PL-ISSN0015-5497(print),ISSN1734-9168(online) FoliaBiologica(Kraków),vol.62(2014),No2 Ó InstituteofSystematicsandEvolutionofAnimals,PAS,Kraków, 2014 doi:10.3409/fb62_2.155 EffectsofAnestheticCompoundsonResponsesofEarthworms toElectrostimulation AgnieszkaPODOLAK-MACHOWSKA,JoannaKOSTECKA,TadeuszLIBROWSKI,MichalSANTOCKI, * JanuszBIGAJ,andBarbaraPLYTYCZ Accepted February 19, 2014 PODOLAK-MACHOWSKA A., KOSTECKA J., LIBROWSKI T., SANTOCKI M., BIGAJ J., PLYTYCZ B. 2014. Effects of anesthetic compounds on responses of earthworms to electrostimulation. Folia Biologica (Kraków) 62: 155-162. Earthworms play an important role in biomedical research, and some surgical procedures require anesthesia. Anesthetic treatments used so far usually induce convulsive body movements connected with extrusion of coelomocyte-containing coelomic fluid that may affect experimental results. Extensive movements connectedwiththeexpulsion ofcoelomic fluid are exploited by immunologists as a method of harvesting immunocompetent coelomocytes from worms subjected to mild electrostimulation (4.5V). The aim of the investigations was to find anesthetic drugs without unintentional coelomocyte depletion. Experiments were performed on adult specimens of Dendrobaena veneta, the coelomocytes of which consist of amoebocytes and riboflavin-storing eleocytes. Earthworm mobility was filmed and extrusion of coelomocytes was quantified by detection of eleocyte-derived riboflavin in immersion fluid. Treatments included earthworms (1) immersed either in physiological saline (controls) or in a solution of one of the tested anesthetic drugs; (2) electrostimulated immediately after anesthesia, and (3) electrostimulated a second time after a 1-hour recovery period. The well-established fish and amphibian anesthetic agent MS-222 induced coelomocyte expulsion. In contrast, solutions of the mammalian local anesthetic drug, prilocaine hydrochloride (0.25-0.5%, 5-10 min) caused temporal earthworm immobilization followed by recovery, thus showing utility as an efficient earthworm anesthetic. Key words: Dendrobaena veneta, immobilization, riboflavin, prilocaine, MS-222. Agnieszka PODOLAK-MACHOWSKA, Joanna KOSTECKA, Department of Biological Bases of Agriculture and Environmental Education, Faculty of Biology and Agriculture, Rzeszów University, Æwikliñskiej 2, 25-601 Rzeszów, Poland. E-mail: [email protected] Tadeusz LIBROWSKI, Department of Radioligands, Faculty of Pharmacy, Medical College, Jagiellonian University, Medyczna 9, 30-688 Kraków, Poland. E-mail: [email protected] Michal SANTOCKI, Janusz BIGAJ,Barbara PLYTYCZ, Department of Evolutionary Immunol- ogy,InstituteofZoology,JagiellonianUniversity,Gronostajowa9,30-387Kraków,Poland. E-mail: [email protected] Available data are consistent with the idea of pain performed on CO2-exposed worms, or other forms in some invertebrates (ANDREW 2011; COOPER 2011; of earthworm anesthesia, like warming, cooling, ELWOOD 2011). In lumbricid earthworms the cen- urethane, and chloretone treatment (OGURO et al. tral nervous system is a highly differentiated neu- 1984). In our hands, worms anesthetized by roendocrine structure producing neurohormones CO2-containing water, cold plate or chlorophorm vapors usually react by convulsive body move- and neurotransmitters (e.g. see HARTENSTEIN 2006; ments leading to extrusion of coelomocyte- HERBERT et al. 2009), thus the feeling of pain containing coelomic fluid through the dorsal pores should be considered and avoided during all kinds in the body wall. The partial loss of coelomocytes, of in vivo experiments, e.g. those on extirpation of the crucial component of the earthworm immune brain or ventral nerve cord ganglia (CSOKNYA et al. system (BILEJ et al. 2011), may affect the experi- 2002; HERBERT et al. 2009). Surgeries are usually mental results. Expulsion of coelomocytes is fol- _______________________________________ *Supported by the Ministry of Science and Higher Education in Poland, grant K/ZDS/3252, and by the National Science Centre inPoland,grantB/NZ4/01640(K/PBO/000178). 156 A.PODOLAK-MACHOWSKA etal. lowed by slow restoration of the initial cell numbers Material and Methods (e.g. EYAMBE et al. 1991; POLANEK et al. 2011; KLIMEK et al. 2012). The transitional depletion of coelomocytes may affect other processes within the organism. Therefore there is an urgent need to Earthworms develop methods of anesthesia leading to tempo- Adult earthworms Dendrobaena veneta (Oligo- rary earthworm immobilization without loss of chaeta; Lumbricidae), purchased from a commer- coelomocytes. cial supplier (EKARGO, S³upsk) were reared in commercial soil (PPUH BIOVITA, Tenczynek) In search of efficient anesthetic drugs for earth- o worms, we have recently tested a local anaesthetic under controlled laboratory conditions (17 C; drug used for human anesthesia (lidocaine and pri- 12:12 LD). The worms were kept in plastic boxes locaine) (LIBROWSKI et al. 2004; STOKES et al. with perforated lids and the moisture level was 2009) and MS-222 which is an efficient anaes- checked weekly. Worms were fed ad libitum with thetic for aquatic animals (NEIFFER &STAMPER a mixed diet comprised of dried/boiled nettle (Ur- 2009; WEBER et al. 2009; VERA et al. 2010). The tica dioica) and dandelion (Taraxacum officinale) findings showed effective immobilization of leaves, boiled/dried tea leaves, and powdered earthworms with prilocaine at a concentration of commercial mouse pellets. 0.25-1%. Lidocaine was less effective at the ap- plied concentrations. In sharp contrast, MS-222 had a strongly irritating effect for earthworms and Anaesthesia and recovery induced convulsive body movements connected with discharge of coelomic fluid (PODOLAK- Adult (clitellate) earthworms were individually -MACHOWSKA et al. 2013). immersed in small Petri dishes (5 cm diameter) filled with 3 ml PBS (controls) or in PBS-solution Convulsive body movements are common in of appropriate concentrations of prilocaine hydro- lumbricid worms under natural conditions, e.g. chloride (Sigma Aldrich) (2%-0.125%), or when irritated by predators or investigators, ther- MS-222 (Tricaine methanesulfonate; Ethyl 3- mal shock, sudden light exposure, or contact with aminobenzoate methanosulfonate salt; Sigma- some chemicals. This ability is used by immunolo- Aldrich; Fluca) (1%-0.0125%) for 5-30 minutes gists for non-invasive quantitative coelomocyte and worm behavior was recorded. Then the earth- retrieval under strictly controlled experimental worms were transferred individually to wells with conditions, i.e. by irritating worms with a mild PBS of the 6-well plate and their mobility was as- electric current (ROCH 1979), ultrasounds (HEN- sessed and filmed (Fig. 1). This was followed by DAWI et al. 2004), or 5% ethanol (COOPER et al. wormrecoveryonwetfilterpapers.Theprocedure 1995). In the present experiments we applied this was repeated one hour later. The coelomocyte- peculiar earthworm response to electrostimulation containing fluid was harvested and analysed in re- for testing the effects of prilocaine and MS-222 on spect of riboflavin content. coelomocyte extrusion. The coelomocytes of lumbricid species consist of amoebocytes (the classical immunocompetent Measurement of earthworm mobility cells) accompanied in some – but not all – species Earthworm mobility was recorded in a 6-well by autofluorescent eleocytes (CHOLEWA et al. 2006). Autofluorescence of the latter cells is de- plate (Nunc) adapted to the application of 4.5V pendent on accumulation of fluorophores includ- electric current and covered with a lid with grids enabling earthworm localization. Adult earth- ing riboflavin (vitamin B2) (KOZIOL et al. 2006; worms were immersed individually in wells filled PLYTYCZ et al. 2006; PLYTYCZ &MORGAN 2011). The fluorescent self-marking of eleocytes with 3 ml of physiological saline (PBS) and their makes them suitable for rapid quantification, ap- mobilitywasfilmedfor15secondsbeforeapplica- plied in the present paper concerning coelomocyte tion of an electric current (spontaneous mobility) expulsion by electrostimulated lumbricid earth- and during 60-second irritation with a 4.5V elec- worms, Dendrobaena veneta. tric current (induced mobility). Changes of earth- worm location within particular areas of the grid The aim of the experiments was to examine the were counted in one-second intervals. After test- effects of prilocaine and MS-222 on earthworms ing, the worms were transferred to plates with measured by temporary inhibition of body move- PBS-soaked filter papers for 1-hour recovery and ments and/or coelomocyte-derived fluorophore the procedure was repeated. The coelomocyte- extrusion during electrostimulation applied imme- containingPBSwasharvestedfromwellsandused diately after drug treatment and after a 1-hour re- for analysis of riboflavin content in coelomocyte covery period. lysates. AnestheticsforEarthworms 157 Riboflavin content Riboflavin contents were spectrofluorimetri- cally quantified in: 1) samples of fluid used for worm anesthesia at various concentrations of anesthetic drugs with 0% concentration as a control; 2) sam- ples of fluid used for electrostimulation immedi- ately after anesthesia; 3) samples of fluid used for electrostimulation after a 1-hour recovery period. Riboflavin was detected and quantified by spec- trofluorometric measurements performed on 2 ml coelomocyte-suspension lysates (lysed with 2% Triton; Sigma-Aldrich) using an LS50B Perkin- Elmer Spectrofluorimeter as described previously (CYGAL et al. 2007) Results