Artemia Embryos Research Articles

Decapsulation of artemia cysts (Artemia sp.) using different versions of sodium hypochlorite solutions (NaOCL)

The article describes the technology of Artemia decapsulation, its features and external factors affecting the efficiency of hatching with subsequent use as a feed composition. Artemia nauplii and its eggs, due to their high nutritional value and high digestibility in the digestive tract, are widely used in growing sturgeon larvae and other fish species. Artemia decapsulation is one of the most important factors in the transition of fish larvae to active feeding, in this regard, it is very important to select the optimal decapsulation conditions, the composition of chemical reagents, temperature and light conditions. The decapsulation process is a chemical process of removing the hard shell that surrounds the Artemia embryo. The procedure involves hydration, during which the outer shell is removed, can be carried out with a number of solutions: sodium hydroxide, sodium carbonate, sodium hypochlorite, hydrochloric acid. However, they all show different availability and efficiency in decapsulation. The most common is the use of sodium hypochlorite solutions, but they do not show stable results, since the process of decapsulation and hatching of nauplii from cysts depends on many factors. The aim of the work was to study the methods of decapsulation of Artemia eggs for their use as a starter feed when transferring to active feeding and feeding of sturgeon larvae. As a result of the experiments, a technology for incubating Artemia eggs in accessible conditions without the use of professional equipment was developed. Cysts are incubated in aerated Weiss apparatuses, under constant illumination, in a volume of 7 liters of water, at a concentration of: NaCl — 30%, artemia cysts 3 g/l, water temperature — 28 °С. The cysts are decapsulated without soaking, with 2.5% NaOCl solution with a total volume of 300 ml (21 g of dry cysts + 150 ml of water + 150 ml of NaOCl) for 4–5 minutes.

Downregulation of a CT10 regulator of kinase (Crk) promotes the formation of diapause embryos in the brine shrimp Artemia

To survive under harsh environments, embryonic development of Artemia was arrested at the gastrula stage and released as the diapause embryo. Cell cycle and metabolism were highly suppressed in this state of quiescence. However, cellular mechanisms underlying diapause remain largely unclear. In this study, we found that the expression level of a CT10 regulator of kinase-encoding gene (Ar-Crk) in diapause embryos was significantly lower than non-diapause embryos at the early embryogenetic stage of Artemia. Knockdown of Ar-Crk by RNA interference induced formation of diapause embryos, while the control group produced nauplii. Western blot analysis and metabolic assays revealed that the diapause embryos produced by Ar-Crk-knocked-down Artemia had similar characteristics of diapause markers, arrested cell cycle, and suppressed metabolism with those diapause embryos produced by natural oviparous Artemia. Transcriptomic analysis of Artemia embryos revealed knockdown of Ar-Crk induced downregulation of the aurora kinase A (AURKA) signaling pathway, as well as energetic and biomolecular metabolisms. Taken together, we proposed that Ar-Crk is a crucial factor in determining the process of diapause in Artemia. Our results provide insight into the functions of Crk in fundamental regulations such as cellular quiescence.

Trends and New Developments in Artemia Research.

Simple SummaryArtemia is an important crustacean group, especially for aquaculture live food and as a model organism for toxicity assessment. The present study aimed to identify the current trends, research gaps, and literature development in the study of Artemia around the world. This primitive Arthropod has undergone significant evolution in terms of its application in various industries as well as relevant literature patterns in terms of scientometric analyses. An increasing number of scientists since 1970 has examined Artemia as an important species in aquaculture-related fields. However, a global scientometric review of Artemia literature is still lacking, which is the objective of this research. Using a CiteSpace analysis, the distribution of core authors and institutions, highly cited keywords and papers, author and journal contributions, and hot topics in the literature, as well as a co-citation analysis, particularly regarding authors, journals, documents, and clusters, were determined. Hence, 8741 relevant publications were generated from the Web of Science Core Collection database. The results revealed that the most significant contributions in Artemia research primarily originated from the USA, Brazil, Spain, India, China, and Belgium. Moreover, Artemia research focused mainly on top keywords such as brine shrimp and antimicrobial activity. Emerging trends related to Artemia research were Atlantic halibut, elongation factor, Artemia salina, lean protein, inert diet, alpha-crystallin protein, and Artemia embryo. At the same time, the study generated a vast total of 45 co-citation clusters. The present study provides the existing body of knowledge on Artemia research by sharing a visual knowledge map. This study offers a valuable perspective and profound understanding for researchers, farmers, and consortia interested in promoting Artemia as a sustainable live food in the global aquaculture industry.

Stable primary embryonic cells of Artemia are suitable for tracing the process of V. anguillarum and V. parahaemolyticus infection

The cell line is an important experimental tool to investigate the mechanism of host-pathogen interactions and to diagnose and cure diseases. However, their application in aquatic invertebrates is limited because they lack immortalized cell lines. The brine shrimp Artemia produces encysted diapause gastrula embryos under stress conditions. The biological advantages of Artemia embryos, such as quick switching on of the cell cycle upon short-term hydration and easy control of microbial contamination via decapsulation, makes them a candidate material for cell culture in vitro. This study identified five subpopulations of embryonic cells at the gastrula stage based on morphological characteristics. After 3–4 h of hydration, embryonic cells were derived from the cysts and successfully cultured for 10 days in 1.2 × L-15 medium containing 15% fetal bovine serum, with a cell viability of more than 90%. Using this primary cell culture system, Vibrio anguillarum and V. parahaemolyticus strains were screened as possible infectious Vibrio strains using laser confocal microscopy observations. The pathological features of viable Vibrio-challenged embryonic cells were further studied by comparing formaldehyde-inactivated and autoclaved Vibrio and polystyrene beads using SEM observation. The results confirmed that both Vibrio strains could challenge embryonic cells and cause some cytopathological features, such as cell aggregation and concaves. Our study provides the first evidence that in vitro cell culture of Artemia embryos can be used to demonstrate the characteristics of Vibrio infection. The primary cell culture system established in this study will facilitate research on the mechanism of host-pathogen interactions in aquatic animals.

Внутри- и межпопуляционная изменчивость цист и взрослых стадий артемии (Branchiopoda: Anostraca) в сибирских популяциях (морфометрия)

Размеры цист артемии являются важным показателем ценности их как кормового ресурса и в некоторой степени позволяют идентифицировать популяции. В статье проанализированы показатели цист артемии партеногенетических популяций (диаметр, толщина хориона, наличие пятен на оболочке), отобранных в гипергалинных озёрах Западной Сибири в разные годы, и морфометрические показатели рачков, выращенных из цист при одинаковой солёности. Установлена значительная внутри- и межпопуляционная изменчивость рассмотренных показателей. Абсолютные значения диаметра цист находились в пределах 210–330 мкм, средние значения по пробам — 243,5–282,9 мкм, средние по популяциям — 257,8–279,6 мкм; абсолютные значения диаметра декапсулированных цист — в пределах 196–294 мкм, средние значения по пробам — 236,5–262,6 мкм, средние по популяциям — 239,9–253,2 мкм; абсолютные значения толщины хориона цист — 3,3–16,9 мкм, средние значения по популяциям — 6,6–12,4 мкм. В основных промысловых озёрах, на которые приходится около 70 % от всего вылова цист артемии в России, цисты имели близкие среднепопуляционные размеры (262–268 мкм). Дано заключение об отсутствии внутрипопуляционной закреплённости таких признаков, как диаметр цист и толщина хориона, то есть они не могут служить надёжными показателями, идентифицирующими сибирские популяции. Установлена статистически значимая связь (r = −0,5) между солёностью материнского водоёма и диаметром эмбрионов артемии. Пятнистость цист, не превышающая 5 % почти во всех их пробах, у цист озера Кучукское составила 24 %. Анализ морфометрических показателей рачков, выращенных из цист, показал, что средняя длина рачков (9,27–11,63 мм), ширина абдомена (0,53–0,69 мм) и расстояние между глазами (1,36–1,52 мм) тесно коррелировали с солёностью материнского водоёма (значения r составили −0,76; −0,62; −0,67 соответственно). Кластерный анализ совокупности морфометрических признаков рачков указывает на объединение популяций по признаку солёности.

Embryonic cuticle from artemia cyst shell displays amyloid-like characteristics and nontoxicity after oral consumption

Artemia cysts are the essential food product for industrial larviculture of fishes. The cyst shell protects the artemia embryo from mechanical damage, ultraviolet light, excessive water loss, thermal variation and anoxia condition. However, the underlying mechanism of such environmental protection is largely unclear. The embryonic cuticle of cyst shell mainly constitutes chitin and proteins. Absence of cyst shell proteins compromises embryo survival. In literature, there are few examples of functional amyloids where proteins adapt amyloid-like structures and act as protective covering. We hypothesized that the proteins from the embryonic cuticle of artemia cyst shell may have amyloid-like properties. Using FTIR and CD analysis, we found that proteins in embryonic cuticle have high β-sheet secondary structures. Embryonic cuticles displayed high Congo red binding affinity and stained samples showed apple-green birefringence under polarized light, confirming the presence of amyloid-like structures. Amyloid structures have a tendency to propagate and cause amyloidosis. However, feeding of amyloid rich embryonic cuticles to zebrafish did not show any signs of discomfort or morbidity and amyloid deposition. Taken together, the study reveals that amyloid-like structures are present in embryonic cuticle of artemia cyst and their consumption does not induce amyloidosis in zebrafish.

Stress-dependent conformational changes of artemin: Effects of heat and oxidant.

Artemin is an abundant thermostable protein in Artemia embryos and it is considered as a highly efficient molecular chaperone against extreme environmental stress conditions. The conformational dynamics of artemin have been suggested to play a critical role in its biological functions. In this study, we have investigated the conformational and functional changes of artemin under heat and oxidative stresses to identify the relationship between its structure and function. The tertiary and quaternary structures of artemin were evaluated by fluorescence measurements, protein cross-linking analysis, and dynamic light scattering. Based on the structural analysis, artemin showed irreversible substantial conformational lability in responses to heat and oxidant, which was mainly mediated through the hydrophobic interactions and dimerization of the chaperone. In addition, the chaperone-like activity of heated and oxidized artemin was examined using lysozyme refolding assay and the results showed that although both factors, i.e. heat and oxidant, at specific levels improved artemin potency, simultaneous incubation with both stressors significantly triggered the chaperone activation. Moreover, the heat-induced dimerization of artemin was found to be the most critical factor for its activation. It was suggested that oxidation presumably acts through stabilizing the dimer structures of artemin through formation of disulfide bridges between the subunits and strengthens its chaperoning efficacy. Accordingly, it is proposed that artemin probably exists in a monomer-oligomer equilibrium in Artemia cysts and environmental stresses and intracellular portion of protein substrates may shift the equilibrium towards the active dimer forms of the chaperone.

Probing heat and oxidation induced conformational changes of molecular chaperone artemin by excitation-emission fluorescence spectroscopy

Artemin is a potent molecular chaperone, which protects Artemia embryos undergoing encystment against extreme environmental stresses. In the present work, we have examined the structural changes of artemin from A. urmiana upon exposure to oxidant and heat, by using CD measurements as well as excitation-emission fluorescence spectroscopy as a powerful tool for monitoring the conformational transitions and molecular interactions in proteins. We have also provided here the first document on reporting the three dimensional fluorescence spectra of a protein using ANS. Totally, the fluorescence results indicated that the microenvironments of tyrosine and tryptophan residues and the hydrophobic pockets as well as the polypeptide backbone or secondary structure of the chaperone were influenced in responses to heat and H2O2 in different degrees. Moreover, the native state of artemin did not induce a considerable exposure of the internal non-polar groups to the solvent. Besides, the excitation-emission spectra of heated artemin by ANS revealed new emission peaks at 430–450 nm when it was excited at 330 nm, which suggests probable exposure of new binding sites for hydrophobic or electrostatic interactions of the protein with ANS. The protein also showed a greater conformational sensitivity to the temperature fluctuations compared to oxidation. Here, we presented some evidence in support of the relation between artemin and its stress dependent activation in vitro and in vivo. This study can expect that the EEM fluorescence spectroscopy could provide a promising tool to study conformational transitions of proteins.

Cyst viability and stress tolerance upon heat shock protein 70 knockdown in the brine shrimp Artemia franciscana.

Females of the brine shrimp Artemia franciscana produce either free-swimming nauplii via ovoviviparous pathway of reproduction or encysted embryos, known as cysts, via oviparous pathway, in which biological processes are arrested. While previous study has shown a crucial role of ATP-dependent molecular chaperone, heat shock protein 70 (Hsp70) in protecting A. franciscana nauplii against various abiotic and abiotic stressors, the function of this protein in diapausing embryos and cyst development, however, remains unknown. RNA interference (RNAi) was applied in this study to examine the role of Hsp70 in cyst development and stress tolerance, with the latter performed by desiccation and freezing, a common method used for diapause termination in Artemia cysts. Hsp70 knockdown was apparent in cysts released from females that were injected with Hsp70 dsRNA. The loss of Hsp70 affected neither the development nor morphology of the cysts. The time between fertilization and cyst release from Artemia females injected with Hsp70 dsRNA was delayed slightly, but the differences were not significant when compared to the controls. However, the hatching percentage of cysts which lacks Hsp70 were reduced following desiccation and freezing. Taken together, these results indicated that Hsp70 possibly plays a role in the stress tolerance but not in the development of diapause-destined embryos of Artemia. This research makes fundamental contributions to our understanding of the role molecular chaperone Hsp70 plays in Artemia, an excellent model organism for diapause studies of the crustaceans.

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.

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.

An inter-subunit disulfide bond of artemin acts as a redox switch for its chaperone-like activity.

Encysted embryos of Artemia are among the most stress-resistant eukaryotes partly due to the massive amount of a cysteine-rich protein termed artemin. High number of cysteine residues in artemin and their intramolecular spatial positions motivated us to investigate the role of the cysteine residues in the chaperone-like activity of artemin. According to the result of Ellman's assay, there are nine free thiols (seven buried and two exposed) and one disulfide bond per monomer of artemin. Subsequent theoretical analysis of the predicted 3D structure of artemin confirmed the data obtained by the spectroscopic study. Native and reduced/modified forms of artemin were also compared with respect to their efficiency in chaperoning activity, tertiary structure, and stability. Since the alkylation and reduction of artemin diminished its chaperone activity, it appears that its chaperoning potential depends on the formation of intermolecular disulfide bond and the presence of cysteine residues. Comparative fluorescence studies on the structure and stability of the native and reduced protein revealed some differences between them. Due to the redox-dependent functional switching of artemin from the less to more active form, it can be finally suggested as a redox-dependent chaperone.

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.

An La-related protein controls cell cycle arrest by nuclear retrograde transport of tRNAs during diapause formation in Artemia.

BackgroundIn eukaryotes, tRNA trafficking between the nucleus and cytoplasm is a complex process connected with cell cycle regulation. Such trafficking is therefore of fundamental importance in cell biology, and disruption of this process has grave consequences for cell viability and survival. To cope with harsh habitats, Artemia has evolved a special reproductive mode to release encysted embryos in which cell division can be maintained in a dormancy state for a long period.ResultsUsing Artemia as a peculiar model of the cell cycle, an La-related protein from Artemia, named Ar-Larp, was found to bind to tRNA and accumulate in the nucleus, leading to cell cycle arrest and controlling the onset of diapause formation in Artemia. Furthermore, exogenous gene expression of Ar-Larp could induce cell cycle arrest in cancer cells and suppress tumor growth in a xenograft mouse model, similar to the results obtained in diapause embryos of Artemia. Our study of tRNA trafficking indicated that Ar-Larp controls cell cycle arrest by binding to tRNAs and influencing their retrograde movement from the cytoplasm to the nucleus, which is connected to pathways involved in cell cycle checkpoints.ConclusionsThese findings in Artemia offer new insights into the mechanism underlying cell cycle arrest regulation, as well as providing a potentially novel approach to study tRNA retrograde movement from the cytoplasm to the nucleus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0239-4) contains supplementary material, which is available to authorized users.

Stress tolerance during diapause and quiescence of the brine shrimp, Artemia.

Oviparously developing embryos of the brine shrimp, Artemia, arrest at gastrulation and are released from females as cysts before entering diapause, a state of dormancy and stress tolerance. Diapause is terminated by an external signal, and growth resumes if conditions are permissible. However, if circumstances are unfavorable, cysts enter quiescence, a dormant stage that continues as long as adverse conditions persist. Artemia embryos in diapause and quiescence are remarkably resistant to environmental and physiological stressors, withstanding desiccation, cold, heat, oxidation, ultraviolet radiation, and years of anoxia at ambient temperature when fully hydrated. Cysts have adapted to stress in several ways; they are surrounded by a rigid cell wall impermeable to most chemical compounds and which functions as a shield against ultraviolet radiation. Artemia cysts contain large amounts of trehalose, a non-reducing sugar thought to preserve membranes and proteins during desiccation by replacing water molecules and/or contributing to vitrification. Late embryogenesis abundant proteins similar to those in seeds and other anhydrobiotic organisms are found in cysts, and they safeguard cell organelles and proteins during desiccation. Artemia cysts contain abundant amounts of p26, a small heat shock protein, and artemin, a ferritin homologue, both ATP-independent molecular chaperones important in stress tolerance. The evidence provided in this review supports the conclusion that it is the interplay of these protective elements that make Artemia one of the most stress tolerant of all metazoan organisms.

A novel model of early development in the brine shrimp, Artemia franciscana, and its use in assessing the effects of environmental variables on development, emergence, and hatching.

The brine shrimp, Artemia (Crustacea, Anostraca), is a zooplankton that is commonly used in both basic and applied research. Unfortunately, Artemia embryos are often cultured under conditions that alter early development, and reports based on these cultures oversimplify or fail to describe morphological phenotypes. This is due in part to the lack of a comprehensive developmental model that is applicable to observations of live specimens. The objective of this study was to build and test a descriptive model of post-diapause development in Artemia franciscana using observations made with a standard dissecting microscope. The working model presented is the first to comprehensively place all known "abnormal" embryonic and naupliar phenotypes within the context of a classic hatching profile. Contrary to previous reports, embryos and nauplii with aberrant phenotypes often recover and develop normally. Oval prenauplii may emerge as normal prenauplii (E2 stage). A delay of this transition leads to incomplete hatching or direct hatching of first instar larvae with a curved thoracoabdomen. When hatching is incomplete, retained cuticular remnants are shed during the next molt, and a "normal" second instar larva is produced. By differentiating between molting events and gross embryonic patterning in live embryos, this new model facilitates fine time-scale analyses of chemical and environmental impacts on early development. A small increase in salinity within what is commonly believed to be a permissive range (20‰-35‰) produced aberrant morphology by delaying emergence without slowing development. A similar effect was observed by decreasing culture density within a range commonly applied in toxicological studies. These findings clearly demonstrate that morphological data from end-point studies are highly dependent on the time points chosen. An alternate assessment method is proposed, and the potential impact of heavy metals, hexachlorobenzene, Mirex, and cis-nonachlor detected in commercial embryos is discussed.

Characterization of PHB1 and its role in mitochondrial maturation and yolk platelet degradation during development of Artemia embryos.

BackgroundTo cope with harsh environments, crustaceans such as Artemia produce diapause gastrula embryos (cysts) with suppressed metabolism. Metabolism and development resume during post-diapause development, but the mechanism behind these cellular events remains largely unknown.Principal FindingOur study investigated the role of prohibitin 1 (PHB1) in metabolic reinitiation during post-diapause development. We found that PHB1 was developmentally regulated via changes in phosphorylation status and localization. Results from RNA interference experiments demonstrated PHB1 to be critical for mitochondrial maturation and yolk degradation during development. In addition, PHB1 was present in yolk platelets, and it underwent ubiquitin-mediated degradation during the proteolysis of yolk protein.Conclusions/SignificancePHB1 has an indispensable role in coordinating mitochondrial maturation and yolk platelet degradation during development in Artemia. This novel function of PHB1 provides new clues to comprehend the roles of PHB1 in metabolism and development.

Regulation of trehalase expression inhibits apoptosis in diapause cysts ofArtemia

Trehalase, which specifically hydrolyses trehalose into glucose, plays an important role in the metabolism of trehalose. Large amounts of trehalose are stored in the diapause encysted embryos (cysts) of Artemia, which are not only vital to their extraordinary stress resistance, but also provide a source of energy for development after diapause is terminated. In the present study, a mechanism for the transcriptional regulation of trehalase was described in Artemia parthenogenetica. A trehalase-associated protein (ArTAP) was identified in Artemia-producing diapause cysts. ArTAP was found to be expressed only in diapause-destined embryos. Further analyses revealed that ArTAP can bind to a specific intronic segment of a trehalase gene. Knockdown of ArTAP by RNAi resulted in the release of cysts with coarse shells in which two chitin-binding proteins were missing. Western blotting showed that the level of trehalase was increased and apoptosis was induced in these ArTAP-knockdown cysts compared with controls. Taken together, these results show that ArTAP is a key regulator of trehalase expression which, in turn, plays an important role in trehalose metabolism during the formation of diapause cysts.

Functional differentiation of small heat shock proteins in diapause‐destined Artemia embryos

Encysted embryos of Artemia franciscana cease development and enter diapause, a state of metabolic suppression and enhanced stress tolerance. The development of diapause-destined Artemia embryos is characterized by the coordinated synthesis of the small heat shock proteins (sHsps) p26, ArHsp21 and ArHsp22, with the latter being stress inducible in adults. The amounts of sHsp mRNA and protein varied in Artemia cysts, suggesting transcriptional and translational regulation. By contrast to p26, knockdown of ArHsp21 by RNA interference had no effect on embryo development. ArHsp21 provided limited protection against stressors such as desiccation and freezing but not heat. ArHsp21 may have a non-essential or unidentified role in cysts. Injection of Artemia adults with amounts of ArHsp22 double-stranded RNA less than those used for other sHsps killed females and males, curtailing the analysis of ArHsp22 function in developing embryos and cysts. The results indicate that diapause-destined Artemia embryos synthesize varying amounts of sHsps as a result of differential gene expression and mRNA translation and also suggest that these sHsps have distinctive functions.

Acetylation of Chromatin-Associated Histone H3 Lysine 56 Inhibits the Development of Encysted Artemia Embryos

BackgroundAs a response to harsh environments, the crustacean artemia produces diapause gastrula embryos (cysts), in which cell division and embryonic development are totally arrested. This dormant state can last for very long periods but be terminated by specific environmental stimuli. Thus, artemia is an ideal model organism in which to study cell cycle arrest and embryonic development.Principal FindingOur study focuses on the roles of H3K56ac in the arrest of cell cycle and development during artemia diapause formation and termination. We found that the level of H3K56ac on chromatin increased during diapause formation, and decreased upon diapause termination, remaining basal level throughout subsequent embryonic development. In both HeLa cells and artemia, blocking the deacetylation with nicotinamide, a histone deacetylase inhibitor, increased the level of H3K56ac on chromatin and induced an artificial cell cycle arrest. Furthermore, we found that this arrest of the cell cycle and development was induced by H3K56ac and dephosphorylation of the checkpoint protein, retinoblastoma protein.Conclusions/SignificanceThese results have revealed the dynamic change in H3K56ac on chromatin during artemia diapause formation and termination. Thus, our findings provide insight into the regulation of cell division during arrest of artemia embryonic development and provide further insight into the functions of H3K56ac.