The most important finding of our study is the demonstration that the metabolic state of mussels sampled from the environment correlates with multiple biologically relevant parameters such as sex and gonadal development, and that it is potentially informative of site-specific environmental stressors, such as chemical contaminants. Our work provides useful knowledge to formulate hypothesis on the molecular basis of important biological processes such as gonadal development and may steer the development of future environmental monitoring tools.
Are sex-specific metabolites important in sex determination?
The five metabolites that are most predictive of sex were glycine, glutamate, gamma-aminobutyric acid (GABA), ATP and UDP-GlcNAc.
Glycine has been previously shown to have higher levels in male mussels (55). ATP is an energy currency of the cell and as it showed higher dynamics in male mussels and was correlated with gonadal development, it is possible that the sex-specificity is related to the content of ATP in sperm. This is supported by previous studies that relate ATP content with sperm quality (56).
In addition to having different dynamics in male and female mussels, ATP was also affected by site. Interestingly, a previous 1H-NMR metabolomics study, which reported models predictive of the scope for growth, showed that pentachlorophenol (PCP, used as a pesticide), a chemical that uncouples oxidative phosphorylation, increases respiration rate and reduces scope for growth in lab-exposed mussels (13). Moreover, using 1H-NMR spectroscopic data for adductor muscle for the same mussels as in the current study, the scope for growth was predicted to be lower in Southampton than in Exmouth (13).
ATP concentrations have been shown to be inversely correlated with respiration rate in sea urchin (57), meaning that site-effects might be due to specific chemicals which affect the oxidative phosphorylation, increase respiration rate and lower ATP concentrations. Unfortunately, we have no data available for the two sites about chemical contaminant concentrations.
Interestingly, gamma-aminobutyric acid (GABA) was linked to gonadal development in the model represented in Fig. 4 and ATP and UDP-GlcNAc were differentially abundant in the Southampton site.
Another interesting property of these metabolites is that three of them (glycine, GABA and glutamate) are known neurotransmitters (reviewed in 58, reviewed in 59, reviewed in 60). In addition to ATP being the ‘energy molecule’ in the cell and controlling a very large number of biological mechanisms, it also operates a wide range of channels involved in modulating the neuronal synapse and changes in its levels can have significant effects in nerve conduction (61, reviewed in 62). Since glycine, glutamate, GABA and ATP are all operating various ionotrophic receptors, it is reasonable to hypothesize that they may be involved in the control of sex determination or at least in the development and differentiation of the gonads.
There are several lines of evidence in support of this hypothesis. A recent gene expression study in the scallop has revealed that sodium- and chloride depending GABA transporters and sodium- and chloride dependent glycine transporters were over-expressed in the ovary (63). Importantly, GABA has been shown to be present and functional in bivalves (64, 65). In oyster, the homolog of glutamic acid decarboxylase (cgCAD), the rate-limiting enzyme for conversion of glutamate into GABA, has been identified and shown to be functional (65). The GABA transporter (GAT2) is also been found to be functional in oyster (64).
These observations raise the question of what the role of GABA in the reproductive system of molluscs may be. Although there are no data in mussels, we know that in vertebrates, GABA regulates GnRH neurons (66). Importantly, GnRH-like peptides play a role in the development of molluscan gonads as well. In the scallop, GnRH-like peptide has been found in the central nervous system where it stimulates spermatogenesis (67). In vivo studies in scallop have showed that GnRH accelerates spermatogenesis in males while inhibiting oocyte development in females whereby shifting sex balance towards males (68). GnRH is also involved in ovarian cell proliferation in abalone (69). In the absence of a real understanding of sex determination and differentiation systems in mussels the possibility that GABA may also regulate GnRH neurons in molluscs represents an exciting hypothesis.
Sex prediction models across geographical sites
We have shown that the metabolic state of male mussels sampled from the Southampton site is similar to females, in contrast to the well differentiated metabolic state of mussel samples from the Exmouth site. Despite the fact that M. edulis and M. galloprovincialis have different reproductive strategies (ref John paper), we could see that the model based on the metabolomics data predicted that organisms sampled from Southampton were females, regardless of the species.
It is possible that chemicals in the water of the Southampton sampling site or other environmental and health factors, may be responsible for changes in the development of the mussel gonads that our model is detecting. This raises the potential that male individuals with the molecular state of a female in the more polluted site might show the detectable changes underlying complex pathological indicators of health, offering a new tool for environmental research. In fact, samples from the Southampton site show a significantly higher prevalence of gonadal pathologies, such as atresia and apoptosis as well as brown cell inflammation. Atresia has been shown to be associated with the presence of pollution and endocrine disrupting chemicals (70), despite the fact that the molecular mechanism or role of these chemicals in mussels is not clear (reviewed in 71–73). Moreover, it has been demonstrated that polluted water downstream of municipal effluents or on shipping routes can alter the male-female ratio(70, 74) or up-regulate vitellogenin (75). Although the notion that the steroidogenic pathway is fully operational in invertebrate species (and its disruption leads to the above reproductive abnormalities) is highly controversial (71, 72), it is certain that more research is needed to characterise the basic physiology and endocrinology of reproduction in key marine ecological species such as mussels (76).