Eugenol, Another Way to Anaesthetize Ascidians, [On Line]

Wilfried Bay-Nouailhat, Anne Bay-Nouailhat, Association Mer et littoral, Melgven, France, contact: [email protected].

Bay-Nouailhat, W. and Bay-Nouailhat, A., november 2015 Eugenol, another way to anaesthetize ascidians, [On line] http://www.mer-littoral.org/eugenol-ascidians-anaesthetic.pdf

Eugenol, another way to anaesthetize ascidians

Laboratory observation of ascidians requires first where the animals are. It may also be combined to collect samples with as less trauma as possible, with menthol for more nervous species which next to anaesthetize them and finally to fix them. can contract easily. In this case, menthol is used The use of crystals of menthols as anaesthetic, as pre-anaesthetic for about 20 minutes and once long proves its effectiveness. Crystallized men- the sea squirt is relaxed, 1 or 2 drops of euge- thol has the advantage of being readily available, nol can be added in the container to complete at a low cost and easy to use: you only need to anaesthesia. let it dissolve in a container full of sea water to anaesthetize the collected ascidians. Detailed protocol: However, this anaesthetic has the disadvantage of being relatively slow-acting (about 1 to 2 hours) Context: most of the samples are collected far as well as being less effective with certain species away from the laboratory. The result is that after which contract under the effect of the fixative, a few hours of travel by boat and/or car, samples that’s why menthol is sometime combined with do not open up once placed into the water MS222, another anaesthetic (Lafargue, 1977). tank, so we prefer carrying out the whole pro- MS222 is used in fish farming to anaesthetize cess of fixation at once out in the field. The use fish. This product is more difficult to get than of menthol alone did not satisfy us because the menthol crystals and more expensive. These two slow action of this product which is about one reasons led us to experiment another anaesthetic hour, doesn’t fit the time available in the field. also used in fish farming, eugenol. So, we’ve experimented eugenol to anaesthetize Eugenol or clove oil is a natural product extrac- ascidians, experiment which gave us complete ted from flowers of Syzygium aromaticum (L.) satisfaction. Merr. & L. M. Perry (=Eugenia caryophyllata). Known for centuries for its anaesthetic powers, Method: as soon as they are collected, each it is particularly used by dentists to relieve sample is put into a 100ml sampling container toothache. It is a cheap easy-to-get product. In with screw cap previously to fixation process. France, it is available in laboratory supplies but They are never taken out of the water. also in pharmacies and even in supermarkets. In summer, sampling containers are stored in a It comes in an oily liquid form and possesses a cool box to avoid increase of water temperature. strong anaesthetizing power: one drop of clove If necessary, some water is pour out of the contai- oil (90% eugenol) in one liter of water (0,4ml/10l) ner to make room for the preservative. We give is enough for a fish to sleep within 2 minutes time to sea squirts to fully open out before begin- with the possibility of latter waking (Chanseau ning anaesthesia which, with samples carefully and all, 2002). collected adhering to their support and without For ascidians, eugenol can be used alone by trauma due to transport for example, can hap- directly adding one or two drops in the water pen very quickly, in less than 10 minutes.

1 For the most sensitive species that readily react their shapes closely resemble those of living spe- to vibrations, we pre-anaesthetize samples with cimens. menthol, by simply adding some crystals of menthol into sea water. After a period of 15-30 Acknowledgements: minutes, when the ascidia under the effect of menthol begins to relax, we add 2 drops of euge- We warmly thank Annaik Cocaud (Bio-Littoral) nol in the container. and Jean-Michel Crouzet (Mer et Littoral) for For less sensitive species, pre-anaesthesia is not their help in the implementation of this protocol. necessary. Note that eugenol badly dissolve in water and it’s conveniently possible to dissolve it References: before hand in a bottle filled with sea water, and to properly shake this mixture before pouring Chanseau, M., Bosc, S., Galiay, E., Oules, G. some of it into the sample container. Anaesthesia 2002. L’utilisation de l’huile de clou de girofle happens within a few seconds and samples can comme anesthésique pour les smolts de saumon then be fixed in formaldehyde. atlantique (salmo salar L.) et comparaison de ses effets avec ceux du 2-phenoxyethanol. Bulletin Advantages: eugenol is a cheap easy-to-find français de la pêche et de la pisciculture, n° 365- product. It is a very powerful anaesthetic for 366, p. 579 - 589 ascidians and 1 to 2 drops for 100ml of water Lafargue, F. 1977 Révision Taxonomique des Di- are enough to definitely anaesthetize samples. demnidae des Côtes de France (Ascidies Com- Sea squirts do not contract with eugenol, on the posées) Synthèse des Résultats Principaux. Ann. contrary, samples anaesthetize that way, show Inst. océanogr., Paris, 1977, 53 (1) : 135-153 particularly well relaxed pharynges. Their cloa- cal apertures are equally widely opened and

Figure 1: Shape of zooids after eugenol anaesthesia and formaldehyde fixation. A, Didemnidae; B, Styelidae (Botrylloides); C, Perophoridae; D, Polyclinidae. Scale = 1 mm. Photo Wilfried Bay-Nouailhat.