A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities.

Brine shrimp (Artemia) are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na+,K+-ATPase (NKA), a heterodimeric (αß) pump that usually exports 3Na+ in exchange for 2 K+ per hydrolyzed ATP. Artemia express several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopus α1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that Artemia express two salinity-independent canonical α subunits (α1NN and α3NN), as well as two ß variants, in addition to the salinity-controlled α2KK. These ß subunits permitted heterologous expression of the α2KK pump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2KK pumps and compared it to that of Xenopus α1 (and its α2KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2KK-like characteristics to Xenopus α1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2KK's Lys308 helps to maintain high affinity for external K+ when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K+-congener 86Rb+, we prove that double-lysine-substituted pumps transport 2Na+ and 1 K+ per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowing Artemia to maintain steeper Na+ gradients in hypersaline environments.

A cytological revisit on parthenogenetic Artemia and the deficiency of a meiosis-specific recombinase DMC1 in the possible transition from bisexuality to parthenogenesis.

Although parthenogenesis is widespread in nature and known to have close relationships with bisexuality, the transitional mechanism is poorly understood. Artemia is an ideal model to address this issue because bisexuality and "contagious" obligate parthenogenesis independently exist in its congeneric members. In the present study, we first performed chromosome spreading and immunofluorescence to compare meiotic processes of Artemia adopting two distinct reproductive ways. The results showed that, unlike conventional meiosis in bisexual Artemia, meiosis II in parthenogenic Artemia is entirely absent and anaphase I is followed by a single mitosis-like equational division. Interspecific comparative transcriptomics showed that two central molecules in homologous recombination (HR), Dmc1 and Rad51, exhibited significantly higher expression in bisexual versus parthenogenetic Artemia. qRT-PCR indicated that the expression of both genes peaked at the early oogenesis and gradually decreased afterward. Knocking-down by RNAi of Dmc1 in unfertilized females of bisexual Artemia resulted in a severe deficiency of homologous chromosome pairing and produced univalents at the middle oogenesis stage, which was similar to that of parthenogenic Artemia, while in contrast, silencing Rad51 led to no significant chromosome morphological change. Our results indicated that Dmc1 is vital for HR in bisexual Artemia, and the deficiency of Dmc1 may be correlated with or even possibly one of core factors in the transition from bisexuality to parthenogenesis.

Identification and characterization of the Doublesex gene and its mRNA isoforms in the brine shrimp Artemia franciscana.

Doublesex (DSX) proteins are members of the Doublesex/mab-3-related (DMRT) protein family and play crucial roles in sex determination and differentiation among the animal kingdom. In the present study, we identified two Doublesex (Dsx)-like mRNA isoforms in the brine shrimp Artemia franciscana (Kellogg 1906), which are generated by the combination of alternative promoters, alternative splicing and alternative polyadenylation. The two transcripts exhibited sex-biased enrichment, which we termed AfrDsxM and AfrDsxF. They share a common region which encodes an identical N-terminal DNA-binding (DM) domain. RT-qPCR analyses showed that AfrDsxM is dominantly expressed in male Artemia while AfrDsxF is specifically expressed in females. Expression levels of both isoforms increased along with the developmental stages of their respective sexes. RNA interference with dsRNA showed that the knockdown of AfrDsxM in male larvae led to the appearance of female traits including an ovary-like structure in the original male reproductive system and an elevated expression of vitellogenin. However, silencing of AfrDsxF induced no clear phenotypic change in female Artemia. These results indicated that the male AfrDSXM may act as inhibiting regulator upon the default female developmental mode in Artemia. Furthermore, electrophoretic mobility shift assay analyses revealed that the unique DM domain of AfrDSXs can specifically bind to promoter segments of potential downstream target genes like AfrVtg. These data show that AfrDSXs play crucial roles in regulating sexual development in Artemia, and further provide insight into the evolution of sex determination/differentiation in sexual organisms.

Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms.

During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers. Small molecule nonsense suppressors, known as TRIDs (translational read-through-inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously [1], we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell's protein synthesis machinery. Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near-cognate tRNA ± TRIDs.

Growth, survival, and myogenic gene expression in the post-larvae of Colossoma macropomum provisioned with Artemia nauplii.

Artemia nauplii are widely used as fish larvae feed due to its beneficial nutritional characteristics for larval development; however, efficient feeding strategies are needed to balance its high costs. Therefore, we evaluated the effects of different densities of Artemia nauplii (100, 250, 500, 750, and 1000 nauplii/post-larvae) on the growth, survival, water quality, and myogenic gene expression of tambaqui (Colossoma macropomum) post-larvae cultivated in a recirculating aquaculture system. After 2 weeks of trial, there was a significant decrease in dissolved oxygen concentration with the increase in nauplii density, but it did not interfere with larval performance and survival. In the first week, larvae fed with fewer than 500 nauplii/post-larvae presented slower growth, while in the second week, larvae fed with 1000 nauplii/post-larvae had the highest final weight and length. Regression analysis suggests that the optimum feeding density of Artemia nauplii during the first week is 411 nauplii/post-larvae, while for the second week, the growth increased proportionally to the feeding densities. The relative expression of the myod, myog, and mstn genes was higher in larvae fed with fewer than 500 nauplii/post-larvae. Although low-growing larvae showed increased expression of myod and myog genes, responsible for muscle hyperplasia and hypertrophy, respectively, mstn expression may have played a significant inhibitory role in larval development. Further research is needed to better determine the effects of the live food on the zootechnical performance and the expression of the myogenic genes in the initial phase of the life cycle of the tambaqui post-larvae.

Expression of Abdominal-B in the brine shrimp, Artemia franciscana, expands our evolutionary understanding of the crustacean abdomen.

The brine shrimp, Artemia franciscana, has a body plan composed of 11 thoracic segments, followed by 2 genital segments, and then 6 additional abdominal segments. Previous studies of Artemia reported that expression of the posterior-most Hox gene, Abdominal-B (Abd-B), is restricted to the genital segments and is not observed posteriorly in the abdomen at any developmental stage. This report was remarkable because it suggested that the Artemia abdomen posterior to the genital segments was a novel body region of 6 segments that bore no homology to any region in other crustaceans and was unique amongst arthropods in being a Hox-free segmented domain outside of the head. In this study, we used RT-PCR, antibody staining, and in situ hybridization on various stages of Artemia nauplii to show that Abd-B mRNA and protein are in fact expressed throughout the abdominal segments during Artemia development, but this expression later retracts to the two genital segments (G1, G2) and the T11 appendages. This suggests that Abd-B does play a role in specifying abdominal segment identity in all crustaceans that have been examined and suggests a common evolutionary origin for the crustacean abdomen.

Prolonged ecological changes can affect morphometrics and gene expression profile? Focusing on Hsp-70 and NLHS-induced Hsp-70 of Artemia urmiana.

BACKGROUND: In recent years, many aquatic ecosystems, including Urmia Lake, have undergone severe ecological tensions. This lake, the largest natural habitat of the brine shrimp Artemia urmiana, has progressively desiccated and its salinity has dramatically increased over the last three decades. In the face of the long period environmental stresses, understanding the adaptation and ecological plasticity mechanisms is the most interesting challenges in genetic and applied ecology. These mechanisms may probably be driven by inducing expression of some genes involved in adaptation such as Hsp-70 and also adjusting morphological parameters. But they are yet to be understood. Hence, the present work aimed to study the mechanisms, along with testing the hypothesis that non-lethal heat shocked nauplii originating from drought period can evoke Hsp-70 expression more than those from rainy period. METHODS: This study measured and analyzed morphometrical characters of adult male and female Artemia urmiana over three decades. Then, the influence of three-decade ecological crisis on Hsp-70 and non-lethal heat shock (NLHS)-induced Hsp-70 expression levels of nauplii of Artemia urmiana habiting Urmia Lake using Real-time PCR technique, based on cyst collections in 1994 (rainy period) to 2020 (drought period), was evaluated. RESULTS: The morphometrics results showed that the morphological characters were significantly shrunk in 2020 compared to 1994 (CI 95%, p < 0.05). Furthermore, our results depicted that, Hsp-70 expression level was significantly upregulated in response to the prolonged ecological crisis, (CI 95%, P < 0.0001), and also interestingly, the nauplii exposed to longe-term ecological crisis (belong to 2020) were able to increase Hsp-70 expression more than other ones in response to environmental stressors including heat. CONCLUSIONS: The present results showed the involvement of Hsp-70 in the adaptation of Artemia urmiana to long term ecological alteration at the cost of shrinking morphometric parameters.

The Origin of Asexual Brine Shrimps.

AbstractDetermining how and how often asexual lineages emerge within sexual species is central to our understanding of sex-asex transitions and the long-term maintenance of sex. Asexuality can arise "by transmission" from an existing asexual lineage to a new one through different types of crosses. The occurrence of these crosses, cryptic sex, variations in ploidy, and recombination within asexuals greatly complicates the study of sex-asex transitions, as they preclude the use of standard phylogenetic methods and genetic distance metrics. In this study we show how to overcome these challenges by developing new approaches to investigate the origin of the various asexual lineages of the brine shrimp Artemia parthenogenetica. We use a large sample of asexuals, including all known polyploids, and their sexual relatives. We combine flow cytometry with mitochondrial and nuclear DNA data. We develop new genetic distance measures and methods to compare various scenarios describing the origin of the different lineages. We find that all diploid and polyploid A. parthenogenetica likely arose within the past 80,000 years through successive and nested hybridization events that involved backcrosses with different sexual species. All A. parthenogenetica have the same common ancestor and therefore likely carry the same asexuality gene(s) and reproduce by automixis. These findings radically change our view of sex-asex transitions in this group and show the importance of considering scenarios of asexuality by transmission. The methods developed are applicable to many other asexual taxa.

The genome of the extremophile Artemia provides insight into strategies to cope with extreme environments.

BACKGROUND: Brine shrimp Artemia have an unequalled ability to endure extreme salinity and complete anoxia. This study aims to elucidate its strategies to cope with these stressors. RESULTS AND DISCUSSION: Here, we present the genome of an inbred A. franciscana Kellogg, 1906. We identified 21,828 genes of which, under high salinity, 674 genes and under anoxia, 900 genes were differentially expressed (42%, respectively 30% were annotated). Under high salinity, relevant stress genes and pathways included several Heat Shock Protein and Leaf Embryogenesis Abundant genes, as well as the trehalose metabolism. In addition, based on differential gene expression analysis, it can be hypothesized that a high oxidative stress response and endocytosis/exocytosis are potential salt management strategies, in addition to the expression of major facilitator superfamily genes responsible for transmembrane ion transport. Under anoxia, genes involved in mitochondrial function, mTOR signalling and autophagy were differentially expressed. Both high salt and anoxia enhanced degradation of erroneous proteins and protein chaperoning. Compared with other branchiopod genomes, Artemia had 0.03% contracted and 6% expanded orthogroups, in which 14% of the genes were differentially expressed under high salinity or anoxia. One phospholipase D gene family, shown to be important in plant stress response, was uniquely present in both extremophiles Artemia and the tardigrade Hypsibius dujardini, yet not differentially expressed under the described experimental conditions. CONCLUSIONS: A relatively complete genome of Artemia was assembled, annotated and analysed, facilitating research on its extremophile features, and providing a reference sequence for crustacean research.

Transitions to asexuality and evolution of gene expression in Artemia brine shrimp.

While sexual reproduction is widespread among many taxa, asexual lineages have repeatedly evolved from sexual ancestors. Despite extensive research on the evolution of sex, it is still unclear whether this switch represents a major transition requiring major molecular reorganization, and how convergent the changes involved are. In this study, we investigated the phylogenetic relationship and patterns of gene expression of sexual and asexual lineages of Eurasian Artemia brine shrimp, to assess how gene expression patterns are affected by the transition to asexuality. We find only a few genes that are consistently associated with the evolution of asexuality, suggesting that this shift may not require an extensive overhauling of the meiotic machinery. While genes with sex-biased expression have high rates of expression divergence within Eurasian Artemia, neither female- nor male-biased genes appear to show unusual evolutionary patterns after sexuality is lost, contrary to theoretical expectations.

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.

The chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) controls cellular quiescence by hyperpolarizing the cell membrane during diapause in the crustacean Artemia.

Cellular quiescence, a reversible state in which growth, proliferation, and other cellular activities are arrested, is important for self-renewal, differentiation, development, regeneration, and stress resistance. However, the physiological mechanisms underlying cellular quiescence remain largely unknown. In the present study, we used embryos of the crustacean Artemia in the diapause stage, in which these embryos remain quiescent for prolonged periods, as a model to explore the relationship between cell-membrane potential (Vmem) and quiescence. We found that Vmem is hyperpolarized and that the intracellular chloride concentration is high in diapause embryos, whereas Vmem is depolarized and intracellular chloride concentration is reduced in postdiapause embryos and during further embryonic development. We identified and characterized the chloride ion channel protein cystic fibrosis transmembrane conductance regulator (CFTR) of Artemia (Ar-CFTR) and found that its expression is silenced in quiescent cells of Artemia diapause embryos but remains constant in all other embryonic stages. Ar-CFTR knockdown and GlyH-101-mediated chemical inhibition of Ar-CFTR produced diapause embryos having a high Vmem and intracellular chloride concentration, whereas control Artemia embryos released free-swimming nauplius larvae. Transcriptome analysis of embryos at different developmental stages revealed that proliferation, differentiation, and metabolism are suppressed in diapause embryos and restored in postdiapause embryos. Combined with RNA sequencing (RNA-Seq) of GlyH-101-treated MCF-7 breast cancer cells, these analyses revealed that CFTR inhibition down-regulates the Wnt and Aurora Kinase A (AURKA) signaling pathways and up-regulates the p53 signaling pathway. Our findings provide insight into CFTR-mediated regulation of cellular quiescence and Vmem in the Artemia model.

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.

DEK terminates diapause by activation of quiescent cells in the crustacean Artemia.

To cope with harsh environments, the Artemia shrimp produces gastrula embryos in diapause, a state of obligate dormancy, having cellular quiescence and suppressed metabolism. The mechanism behind these cellular events remains largely unknown. Here, we study the regulation of cell quiescence using diapause embryos of Artemia We found that Artemia DEK (Ar-DEK), a nuclear factor protein, was down-regulated in the quiescent cells of diapause embryos and enriched in the activated cells of post-diapause embryos. Knockdown of Ar-DEK induced the production of diapause embryos whereas the control Artemia released free-swimming nuaplii. Our results indicate that Ar-DEK correlated with the termination of cellular quiescence via the increase in euchromatin and decrease in heterochromatin. The phenomena of quiescence have many implications beyond shrimp ecology. In cancer cells, for example, knockdown of DEK also induced a short period of cellular quiescence and increased resistance to environmental stress in MCF-7 and MKN45 cancer cell lines. Analysis of RNA sequences in Artemia and in MCF-7 revealed that the Wnt and AURKA signaling pathways were all down-regulated and the p53 signaling pathway was up-regulated upon inhibition of DEK expression. Our results provide insight into the functions of Ar-DEK in the activation of cellular quiescence during diapause formation in Artemia.

Activin A affects feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish (Danio rerio) larvae.

Activin A belongs to the superfamily of transforming growth factor-ß and plays an important role in hormone regulation and tissue development. However, few research studies have been conducted on the effect of activin A on feeding organs in fish. In this study, the zebrafish (Danio rerio) larvae were treated with 1 ng ml-1 activin A for 8 days continuously. The haematoxylin and eosin (H&E) staining section results revealed that the transverse inner diameter of the pharynx and oesophagus significantly increased on the third and eighth days after treatment compared with the control group (P < 0.05). On the eighth day, the cross-sectional area of the pharyngeal muscle increased by 8638 µm2 compared to the control group (P < 0.05). The RNA in situ hybridization results also showed that the expression of skeletal muscle-specific genes (myog and myod) was significantly increased in pharyngeal muscle on the eighth day. Furthermore, the qRT-PCR results showed the expression of gh gene was significantly increased on the eighth day (P < 0.05). At the same time, more larvae in activin A group were able to feed larger brine shrimp (Artemia) than in the control group on the eighth day. In conclusion, activin A could affect feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish larvae. This study is the first to report that the development of the pharynx and oesophagus can directly affect food intake in fish larvae, which provides a theoretical basis for the study of food intake of fish at an early stage.

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.

ArHsp40 and ArHsp40-2 contribute to stress tolerance and longevity in Artemia franciscana, but only ArHsp40 influences diapause entry.

Embryos of the crustacean Artemia franciscana develop either ovoviviparously or oviparously, yielding swimming larvae (nauplii) or encysted gastrulae (cysts), respectively. Nauplii moult several times and become adults whereas cysts enter diapause, a state of dormancy characterized by exceptionally low metabolism and high stress tolerance. Synthesis of molecular chaperones such as the J-domain proteins ArHsp40 and ArHsp40-2 occurs during embryo development and post-diapause growth of A. franciscana and they influence development and stress tolerance. To further investigate J-domain protein function, ArHsp40 and ArHsp40-2 were each knocked down by RNA interference. Reductions in ArHsp40 and ArHsp40-2 had no effect on adult survival, time to release of cysts and nauplii from females and first-brood size. However, knockdown of both A. franciscana J-domain proteins reduced the longevity and heat tolerance of nauplii, with the loss of ArHsp40 having a greater effect. The knockdown of ArHsp40, but not of ArHsp40-2, caused approximately 50% of cysts to abort diapause entry and hatch without exposure to an exogenous signal such as low temperature and/or desiccation. Cysts lacking ArHsp40 that entered diapause exhibited decreased stress tolerance as did cysts with reduced ArHsp40-2, the latter to a lesser degree. The longevity of nauplii hatching prematurely from cysts was less than for nauplii arising by other means. The results expand our understanding of Hsp40 function in A. franciscana stress tolerance and development, especially during diapause, and they provide the first example of a molecular chaperone that influences diapause entry.

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.

The Potential Roles of the Apoptosis-Related Protein PDRG1 in Diapause Embryo Restarting of Artemia sinica.

High salinity and low temperatures can induce Artemia sinica to enter the diapause stage during embryonic development. Diapause embryos stop at the gastrula stage, allowing them to resist apoptosis and regulate cell cycle activity to guarantee normal development after diapause termination. P53 and DNA damage-regulated gene 1 (pdrg1) is involved in cellular physiological activities, such as apoptosis, DNA damage repair, cell cycle regulation, and promotion of programmed cell death. However, the role of pdrg1 in diapause and diapause termination in A. sinica remains unknown. Here, the full-length A. sinica pdrg1 cDNA (As-pdrg1) was obtained and found to contain 1119 nucleotides, including a 228 bp open reading frame (ORF), a 233 bp 5'-untranslated region (UTR), and a 658-bp 3'-UTR, which encodes a 75 amino acid protein. In situ hybridization showed no tissue specific expression of As-pdrg1. Quantitative real-time PCR and western blotting analyses of As-pdrg1 gene and protein expression showed high levels at 15-20 h of embryo development and a subsequent downward trend. Low temperatures upregulated As-pdrg1 expression. RNA interference for the pdrg1 gene in Artemia embryos caused significant developmental hysteresis. Thus, PDRG1 plays an important role in diapause termination and cell cycle regulation in early embryonic development of A. sinica.