Effects of anesthetic compounds on responses of earthworms to electrostimulation.

Agnieszka Podolak-Machowska,Tadeusz Librowski,Barbara Plytycz,Joanna Kostecka,Michal Santocki,Janusz Bigaj

doi:10.3409/fb62_2.155

Abstract

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 connected with the expulsion of coelomic 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.

Highlights

Available data are consistent with the idea of pain in some invertebrates (ANDREW 2011; COOPER 2011; ELWOOD 2011)
In search of efficient anesthetic drugs for earthworms, we have recently tested a local anaesthetic drug used for human anesthesia (LIBROWSKI et al 2004; STOKES et al 2009) and MS-222 which is an efficient anaesthetic for aquatic animals (NEIFFER & STAMPER 2009; WEBER et al 2009; VERA et al 2010)
MS-222 was noxious for earthworms and induced extrusion of coelomocytes including riboflavin-storing eleocytes, this drug seems to be inappropriate for earthworm anesthesia

Summary

Introduction

Available data are consistent with the idea of pain in some invertebrates (ANDREW 2011; COOPER 2011; ELWOOD 2011). Convulsive body movements are common in lumbricid worms under natural conditions, e.g. when irritated by predators or investigators, thermal shock, sudden light exposure, or contact with some chemicals. This ability is used by immunologists for non-invasive quantitative coelomocyte retrieval under strictly controlled experimental conditions, i.e. by irritating worms with a mild electric current (ROCH 1979), ultrasounds (HENDAWI et al 2004), or 5% ethanol (COOPER et al 1995). The coelomocytes of lumbricid species consist of amoebocytes (the classical immunocompetent cells) accompanied in some – but not all – species by autofluorescent eleocytes (CHOLEWA et al 2006). The fluorescent self-marking of eleocytes makes them suitable for rapid quantification, applied in the present paper concerning coelomocyte expulsion by electrostimulated lumbricid earthworms, Dendrobaena veneta

Methods

Results

Discussion

Conclusion