Linalool induces relaxation of the mantle of golden apple snail ( Pomacea canaliculata )

: The objective of this study was to evaluate the possible relaxing effect of essential oils (EOs) ( Aloysia triphylla and Lippia alba ) and phytochemicals (citral and linalool) in the gastropod Pomacea canaliculata . Animals were exposed to compounds at the concentrations range of 25-750 µL L –1 . Magnesium chloride (MgCl 2 , 10-50 g L –1 ) and control group (ethanol 6.75 mL L –1 , highest concentration used for treatment dilution) were also tested. The EOs, citral and MgCl 2 had no relaxing effect at the concentrations range tested, and citral caused aversive behavior (closure of the operculum) from 90 μL L –1 . Exposure to linalool at 25, 50, 100, 200 and 400 µL L -1 relaxed 28, 76, 88, 96 and 100% of the animals, respectively. The concentrations of 25, 50 and 400 µL L -1 differed statistically from each other, while 100 and 200 µL L -1 were equal to 50 and 400 µL L -1 . All animals recovered up to 40 min, except at of 400 µL L -1 . Linalool is effective for relaxing P. canaliculata and can be useful in management techniques that require relaxation. However, further studies are needed to certify whether linalool is appropriate for maintaining animal welfare in invasive procedures that require total insensitivity.

Natural products constitute an important source of bioactive and biodegradable molecules (Bakkali et al. 2008).Among the different vegetable extractives, essential oils (EOs) extracted from aromatic plants and/or isolated compounds are successfully used as anesthetics in fish (Baldisserotto et al. 2018, Bianchini et al. 2017b, Hoseini et al. 2019) and crustaceans (Li et al. 2018).Eugenol is effective as an anesthetic/relaxant in marine Haliotis tuberculata coccinea (Bilbao et al. 2010) and freshwater Pomacea poludosa and Pomacea canaliculata gastropods (Bianchini et al. 2017a, Garr et al. 2012), but not in the bivalve Pinctada maxima (Mamangkey et al. 2009).Recently, the anesthetic/relaxant action of Origanum majorana and Ocimum americanum in P. canaliculata have been described (Bianchini et al. 2017a).
In the current study, the possible relaxing effects of EOs from Lippia alba and Aloysia triphylla were evaluated.Both have anesthetic effects in fish (Almeida et al. 2018, Batista et al. 2018, Gressler et al. 2014) and shrimp (Litopenaeus vannamei;Parodi et al. 2012).The effectiveness of linalool and citral were also evaluated.These compounds are major constituents of the EOs of L. alba and A. triphylla, respectively, collected in certain regions (Almeida et al. 2018).In view of the anesthetic activity proven in other species, we believe that these EOs, as well as their isolated phytochemicals, can also be used in mollusk species.For this reason, we used the gastropod P. canaliculata as the study organism.

Animals
The animals were obtained from a fish culture in São João do Polêsine city (Rio Grande do Sul, Brazil) and then acclimated in 250-L tanks in a water re-circulation system with activated charcoal/stone filters (temperature 25 °C, pH 7.32, and dissolved oxygen levels 7.43 mg L -1 ).Animals were fed "ad libitum" with fresh lettuce.The identification of animals was carried out by Dr. Carla Bender Kotzian (Department of Biology, Federal University of Santa Maria, UFSM).Invertebrate experiments do not require approval by the ethics committee.

Essential oil and phytochemicals
The EOs were extracted from the leaves of A. triphylla (EOA) and L. alba (EOL) cultivated at the campus of the Universidade Federal de Santa Maria in the city of Frederico Westphalen, southern Brazil.The plant material was subjected to extraction by hydro-distillation in a Clevenger type apparatus.Subsequent chemical characterization was done by gas chromatography using an Agilent 7890A gas equipment coupled to an Agilent 5975C mass selective detector (GC-MS).The majoritarian constituents identified were geranial or citral A (24.32%), limonene (21.69%) and cis-Carveol (18.53%) in the EOA, and S-(+)-Linalool (66.34%), eucalyptol (10.63%) and aromadendrene (3.48%) in the EOL.More details on chromatographic conditions and chemical characterization can be found in Almeida et al. (2018).

Experimental design: relaxation
Animals (shell: 3.43 ± 0.28 cm) were placed in continuously aerated 1-L aquaria (n = 5/ aquarium, 5 replicates) for induction of relaxation and recovery.EOA, EOL, linalool, and citral, previously dissolved in 95% ethanol (1:10) were tested at concentrations range of 25-750 µL L -1 .Magnesium chloride (MgCl 2 , diluted in water) was tested at 10, 20, 40 and 50 g L -1 .A control group was exposed to ethanol (vehicle used for the dilution of EOs and phytochemicals) at the highest concentration used for treatment dilution (6.75 mL L -1 ).The protocol used to evaluate the relaxing activity of these animals was performed as described previously by Bianchini et al. (2017a) and adapted from Garr et al. (2012).Briefly, animals were considered relaxed when they did not show any resistance to the pulling of the operculum with the aid of a forceps.The animals were evaluated every 10 min for up to 40 min.The relaxed animals were transferred to anesthetic-free aquaria and the recovery time was evaluated at 10-min intervals.The animal was considered recovered when it presented resistance to pulling of the operculum.Mortality was evaluated 24 h after the experiment.

Aversive behavior assessment
The animals exposed to citral had aversive behavior characterized by immediate closure of the operculum (Figure 1).This behavior may have hindered the absorption of citral by the animal.Therefore, a second experiment with gradual exposure of the animals to citral was carried out.For this, a re-use system with Aquaria 1 and 2 (total 4 L) was set up.Into Aquarium 1, 5 μL L -1 citral was added every 5 min, which slowly came into equilibrium with Aquarium 2 in which the animals (n = 5, duplicate) were present.Citral concentration gradually increased until the animals (60%) closed the operculum.This concentration was considered as the minimum concentration of citral capable of causing aversive behavior.The objective of this second experiment was also to verify whether citral would have relaxing activity when its concentration increased gradually.Therefore, the verification of a possible relaxing effect was also performed according to the methodology used in the first experiment.

Statistical analysis
Data were evaluated by Kruskal-Wallis test followed by Dunn post-hoc.The statistical tests were performed using GraphPad Prism software (San Diego, CA, EUA, version 6.0), and the minimum significance level for all analyses was set at p < 0.05.Data are reported as mean ± SEM.

RESULTS
Relaxing effect EOL, EOA, MgCl 2 and ethanol control did not cause a relaxing effect up to the highest concentration tested (750 µL L -1 for Eos, 50 g L -1 for MgCl 2 and 6.75 mL L -1 for ethanol).In contrast, linalool caused relaxation in this species with maximum effect (100% relaxation) at 400 µL L -1 (Figures 1b and 2a).All animals recovered within the maximum observation period (40 min), except those exposed to the highest concentration (400 µL L -1 ).The lowest concentration tested (25 µL L -1 ) relaxed only 28 % (± 10.19%) of the animals tested.Regarding relaxation time, there was no correlation between time and concentration.At the most effective concentrations (50, 100, 200 and 400 µL L -1 ), the relaxation time did not differ significantly between them, with the exception of the 100 µL L -1 concentration which differed from 50 µL L -1 .
Citral did not cause relaxation.However, it was observed that exposure to all citral concentrations caused an aversive behavior in the animals characterized by the immediate closure of the operculum, which remained in this way for all the time the animals were in contact with the compound (Figure 1c).In relation to other adverse effects, no mortality occurred with the compounds tested in the 24-h period after the experiments.However, in animals exposed to linalool 400 µL L -1 loss of mucus was observed.

Aversive behavior assessment
The minimum citral concentration that promoted an aversive effect in the animals was 90 μL L -1 (the same value in the two duplicates).Citral also did not promote a relaxing effect in this exposure protocol.Animals returned to normal (opening of the operculum) quickly after citral removal.No mortality occurred after 24 hours.

DISCUSSION
Anesthetic substances can produce muscle relaxation, analgesia, sedation and even general anesthesia that involves general CNS depression (Winlow et al. 2018).In this study, the effectiveness of the compounds was based on the ability to induce relaxation of the mantle, identified by the lack of resistance to the act of pulling the operculum (Garr et al. 2012).Therefore, we use the term relaxation to designate this effect, a standard term used in other studies with bivalves (Acosta-Salmón et al. 2005, Acosta-Salmón & Davis 2007, Butt et al. 2008, Mamangkey et al. 2009) and gastropods (Aquilina & Roberts 2000, Bianchini et al. 2017a, Garr et al. 2012) where a similar methodology was used.The assessment of general anesthetic effect in molluscs may require non-behavioral methodologies.In an ex vivo study, for example, it was found that 2-phenoxyethanol, in addition to decreasing the muscle contraction force in isolated deganglionized oral masses, also reduces the excitability in central neurons of Hermissenda crassicornis (Wyeth et al. 2009), which is indicative of a general anesthetic agent.MgCl 2 is used as anesthetic in different species of mollusks and other invertebrates (Lewbart & Mosley 2012).Low concentrations usually cause relaxation in many species.In the gastropod P. paludosa, for example, 20 g L -1 of MgCl 2 was 80% effective (Garr et al. 2012).Contrary to expectation, for P. canaliculata it was not effective up to 50 g L -1 .It is likely that P. canaliculata has higher resistance to MgCl 2 compared to other species, due to some unknown factor.Adami et al. (2019) recently reported the difficulty in anesthetizing giant African snails (Acathina fulica) with alfaxalone and ketamine, well-known general anesthetics.It has also been demonstrated the existence of genetic variation for the phenotype of anesthesia efficacy in Potamopyrgus antipodarum, a New Zealand freshwater snail (Song et al. 2021).
Relaxation caused by linalool was expected, as this monoterpenoid has depressant activity in fish (Rhamdia quelen and Cyprinus carpio) and shrimp (Litopenaeus vannamei) species (Becker et al. 2015, Heldwein et al. 2014, Taheri Mirghaed et al. 2016).In addition, linalool is also known to act on the nervous system of mammals and rodents through mechanisms of action that include inhibition of glutamatergic receptors (Aprotosoaie et al. 2014), which can also be found in gastropods (Greer et al. 2019).Compared to other natural compounds previously tested in this species, linalool is as effective as eugenol (both promoted 100% relaxation) and more effective than the O. americanum EO (70%) and Origanum majorana EO (80%).Besides that, it was the most potent compound tested so far (Bianchini et al. 2017a).
Exposure to 50 µl L -1 linalool promoted relaxation in 76% or more of the animals, reaching 100% with 400 µl L -1 .However, at this concentration it did not occur total recovery of the animals up to 40 min, and besides there was loss of mucus.The increase in mucus production and release by gastropods is one of the first reactions to stressors, such as chemical irritation caused by molluscicidal agents.Its function is to create a protective barrier between the irritating agent and the animal's skin (Barker 2002).Therefore, due to the adverse effects observed with 400 µl L -1 linalool, concentrations higher than 200 µl L -1 are not recommended in this species.In addition, it was possible to achieve satisfactory efficacy, fast recovery time, and absence of the noticeable adverse effects at concentrations up to 200 µl L -1.
While linalool promoted relaxation, EOL (around 66% S-(+)-linalool) had no relaxing effect on golden apple snails, even at the highest concentration tested, in contrast to the results obtained in fish (Almeida et al. 2018, Heldwein et al. 2014).The other constituents of EOL may have antagonized the relaxing effect of S-(+)-linalool in golden apple snails by some unknown mechanism (Bakkali et al. 2008).The EOL has the S enantiomer, and the linalool we tested is made up of a racemic mixture (S-(+) and R-(-)-linalool).According to this information, we should also consider the hypothesis that the R enantiomer may be more potent than the S in producing relaxation in this species, which would explain in part the lack of effect of the EOL.On the other hand, S-(+)linalool is known for its anesthetic effect on R. quelen (Heldwein et al. 2014).Therefore, further studies are necessary to clarify this doubt.EOA was also not effective in P. canaliculata despite causing sedation/anesthesia in fish (Almeida et al. 2018, Gressler et al. 2014) and shrimp (Parodi et al. 2012).
A relaxing effect was also absent for citral, and aversive-like behavior was observed in the animals shortly after exposure.The closure of the operculum characterizes a defensive behavior against substances identified by the animal as potentially noxious.Similar behavior was observed in gastropods or bivalves exposed to sudden changes of environmental conditions (e.g.temperature) (McAlister & Fisher 1968).This same behavior was also observed during the attempt to anesthetize the giant African snails with ketamine and alfaxalone by different routes (Adami et al. 2019).The shell of mollusks, mainly bivalves and gastropods, has a protective function.Thus, the retraction into the shell and closing the operculum is one of the defense mechanisms and reflects the animal's perception of an unknown/dangerous situation, or chemical or physical stimulus (Goodchild et al. 2020).Considering that citral is an irritating substance for skin / mucous membranes in certain concentrations (OECD 2001), it is possible that citral caused irritation or discomfort in the mantle mucosa, triggering this aversive behavior.The absorption of citral was probably hindered by the closure of the operculum, and even when the animals came into contact with gradual concentrations, no relaxing effect was observed, possibly due to low concentrations (<90 µL L -1 ).Studies with other mollusks, particularly those devoid of shell, would be of great interest to better elucidate the effects of citral in these animals.
In conclusion, linalool may be useful as relaxant in gastropods.Nevertheless, other studies needed to be performed in order to test the effectiveness of linalool on other mollusks species, as well as the ideal concentrations.Not less important, the effectiveness of linalool in procedures that require a state of deep anesthesia must be confirmed in order to maintain animal welfare.

Figure 1 .
Figure 1.Different stages of Pomacea canaliculata exposed or not to substances with relaxing properties.(a) Normal stage or with essential oil without effect, (b) Relaxation stage, (c) Aversive-like effect observed for citral.

Figure 2 .
Figure 2. Percentage of relaxation (a) and recovery of Pomacea canaliculata exposed to linalool and their respective times (b).Lowercase letters indicate statistical difference between linalool concentrations in the relaxation stage, and uppercase letters indicate statistical difference between linalool concentrations in the recovery stage (Kruskal-Wallis test and Dunn post-hoc, p < 0.05, as mean ± SEM).