Integrating ATAC-Seq and RNA-Seq Reveals the Signal Regulation Involved in the Artemia Embryonic Reactivation Process.

Embryonic diapause is a common evolutionary adaptation observed across a wide range of organisms. Artemia is one of the classic animal models for diapause research. The current studies of Artemia diapause mainly focus on the induction and maintenance of the embryonic diapause, with little research on the molecular regulatory mechanism of Artemia embryonic reactivation. The first 5 h after embryonic diapause breaking has been proved to be most important for embryonic reactivation in Artemia. In this work, two high-throughput sequencing methods, ATAC-seq and RNA-seq, were integrated to study the signal regulation process in embryonic reactivation of Artemia at 5 h after diapause breaking. Through the GO and KEGG enrichment analysis of the high-throughput datasets, it was showed that after 5 h of diapause breaking, the metabolism and regulation of Artemia cyst were quite active. Several signal transduction pathways were identified in the embryonic reactivation process, such as G-protein-coupled receptor (GPCR) signaling pathway, cell surface receptor signaling pathway, hormone-mediated signaling pathway, Wnt, Notch, mTOR signaling pathways, etc. It indicates that embryonic reactivation is a complex process regulated by multiple signaling pathways. With the further protein structure analysis and RT-qPCR verification, 11 GPCR genes were identified, in which 5 genes function in the embryonic reactivation stage and the other 6 genes contribute to the diapause stage. The results of this work reveal the signal transduction pathways and GPCRs involved in the embryonic reactivation process of Artemia cysts. These findings offer significant clues for in-depth research on the signal regulatory mechanisms of the embryonic reactivation process and valuable insights into the mechanism of animal embryonic diapause.

A novel group 6 LEA protein from diapause embryos of Artemia franciscana is cytoplasmically localized.

The expression of late embryogenesis abundant (LEA) proteins is one mechanism by which anhydrobiotic organisms survive periods of severe water loss. Artemia franciscana is an animal extremophile that uniquely expresses LEA proteins concurrently from Groups 1, 3, and 6. In this study we examine the subcellular localization of AfrLEA6, a Group 6 LEA protein from embryos of A. franciscana. Immunohistochemistry reveals that AfrLEA6 is located in the cytoplasm of diapause embryos and does not co-localize with nuclei or mitochondria. Due to a trace contaminant arising from chitin-based affinity chromatography during AfrLEA6 purification, the primary antiserum displayed affinities for both AfrLEA6 as well as Artemia chitin-binding proteins. This contaminant (fusion protein of intein plus chitin binding domain) co-migrates with AfrLEA6 during SDS-PAGE. Pre-adsorption of the antiserum with dechorionated embryos was required to remove the non-specific fluorescence in the embryonic cuticular membrane. Results of this study are consistent with the apparent importance of distributing multiple types of LEA proteins across many subcellular locations in anhydrobiotic organisms.

Structural properties and cellular expression of AfrLEA6, a group 6 late embryogenesis abundant protein from embryos of Artemia franciscana.

Late embryogenesis abundant (LEA) proteins are intrinsically disordered proteins (IDPs) commonly found in anhydrobiotic organisms and are frequently correlated with desiccation tolerance. Herein we report new findings on AfrLEA6, a novel group 6 LEA protein from embryos of Artemia franciscana. Assessment of secondary structure in aqueous and dried states with circular dichroism (CD) reveals 89% random coil in the aqueous state, thus supporting classification of AfrLEA6 as an IDP. Removal of water from the protein by drying or exposure to trifluoroethanol (a chemical de-solvating agent) promotes a large gain in secondary structure of AfrLEA6, predominated by α-helix and exhibiting minimal ß-sheet structure. We evaluated the impact of physiological concentrations (up to 400 mM) of the disaccharide trehalose on the folding of LEA proteins in solution. CD spectra for AfrLEA2, AfrLEA3m, and AfrLEA6 are unaffected by this organic solute noted for its ability to drive protein folding. AfrLEA6 exhibits its highest concentration in vivo during embryonic diapause, drops acutely at diapause termination, and then declines during development to undetectable values at the larval stage. Maximum cellular titer of AfrLEA6 was 10-fold lower than for AfrLEA2 or AfrLEA3, both group 3 LEA proteins. Acute termination of diapause with H2O2 (a far more effective terminator than desiccation in this Great Salt Lake, UT, population) fostered a rapid 38% decrease in AfrLEA6 content of embryos. While the ultimate mechanism of diapause termination is unknown, disruption of key macromolecules could initiate physiological signaling events necessary for resumption of development and metabolism.

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.

The synthesis of diapause-specific molecular chaperones in embryos of Artemia franciscana is determined by the quantity and location of heat shock factor 1 (Hsf1).

The crustacean, Artemia franciscana, displays a complex life history in which embryos either arrest development and undertake diapause as cysts or they develop into swimming nauplii. Diapause entry is preceded during embryogenesis by the synthesis of specific molecular chaperones, namely the small heat shock proteins p26, ArHsp21, and ArHsp22, and the ferritin homolog, artemin. Maximal synthesis of diapause-specific molecular chaperones is dependent on the transcription factor, heat shock factor 1 (Hsf1), found in similar amounts in cysts and nauplii newly released from females. This investigation was performed to determine why, if cysts and nauplii contain comparable amounts of Hsf1, only cyst-destined embryos synthesize diapause-specific molecular chaperones. Quantification by qPCR and immunoprobing of Western blots, respectively, demonstrated that hsf1 mRNA and Hsf1 peaked by day 2 post-fertilization in embryos that were developing into cysts and then declined. hsf1 mRNA and Hsf1 were present in nauplii-destined embryos on day 2 post-fertilization, but in much smaller amounts than in cyst-destined embryos, and they increased in quantity until release of nauplii from females. Immunofluorescent staining revealed that the amount of Hsf1 in nuclei was greatest on day 4 post-fertilization in cyst-destined embryos but could not be detected in nuclei of nauplius-destined embryos at this time. The differences in quantity and location of Hsf1 explain why embryos fated to become cysts and eventually enter diapause synthesize p26, ArHsp21, ArHsp22, and artemin, whereas nauplius-destined embryos do not produce these molecular chaperones.

Stress tolerance in diapausing embryos of Artemia franciscana is dependent on heat shock factor 1 (Hsf1).

Embryos of the crustacean, Artemia franciscana, may undergo oviparous development, forming encysted embryos (cysts) that are released from females and enter diapause, a state of suppressed metabolism and greatly enhanced stress tolerance. Diapause-destined embryos of A. franciscana synthesize three small heat shock proteins (sHsps), p26, ArHsp21 and ArHsp22, as well as artemin, a ferritin homologue, all lacking in embryos that develop directly into nauplii. Of these diapause-specific molecular chaperones, p26 and artemin are important contributors to the extraordinary stress tolerance of A. franciscana cysts, but how their synthesis is regulated is unknown. To address this issue, a cDNA for heat shock factor 1 (Hsf1), shown to encode a protein similar to Hsf1 from other organisms, was cloned from A. franciscana. Hsf1 was knocked down by RNA interference (RNAi) in nauplii and cysts of A. franciscana. Nauplii lacking Hsf1 died prematurely upon release from females, showing that this transcription factor is essential to the survival of nauplii. Diapause cysts with diminished amounts of Hsf1 were significantly less stress tolerant than cysts containing normal levels of Hsf1. Moreover, cysts deficient in Hsf1 possessed reduced amounts of p26, ArHsp21, ArHsp22 and artemin, revealing dependence on Hsf1 for expression of their genes and maximum stress tolerance. The results demonstrate an important role for Hsf1, likely in concert with other transcription factors, in the survival and growth of A. franciscana and in the developmentally regulated synthesis of proteins responsible for the stress tolerance of diapausing A. franciscana cysts.

Target and non-target toxicity of fern extracts against mosquito vectors and beneficial aquatic organisms.

Dengue and malaria are significant mosquito-borne diseases that are rapidly spread worldwide, mainly in temperate countries. Pteridophytes were identified to be a significant source of novel mosquitocidal agents. The present research was to explore the eco-friendly larvicides from methanol extracts of ferns, viz., Actiniopteris radiata, Adiantum caudatum, Cheilanthes swartzii, Hemionitis arifolia and Lycopodium clavatum. The larvicidal potential of the extracts screened using larvae of dengue vector Aedes aegypti (III and IV instar) and malarial vector Anopheles stephensi (III and IV instar), showed 10-100% mortality rates. Biosafety assessment was made on embryos of Danio rerio and Artemia nauplii. The phyto-constituents of the methanol extract of A. radiata leaves were identified through gas chromatography-mass spectrometry (GC-MS). Methanolic leaf extracts of A. radiata, A. caudatum and C. swartzii exhibited larvicidal activity against III and IV instar larvae of Ae. aegypti (LC50: 37.47, 74.51 and 152.38 and 67.58, 95.89 and 271.46 ppm) and An. stephensi (LC50: 70.35, 112.12 and 301.05 and 113.83, 175.30 and 315.19 ppm), respectively. The GC-MS of the methanol extract of A. radiata leaves revealed the presence of 7 phyto-components among which, Carbamic acid, phenyl-, (2-Nitrophenyl) methyl ester (1), Benzoic acid, 3- methylbenzoate (2) and 4-(benzylimino)- 1,4-dihydro-1-(p-toluoylmethyl) pyridine (3) were dominant. Biosafety assessment of methanol extract of A. radiata leaves on embryos of Danio rerio (Zebra fish) and Artemia nauplii (micro crustacean) revealed that there were no destructive or teratogenic effects. To conclude, the larvicidal activity and insignificant toxicity to non-target aquatic organisms of A. radiata leaves makes it a potential and environment safe biocontrol agent against dengue and malarial vectors.

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.

Cloning, expression pattern, and potential role of apoptosis inhibitor 5 in the termination of embryonic diapause and early embryo development of Artemia sinica.

During the embryonic development of Artemia sinica, the diapause phenomenon can be induced by high salinity or low temperature conditions. The diapause embryo at the gastrula stage is maintained under the threat of apoptosis to guarantee the embryo's normal development. In this process, apoptosis inhibitor proteins play vital roles in protecting embryos against apoptosis. Apoptosis inhibitor5 (API5) plays a pivotal role in regulating the cell cycle and preventing programmed cell death after growth factor starvation. In the present study, we cloned the full-length cDNA representing the api5 gene from A. sinica (As-api5), which encodes a 372-amino acid protein. In situ hybridization experiments revealed that As-api5 expression is not tissue or organ specific. Quantitative real-time PCR analyses of the developmental expression of As-api5 showed that it reached its highest level at 10h, after which its expression decreased. High salinity and low temperature treatments increased the expression of As-api5. Western blotting was used to assess the abundance of As-API5 and related proteins (As-CyclinA, As-CyclinE, As-E2F1, As-CDK2, As-APAF1, and As-Caspase9). Downregulation of As-api5 expression using a short interfering RNA resulted in increased mortality and embryo malformation of A. sinica. Taken together, the results indicated that API5 plays a crucial role in embryonic diapause termination and early embryo development of A. sinica.