Formation of Diapause Cyst Shell in Brine Shrimp, Artemia parthenogenetica, and Its Resistance Role in Environmental Stresses
2009; Elsevier BV; Volume: 284; Issue: 25 Linguagem: Inglês
10.1074/jbc.m109.004051
ISSN1083-351X
AutoresYu-Lei Liu, Yang Zhao, Zhong‐Min Dai, Han‐Min Chen, Wei‐Jun Yang,
Tópico(s)Aquaculture disease management and microbiota
ResumoArtemia has attracted much attention for its ability to produce encysted embryos wrapped in a protective shell when subject to extremely harsh environmental conditions. However, what the cyst shell is synthesized from and how the formative process is performed remains, as yet, largely unknown. Over 20 oviparous specifically expressed genes were identified through screening the subtracted cDNA library enriched between oviparous and ovoviviparous Artemia ovisacs. Among them, a shell gland-specifically expressed gene (SGEG) has been found to be involved in the cyst shell formation. Lacking SGEG protein (by RNA interference) caused the cyst shell to become translucent and the chorion layer of the shell to become less compact and pultaceous and to show a marked decrease of iron composition within the shell. The RNA interference induced defective diapause cysts with a totally compromised resistibility to UV irradiation, extremely large temperature differences, osmotic pressure, dryness, and organic solvent stresses. In contrast, the natural cyst would provide adequate protection from all such factors. SGEG contains a 345-bp open reading frame, and its consequentially translated peptide consists of a 33-amino acid residue putative signal peptide and an 81-amino acid residue mature peptide. The results of Northern blotting and in situ hybridization indicate that the gene is specifically expressed in the cells of shell glands during the period of diapause cyst formation of oviparous Artemia. This investigation adds strong insight into the mechanism of cyst shell formation of Artemia and may be applicable to other areas of research in extremophile biology. Artemia has attracted much attention for its ability to produce encysted embryos wrapped in a protective shell when subject to extremely harsh environmental conditions. However, what the cyst shell is synthesized from and how the formative process is performed remains, as yet, largely unknown. Over 20 oviparous specifically expressed genes were identified through screening the subtracted cDNA library enriched between oviparous and ovoviviparous Artemia ovisacs. Among them, a shell gland-specifically expressed gene (SGEG) has been found to be involved in the cyst shell formation. Lacking SGEG protein (by RNA interference) caused the cyst shell to become translucent and the chorion layer of the shell to become less compact and pultaceous and to show a marked decrease of iron composition within the shell. The RNA interference induced defective diapause cysts with a totally compromised resistibility to UV irradiation, extremely large temperature differences, osmotic pressure, dryness, and organic solvent stresses. In contrast, the natural cyst would provide adequate protection from all such factors. SGEG contains a 345-bp open reading frame, and its consequentially translated peptide consists of a 33-amino acid residue putative signal peptide and an 81-amino acid residue mature peptide. The results of Northern blotting and in situ hybridization indicate that the gene is specifically expressed in the cells of shell glands during the period of diapause cyst formation of oviparous Artemia. This investigation adds strong insight into the mechanism of cyst shell formation of Artemia and may be applicable to other areas of research in extremophile biology. Salt lakes on plateaus, are widely known as "seas of death," because they represent one of the most hostile environments on the earth in terms of extreme salinity, high pH, anoxia, large temperature differences, and intermittent dry conditions. Hardly any animal can survive such extremes. However, one notable exception lies in the shape of a small crustacean, Artemia. Artemia, also called the brine shrimp, is an ancient species that first appeared ∼400 million years ago (1Dattilo A.M. Bracchini L. Carlini L. Loiselle S. Rossi C. Int. J. Biometeorol. 2005; 49: 388-395Crossref PubMed Scopus (30) Google Scholar). To cope with harsh and complex habitats such as salt lakes, Artemia are able, when the circumstances become adverse, to release their offspring into a dormant, encysted state, rather than simply releasing swimming nauplius, to ensure survival. Such adverse conditions include environments where the Artemia may experience high salinity, low oxygen levels, short days, or conditions of extreme temperature variation (2Clegg J.S. Trotman C.N.A. Abatzopoulos TH.J. Beardmore J.A. Clegg J.S. Sorgeloos P. Artemia: Basic and Applied Biology. Kluwer Academic Publishers, Dordrecht, The Netherlands2002: 129-170Google Scholar, 3Nambu Z. Tanaka S. Nambu F. J. Exp. Zool. Part A. Comp. Exp. Biol. 2004; 301: 542-546Crossref PubMed Scopus (38) Google Scholar). These dormant cysts will keep diapause until the state is terminated by activation (triggered by factors such as desiccation, dehydration, cold or chemical treatment), at which point they resume development when appropriate and stable environmental conditions have arisen (4Nambu Z. Tanaka S. Nambu F. Nakano M. J. Exp. Zool. Part A. Ecol. Genet. Physiol. 2008; 309: 17-24PubMed Google Scholar, 5Drinkwater L.E. Clegg J.S. Browne R.A. Sorgeloos P. Trotman C.N.A. Artemia Biology. CRC Press, Boca Raton, FL1991: 93-118Google Scholar, 6Lavens P. Sorgeloos P. Sorgeloos P. Bengtson D.A. Decleir W. Jaspers E. Artemia Research and Its Applications. Universa Press, Wetteren, Belgium1987: 27-63Google Scholar, 7Versichele D. Sorgeloos P. Persoone G. Sorgeloos P. Roel O. Jaspers E. The Brine Shrimp Artemia, Ecology, Culturing, Use in Aquaculture. Universa Press, Wetteren, Belgium1980: 231-246Google Scholar). The diapause cysts, with their greatly reduced metabolic activity, contain embryos existing as late gastrulae and are composed of ∼4000 cells that are arrested at the G2/M phase with a complete turning off of RNA and protein synthesis (8Clegg J.S. J. Cell. Physiol. 1978; 94: 123-137Crossref PubMed Scopus (39) Google Scholar, 9Clegg J.S. J. Exp. Biol. 1997; 200: 467-475PubMed Google Scholar). Previous studies indicate that the resistance and resumption ability of Artemia cysts have several causes. In addition to the arrested cell cycle, it has been noted that large amounts of two molecular chaperone proteins, namely p26 and artemin, are synthesized (10MacRae T.H. Semin. Cell Dev. Biol. 2003; 14: 251-258Crossref PubMed Scopus (95) Google Scholar, 11Liang P. Amons R. Clegg J.S. MacRae T.H. J. Biol. Chem. 1997; 272: 19051-19058Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 12Warner A.H. Brunet R.T. MacRae T.H. Clegg J.S. Arch. Biochem. Biophys. 2004; 424: 189-200Crossref PubMed Scopus (56) Google Scholar), and a high concentration of trehalose is also accumulated (13Hand S.C. Gnaiger E. Science. 1988; 239: 1425-1427Crossref PubMed Scopus (74) Google Scholar, 14Crowe J.H. Hoekstra F.A. Crowe L.M. Annu. Rev. Physiol. 1992; 54: 579-599Crossref PubMed Scopus (1188) Google Scholar, 15Crowe L.M. Reid D.S. Crowe J.H. Biophys. J. 1996; 71: 2087-2093Abstract Full Text PDF PubMed Scopus (509) Google Scholar). Moreover, a complicated enzyme system is also involved in the diapause and resumption mechanism, including AMP-activated protein kinase (16Zhu X.J. Feng C.Z. Dai Z.M. Zhang R.C. Yang W.J. Stress. 2007; 10: 53-63Crossref PubMed Scopus (22) Google Scholar) and p90 ribosomal S6 kinase regulatory pathway (17Dai J.Q. Zhu X.J. Liu F.Q. Xiang J.H. Nagasawa H. Yang W.J. J. Biol. Chem. 2008; 283: 1705-1712Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Nakanishi Y.H. Iwasaki T. Okigaki T. Kato H. Annot. Zool. Jap. 1962; 35: 223-228Google Scholar, 19Finamore F.J. Clegg J.S. Padilla G.M. Whitson G.L. Cameron I.L. The Cell Cycle: Gene-Enzyme Interactions. Academic Press, New York1969: 249-278Crossref Google Scholar). In addition to falling into diapause, Artemia themselves secrete a rigid noncellular shell to cope with the extreme environmental stresses before they release the diapause cysts. The complex noncellular cyst shell consists of two main regions; the outer region, secreted by the shell gland, is of hypochlorite-soluble chorion, whereas the hypochlorite-resistant inner region is formed by blastoderm cells and comprises the embryonic cuticle (5Drinkwater L.E. Clegg J.S. Browne R.A. Sorgeloos P. Trotman C.N.A. Artemia Biology. CRC Press, Boca Raton, FL1991: 93-118Google Scholar, 20Morris J.E. Afzelius B.A. J. Ultrastruct. Res. 1967; 20: 244-259Crossref PubMed Scopus (98) Google Scholar, 21Clegg J.S. Jackson S.A. Liang P. MacRae T.H. Exp. Cell Res. 1995; 219: 1-7Crossref PubMed Scopus (69) Google Scholar). The shell glands, which are composed of clusters of secretory cells, are situated at the ovisac and open into the uterus. There are many dark brown secretory granules, which probably contain chorion material and pigments such as hematin formed in the cells of the shell glands at the point where the oocytes emerge in the ovaries during the reproductive period. These are secreted out at the second day after the oocytes enter the uterus. Therefore the shell glands vary from dark brown to white, even to colorless, as reproductive cycles differ (22Criel G.R.J. Macrae T.H. Abatzopoulos TH.J. Beardmore J.A. Clegg J.S. Sorgeloos P. Artemia: Basic and Applied Biology. Kluwer Academic Publishers, Dordrecht, The Netherlands2002: 1-38Google Scholar, 23Dutrieu J. Arch. Zool. Exp. Gen. 1960; 99: 1-134Google Scholar). Microphotographs shot by Sugumar and Munuswamy (24Sugumar V. Munuswamy N. Microsc. Res. Tech. 2006; 69: 957-963Crossref PubMed Scopus (17) Google Scholar) reveal that both the chorion and the embryonic cuticle have an exquisite structure (21Clegg J.S. Jackson S.A. Liang P. MacRae T.H. Exp. Cell Res. 1995; 219: 1-7Crossref PubMed Scopus (69) Google Scholar). Chorion consists of two distinct layers. First, a compact outer covering is over the cyst with many radially aligned aeropyles penetrating through. This is known as the cortical layer. Second, in a cavernous region below the cortical layer is the alveolar layer, which may act as a float for the newly laid cysts. A thin supra cortical layer, probably consisting of cuticulin, covers the outer surface of the cortical layer. The embryonic cuticle, which is impermeable to nonvolatile solutes, is otherwise composed of a broad multilamellar region as a fibrous layer sandwiched between the outer and inner cuticular membranes and constructed as a tripartite structure. This forms an area of relative independence from the external environment and serves to maintain the homeostasis of inorganic ions (2Clegg J.S. Trotman C.N.A. Abatzopoulos TH.J. Beardmore J.A. Clegg J.S. Sorgeloos P. Artemia: Basic and Applied Biology. Kluwer Academic Publishers, Dordrecht, The Netherlands2002: 129-170Google Scholar). The molecular formulation of the cyst shell is complex, and details remain unclear, although it is known that the cyst shell does contain chitin, lipoprotein, hematin, and some metal elements (25Yosefali A. Abbasali M. Amin E. Biotechnological Approach to Produce Chitin and Chitosan from the Shells of Artemia urmiana Günther, 1899 (Branchiopoda, Anostraca) Cysts from Urmia Lake, Iran. Koninklijke Brill NV, Leiden, Holland1999Google Scholar, 26Adamkova I. Hatching Quality of Artemia (Artemia salina) Cysts Treated with Commercial Hypochlorite Product Savo. Jihoceska University, Vodnany, Czech Republic1999Google Scholar, 27Van der Linden A. Blust R. Cuypers K. Thoeye C. Bernaerts F. Artemia Research and Its Applications: 2. Physiology, Biochemistry, Molecular Biology.in: Decleir W. Moens L. Slegers H. Sorgeloos P. Jaspers E. Proceedings of the Second International Symposium on the Brine Shrimp Artemia. Universa Press, Wetteren, Belgium1987: 181-188Google Scholar). Besides preventing mechanical damage (28Clegg J.S. Conte F. Persoone G.P. Sorgeloos P. Roels O. Jaspers E. The Brine Shrimp Artemia. Universa Press, Wetteren, Belgium1980: 11-54Google Scholar), the cyst shell also plays an important role in protecting the embryo within from other lethal environmental stresses. Previous experimental data have confirmed the protective capabilities of the cyst shell. Tanguay et al. (29Tanguay J.A. Reyes R.C. Clegg J.S. J. Biosci. 2004; 29: 489-501Crossref PubMed Scopus (57) Google Scholar) indicated that the hatching rate of intact cysts is significantly higher than the decapsulated ones after ultraviolet irradiation treatment. Hematin, the hemopigment of the cyst shell, is also demonstrated to have a light-screening function (27Van der Linden A. Blust R. Cuypers K. Thoeye C. Bernaerts F. Artemia Research and Its Applications: 2. Physiology, Biochemistry, Molecular Biology.in: Decleir W. Moens L. Slegers H. Sorgeloos P. Jaspers E. Proceedings of the Second International Symposium on the Brine Shrimp Artemia. Universa Press, Wetteren, Belgium1987: 181-188Google Scholar). Clegg (30Clegg J.S. Integr. Comp. Biol. 2005; 45: 715-724Crossref PubMed Scopus (98) Google Scholar) indicated that the cyst shell plays a critical role in desiccation tolerance, because the rate of dehydration of decapsulated cysts is much higher than intact ones in the dehydration study, and rapid water loss significantly reduces the hatching level of dehydrated cysts. Liu et al. (31Liu F.Q. Ji B.C. Xuan C.H. Anim. Sci. Vet. Med. 2002; 83: 10-11Google Scholar) also found that intact cysts have better thermotolerance than decapsulated ones in both dry and water heating studies. In our experiments, through the in vivo gene knockdown by RNA interference, a shell gland-specifically expressed gene (SGEG) has been found to be involved in the cyst shell formation. The formed cyst shell has been demonstrated to play an important role in resistance to UV irradiation, large temperature differences, osmotic pressure, dryness, and organic solution stresses. The oviparous Artemia were reared in 8% (w/v) artificial seawater (Blue Starfish, China) under light and dark cycles of 4 h (12:00–16:00) and 20 h, respectively. Otherwise the ovoviviparous Artemia were reared in 3% (w/v) artificial seawater under light and dark cycles of 16 h (7:00–23:00) and 8 h, respectively. The water temperature was kept at 28 °C. Chlorella powder was supplemented as brine shrimp food (Fuqing King Dnarmsa Spirulina Co. Ltd., Fuqing, China). A sufficient amount of both oviparous and ovoviviparous Artemia, at the stage where oocytes are stored in lateral pouches (the stage usually only lasting less than 6 h), were collected and temporarily cultured. Both types were used for isolating ovisacs each day throughout the reproductive cycle (for 5 days). The collected ovisacs were marked as either oviparous or ovoviviparous samples, and the day of collection was noted (from day 1 to day 5). The Artemia used for isolating ovisacs were placed in an ice bath for 1–2 min until they were lightly anesthetized. Then the ovisac was dissected, snap-frozen in liquid nitrogen, and stored at −80 °C until RNA preparation. The total RNAs of both oviparous and ovoviviparous ovisacs were extracted individually by TRIzol reagent (Invitrogen). One microgram of each total RNA was used to synthesize the first strand cDNA by PowerScriptTM reverse transcriptase (Takara Bio Company, Otsu, Japan) at 42 °C for 90 min, and two adaptors were added to both endings by 3′ SMART CDS Primer II A and SMART II A Oligonucleotide (Super SMARTTM PCR cDNA synthesis kit; Takara Bio Company, Japan). The first strand cDNAs, purified by NucleoSpin® columns, were amplified using 5′ PCR Primer II A for optimized cycles, and the double-stranded cDNAs were finally purified by NucleoSpin® columns once again. For SSH, 2The abbreviations used are: SSHsuppression subtractive hybridizationDIGdigoxingeninRACErapid amplification of cDNA endsdsdouble-strandedRNAiRNA interferenceSEMscanning electron microscopeFE-SEM/EDSfield emission SEM/energy dispersive system. the double-stranded cDNAs of oviparous ovisac samples from day 1 to day 5 were mixed in equal parts as a tester, whereas the double-stranded cDNAs of ovoviviparous ovisac samples from day 1 to day 5 were mixed in equal parts as a driver. The subtractive library was then generated using the Clontech PCR-Select cDNA subtraction kit (Takara Bio Company), according to established protocols. After two cycles of hybridization and two cycles of PCR amplification, a total of 84 individual recombinant clones in pUCm-T vector (Bio Basic Inc., Markham, Canada) were picked and used as templates for PCR amplification using vector primers M13F and M13R. Each PCR product (1 μl) was spotted onto a nylon membrane (Hybond-N; Amersham Biosciences), hybridized with the probes of tester and driver cDNA in order, and detected by the DIG chemiluminescent detection system (DIG High Prime DNA labeling and detection starter kit II; Roche Applied Science). suppression subtractive hybridization digoxingenin rapid amplification of cDNA ends double-stranded RNA interference scanning electron microscope field emission SEM/energy dispersive system. All of the clones that exhibited distinct expression differences according to the result of SSH were sequenced (Sangon, Shanghai, China). One full-length cDNA of them (SGEG) was achieved by 3′- and 5′-RACE methods. The gene-specific primers CRaceF for 3′-RACE and CRaceR for 5′-RACE (Table 1) were designed based on the nucleotide sequences of SGEG, and the cDNAs for RACE were synthesized from the total RNA of oviparous ovisacs. All of the processes of 3′- and 5′-RACE were following the manufacturer's protocol of FirstChoiceTM RLM-RACE kit (Ambion, Austin, TX). The sequenced cDNA was analyzed by using Lasergene software (DNAStar Inc.), and the deduced amino acid sequence of the peptide was predicted by the PredictProtein and Scratch Protein Predictor website. Otherwise the Blast (blastx and blastn) program was performed using the NCBI website. The nucleotide sequence of SGEG was submitted to GenBankTM under the accession number EU683079.TABLE 1Nucleotide sequences and positions of primers used in polymerase chain reactionsPrimerLengthPositionDirectionSequence (5′ → 3′)bpCRaceF20273–292FTTTGACGGACACCAACTTAGCRaceR20121–140RTTCTTTGGCAGCTTCCTCTGCExpF3037–57FCGCGGATCCATGGGGGTAAAGGAAGTTTTGCRiR31410–432RGCTCTAGACTAAATTTGCATCTGTTTAATCCCExpR30400–420RCCGCTCGAGCTGTTTAATCCAGTAAGCAACGFPF30122–144FGGAATTCAACTTACCCTTAATTTTATTTGCGFPR28461–480RGCTCTAGAGCCATTCTTTGGTTTGTCTCCQF20121–140FCAGAGGAAGCTGCCAAAGAACQR20273–292RCTAAGTTGGTGTCCGTCAAATubF20532–551FTCTACTGCCGTTGTTGAGCCTubR20694–713RATGGAGGAAACGATTTGACC Open table in a new tab A pair of primers (CExpF and CRiR in Table 1) were designed for the preparation of SGEG dsRNA. A 361-bp cDNA fragment was subcloned into the plasmid pET-T7 (17Dai J.Q. Zhu X.J. Liu F.Q. Xiang J.H. Nagasawa H. Yang W.J. J. Biol. Chem. 2008; 283: 1705-1712Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) at XbaI and EcoRI sites (the EcoRI site exists in the SGEG sequence, positions 72–77). For negative control, a 359-bp GFP cDNA fragment was amplified using the primers GFPF and GFPR (Table 1) and subcloned into the pET-T7 vector at the same restriction sites. The recombinant plasmids were transformed into Escherichia coli HT115, and the dsRNAs were produced and purified as described by Yodmuang et al. (32Yodmuang S. Tirasophon W. Roshorm Y. Chinnirunvong W. Panyim S. Biochem. Biophys. Res. Commun. 2006; 341: 351-356Crossref PubMed Scopus (158) Google Scholar). Based upon the basic and quantitative controls (supplemental Fig. S1), 300 ng of SGEG dsRNA was injected into the reproductive segments of Artemia at the instar XII stage (before ovarian development). An UltraMicroPump II equipped with the Micro4TM microsyringe pump controller was used for the microinjection. Both RNAi and control Artemia were cultured in 8% artificial seawater under the condition of light/dark cycles of 4/20 h. The RNAi-induced and control cysts were observed by light microscopes and collected for the following experiments. Total RNAs were extracted from ovisacs (lateral pouches filled with oocytes) of RNAi-induced Artemia and controls. After reverse transcription, all real time PCRs were performed on the Bio-Rad MiniOpticonTM real time PCR System using the SYBR® Premix Ex TaqTM (Takara Bio Inc.) and 200 nm SGEG-specific primers (CQF/CQR and TubF/TubR in Table 1). Relative transcript levels are presented as fold change calculated using the comparative CT method as described by Livak and Schmittgen (33Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (124899) Google Scholar, 34Schmittgen T.D. Livak K.J. Nat. Protoc. 2008; 3: 1101-1108Crossref PubMed Scopus (17485) Google Scholar) with α-tubulin cDNA as the internal reference. All of the data are given as the means ± S.E. of independent experiments from six separate RNA pools. All statistical analyses were performed using the one-way analysis of variance, and the difference was considered significant for p < 0.01. A transmission electron microscopic study was carried out to search for differences between the shells of RNAi-induced and those of control cysts, following the method described by Hofmann et al. (35Hofmann G.E. Hand S.C. J. Exp. Zool. 1990; 253: 287-302Crossref Scopus (30) Google Scholar). Both cysts were nicked and fixed in 2.5% glutaraldehyde prepared in 3% NaCl for 12 h. Then they were washed and postfixed in 1% osmium tetroxide in 3% NaCl solution, dehydrated in graded acetone series, and embedded in spurr resin. Sections of ∼70 nm were cut with microtome (UC6; Leica), stained with 2% uranyl acetate and Reynold's solution (0.2% sodium citrate and 0.2% lead nitrate), then viewed in a transmission electron microscope (JEM-1230; JEOL), and photographed at a voltage of 70 kV. For SEM and FE-SEM/EDA analysis, the RNAi-induced and control cysts were individually fixed by 2.5% glutaraldehyde for 2 h. Then these cysts were washed and dehydrated in graded acetone series and critical point dried with CO2 using a critical point dryer (Hitachi HCP-2). These were used for FE-SEM/EDS (Sirion SEM System, FEI Co., Hillsboro, OR) analysis directly or sputter-coated with gold (Hitachi E-1010) for scanning electron microscopy (Hitachi S-3000N). Equal numbers of the RNAi induced and control cysts were homogenized in 8 m urea individually and boiled for 10 min. After centrifugation to remove insoluble shell fragments, the supernatants were quantified using the Bradford method (36Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). Twenty micrograms of protein of each sample were electrophoresed on 15% polyacrylamide gels and detected by Coomassie Brilliant Blue. The open reading frame of SGEG cDNA was amplified using the primers CExpF and CExpR (Table 1) and cloned into the pET-32 vector with an N-terminal His6 tag. Recombinant SGEG protein was expressed in E. coli BL21 (DE3) and purified by nickel-nitrilotriacetic acid-agarose (Qiagen), and the anti-SGEG antibody was raised in rabbit (HuaAn Biotechnology Co. LTD.). Fifty micrograms of protein of each sample were separated on 12.5% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore) for Western blotting analysis. The membranes were incubated with anti-SGEG antibody and anti-α-tubulin antibody (Beyotime, Shanghai, China) overnight at 4 °C and detected using the BM Chemiluminescence Western blotting kit (Roche Applied Science). A sufficient number of RNAi-induced and control cysts were collected for the anti-environmental stress test, which included dryness, UV irradiation, high temperature, freezing, extreme osmotic pressure, and organic solvent tests. Triplicate experiments were performed for each of these physiological stresses. For the test of dryness, both the RNAi-induced and control cysts were air dried and kept at room temperature with continual observation. For the UV irradiation test, both the RNAi-induced and control cysts were exposed to ultraviolet light (∼310 nm) for 3.6 J/cm2. For the high temperature and freezing stress test, the RNAi-induced and control cysts were incubated in the water bath at 50 °C for 5 min or frozen at −30 °C for 3 days. For the test of coping with extreme osmotic pressure, the RNAi-induced and control cysts were soaked in 6 m NaCl for 450 min or in deionized water for 3 days. After the above tests, all RNAi-induced cysts were incubated in 3% artificial sea water with continual illumination at 25 °C for hatching. The hatching rates were then investigated for a period of 72 h. In contrast all the control cysts were activated before hatching by soaking in saturated brine for 24 h and freezing in −20 °C for 3 months. In addition, for the test of resistance to organic solvent, both the RNAi-induced and activated control cysts were incubated in 3% artificial sea water with 0.5 m methanol for investigating hatching rates. A DIG-labeled cDNA fragment (384 bp) was amplified using the primers CExpF and CExpR (Table 1) and used as a probe for the Northern blotting analyses. The total RNA of both oviparous and ovoviviparous Artemia, the total RNA of both thoracic segments and ovisacs of oviparous Artemia, and the total RNA of oviparous ovisacs from days 1–5 were extracted. All of the samples (15 μg corresponding to each tissue) were separated by agarose gel electrophoresis, then transferred to a nylon membrane (Hybond-N; Amersham Biosciences) for overnight hybridization at 45 °C, washed twice at 55 °C, and finally detected by the DIG chemiluminescent detection system (Roche Applied Science). The DIG-labeled sense and antisense RNA probes, corresponding to nucleotides 80–432 of SGEG cDNA, were amplified (primed by CExpF and CExpR; Table 1) and cloned into plasmid vector pSPT18 at EcoRI and XbaI sites (the EcoRI site exists in the SGEG sequence). They were then transcribed in vitro from the EcoRI- and XbaI-linearized templates, respectively (according to the manufacturer's instruction of DIG RNA Labeling kit SP6/T7; Roche Applied Science). For tissue slice preparation, Artemia were anesthetized as mentioned previously, snap-frozen in liquid nitrogen, and embedded in Tissue-TekTM (Sakura Finetechnical Co. Ltd). Then 10-μm-thick frozen sections were prepared by frozen ultramicrotome. Dry sections were fixed with paraformaldehyde, digested with proteinase K and hybridized at 42 °C overnight. Then these slices were washed at 52 °C and blocked by blocking solution (Roche Applied Science). They were then treated with anti-DIG-AP conjugate (Roche Applied Science; 1:500) and visualized with the colorimetric substrates nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate (Promega) according to the manufacturer's instructions. Finally, the photographs were taken on an inverted microscope (ECLIPSE TE2000-S; Nikon). A SSH library was constructed, and the double-stranded cDNA from oviparous and ovoviviparous ovisacs of Artemia were used as tester and driver, respectively. Eighty-four clones were obtained from the SSH library enriched for the oviparous-specific transcripts. Twenty-two clones exhibiting distinct expression differences were selected for sequencing and obtaining of the complete cDNA sequence by RACE. A differentially expressed gene, which contained a 450-bp complete cDNA sequence, was the focus, and this was named the shell gland-specifically expressed gene (SGEG). The conceptually translated peptide from the gene consists of a 33-amino acid putative signal peptides and an 81-amino acid putative residue mature peptides (supplemental Fig. S2). It was considered to be a novel gene, having no apparent identity with other known genes or proteins when alignments were compared with sequences in the DDBJ/EMBL/GenBankTM data base. RNA interference was performed by dsRNA microinjection into the Artemia at instar XII stage, and the SGEG transcript level was reduced to less than 20% (Fig. 1A). Although both the RNAi and control Artemia showed no significant abnormal phenotypes, the cysts, which were oviposited by RNAi-treated Artemia, stuck on bottom of the culture tank having a characteristically soft cyst shell in direct contrast to control cysts, which could be observed sinking, floating, and suspended in the same tank. For the RNAi-induced cysts, more than 20% were found to be able to develop into swimming nauplii by 72 h without any activation necessary. The control cysts remained unable to be hatched out directly without a procedure of diapause termination even in the presence of appropriate hatching conditions. The observations using the inverted microscope revealed that the shells of RNAi-induced cysts were transparent, whereas the control cysts remained opaque. Thus the embryo and blastopore inside could be observed clearly for the RNAi-induced cysts (Fig. 1B, panels a and a′). Moreover, in contrast to the control cyst shell, the RNAi-induced cyst shell had changed to light yellow when observed by anatomical microscopes (Fig. 1B, panels b and b′). Furthermore, the cortical layer and alveolar layer (chorion layer) of the RNAi-induced cyst shell was contorted and obviously lighter (more electron-transparent) than that of control cysts as shown in the transmission electron micrographs (Fig. 1C, panels a and a′). In addition, in the RNAi-induced cysts, the thin supra cortical layer had disappeared, the aeropyles in the cortical layer were blurry and syncretized, and the cortical layer of RNAi-induced cyst shell was less than half the thickness of the control cortical layer (Fig. 1C, panels b and b′). The scanning electron micrographs revealed that the shell surface of cysts reproduced by RNAi-treated Artemia was lamellar and rough (Fig. 1D, panel a), instead of the compact and smooth shell of the control cyst (normal cortical layer; Fig
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