INSULATION STRATEGIES IN THE PACIFIC ELECTRIC RAY TORPEDO CALIFORNICA Lara Rosenthal Biology 175 H June 9, 1989 ABSTRACT This paper explores three aspects of the Pacific electric ray Torpedo californica: the basic anatomy of this fish, how the torpedo insulates itself from its own shock, and whether the torpedo's electroreception can be used as à tool for learning. Using anatomical observations and skin resistance méasurements, I set out to determine whether the torpedo's skin was different over the electric organs and non electric organ areas and how the torpedo insulates itself. I could find no difference in appearance, texture or color from the electric organ to non electric organ. I did, however, find that thère are many more layers of connective tissue over and under non electric organ areas than over the electric organs. Furthermore, I measured the dorsal skin to have roughly twice the resistivity as ventral skin. I believe that the other important insulation tactics are use of cartilege to protect the brain, heart and nerves, connective tissue to protect peripheral nerves, and placement of muscles as far from the electric organs as possible. I have also described the placement of the ampullae of Lorenzini and applied Pavlovian conditioning techniques using the ampullae. During the time we tried to condition the torpedo, it was not responsive. INTPODUCTION The torpedo is a nocturnal fish that spends most of its time lying on the bottom of the subtidal zone under a loose covering of sand or mud. Although the torpedo looks similar to many rays and skates, internally they are quite different from their non-electric relatives. The two large electric organs comprise 228 of the body mass and 322 of the surface area in the torpedo. Thus, this ray has become highly specialized to accomodate the large size of these organs and to insulate themselves from their own shock. Torpedo discharges can be as powerful as 50 volts and 50 amps, or 2,500 Watts (Keynes and Martins-Ferreira). The small rays in the Monterey bay subtidal have a much smaller discharge but even so, this shock is powerful énough to stun a prey fish at close range or issue a warning to a predator (Fong). The electric field applied by the torpedo on a victim upsets the delicate electrochemical balance that controis the victim's nerves and muscles, rendering the victim helpless as his nerves fire and muscles contract. While the electric organ is not discharging, the dorsal and ventral faces of the electric organ are at the same potential. During discharge, the ventral face depolarizes creating an electric field (figure 1). High ion concentration gives sea water low resistance and high conductivity, therefore, most of the current generated will travel the least resistive pathway from one face, through the water, around the wing tip to the other face as illustrated by the dark field lines (figure 1). Some current must travel the longer alternate pathways through the water as illustrated by the lighter field lines. Finally, since the body fluid of the torpedo also has high ion concentration there is the probability that some current could travel through the inside of the body and wreak internal havoc. How does the torpedo protect itself from its own discharge? To answer this question I performed several dissections and developed a system for measuring skin resistance. I set out to test the hypothesis that skin above and below the electric organs would be of low resistance to permit easy current flow and the rest of the skin would be highly resistive for insulation. I found that the torpedo uses three primary tactics to insulate itself; layering of connective tissue, use of cartilege, and strategic placement of nerves and muscles. Another interesting field of study is electroreception. All elasmobranchs and many teleosts have the ability to sense electric fields. The electroreceptive organs in the torpedo are the ampullae of Lorenzini. The ampullae are able to sense thermal, mechanical, and salinity changes as well as electric fields (Kalmijn), and have many different uses in different fish such as prey detection (Kalmijn), communication (Moller) propioreception (Lissman), geomagnetic navigation (Rayan) and sexual signals (Hopkins) Littie is known about the uses of electroreception in the torpedo, but studies of other elasmobranchs have shown electroreception is useful in prey detection and geomagnetic navigation (Kalmijn). Also, while the location of the ampullae have been described in the skate Kaya (Murray), they have not been described in the torpedo. I have mapped the location and distribution of the ampullae in the torpedo, laying the groundwork for more experiments on ampullae in the torpedo. This has proven necessary because the location and distribution of the ampullae in the torpedo is quite different from that of the skate. If the torpedo is able to sense electric fields, this electric sense could be used with Pavlovian conditioning techniques to condition a ray to exhibit a specific behavior on an electrical cue. We decided to test this hypothesis to see if the torpedo can integrate information from the electroreceptors to the point of learning. The conditioned response we sought was a discharge in response to an electric stimulus. The unconditioned response was a poke on the wing with a wood stick (the wing stomp). We hoped that by coupling the wing stomp with an electric stimulus, the ray would eventually learn to discharge when the unconditioned stimulus (the electric stimulus) was presented without the wing stomp MATERIALS AND METHOD Nine rays were obtained in 30-40 feet of water off Wharf 2 in Monterey Bay. Five of these (including the one we used for conditioning were kept in a tank shielded from sunlight by a tarp in order to approximate the dim lighting conditions at their usual depth. The five rays used to measure skin resistance were stunned with an ice/sea water bath and killed by decerebration. One ray was killed with an overdose of quinaldine anesthetic (208 quinaldine in isopropyl alcohol). Two were found dead, the probable cause of death being nitrogen gas bubbles in their tissue. Dissection: Standard dissecting tools were used, aided by a Bausch and Lomb dissecting microscope. Dissected samples were kept in a solution of 508 sand filtered sea water and 508 tap water. Skin Resistance: A Grass DC stimulator was used as the voltage source. Recordings were amplified by 10X and then recorded on a Nicolet digital oscilloscope. The apparatus (figure 2) was arranged with the voltage source grounded on one side and two resistances in series to the circuit. The first resistance (Ki) was exactiy 100 kohms and the second resistance (R2) was unknown. The measuring electrode from the oscilloscope was placed between the two resistances. Rz was calculated using the following equation. R2 - (Ri * Vout) / (Vin - V out) Vin is the voltage provided by the stimulator measured on one channel of the oscilloscope, Vout is the voltage after the 100 Kohm resistor measured on the other channel of the oscilloscope. The resistance of the skin and the electrodes act as two resistors in series, Relectrode and Rekin. To solve for Eskin, 1 subtracted Reelectrode (no skin in the circuit) from R2(electrodet skin) (with skin in the circuit). In measuring skin resistance there were several logistical problems that needed to be considered in making the electrodes. First, I needed to créate a system that would not poke holes in the skin, because even the smallest hole would allow enough ion leakage to lower the skin resistance to nearly zero ohms. Second, it was very important that the charge travel through the skin and not around the skin through sea water. Thus I needed a way block the path of current around the section of tissue being measured Third, resistance depends not only upon the properties of the material being measured (resistivity), but also upon the cross sectional area in contact with the current source, and the distance through the material that current has te travel. resistance - resistivity » (length/ cross sectional area) Therefore, I needed to be able to align my electrodes perfectly each time to keep the cross sectional area constant and I needed to stretch the tissue to the same thickness for every measurement to keep the distance through the material constant. To remedy these problems I used a chamber (figure 3) made from one petri dish and two petri dish lids with 6.0 mm holes drilled through them. The electrodes were Ag/AgCl wires submerged in sea water as the conducting medium. The petri dish and lid were sealed with 5 minute epoxy with the Ag/AgCl wire running from the inside of the dish over the edge of the dish to the outside. In order to minimize the possibility of current flowing around the skin through the sea water instead of through the skin the tissue was pat dry with kimwipes until no wetness was visible and briefly air dried with an aquarium aerator. Both lids were covered with vaseline to form a tight seal to the skin to prevent water leakage and to further impede current flow around the skin. Before placing the skin onto the vaseline covered bottom electrode, enough sea water was added to insure that the meniscus would contact the skin. The bottom electrode aiso served as a stage to lay the skin upon. To control for inconsistencies in how far the skin was stretched, I measured the area of the skin before removing it and stretched the skin once on the stage to the measured area. The other lid, with vaseline on the base, was placed on top of the skin, pressed down until the skin bulged slightly in the center hole to further insure good water contact, then the Ag/AgCl wire was placed in the upper lid and the lid filled with sea water. Unfortunately, the Ag/AgCl wires often polarized, so it was difficult to obtain valid measurements. The criteria used for determining whether the data was valid was whether the electrodes had drifted significantly during the skin measurements. To determine this I measured the Relectrode before and after measuring Rekin- Skin measurements were considered valid if the electrodes had drifted less than 5.0 2 of the original resistance. Only the epithelial tissue was used in resistivity experiments. Valid data included pulses of 10.9 mV, 100 mV and 100.7 mV across dorsal electric organ skin, and 100 mV and 101.5 mV across ventral electric organ skin. Puises lasted 5.00 seconds in order to measure the steady component of the voltage drop. On dorsal skin, fourteen valid recordings were made from thrée separate pieces of skin. On ventral skin, two pieces of skin were used to make seven different recordings. The measurements from each piece of tissue were averaged. Thus, the sample size for dorsal skin was 3 and for ventral skin was 2. Since resistance depends upon the thickness of the tissue, dorsal and ventral skin were measured using calipers to calculate the resistivity of the tissue. Results were statistically analyzed with the Student's T-test. Conditioning: A Grass DC stimulator was used to create the electric stimulus, and a Nicolet digital oscilloscope measured the preamplified discharges. One measuring lead was glued (with crazy glue and electrical tape) to the dorsal skin over one elecroplaque, the other was in the water. During the training period, every two minutes we applied an electric stimulus of 10 volts at a frequency of 1000 Hz for one second duration. Immediately following the electric stimulus we poked the ray on the wing with a wood stick. The unconditioned and conditioned stimuli (the electrical pulse and the wing stomp, respectively) were seperate in time so that the torpedo would come to expect the wing stomp after experiencing the electric stimulus and learn to discharge in expectation of the wing stomp. For one hour on four consecutive days and again for two consecutive days we trained the torpedo. Each day we tested the uncoupled electric stimulus at least twice. When testing the response to the electric stimulus without the wing stomp, we controlled for the movements in the water that the stomp creates by coupling the electric stimulus to a stomp right next to the wing. rESU Dissection: An orientation to the anatomy is shown in a ventral view of internal organs (figure 4), a dorsal view of internal organs and central nervous system (figure 5), and the cartilege skeleton of the torpedo (figure 6). The sex of the torpedo (figure 7) can be identified by the presence of claspers at the ventral base of the male's tail. From dissection, we find that specific structures (the central nervous system, peripheral nerves, heart and muscle) that are highly sensitive to electrical stimuli use cartilege, connective tissue and careful placement to mitigate the internal effect of the discharge. Central Nervous System. The brain and spinal cord are enclosed by cartilege shells (figure 6). In certain areas the cartilege shell has two layers seperated by a fluid pocket. There are three pockets (shown in blue in figure 6). The first is located in front of the brain and is shaped roughly like a rectangular prism tapered towards the snout. The other two pockets are located on the lateral sides of the electromotor cortex of the brain. At the snout of the torpedo (roughly from the nostrils forward and above the cartilege) there is an area of loose connective tissue that is organized into columns, much like the way the electric organ tissue is structured Peripheral nerves. The nerve fibers that innervate the electric organ (figure 5) are heavily insulated by longitudinal sheaths of fibrous connective tissue. A cross section of this fiber from a 30 em ray measured 4mm diameter. In addition to heavily insulating fibrous connective tissue on the nerves, some fibers are encased by continuous portions of the cartilege skeleton. Specifically, two nerve trunks that innervate the posterior portion of the body runs through the cartilege that houses the belly cavity (figure 5). Also, the nerves that encircle the electric organ run through the cartilege frame of the electirc organ. Musde Hber: The heart is an organ particularly sensitive to electrical activity. It is encased on all sides by the cartilege skeleton that provides excellent insulation. Other muscle on the body is sequestered away from the électric organs. The torpedo is not a muscular animal. The electric organs are composed of modified muscle but they do not contract and release Electric organ muscle fibers are arranged into vertical colunns, and the tendons that normally link muscle to cartilege are absent. Non-electric organ muscle is carefully placed and insulated with varying degrees of connective tissue. Thère is no trace of non-electric organ muscle above or below the electric organs. Non-electric organ muscle is dense down the central axis of the body and more sparse on the fins (figure 8). Near the electric organs thère is a muscle beneath the mouth that mediates mouth movement. There are two muscles that run from between the electric organ and the dorsal nostril and connect to the horizontal bar of cartilege in the center of the torpedo. The fin muscle is thicker at the base of the fin than it is at the tip and the muscle is thicker on the fins at the posterior end of the fish than at the anterior end. Connective tissue plays an important role in insulating muscle Above and below the electric organ there is a layer of epithelial tissue and one layer of fibrous connective tissue. The dorsal epithelium seemed constant all over the dorsal side, and the ventral epithelium seemed constant all over the ventral side of the body. There was no noticable difference between epithelium over the electric organs and on the rest of the body. The difference between the covering of the electric organs and of the rest of the body is the layering of connective tissue. The rest of the body is protected by layers of epithelial and fibrous connective tissues similar to those over the electric organs. However, in contrast to the electric organs, the body is further protected by an additional layer of loose connective tissue and another dense layer of fibrous connective tissue (figure 9). All of the muscles on the central axis are protected by a thick wrapping in fibrous connective tissue. The fibrous connective tissue on the fins thins out toward the edge of the ray and there is an additional layer of loose connective tissue in the vertical plane between the cartilege and the fin muscle. Skin Resistance: The mean resistance for dorsal electric organ skin was 230 Kohms with standard deviation equal to 62 Kohms. For ventral electric organ skin the mean resistance was 94 Kohms with standard deviation equal to 19 Kohms The Student's T-test shows this data to be statistically significant (P 60.05). Measurements of the skin thickness in a 30 cm ray were: dorsal skin =0.9 mm and ventral skin = 0.7 mm. The cross sectional area = 28.26mm. This gives the resistivity of dorsal skin - 7.2 Mohmsemm and for ventral - 3.8 Mohmsemm. The resistance of cartilege was infinite by my measurements. Electroreception: Location of Ampullae of Lorenzini. The tiny openings on the exterior of the torpedo lead into the lateral line system or into ampullae of Lorenzini (figure 10). The distinguishing factor is that the ampullar pores are slightly larger than the lateral line pores. The jelly tubes run along the electric organ side of the cartilege that separates the electric organ from rest of the fin (figure 6). The ampullar bulbs (figure 11) are bundled in two locations (figure 6). The first is directly in front of the eye socket cupped in an indentation in the cartilege, the second is on the electric organ side of the cartilege around the electric organ. The jelly tubes that lead from the anterior end of the torpedo end in bulb cluster I, which is surrounded by loose connective tissue. The ampullae that begin around the lateral periphery of the electric organ lead to bulb cluster II which lies between the electric organ and cartilege. The afferent nerves from the ampullar bulbs are concentrated into a few fibers (figure 5). These nerves do connect to the lateral line system, but it would be entirely possible to sever only the ampullar nerves to perform a sensory denervation for behavioral studies. conditioning: In the time we spent trying to condition the torpedo, it did not seem to condition to the electric stimulus as a cue to discharge. The torpedo did discharge for about 703 of the wing stomps. The other 302 resulted from 1. wing stomps that missed because the ray was swimming (202), 2. the ray not discharging towards the end of the one hour sessions (53) or 3. for no obvious reason (52). Of the many times we attempted to solicit the unconditioned response only once did we observe a discharge. DISCUSSION The ability to generate electric shocks gives the torpedo a definite advantage out in the ocean, but this is not without cost. The electric organ muscle fiber, as arranged into vertical columns with no tendon structure, is quite useless for movement. The electric organ goes all the way through the width of the torpedo, with no other structures intervening. This is in contrast to non-electric skates and rays where the area occupied by the electric organ is composed of cartilagenous fin rays and muscle to form the pectoral fins (Daniel). Furthermore, the cartilege fins at the most anterior 1/4 of the body are continuous with the cartilège around the electric organ The lack of a joint at the fin junction inhibits movement even more. Thus the torpedo is a rather clumsy creature unable to propell itself along with graceful sweeps of the wings as its nimble skate and ray relatives can. Instead, it swings its tail back and forth for a very slow and awkward mode of locomotion (figure 12). The idea that the external skin of the torpedo is the key factor in insulation seems false in light of observations from the dissection. Instead, what seems to be most important to torpedo insulation are the layering of connective tissue below the epithelial layer, clever use of cartilege and careful placement of muscle. Cartilege is probably the best insulating tissue in the torpedo, its resistance being close to infinite. Current is conducted through tissue because of the availability and mobility of ions from the torpedos high- osmolarity body fluids. Epithelial and connective tissue are far more fluid¬ saturated than cartilege, this might explain its high resistivity to elective flow. The torpedo's body takes advantage of the insulating properties of cartilege by shielding the brain, heart, spinal cord and some peripheral nerves from the electric field evolved during the discharge The careful placement of connective tissue is important in general body insulation. It is placed beneath the epithelial skin on non electric organ areas of the body and insulates specific sensitive structures like peripheral nerves and muscles especially close to the electric organs. The jelly tubes from the ampullae of Lorenzini run along the periphery of the electric organ in a thin layer of loose connective tissue. Each tube contains a highly conductive mucopolysaccharide jelly bounded by connective tissué that could be of the fibrous breed. Fish that give off a constant low grade electric discharge have ampullae that are not affected by the fish's own electric organ discharge (Suga). If the tubing is indeed fibrous connective tissue, this would show the potential value of fibrous connective tissue as a powerful insulator. The results of the skin resistance experiment show that dorsal skin has about double the resistivity of ventral skin. This may be partially due to the less than ideal accuracy of the calipers that were used to measure skin thickness. The dorsal skin is probably still more resistive than the ventral skin, even allowing for some inaccuracy in thickness measurements. This is curious, the torpedo does not endanger its dorsal side more than its ventral side during discharge (figure 1b) Nor is it likely that the torpedo has developed a passive defense mechanism to guard against the electrical onslaught of other electric fish, stray lightening bolts, or live wires thrown into their environment by careless humans. Instead, the difference in resistivity between dorsal and ventral skin might be explained by entirely different factors such as the resistive properties of the black pigment cells that cover the torpedo on its dorsal side. Maybe the dorsal side has tighter packing of cells for better thermal insulation since the torpedo spends most of its time on the muddy bottom, and water conducts heat away from the body faster than mud. Or other reasons than these. More accurate measurements of skin thickness need to be made, perhaps by embedding a section of tissue in wax and taking a longitudinal slice with a sharp razor or vibratome. Once better thickness measurements are available, better discussion of the inherent properties of the tissue will be possible. More data needs to be collected on skin resistance, particularly on skin from different areas of the body. The original hypothesis of whether or not skin covering the electric organs has different resistance than other skin still needs to be tested rigorously. And, the resistance of the fibrous connective tissue needs to be measured to determine whether it is actually the amazing insulator I think it is. Our attempts to conditioning our torpedo using its electroreception were not successful, but this does not mean that its electric sense is weak or intermittent, or that the torpedo is dumb. There were several problems with our setup. First was the fact that the wing stomp was an undesirable stimulus. As the training time wore on each day, the ray's liklihood of responding to a wing stomp with a discharge decreased. This pattern could show habituation to the wing stomp, which was exactly what we did not want. Second, if there was habituation, our results were further skewed by our testing the unconditioned response towards the end of the hour. Third, we may not have persisted with the training for long enough. A better experiment would be to use a positive unconditioned stimulus such as a good food. This is difficult because torpedos are not very receptive to anything but lying on the bottom of the tank covered with sand. Further directions for research: On the topic of insulation, it would be interesting to see if the relative thickness of fibrous tissue surrounding nerve fiber decreases farther away from the electric organs, as the fibrous tissue on muscle seems to. This could be done by again embedding the tissue in wax and cutting sections to be viewed under a powerful light microscope. The connective tissue, especially the fibrous tissue, the jelly tube tissue, and the tissue at the snout of the torpedo, needs to be further characterized using histological techniques to stain for collagen fiber organization and biochemical structure. Unfortunately, time did not permit comparison of the torpedos musculature and connective tissue to that of its non electric relatives. This comparison would show whether the connective tissue that seems to serve the purpose of insulation in the torpedo was actually evolved for that specific purpose. On the topic of electroreception, I hope that my map of the ampullae of Lorenzini will prove useful to someone able to pursue the topic, especially since the only remotely similar map of ampullae was in the skate Kaya and its ampulsar distrubution is quite different from the torpedo's. Several types of desensitization experiments are possible for studying the behavioral uses of electroreception without cutting off the lateral line system. Selective denervation is definitely possible but not simple. An alternative method would be to destroy the ampullar bulbs with four incisions, one above each bulb cluster. Another possibility would be to cut the jelly tubes just posterior to the bulb clusters. More humane experiments could use Kalmijn's chambers of ion concentrated agar to conceal stimuli visually, chemically and mechanically without disturbing the bioelectric field. FIGURE LEGEND figure 1 — a. field lines around torpedo b. schematic of discharge circuit figure 2 — apparatus for skin resistance experiment figure 3 - electrode chamber for measuring skin resistance figure 4 — ventral view of internal organs figure 5 -dorsal view of internal organs and central nervous system figure 6 — cartilege skeleton and ampullae of Lorenzini figure 7 — external differentiation of male versus female figure 8 — musculature figure 9 - comparison of electric organ skin and non-electric organ skin figure 10— lateral line system and ampullae of Lorenzini figure 11— close up of ampullae of Lorenzini WORKS CITED Bennett, M.V.L., Wurzel, M., and Grundfest, H. (1977) "The Electrophysiology of Electre Organs of Marine Electric Fishes," J.Gen. Phys. 44.757-803. Daniel, Frank J. (1922) The Elasmobranch Fiches, University of California Press. Berkeley, California. pp 2 1, 117-118 Fong, Loren H. (198 1) Predatory Behavior of the Pacific Electric Ray Torpedo Californica, Stanford University Hopkins Marine Station Journal Garman, Samuel. (1686) Lateral System, Bulletin Museum of Comparative Zoology at Harvard. Volume XVII No. 2. Cambridge, Mass. plate XXXI Hopkins, C. D. (1974) Electric Communication: Functions in the Social Behavior of Egenmannis virescena“ Behavior 50: 270-305. Kalmijn, J. (1976) Electric and Magnetic World of Sharks, Skates and Rays, Sensory Biol y of Sharks, Skates and Rays, Ed. Hodgson, E. B. and Mathewson, R. F.; Office of Naval Research, Arlington, VA. pp 507-28. Keynes, R.D. and Martins-Ferreira, H. (1953) Membrane Potentials in the Electroplates of the Electric Eel," J. of Phys. 119:315-351. Lissman, H.W. (1958) On the function and Evolution of Electric Organs in Fish, J. Exp. Bio,“ 35: 156-91. Moller, P. (1976) Electrical Signals and Schoolong Behavior in a Weakly Electric Fish Marcusenius cyprinoides L. (Mormyriformes) Science 193: 697-699. Murray, R. W. (1962) The Response of the Ampullae of Lorenzini of Elasmobranchs to Electrical Stimulation" J. Exp. Bio. 39: 119-128. Rayan, PR. (1980) "Geomagnetic Guidance Systems in Bacteria and Sharks Skates and Rays," Oceanus 23(3): 55-60. Suga, N. 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