Carbofuran accelerated yolk opaqueness and SA‐β‐galactosidase activity in spns1 mutant zebrafish. A, Incross among heterozygote (spns1^+/−) fishes. Around 25% of resultant embryos are spns1^−/− homozygous mutant, whereas the remaining 75% of embryos consist of wild (spns1^+/+) and heterozygous (spns1^+/−) embryos. B, Yolk opaqueness phenotype in homozygote (spns1^−/−) fish. The opaqueness was accelerated by carbofuran exposure. C, The SA‐β‐gal activity was high in homozygote (spns1^−/−) fish and accelerated by exposure to carbofuran. The SA‐β‐gal staining at the head was expanded, as shown by a red box and red arrow. The SA‐β‐gal staining of whole fish was observed under the bright field (BF) condition of the stereo‐microscope. The cellular SA‐β‐gal staining in the head (shown by green box and arrow) was observed by confocal microscopy. The scale bars are 250 mm (stereo microscopic images) and 10 mm (confocal microscopic images). D, Quantification of SA‐β‐gal staining intensity of whole fish. E, Quantification of cellular SA‐β‐gal staining signals in the head region. The number of animals was 10 (n = 10). ***P ≤ .005

Carbofuran exacerbated the spns1 deficiency phenotype and shortened the survival of spns1 mutant zebrafish. A, Sorts of yolk opaqueness phenotypes in spsn1^−/− (homozygous) zebrafish based on the extent of opacity in the yolk and/or yolk extension: partially opaque (yellow colour) and mostly opaque (red colour). Wild (spns1^+/+) and heterozygous (spns1^+/−) zebrafish do not show such opaqueness (dark blue colour). B, Effect of carbofuran on the opaqueness phenotype of spns1 mutant fish and death record. The opaqueness phenotype and death records were not affected by 10 μmol/L carbofuran exposure. However, the mostly opaque phenotype and death of embryos of 100 μmol/L carbofuran treated group were found at earlier times than fishes of the control group (egg water treatment). C, Survival curves of spns1 mutant fishes for carbofuran treatment at 10 and 100 μmol/L concentrations. By 100 μmol/L carbofuran exposure, the survival of spsn1 mutant fishes was significantly declined (P = .0003)

Carbofuran accelerated autolysosomal puncta formation in spns1 mutant zebrafish. A, Carbofuran treatment worsened the yolk opaqueness phenotype of spns1 mutant fishes, and most embryos lost their yolk extension. Loss of the spns1 gene increased autophagosomal EGFP‐LC3 and lysosomal LysoTracker (LysoT) red expressions, which were further accelerated by carbofuran exposure. Most LysoTracker red expressions were co‐localized with EGFP‐LC3 expressions (bottom row; yellow colour). B, Quantification of autophagosomal EGFP‐LC3 expression. C, Quantification of LysoTracker red expression. D, Quantification of the merged expression (co‐localization). These expressions were significantly increased by the loss of spns1 gene and were further significantly up‐regulated by carbofuran exposure. The scale bar is 10 mm; n = 6; *P ≤ .05; **P ≤ .01; ***P ≤ .005

Carbofuran accelerated autolysosomal puncta formation in a double‐transgenic zebrafish line of spns1 mutant background, expressing EGFP‐LC3 and mCherry‐Lamp1. A, Carbofuran exposure accelerated both autophagosomal EGFP‐LC3 and lysosomal mCherry‐Lamp1 expressions in spns1 mutant zebrafish. Lysosomal mCherry‐Lamp1 expression was merged to EGFP‐LC3 expression (bottom row; yellow colour). B, Quantification of autophagosomal EGFP‐LC3 expression. C, Quantification of lysosomal mCherry‐Lamp1 expression. D, Quantification of the co‐localization of expressions. Both EGFP‐LC3 and mCherry‐Lamp1 expressions (with their co‐localization) were significantly increased by the loss of the spns1 gene and were further significantly up‐regulated by carbofuran exposure. The scale bar is 10 mm; n = 6; *P ≤ .05; **P ≤ .01; ***P ≤ .005

RT‐PCR analysis to determine the effect of carbofuran on pai1, p21 and smp30 mRNA levels. The expressions levels of pai1 and p21 were increased by the loss of the spns1 gene. Carbofuran treatment further increased their expressions. The expression of smp30 was significantly down‐regulated by carbofuran exposure in spns1 mutant zebrafish. n = 5; *P ≤ .05; ***P ≤ .005

A proposed schematic model for the senescence and ageing effect of carbofuran under spns1 defective condition. Cellular untoward events are prevented at low to medium stress conditions by epigenetic adjustment. The Spns1 defect via strong genotoxic stress induces untoward events, such as senescence, ageing, ageing‐related diseases and cell death. Carbofuran‐induced oxidative stress accelerates these untoward cellular events in spns1 defective animals