Advances in toxicological studies demand the extension of toxicity tests to additional levels beyond traditional endpoints such as survival, reproduction, or development. Nowadays, a molecular approach to toxicity evaluation is frequently taken, and it requires additional putative biomarkers that assess the modulation of different cellular processes and physiological mechanisms (Lee et al. 2015; Martins et al. 2019; Steiblen et al. 2020). In this sense, adding new genes to the battery of biomarkers extends the number of processes studied and the levels of response, depending on the pathway analyzed. Therefore, the description new genes is a step toward extending the value of Physella acuta in toxicological studies. Here we have described nine new sequences that code for different hormone receptors, an enzyme involved in regulating the concentration of active estrogens and androgens (estradiol 17-beta-dehydrogenase 8), one stress protein, and three proteins involved in the DNA repair. These genes can improve the analysis of different processes related to endocrine disruption, genotoxicity, and development. Furthermore, all of them can help us to understand the response on different levels of organization, from molecular to ecological, providing insights into the mechanisms of the toxicant and the responses of the organisms to maintain homeostasis in the face of a changing environment. On the other hand, these putative biomarkers open new ways to assess toxicity prior to its observation at the individual level, preventing irreversible damage that affects the population. Thus, as in clinical practice, new tools are required to better identify molecular events and obtain an earlier diagnosis that will help to detect pollution before it causes irreversible damaging effects on ecosystems.
For a long time, the search for pesticides with either low or no impact on non-target species has been a key agricultural aim. Analogs of the juvenile hormone have been one such pesticide since they mimic one specific hormone of arthropods, drastically reducing the risk to other species (Wilson 2004). It is known that Fenoxycarb affects the development and different cell processes in insects, arachnids, and crustaceans (Jungmann et al. 2009; Navis et al. 2018; Lee et al. 2020). However, the current poor knowledge of invertebrate physiology makes it necessary to test them in non-target species to ensure their low impact. The first element to consider is the fact that the response is observed at 1 µg/L of Fenoxycarb, which is the intermediate concentration used. It is very low compared to those that have an effect on insects and crustaceans (Cripe et al. 2003; Mahmoudvand and Moharramipour 2015; Hu et al. 2019). The lack of response at higher concentrations can be due to an earlier response, recovering the normal condition to the time of the analysis by the action of detoxification mechanisms. At the lower concentration, the lack of observed response could be due to the amount of toxicant being below the threshold concentration needed to trigger an effect. Another possibility could be that more time is necessary to reach the threshold concentration. Additional research employing different response times would allow elucidation of the cellular processes affected and the concentration-dependent consequences.
In this work, we have tested the response at the gene expression level of the analog of juvenile hormone, Fenoxycarb and observed a response that, although weak, demands additional studies to ensure the lack of toxicity in non-arthropods. As expected, no effect was observed in genes related to DNA repair mechanisms or the stress response. There are no data in the literature on studies of these genes, even in insects. A similar situation happens with energy metabolism, although there are some reports about the impact of fenoxycarb in lipids and carbohydrates of crustaceans (Arambourou et al. 2018; Hu et al. 2019, 2020). As far as we know, there is no previous report analyzing the response of detoxification mechanisms in the presence of Fenoxycarb. In P. acuta there is no change in the genes analyzed involved in phase I, phase II, and phase III of detoxification, suggesting that other proteins different from those analyzed here are responsible for the biotransformation of this chemical. In contrast to our results, with no changes in genes related to epigenetic regulation, it has been described that exposure for three days at 50 µg/L of Fenoxycarb can upregulate histone deacetylase in the water flea Moina macrocopa (Hu et al. 2020). This could reflect differential sensitivity to the compound but also the fact that the epigenetic changes in the water flea are related to mimicking of the juvenile hormone effects.
The effect on the acetylcholinesterase gene suggests some impact on the nervous system. As stated in the introduction, inhibition on rat brain acetylcholinesterase activity and nicotinic receptors have been observed (Smulders et al. 2003), suggesting that Fenoxycarb can have nervous effects on non-target organisms. Studies in nicotinic receptors showed that the mechanism of Fenoxycarb was noncompetitive (Smulders et al. 2004). Although Fenoxycarb has been used as an analog of juvenile hormone, it seems to also have some effect as the rest of carbamates by affecting the nervous system. The response observed in P. acuta supports a nervous effect and suggests that it could affect the ability of the snail to survive by altering the central nervous system. The increase observed in the transcription could be reflecting and attempt to compensate the inhibition on enzyme activity. Additional studies would help elucidate the putative effect on the snail's behavior or ability to respond to situations involving the nervous system. In any case, it is a fact to consider in the impact that Fenoxycarb can have in non-target species in the mid- and long-term.
On the other hand, the modulation of sHSP17.9 suggests some effect, but to determine the real impact on the cell is a complex matter. Small heat shock proteins are diverse proteins involved in the stress response and related to multiple cellular processes, including neural functions (de Los Reyes and Casas-Tintó 2022). In this sense, it is tempting to speculate that sHSP17.9 could encode some sHSP involved in neural physiology but the difficulties to establish the homology demand caution. Additional research will provide more information and could help to define the role of this protein in the cell. As biomarkers, the fact that sHSPs share the alpha-crystallin domain makes it easy to identify them. However, their high diversity in the N- and C-terminal regions complicate the identification of homologies between species. Consequently, a deeper study of this protein family is required to determine their roles in cell metabolism and to establish functional homologies between them. In any case, the alteration observed suggests that Fenoxycarb has some effect in the mid-term in Physella acuta, raising the possibility that it causes some reduction in the wellness of the snail.
Aplysianin-A is a protein involved in the immune response by acting as an antibacterial. This antibacterial glycoprotein inhibits both gram-positive and gram-negative bacteria in Aplysia kurodai (Kamiya et al. 1986). The alteration in transcriptional activity can produce a modulation of the response to bacterial infections, making P. acuta more sensitive to them. The impact observed in P. acuta suggests a putative alteration in immunity, being the first time that this possibility is suggested for Fenoxycarb. Additional studies involving more immune related genes are needed to confirm it and determine the effect in the long term survival of the population.