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Using non-reducing Western blotting to assess protein thiol redox state is challenging because most reduced and oxidised forms migrate at the same molecular weight and are, therefore, indistinguishable. While copper catalysed Click chemistry can be used to ligate a polyethylene glycol (PEG) moiety termed Click PEGylation to mass shift the reduced or oxidised form as desired, the potential for copper catalysed auto-oxidation is problematic. Here we define a catalyst-free trans -cyclooctene-methyltetrazine (TCO-Tz) inverse electron demand Diels Alder chemistry approach that affords rapid ( k ~2000 M −1 s −1 ), selective and bio-orthogonal Click PEGylation. We used TCO-Tz Click PEGylation to investigate how fertilisation impacts reversible mitochondrial ATP synthase F 1 -F o sub-unit alpha (ATP-α-F 1 ) oxidation—an established molecular correlate of impaired enzyme activity—in Xenopus laevis. TCO-Tz Click PEGylation studies reveal substantial (~65%) reversible ATP-α-F 1 oxidation at evolutionary conserved cysteine residues (i.e., C 244 and C 294 ) before and after fertilisation. A single thiol is, however, preferentially oxidised likely due to greater solvent exposure during the catalytic cycle. Selective reduction experiments show that: S-glutathionylation accounts for ~50–60% of the reversible oxidation observed, making it the dominant oxidative modification type. Intermolecular disulphide bonds may also contribute due to their relative stability. Substantial reversible ATP-α-F 1 oxidation before and after fertilisation is biologically meaningful because it implies low mitochondrial F 1 -F o ATP synthase activity. Catalyst-free TCO-Tz Click PEGylation is a valuable new tool to interrogate protein thiol redox state in health and disease.

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Internal electronic tagging is a major issue in flatfish species due to the small size of abdominal cavity. In this study, three tag types, referred to as Nonatec, nano and mini, were evaluated in three weight classes of Senegalese sole: small (0.3 g), middle (0.8 g) and large (2.0 g). Tags were injected from the blind side and fish were carefully handled to minimize sharp movements. Tag losses were 8% in the small size class, between 5.2 and 15.1% in the middle size class and 2-4% in the large size class. The mortality rates ranged between 2.0 and 15.0% with the lowest values in the large size class. No negative effects of tags on growth (tagged vs non-tagged fish using a middle size class) were found after 57 days of culture. Four additional trials using mini tags in a large size class at industrial scale validated our experimental results. With respect to morphology, no differences in the area, ellipticity and circularity were found except for a slight higher aspect ratio index in mini-and nano-tagged soles when compared with untagged fish. A longitudinal analysis of growth using the tag type, sex and tag position (anterior, medium or posterior) as fixed factors revealed a significant and strong effect of sex, with females appearing significantly heavier (13.6%) than males. In addition, the significant interactions between tag position and tag type with the time indicated a delayed growth of Nonatec-tagged fish and specimens with tags in the posterior section of abdomen. Expression analysis of stress-related genes revealed an activation of HPI axis and cellular stress defenses at 2 days just after tagging (dat) not evident at 11 dat. All these data indicate that soles can be successfully tagged at very small sizes both at experimental and industrial scales if tag type is properly selected and fish correctly handled. Moreover, sex and tag position are significant factors affecting growth that need to be controlled in longitudinal studies and selective breeding programs.Highlights ► A methodology for internal tagging of small soles was developed and validated at experimental and industrial scales. ► Optimal tag retention and mortality rates were obtained and no negative effects on growth or morphology were detected. ► Longitudinal analysis using internal tags revealed a major effect of sex, tag type and tag position on growth. ► Expression analysis indicated no signs of chronic stress linked to internal tags.

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