The development of massive sequencing has provided a relatively cheap method to obtain the transcriptome of a species. Taking advantage of it, we have used a previously obtained transcriptome to identify eighteen genes related to different pathways of interest in ecotoxicology. With others yet described, the genes identified allow us to analyze these pathways' response to different chemicals. The genes identified cover processes of interest in ecotoxicology such as DNA repair mechanisms, stress, detoxification, apoptosis, immunity, energy reserves, and lipid transportation. There is a growing interest in combining ecologically relevant endpoints with biochemical and molecular parameters to seek a more integrative analysis. In this sense, to increase the number of genes described will allow for the design of standard arrays that could be used in combination with toxicity tests. In this way, initiatives such as the Adverse Outcome Pathway wiki23 will increase its relevance in assessing old and new compounds and provide putative mechanisms of action to explain the differences to the animals' specific physiology. Furthermore, increasing knowledge at the molecular level in this species supports its use as a representative of freshwater gastropods in toxicity analysis. There is a lack of model organisms in freshwater mollusk, being one of the animal groups whose pollution response is currently less known.
The eighteen genes evaluated in this work show homology with those previously described in other species, as expected. Rad21 and rad50 are both genes involved in DNA repairing mechanisms. The first one is an essential gene encoding a DNA double-strand break (DSB) repair protein20. In contrast, the second is a member of the protein complex MRN (including Mre11, Rad50, and Nbs1) that functions in DNA double-strand break repair to recognize and process DNA ends as well as a signal for cell cycle arrest24. Practically, there is no information about these genes in mollusks, with only one report in Carssostrea gigas for rad5025. The relevance of these genes is that they can be combined with other methodologies, such as the comet assay, to perform an integrated study to know if a compound is genotoxic and if the organism has the ability to compensate for the damage.
The catalase and the Mn superoxide dismutase genes allow us to analyze the impact of oxidative stress. Usually, oxidative stress analysis is focused on biochemical parameters, such as the enzyme's activity. However, it should also include a transcriptional activity study since it can provide additional information about the mid- and long-term response. The protein turnover can also be relevant in the response, especially in chronic exposure to toxicants. It is similar to that concerning detoxification mechanisms. The GST activity is one of the most used methods to assess detoxification26, but it does not differentiate between the members involved. A similar situation is found with the cytochromes P450, which show high diversity with many roles in the cell27. The Cyp72a15 gene increases the number of Cyp450s described in P. acuta and helps to elucidate how the organism can process the toxicants.
The small heat shock gene and the heat shock cognate gene extend the battery of genes available to assess this species' stress response. The small heat shock gene is difficult to match with other species' genes because the alpha-crystallin domain characterizes them, but there is no other sequence that, presently, allows for the homology to be established. Additional functional studies will help search for it. However, it is worth mentioning that HIF1α offers a new aspect of stress related to hypoxia28. The stress response is mainly focused on the canonical heat shock proteins, so other mechanisms involved in specific stresses, such as hypoxia, usually are neglected. The description of the factor inducible by low oxygen levels will help to know the effect of the toxicant on oxygen intake.
Similarly, the rest of the genes identified allow for the analysis of additional pathways that can also be altered by toxicants, like apoptosis (AIF3), the immune system (ApA), energy reserves (PYGL), and lipid transport (ORP8). To our knowledge, these genes are analyzed for the first time concerning pollution in freshwater mollusks. The last three genes, DNMT1, KATB6, and HDAC1, are involved in epigenetic mechanisms. Epigenetic regulation is arising as one of the long-term effects of toxicants. However, the genes involved in invertebrates are still poorly represented in toxicity analysis. The description of these three genes opens the possibility of analyzing their role in the epigenetic response and the relevance that they have in the transgenerational effects that have started to be described with different toxicants29–31.
Plastics in the environment are a growing problem that involves the release of polymers themselves and the compounds used as catalysts and additives during the degradation process. Phthalates are such additives that are increasing their presence in the environment5,32,33. Three phthalates, BBP, DEP, and DEHP, have been analyzed in this work, which showed a differential impact in Physella acuta. Two of the compounds, DEP and DEHP, did not show any change in the genes' mRNA levels. It has been described previously that both phthalates can alter the physiology of invertebrates15,34−37, including mollusks38–40. The differences observed here can be assigned to the type of analysis (molecular vs. physiological), the exposure time (one week vs. a few hours or days), the concentration used (µg/L vs. mg/L), and evidently, the species used. Additional research will help elucidate the putative relationship between the different data obtained and those from other organisms. However, it is essential to highlight that the obtained results suggest that Physella acuta can manage the environmentally relevant doses used in this work for DEP and DEHP. This species may be less sensitive to these phthalates, but this eventually will require further research, including the use of other methodological approaches, to confirm it.
On the other hand, BBP showed a very different picture, with almost all genes increasing the mRNA levels in response to the compound. Previous studies in other organisms have confirmed that BBP can induce different types of damage such as apoptosis41, genotoxicity42, oxidative stress43, stress response activation44, or endocrine disruption13. Although there are studies in invertebrates showing the impact on development and other physiological processes38,45, most of them did not focus on the putative mode of action, with only a few of them trying to delve into the mechanisms of response. BBP can affect very diverse processes in the organisms, and here we have found that all the pathways analyzed are modulated. The results can be considered in two ways. The first is that the changes are reflecting specific alterations on these pathways. It would mean that BBP is the most active phthalate in P. acuta, with a broad spectrum of action and a potential activity on many pathways. The second possibility is that BBP induces some change in other non-tested processes, like the hormonal system, that increases the transcriptional rate of the genes because of its regulatory role in the cell. It is relevant to keep in mind its known role as an endocrine disruptor of BBP46. However, a recent study in Daphnia magna can help elucidate between both possibilities since, by RNA-Seq, it has been observed that genes involved in signal transduction, cell communication, and embryonic development were significantly down-regulated, while those related to biosynthesis, metabolism, cell homeostasis, redox homeostasis were remarkably upregulated upon BBP exposure47. Although the organism and the stage analyzed are different than in our study, those results support the idea that BBP can have multiple effects on the cell metabolism, altering the pathways analyzed.
It is also essential to consider that most of the studies on invertebrates that involve transcriptional activity analysis use short exposure times and use arthropods13,44,47. Limited data are available on mollusks and, usually, they are on the marine representatives39,46. To our knowledge, this is the first study on a freshwater snail that shows that BBP can produce a substantial effect on cell metabolism. Several of the altered pathways can explain, in some way, the effects observed in other organisms, like the DNA repairing mechanisms, which are related to DNA damage, or the alteration of the genes involved in histone and DNA modification, which are related to epigenetic regulation. However, other genes open new possibilities, such as ApA and ORP8, involved in pathways not studied in other organisms. The changes in genes involved in oxidative stress, stress response, and detoxification back previous analysis, adding new light about the mechanisms involved in modulating these processes. The absence of changes in GSTm1 supports a differential role for each GST family in response to toxicants. On the other hand, the differences concerning concentrations for some of the genes suggest subtle differences requiring additional kinetic analysis to elucidate early and late activated genes.
As stated before, the results obtained in this work show that DEP and DEHP have no apparent effect in a week exposure at environmentally relevant concentrations. However, BBP shows a strong effect in comparison. It could be due to several reasons that need to be explored in future works. One possibility is the structure of each compound. In this sense, BBP has two rings while DEP and DEHP have only one. This factor could determine the biological activity that they have. Another possibility is that DEP and DEHP have effects earlier than the time studied, and the cell returned to the basal state, being able to process and remove the compounds. Finally, it cannot be dismissed that DEP and DEHP are not toxic for P. acuta, at least at environmentally relevant concentrations. In any case, BBP can alter the metabolism of this species. Additional research should be done in P. acuta and other freshwater species to know the impact on organisms based on freshwater ecosystems' food web.