7. exp. Biol. (1975), (a, 389-404 389mVith 14 figures

Printed in Great Britain

ELECTROPHYSIOLOGY AND CO-ORDINATEDBEHAVIOURAL RESPONSES IN THE COLONIAL

BRYOZOAN MEMBRANIPORA MEMBRANACEA (L.)

BY J. P. THORPE,* G. A. B. SHELTON ANI> M. S. LAVERACK

Gatty Marine Laboratory and Department of Natural History,University of St Andrews, Scotland

(Received 27 August 1974)

SUMMARY

1. There is a colonial retraction response in the Bryozoans Membraniporamembranacea and Electra pilosa.

2. The conduction velocity of the response is about 100 cm sec"1.3. The colonial response will circumnavigate the end of a cut, but will not

cross it.4. The lophophore retraction time is 60-80 msec.5. The lophophore retractor muscle with a peak contraction rate of 20 +

muscle lengths per second is probably one of the fastest contracting musclesknown.

6. The colonial responses to successive stimuli under certain circum-stances are similar to those of some corals.

7. Nervous pulses can be recorded travelling across the colony at the samevelocity as the colonial response.

8. Increases and decreases in the number and frequency of T i pulsescorrespond with increases and decreases in the area and duration of thecolonial response and are produced in response to the same stimuli.

9. Other pulses can be recorded which correspond tp the retraction of thelophophore retractor muscle.

10. The lophophore retractor muscle is apparently under the control ofa giant axon from the zooidal ganglion.

11. The colonial nervous system has many of the properties expected of anerve plexus.

INTRODUCTION

Membranipora membranacea belongs to the largest class of living Bryozoa (= Ecto-procta), the Gymnolaemata, and to the dominant order, the Cheilostomata. It formsextensive flat colonies on the fronds of various kelps (Laminaria spp.). The zooids areelongate and rectangular, each measuring about 800 x 300 x 200 /rai. They arearranged quincuncially, or as bricks in a wall, and each is thus contiguous to six others.All adjacent zooids are inter-connected by a pair of communication organs (poreplates) in the dividing wall.

Bryozoa are filter feeders depending on an extensible lophophore of slender ciliatedtentacles for the collection of food particles. This apparatus is delicate and presumably

• Present address: University College of Swansea, Swansea, U.K.25-3

390 J. P. THORPE, G. A. B. SHELTON AND M. S. LAVERACK

CJS.

Fig. i. Diagram of Membrardpora manbranacea to show the main anatomical features. Scale500/un. L. =3 Lophophore; O. = 0perculum;2.te. = Zooid walljF.m. = Frontal membrane;P.m. = Parietal muscles; O.o.m = Opercular occlusor muscles; L.r.nu = Lophophore re-tractor muscles; A.c. = Alimentary canal; C.t. = Cut side of zooid wall.

Fig. 2. Diagram to show the colonial nervous system of Electra pilosa. Modified after a photo-graph by Lutaud (1969). Scale 150 fun. N.g. = Nerves to ganglion; C.n. = Colonial nerves;Z.w. = zooid walL

highly vulnerable to damage. The main defensive mechanism is the rapid withdrawalof the lophophore into the zooecium and (in cheilostomes only) the subsequentclosure of the operculum (see Fig. 1). In Membranipora and other anascan cheilo-stomes, extension of the lophophore is associated with eversion of the tentacle sheath.This follows the increase in hydrostatic pressure caused by the downward pull of theparietal muscles on the frontal membrane.

Colonial behaviour in Bryozoa 391

Until recently very little was known about the nervous systems of Bryozoa andthere was some disagreement between authors (Nitsche, 1868; Gerwerzhagen, 1913,Marcus, 1926; Graupner, 1930; Bronstein 1937). Moreover there was no work of noteon the Cheilostomata. The basis of present knowledge is the recent detailed studies byLutaud (1969, 1971, 1973) on the anascan cheilostome Electra pilosa (L.).

Each zooid contains a circumpharyngeal nerve ring with a well developed ganglionon the dorsal (anal) side. From this ring at least four nerves run up into each tentacle(Lutaud, 1973; Thorpe & Laverack unpublished) and other nerves connect withvarious organs. Gordon (in press) states that in Cryptosula paUasiana there are sixnerves running up each tentacle.

Marcus (1926) in a major study concluded that there was no nervous connexionbetween zooids and that no zooid responded to a stimulus applied to a neighbour.There has been no work of substance to contradict this, although Bronstein (1937)reported colonial responses in Bowerbankia and interzooidal connecting nerves in thestolons of this species. Recent work using the electron microscope has failed to confirmthe existence of any nerves in the stolons of this species (personal communication -D. P. Gordon). Hiller (1939) wrote a short note claiming to have observed inter-zooidal nervous connexions in Electra pilosa. Reviewers have regarded this evidence asinsufficient for them to disagree with the conclusions of Marcus (1926) (Hyman,1959; Brien, i960; Bullock & Horridge, 1965; Ryland, 1970).

In 1969, however, Lutaud, using methylene blue staining, produced evidence ofwhat appears to be a colonial nervous system in E. pilosa (Fig. 2). She described ineach zooid a nerve which runs around the basal border of the interzooidal walls andconnects via the pore plates with a similar nerve in each adjacent zooid. This networkis connected to the ganglion of each zooid via two nerves running along the tentaclesheath. Ryland (1970) concluded that the physiology and role of this nervous systemrequired investigation; and such was the main purpose of our work.

MATERIALS AND METHODS

Selection of species

The species Membranipora membranacea was chosen because it is readily availablelocally throughout the year and the large, flat colonies are convenient for experi-mentation. The closely related Electra pilosa used by Lutaud (1969), Marcus (1926)and Hiller (1939) was rejected because of the small size of the colonies compared tothose of Membranipora, although much of our work was subsequently repeated onthis species.

The colonies were collected by pulling up fronds of Laminaria from the sublittoralzone. New stocks had to be obtained every two or three days because survival in thelaboratory was poor even in running sea water.

Mechanical and electrical stimulation

For these experiments parts of colonies of Membranipora on Laminaria were cut offand mounted, using pins, on a sheet of cork glued to the bottom of a dish of cold seawater. The sea water in the dish was changed at frequent intervals to maintain atemperature of about 10 °C. Every effort was made to keep the period of emersion as

392 J. P. THORPE, G. A. B. SHELTON AND M. S. LAVERACK

short as possible when transferring colonies to the dishes and also to avoid stimulatingthem by vibrations during experiments.

Mechanical stimulation was given using a glass micropipette, of tip diameter about20 /an, mounted on a micromanipulator.

Electrical stimulation was by means of a silver stimulating electrode of tip diameterabout ioo/un. A Tektronix type 161 pulse generator was used in conjunction with aTektronix type 162 waveform generator to give square-wave pulses, the amplitude andduration of which could be adjusted as required. After placing the electrode againstthe surface membrane of a zooid, time was given for the colony to recover frompossible effects of the mechanical disturbance before any electrical stimulus was given.

To find out what effect they had on the transmission of the colonial response,incisions were made in the colony, using a new scalpel blade. After cutting, theanimals were left to recover before experimentation was commenced.

Measurement of lophophore retraction times

The most instructive technique for measuring lophophore retraction times wouldprobably be high speed cinephotomicrography, but as suitable equipment was notavailable an alternative method was devised.

Because of the microscopic size of the zooids, any apparatus using physical means tomonitor movement was unsuitable. An optical method was, therefore, employed.Zooids were so positioned that, when the lophophore was extended, a light beam to aphotodiode (Texas Instruments type H-38 NPN photo duo-diode) was partiallyobscured. The resulting change in light intensity altered the resistance of the photo-diode, which produced a variation in the voltage drop across it. This voltage changewas amplified and then monitored on a pen recorder and oscilloscope.

The apparatus was built around a binocular microscope with a moving stage. Thephotodiode was positioned at the focal point of one eyepiece, which was about 1-5 cmabove the lens. (Modified from Campbell, 1972.)

Strips of Membranipora on Laminaria were cut to about 3 cm x 0-2 cm. These weremounted in a glass bottomed Perspex cell rilled with cold sea water. The animals wereheld in position by placing the ends of the strips into slots cut in the Perspex. Thesewere so positioned that the light beam to the objective lens was parallel to the surfaceof the colony and at right angles to any extended lophophores. Extended lophophoreswere located using the free eyepiece (which did not have the photodiode attachedto it).

This apparatus was subsequently modified by the addition of a stimulating elec-trode. The input to this was also fed into the pen recorder, thus making it possible tomeasure the time elapsing between stimulus and response (reaction time).

Electrical recordings

Standard polythene suction electrodes, modified from McFarlane (1969), of tipdiameter about 100 fim were positioned on the frontal membranes of individual2ooids. Recordings, amplified by differential pre-amplifiers and monitored on oscillo-scopes (Tektronix types 564B and 561B), were made of both spontaneous and stimu-lated activity from various points on the colony. Electrical stimuli were given usingsuction electrodes.

Colonial behaviour in Bryozoa 393

Fig. 3. Approximate shape of the area of the colonial response. This represents the idealizedshape derived from many visual observations, but which in any single experiment may showgreat variation. The black spot marks the point of stimulation. Scale 2 cm.

4000 -

2000 -

2 3 4Stimulus

2 3 4Stimulus

Fig. 4 (a) Numbers of rooids responding to successive stimuli. Stimuli just above thresholdat 2 sec intervals. (6) Numbers of zooids responding to successive stimuli. Stimuli well abovethreshold at 2 sec intervals.

RESULTS

Mechanical and electrical stimulation

It was found that mechanical stimulation of an extended lophophore ofMembranipora resulted solely in the retraction of that lophophore. If, however, not thelophophore but the frontal membrane was stimulated, the result was the immediaterapid withdrawal of all the extended lophophores within some distance of the zooidstimulated. The response was the same to electrical stimuli.

The shape of the area covered by the response is very variable but is often approxi-mately as shown in the accompanying diagram (Fig. 3), being greatest in a directionparallel to the longitudinal axis of the zooids.

The area affected increases with the strength of the stimulus up to a maximum ofabout 10 cm by 5 cm. This was never exceeded in the colonies we studied.

The time taken by a colony to re-extend its lophophores following a single stimulusdepends on the extent of the response, but is usually not more than about 2 min.After a colonial response it was always the zooids furthest from the point of stimu-lation which re-extended their lophophores most quickly, and those nearest which

394 J- p - THORPE, G. A. B. SHELTON AND M. S. LAVERACK

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Fig. 5. Graph of time against stimulus number to show response duration for successivestimuli (twice threshold) at 2 min intervals.

reappeared last. Those on the edge of the area of a response often withdrew onlymomentarily and reappeared almost immediately.

The sensitivity of the animals to mechanical stimulation depended also upon thepart of the surface membrane touched. However, it was difficult to investigate thisbecause of the extremely small size of the zooids. The tentacle sheath, the area of thefrontal membrane immediately surrounding this and the operculum appeared to bethe most sensitive, with no great variation in sensitivity over the rest of the membrane.

Another factor governing the size of the colonial response was the velocity of thestimulus. A rapid mechanical stimulus produced a greater response from the colonythan a slow one, although the degree of membrane deformation was about the samein each case. If the stimulator was left in contact with the membrane, the animalsreextended their lophophores after a short time.

In the case of electrical stimulation, the threshold values for the duration and voltageof the stimulus were found to vary considerably, depending on the state of the colony.Similarly the response to stimuli of fixed voltage and duration varied between stimuli.The threshold voltage was typically about 1*5 volts at 10 msec.

Area and duration of colonial responses

Whether a decrease or an increase was obtained in the area of a colonial responsewas found to depend upon the size and frequency of successive stimuli. The resultswere comparable for both mechanical and electrical stimuli.

If stimuli just above threshold at short interstimulus intervals (e.g. one or twoseconds) were used, a marked initial increase in response was shown. The response tothe second stimulus was far greater than that to the first, and was maximal or nearmaximal. Subsequent stimuli produced very little increase in the area of response

Colonial behaviour in Bryozoa 395120 i

80

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7 9Stimulus

11 13 15

Fig. 6. As Fig. 5. Note the partial return of the colonial response followinga change of stimulus position.

(Fig. 4a). If stimuli well above threshold were used, a near maximal response wasobtained to the initial stimulus, and subsequent stimuli had little or no further effects(Fig. 46). If the stimuli were continued the response diminished and eventually mostof the polypides re-emerged.

With long interstimulus intervals (e.g. 2 min) and stimuli well above thresholdthere was no increase in response area and duration following the second stimulus(Fig. 5). On the contrary, the area and the duration of the response following successivestimuli were found to decrease. With stimuli just above threshold at long interstimulusintervals, the response to each stimulus was about the same as to the one before it.

As stated earlier, after a burst of stimuli well above threshold at long interstimulusintervals, the colony ceases to respond. If, however, the point of stimulation is movedfrom the zooid previously stimulated to one of its immediate neighbours there is asudden partial return of excitability with the response increasing to about half of thatobtained to the initial stimulus. With successive stimuli this response also diminishes(Fig. 6).

Effects of cuts across the colony

In the case of either electrical or mechanical stimulation, a cut across the colony willprevent the spread of a colonial response. The response will spread to a limited extentaround the end of a cut (Fig. 7).

Speed of retraction response

Using the light beam apparatus it was found that the time for the complete retrac-tion of the lophophore in Membranipora membranacea was usually 60-80 msec. Thisis the normal withdrawal time for the 'escape' response. Other withdrawals at slowerspeeds were recorded, taking anything up to 500 msec. The animals are also capableof stopping the withdrawal before completion and of changing the speed of a

396 J. P. THORPE, G. A. B. SHELTON AND M. S. LAVERACK

(6)-

Fig. 7 (a) Unhindered spread of colonial response from point of stimulation. Scale 2 cm.(6) Failure of response to cross a cut in the colony, (c) Circumnavigation of the end of a cut bythe colonial response.

1 Mr tFig. 8. Type i (Ti) pulses. 'Spontaneous' activity in the colonial nervous system.

Scale = io /*V, 25 msec.

withdrawal whilst it is taking place. The great majority of recorded withdrawalswere, however, of the very rapid type.

Extension rates are far more variable, frequently not at an overall constant speedand may last from about 200 msec up to a few seconds.

With Electra pilosa the results obtained were very similar, although the fast with-drawals were slightly slower than those of Membranipora.

Often when several lophophores were in the field of view they would all be retractedapparently in synchrony. The time taken for this colonial response was the same(approximately) as that for a single zooid. Again this was also found to be the case withElectra.

The latency between stimulus and response in Membranipora is about 20-30 msec.This increases by about 10 msec cm"1 with increasing distance of the zooid from thepoint of stimulation.

Colonial behaviour in Bryozoa 397

ir

Fig. 9(0) Initiation of a burst of Ti pulses in response to an electrical stimulus (is V, 10 ms).The total burst duration was 13 sec and peak frequency 220 Ti pulses/sec. Triangle marks thestimulus artifact, arrows indicate Ta pulses with Ti pulses superimposed upon them. Becauseof their large amplitude parts of the T2 pulses have been lost. Scale •= 20 /*V, 50 msec.(6) Same burst after 3 seconds, (c) Same burst after 11 seconds. Note that Ta pulses occurnear the peak Ti pulse frequency.

Electrical responses

Two types of electrical pulses were recorded from Membranipora. These we havenamed 'type one' (Ti) and 'type two' (T2). Very similar pulses can also be recordedfrom Electro pilosa.

Ti pulses are of very short duration (about 3 msec) and are biphasic with an ampli-tude of about 10 fiV (Fig. 8). They occur occasionally singly, but more often inbursts, during which the frequency may reach a maximum well in excess of twohundred pulses per second.

Simultaneous recordings at various points on the colony show that T i pulses areconducted between zooids over an area greater than that covered by the colonialresponse. There is a short delay between the arrival of Ti pulses at different recordingelectrodes. From this the conduction velocity can be shown to be about 100 cm sec"1

in a direction parallel to the longitudinal axis of the zooids and about 50 cm sec"1 atright angles to this. Similar figures for conduction velocities are obtained using thedelay between stimulation and the arrival of the first Ti pulse at electrodes a knowndistance from the point of stimulation.

In response to an electrical stimulus large numbers of Ti pulses are produced athigh frequency (Fig. 9). The variation of frequency with time is shown in Figs. 10 and11. The Ti frequency is initially high; this frequency then increases slightly (up to250 pulses sec"1) followed by a decrease to a tonic level. There is subsequently arelatively sudden drop in frequency with the discharge eventually ceasing entirely. Thebursts with the greatest total duration are those with the highest initial T i frequency.

J. P THORPE, G. A. B. SHELTON AND M. S. LAVERACK

140 r

2000 25001000 1500Time (msec)

Fig. io. Graph of frequency against time for a stimulated burst of Ti pulse*.

3000

140 r-

120

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o

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500 1000 1500Time (msec)

2000 2500 3000

Fig. 11. Graph of frequency against time for a series of T i bursts in response to electrical stimuliat a min intervals. N.B. For clarity the curves only have been drawn and not the experi-mentally determined points.

There is a significant decrease in both overall and peak frequency (Fig. 12) ofbursts with increasing distance from their point of origin. This effect is most pro-nounced with short bursts. With successive stimuli the discharge is continued for amarkedly decreasing length of time (Fig. 13).

With a stimulus interval of 2 min the T i bursts in response to each stimulus

Colonial behaviour in Bryozoa120

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Distance (cm)

Fig. i s

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Stimulus number

Fig. 13Fig. 12. Reduction in peak frequency of a high frequency Ti burst with increasing distancefrom the point of stimulation.Fig. 13. Reduction in the number of Ti pulses produced in response to successive stimuli at2 min intervals.

rFig. 14. Type a (T2) pulse. At this amplification part of the initial phase of the T2 pulse hasbeen lost because of the size of the oscilloscope screen. Its true amplitude is therefore greaterthan is apparent Scale 20 /tV, 50 msec.

terminates before the next stimulus arrives and the number of Ti pulses decreaseswith successive stimuli. In an experiment in which repetitive stimuli were given(2 msec, 25 V) at 630 msec intervals, the Ti response to the first shock was not completedby the time the second shock arrived. The peak Ti pulse frequency after the secondstimulus increased by twenty-five per cent over the peak Ti pulse frequency followingthe first shock. There was a further small increase after the third shock. The peak Tifrequency after the fourth shock was the same as that after the third but the fifth,sixth and seventh shocks produced a steadily declining response.

T2 pulses (Fig. 14) are of much greater duration (100-120 msec) than T i pulsesand consist of two distinct parts. The first part is a biphasic pulse of large amplitude(up to 200/iV) and short duration (5-10 msec); the second is not biphasic and is ofsmall amplitude (10-15 fiV), but long duration (about 100 msec).

400 J. P. THORPE, G. A. B. SHELTON AND M. S. LAVERACK

It can be shown by observation that a T2 pulse is recorded whenever the lophophoreis retracted. It is also recorded when, as often happens, the retracted lophophore ispulled further down inside the tentacle sheath.

T2 pulses can normally be seen to accompany high frequency Ti activity, butoccasional 'spontaneous' T2 pulses may occur when the number of T i pulses re-corded is few or none.

The possibility that the second part of the T2 pulse is an artifact caused by move-ment of the frontal membrane of the zooid under the electrode tip was investigated.It was established that the amount of movement necessary to produce an artifact of thesame amplitude and duration as the recorded pulse was far larger than any movementthat could have been produced by the animal.

DISCUSSION

The present results show that there is a colonial retraction response in both Mem-branipora membranacea and Electro pilosa. The conclusion of Marcus (1926) that nozooid responds to a stimulus applied to a neighbour holds only in the case of stimuliapplied to a single lophophore.

Stimulation of the surface membrane does produce a colonial response. There areseveral possible methods by which this response may be coordinated, but the mostobvious and in our view most likely is that there is a colonial nervous system, probablyas described in Electro, by Lutaud (1969). This links contiguous zooids by through-conducting nerve pathways.

An alternative hypothesis is that hydrostatic pressure changes caused by retractionof the lophophore are transmitted between zooids through holes or thin membranesin the rosette (pore) plates in the interzooidal walls. Using a scanning electron micro-scope (Thorpe and Laverack, unpublished) no apparent open pores were observed inthe rosette plates or in any other parts of the interzooidal walls. Similarly transmissionelectron-micrographs of pore plates (Thorpe and Laverack, unpublished) do not showany open pores or possibly flexible membranes. As described by Banta (1969) therosette plates are rigid and calcified with balls of cells completely occluding the pores.No hydrostatic pressure sensors have been described in Bryozoa and there is noevidence to suggest their presence. Finally, the involvement of pressure changesappears to be disproved by the fact that a response can be initiated by stimulating themembrane of a marginal zooid still lacking a functional polypide.

Alternative hypotheses involving the mechanical stimulation of neighbours by aretracting lophophore do not explain either the failure of the response to cross cuts inthe colony or how such a response can traverse areas of retracted zooids. Only thepresence of a colonial nervous or alternatively a neuroid system could adequatelyexplain the limitation of a response to a particular area.

The presence of a neuroid system cannot be discounted, but Bullock & Horridge(1965) have pointed out the extreme difficulty in determining unequivocally whethernervous or non-nervous pathways conduct recorded electrical activity. Many epitheliahave structural modifications between adjacent cell walls but on its own this propertyis not enough to prove that such epithelia conduct spiking activity. As yet, there is verylittle data available on the fine structure of epithelia in Membranipora. There is good

Colonial behaviour in Bryozoa 401

evidence for a colonial nervous system, however (Lutaud, 1969), and the results re-ported here accord closely with the expected properties of that system. In the absenceof further data, we suggest that the T i system we have recorded from corresponds tothe histologically demonstrated nerve plexus. Our arguments in favour of this vieware set out in the following paragraphs.

The shape of the area responding to a stimulus applied to one zooid (Fig. 3) is con-sistent with predictions based on Lutaud's (1969) anatomical studies, if bidirectionalpolarization is assumed, with the greatest polarization along the longitudinal axis ofthe zooids. This assumption is also consistent with the variations in conductionvelocity of T i pulses in different directions across the colony. The large variation inshape of the area of response is probably due to local variations in the sensitivity ofdifferent parts of the colony affected by any one stimulus.

The non-linear increases in the area of the colony responding to successive stimuliare not easily explained in terms of a simple nerve network. If the 'all or none' natureof neuronal conduction is accepted, then most simple models which have been pro-posed for nerve nets (Pantin 1935; Ramsay 1952) and even the complex computermodel of Josephson (1964) will not account for the observed results in Membrampora.

Some of our results are, however, very similar to those obtained by Horridge (1957,1968) working on the spread of colonial excitation in Madreporarian and Alcyonariancorals. He concluded that such responses could be explained by multiple firing fromthe point of excitation in response to a single stimulus. The small increase in theresponse to the second and subsequent stimuli, which Horridge found in Porites andwe have found in Membrampora, occurs, Horridge suggests, because a rapid burst ofimpulses follows the initial stimulus and subsequently the probability of repetitivefiring in the nerve net diminishes.

Horridge explained 'facilitation', during which there is a more than linear increasein response to successive stimuli, in terms of repetitive firing at other places on thenerve net as well as at the point of stimulation. In Membrampora, the increase inresponse to stimuli just above threshold can be explained more simply as the summa-tion of the two stimuli (or conversly a lowering of threshold in response to the first one).

The partial return of the colonial response on changing the point of stimulationfrom one zooid to one of its immediate neighbours is compatible with Horridge's(1968) model. Decreases in the area and duration of colonial responses are caused by alessening of repetitive T i pulse firing from the zooid stimulated; thus there should bea substantial increase in response if another zooid is stimulated. This is, in practice,found to be the case.

The effect of cuts across the colony in Membrampora resembles their effect onepithelial conduction in echinoderms. Kinosita (1941), Smith (1950), Bullock andHorridge (1965) all indicated that in certain asteroids and echinoids epithelialtransmission occurs in straight lines, and it is a matter for debate as to how cuts arecircumnavigated, if at all. In both the Echinodermata and the Bryozoa the abilityof nervous activity to travel around the ends of cuts may be taken to indicate thepresence of a nerve net of some kind. In Bryozoa such a nervous system has beendescribed by Lutaud (1969).

The contraction times for the lophophore retractor muscle in Membrampora arethemselves of interest since the peak contraction rate is in excess of twenty times their

402 J. P. THORPE, G. A. B. SHELTON AND M. S. LAVERACK

own length per second. This most exceptional rapidity is less surprising, however,when the extremely small size of the animal is taken into account. It would not besurprising if these muscles showed interesting adaptations to contraction at highvelocity under very light load. From electromicrographs (Gordon, in press; Thorpeand Laverack, unpublished) the tissue appears to be smooth muscle.

The latency of the contraction response is apparently about 25 msec for zooids at anegligible distance from the point of stimulation. For zooids further away this isincreased by the time taken for the stimulus to be transmitted to the animal. This isapproximately 10 msec cm"1 (or 100 cm sec"1).

Implications of electrical recordings

The properties of the T i system lead us to believe it is nervous for the followingreasons:

(i) Ti pulses are of very short duration: 3 msec.(ii) they are conducted across the colony, arguing for a system which links all, or

many, zooids together.(iii) the conduction velocity (100 cm sec"1) is the same as that calculated for the

speed of transmission across the colony, as measured by lophophore retraction.(iv) they are generated after electrical and mechanical stimulation.(v) they reach high frequencies (occasionally > 200 sec"1) and occur in bursts of

varying length.We suggest that T i pulses are nerve pulses travelling through the colonial nervous

system. They are conducted across the colony at the same rate (100 cm sec"1) as thespread of lophophore retractions. They are produced in response to stimulation andapparently influence lophophore retraction. The reduction in the number of Ti pulsesfollowing successive stimuli corresponds with the reduction of the colonial response.They are produced in a burst (i.e. multiple firing) in response to a single stimulus aspredicted from the model proposed by Horridge (1968). The fit with this model is notcomplete however since the number of pulses in a burst does not decrease linearlywith distance from the point of stimulation and Ti pulses can be recorded outside thearea of the colonial response. Clearly, therefore, Horridge's model is not adequate toexplain our results.

It seems probable that in Membranipora the spread of response is limited in someway by the reduction in peak frequency and increase in total duration of a burst of T ipulses as it travels across the colony.

The T2 pulses are probably of a compound nature. The initial phase is rapid(5-10 msec) and of large amplitude (up to 200 /*V) whilst the latter stage is compara-tively slow (about 100 msec). In our opinion these two responses are due to the motorinnervation and subsequent contraction of the lophophore retractor muscle. T2pulses occur whenever a lophophore is retracted and their latency is similar to that oflophophore retraction. The retraction time of the lophophore is somewhat faster thanthe second phase of a T2. This difference can, however, be accounted for if allowanceis made for the extra time taken to retract the lophophore further down inside thezooecium after it has passed through the orifice, and continuation of electrical activityin the muscle after the cessation of visible contraction.

Colonial behaviour in Bryozoa 403

The initial phase of a T2 occurs only before a muscle contraction and has such ashort duration that it would seem to be a result of nervous activity. This activity couldbe the firing of a motor nerve innervating the lophophore retractor muscle, probablyrunning from the suprapharyngeal ganglion. Its very large amplitude strongly suggeststhat some kind of giant fibre system is involved. A structure which could be a giantaxon is described by Lutaud (1971) in the main tentacle sheath nerve in Electra.

There are several unusual features shown in the electrophysiological responses ofMembranipora (and Electra). The high frequency of firing in the colonial nervoussystem (200 + sec"1) is unusual but not unique. The very long periods (up to 10 sec)for which these small nerves will continue to fire at high frequency are most surprising.This suggests that the axons of the colonial nervous system have an exceptional degreeof tolerance to changes in internal ionic concentration.

Integration in the nervous system of Membranipora

The activity of an individual zooid of Membranipora consists mainly of extensionand retraction of the lophophore. This movement, however, is now seen to be de-pendent on the integration of two distinct nervous systems.

Each zooid has a well-developed ganglion and its own discrete nervous system. Atthe same time the zooid is connected to its neighbours and is influenced by them viathe colonial nervous system. The contraction of the lophophore retractor muscle iscontrolled, probably via a giant axon, by the ganglion. Whether the lophophore isextended or withdrawn depends upon the input to the ganglion of Ti pulses from thecolonial nervous system and also information from the zooidal sensory nervoussystem.

In addition it appears that the physiological state of the ganglion can change withtime. Responses of individual zooids are therefore governed by both internal andexternal conditions.

The great regularity of the T i pulses in a burst suggests that each ganglion has cellscapable of acting as pacemakers. Since no extra pulses are found during a burst it isalso probable that the firing of the colonial nervous system by one ganglion inhibits theproduction of any other pulses of lower frequency from other ganglia. Therefore thesame pulses within the colonial nervous system may simultaneously produce bothexcitatory and inhibitory effects on other ganglia.

We wish to thank Dr J. S. Ryland for reading and criticizing the manuscript, DrsJ. L. S. Cobb and I. D. McFarlane, Mr P. R. Balch and the staff of the Gatty MarineLaboratory for their assistance.

G.A.B.S. is supported by a Science Research Council Research Studentship.

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404 J. P. THORPE, G. A. B. SHELTON AND M. S. LAVERACK

REFERENCES

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