Expression pattern of mitogen-activated protein kinase kinase 4 and regulation to antibacterial factor ABF-1/2 in response to bacterial challenge from Artemia parthenogenetica.

Mitogen-activated protein kinase 4, MKK4, is a key upstream kinase in the JNK/p38 MAPK pathway that has been reported to participate in multiple immune responses. In this study, the gene that encodes ApMKK4 was isolated and identified from Artemia parthenogenetica. It was found to contain a 1134 bp open reading frame encoding 378 amino acids. The predicted protein contains D domain, DVD domain and kinase domain. Homology analysis revealed that ApMKK4 shares 38-69% identity with MKK4 homologs from other species. Results revealed that ApMKK4 was mainly expressed during early development of which highest at the gastrula stage. After challenged by Vibrio harveyi and Micrococcus lysodeikticus, ApMKK4 was remarkably upregulated at 10 and 103 cfu/mL bacterial concentrations, respectively. Through siRNAi, the transcript level of ApMKK4 was significantly decreased by 46-67%. Intriguingly, when the ApMKK4-knockdown nauplii faced with bacterial stimulation, the expression of ApMKK4 was completely restored in a short time. Moreover, this phenomenon also occurred in related antimicrobial peptide genes, ABF-1 and ABF-2. Our research reveals that ApMKK4 plays a pivotal role during early development and immune responses against bacterial infections.

Peculiarities of innate immune memory in crustaceans.

Classical characteristic of the innate immune system is the lack of ability to build up immunological memory, contrast to the adaptive immune system that is capable of "remembering" antigens, and rapidly mount a greater magnitude of immune response upon subsequent exposure to the same antigens. Peculiarly, immunological memory of innate immunity is evidenced in invertebrates. At least three different memory phenomena have been described, namely sustained unique response, recalled response, and immune shift. Studies attended to decipher the mechanistic biology of the innate immune memory reveals the role of epigenetics, which modulates the response of immune memory, and the heritability of immune memory to subsequent generations. A parthenogenetic Artemia model demonstrated successful transgenerational epigenetic inheritance of resistance trait against Vibrio campbellii. Following, the role of invertebrate hemocytes and Down syndrome cell adhesion molecule (Dscam) in innate immune memory is reviewed. While there is no vertebrate antibody homolog found in invertebrates, Dscam was found to resemble the functionality of vertebrate antibody. Insight of Dscam as immune factor was illustrated further in the current review.

Beta-glucan's varying structure characteristics modulate survival and immune-related genes expression from Vibrio harveyi-infected Artemia franciscana in gnotobiotic conditions.

ß-Glucans have long been used as an immunostimulant in aquaculture. However, the relationship of its structure to its immunomodulatory properties are poorly understood. In this study, the particle size and chemical structure of ß-glucans extracted from wild-type strain of baker's yeast (Saccharomyces cerevisiae) and its null-mutant yeasts Gas1 were characterised. Using Sigma ß-glucan as a reference, the immunomodulatory properties of these polysaccharides in the germ-free Artemia franciscana model system in the presence of Vibrio harveyi bacterial challenge were investigated. The survival of the A. franciscana nauplii, upon challenge with V. harveyi, was significantly higher in all three glucan-treated groups compared to the control. The glucan Gas1 with a lower degree of branching and shorter side chain length had the most prominent V. harveyi-protective effects. The particle size did not affect the nauplii survival when challenged with V. harveyi. Results also showed that the salutary effect of the tested glucans was associated with the upregulation of innate immune genes such as lipopolysaccharide and ß-1,3-glucan-binding protein (lgbp), high mobility group box protein (hmgb), and prophenoloxidase (proPO). Interestingly, the up-regulation of superoxidase dismutase (sod) and glutathione-s-transferase (gst) was only observed in Gas1 treated group, indicating that Gas1 could function to induce higher reactive oxygen species and stronger immunomodulatory function in A. franciscana, and therefore higher survival rate. The expression of heat shock protein 70 (hsp70), peroxinectin (pxn), and down syndrome cell adhesion molecule (dscam) remain unaltered in response to glucan treatment. Taken together, this study provides insights into the structure-function relationship of ß-glucan and the results confirmed that ß-glucan can be an effective immunostimulant in aquaculture, especially the Gas1 glucan.

Osmoregulatory performance and immunolocalization of Na+/K+-ATPase in the branchiopod Artemia salina from the Sebkha of Sidi El Hani (Tunisia).

Artemia salina is an extremophile species that tolerates a wide range of salinity, especially hypertonic media considered lethal for the majority of other aquatic species. In this study, A. salina cysts were hatched in the laboratory and nauplii were acclimated at three different salinities (60, 139 and 212 ppt). Once in the adult phase, their hemolymph osmolality was measured. The animals were strong hypo-osmoregulators in the entire range of tested salinities, with up to 10 fold lower hemolymph osmolalities than their surrounding environment. Immunostaining of Na+/K+-ATPase was done on sections and on whole body mounts of adults in order to localize the ionocytes in different organs. An intense Na+/K+-ATPase immunostaining throughout the cells was observed in the epithelium of the ten pairs of metepipodites. A positive immunoreactivity for Na+/K+-ATPase was also detected in the maxillary glands, in the epithelium of the efferent tubule and of the excretory canal, as well as in the anterior digestive tract. This study confirms the strong hypo-osmotic capacity of this species and affords an overview of the different organs involved in osmoregulation in A. salina adults.

Comparative analysis of two brine shrimps revealed differential expression pattern and functional characterization of CK2α under bacterial stimulation from different geographical distribution.

Understanding how the brine shrimp responds to different geographical populations can provide novel insights on response to bacterial stimulation. In the paper, Artemia sinica from lower altitudes and Artemia parthenogenetica from higher altitudes of the Tibetan Plateau, were used to illustrate different defense against bacteria mechanisms that these organisms used to adapt to different geographical environments. Protein kinase CK2 is a serine/threonine kinase with a multitude of protein substrates. It is a ubiquitous enzyme essential for the viability of eukaryotic cells, where its functions in a variety of cellular processes, including cell cycle progression, apoptosis, transcription, and viral infection. The gene encodes the same mRNA sequence in A. sinica and A. parthenogenetica, named AsCK2α and ApCK2α, respectively. The open reading frame was obtained, a 1047-bp sequence encoding a predicted protein of 349 amino acids. To systematically analyze the expression of AsCK2α and ApCK2α during embryonic development and bacterial challenge, real-time PCR, Western blotting and immunohistochemistry were performed. The results showed that AsCK2α was higher than ApCK2α at different developmental stages. Under bacterial challenge, the expression of ApCK2α was significantly higher than AsCK2α. Protein localization analysis showed that AsCK2α and ApCK2α were mainly distributed in the head and chest. Our research revealed that CK2α plays a vital role in the growth, development and bacterial stimulation of the brine shrimp.

Phloroglucinol Treatment Induces Transgenerational Epigenetic Inherited Resistance Against Vibrio Infections and Thermal Stress in a Brine Shrimp (Artemia franciscana) Model.

Emerging, infectious diseases in shrimp like acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus and mortality caused by other Vibrio species such as Vibrio harveyi are worldwide related to huge economic losses in industrial shrimp production. As a strategy to prevent disease outbreaks, a plant-based phenolic compound could be used as a biocontrol agent. Here, using the brine shrimp (Artemia franciscana) as a model system, we showed that phloroglucinol treatment of the parental animals at early life stages resulted in transgenerational inherited increased resistance in their progeny against biotic stress, i.e., bacteria (V. parahaemolyticus AHPND strain and V. harveyi) and abiotic stress, i.e., lethal heat shock. Increased resistance was recorded in three subsequent generations. Innate immune-related gene expression profiles and potential epigenetic mechanisms were studied to discover the underlying protective mechanisms. Our results showed that phloroglucinol treatment of the brine shrimp parents significantly (P < 0.05) enhanced the expression of a core set of innate immune genes (DSCAM, proPO, PXN, HSP90, HSP70, and LGBP) in subsequent generations. We also demonstrated that epigenetic mechanisms such as DNA methylation, m6A RNA methylation, and histone acetylation and methylation (active chromatin marker i.e., H3K4Me3, H3K4me1, H3K27me1, H3 hyperacetylation, H3K14ac and repression marker, i.e., H3K27me3, H4 hypoacetylation) might play a role in regulation of gene expression leading toward the observed transgenerational inheritance of the resistant brine shrimp progenies. To our knowledge, this is the first report on transgenerational inheritance of a compound-induced robust protected phenotype in brine shrimp, particularly protected against AHPND caused by V. parahaemolyticus and vibriosis caused by V. harveyi. Results showed that epigenetic reprogramming is likely to play a role in the underlying mechanism.

Defining specific allergens for improved component-resolved diagnosis of shrimp allergy in adults.

Shrimp is one of the predominant causes of food allergy among adults, often presenting with severe reactions. Current in vitro diagnostics are based on quantification of patient specific-IgE (sIgE) to shrimp extract. Tropomyosin is the known major shrimp allergen, but IgE sensitisation to other allergens is poorly characterised. In this study, the binding of IgE to various shrimp allergens, additional to tropomyosin, was investigated using sera from 21 subjects who had clinical reactions to one or more shellfish species. Total shrimp-sIgE was quantified using ImmunoCAP, while allergen-sIgEs were quantified using immunoblotting and mass spectrometry, and immuno-PCR to recombinant shrimp tropomyosin. Sixty-two percent of subjects (13/21) were positive to shrimp by ImmunoCAP. IgE from 43% of subjects (9/21) bound tropomyosin, while an additional 29% of subjects (6/21) demonstrated IgE-binding solely to other shrimp allergens, including sarcoplasmic calcium-binding protein, arginine kinase and hemocyanin. Furthermore, IgE sensitisation to other shrimp allergens was demonstrated in 50% of subjects (4/8) who were ImmunoCAP negative. The lack of standardised shrimp allergens and inadequacy of current extracts for shrimp allergy diagnosis is highlighted by this study. Comprehensive knowledge of less studied allergens and their inclusion in component-resolved diagnostics will improve diagnostic accuracy, benefitting the wider population suffering from shellfish allergy.

Molecular characterization and expression analysis of protein enhancer of sevenless 2B from Artemia sinica at early embryonic development and during immune response to bacterial stimulation.

Protein enhancer of sevenless 2B, E(sev)2B, is a key adapter protein in the Ras/MAPK signaling pathway which has been reported to be involved in innate immunity. In this study, the gene that encodes AsE(sev)2B was isolated from A. sinica. It was found to contain a 636 bp open reading frame encoding 211 amino acids with a calculated molecular mass of 24.357 kDa and a predicted isoelectric point of 5.39. The predicted protein contains a N-terminal Src homology 3 domain (SH3), a central Src homology 2 domain (SH2), and a C-terminal Src homology 3 domain (SH3). Homology analysis revealed that AsE(sev)2B shares 49%-95% identity with E(sev)2B homologs from other species. In this study, the expression pattern and location of AsE(sev)2B during different stages of embryonic development and bacterial challenge were investigated by means of real-time qPCR, Western blotting and immunohistochemistry. Results showed that the highest expression level of AsE(sev)2B was at 0 h. After challenged by Gram-positive bacteria and Gram-negative bacteria, AsE(sev)2B was remarkably upregulated at 106 cellsL-1 bacterial concentrations. These results suggested that AsE(sev)2B plays a vital role during early embryonic development and in immune responses against bacterial challenge.

Teaching Shrimps Self-Defense to Fight Infections.

A paradigm shift in our understanding of shrimp immunity offers the potential to develop novel disease-control strategies. We summarize cutting-edge findings on the phenomenon of trained immunity in shrimps and discuss how it may contribute to new avenues for controlling disease in these aquaculturally important animals.

High doses of sodium ascorbate act as a prooxidant and protect gnotobiotic brine shrimp larvae (Artemia franciscana) against Vibrio harveyi infection coinciding with heat shock protein 70 activation.

Ascorbate is an essential nutrient commonly regarded as an antioxidant. In this study, using axenic brine shrimp and pathogenic strain Vibrio harveyi as the host-pathogen model, we confirmed that pretreatment of sodium ascorbate (NaAs), at an optimum concentration, was a prooxidant by generation of hydrogen peroxide, inducing protective effects in the brine shrimp against V. harveyi infection. Such a protective effect could be neutralized by the addition of an antioxidant enzyme catalase. We further showed that generation of oxygen radicals is linked to the induction of heat shock protein 70 (Hsp70), which is involved in eliciting the antioxidant protection system including superoxidase dismutase (SOD) and possibly many other immune responses. Furthermore, using RNA interference technique, we found that the pretreatment of sodium ascorbate increased the survival significantly in the control knockdown groups (using green fluorescent protein, GFP) but not in Hsp70 knockdown groups and the result directly suggested that the up-regulated Hsp70 induced by sodium ascorbate pretreatment induced the protective effect. These results provide a mechanistic rationale for exploring the further use of ascorbate for antimicrobial therapy in aquaculture.

Interactions among yeast and probiotic bacteria enhance probiotic properties and metabolism offering augmented protection to Artemia franciscana against Vibrio anguillarum.

The interactions of the probiotics Bacillus subtilis, Lactococcus lactis and Lactobacillus plantarum with the yeast Saccharomyces cerevisiae were examined in terms of probiotic and biochemical characteristics. Yeast supernatant had a positive effect on the aggregation biofilm formation capacity and hydrophobicity of probiotics, and resulted in increased lactic acid levels, reduced pH values as well as lower RS and FAN levels of probiotics. The effect of probiotics supernatants on yeast was more complex but best results were obtained in the yeast: probiotic CFS ratio of 1:2 for B. subtilis and of 2:1 for the other probiotics. The observed effects depended on the volume ratio of the cell free supernatant to the culture it was applied on. Best results were obtained by the volume ratio probiotic: yeast of (2:1) for B. subtilis and of (1:2) probiotic: yeast for L. plantarum and L. lactis. These ratios were used for further evaluation in vitro against V. anguillarum, resulting in reduced survival and attachment properties of the pathogen. Moreover, the administration of the corresponding combination of bacteria and yeast to Artemia nauplii greatly improved their survival following a challenge with the pathogen. Our results demonstrate that yeast enhances the protective effect of probiotics in a strain specific manner.

Searching for crab-borne antimicrobial peptides: Crustin from Portunus pelagicus triggers biofilm inhibition and immune responses of Artemia salina against GFP tagged Vibrio parahaemolyticus Dahv2.

Marine organisms represent a huge source of novel compounds for the development of effective antimicrobial drugs. The present study focus on the purification of the antimicrobial peptide crustin from the haemolymph of the blue swimmer crab, Portunus pelagicus, by blue Sepharose CL-6B matrix assisted affinity column chromatography. Crustin showed a single band with a molecular mass of 17 kDa in SDS-PAGE analysis. The XRD analysis exhibited peaks at 32° and 45° while a distinct peak with a retention time of 1.8 min resulted in high performance liquid chromatography (HPLC) pointing out the crystalline nature and purity of crustin, respectively. Crustin purified from P. pelagicus (Pp-Cru) showed immunological activities, triggering encapsulation, phagocytosis on Sepharose beads and yeast (Saccharomyces cerevisiae) respectively. Furthermore, encapsulation of GFP tagged V. parahaemolyticus in Artemia salina and challenging study were assessed under CLSM and the potential of Pp-Cru was examined in vivo. In addition, the growth reduction and biofilm inhibition potential of Pp-Cru on Staphylococcus aureus, Enterococcus faecalis (Gram- positive bacteria) and Pseudomonas aeruginosa, Escherichia coli (Gram-negative bacteria) was evidenced by inverted and confocal laser scanning microscopic analysis, revealing that 100 µg/ml of Pp-Cru can disrupt the biofilm matrix thereby the thickness of biofilm was significantly reduced. Overall, the present investigation might provide a sensitive platform to realize the significant function of Pp-Cru in crustacean immune mechanism as well as its potential to bacterial growth inhibitor. The functional properties of purified Pp-Cru antimicrobial peptide may lead to a superior understanding of innate immune response in P. pelagicus species, which suggest the promising application for drug development in aquaculture.

Transcriptome analysis of Artemia sinica in response to Micrococcus lysodeikticus infection.

To enhance genomic resources and understand the molecular immune mechanisms underlying the response topathogens, we first performed a comparative gene transcription analysis from Micrococcus lysodeikticus-immunized Artemia sinica and from a control group through RNA-Seq technology, meanwhile the differentially expressed genes (DEGs) were investigated. In total, 80, 113, 984 clean reads were obtained and then assembled into 71,536 unigenes with an average length of 1115 bp and an N50 of 1783 bp. Unigenes were annotated by comparing against nr, Swiss-Prot\KEGG\ COG\ KOG\ GO and Pfam databases, and 27,689 unigenes (38.7%) were annotated in at least one database. After bacterial challenge, 183 and 298 genes were identified as remarkably up-regulated or down-regulated, respectively, amongst 481 were associated with 168 pathways, including classical immune-related pathways, such as 'Toll-like receptor signaling', 'the complement cascades', 'MAPK signaling pathway' and 'Apoptosis'. Besides, eight genes which were differently expressed immune-related were confirmed by using quantitative real-time PCR. This study characterized a gene expression pattern for normal and M. lysodeikticus -immunized A. sinica for the first time and sheds new light on the molecular mechanisms thus enabling future efforts on disease control programs in this valuable aquaculture species.

Differential proteome of haemocyte subpopulations responded to white spot syndrome virus infection in Chinese shrimp Fenneropenaeus chinensis.

In our previous study, the differentially expressed proteins have been identified by proteomic analysis in total haemocytes of shrimp (Fenneropenaeus chinensis) after white spot syndrome virus (WSSV) infection. To further investigate the differential response of haemocyte subpopulations to WSSV infection, granulocytes and hyalinocytes were separated from healthy and WSSV-infected shrimp by immunomagnetic bead (IMB) method, respectively. Then two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to analyze the differentially expressed proteins in haemocyte subpopulations between healthy and WSSV-infected shrimp. The results of flow cytometry (FCM) showed that about 98% of granulocytes and about 96% of hyalinocytes in purity were obtained. Quantitative intensity analysis revealed that 26 protein spots in granulocytes and 24 spots in hyalinocytes were significantly changed post WSSV infection. Among them, 24 proteins in granulocytes and 23 proteins in hyalinocytes were identified by MS analysis, which could be divided into eight categories according to Gene Ontology. The identification of prophenoloxidase (proPO), proPO 2 and peroxiredoxin in WSSV-infected granulocytes was consistent with the facts that the proPO-activating system and peroxiredoxin were mainly existed in granulocytes. The phagocytosis of hyalinocytes seemed to be enhanced during the infection, because several proteins that involved in phagocytosis, including clathrin heavy chain, ADP ribosylation factor 4 and Alpha2 macroglobulin were up-regulated in hyalinocytes upon WSSV infection. Our results also reflected the vital biological significance of calcium ion binding proteins in granulocytes and ATPase/GTPase in hyalinocytes during WSSV infection. The data in this study verified the roles of granulocytes and hyalinocytes involved in WSSV infection, and differentially expressed proteins identified in granulocytes and hyalinocytes had a close correlation with their function characteristics.

Lipopolysaccharide- and ß-1,3-glucan-binding protein from Litopenaeus vannamei: Purification, cloning and contribution in shrimp defense immunity via phenoloxidase activation.

Lipopolysaccharide- and ß-1,3-glucan-binding protein (LGBP) existed in diversity of invertebrates including shrimp plays a crucial role in an innate immunity via mediating the recognition of invading pathogens. In this study, LGBP was cloned and characterized from the hepatopancreas of Litopenaeus vannamei, named as LvLGBP. Its full-length cDNA of 1282 bp contained an open reading frame (1101 bp) encoding a peptide of 367 amino acids. The LGBP primary structure contained a glycosyl hydrolase domain, two integrin binding motifs, two kinase C phosphorylation sites, and two polysaccharide recognition motifs which were identified as a polysaccharide binding motif and a ß-1,3-glucan recognition motif. The LvLGBP transcripts were expressed mainly in the hepatopancreas. Upon challenge with Vibrio parahaemolyticus or white spot syndrome virus (WSSV), the LvLGBP mRNA expression was significantly up-regulated to reach a maximum at 48 h post injection. Its expression was also induced by lipopolysaccharide (LPS) or ß-1,3-glucan stimulation. RNAi-based silencing resulted in the critical suppression of LvLGBP expression. Knockdown of LvLGBP gene with co-inoculation by V. parahaemolyticus or WSSV led to increase in the cumulative mortality and reduce in the median lethal time. Native LGBP was detected only in the hepatopancreas as verified by Western blotting. Purified LGBP from the hepatopancreas exhibited the agglutinating and binding activity towards Gram-negative bacterium V. parahaemolyticus with calcium-dependence. Its agglutinating activity was dominantly inhibited by LPS with higher potential than ß-1,3-glucan. Purified LvLGBP could significantly activate the hemocyte phenoloxidase activity in the presence of LPS (12.9 folds), while slight activation was detected with ß-1,3-glucan (2.0 folds). It could enhance the encapsulation by hemocytes but did not have antibacterial activity. These results provided evidence that LvLGBP might act as a pathogenic recognition protein to activate shrimp immune defense against invading pathogens via the agglutination, binding and enhancing encapsulation and phenoloxidase activity of the hemocytes.

A cuticle protein from the Pacific white shrimp Litopenaeus vannamei involved in WSSV infection.

White spot syndrome virus (WSSV) is a major viral pathogen in global shrimp farming, causing huge economic damage. Through penetrating the outer surface of the target tissues, WSSV enters into the cells of the target tissue to complete the replication process in the host. In the present study, a cuticle protein gene from Litopenaeus vannamei, designated as LvAMP13.4, was identified and proved to be involved in WSSV invasion. The deduced amino acid sequence of LvAMP13.4 contained a signal peptide and a conserved chitin-binding domain type 4 (ChBD4). This cuticle protein gene was mainly expressed in stomach, gill and epidermis. The expression level of LvAMP13.4 was significantly changed during WSSV infection. Silencing of LvAMP13.4 by dsRNA interference apparently reduced the mortality rate and the WSSV copy number in shrimp upon WSSV infection. Furthermore, yeast two-hybrid system and Co-IP assay were performed to confirm that LvAMP13.4 could interact with the major envelop protein VP24 of WSSV. These data indicated that LvAMP13.4 was involved in the invasion process of WSSV through interaction with VP24. The present results could provide new insights for us in understanding the role of host cuticle proteins during virus invasion.

RNA-seq identifies integrin alpha of kuruma shrimp Marsupenaeus japonicus as a candidate molecular marker for phagocytic hemocytes.

Phagocytosis is main cellular immunity, however, it is still unknown or debated upon which types of hemocyte contributes phagocytosis in penaeid shrimps. The hemocyte characterization in kuruma shrimp have been mainly performed based on its morphology by microscopic observation. Therefore, establishment of molecular markers to distinguish phagocytic hemocytes is required. In this study, using magnetic fluorescent beads, we enriched phagocytic hemocytes and conducted RNA-seq analysis between total and enriched phagocytic hemocytes. The data demonstrated functional difference between total and phagocytic hemocytes. In addition, a transcript homologous to integrin-alpha was highly expressed in phagocytic hemocytes, and named Mj-Intgα. Using anti-serum against Mj-Intgα revealed that around 60% of total hemocytes and more than 90% of phagocytic hemocytes showed positive for Mj-Intgα. This study presents Mj-Intgα as a candidate molecular marker for future functional characterization of hemocytes.

An immune-related gene expression atlas of the shrimp digestive system in response to two major pathogens brings insights into the involvement of hemocytes in gut immunity.

Much of our current knowledge on shrimp immune system is restricted to the defense reactions mediated by the hemocytes and little is known about gut immunity. Here, we have investigated the transcriptional profile of immune-related genes in different organs of the digestive system of the shrimp Litopenaeus vannamei. First, the tissue distribution of 52 well-known immune-related genes has been assessed by semiquantitative analysis in the gastrointestinal tract (foregut, midgut and hindgut) and in the hepatopancreas and circulating hemocytes of shrimp stimulated or not with heat-killed bacteria. Then, the expression levels of 18 genes from key immune functional categories were quantified by fluorescence-based quantitative PCR in the midgut of animals experimentally infected with the Gram-negative Vibrio harveyi or the White spot syndrome virus (WSSV). Whereas the expression of some genes was induced at 48 h after the bacterial infection, any of the analyzed genes showed to be modulated in response to the virus. Whole-mount immunofluorescence assays confirmed the presence of infiltrating hemocytes in the intestines, indicating that the expression of some immune-related genes in gut is probably due to the migratory behavior of these circulating cells. This evidence suggests the participation of hemocytes in the delivery of antimicrobial molecules into different portions of the digestive system. Taken all together, our results revealed that gut is an important immune organ in L. vannamei with intimate association with hemocytes.

Cloning, expression pattern and functional characterization of interleukin-1 receptor-associated kinase 4, an important mediator of the Toll-like receptor signaling pathway, from Artemia sinica.

As a crucial component of Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) signaling pathways, IL-1R--associated kinase 4 (IRAK-4) plays a central role in innate immunity and embryonic development. Herein, we have characterized the full length cDNA of IRAK4 from Artemia sinica. Molecular characterization revealed that the sequence includes a 2550 bp open reading frame, encoding a predicted protein of 849 amino acids. The predicted protein contains a death domain in the N-terminus and two serine/threonine/tyrosine protein kinasedomains. Bioinformatics analysis showed that it belonged to a new member of the IRAK-4 family. The expression of AsIRAK-4 was researched in various stages during embryonic development by several molecular biology methods including real time PCR, Western blotting and immunohistochemistry. The results showed that AsIRAK-4 was constitutively expressed at all developmental stages from embryo to adult, and it was mainly expressed in the head and thorax at the early stages and on the surface of the alimentary canal at later stages. The highest expression level was at the 0 h, 15 h and 5 d stages of A. sinica. While challenged by Gram-positive and Gram-negative bacteria, AsIRAK-4 was remarkably upregulated with the rising concentration of bacteria. Our research revealed that AsIRAK-4 plays a vital role in growth, development and innate immunity of A. sinica.