Figure 1.
Two developmental pathways of Artemia and structure of the cyst shell.
a, Two developmental pathways of Artemia. b–d, The structure of the cyst shell. Inverted microscope at ×400 magnification, scale bar, 30 µm (b). TEM at ×8,000 magnification, scale bar, 1.5 µm (c). TEM at ×30,000 magnification, scale bar, 0.2 µm (d). TSCL, thin supra cortical layer; CL, cortical layer; AL, alveolar layer.
Figure 2.
The characteristic and localization of SGEG2 mRNA.
(a) Northern blot analysis of SGEG2 in various tissues of A. parthenogenetica. (b) Northern blot analysis in various oviparous developmental stages. Adults, cyst-destined female adults without oocytes; early oocytes, with the oocytes in the ovaries; middle oocytes, with the oocytes in the lateral pouches; late oocytes, with the oocytes in the uterus for 2 days and 4 days. The ethidium bromide-stained 28S/18S rRNA bands were used as a control for loading variation. c, d, The localization SGEG2 mRNA in the ovisac of oviparous Artemia. The 297 bp DIG-labelled sense and antisense RNA probes were used for in situ hybridization. The distribution of the positive brown signals in cells of shell glands are indicated by black arrow (c). Control (d) was with sense RNA probe (cross section ×40). U, uterus; Sg, shell gland; O, oocyte. Scale bar, 200 µm.
Figure 3.
Expression and localization of the SGEG1, 2a and 2b.
a, Western blotting analysis. Western blots were probed with the indicated antibodies. α-tubulin served as the internal standard. 50 µg of proteins were loaded for each lane. b, Schematic representation of deduced amino acid sequences of SGEG1, 2a and 2b. The red boxes are putative signal peptides. The yellow box is SGEG1 peptide. The green box is SGEG2a peptide and blue is SGEG2b peptide. c, Immunohistochemical localization in TEM image analysis using gold nanoparticles at ×20,000 magnification. No primary antibody was added in the control. The slices of cyst shell incubated with antibody to SGEG1, 2a and 2b. Arrows indicate the gold nanoparticles. Scale bars, 0.25 µm.
Figure 4.
RNAi treatment and phenotypes of cysts.
a, The expression levels of SGEG1 and 2 mRNA were determined with real-time quantitative PCR after RNAi treatments. The expression levels of the GFP dsRNA injected group are attributed a relative value of 100 as a control. Plotted is the mean ± s.d (n = 10, ** P<0.001, one-tailed t-test). The peptides in cysts were analyzed by Western blotting and alpha-tublin was used for loading control. b, c, The phenotypes of all RNAi-treated cysts. b, Observation by anatomical microscopy (AM) at ×80 magnification, scale bar, 100 µm; Inverted microscopy (IM) at ×100 magnification, scale bar, 80 µm; SEM at ×400 magnification, scale bar, 60 µm; and SEM at ×2,000 magnification (the lower panel) , scale bar, 12 µm. c, Observation by TEM at ×8,000 magnification, scale bars, 3 µm.
Figure 5.
Assay of resistance of the cysts to UV radiation and osmotic stress.
a, Comet assay. DNA damage in the cells of all RNAi-treated cysts after UV irradiation with 3.6 joules/cm2 for 30 min. Damage is expressed as DNA in tail (%). Plotted is the mean ± s.d (n = 50, ** P<0.001, one-tailed t-test). b, c, The cysts were incubated in 6 M NaCl solution (b) and in fresh water (c).
Figure 6.
Resistance of encysted embryos to environmental stress of freezing, heat shock, UV radiation, and organic solvent.
Triplicate experiments were performed for each of these physiological stresses. In every group, 100 cysts were taken to hatch; the hatch rate was calculated by the number of live nauplius resulting.