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.

Effects of doping on photocatalytic water splitting activities of PtS2/SnS2 van der Waals heterostructures.

Photocatalytic water splitting is a promising technology to solve serious energy and environmental problems. The PtS2 monolayer has been previously predicted to be a water splitting photocatalyst. But the high efficiency of carrier recombination in the monolayer results in poor photocatalytic performance. It is well known that the construction of van der Waals (vdW) heterojunctions can improve the photocatalytic performance of a monolayer. In this investigation, we constructed a PtS2/SnS2 vdW heterojunction and systematically investigated the influence of the doping position and doping ratio on its performance using density functional theory calculations. Interestingly, the band alignment transforms from Type-II to Type-I and from Type-I to Type-II when the S in SnS2 is replaced with Se in the PtS2/SnS2 vdW heterojunction and the S in PtS2 is replaced with Se in the PtS2/SnSe2 vdW heterojunction, respectively. More importantly, from the PtS2/SnS2 to PtSe2/SnSe2 vdW heterojunction, the decomposition of water also changes from semi-decomposed water to fully decomposed water. Furthermore, the results show that the direct Z-scheme photocatalytic mechanism exists in the PtSSe/SnSe2 vdW heterojunction by analysis of the migration paths of photoinduced electrons and holes. And compared with the PtS2/SnS2, the PtSSe/SnSe2 heterostructure exhibits better photocatalytic water splitting activities. These results can provide a direction that doping can improve the photocatalytic water splitting performance of heterojunction photocatalysts.

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.

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.

Identification and characterization of a symbiotic agglutination-related C-type lectin from the hydrothermal vent shrimp Rimicaris exoculata.

Rimicaris exoculata (Decapoda: Bresiliidae) is one of the dominant species of hydrothermal vent communities, which inside its gill chamber harbors ectosymbioses with taxonomic invariability while compositional flexibility. Several studies have revealed that the establishment of symbiosis can be initiated and selected by innate immunity-related pattern recognition receptors (PRRs), such as C-type lectins (CTLs). In this research, a CTL was identified in R. exoculata (termed RCTL), which showed high expression at both mRNA and protein levels in the scaphognathite, an organ where the ectosymbionts are attached outside its setae. Linear correlationships were observed between the relative quantities of two major symbionts and the expression of RCTL based on analyzing different shrimp individuals. The recombinant protein of RCTL could recognize and agglutinate the cultivable γ-proteobacterium of Escherichia coli in a Ca2+-dependent manner, obeying a dose-dependent and time-cumulative pattern. Unlike conventional crustacean CTLs, the involvement of RCTL could not affect the bacterial growth, which is a key issue for the successful establishment of symbiosis. These results implied that RCTL might play a critical role in symbiotic recognition and attachment to R. exoculata. It also provides insights to understand how R. exoculata adapted to such a chemosynthesis-based environment.

An H-ferritin from the hydrothermal vent shrimp Rimicaris exoculata and its potential role in iron metabolism.

Rimicaris exoculata (Decapoda: Bresiliidae) is one of the dominant species among hydrothermal vent communities along the Mid-Atlantic Ridge. This shrimp can tolerate high concentrations of heavy metals such as iron, but the mechanisms used for detoxification and utilization of excess metals remain largely unknown. Ferritin is a major iron storage protein in most living organisms. The central heavy subunit of ferritin (H-ferritin) possesses ferroxidase activity and converts iron from Fe2+ to Fe3+, the non-toxic form used for storage. In the present study, the H-ferritin RexFrtH was identified in the hydrothermal vent shrimp R. exoculata, and found to be highly expressed in the gill, the main organ involved in bioaccumulation of metals, at both RNA and protein levels. Accumulation of RexFrtH decreased from efferent to afferent vessels, coinciding with the direction of water flow through the gills. Fe3+ was localized with RexFrtH, and in vitro iron-binding and ferroxidase assays using recombinant RexFrtH confirmed the high affinity for iron. Based on these results, we propose a model of iron metabolism in R. exoculata gills; ferrous iron from ambient hydrothermal water accumulates and is converted and stored in ferric form by RexFrtH as an iron reservoir when needed for metabolism, or excreted as an intermediate to prevent iron overload. The findings expand our understanding of the adaptation strategies used by shrimps inhabiting extreme hydrothermal vents to cope with extremely high heavy metal concentrations.

[Transcriptome analysis reveals candidate genes involved in flavonoid biosynthesis in Ziziphora bungeana].

Ziziphora bungeana is a kind of medicinal plants belongs to Labiatae,and it also a kind of geoherbs in Xinjiang. The main active ingredient linarin has a higher content in inflorescence than in other parts. In this study,high-throughput sequencing technology was used to reveal the transcriptome of the inflorescence of Z. bungeana,77 366 unigenes were acquired,of which 56 375 unigenes were annotated based on search of the database and classification. Through the analysis of metabolic pathways,sixty unigenes were probably encoding some enzymes involved in the flavonoid biosynthesis pathways. The contents of linarin in different parts were determined and the key genes were verified by qRT-PCR. The discovery provides the research basis for further analysis of the enzyme genes involved in the biosynthesis of the major flavonoid components in Z. bungeana.

[Determination of 3 components in Xinjiang Ziziphora bungeana by HPLC].

This study aimed to develop an HPLC method for simultaneous determination of the 3 components in Ziziphora bungeana. The optimum HPLC condition was as follows：ZORBAX SB-C18 column（4.6 mm×250 mm, 5.0 µm）with gradient elution of methanol (A)-0.2% glacial acetic acid (B), detection wavelength 340 nm,column temperature 30 °C, flow rate 1 mL·min⁻¹. There were good linearity between peak areas and injection quantity of caffeic acid, rosmarinic acid, and linarin in the range of 2-40 mg·L⁻¹ï¼ˆr=0.999 9）, 3-60 mg·L⁻¹ï¼ˆr=1）, 7-140 mg·L⁻¹ï¼ˆr=0.999 9）, respectively. The average recoveries were 100.9%（RSD 1.3%）,98.25%（RSD 2.0%）,and 98.73%（RSD 1.5%）, respectively. The HPLC method was stable and accurate, which could be used to detect caffeic acid,rosmarinic acid, and linarin in Ziziphora bungeana.

[Classification, distribution and change of Uighur medicinal materials resources].

With the development and change of Uygur medicine, The sources,medicinal parts and producing area of some Uygur Medicine have changed. It is more important to master the classification, distribution and change of Uygur medicinal materials. These were more than 1 200 kinds Uygur medicine in history were resaerched by field investigation, philological research, and textual research, which main source of original plant were 140 families, 510 genera, 840 species; and source of original animal were 76 families, 107 genera, the original animal 141 species; 55 kinds of original mineral, which main producer were Xinjiang and Central Asia, West Asia, the Mediterranean, and North Africa, Southeast Asia and other provinces in China, there are individual medicinal materials from the Americas, Europe and other places. Through this study the classification, distribution, source and evolution of specific families and genera of Uygur medicine resources have mastered.It is hoped to provide theoretical basis for further research and development of Uygur medicinal materials.

The Transcription Factor p8 Regulates Autophagy in Response to Palmitic Acid Stress via a Mammalian Target of Rapamycin (mTOR)-independent Signaling Pathway.

Autophagy is an evolutionarily conserved degradative process that allows cells to maintain homoeostasis in numerous physiological situations. This process also functions as an essential protective response to endoplasmic reticulum (ER) stress, which promotes the removal and degradation of unfolded proteins. However, little is known regarding the mechanism by which autophagy is initiated and regulated in response to ER stress. In this study, different types of autophagy were identified in human gastric cancer MKN45 cells in response to the stress induced by nutrient starvation or lipotoxicity in which the regulation of these pathways is mammalian target of rapamycin (mTOR)-dependent or -independent, respectively. Interestingly, we found that p8, a stress-inducible transcription factor, was enhanced in MKN45 cells treated with palmitic acid to induce lipotoxicity. Furthermore, an increase in autophagy was observed in MKN45 cells stably overexpressing p8 using a lentivirus system, and autophagy induced by palmitic acid was blocked by p8 RNAi compared with the control. Western blotting analyses showed that autophagy was regulated by p8 or mTOR in response to the protein kinase-like endoplasmic reticulum kinase/activating transcription factor 6-mediated ER stress of lipotoxicity or the parkin-mediated mitochondrial stress of nutrient starvation, respectively. Furthermore, our results indicated that autophagy induced by palmitic acid is mTOR-independent, but this autophagy pathway was regulated by p8 via p53- and PKCα-mediated signaling in MKN45 cells. Our findings provide insights into the role of p8 in regulating autophagy induced by the lipotoxic effects of excess fat accumulation in cells.

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

BACKGROUND: In 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. RESULTS: Using 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. CONCLUSIONS: These 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.

[The present situation and countermeasures of Uighur medicine compound].

Uighur medicine compound, which created and used by Uighur nationality, is under the guidance of the Uighur medical theory system of herbal formula and dialectical use of minority nationality conventional medicines. In recent years, Uighur medicine attracted more and more attention of people which have used and were using it. Combining the history of Uighur medicine, this article summarizes the Uighur resources, medicinal materials, drugs preparation, ancient documents, and the establishment of the clinical evaluation system and so on, and then analyzes the status quo and the existing problems in Uighur medicine compound research and industry. On this basis, we put forward countermeasures and suggestions for the development of Uighur medicine.

The RNA-editing deaminase ADAR is involved in stress resistance of Artemia diapause embryos.

The most widespread type of RNA editing, conversion of adenosine to inosine (A→I), is catalyzed by two members of the adenosine deaminase acting on RNA (ADAR) family, ADAR1 and ADAR2. These enzymes edit transcripts for neurotransmitter receptors and ion channels during adaption to changes in the physical environment. In the primitive crustacean Artemia, when maternal adults are exposed to unfavorable conditions, they release diapause embryos to withstand harsh environments. The aim of the current study was therefore to elucidate the role of ADAR of Artemia diapause embryos in resistance to stress. Here, we identified Artemia ADAR (Ar-ADAR), which harbors a putative nuclear localization sequence (NLS) and two double-stranded RNA-binding motifs (dsRBMs) in the amino-terminal region and an adenosine deaminase (AD) domain in the carboxyl-terminal region. Western blot and immunofluorescence analysis revealed that Ar-ADAR is expressed abundantly in post-diapause embryos. Artemia (n = 200, three replicates) were tested under basal and stress conditions. We found that Ar-ADAR was significantly induced in response to the stresses of salinity and heat-shock. Furthermore, in vivo knockdown of Ar-ADAR (n = 100, three replicates) by RNA interference induced formation of pseudo-diapause embryos, which lack resistance to the stresses and exhibit high levels of apoptosis. These results indicate that Ar-ADAR contributes to resistance to stress in Artemia diapause embryos.

Expression profiles of miRNAs and involvement of miR-100 and miR-34 in regulation of cell cycle arrest in Artemia.

Regulation of the cell cycle is complex but critical for proper development, reproduction and stress resistance. To survive unfavourable environmental conditions, the crustacean Artemia produces diapause embryos whose metabolism is maintained at extremely low levels. In the present study, the expression profiles of miRNAs during Artemia diapause entry and termination were characterized using high-throughput sequencing. A total of 13 unclassified miRNAs and 370 miRNAs belonging to 87 families were identified; among them, 107 were differentially expressed during diapause entry and termination. We focused on the roles of two of these miRNAs, miR-100 and miR-34, in regulating cell cycle progression; during the various stages of diapause entry, these miRNAs displayed opposing patterns of expression. A functional analysis revealed that miR-100 and miR-34 regulate the cell cycle during diapause entry by targeting polo-like kinase 1 (PLK1), leading to activation of the mitogen-activated protein kinase kinase-extracellular signal-regulated kinase-ribosomal S6 kinase 2 (MEK-ERK-RSK2) pathway and cyclin K, leading to suppression of RNA polymerase II (RNAP II) activity respectively. The findings presented in the present study provide insights into the functions of miR-100 and miR-34 and suggest that the expression profiles of miRNAs in Artemia can be used to characterize their functions in cell cycle regulation.

Two p90 ribosomal S6 kinase isoforms are involved in the regulation of mitotic and meiotic arrest in Artemia.

There are multiple isoforms of p90 ribosomal S6 kinase (RSK), which regulate diverse cellular functions such as cell growth, proliferation, maturation, and motility. However, the relationship between the structures and functions of RSK isoforms remains undetermined. Artemia is a useful model in which to study cell cycle arrest because these animals undergo prolonged diapauses, a state of obligate dormancy. A novel RSK isoform was identified in Artemia, which was termed Ar-Rsk2. This isoform was compared with an RSK isoform that we previously identified in Artemia, termed Ar-Rsk1. Ar-Rsk2 has an ERK-docking motif, whereas Ar-Rsk1 does not. Western blot analysis revealed that Ar-Rsk1 was activated by phosphorylation, which blocked meiosis in oocytes. Knockdown of Ar-Rsk1 reduced the level of phosphorylated cdc2 and thereby suppressed cytostatic factor activity. This indicates that Ar-Rsk1 regulates the cytostatic factor in meiosis. Expression of Ar-Rsk2 was down-regulated in Artemia cysts in which mitosis was arrested. Knockdown of Ar-Rsk2 resulted in decreased levels of cyclin D3 and phosphorylated histone H3, and the production of pseudo-diapause cysts. This indicates that Ar-Rsk2 regulates mitotic arrest. PLK and ERK RNAi showed that Ar-Rsk2, but not Ar-Rsk1, could be activated by PLK-ERK in Artemia. This is the first study to report that RSK isoforms with and without an ERK-docking motif regulate mitosis and meiosis, respectively. This study provides insight into the relationship between the structures and functions of RSK isoforms.

Gastric cancer-associated enhancement of von Willebrand factor is regulated by vascular endothelial growth factor and related to disease severity.

BACKGROUND: von Willebrand factor (vWF) is a potent regulator of angiogenesis, tumor growth, and metastasis. Yet, the expression pattern of vWF in human gastric cancer (GC) tissues and its relation to clinicopathological features of these cases remains unknown. METHODS: Tumor and 5-cm adjacent non-tumoral parenchyma specimens were collected from 99 patients with GC (early stages I/II and late stages III/IV), and normal specimens were collected from 32 healthy controls (reference group). Plasma vWF antigen (vWF:Ag) and vWF activity were assessed by ELISA. The role of vascular endothelial growth factor (VEGF) in differential vWF expression was investigated using cultured human umbilical vein endothelial cells (HUVECs). vWF and VEGF protein and mRNA expression levels were investigated by qRT-PCR, western blotting and immunohistochemistry (IHC) respectively. The correlation of IHC-detected vWF expression with patient clinicopathological characteristics was analyzed. RESULTS: Compared to the reference group, the patients with late GC showed significantly higher levels of vWF:Ag (72% (21-115) vs. 101% (40-136)) and vWF activity (62% (20-112) vs. 117% (33-169)) (both P < 0.001). The GC tumor tissues also showed higher vWF mRNA and protein levels than the adjacent non-tumoral parenchyma. Patients at late GC stage had significantly higher median number of vWF-positive cells than patients at early GC stage (P < 0.05). VEGF induced vWF mRNA and protein expression in HUVECs in dose- and time-dependent manners. Patients with late GC stage also had significantly higher serum VEGF than patients at early GC stage (23 ± 26 vs. 10 ± 12 pg/mL, P < 0.01). Most of the undifferentiated GC tumor tissues at late disease stage exhibited strong VEGF and VEGFR2 protein staining, which co-localized with the vWF protein staining pattern. CONCLUSIONS: GC-related plasma vWF:Ag and vWF activity levels become substantially elevated in the late stage of disease. The higher mRNA and protein expression of vWF in GC tumor stroma may be regulated by the VEGF-VEGFR2 signaling pathway in vitro and may contribute to GC progression in vivo.

[Influence on Strychni Semen's Analgesic Effect and Toxicity of Milk-Impregnated- Processing-Technology of Traditional Uighur Medicine].

OBJECTIVE: To investigate the effects of different processing methods on analgesic effect and toxicity of Strychni Semen, and to provide the scientific basis of milk-impregnated-processing-technology of traditional Uighur medicine for toxic medicines. METHODS: Three different methods, frying with sand of traditional Chinese medicine (1), cooled milk-impregnated-processing-technology of traditional Uighur medicine (2), and heated milk-impregnated-processing-technology of traditional Uighur medicine (3) were used to process raw Strychni Semen and the processed products were compared with those in the raw drug (4). Karber method was used to caculate the LD50. RESULTS: The LD50 of (4), (1), (2) and (3)were 273.0, 289.3, 289.3 and 339.2 mg/kg,successively. Low dose group of heated milk-impregnated-processing-technology, low and high dose groups of cooled milk-impregnated-processing-technology had significant inhibition on the acetic acid-induced writhing reaction in mice (P < 0.05). CONCLUSION: Strychni Semen processed by cooled and heated milk can enhance the analgesic effect in mice while improving the LD50 of mouse, which illustrates milk-impregnated-processing- technology's pharmacological mechanism on efficacy enhancing and toxicity reducing of Strychni Semen.

When did decapods invade hydrothermal vents? Clues from the Western Pacific and Indian Oceans.

Hydrothermal vents are typically located in midocean ridges and back-arc basins and are usually generated by the movement of tectonic plates. Life thrives in these environments despite the extreme conditions. In addition to chemoautotrophic bacteria, decapod crustaceans are dominant in many of the hydrothermal vents discovered to date. Contrary to the hypothesis that these species are remnants of relic fauna, increasing evidence supports the notion that hydrothermal vent decapods have diversified in more recent times with previous research attributing the origin of alvinocarid shrimps to the Miocene. This study investigated seven representative decapod species from four hydrothermal vents throughout the Western Pacific and Indian Oceans. A partitioned mix-model phylogenomic analysis of mitochondrial DNA produced a consistent phylogenetic topology of these vent-endemic species. Additionally, molecular dating analysis calibrated using multiple fossils suggested that both bythograeid crabs and alvinocarid shrimps originated in the late Mesozoic and early Cenozoic. Although of limited sampling, our estimates support the extinction/repopulation hypothesis, which postulates recent diversification times for most hydrothermal vent species due to their mass extinction by global deep-water anoxic/dysoxic events during the Late Cretaceous and Early Tertiary. The continental-derived property of the West Pacific province is compatible with the possibility that vent decapods diversified from ancestors from shallow-water regions such as cold seeps. Our results move us a step closer toward understanding the evolutionary origin of hydrothermal vent species and their distribution in the Western Pacific-Indian Ocean Region.

Chitin-binding proteins of Artemia diapause cysts participate in formation of the embryonic cuticle layer of cyst shells.

The brine shrimp Artemia reproduces either ovoviviparously, producing free-swimming nauplii, or oviparously, producing encysted embryos (diapause cysts) able to cope with harsh and complex habitats. When the cysts enter diapause they are encased in a complex external shell that protects them from certain extreme environments. The genomic comparison of oviparous and ovoviviparous ovisacs has been described previously. We isolated three significantly up-regulated genes in oviparous oocytes and identified them as Arp-CBP (Artemia parthenogenetica chitin-binding protein) genes. Quantitative real-time PCR indicated that the expression of Arp-CBP genes gradually increases during diapause cyst formation and significant mRNA accumulation occurs during the ovisac stage of oviparous development. Moreover, in situ hybridization results demonstrated that Arp-CBP mRNAs are expressed in the embryo. Interestingly, the results of immune electron microscopy showed that all three Arp-CBPs are distributed throughout the cellular ECL (embryonic cuticle layer) of the cyst shell. Furthermore, knockdown of Arp-CBP by RNA interference resulted in marked changes in the composition of the embryonic cuticular layer. The fibrous layer of the cyst shell adopted a loose conformation and the inner and outer cuticular membranes exhibited marked irregularities when Arp-CBP expression was suppressed. Finally, an in vitro recombinant protein-binding assay showed that all three Arp-CBPs have carbohydrate-binding activities. These findings provide significant insight into the mechanisms by which the ECL of Artemia cyst shell is formed, and demonstrate that Arp-CBPs are involved in construction of the fibrous lattice and are required for formation of the ECL of the cyst shell.