Some Contemporary Studiea in Marine Science, pp. 395-405 (1966) Harold Ba.mes, Ed. George Allen and Unwin Ltd., London

SOME RECENTLY DISCOVERED UNDERWATER VIBRATION RECEPTORS IN INVERTEBRATES

G. A. HORRIDGE Gatty Marine Laboratory and Department of Natural Hi~tory, University of St. Andrewa

As every submarine commander knows, the best and almost the only underwater sense organs which are effective at long range utilize the excellent transmission of mechanical waves through water. And yet when the known examples of sense organs which are adapted to the detection of underwater vibrations were recently reviewed by Frings (1964), almost no substantiated examples could be found among marine invertebrates. Even in shallow water, large animals with excellent eyes spend about half of their time in dim moonlight or in the dark, and the range of vision falls off rapidly with depth, so that objects cannot be seen until they are relatively close. However, most invertebrates have eyes which are not well adapted for the detection of the direction and range of a moving prey. On the other hand, vibrations are set up in the water by any animal which propels itself along, as well as by waves at the surface or choppy water at a shore line. Therefore we can expect receptors for vibrations to be distributed ubiquitously through the marine groups, and to be used to detect prey or enemies, or to allow avoidance of environ­ mental sources of vibration. Perhaps one of the reasons why more examples of sense organs of this type are not known is because we ourselves have nothing similar, because the receptors are inconspicuous, and because the responses depending on them are usually not obvious escape reactions. However, in the last two or three years a number of examples have appeared in different phyla so that the general features of invertebrate underwater vibration receptors have now become apparent. Several of these examples have been discovered at St. Andrews.

VIBRATION RECEPTORS

Fingers of Leucothea (Ctenophora) This is an example of the vibration sense used by a carnivore in locating its prey. Leucothea ( = Eucharis) multicornis is a large fragile carnivorous Mediter­ ranean ctenophore which has numerous finger-like organs about 1 cm long scat- 396 G . .A. HORRIDGE tered over its external surface (Fig. lA). The epithelium of the finger tips is com­ posed of large glandular cells between which are sensory cells (Chun, 1880). Some of the sensory cells bear single straight stiff, non-motile cilia and these are receptors for any small displacement of the surrounding water. When a copepod or similar small planktonic animal swims within range, the finger shoots out with a sudden extension, caused by contraction of circular muscles which run through the mesogloea of the finger. The fingers extend to about 1 cm from a resting length of about half this value. The bottom of any dish in which the animal is supplied with copepods is soon littered with immobilized copepods. In 1844 Dr. Will, working in Trieste, claimed that the copepods were caught on small hooks on the ends of the fingers, but I have not yet seen this, and have not yet discovered how the Leucothe,a makes use of its victims. There are neither nematocysts nor similar organs on the fingers and the poison which kills the copepods presumably originates in the gland cells.

A C B

2cm L---.J

Fig. 1.-Leucothea ( = Etwharis) multicornis. A, the apical part of the ctenophore showing the finger organs scattered over the general surface. B, two fingers, one in the relaxed, and the other in the extended, position. C, the terminal epithelium of a finger showing the gland cells, thick pogs (function unknown) and a long non-motile cilium of the vibration sensory neurone. D, the base of a non-motile cilium with concentric lamellae which are surrounded by a mass of tubules (after Horridge, 1965a).

The sense organs have been directly demonstrated to be the non-motile cilia in the following way. When an isolated finger is observed on a slide with a high power objective of long working distance, the cilia are found to be extremely sensitive mechanically. They cannot be touched without exciting the response. A glass needle mounted on the diaphragm of a loudspeaker, vibrated at a fre­ quency of 10 cycles/sec with a tip excursion of a few microns, can be manoeuvred into position near the cilium with a micromanipulator, but cannot be brought closer than about 100 µ, before the extension response of the whole finger is evoked. The structure of the sensory cilium itself is not peculiar, but the basal body and root is modified into a solid banded core which fits inside a spherical shell. The latter fits concentrically inside another spherical shell which is attached to a mass UNDERWATER VIBRATION RECEPTORS 397 of tubules filling the cytoplasm of the distal end of the cell (Fig. ID). It is clear that the non-motile cilium is mechanically coupled with the water so that any lateral movement of the water will be conveyed into a rocking movement of the basal body inside its spherical cup. This assumes that there is a plane of shearing between the concentric shells. A tentative theory of the mechanism is that shearing between the spherical shells causes current to flow through the mass of tubules which in turn depolarize the distal end of the cell membrane. The proximal end of the sensory neurone bears an axon which connects with other axons of the nerve net, which in turn are inferred to connect specifically with the circular muscle fibres causing extension. For further details, with electron micrographs, see Horridge (1965a).

Tentacles of Pleurobrachia This is an example of a vibration receptor used as a detector of adverse environ­ mental conditions. The common St. Andrews ctenophore Pleurobrachia pileus, after being caught in a plankton net, usually swims downwards when brought into the laboratory. However, when the animals are carefully dipped out of the sea, and are handled

. r ! ~

Fig. 2.-A, Pleurobrachia pilet,s turning over when vibration receptors of the tentacles and elsewhere are stimulated. B, Eutonina indicans turning to swim downwards and away from a source of low frequency vibration or ripples. Approximately natural size. gently without being lifted from the water, they commonly swim upwards. Speci­ mens which are swimming up respond by turning over and swimming downwards when one runs one's fingers to and fro over them in the tank, so causing ripples on the water surface. This stimulus causes a strong vibration which shakes the whole animal. The first sign of the responsei s a contraction of the two long tenta- 398 G. A. HORRIDGE

cles, which normally trail in a relaxed fashion behind the animal. A vibrating needle, attached to a loud-speaker diaphragm, can stimulate a single tentacle when brought close to it. The response then starts as a contraction of the tentacle and spreads to the body of the Pleurobrachia, which changes the rate of its comb­ plates on one side relative to the other and turns over to swim downward (Fig. 2A). Isolated tentacles will respond by contracting and animals without tentacles will still invert when stimulated with the vibrating probe. The sense organ has not been identified. However, Hertwig, R. (1880, p. 326) mentioned that "Sinnesborsten" , similar to those of Leucothea, occur on the tenta­ cles of ctenophores. These are non-motile cilia which have not yet been found with the electron microscope on tentacles, but they may well turn out to be similar to the vibration receptors of Eucharis. The statolith in the ctenophore apical organ functions as a receptor of the direction of gravity because the weight of the statolith bears on the four groups of balancer cilia. These are strong groups of cilia which stand at the head of the ciliated grooves which in turn lead down to the comb-plates. Each beat of the balancer cilia sets off a wave of beats along the pair of ciliated grooves, which run from that balancer tuft and this wave travels along the comb-plates. In this way the balancer cilia are in control of the geotactic response (Horridge, 1965b). When a ctenophore turns over and swims downward in response to disturbance of the water, the sign of the response of the balancer cilia is reversed by excitation in the general nerve net. As a vibration receptor the statolith is not very effective. A large vibration in the water will jerk the statolith and then the balancer cilia respond, but usually all four respond together so that the additional waves in the comb-plates are of no consequence.

Margin of Eutonina indicans (Hydromedusae) This is another example of the avoidance of surface ripples by a delicate plank­ tonic animal, which at the same time presents its mouth to a source of vibration. Eutonina is a hemispherical medusa about 3 cm. diameter, with a manubrium which reaches as far as the edge of the bell. Although the response is not typical of Hydromedusae the manubrium can be directed to any point on the subumbrellar surface where a stimulus is applied (Romanes, 1877). As was shown by making cuts, the manubrium is controlled by radial pathways which transmit inwards from the edge of the bell, as in Geryonia (Horridge, 1955). When a Eutonina is swimming in an upright position in a dish of sea water, it can be stimulated with a vibrating needle mounted on a loudspeaker diaphragm. The Eutonina turns over in a direction away from the stimulus and it swims downwards for several seconds or minutes (Fig. 2B). Like ctenophores, individuals of Eutonina which are damaged when collected from the sea usually swim down­ wards when brought into the laboratory, and upwardly swimming specimens must be dipped, not netted, out of the sea. As illustrated in Figure 2B, the response presents the mouth towards the stimulus, and if this originated from a copepod or similar prey the new direction UNDERWATER VIBRATION RECEPTORS 399 The response of the water movement into the bell would lead to the capture of food. also takes the Eutonina into deeper water when the surface is disturbed.

Hair-fan Organs of the Lobster organs of fish. Among invertebrates these sensilla are analogous to the lateral line (1962) During a survey of receptors of the common lobster Homarus, Laverack composed of discovered a class of cuticular receptors which are fan-shaped organs and widely bristles, about 75 µ in overall size, contained in shallow depressions but not on the distributed over the anterior of the body, especially on the chela, disturbances antennae or antennules (Fig. 3). These receptors are sensitive to water

the hair-fan organs, represented Fig. 3.-Homarus vulgaris. A, typical posture showing how a way that a wide vibration as dots, are distributed on the claws and carapace in such receptor lying in its pit (after receptor area is presented towards the front. B, a single the larger spike of a. rapidly Laverack, 1962). C, typical response from a receptor showing the unit with lower threshold. adapting unit, and the more numerous smaller spikes of of the immediately adjacent The oscillations in the lower record show the movements of the receptor. surface of the water, and therefore are related to the movements

open surface, by such as those caused by tapping the container, which has an vibrations set up drops of water falling into the dish containing the lobster, or by receptor can be from a diaphragm in one wall at the end of the dish. A single and so isolated contained in a small drop of sea water on the surface of the cuticle In a single for study while impulses are recorded from its axon behind the cuticle. observed that sensillum, Laverack found two units, one rapidly adapting, and of the receptor after it had been displaced, impulses are not set off by the return base so that they to its resting position. The hair-fan organs are pivoted at their so that theoretic­ move in one plane and different receptors are variously oriented, the direction ally the animal has available a great deal of information concerning falling 2 · 5 cm and intensity pattern of a disturbance in the water. A water drop a spike and on to the surface of the dish 15 cm away from the chela will arouse this by averaging doubtless the whole animal can achieve a lower sensitivity than a dark hole, the over a large number of receptors. To an animal at night, or in actual behaviour value of this system as a distance receptor may be very great, but in which the animal makes use of it has not yet been observed. 400 G. A. HORRIDGE

Selae and Non-motile Cilia of the Chaetognatha This is a further example of a highly specialized prey detection by underwater waves which are sensed by a system analogous to the lateral line of fish. The arrow-worms feed by grabbing their prey with the armature of the mouth. Notwithstanding the fact that their prey consists mainly of copepods and larval fish, which must swim by them in the water at a relatively considerable speed, they feed mainly at night, and the amount of food taken is independent of the illumination (Reeve, this Volume, p. 613). In any case, each eye, as described recently by Eakin and Westfall (1964), consists of a group of modified receptor neurones which surround a pigment spot. There is no system which could form an image or even an accurate localization of a passing small object. It was this fact which led us to investigate the Chaetognatha as animals which rely completely

C

L..___J 10}-J

Fig. 4.-Spadella cephaloptera. A, the whole animal turning to grab at a vibrating glass probe. B, two tufts of setae and a fan of ciliated neurones (centre); these structures alternate all round the margin of Spadella and probably of all chaetognaths. C, a single sensory neurone showing the base of the single non-motile cilium. on vibration receptors to locate their food. Recently, in this laboratory, Miss S. Boulton has demonstrated that Spadella will grab the end of a glass needle which is brought near while it is vibrating with an amplitude of about 300 µ, at a fre­ quency of about 10/sec (Fig. 4A). Only small local stimuli will elicit the response: strong stimuli, or a general vibration in the water, are ineffective. When some of the receptors are clogged with viscous methyl cellulose the response to vibrations fails in that sector but persists in other sectors. Alternating with the groups of setae, which have long been known, there are fan-shaped groups of non-motile stiff cilia borne by cells with large nuclei which we interpret as sensory nerve cells (Fig. 4B). These cells each have an apical dendrite which terminates in a single cilium (Fig. 4C). So far it has proved impossible to trace axons from these cells but lying in the ectoderm peripheral to the basement membrane there are UNDERWA'l'ER VIBRATION RECEPTORS 401 small groups of axons which may arise from these cells. The fans of cilia are orientated in various directions in different parts of the animal. The bunches of straight setae, figured in all texts on chaetognaths, are the processes of ordinary epithelial cells. Several setae grow from a single cell, whereas each of the non-motile cilia stands upon its own elongated cell. Hertwig, 0. (1880) suggested that the setae or bristles are touch sensilla, but he and all later workers have missed the non-motile cilia which lie between the bristles. However, the groups of deeply staining cells from which the cilia arise have been mistaken for the cells which bear the bristles. In fact the cilia are extremely thin and their true relations can be seen only with the electron microscope. Under an ordinary micro­ scope they are not seen in life unless a special search is made.

l>

A B O·Smm

Fig. 5.-Syncoryne polyp. A, before and then B, after lightly tapping the manipulator which holds the thin glass probe (after Josephson, 1961). C, typical sensory neurone from the tentacle of an anemone showing the peripheral cilium, which should be investigated as a possible receptor of movements at a distance (after Hertwig and Hertwig, 1879).

Planktonic chaetognaths do not behave well or live long in the laboratory. Therefore all observations have been made upon Spadella which is a benthic form. However, there is every reason to suppose that what has been found is character­ istic of the whole group. From their situation on cells which look like sensory neurones, and from the expectancy that non-motile cilia might be vibration receptors, it appears probable that the fans of stiff cilia are the receptors for the feeding response. As yet there is little reason to give the bristles themselves a receptor function, as has been assumed since 1880.

Feeding by Syncoryne mirabilis The behaviour of this hydroid, in the capture of small prey, resembles that of Leucothea fingers, but the receptors are not yet known. 402 G. A. HORRIDGE Polyps of the hydroid Syncoryne respond to a vibrating object in their vicinity by bending rapidly in the direction of the stimulus (Josephson, 1961). If the source of the vibration is a copepod or similar suitable prey, it will very likely be caught on the capitate tentacles as a result of this movement. If a finely tipped rod is brought near to the hydroid, the response is elicited by a tap to the manipu­ lator which holds the rod (Fig. 5). A displacement of 2 to 3 µ is effective at a distance of O ·5 mm. As with all the vibration receptors mentioned, rested animals are more sensitive than those recently disturbed. The response rapidly fatigues and therefore the animals are insensitive to general water currents. In fact, there is no response to repetition frequencies greater than about 3/sec, and there is a progressive rise in threshold when a steadily increasing water current flows over the whole polyp. There is no response to the current and its associated eddies, but a sudden pulse superimposed on this can still produce a response. These are also features of the fingers of Leucothea. The receptor has not been identified and there are no experiments which even suggest its nature. The hydroid need not be attached by its stolon, and when deprived of its rather short capitate tentacles the sensitivity is reduced. When Dr Josephson was searching for possible receptors, there were no suggestions from other coelenterates or ctenophores that sensory cilia might be involved. However, it has been known since the work of the Hertwig brothers that in hydromedusans, anemones and ctenophores sensory neurones bear cilia. Although repeatedly mentioned by later workers, these processes of sensory cells have never been thoroughly examined in life, and in most instances we do not know whether they are motile or not, even if they are tnlly cilia. Similar processes on cells of the tentacles of anemones have been discussed by Pantin (1942) in relation to the discharge of nematocysts. There is one suggestion in the literature that underwater vibrations may play a part in the life of anemones. Passano and Pantin (1955) observed that move­ ments of a probe above the disc of Oalliactis can cause twitches of the tentacles. Again no particular receptor was identified.

Feeding by Chaoborus Larvae (Diptera) - This is an astonishing convergence between the aquatic larvae of a group of insects and the chaetognaths. Although it is not my purpose to survey the possi­ bilities of underwater vibration receptors in aquatic insects and their larvae, this example will emphasize the probable widespread distribution of such receptors. The transparent larva of the corethrid mosquito Ohaoborus (Culicidae, Diptera) usually hangs motionless in mid-water in freshwater ponds in St. Andrews. A variety of modified hairs project from the body (Fig. 6) and quiver with all water movements. An air cavity at each end adjusts the buoyancy appropriately. The mouth is armed with sharp grasping mandibles, and prey, such as copepods, are caught by the prehensile antennae. The animal is difficult to see on account of its transparency and it grabs at passing small animals exactly as does Spadella. Ohaoborus has small compound eyes, which in the light may be of significance in feeding, but it feeds well in total darkness. UNDERWATER VIBRATION RECEPTORS 403 When the probe attached to the loudspeaker diaphragm is brought near to a Ghaoborus larva, the animal may at first attack but then jumps away when the probe touches it. If the probe is vibrated at a low frequency the animal will now jump away before the probe reaches it. The response is clearly directional and changes with repetition. If, however, the frequency of vibration of the probe is raised and the amplitude reduced, the larvae are now attracted by it, and make grabbing movements at the end. At a frequency of 200/sec and a tip amplitude of 5 µ, the larvae respond over distances up to 5 mm, which is the normal range of their grabbing action. Strong vibrations of any kind disturb the animals and they swim away.

Fig. 6.-CJhnobortt8 larva, with head on the left, showing the variety of modified hairs (exaggerated in this drawing), the prehensile antennae used for grabbing prey, and the posterior flat paddle formed by branched hairs. The two types of modified bristles drawn larger below tremblP with every movement of the water.

No reference to this type of feeding behaviour in any aquatic insect has come to hand, but, considering related phenomena, there are many reasons for in­ ferring that a vibration sense plays a large part in aquatic insects. Fine bristle and hair sensilla are abundant; both single and elaborate chordotonal organs are common and are known in certain adult insects to be receptors of vibrations through the substrate or through the air (Autrum, 1959). There is evidently a large field for further study.

DISCUSSION

In this paper I am leaving out of account all the difficulties of distinguishing between pressure changes and water movements as the stimulus to receptors of underwater ripples. These important physical problems are reviewed by Frings (1964) and by other authors in the same review. The examples listed now reveal a class of receptors which are typically small projections from the surface of aquatic animals. They are usually less than 100 µ, long, thin in proportion, and therefore easy to miss in a superficial search. The mobile projection is coupled to the surrounding water and moves with it, thereby allowing a sensitivity to bending at the base to be interpreted as a movement of the water. Adaptation is rapid and fatigue is marked, being accompanied by very wide changes in threshold. Responses are sometimes directional by virtue of the receptors occurring in fans, ridges, or along the bottom of a canal. Most aquatic 404 G. A. HORRIDGE animals probably have receptors of this type but they have not been identified because the relevant tests have in most cases never been made. Responses by invertebrates to moving prey are often attributed to the eyes without further consideration. The typical behavioural responses are (a) of an eyeless predator towards movements of its prey and (b) of a delicate planktonic animal which moves away from disturbed water. We have electrophysiological data from the lobster but no behavioural significance is known in that instance. The significance in the change in geotactic sign is not understood but a reason­ able theory is that the response is highly adaptive when these planktonic animals are blown into surf by an onshore wind. As they become stimulated by the approaching disturbed water they will swim down from the surface water which is drifting onshore and come into bottom water which is drifting away from the shore. Certainly many planktonic animals only come to the surface on calm days. The nature of the sensory projection is of no significance so long as it is light and connected with a nerve terminal of some kind at its base. Among coelenterates, annelids, molluscs and most of the smaller phyla, non-motile cilia on apical dendrites of sensory neurones can be expected to be the receptors where there are responses to underwater ripples; among crustacea or insects, modified cuticular hairs or fans will be suspected of the same function. However, few examples have so far been examined. For further new underwater vibration receptors, some places to look are rather obvious. For example, the tips of the tentacles of tubiculous animals of all kinds, whether phoronids, polychaetes or echiurids, can be expected to bear groups of sensory cilia with this function. Aquatic insects and their larva com­ monly bear numerous fine hairs which cannot fail but be receptors of low fre­ quency vibrations in the water. Most trochophore larvae of annelids, molluscs and others, have tufts of apical cilia from which nerve fibres run; many rotifers have setigerous papillae and there are numerous similar examples which should be investigated. Except for the lateral line organs, it is only in recent years that distance reception by underwater vibration has been studied in vertebrates; we can expect that many curious examples will turn up among the invertebrate phyla. The small extent of the movement at the base of the receptor is not a limiting factor in detection by the neurone.In our own ears and in othervibrationreceptors such as the subgenual organs of insects, it has already been shown that relative movements of about 4 X 10 -rn cm are adequate to excite (Autrum, 1959). These are movements which approach atomic dimensions. In any case, the highest sensitivity is not at a premium in the examples considered, because most of the background disturbance will be irrelevant, and a copepod as a potential prey, or a water disturbance as a potential danger, must be distinguished only when they come within the appropriate range. The ctenophores and the chaetognaths together are the chief rivals of the larval fish as predators upon planktonic copepods, and upon each other, and therefore vibration receptors in all three of these groups must play an overwhelm­ ing part in the contest for the resources of the sea. UNDERWATER VIBRATION RECEPTORS 405

SUMMARY

In the last year or two a new class of receptors has been found in several marine invertebrate phyla, namely chaetognaths, crustaceans, coelenterates and cteno­ phores; they are small innervated projections, commonly non-motile cilia or modified bristles, which are sensitive to small disturbances in the water. In planktonic predators they can serve to locate and capture prey, such as copepods, which set up vibrations as they swim, or they serve as detectors of ripples at the water surface. Some are inferred only from responses; in others, the receptor is identified and its structure described, and direct records from the receptor neurones have been made in the lobster.

REFERENCES Autrum, H., 1959. Nonphotic receptors in lower forms. In, Handbook of Physiology, Sect. 1, Vol. 1, edited by J. Field, Puhl. Am. Physiol. Soc., Washington, D.C., pp. 369-385. Chun, C., 1880. Die Otenophoren des Goljes van Neapel und der angrenzenden Meeres-Abschnitte. Engelmann, Leipzig, 313 pp. Eakin, R. M. and Westfall, J. A., 1964. Fine structure of the eye of a chaetognath. J. Gell Biol., Vol. 21, pp. 115-132. Frings, H., 1964. Problems and prospects in research on marine invertebrate sound pro­ duction and reception. In, 111arine Bioacoustics, edited by W. N. Tavolga, Pergamon Press, Oxford, pp. 155-173. Hertwig, 0., 1880. Die Chaetognathen, eine Monographie. Jena Z. Naturw., Vol. 14, pp. 1-111. Hertwig, 0. and Hertwig, R., 1878. Das Nervensystem und die Sinnesorgane der llfedusen, monographisch dargestellt. Vogel, Leipzig, 189 pp. Hertwig, 0. and Hartwig, R., 1897. Die Actinien, eine Monographie. Jena Z. Naturw., Vol. 13, pp. 457-640. Hertwig, R., 1880. Ueber den Bauder Ctenophoren. Jena Z . Naturw., Vol. 14, pp. 313-457. Horridge, G. A., 1955. The nerves and muscles of medusae. II. Geryonia proboscidalis Esch­ scholtz. J. exp. Biol., Vol. 32, pp. 555-568. Horridge, G. A., 1965a. Non-motile sensory cilia and neuromuscular junctions in a ctenophore independent effector organ. Proc. ray. Soc. B, Vol. 162, pp. 330- 350. Horridge, G. A., 1965b. Rolations between nerves and cilia in ctenophores. Amer. Zool., Vol. 5, pp. 357- 375. Josephson, R. K., 1961. The response of a hydroid to weak water-borne disturbances. J. exp. Biol., Vol. 38, pp. 17- 27. Laverack, M. S., 1962. Responses of cuticular sense organs of the lobster Hornarus vulgaris (Crustacea). II. Hair-fan organs as pressure receptors. Comp. Biochem. Physiol., Vol. 6, pp. 137-145. Pantin, C. F. A., 1942. The excitation of nematocysts. J . exp. Biol., Vol. 19, pp. 294-310. Passano, L. l\L and Pantin, C. F . A., 1955. l\Icchanico1 stimulation in the sea-anemone Oalliactis parasitica. Proc. ray. Soc. B, Vol. 143, pp. 226-238. Romanes, G. J., 1877. Further ob ervations on the locomotor system of medusae. Phil. Trans., Vol. 167, pp. 659-751. Will, J. G. F., 1844. Horae '1.'ergestinae, Beschreibung und Anatomie der im Herbste 1843 bei Trieste beobachteten Akalephen. Voss. Leipzig, 81 pp.