5%YC increased Chao1 index in the digestive tract of juvenile shrimp. In addition, compared with the control, dietary 0.5% and 1.0%YC significantly increased relative abundance of Lactobacillus (P < 0.05). It can be concluded that dietary YC made positive contribution to health status in digestive tract of juvenile shrimp through improving morphology and microbiota, enhancing immune function, and inhibiting inflammation of digestive tract.

A TRIM-like protein restricts WSSV replication in the oriental river prawn, Macrobrachium nipponense

Tripartite motif (TRIM) proteins are a multifunctional family of ubiquitin E3 ligases involved in multiple biological processes. Studies have shown that many TRIM proteins in mammals play vital roles in the host defense against viral pathogens. In the present study, we identified a novel TRIM gene (MnTrim-like) from the oriental river prawn, Macrobrachium nipponense. Predicted MnTrim-like protein contains the characteristic RING finger domain. MnTrim-like was abundantly distributed in hepatopancreas, intestine, stomach, and gills. Upon white spot syndrome virus (WSSV) challenge, transcripts of MnTrim-like in the stomach were significantly up-regulated. Knockdown of MnTrim-like increased the expression of VP28 and decreased the synthesis of several antimicrobial peptides, including two crustins and one anti-lipopolysaccharide factor. Besides, silencing of these three antimicrobial peptides (AMPs) led to an increase in the expression of VP28 and WSSV copies. Moreover, it was found that injection of recombinant MnTrim-like protein with WSSV could decrease the transcription of VP28 and the number of virus particles. These results suggest that this MnTrim-like may restrict WSSV infection by positively regulating the expression of AMPs with antiviral activities and directly interacting with viral components. This study will broaden our understanding about the function of TRIM in crustacean during viral infection.

Marine Arthropods as a Source of Antimicrobial Peptides.

Peptide therapeutics play a key role in the development of new medical treatments. The traditional focus on endogenous peptides has shifted from first discovering other natural sources of these molecules, to later synthesizing those with unique bioactivities. This review provides concise information concerning antimicrobial peptides derived from marine crustaceans for the development of new therapeutics. Marine arthropods do not have an adaptive immune system, and therefore, they depend on the innate immune system to eliminate pathogens. In this context, antimicrobial peptides (AMPs) with unique characteristics are a pivotal part of the defense systems of these organisms. This review covers topics such as the diversity and distribution of peptides in marine arthropods (crustacea and chelicerata), with a focus on penaeid shrimps. The following aspects are covered: the defense system; classes of AMPs; molecular characteristics of AMPs; AMP synthesis; the role of penaeidins, anti-lipopolysaccharide factors, crustins, and stylicins against microorganisms; and the use of AMPs as therapeutic drugs. This review seeks to provide a useful compilation of the most recent information regarding AMPs from marine crustaceans, and describes the future potential applications of these molecules.

Identification of three novel Spätzle genes in Eriocheir sinensis and their roles during white spot syndrome virus infection

Proteins of Spätzle family play an essential role in innate immunity in invertebrates by activating the Toll pathway to induce the expression of antimicrobial peptides. However, little is known about the function of Spätzle in in the immune response of the Chinese mitten crab. In the present study, three novel Spätzle genes (named as EsSpz1, EsSpz2, and EsSpz3) were identified from Eriocheir sinensis. The genome structure of EsSpz1 contains two exons and an intron. Three Spätzle proteins all contain a Pfam Spaetzle domain. In the evolution, EsSpz1-3 cluster with other Spätzle proteins from crustaceans. EsSpz1-3 were widely distributed in multiple immune tissues. The expression levels of EsSpz1-3 in the intestine were remarkably upregulated after white spot syndrome virus (WSSV) challenge. The knockdown of EsSpz1-3 remarkably decreased the expressions of crustins and anti-lipopolysaccharide factors during WSSV infection. Moreover, EsSpz1-3 silencing remarkably increased the expression of WSSV envelope protein VP28. These findings suggest that new-found EsSpz1-3 in E. sinensis could promote the synthesis of antimicrobial peptides and inhibit the expression of VP28 during WSSV infection. Our study indicates that EsSpz1-3 in E. sinensis may participate in the innate immune defenses against WSSV by inducing the expression of antimicrobial peptides. This study provides new knowledge for the function of Spätzle in the antiviral immune defense in crustacean.