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

Abstract

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 (MgCl2, 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 MgCl2 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.

Key words
anesthetics; essential oil; gastropods; mollusks; relaxation

INTRODUCTION

Mollusks are important for various sectors of the economy, such as pearl production and food. The Food and Agriculture Organization (FAO) estimates that in 2016 mollusk production reached 17.1 million tons (metric) (FAO 2018FAO - FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS. 2018. The State of World Fisheries and Aquaculture: Meeting the sustainable development goals. http://www.fao.org/3/i9540en/I9540EN.pdf.
http://www.fao.org/3/i9540en/I9540EN.pdf...
). In addition, mollusks are extensively used for research purposes due to the anatomy of their nervous system (Winlow et al. 2018WINLOW W, POLESE G, MOGHADAM HF, AHMED IA & DI COSMO A. 2018. Sense and insensibility - An appraisal of the effects of clinical anesthetics on gastropod and cephalopod molluscs as a step to improved welfare of cephalopods. Front Physiol 9: 1147.). In this context, anesthetics are used to perform many procedures (Garr et al. 2012GARR AL, POSCH H, MCQUILLAN M & DAVIS M. 2012. Development of a captive breeding program for the Florida apple snail, Pomacea paludosa: Relaxation and sex ratio recommendations. Aquaculture 370-371: 166-171.), to ensure animal welfare, to assist in performing neurobiological research (Lewbart & Mosley 2012LEWBART GA & MOSLEY C. 2012. Clinical anesthesia and analgesia in invertebrates. J Exot Pet Med 21: 59-70.), by promoting animal relaxation and/or complete anesthesia. Among anesthetics/relaxants for mollusks, magnesium hydrochloride (MgCl2) is widely used in gastropods (Garr et al. 2012GARR AL, POSCH H, MCQUILLAN M & DAVIS M. 2012. Development of a captive breeding program for the Florida apple snail, Pomacea paludosa: Relaxation and sex ratio recommendations. Aquaculture 370-371: 166-171., Zeidan et al. 2018ZEIDAN GC, BARROSO CM & BOEHS G. 2018. Efficiency of anesthetics on female gastropod Littoraria angulifera. Bol Inst Pesca 44(3): e304.), bivalves (Puchnick-legat et al. 2015PUCHNICK-LEGAT A, LEGAT JFA, GOMES CHAM, SUHNEL S & MELO CMR. 2015. Anesthesia in oysters of the genus Crassostrea cultured in Brazil. Bol Inst Pesca 41: 785-793.), and cephalopods (Winlow et al. 2018WINLOW W, POLESE G, MOGHADAM HF, AHMED IA & DI COSMO A. 2018. Sense and insensibility - An appraisal of the effects of clinical anesthetics on gastropod and cephalopod molluscs as a step to improved welfare of cephalopods. Front Physiol 9: 1147.). Other substances used are ethanol (Lewbart & Mosley 2012LEWBART GA & MOSLEY C. 2012. Clinical anesthesia and analgesia in invertebrates. J Exot Pet Med 21: 59-70.), 1-phenoxy-2-propanol (Wyeth et al. 2009WYETH RC, CROLL RP, WILLOWS AOD & SPENCER AN. 2009. 1-Phenoxy-2-propanol is a useful anaesthetic for gastropods used in neurophysiology. J Neurosci Methods 176(2): 121-128.), 2-phenoxyethanol, benzocaine (Mamangkey et al. 2009MAMANGKEY NGF, ACOSTA-SALMON H & SOUTHGATE PC. 2009. Use of anaesthetics with the silver-lip pearl oyster, Pinctada maxima (Jameson). Aquaculture 288: 280-284.), and isoflurane (Polese et al. 2014POLESE G, WINLOW W & DI COSMO A 2014. Dose-Dependent Effects of the Clinical Anesthetic Isoflurane on Octopus vulgaris : A Contribution to Cephalopod Welfare. J Aquat Anim Health 26: 285-294.).

Natural products constitute an important source of bioactive and biodegradable molecules (Bakkali et al. 2008BAKKALI F, AVERBECK S, AVERBECK D & IDAOMAR M. 2008. Biological effects of essential oils - A review. Food Chem Toxicol 46: 446-475.). 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. 2018BALDISSEROTTO B, BARATA LES, SILVA AS, LOBATO FF, SILVA LL, TONI C & SILVA LVF. 2018. Anesthesia of tambaqui Colossoma macropomum (Characiformes: Serrasalmidae) with the essential oils of Aniba rosaeodora and Aniba parviflora and their major compound, linalool. Neotrop Ichthyol 16(1): e170128., Bianchini et al. 2017bBIANCHINI AE, GARLET QI, DA CUNHA JA, BANDEIRA JUNIOR G, BRUSQUE ICM, SALBEGO J, HEINZMANN BM & BALDISSEROTTO B. 2017b. Monoterpenoids (thymol, carvacrol and S-(+)-linalool) with anesthetic activity in silver catfish (Rhamdia quelen): evaluation of acetylcholinesterase and GABAergic activity. Braz J Med Biol Res 50(12): e6346., Hoseini et al. 2019HOSEINI SM, MIRGHAED AT & YOUSEFI M. 2019. Application of herbal anaesthetics in aquaculture. Rev Aquac (3): 550-564.) and crustaceans (Li et al. 2018LI Y, SHE Q, HAN Z, SUN N, LIU X & LI X. 2018. Anaesthetic effects of eugenol on Grass Shrimp (Palaemonetes sinensis) of different sizes at different concentrations and temperatures. Sci Rep 8: 1-9.). Eugenol is effective as an anesthetic/relaxant in marine Haliotis tuberculata coccinea (Bilbao et al. 2010BILBAO A, DE VICOSE GC, PINO VIERA MD, SOSA B, FERNÁNDEZ-PALACIOS H & HERNÁNDEZ MDC. 2010. Efficiency of Clove Oil as Anesthetic for Abalone (Haliotis Tuberculata Coccinea). J Shellfish Res 29: 679-682.) and freshwater Pomacea poludosa and Pomacea canaliculata gastropods (Bianchini et al. 2017aBIANCHINI AE, CUNHA JA, BRUSQUE ICM, PINHEIRO CG, SCHINDLER B, HEINZMANN BM & BALDISSEROTTO B. 2017a. Relaxing effect of eugenol and essential oils in Pomacea canaliculata. Ciênc Rural 47(10): e20170210., Garr et al. 2012GARR AL, POSCH H, MCQUILLAN M & DAVIS M. 2012. Development of a captive breeding program for the Florida apple snail, Pomacea paludosa: Relaxation and sex ratio recommendations. Aquaculture 370-371: 166-171.), but not in the bivalve Pinctada maxima (Mamangkey et al. 2009MAMANGKEY NGF, ACOSTA-SALMON H & SOUTHGATE PC. 2009. Use of anaesthetics with the silver-lip pearl oyster, Pinctada maxima (Jameson). Aquaculture 288: 280-284.). Recently, the anesthetic/relaxant action of Origanum majorana and Ocimum americanum in P. canaliculata have been described (Bianchini et al. 2017aBIANCHINI AE, CUNHA JA, BRUSQUE ICM, PINHEIRO CG, SCHINDLER B, HEINZMANN BM & BALDISSEROTTO B. 2017a. Relaxing effect of eugenol and essential oils in Pomacea canaliculata. Ciênc Rural 47(10): e20170210.).

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. 2018ALMEIDA APG, HEINZMANN BM, VAL AL & BALDISSEROTTO B. 2018. Essential oils and eugenol as anesthetics for Serrasalmus rhombeus. Bol Inst Pesca 44: 44-50., Batista et al. 2018BATISTA ES, BRANDÃO FR, MAJOLO C, INOUE LAKA, MACIEL PO, DE OLIVEIRA MR, CHAVES FCM & CHAGAS EC. 2018. Lippia alba essential oil as anesthetic for tambaqui. Aquaculture 495: 545-549., Gressler et al. 2014GRESSLER LT ET AL. 2014. Silver catfish Rhamdia quelen immersion anaesthesia with essential oil of Aloysia triphylla (L’Hérit) Britton or tricaine methanesulfonate: effect on stress response and antioxidant status. Aquac Res 45: 1061-1072.) and shrimp (Litopenaeus vannamei; Parodi et al. 2012PARODI TV ET AL. 2012. The anesthetic efficacy of eugenol and the essential oils of Lippia alba and Aloysia triphylla in post-larvae and sub-adults of Litopenaeus vannamei (Crustacea, Penaeidae). Comp Biochem Physiol C Toxicol Pharmacol 155: 462-468.). 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. 2018ALMEIDA APG, HEINZMANN BM, VAL AL & BALDISSEROTTO B. 2018. Essential oils and eugenol as anesthetics for Serrasalmus rhombeus. Bol Inst Pesca 44: 44-50.). 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.

MATERIALS AND METHODS

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)ALMEIDA APG, HEINZMANN BM, VAL AL & BALDISSEROTTO B. 2018. Essential oils and eugenol as anesthetics for Serrasalmus rhombeus. Bol Inst Pesca 44: 44-50..

Linalool (purity: 98%, density: 0.86 g mL–1, Sigma-Aldrich®, Brazil), citral (purity ³ 96% density: 0.89 g mL–1, Sigma-Aldrich®, Brazil) (racemic mixtures), and magnesium chloride (MgCl2, PA, Synth®, Brazil) were obtained commercially.

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 (MgCl2, 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)BIANCHINI AE, CUNHA JA, BRUSQUE ICM, PINHEIRO CG, SCHINDLER B, HEINZMANN BM & BALDISSEROTTO B. 2017a. Relaxing effect of eugenol and essential oils in Pomacea canaliculata. Ciênc Rural 47(10): e20170210. and adapted from Garr et al. (2012)GARR AL, POSCH H, MCQUILLAN M & DAVIS M. 2012. Development of a captive breeding program for the Florida apple snail, Pomacea paludosa: Relaxation and sex ratio recommendations. Aquaculture 370-371: 166-171.. 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.

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.

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, MgCl2 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 MgCl2 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.

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).

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. 2018WINLOW W, POLESE G, MOGHADAM HF, AHMED IA & DI COSMO A. 2018. Sense and insensibility - An appraisal of the effects of clinical anesthetics on gastropod and cephalopod molluscs as a step to improved welfare of cephalopods. Front Physiol 9: 1147.). 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. 2012GARR AL, POSCH H, MCQUILLAN M & DAVIS M. 2012. Development of a captive breeding program for the Florida apple snail, Pomacea paludosa: Relaxation and sex ratio recommendations. Aquaculture 370-371: 166-171.). Therefore, we use the term relaxation to designate this effect, a standard term used in other studies with bivalves (Acosta-Salmón et al. 2005ACOSTA-SALMÓN H, MARTÍNEZ-FERNÁNDEZ E & SOUTHGATE PC. 2005. Use of relaxants to obtain saibo tissue from the blacklip pearl oyster (Pinctada margaritifera) and the Akoya pearl oyster (Pinctada fucata). Aquaculture 246: 167-172., Acosta-Salmón & Davis 2007ACOSTA-SALMÓN H & DAVIS M. 2007. Inducing relaxation in the queen conch Strombus gigas (L.) for cultured pearl production. Aquaculture 262: 73-77., Butt et al. 2008BUTT D, O’CONNOR SJ, KUCHEL R, O’CONNOR WA & RAFTOS DA. 2008. Effects of the muscle relaxant, magnesium chloride, on the Sydney rock oyster (Saccostrea glomerata). Aquaculture 275: 342-346., Mamangkey et al. 2009MAMANGKEY NGF, ACOSTA-SALMON H & SOUTHGATE PC. 2009. Use of anaesthetics with the silver-lip pearl oyster, Pinctada maxima (Jameson). Aquaculture 288: 280-284.) and gastropods (Aquilina & Roberts 2000AQUILINA B & ROBERTS R. 2000. A method for inducing muscle relaxation in the abalone, Haliotis iris. Aquaculture 190: 403-408., Bianchini et al. 2017aBIANCHINI AE, CUNHA JA, BRUSQUE ICM, PINHEIRO CG, SCHINDLER B, HEINZMANN BM & BALDISSEROTTO B. 2017a. Relaxing effect of eugenol and essential oils in Pomacea canaliculata. Ciênc Rural 47(10): e20170210., Garr et al. 2012GARR AL, POSCH H, MCQUILLAN M & DAVIS M. 2012. Development of a captive breeding program for the Florida apple snail, Pomacea paludosa: Relaxation and sex ratio recommendations. Aquaculture 370-371: 166-171.) 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. 2009WYETH RC, CROLL RP, WILLOWS AOD & SPENCER AN. 2009. 1-Phenoxy-2-propanol is a useful anaesthetic for gastropods used in neurophysiology. J Neurosci Methods 176(2): 121-128.), which is indicative of a general anesthetic agent.

MgCl2 is used as anesthetic in different species of mollusks and other invertebrates (Lewbart & Mosley 2012LEWBART GA & MOSLEY C. 2012. Clinical anesthesia and analgesia in invertebrates. J Exot Pet Med 21: 59-70.). Low concentrations usually cause relaxation in many species. In the gastropod P. paludosa, for example, 20 g L–1 of MgCl2 was 80% effective (Garr et al. 2012GARR AL, POSCH H, MCQUILLAN M & DAVIS M. 2012. Development of a captive breeding program for the Florida apple snail, Pomacea paludosa: Relaxation and sex ratio recommendations. Aquaculture 370-371: 166-171.). 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 MgCl2 compared to other species, due to some unknown factor. Adami et al. (2019)ADAMI C, MONTICELLI P & D’OVIDIO D. 2019. Challenges encountered while attempting anaesthesia of giant African snails (Acathina fulica). Vet Anaesth Analg 46: 713-715. 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. 2021SONG Q, MAGNUSON R, JALINSKY J, ROSEMAN M, & NEIMAN M. 2021. Intraspecific genetic variation for anesthesia success in a New Zealand freshwater snail. Genetica 149: 47-54.).

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. 2015BECKER A, JENSEN L, GARCIA LO, SILVA LL, MONSERRAT JM, WASIELESKY W, HEINZMANN BM & BALDISSEROTTO B. 2015. The efficacy of linalool on transportation and anesthesia of white shrimp Litopenaeus vannamei (Crustacea, Penaeidae). FENACAN 54-60., Heldwein et al. 2014HELDWEIN CG, SILVA LL, GAI EZ, ROMAN C, PARODI TV, BÜRGER ME, BALDISSEROTTO B, FLORES MDM & HEINZMANN BM. 2014. S-(+)-Linalool from Lippia alba: Sedative and anesthetic for silver catfish (Rhamdia quelen). Vet Anaesth Analg 41: 621-629., Taheri Mirghaed et al. 2016TAHERI MIRGHAED A, GHELICHPOUR M & HOSEINI SM. 2016. Myrcene and linalool as new anesthetic and sedative agents in common carp, Cyprinus carpio - Comparison with eugenol. Aquaculture 464: 165-170.). 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. 2014APROTOSOAIE AC, HǍNCIANU M, COSTACHE II & MIRON A. 2014. Linalool: A review on a key odorant molecule with valuable biological properties. Flavour Fragr J 29: 193-219.), which can also be found in gastropods (Greer et al. 2019GREER JB, MAGER EM & FIEBER LA. 2019. Altered expression of ionotropic L-Glutamate receptors in aged sensory neurons of Aplysia californica. PLoS ONE 14(5): e0217300.). 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. 2017aBIANCHINI AE, CUNHA JA, BRUSQUE ICM, PINHEIRO CG, SCHINDLER B, HEINZMANN BM & BALDISSEROTTO B. 2017a. Relaxing effect of eugenol and essential oils in Pomacea canaliculata. Ciênc Rural 47(10): e20170210.).

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 2002BARKER GM. 2002. Molluscs as Crop Pests. New Zealand: ABI publishing, 400 p.). 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. 2018ALMEIDA APG, HEINZMANN BM, VAL AL & BALDISSEROTTO B. 2018. Essential oils and eugenol as anesthetics for Serrasalmus rhombeus. Bol Inst Pesca 44: 44-50., Heldwein et al. 2014HELDWEIN CG, SILVA LL, GAI EZ, ROMAN C, PARODI TV, BÜRGER ME, BALDISSEROTTO B, FLORES MDM & HEINZMANN BM. 2014. S-(+)-Linalool from Lippia alba: Sedative and anesthetic for silver catfish (Rhamdia quelen). Vet Anaesth Analg 41: 621-629.). 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. 2008BAKKALI F, AVERBECK S, AVERBECK D & IDAOMAR M. 2008. Biological effects of essential oils - A review. Food Chem Toxicol 46: 446-475.). 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. 2014HELDWEIN CG, SILVA LL, GAI EZ, ROMAN C, PARODI TV, BÜRGER ME, BALDISSEROTTO B, FLORES MDM & HEINZMANN BM. 2014. S-(+)-Linalool from Lippia alba: Sedative and anesthetic for silver catfish (Rhamdia quelen). Vet Anaesth Analg 41: 621-629.). 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. 2018ALMEIDA APG, HEINZMANN BM, VAL AL & BALDISSEROTTO B. 2018. Essential oils and eugenol as anesthetics for Serrasalmus rhombeus. Bol Inst Pesca 44: 44-50., Gressler et al. 2014GRESSLER LT ET AL. 2014. Silver catfish Rhamdia quelen immersion anaesthesia with essential oil of Aloysia triphylla (L’Hérit) Britton or tricaine methanesulfonate: effect on stress response and antioxidant status. Aquac Res 45: 1061-1072.) and shrimp (Parodi et al. 2012PARODI TV ET AL. 2012. The anesthetic efficacy of eugenol and the essential oils of Lippia alba and Aloysia triphylla in post-larvae and sub-adults of Litopenaeus vannamei (Crustacea, Penaeidae). Comp Biochem Physiol C Toxicol Pharmacol 155: 462-468.).

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 1968MCALISTER RO & FISHER FM. 1968. Responses of the false limpet, Siphonaria pectinata Linnaeus (Gastropoda, Pulmonata) to osmotic stress. Biol Bull 134(1): 96-117.). 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. 2019ADAMI C, MONTICELLI P & D’OVIDIO D. 2019. Challenges encountered while attempting anaesthesia of giant African snails (Acathina fulica). Vet Anaesth Analg 46: 713-715.). 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. 2020GOODCHILD CG, SCHMIDT LM & DURANT SE. 2020. Evidence for the ‘behavioural character’ hypothesis: does boldness programme disparate antipredator strategies? Anim Behav 164: 123-132.). Considering that citral is an irritating substance for skin / mucous membranes in certain concentrations (OECD 2001OECD. 2001. SIDS Initial Assessment Report for 13th SIAM. Citral. CAS N°:5392-40-5. Japan.), 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.

ACKNOWLEDGMENTS

A.E. Bianchini received a Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) scholarship. B. Baldisserotto and B.M. Heinzmann received Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) research fellowships, and S. Descovi FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul) fellowship.

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Publication Dates

  • Publication in this collection
    22 Oct 2021
  • Date of issue
    2021

History

  • Received
    19 Jan 2021
  • Accepted
    4 July 2021
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