What do oysters smell? Electrophysiological evidence that the bivalve osphradium is a chemosensory organ in the oyster, Magallana gigas

As in the olfactory system of fishes (Caprio 1978; Hara 1994; Kasumyan 2004), the bivalve osphradium proved to be sensitive to amino acids, albeit with slightly higher thresholds of detection. The EOsG responses in oysters are distinct from those in vertebrates, being slower and of lower amplitude than those of fishes and lacking the fast-adapting initial phasic response. For example, in the chameleon cichlid (Australoheros facetus), a freshwater fish, the standard stimulus l-serine (10− 5 M) evoked EOG amplitudes about 4.5 times higher (3.77 ± 1.38 mV) (Hubbard et al. 2017) than those evoked by l-serine (10− 3 M) (0.85 ± 0.208 mV) in oysters. However, if one compares the responses evoked by oysters (0.53 ± 0.09 mV) to those evoked by saltwater fishes, as is the case of Senegalese sole (Solea senegalensis) (Velez et al. 2005), the responses evoked by the standard stimulus (l-cysteine, 10− 3 M) are similar in amplitude, around 0.5 mV for sole and 0.53 ± 0.09 mV in oysters. This is due to the fact that EOG carried out in seawater is less sensitive than that in freshwater, as a result of the water conductivity—the higher the conductivity, the lower the resistance and therefore the amplitude will be lower (Hubbard et al. 2011; Hubbard and Velez 2020). Given that the electro-osphradiogram is a DC voltage signal measured in seawater, the method may slightly underestimate the true sensitivity, as has been shown in marine fish (Hubbard et al. 2011).

In vertebrates, the EOG is characterized by a phasic response with a rapid negative deflection at the beginning of stimulus exposure, followed by an adaption period and a slower-adapting tonic response (e.g. Chaput 2000; Frade et al. 2002; Hubbard et al. 2003b, 2011; Lalloué et al. 2003; Velez et al. 2005; Eom et al. 2009; Lapid and Hummel 2013), whereas in oysters, the EOsG was characterized by a slow negative deflection at stimulus onset, followed by a tonic response with little or no sign of accommodation. The return to baseline levels occur within seconds. This difference may be because oysters are sessile organisms, and the only decision to make in a presence of a certain odorant is to open or close the valves, rather than actively follow an odour plume (Atema 2012), such as mobile animals.

The electrode position within the osphradium, as well as the variability between individuals, may have caused some fluctuation in the recorded responses for the same amino acid. This variation highlights the importance of normalizing the recorded responses to the standard stimulus, in this case, l-cysteine (10− 3 M).

The use of magnesium chloride (MgCl2), a muscle relaxant widely used as anaesthetic in bivalves (Culloty and Mulcahy 1992; Butt et al. 2008; Suquet et al. 2009; Alipia et al. 2014), was efficient to prevent the contraction of the adductor muscle and therefore keep the shell open (Butt et al. 2008; Suquet et al. 2009; Azizan et al. 2021). However, MgCl2 blocks calcium channels in the membrane of presynaptic terminals (Namba et al. 1995; Azizan et al. 2021) and consequently interferes with electrical signal transduction. Indeed, there was little or no response to amino acids immediately after exposure to MgCl2 (data not shown). However, after overnight recovery in clean seawater, it was possible to record from the osphradium; oysters are able to recover within 24 h with no physiological effects caused by MgCl2 (Namba et al. 1995).

Like fishes, oysters were highly sensitive to aliphatic (e.g. l-leucine, l-valine, glycine), hydroxylic (e.g. l-serine and l-threonine), amidic (l-asparagine and l-glutamine), and to sulphur-containing amino acids such as l-cysteine and l-methionine (Velez et al. 2005; Hubbard et al. 2011). Apparently, and in contrast to fishes, oysters are more selective in which amino acids they detect. The rank order of potency in oysters also differs from that described for fishes. For instance, in blackspot seabream (Pagellus bogaraveo) and in Senegalese sole (Solea senegalensis), l-proline proved to be the least potent amino acid (Velez et al. 2005; Hubbard et al. 2011), whereas, in oysters, it was highly potent. l-Arginine, which evoked a strong response in the upper epithelium of Senegalese sole as well as is pointed as one of the most stimulative amino acids for goldfish (Rolen et al. 2003; Velez et al. 2005), did not evoke any response in oysters (Fig. 4). Neither did l-glutamate nor l-aspartic acid (Fig. 4) evoke any response in oysters. This suggests that, like marine fishes, oysters are less responsive to acidic amino acids (l-glutamic acid and l-aspartic acid) (Velez et al. 2005; Hubbard et al. 2011). However, in the marine gastropod Buccinum undatum, these acidic amino acids were the most effective chemical stimuli, inducing strong responses even at low concentrations (Bailey and Laverack 1966).

The apparent sensitivity to amino acids was lower than that in fishes, with thresholds of detection between 10− 6.36 to 10− 5.27 M, while in fishes, like Mozambique tilapia (Oreochromis mossambicus) and other teleosts, and blackspot seabream, the threshold of detection ranged from 10− 9 to 10− 5 M (Kasumyan 2004; Hubbard et al. 2011; Kutsyna et al. 2016). Moreover, Kutsyna et al. (2016) observed that amino acids with lower thresholds of detection elicited higher EOG amplitudes, in the Mozambique tilapia. The same pattern was seen in oysters. For example, l-asparagine and l-cysteine evoked larger amplitude EOG responses and the thresholds of detection were correspondingly lower. However, some amino acids, such as l-tyrosine and l-phenylalanine, evoked lower EOG amplitudes but had relatively low thresholds of detection (Fig. 5).

Similar to fishes, oysters seem to be more responsive to l-amino acids, probably due to their ubiquity in nature and their involvement in food identification and location (Hara 1994; Velez et al. 2007). Amino acids are potent odorants for aquatic organisms, inducing strong responses and triggering feeding behaviour in a wide variety of species, such as fishes (e.g. Hara 2006), crustaceans (e.g. Fuzessery and Childress 1975), gastropods (e.g. Bailey and Laverack 1966; Croll 1983; Wedemeyer and Schild 1995; Magel et al. 2007), larval amphibians (Arzt et al. 1986; Heerema et al. 2018) and, in the current study, bivalves. The fact that several groups of invertebrates (e.g. gastropods and crustaceans) detect amino acids may indicate that amino acid chemoreceptors may be a common feature among invertebrates (Bailey and Laverack 1966). However, due to the use of slightly different experimental approaches, it is not possible to directly compare oysters with other invertebrates.

As in fishes (Hubbard et al. 2003a), the osphradium of oysters proved to be highly sensitive to conspecific milt. In fact, in spawning trials, besides physical stimulation (e.g. thermal shock), conspecific sperm is widely used as an additional stimulus to induce oysters, namely females, to spawn. Since oysters are external fertilizing broadcast spawners, it is crucial for conspecifics to be able to detect the gametes released in order to synchronize spawning and therefore maximize fertilization. This may suggest a role of the osphradium in such spawning synchronization, as proposed by Haszprunar (1987a). However, further research is needed including, but not limited to, identifying the chemicals involved.

The neurotransmitter serotonin is known to act as a spawning inducer in bivalves (Gibbons and Castagna 1984), while γ-aminobutyric acid (GABA) is known for its role as an inducer of settlement and metamorphosis in bivalve larvae (García-Lavandeira et al. 2005; Mesías-Gansbiller et al. 2008, 2013). That these two compounds did not evoke any EOsG response in oysters may suggest a direct effect on the reproductive and/or nervous system of bivalves, rather than via the osphradium. Thus, although serotonin is known as a spawning inducer in bivalves (Gibbons and Castagna 1984), it cannot be considered a pheromone.

The knowledge of the chemosensory role of the osphradium in bivalves may be relevant in the development of aquaculture techniques. Recently, there has been an increasing demand for alternative diets for bivalves (Knauer and Southgate 1996; McCausland et al. 1999; Arney et al. 2015; Rato et al. 2018). This technique may prove useful in the formulation of alternative diets, by seeking to include, in the formulation, food-related odours with higher olfactory potency and therefore improve the overall acceptance by bivalves.

To our knowledge, this was the first time an EOsG recording was successfully carried out in any bivalve and strongly supports the hypothesis that the osphradium is a chemosensory organ (Haszprunar 1987a) in this taxon, as it is in other molluscs. Subsequently, a whole series of questions about chemoreception in bivalves may finally be answered. How do bivalves perceive the surrounding environment? What is the role of chemical cues in reproduction and predator avoidance? Are they able to detect predators or conspecifics nearby? The ‘electro-osphradiogram’ (EOsG) may prove to be a powerful tool in the isolation and characterization of pheromones and other important chemical cues for bivalves. Future research on bivalve chemoreception, as well as establishing how widely applicable the ‘electro-osphradiogram’ is to other bivalves, is needed to fully understand the role of the osphradium as a chemosensory organ.

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