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.

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.

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.

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.

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.

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.

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.

Defense systems in developing Artemia franciscana nauplii and their modulation by probiotic bacteria offer protection against a Vibrio anguillarum challenge.

The alterations of immune responses of Artemia franciscana nauplii as a function of culture time and after a challenge with the pathogen Vibrio anguillarum were studied. The effect of the administration of the probiotic bacteria Bacillus subtilis, Lactobacillus plantarum and Lactococcus lactis either alone or in combination with the pathogen was evaluated. The activity of the antioxidant enzymes superoxide dismutase (SOD), Glutathione reductase (GRed), Glutathione transferase (GST) and Phenoloxidase (PO) presented a significant increase as a function of culture time, appeared elevated following probiotic administration and were depleted 48 h following the experimental challenge. Lipid peroxidation reached peak levels at 48 h of culture, when nauplii start feeding and returned to lower values at 144 h, remaining however significantly higher than control (P < 0.05). The three probiotics significantly reduced lipid peroxidation in comparison with the corresponding control, while challenge with the pathogen resulted in its threefold increase. Survival of nauplii remained high throughout culture and was either increased or remained at control levels following the administration of the probiotics. The challenge with the pathogen resulted in a significantly decreased survival of 15.3% for the positive control, while in the probiotic treated series survival values were not significantly different from the negative control (P > 0.05). Following a combined administration of each probiotic and the pathogen the activities of all enzymes tested were significantly lower (P < 0.001) than the negative control (no treatment), but higher than the positive control (challenge, no probiotic). Lipid peroxidation was significantly lower in the probiotic treated series in comparison to the positive control (P < 0.001). The results of the present study provide evidence that major alterations take place as a function of culture time of Artemia nauplii. In addition the pathogen induces an oxidative stress response. The probiotics B. subtilis, L. plantarum and L. lactis protect Artemia against a V. anguillarum challenge by enhancing its immune responses thus contributing to reduced oxidative damage and increased survival.

Small creatures use small RNAs to direct antiviral defenses.

Antiviral RNA silencing has been recognized as an important defense mechanism in arthropods against RNA viruses. However, the role of this pathway in DNA virus infection remains largely unexplored. A report in this issue of the European Journal of Immunology provides new insight into the role of RNA silencing in antiviral defense against DNA viruses. Huang and Zhang [Eur. J. Immunol. 2013. 137-146] found that the dsDNA virus white spot syndrome virus, an agriculturally important pathogen of shrimp, is targeted by the shrimp RNA-silencing machinery via the production of virus-derived siRNAs. Furthermore, the authors show that the RNA-silencing pathway, and crucially, Dicer-2, is important for restricting viral infection. This study provides novel insights not only into shrimp antiviral defenses but also potentially into antiviral immunity against DNA viruses in a larger spectrum of hosts, as discussed in this Commentary. Furthermore, this study may contribute to the future development of immune-based therapeutics to combat viral pathogens, not only in aquaculture, but also in insect vectors of human diseases.

Host defense against DNA virus infection in shrimp is mediated by the siRNA pathway.

The RNA interference (RNAi) system of eukaryotes using siRNAs has been documented as an immune response against invasion by RNA viruses. However, whether the siRNA pathway can be triggered by the infection with DNA viruses in animals remains to be investigated. In the present study, we show that Marsupenaeus japonicus shrimp can generate an antiviral siRNA (vp28-siRNA) in response to infection by a double-stranded DNA virus, white spot syndrome virus (WSSV). After challenging with WSSV, vp28-siRNA is detected in all the WSSV-infected organs and tissues of shrimp as early as 24 h postinfection (p.i.). The results indicate that the host Dicer2 and Ago2 proteins are required for the biogenesis and function of vp28-siRNA, respectively. We show further that vp28-siRNA predominates in the cytoplasm of shrimp hemocytes at 48 h p.i. Knockdown of Dicer2 by special siRNA or inhibition of vp28-siRNA with locked nucleic acid antisense oligonucleotides both lead to a significant increase in WSSV copy number at 24-48 h p.i. Our study highlights a novel aspect of the siRNA pathway in the immune response of animals against infection by DNA viruses.

Feeding truncated heat shock protein 70s protect Artemia franciscana against virulent Vibrio campbellii challenge.

The 70 kDa heat shock proteins (Hsp70s) are highly conserved in evolution, leading to striking similarities in structure and composition between eukaryotic Hsp70s and their homologs in prokaryotes. The eukaryotic Hsp70 like the DnaK (Escherichia coli equivalent Hsp70) protein, consist of three functionally distinct domains: an N-terminal 44-kDa ATPase portion, an 18-kDa peptide-binding domain and a C-terminal 10-kDa fragment. Previously, the amino acid sequence of eukaryotic (the brine shrimp Artemia franciscana) Hsp70 and DnaK proteins were shown to share a high degree of homology, particularly in the peptide-binding domain (59.6%, the putative innate immunity-activating portion) compared to the N-terminal ATPase (48.8%) and the C-terminal lid domains (19.4%). Next to this remarkable conservation, these proteins have been shown to generate protective immunity in Artemia against pathogenic Vibrio campbellii. This study, aimed to unravel the Vibrio-protective domain of Hsp70s in vivo, demonstrated that gnotobiotically cultured Artemia fed with recombinant C-terminal fragment (containing the conserved peptide binding domain) of Artemia Hsp70 or DnaK protein were well protected against subsequent Vibrio challenge. In addition, the prophenoloxidase (proPO) system, at both mRNA and protein activity levels, was also markedly induced by these truncated proteins, suggesting epitope(s) responsible for priming the proPO system and presumably other immune-related genes, consequently boosting Artemia survival upon challenge with V. campbellii, might be located within this conserved region of the peptide binding domain.

Priming the prophenoloxidase system of Artemia franciscana by heat shock proteins protects against Vibrio campbellii challenge.

Like other invertebrates, the brine shrimp Artemia franciscana relies solely on innate immunity, which by definition lacks adaptive characteristics, to combat against invading pathogens. One of the innate mechanisms is melanisation of bacteria mediated by the activation of the prophenoloxidase (proPO) system. The 70 kDa heat shock proteins (Hsp70) derived from either prokaryote (Escherichia coli) or eukaryote (Artemia), well conserved and immune-dominant molecules, protect Artemia against Vibrio campbellii. However, the molecular mechanisms by which these proteins protect Artemia against Vibrio campbellii infection are unknown. Here we demonstrated that feeding gnotobiotically grown Artemia with either Artemia Hsp70 or the E. coli Hsp70 equivalent DnaK, each overproduced in E. coli, followed by V. campbellii challenge enhanced the proPO system, at both mRNA and protein activity levels. Additionally, the Artemia fed with these proteins survived well in a Vibrio challenge assay. These results indicated that Hsp70s derived from either prokaryotic or eukaryotic sources generate protective immunity in the crustacean Artemia against V. campbellii infection by priming the proPO system. This is apparently the first in vivo report on priming activity of Hsp70 in an invertebrate.

A prophenoloxidase from Artemia sinica: cDNA cloning, expression and activity analysis during early development.

To understand the defense mechanisms of Crustacean animals, brine shrimp Artemia sinica prophenoloxidase (AsproPO) cDNA was cloned and its expression at early developmental stages was examined by reverse-transcription PCR (RT-PCR) and semi-quantitative RT-PCR, and activity of phenoloxidase (PO) at different developmental stages was further detected by using l-3,4-dihydroxyphenylalanine (l-DOPA) as a specific substrate in this study. It was found that the full-length of AsproPO cDNA is 2125 bp and it contains an open reading frame of 2100 bp encoding a protein of 699 amino acids. The deduced amino acid sequence of AsproPO has two putative copper binding sites highly conserved in Arthropods. Semi-quantitative RT-PCR analyses showed that the gene of AsproPO expressed at Emergence, Instar I and Instar II stages but did not at 0 h and 6 h stages. Activity measurement showed that PO activity could only be detected at Instar II stage but the other measured stages. All these implied that Artemia proPO immune system was complexly modulated during early development.

Efficacy of heterologous and homologous heat shock protein 70s as protective agents to Artemia franciscana challenged with Vibrio campbellii.

The Hsp70 class of heat shock proteins (Hsps) has been implicated at multiple points in the immune response of both vertebrates and invertebrates. This class of chaperones is highly conserved in both sequence and structure, from prokaryotes to higher eukaryotes. In view of their high degree of homology, it was assumed that these Hsp70 proteins derived either from the prokaryotes or eukaryotes would have similar functions, especially in relation to their protective ability in a challenge assay. To verify this, we compared two evolutionary diverse Hsp70s, Artemia Hsp70 and Escherichia coli Hsp70 equivalent DnaK (each overproduced in E.coli), for their ability to protect Artemia against Vibrio challenge. Results showed that Artemia fed with E. coli producing Artemia Hsp70 or DnaK proteins, as assessed by immune-probing in western blots, survived better in a Vibrio challenge assay. The observed effects could be due to enhancement of the Artemia immune system as phenoloxidase activity was found to be increased by these proteins. These two Hsp70 proteins exhibit a high degree of homology, particularly in the peptide-binding domain (the putative innate immunity-activating portion) with 59.6% identity, indicating that the observed protective capacity of homologous or heterologous Hsp70 proteins might reside within this peptide-binding domain.

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.

Feeding Artemia franciscana (Kellogg) larvae with bacterial heat shock protein, protects from Vibrio campbellii infection.

Among their numerous physiological effects, heat shock proteins (Hsps) are potent immunomodulators, a characteristic reflecting their potential as therapeutic agents and which led to their application in combating infection. As an example, the up-regulation of endogenous Hsp70 in the branchiopod crustacean Artemia franciscana (Kellogg) is concurrent with shielding against bacterial infection. To better understand this protective mechanism, gnotobiotic Artemia were fed with Escherichia coli treated to over-produce different prokaryotic Hsps. This was shown to increase larval resistance to experimental Vibrio campbellii exposure. Immunoprobing of Western blots showed that the enhanced resistance to V. campbellii correlated with DnaK production in E coli. A definitive role for DnaK was then demonstrated by feeding Artemia larvae with transformed bacteria over-producing only this protein, although other Hsps such as DnaJ and grpE also provided tolerance against Vibrio infection. Feeding of bacteria synthesizing selected Hsps is therefore suggested as an alternative to antibiotic use as a means of enhancing resistance of Artemia larvae to bacterial infection, which may have potential applications in aquaculture.

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.