Probes to detect and quantify reactive oxygen species (ROS) are typically used in isolated cells or tissues. In shellfish, previous studies have quantified ROS in extracted haemolymph (e.g. Delorme et al., 2021b, Rolton and Ragg, 2020) or excised gill tissue (e.g. Rivera-Ingraham et al., 2016). The present study shows a novel technique that was developed to determine and quantify ROS in individual living shellfish. The protocol is appropriate for translucent juvenile mussels that do not have a heavily calcified or coloured shell (up to ∼4-5 mm of shell length in Perna canaliculus). This protocol could also be used in response to other biotic or abiotic stressors, e.g. hypoxia-reoxygenation stress, temperature fluctuations, bacterial infections, algal toxins.

Pilot trials using P. canaliculus determined that a standard staining incubation time of 30 min is sufficient to successfully detect and quantify ROS in haemocytes (Delorme et al., 2021b; Rolton and Ragg, 2020). However, when assessing P. canaliculus veliger larvae, increasing incubation time to 1 h resulted in an improved fluorescent signal (Delorme, unpublished data). Juvenile P. canaliculus typically closed their valves immediately after addition of the CellROX™ Green dye, reducing tissue exposure and further limiting the fluorescent signal. A non-toxic anaesthetic applied prior to staining and extending to 1 h the incubation time is therefore recommended for bivalves that can reactively isolate from their surrounding environment by closing their shells.

The distribution of the fluorogenic ROS signal observed inside the juvenile mussels shows that a larger part of the signal is located in the mussel mantle tissue. The function of the mantle in molluscs is to form the shell by secreting and depositing conchiolin and calcium carbonate as mussels grow (Chapman and Barker, 1964). Therefore, it is possible that a higher ROS signal in the mantle of juvenile mussels is the result of a higher metabolic activity of the mantle epithelial cells associated with shell formation in actively growing juveniles. Additionally, fluorescence intensity may depend on the volume or mass of the stained tissue. Using a different approach in which the quantification of the ROS signal is standardized by tissue volume or mass could perhaps result in a better understanding on how different tissues are being specifically impacted by the applied stress.

Juvenile P. canaliculus can be stranded by the receding tide or exposed to air during transfer from the hatchery to grow-out facilities in aquaculture (Delorme et al., 2020; Jeffs et al., 2018). Additionally, emersion in juvenile P. canaliculus has shown to result in high oxidative damage, particularly after 1 h of rehydration after emersion (Delorme et al., 2021a). Hence, the present study applied emersion and relative humidity treatments as relevant stressors likely to induce a range of ROS levels in P. canaliculus juveniles (Figs 1 and 2). Here, emersion and relative humidity had a significant effect on the ROS signal quantified in juvenile mussels (nested analysis of variance, ANOVA, F8,154=8.391, P=0.0048). Mussels that did not experience an emersion and relative humidity stress show significantly lower ROS signal than the other treatment groups (Fig. 2). There were no significant differences in ROS signal when juvenile mussels were exposed to air at high RH (Tukey, P=0.985; Fig. 2). However, when mussels were exposed to moderate RH during emersion, the ROS signal was ca. 60% higher in mussels that experienced emersion for 1 h than mussels exposed to air for 20 h (Tukey, P<0.001; Fig. 2). This response was unexpected and could be explained by homeostatic stress response activating antioxidant mechanisms to neutralise ROS in the organism (Espinosa-Diez et al., 2015). Since only living, apparently vigorous juvenile mussels were selected for staining after treatment, it is possible that under prolonged emersion conditions the antioxidant mechanisms in the selected mussels overcompensated for the large accumulation of ROS, resulting in a relatively low ROS signal (mean of 16% ROS) compared with the other treatments (mean between 26-38% ROS), but still higher than control mussels (mean of 5% ROS). Previous research has shown that the antioxidant activity increases greatly in juvenile P. canaliculus after exposure to different emersion times and relative humidity levels, especially when mussels are exposed to mid humidity conditions (Delorme et al., 2021a). This result suggests that selected (living) mussels were able to cope with the prolonged stress caused by a 20 h emersion and 1 h of recovery in seawater, but their subsequent capacity to completely recover and survive in the long term remains unknown. Recent studies in juvenile P. canaliculus have shown that survival during recovery (up to 10 h) is greatly compromised as relative humidity decreases (Delorme et al., 2021a). It is also worth noting that the strength of the ROS signal observed in dead mussels was comparable to that of stressed mussels. This may be attributable to the cold shock that the mussels suffered during the freezing, inducing ROS production in the process. The ROS signal found in dead (freeze-killed) mussels indicates that the ROS dye used in this study can penetrate dead cells and tissues and can be oxidised by the presence of preformed ROS or other oxidants. It is also important to note that oxidation reactions can still occur in dead tissues (Domínguez et al., 2019), and that some of these oxidation products could react with fluorogenic probes to give a fluorogenic signal. Therefore, it may be possible that a fluorogenic ROS signal could still be visible if mussels died during exposure to a stressor, but the production and lifespan of fluorogenic ROS signals in dead or frozen, and then thawed, tissues require further investigation.

In this study, juvenile mussels that were exposed to 1 h of emersion at moderate RH showed an increase of ca. 30% in ROS signal compared to mussels experiencing 1 h emersion at high RH (Tukey, P<0.022; Fig. 2). The opposite trend was observed when mussels were exposed to 20 h of emersion, where mussels experiencing high RH had a ca. 48% higher ROS signal compared to those that experienced 20 h of emersion at moderate RH (Tukey, P=0.001; Fig. 2). The differential ROS signal between treatments may be explained by differences in behaviour between mussels maintained under different conditions during emersion. It is possible that mussels opened their valves during emersion while others closed their valves, as observed in adult mussels showing different gaping behavioural strategies in response to emersion (Powell et al., 2017; Zamora et al., 2019). Mussels closing their valves would be potentially under anaerobic metabolism, presumably reducing ROS production compared to mussels attempting to maintain aerobic respiration under emersion conditions.

Overall, this study showed that the ROS detection protocol performed on juvenile P. canaliculus effectively distinguished differences in environmentally induced stress levels through quantification of the ROS signal in terms of area of coverage. While emersion stress in juvenile P. canaliculus has previously been shown to result in the accumulation of oxidative damage markers (Delorme et al., 2019, 2021a), as a consequence of increased ROS in the cells (Freire et al., 2012), the direct ROS quantification presented in the present study reveals complex kinetics that warrant further investigation. Additionally, future research would also benefit from exploring alternative fluorogenic probes to gain more insight to the origins of ROS production. Probes that detect different ROS types could be used to identify tissue-specific ROS production as previously seen in bivalve gill tissue (Rivera-Ingraham et al., 2016). Other probes that target mitochondrial ROS could be used to better understand the origins of ROS production under different stressful conditions. Furthermore, probes that detect either the effects of ROS accumulation in the cells, such as lipid peroxidation probes, or fluorogenic probes that detect antioxidants such as glutathione (indicating the ability of the cell to prevent oxidative stress) would complement this research and would allow us to better understand the fundamentals of the redox biology of juvenile P. canaliculus.