Spat 6.0 7 Full Version
effects of no donors as metamorphosis inhibitor were tested in co-exposures of larvae (18 dpf, march 2019) with an inducer such as epi, mk-801 or smis together with a potential inhibitor, the no donor 3-morpholinosydnonimine (sin-1) or sodium nitroprusside (snp) for 3h, followed by a continuous exposure to the no donor alone (fig. 2 ). the no donor snp significantly inhibited metamorphosis at concentrations of 105m and 104m, while the effect of sin-1 did not significantly decrease metamorphosis percentages compared to inducers alone. besides the reduction of spat in the high concentration treatments of snp, spat exposed to snp at 104m displayed limited adult shell growth compared to single inducer exposed spat and remaining larvae were immobile on the bottom.
effects of different no donors as metamorphosis inhibitor were tested in co-exposures of larvae (18 dpf, march 2019) with an inducer such as epi, mk-801 or smis together with a potential inhibitor, the no donor 3-morpholinosydnonimine (sin-1) or sodium nitroprusside (snp) for 3h, followed by a continuous exposure to the no donor alone (fig. 2 ). the no donor snp significantly inhibited metamorphosis at concentrations of 105m and 104m, while the effect of sin-1 did not significantly decrease metamorphosis percentages compared to inducers alone. besides the reduction of spat in the high concentration treatments of snp, spat exposed to snp at 104m displayed limited adult shell growth compared to single inducer exposed spat and remaining larvae were immobile on the bottom.
effect of the no donor snp on a sustained larval induction protocol was then tested. larvae were co-exposed to a 1 h elicitor treatment with epi (1 mm), followed by a constant 48 h inducer exposure of epi (1 mm). at 24 h post epi exposure, larvae were treated with 105m snp or the same volume of milliq. all larvae except those in the control group exhibited a metamorphic response at 48 h post-inducer.
the no donor snp can be used in a continuous exposure in order to reduce the response of the larvae to the inducer at the same time, thus preventing metamorphosis. for example, in order to reduce metamorphosis induced by a 48 h exposure to epi (1 mm), half of the larvae were previously exposed to 1 mm epi for 1 h, followed by treatment with 105m snp for 48 h after removal of the inducer. larvae treated with snp alone (without epi) remained full-inducable and most were metamorphic (fig. 3 ). in order to reduce epi-induced metamorphosis by no donor treatment (fig. 2 ), larvae were exposed to epi (1 mm) for 1 h followed by a continuous exposure to snp (105m) for an additional 48 h after removal of epi.
using qrt-pcr, the expression of acspat (fig. 8 b) and accs (fig. 8 d) in ac larvae treated with epi, agh, agh/7-ni, l-name, l-nna and odq showed increases in gene expression after exposure to the treatments as measured by the comparative ct method [ 89 ]. gene expression changes were more variable for acgdh (fig. 8 a). expression of acgdh mrna in 17 dpf spat showed no significant increase in expression at 6hpe of agh exposure compared to the epi treatment, and acgdh expression after smis and agh/7-ni exposure was not significantly different from unexposed controls (fig. 8 c). expression of acgdh in spats sampled at 24 hpe showed increases in expression for smis and odq treated larvae, as well as 7-ni treated larvae. surprisingly, agh treatment reduced gene expression in spat at 24 hpe, to 48 hpe to values similar to the smis exposure. at 48 hpe, acgdh expression in 7-ni treated spat was significantly higher than smis treated spat (p = 0.019). expression of acgdh after l-name treatment at 48 and 66 hpe (p = 0.33) was significantly reduced compared to the dmso control and to exposure to smis and odq at the same time points. spat expression of acgdh after l-nna treatment at 24, 48 and 66hpe was not significantly different from all other treatment or controls (fig. 8 d). table 3 shows the qrt-pcr used in this experiment and also the relative fold change of expression at each time point
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