J. Exp. Biol. (1965), 43. 79-106 jgfVith 1 plate and 10 text-figures

Printed in Great Britain

THE OCCURRENCE AND FUNCTIONAL CHARACTERISTICSOF HETEROMORPH ANTENNULES IN AN EXPERIMENTALPOPULATION OF SPINY LOBSTERS, PANULIRUS ARGUS*

BY D. M. MAYNARD

Bermuda Biological Station and Department of Zoology,The University of Michigan, Aim Arbor

{Received 29 September 1964)

INTRODUCTION

The occurrence of heteromorph antennules growing in place of amputated eye-stalks is a well-documented phenomenon among the higher Crustacea, and there areextensive treatments of the associated anatomy (Milne Edwards, 1864; Herbst, 1896,1900, 1917; Hofer, 1894; Wolsky, 1931). There are also scattered physiological studies(Herbst, 1910; Lissmann & Wolsky, 1933; Maynard & Cohen, 1965) which demon-strate functional connexions between sensory elements on the heteromorph and thecentral nervous system. In some cases, the behaviour and neuromuscular activityelicited by these connexions is very like that caused by normal antennule stimulation.

In each of the physiological studies, however, only a few individuals were analysed,and in none were responses followed over any period of time. Interpretation of thefindings therefore suffered because one has been unable to say how representative theindividuals studied may have been, or whether the responses described representedstable or transient patterns in the individual. The need to have some answer to suchquestions became particularly evident in the course of a detailed analysis of theneurophysiology of a ' naturally occurring' heteromorph in a spiny lobster, Panulirusargus (Maynard & Cohen, 1965).

The present paper represents an initial attempt to approach these two problems.It presents a description of the heteromorphs which developed in about 50 % of anexperimental population of P. argus whose eyestalks were removed unilaterally.Functional connexions between heteromorph and brain were found in some form inevery instance tested. They often appeared to be quite specific, and, in at least someindividuals, relatively stable for periods of 12 months or more.

METHODS

Eyestalks were removed unilaterally from three series of adult or near-adult P. argusLatreille. In Series O eyestalks were removed from eighteen lobsters on 17 March1962. In six of these eighteen, only eyestalks were ablated; but in the remainingtwelve, the distal segment and flagella of an antennule were also removed, six ipsi-lateral, six contralateral. In addition, six more lobsters with only antennules removed

• Contribution number 365 from the Bermuda Biological Station, St George's West, Bermuda. Thefirst modern report of the occurrence of a heteromorph antennule in a spiny lobster was made 24 October1864 by Alphonse Milne Edwards before the Academy of Sciences, Paris.

80 D. M. MAYNARD

were used as controls. In Series P, eyestalks only were removed from six lobsters oq8 August 1962. In Series Q, eyestalks were removed from twenty-five lobsters on16 March 1963. After operation the lobsters were maintained in running sea water inlarge cement tanks at the Bermuda Biological Station. Water temperatures rangedfrom below 150 C. during February and March to over 300 C. during August andSeptember. The lobsters were fed periodically on fresh or frozen fish.

Eyestalks were removed by cutting across the eyestalk with heavy scissors betweenthe proximal and distal chitinous segments. This removed the medulla terminalis andall distal optic ganglia (see Maynard & Dingle, 1963). The stump was crushed withhaemostats to prevent excessive bleeding.

Mortality at the operation was nil, but subsequently a number of lobsters were lostin the holding tanks, often at ecdysis. Animals were also killed periodically for histo-logical study and in the course of acute experiments. One animal from Series O wasmaintained for over 2 years 3 months, one from series P for more than 1 year 8 months,and ten from series Q for over 1 year.

For behavioural observation individuals were usually isolated in smaller, glass-fronted aquaria. For electrophysiological observations lobsters were removed fromthe tanks and immobilized by tying to a partially submerged board. The anteriorcarapace and appendages remained out of water. When moistened periodically theanimal remained in good condition for several hours and at the end of the experimentcould be returned to the tanks without apparent damage. Conventional amplifiers,oscilloscopes, cameras and stimulators were used. Initially, recording electrodes forelectromyography were connected, via cuts in the antennular cuticle, to the musclesinvolved, but in subsequent experiments surface electrodes placed on undamagedcuticle gave comparable records. Surface stimulating electrodes were used.

Carapace (cape) lengths were measured from the most anterior edge of the carapace,between the horns and above the optic yoke, back to the posterior, mid-dorsal margin(Travis, 1954). The relation between cape length and body weight is given in a previouspublication (Maynard, i960).

RESULTS

Ecdysis and growth

Thirty-seven of the forty-three lobsters of Series O and Q remained alive to thefirst moult. Fig. 1 gives the size distribution of the population at the time of theoperation together with the size distributions of the subpopulations moulting withinthe first 3 months of the operation (before 18 June); during the fourth month (18 Juneto 18 July); and during or after the fifth month. The smaller animals tended to moultearlier in the season than did the larger animals.

All except one lobster—the largest—moulted within 5 months of the operation, i.e.152 days. Subsequent intermoult intervals were measured precisely for only a fewanimals. In one lobster with a cape length of 11*35 cm., the interval between thefirst post-operative moult in August and the second in March was over 6 months,194 days. In four individuals, with cape lengths ranging from 10-4 to 12a cm., theinterval between the first in August and the third post-operative moults in June rangedfrom 314 to 339 days, an average intermoult period of slightly over 5 months,157-170 days. Comparable data for intermoult intervals of normal adult lobsters of

Heteromorph antermules in Panulirus argus 81

this size are not available, but incidental observations suggest about two moults peryear (Sutcliffe, 1952). Slightly smaller (8-O-8-9 cm cape length) immature lobsterskept in the Bermuda laboratory moulted 3-4 times per year with intervals varyingaccording to temperature from 60 to 232 days (Travis, 1954). Apparently, therefore,the loss of the neurosecretory apparatus of a single eyestalk did not have major effectson the intermoult interval in these lobsters.

The incremental increases in carapace length are plotted for fifty-two moults inthirty-two lobsters in Fig. 2. Although a few individuals showed very little or no

8 9 10 11 12 13 14Cape length (cm.)

6 7 8 9 10 11 12 13 14

Mean

2 -

- 1 0 1 2 3 4 5 6 7Cape increase (mm.)

Fig. 2

Fig. 1. A. Size distribution, in premoult cape lengths, of experimental population alive afterfirst moult. B. Size distribution, in premoult cape lengths, according to interval betweenoperation and first moult (O-E interval), see text. The open histograms represent animals inwhich no heteromorph appeared at the first moult; the hatched histograms represent animalswhich produced a heteromorph at the first moult.Fig. 2. Distribution of growth increments, in millimetres, at moult of experimental popula-tion; fifty-two moults, thirty-four lobsters. Open histogram, Series O; hatched histogram,Series Q.

growth, most exhibited significant increases in size. The mean, 0-35 cm., was com-parable to or greater than that reported by Travis for slightly smaller animals (1954).Growth increments of Series O lobsters tended to be greater than those of Series Q(see Fig. 2). The reasons are not altogether clear, and may represent a combination offactors: size, crowding, nutrition, temperature. The last may be particularly relevant,for spring-water temperatures in 1962 (Series O) were slightly higher than in 1963(Series Q) (see Travis, 1954).

Since lobsters in crowded or adverse conditions often show little or no growth at6 Exp. BioL 43, 1

82 D. M. MAYNARD

ecdysis (Travis, 1954), the above observations suggest that the lobsters used in theseexperiments were in reasonably good health. There was no significant difference ingrowth at ecdysis between lobsters which did not regenerate a heteromorph at firstmoult (mean increment, 0-37 cm.) and those which did (mean increment, 0-34 cm.).

Occurrence and growth of heteromorph

Table 1 lists thirty-eight of the forty-three lobsters from Series O, P and Q whichwere alive at the first post-operative moult. Five lobsters from Series P which lacked

Table 1. Moult interval and heteromorph growth

ist moult 2nd moult 3rd moult 4th moult

mobster

Q-4O-i*O-21 •O - 2O-io*

O-6*O-i9#

O-i7»O-18Q - 2

Q-24O-i3»Q-7O-5 *Q-I3Q-15Q-20Q-6O-23»

O-3*Q-5Q - i

Q-21Q-8

O-9»Q-19Q-12Q-23Q-14

Q-17O-15Q-25Q-22O-7Q-16Q-10P - i

Sex

MFFMM

FFFFM

FMMMM

MFFMF

FMFFF

MFMMM

FFFFF

FMM

an heteromorph

Initialsize

Dayspost-op.

9-4 cm. 889 48-27 67 0

9 47'58 96 99 9

11-359 5

io-6I O I

1 0 3

9 99759 2 5

io-8

9 5IO-2n o1 0 4

n-351 2 0

I O I

II-451 2 3

10051185

n-45I I - I

I I - 2

io-6IO-O

1 2 1

1 3 79 1

< 93< 93< 93< 93< 93< 93< 93

9797

991 0 0

1 0 4

1 0 4

i°51 0 7

1 0 8

1 1 2

1 1 2

1 1 6

1 2 0

1 2 1

1 2 7

1 2 7

1 2 8

1 2 9

1 3 01 3 0

1 3 1

1 3 1

1391 4 0

1 4 1

1 4 2

1 4 2

144> 152< 202

length (cm.)—

Het.size0

0

0

0

0

0

0

0

a-60 6 5

0

I - I

2 9

I - I

0

0

2 40

3 33 0

2-6

i - 5I - I

2 9

3-4

2 70

3"5I - I

2-9

0

2 3

3'50

2-5

0

2 13 0

2 4

Dayspost-op.

145145

1 5 2

154170-190

.

152-321

152-3213 2 1

g

152-321

152-321

152-321

290-321152-321

152-321

'306

Het.size

0

0

0

0

3'4

.2-1

2-7

36 +

0

4 0

0

4 10

0

3-8

3 4

Dayspost-op.

514

452-456

469

469

458

355-524

Het.size

3:8

3"5

0

0

0

4 9

4-1

Dayspost-op.

514-685

—

Het.size

.4 1

Heteromorph length is given in cm.• Animals of Series O in which an antennule was removed along with an eyestalk. Mean hetero-

morph length is calculated from those animals possessing heteromorphs.

Heteromorph antetmules in Panulirus argus 83

heteromorphs are omitted. A heteromorph antennule formed within the stump of theeyestalk and appeared in place of the ablated eyestalk in twenty-two lobsters at thefirst moult.

Heteromorph occurrence and the operation-ecdysis (O-E) interval

Heteromorphs were completely lacking in the eight animals moulting within lessthan 3 months after eyestalk ablation, but they occurred in over two-thirds of thelobsters moulting during the fourth and fifth months after the operation. Heteromorphfailures were evenly distributed over these latter two months. Presumably there wasa critical period in the intermoult interval—apparently just preceding ecdysis—in whicheyestalk removal did not initiate heteromorph regeneration. When the operationoccurred at any time outside this period, however, the probability of heteromorphregeneration seemed to remain essentially constant.

Nine lobsters lacking heteromorphs after the first moult were followed through thesecond moult; only one developed a heteromorph. Three of these animals were thenfollowed through the third moult, 15 months post-operative; none developed hetero-morphs. If only O-E intervals of more than 3 months are considered, twenty-two outof thirty first moults produced heteromorphs, while only one out of twelve second orthird moults produced new heteromorphs. Apparently heteromorph flagella mustdevelop before the first moult in the normal course of events if they are to occur at all.The effects of secondary injuries to eyestalk stumps lacking heteromorphs were notexamined.

Regeneration of heteromorph and normal antennule

In eleven of the fifteen Series O lobsters of Table 1 (indicated by (•)), the distalportion of either the ipsilateral or the contralateral antennule was also removed at thetime of eyestalk ablation. The normal antennule regenerated within the stump in allof these animals and appeared at first moult. It even occurred in those moultingwithin 3 months of the operation and in which heteromorphs failed to appear.Such simultaneous regeneration of normal antennules, however, did not obviouslyaffect either the probability of heteromorph occurrence or the length of the hetero-morph in the fourth and fifth months.

Certain features of normal antennule regeneration differed from heteromorphformation. Although one individual of a control series with antennule ablation onlyfailed to regenerate on the first moult it did so on the second, and in most cases thecritical period for antennule regeneration was obviously less than that required forheteromorph development. Regeneration of the normal antennule was also moreextensive than heteromorph formation; all lost parts, both flagella and segments,reappeared together, although usually in reduced size and distorted form (see Fig. 3).

Heteromorph size

The length of the heteromorph flagellum on the first moult averaged 2-4 cm. andranged from 0-65 to 3-5 cm. It increased at the second moult to an average of 3-4 cm.,and at the third to 4-1 cm. The longest heteromorph observed in these experimentswas 4-9 cm.

These values and those of Table 1, however, are not completely reliable as measuresof incremental growth at moult. In many instances the tip of the heteromorph became

6-2

84 D. M. MAYNARD

broken or damaged before the next ecdysis, so that the increases shown are minimaland include minor heteromorph regeneration as well as simple growth.

The heteromorph lengths at first moult of animals of Series O averaged 2-7 cm.and were more consistent than those of Series Q. They also showed no progressiveincreases with longer O-E intervals. In lobsters of Series Q heteromorphs appearingduring the 4th post-operational month averaged 1 -7 cm.; those appearing during the5th month averaged significantly longer, 2-6 cm.

Normaleye-stalk

Interocularyoke (cut)

Normal outer flagellum

Fig. 3. Eyestalk, heteromorph flagellum, regenerated outer flagellum (distal portion only) ofnormal antennule, and original outer flagellum (distal portion only) of normal antennule onopposite side: all from same lobster. Animal killed after first post-operative moult and tracingmade from photograph of preserved specimens. See text for further discussion.

Heteromorph form

As in other Palinuridae (Herbst, 1900; Milne Edwards, 1864) the heteromorphantennule of Panulirus argus resembled the outer flagellum of a normal antennule.It characteristically lacked structures analogous to either basal segments or innerflagellum.

The heteromorph arose as a ringed flagellum directly from the remnants of theheavy chitinous basal segment of the eyestalk (Fig. 3). There was some variation inthe direction taken by the flagellum, but in most cases it was oriented horizontally orat a slight upward angle and pointed straight ahead (Figs. 4, 5, PI. 1). There was noindependent movement of the heteromorph. However, since it was attached rigidlyto the interocular yoke, it did tip up and down in concert with similar movements of

Heteromorph antennules in Panulirus argus 85

contralateral eye (Maynard & Cohen, 1965). The form of the chitinous base of theheteromorph varied considerably. There were occasional protuberances or sculpturingsbut no true segmentation.

The flagellum itself consisted essentially of two parts. The proximal portion wasmade up of wider annuli that bore a scattered assortment of relatively short sensoryhairs (see Laverack, 1964). In the distal portion the annuli tended to be narrower, andin addition to the sensory hairs mentioned above bore guard, companion and aesthetaschairs on the ventral side. The aesthetasc hairs were situated in two transverse rows atthe forward and rear edge of each annulus, and were bordered laterally on each sideby one large guard hair and one or two companion hairs. This gave a toothbrush-likeappearance to the flagellum.

The aesthetasc hairs are apparently chemoreceptors (Laverack, 1964), while manyof the other hairs mediate various kinds of mechanical stimuli. The brush of aesthetaschairs and associated companion and guard hairs distinguished the outer from the innerflagellum in the normal antennule. In all major respects, therefore, except number ofannuli and proportion of individual annuli, the heteromorph flagellum was like theouter flagellum of the normal antennule.

The essential structure of the heteromorph flagellum as described above was presentin all twenty-two heteromorphs observed in this study. There was individual varia-tion, however, with respect to length-breadth proportions of the flagellum, and withrespect to the relative number of proximal and distal rings. There were also changeswith time. After the first moult, the flagellum tended to retain bends and folds whichhad formed during the course of its development beneath the scab of the eyestalk stump.Imperfect or incomplete annuli were also common (Fig. 3; Fig. 4, PL 1). These werelost on the second moult (Fig. 5, PL 1). In the shorter heteromorphs the distal annuliwere fewer in number and bore fewer aesthetascs. For example, in one 1 • 5 cm. flagellum,there were eighteen proximal annuli and ten distal annuli. In a more characteristicflagellum measuring 2-9 cm., there were twenty-four proximal and twenty-eight distalannuli. Increases in flagellar length, both during primary growth and betweenmoults, appeared to involve disproportionate increase in the number of distal rings.In a typical instance, the distal annuli increased from twenty-seven to well over thirty-eight in one moult, while the proximal annuli increased by one, from twenty-four totwenty-five. In none of the animals observed, however, did the heteromorph flagellumapproach the normal flagellum in length or number of segments. The ' oldest' animal,examined 27 months and four moults after operation, had twenty-six proximal andsixty distal annuli in the heteromorph compared with sixty-seven proximal and over116 distal annuli in the normal outer flagellum. Moreover, in lobsters simultaneouslyregenerating normal flagella and heteromorphs the normal regenerate formed moredistal rings bearing the sensory brush than did the heteromorph in the same animal(Fig. 3). In a typical case, there were fifty-seven distal annuli in the normal regenerateat first moult, but only twenty-five in the heteromorph.

When the heteromorph flagellum failed to appear the eyestalk scar generally healedover in a smooth stump, and often formed a hard mound of heavy chitin (Fig. 4E,PL 1). In one individual a 1-2 mm, non-segmented nubbin appeared after the secondmoult, possibly the forerunner of a heteromorph flagellum (Fig. 4F, PL 1).

86 D. M. MAYNARD

Behavioural responses to heteromorph stimulation

General observations

Stimulation of the heteromorph flagellum may evoke three general classes ofresponse (Herbst, 1910; Lissman & Wolsky, 1933; Maynard & Cohen, 1965): (1)general escape activity and bodily withdrawal from the stimulus; (2) cleaning of thestimulated heteromorph with ipsilateral walking legs—these motions are analogous tothose employed in cleaning the normal eyestalk; (3) movements of the ipsilateralantennule. The latter have been of particular interest because they often resembleresponses normally specific to stimulation of the outer flagellum of the normalantennule (Maynard & Dingle, 1963; Maynard & Cohen, 1965).

Table 2. Behavioural responses to heteromorph stimulation

Stimulus

Mechanical Chemotactile Manipu-

Lobster

Q-i#

Q-2I*P - I

Q-I4*Q-io*Q-5Q-7Q-8O-12Q - 2Q-15Q-23Q-25*Q-6Q-24O-i7#

Total no.Total no.Total no.

Hetero-morphlength(cm.)

i ' i

2-93-82 92-1

i'5I - I

3'43'50 6 52-4i ' i

3'53 4i ' i

3 4

DaysO-E

1 2 71 2 7

< 202

1 3 1

152 +1 2 1

1 0 4

1281 3 0

971 0 8

131141

1 1 2

1 0 0

170—190

lobsters respondingquestionable 1•esponsesnot responding

gen.

X

X

X

X

X

-

X

X

X

X

X

X

X

X

X

X

150

0

ant.

X

X

X

X

X—

X

X

?X

0

?

?0

0

0

834

cl.

X

0

0

0

0

—

0

0

0

0

X

X

0

0

0

?31

1 1

Response

gen.X

X

X

X

X—

X

X

X—

X

X

X

X

—

0

1 2

0

I

ant.X

X

X

X

?—0

X

X—

?0

?0

—

0

634

cl.

X

X

X

X

0

-

X

0

0

-

X

0

X

0

—

0

70

6

lat

an

X

X

X

X

X

X

0

0

0

0

0

0

0

0

0

0

60

1 0

Days O-E, period in days between eyestalk removal and moult at which heteromorph appeared;gen., generalized, non-specific response; ant., specific antennular movement; cl., specific cleaning ofstimulated heteromorph. See text for further details on responses.

• Animals examined in detail in subsequent experiments.x , Occurrence of specific response to indicated stimulus; ?, occurrence of the specified response

questionable, or exact nature of response unclear; —, stimulus not given; o, no response to the specifiedstimulus.

Table 2 gives the responses to various kinds of heteromorph stimulation as observedin sixteen lobsters—fourteen first-moult heteromorphs from Series Q, one second-moult heteromorph from Series P, and one second-moult heteromorph from Series O—in August 1963. Four kinds of stimuli were used: (1) tap, press, or bend hetero-morph with a clean glass rod; (2) touch or stroke heteromorph with a clean cloth swab;(3) touch or stroke heteromorph with a cloth swab dipped in a fresh homogenate of raw

Heteromorph antennules in Panulirus argus 87

pish in sea water; (4) gently roll and stroke heteromorph between fingers while thelobster is held by hand out of water. In Table 2 the first two stimulus types arecombined under ' mechanical stimulation', while the third and fourth types are labelled'chemotactile' and 'manipulate' respectively. Because of the complex nature of thestimuli used and the variability in heteromorph anatomy, stimuli to different animals,or to the same animal at different times, cannot be considered quantitatively equiva-lent. None the less, the range of stimulus intensities used was usually sufficient toassure responses if any could be evoked by non-injurious stimuli (see howeverO-17 below), and the failure of a response could not generally be ascribed to abnorm-ally weak stimuli. Responses were variable, but fell roughly into the three classesmentioned above: (1) non-specific response, usually escape activity and bodilywithdrawal; (2) specific cleaning of stimulated heteromorph with 4th ipsilateral walkingleg; (3) specific movement of ipsilateral antennule; the response to manipulation inthe hand-held animal generally involved depression of the outer flagellum (Maynard &Cohen, 1965). Within any one response class individual lobsters differed both inthreshold strength of the effective stimulus and in the exact form and intensity of theevoked motor activity. Some of these variations will be detailed for selected indivi-duals in a later section.

The major points of Table 2 can be summarized as follows:A. Non-specific functional connexions between heteromorph sensory neurons and

the brain usually occur. Behavioural responses to heteromorph stimulation werepresent in all lobsters of this population.

B. Specific connexions between heteromorph sensory neurons and ipsilateralantennular motor neurons are common but not universal. On the basis of clearbehavioural evidence they occurred in over 60% of the population. In another 20%the evidence was suggestive but not conclusive.

C. Connexions necessary to mediate an accurate 'place sense' are also prevalent.Appropriately directed cleaning activity indicated their presence in over 50 % of thepopulation.

D. Chemoreceptors sending afferents from the heteromorph often formed func-tional connexions in the brain. This is implied by the increase of heteromorph cleaningactivity following chemotactile stimulation over that following mechanical stimulationalone.

E. Functional effectiveness does not obviously correlate with the length of theheteromorph.

Specific responses

Several lobsters (indicated by (•) in Table 2) were re-examined in detail in June1964 (see Fig. 5, PI. 1). Particular attention was given to the ipsilateral antennulairesponses, and to the comparison of these with responses evoked by stimulation of thenormal antennule. Over a period of 4 days there were three or four sessions with eachof the four kinds of stimulation: glass rod, cloth swab, hand manipulation, and fishextract on cloth swab. Responses to the glass rod and cloth swab, both mechanicalstimuli, were similar and are considered together (Table 3). Antennular responses tochemotactile stimulation of the heteromorph with the fish extract swab were generallyJike those evoked by mechanical stimuli; four of the six lobsters, however, cleaned the

88 D. M. MAYNARD

heteromorph with the 4th ipsilateral walking leg after contact with the fish extraciwhile mechanical stimulation alone never evoked heteromorph cleaning in this seriesof observations (see, however, Table 2). Responses to heteromorph manipulation inthe lobsters held out of water differed slightly from other responses to mechanicalstimuli, and are treated separately in Table 4. The distal portion of the heteromorphflagellum was generally more effective than the proximal portion in eliciting thisresponse.

Mechanical stimulation. The major responses of an unrestrained lobster to mechanicalstimulation of the normal antennule were described by Maynard & Dingle (1963).The fast withdrawal reflex of the antennule (antennular reflex) and the bodily with-drawal responses are relevant here. The antennular reflex following stimulation of theouter flagellum involves an inward and downward twitch of the first antennular seg-ment, lateral movement of the second segment, depression of the distal segment, and

Table 3. Behavioural responses to mechanical stimulation

Specific antennular response(% stimulus sessions responding: n = 6—8)

0-17Q-25

Q-21

Q - iQ-10

Q-14

Non-specific(% stimulus sessions

responding:n =

Startle

0

62

0

2967

33

= 6-8)A

Crouch

0

87

1 0 0

7183

67

Average

,1

Any ant.response

0

38

57

861 0 0

1 0 0

response

Segmental 1

Basal(in-

down)

0

38

2 9

4383

1 0 0

A

Second(straight)

0

0

0

2916

67

*—novements

Distal(down)

0

0

43

5750

5°

to normal outer flagellum

— In-down Lateral(usually)

Down

Outerflagellum(down)

0

0

29

5783

50

Comment

Basal twitch veryslight

Slight, slow move-ments

Basal twitchSlow (single basaltwitch)

Basal twitch, othermovements slow

stimulation

Down All twitches

depression of the outer flagellum of the antennule stimulated (Table 3). The con-tractions are fast and twitch-like. Bodily withdrawal may have two or more com-ponents: an initial startle reaction characterized by a synchronized twitch-like con-traction or extension of most of the appendages, and subsequent tonic crouch orstepping withdrawal from the locus of the stimulus.

Table 3 compares responses to normal outer flagellar stimulation with those observedfollowing heteromorph stimulation. Response frequencies were calculated from thenumber of sessions—out of a total of six to eight—in which at least one response ofthe kind indicated occurred; they are expressed in percentages. Usually several pres-entations were made in any one session. Individual differences are apparent. Forexample, in this particular series of tests, O-17 gave no obvious non-specific responseof any kind—its reaction to normal antennular stimulation, however, was prompt andappropriate—while Q-25 and Q-10 usually showed both startle and withdrawalreflexes. Of particular interest were the specific antennular responses. Some lobster^

Heteromorph antennules in Panulirus argus 89

responded rarely, and with weak motions (Q-25, Q-21), while others, Q-14 par-ticularly, always responded with strong reflexes. The pattern of response also variedso that, although all responding lobsters included an inward and downward movementof the basal segment in some of their responses, this appeared as a rapid, twitch-likecontraction in some individuals (Q-25, Q-i, Q-14), or as a slower, more prolongedmovement in others (Q-21, Q-10). The relative frequency of movements of the moredistal antennular segments also differed among individuals. In general all theseresponses resembled the normal antennule reflex, but in addition to individual varia-tion seemed to have two consistent discrepancies: (1) most of the antennular contrac-tions elicited by heteromorph stimulation, and particularly those involving the distalsegments and outer flagellum, tended to be slower and more prolonged than the fastwithdrawal reflex; and (2) where movement of the second segment occurred it com-monly took the form of forward extension rather than the lateral flexion more usualwith normal outer flagellar stimulation. Elevation of the distal segment and outerflagellum, which characterized the reflex evoked by inner flagellar stimulation, was notobvious in these animals.

Table 4. Behavioural responses to flagellar manipulation

Specific antennular response(% stimulus sessions responding: n = 3)

-obster

O-i7

Q-25Q-21Q - i

Q-10Q-14

Any ant.response

0

0

3367

331 0 0

Segmental movements

Basal(in-down)

0

0

0

0

0

67

Second(straight)

0

0

0

0

0

1 0 0

Distal(down)

0

0

3367

331 0 0

Outerflagellum(down)

0

0

3367

331 0 0

Average response to manipulation of normal outer

In-down,or up

and down

Lateraland

straight

Down Down

Comment

No movement of basal orsecond segment

Weak responseStrong response, = normalantennular response

flagellum

Manipulation. Maynard & Cohen (1965) found that flagellar depression in a normalhand-held lobster was specifically induced by manipulation of the outer flagellum of theantennule. Similar stimulation of the heteromorph discovered on a lobster' in nature'evoked the same response. Table 4 compares the response of the antennule to suchstimulation of its outer flagellum with responses resulting from stimulation of experi-mentally induced heteromorphs. The slow depression of distal segment and outerflagellum found in the response to normal flagellar stimulation was often associated withmovements of the basal segments in these animals. An inward and downward movementof the basal segment and lateral flexion of the second segment were common, but inmany cases an up-down oscillation of the basal segment and an extension of the secondsegment also occurred. It should be stated, perhaps, that although flagellar depression

90 D. M. MAYNARD

was the usual and most consistent response it did not invariably follow every flagellarmanipulation, and that, if strong stimulation was continued after depression, elevationand other avoiding movements often followed.

Only one of the six experimental lobsters (Q-14) gave flagellar depression followingheteromorph stimulation during each of the three stimulus sessions. The detailedform of the response was very similar to that evoked by stimulation of the normaluterflagellum, differing only in greater tendency to extend the second segment.Heteromorph-evoked flagellar depression in the other lobster giving a strong response(Q-i) differed from the normal response in that only the distal segment and outerflagellum were involved; in this series of experiments the basal and second segmentsremained motionless.

Tables 3 and 4, together with Table 2, seem to suggest that, although heteromorphsdo occur whose stimulation evokes several responses essentially like those caused bystimulation of the outer flagellum of the normal antennule, animals bearing them arenot very common. Moreover, there may be significant variations in certain com-ponents of the response such as time course or direction of movement at specific joints.This does not imply, however, that heteromorph connexions are random or neces-sarily unorganized. Most lobsters gave some indication of specific responses, andthese generally seemed to resemble components or incomplete sequences of activitynormally elicited by the normal outer flagellum more closely than any other kind ofbehaviour observed thus far. Inappropriate responses such as those specific for theinner flagellum were not obvious.

Responses in lobsters lacking heteromorphs

Six lobsters of Series Q that failed to develop heteromorphs were examined, some(as indicated below) repeatedly over the course of a year. All showed evidence ofwithdrawal activity upon mechanical stimulation of the stump, and one demonstrated' place sense' with appropriate cleaning motions by the fourth ipsilateral walking legdirected at the stump following strong mechanical stimulation. In none of theseanimals, however, was the response potentiated by chemotactile stimuli, nor werespecific antennular responses evident.

Stability of behavioural responses over time

In an attempt to determine whether progressive changes in heteromorph functionoccur with time or ecdysis, the behaviour of the six lobsters of Tables 3 and 4, to-gether with two more with heteromorphs (P-i, Q-7) and four lacking heteromorphs(Q-16, Q-17, Q-19, Q-22) was examined periodically for about a year (July-August1963, February, April, June 1964). O-17 was followed over a longer period of20 months and two moults after attaining a heteromorph, and P-i for almost as long.In none of these lobsters was there any indication of significant change in the be-havioural responses evoked by heteromorph stimulation. The population is somewhatsmall, however, and discrimination of small differences by the behavioural methodsused is difficult so I cannot exclude the possibility of subtle changes with time in someor all of the lobsters tested, or indeed, the possibility of major changes in the occasionalindividual. This is particularly true since electrophysiological observations reported

Heteromorph antennules in Panulirus argus 91

below indicated good functional connexions between heteromorph and brain in O-17,and yet this animal was behaviourally unresponsive to ' natural' stimuli. Nevertheless,the consistency of the behaviour in individual lobsters, with the range of behavioursincluded—from non-responsive to very responsive individuals—does suggest thatheteromorph-induced behaviour is reasonably stable, and is consistent with thetentative hypothesis that once connexions are established in the course of initialdevelopment further modification of connexions is normally insignificant or very slow.

Fig. 6. Compound action potentials recorded in vivo from flagellar nerves. A, Normal nervefrom antennular inner flagellum (lobster Q-i), rapid trace to show two major fast components.B, Normal nerve from antennular outer flagellum (Lobster Q-i), rapid trace to show subdivisions ofmajor fast component; slow components are not visible. C. As in B, but with slow trace to showlater slow components; only component'c' is obvious; see Table 5 and Fig. 10. D-I, Nervesfrom heteromorph flagella: D, lobster O-i7; E, lobster Q-i ; F, lobster Q-25; G, lobster Q-10;H, lobster Q—21; I, lobster Q—14. In all records the initial upward deflection rising above the base-line represents the first component of the compound action potential. Calibration, 60 eye./sec.

Table 5. Conduction velocities in hcteromorph and flagellar nerves

Lobster

O-17Q-25Q-21Q - iQ-10Q-14

Temp.(° C.)

26-7-27-526-4-27-2260-26-72 7 026-8-2727-3

Mean (m/sec)

a

5'44 16-2

3 73 73 9

4 5

Heteromorph nerve

b

1 9—2 61 9—

i-5

2 0

c

0 7 6o-86——

o-6i—

0-72

d

0-410 4 80 4 20 3 20 2 80 3 7

0-37

Flagellar

Ipsilateral

Outer flagellum

a

5 65'37 67-47'47-0

6 7

b

0 4 70 5 8

O-54°5 50-560 4 9

0 5 4

c

o-330-410-330 3 8

——

0-38

nerve

Innerflagellum

a

5 65'56-86 98-37-0

6 7

b

3 94-4—3-85'55 1

4-5

Contralateral

Outer flagellum(regen.)

a b c— — —4-4 — O-2.— _ —

— —— —

—— —

92 D. M. MAYNARD

Conduction in heteromorph nerve

Nerve fibres originating in sensory neurons of the heteromorph run down theflagellum and on toward the cerebral ganglia along the course of the former optic tract.Direct evidence for conducting pathways was obtained by recording compound actionpotentials in vivo in the proximal part of the flagellum while stimulating the distalregion. Conduction velocities were calculated from response latencies which re-mained constant over wide variations in stimulus intensity.

Typical compound action potentials recorded from six heteromorphs (see Fig. 5,PI. 1) are illustrated in Fig. 6. The responses of these lobsters are discussed undersections on Specific responses and Electromyography. There are two major components:a group of fast fibres, ' a ' component, conducting at an average of 4-5 m./sec. at 270 C ,and a slow group, 'd' component, conducting at an average of 0-37 m./sec. (Table 5).In some individuals intermediate subcomponents, 'b' and 'c', also occur; these areparticularly prominent in O-17 and Q-10. In view of the unresponsiveness of O-17to natural heteromorph stimulation the demonstration of functional conductingelements in the heteromorph nerve is of particular interest.

Fig. 6 and Table 5 also illustrate the conduction velocity spectra of nerves in theinner flagellum and outer flagellum of normal antennules, and in one regeneratedantennule. The inner flagellar nerve is characterized by two major fast components,'a' and 'b\ with velocities of 6-7 and 4-5 m./sec. It may also include some slightlyslower elements, but apparently lacks a major slow component. The outer flagellumis characterized by a major fast component, 'a', often with several slower sub-components, averaging 6-7 m./sec. and two major slow subcomponents, '£ ' and 'c ' ,averaging 0-54 and 0-38 m./sec. The failure to observe the slowest component inQ-10 and Q-14 might be ascribed to the position of recording electrodes. In therecord from the single regenerate outer flagellum both a slow and a fast componentwere present, but both were slower than any recorded from normal outer flagella. Ingeneral, the conduction velocity spectrum of the outer flagellum resembles that of theheteromorph more closely than does that of the inner flagellum.

Antenrndar muscle responses to heteromorph stimulation

Electromyography

After behavioural observation (Tables 3 and 4) recordings of antennular muscleactivity during heteromorph and normal antennular stimulation were made from the sixlobsters of Fig. 5 (PI. 1). External electrodes clipped to the intact cuticle of an anten-nular segment recorded action potentials in the muscles of that segment only. Simul-taneous recordings (Fig. 7) illustrate independent activity in muscles of the con-tiguous second and distal segments. In the second segment functional interpretationis complicated because there are both depressor and elevator muscles, but in the distal,third segment, there is only one muscle, musculus reductor4, and that one depressesthe outer flagellum; flagellar elevation is passive. In the following experimentssimultaneous recordings were made from the distal segments of both antennules(Fig. 7C). Two kinds of action potential were usually evident in recordings fromm. reductor4. Two patterns of activity were also present. The first, a brief burst with

Heteromorph antenmdes in Panulirus argus 93

frequencies reaching 300/sec., occurred at intervals and caused the characteristic flickof the outer flagellum (Maynard & Dingle, 1963). It appeared in partially contractedas well as in completely relaxed muscle. The second pattern was simply a maintainedbarrage of potentials at varying frequencies; the higher the frequency, the greater thedepression of the outer flagellum. During strong contractions instantaneous fre-quencies occasionally approached 200/sec.

1.3

0-2

Fig. 7. Action potentials recorded in vivo from antennular muscles. A, Lobster Q—21. Uppertrace recorded from second segment of antennule, lower trace recorded from third or distalsegment of antennule (= m. reductor«). Manual stroke of ipsilateral heteromorph flagellum—indicated by marker trace above upper trace—produced increased activity in both segments.B, Lobster Q—10. Upper trace recorded from second segment, lower trace recorded from thirdor distal segment of antennule. Lifting inner flagellum of the same antennule—indicated bymarker trace—produced increased activity in muscles of the second segment, but inhibitedon-going discharges from the muscle of the third segment. Note that electrodes only recordedactivity tvithin one segment; no 'cross-talk' was evident. C, Lobster Q—10. Upper tracerecorded from third segment of left antennule. Note on-going activity in muscle of rightantennule, action potentials of two amplitudes and apparently independent discharge fre-quencies are present. In simultaneous recording from muscle of left antennule backgroundactivity is absent, but a single burst of action potentials mediating an antennular flick occurs.Calibration, o-z sec.

Responses to mechanical stimulation

Lobsters were immobilized and partially immersed in sea water. Antennularflagella, eyestalks and heteromorphs remaining out of water were moistened periodi-cally. For stimulation, flagella were lifted or bent with a glass rod, or were pinchedbetween rubber-tipped forceps. Stimuli were obviously imprecise, and varied withtime and individual; the general excitability of the animal also varied. Analyses offine detail in the responses are therefore inappropriate. Nevertheless, in individuallobsters re-examined after an interval of days, responses were usually consistent, andsome general statements are possible. As a basis for comparison normal responses areconsidered first.

Stimulation of the normal flagellum. Maynard & Cohen (1965) reported thatmanipulation of the outer flagellum usually resulted in increase, and manipulation of

94 D. M. MAYNARD

the inner flagellum in decrease, of ipsilateral muscle action-potential frequencies!A similar dichotomy tended to occur in the present experiments, but with the slightlydifferent stimuli used several complications appeared.

In all six lobsters mechanical elevation of the inner flagellum always caused a reductionin action-potential frequency in m. reductor4, and, sometimes, nearly completeinhibition. A typical response appears in Fig. 8C, G. In some animals this wasassociated with contralateral excitation and with augmented frequencies as theflagellum dropped at the end of stimulation. In most, but not all lobsters, mechanicaldepression or pinching of the inner flagellum caused excitation, not inhibition; when inhibi-tion did occur it was less than that following flagellar elevation (Fig. 8 D). In contrast,stimulation of the outer flagellum, particularly elevation, usually caused excitation of theipsilateral muscle; sometimes there were contralateral effects (Fig. 8A, 8F). In twoof the six lobsters, however, responses to outer flagellar stimulation, particularlyflagellar depression, were complicated by inhibition following an initial excitation, oroccasionally by inhibition only (Fig. 8B).

These observations demonstrate that both excitatory and inhibitory reflexes can beelicited from both antennular flagella. Complex sequences of inhibition and excitationcan also occur with proper stimulation of either flagellum. Thus far, however, theevidence seems to confirm the initial impression that net excitation is usually charac-teristic of the responses to gross stimulation of the outer flagellum, while net inhibitionis more prevalent upon stimulation of the inner flagellum. This also is in accordancewith behavioural observation (Maynard & Dingle, 1963).

Stimulation of the heteromorph. Responses of the antennular muscle to heteromorphstimulation were observed in all six lobsters, even those which were behaviourallyunresponsive, lobster O-17 (Fig. 9F, G) and lobster Q-25 (Fig. 9J). The form of theresponse, however, differed between individuals, and often within one individualaccording to the kind of stimulus. The latter is particularly well illustrated in lobsterQ-14 (Fig. 9D, E), where an inward bend of the heteromorph caused ipsilateral, andto a much lesser extent, contralateral excitation, while an outward bend caused ipsi-lateral inhibition. Lobster Q-10 (Fig. 9H, I) is another example in which an upwardbend again caused bilateral, asymmetrical excitation, while pinching the distal portionof the heteromorph caused ipsilateral inhibition and contralateral excitation. In fact,in all lobsters tested except lobster Q-21 (Fig. 9C), certain kinds of mechanicalstimulation tended to produce inhibition while others favoured excitation. In manycases (see lobsters Q-i, Q-25 and O-17 in Fig. 9 for examples) both excitation andinhibition were apparent in single response sequences, excitation commonly beingfollowed by inhibition. In at least one lobster, O-I7, relatively strong ipsilateralexcitation was associated with contralateral inhibition, while in another, Q-i, bilateralinhibition occasionally occurred.

Individual variation was difficult to evaluate. Heteromorph responses were notspecific enough to be identified with either 'inner flagellar responses' or 'outerflagellar responses', and direct intra-lobster comparison of heteromorph and normalresponses showed almost no consistent pattern of difference. The muscle responsesof lobsters O-17 and Q-25, lobsters with little or no apparent heteromorph-induced be-haviour, were somewhat weaker than others, but further correlation between individualbehaviour and electromyography was not obvious. Nevertheless, two generalizations

Heteromorph antennules in Panulirus argus 95

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0-5Fig. 8. Responses of m. reductor4 in third antennular segment to mechanical stimulation ofantennular flagella. In each record the upper trace is taken from the right antennule, thelower trace from the left antenule, simultaneous recording. The marker signal above the uppertrace in each case indicates the approximate duration of the stimulus, but is displaced to theright by o- i-o-2 sec. Arrows indicate the actual beginning of stimulation. A-D, Lobster Q-14:A, right outer flagellum lifted; B, right outer flagellum depressed; C, right inner flagellumlifted; D, right inner fkgellum depressed. E-H, Lobster Q-10: E, left outer flagellum Lifted;F, left outer flagellum depressed; G, left inner flagellum lifted; H, left inner flagellumdepressed. Note that responses are largely, although not completely, unilateral. R and Lindicate antennule stimulated. Calibration, C5 sec.

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Fig. 9. Responses of m. reductorj in third antennular segment to mechanical stimulation ofheteromorph flagellum. Upper trace, right antennule; lower trace, left antennule; simul-taneous recording. Marker signal indicating stimulus duration (see Fig. 8) is displaced to right;vertical lines indicate actual beginning of stimulation. A, Lobster Q-i, bend left heteromorphinward, B, lobster Q-i , pinch left heteromorph; C, lobster Q-21, manipulate left hetero-morph; D, lobster Q—14, bend right heteromorph inward; E, lobster Q—14, bend right hetero-morph outward; F, lobster O-17, bend left heteromorph backward; G, lobster O-17, pinchleft heteromorph; H, lobster Q-10, bend left heteromorph upward; I, lobster Q-10, pinchleft hetermorph; J, lobster Q—25, bend left heteromorph downward. R and L indicate side ofstimulated heteromorph. Calibration, 0-5 sec.

Heteromorph antemtules in Panulirus argus 97

seem possible for the responses of these particular six lobsters: (1) Although contra-lateral effects occurred the primary excitatory response to heteromorph stimula-tion was ipsilateral. (2) None of the excitatory responses was as vigorous as thoseelicited by normal antennular stimulation. In this respect these six lobsters contrastwith the lobster described by Maynard & Cohen (1965) in which heteromorphstimulation produced very strong excitation. The extent to which gross and uncon-trolled stimuli contributed to the variability of responses discussed here is not known.

60~

Fig. 10. Simultaneous records from flagellar nerves and m. reductor^, in vivo. Upper trace,compound action potential of flagellar nerve recorded from surface of flagellum; lower trace,muscle action potentials in m. reductor4 recorded from surface of third segment. Singleelectrical stimulus given to distal flagellum at artifact. Lobster Q— i. A, Stimulus and uppertrace from outer flagellum, stimulus subthreshold for slow components (6 and c) of outerflagellar nerve. B, As in A, but stimulus suprathreshold for slow components. Early response inmuscle ( i ) is more apparent. C, Stimulus and upper trace from inner flagellum. Stimulus suprathreshold, but only fast components (a-b) are evident. Slight elevation coming later is artifact.Portions of reflex response in m. reductor4 are indicated as follows: I, initial reflex burst; a,reflex inhibition; 3, second reflex burst. Seetext for further discussion. Calibration, 60 cyc./sec.

Stimulation of the eyestalk stump. Two lobsters which did not regenerate hetero-morphs, Q-16 and Q-22, were examined. Responses to stimulation of the normalantennular flagellum resembled those described above. Antennular responses tomanipulation of the stump of the ablated eyestalk were lacking in one (lobster Q-16)but appeared as slight ipsilateral excitation in the other (lobster Q-22).

Stimulation of the eyestalk. Responses to eyestalk manipulation were not particularlystrong and were somewhat variable. They were usually bilateral, and in at least halfthe cases were inhibitory. There were also excitatory responses however (see Maynard& Cohen, 1965) and occasionally potentiation of burst frequency associated withantennular flicks occurred. In lobster Q-14 ipsilateral excitation was associated withcontralateral inhibition. In general, responses to eyestalk manipulation did not closelyresemble responses to antennular or heteromorph manipulation.

Responses to electrical stimulation

Stimulation of outer flagellum. A single stimulus applied to an outer flagellum justproximal to the distal brush of sensory hairs characteristically elicited a four-partresponse in the ipsilateral m. reductor^. An initial burst of action potentials (1) wasfollowed first by inhibition (2), then by a subsequent second burst (3) and, often, bya final prolonged discharge at increased frequency (4). Contralateral responses werenot observed. The afferent fibres mediating this response apparently belong to thefast component of the antennular nerve because the response occurred before thearrival of the slow component at the brain, and also occurred at stimulus strengthssubthreshold for the slow component (Fig. 10). The response is like that described by

7 Exp. Biol. 43, 1

98 D. M. MAYNARD

R if of het

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I'Hjrkiii

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I . J L . i l . i l . I . I L I L L . , I . J . . L . I . J . I . J . I . I , L J 1 . J . L . I . I . . . 1 . . L .

1 lFig. 11. Responses of m. reductor4 in third antennular segment to electrical stimulation—singlestimulus—of the ipsilateral antennular inner flagellum (if), the ipsilateral antennular outerflagellum (of), and the heteromorph flagellum (het). Upper trace, right antennule; lowertrace, left antennule, simultaneous recording. Dots (•) indicate stimulus. R and L indicateside stimulated. A, Lobster Q—22, right eyestalk removed, heteromorph failed to regenerate.Right antennular nagella and stump of right eyestalk stimulated. B, Lobster Q-i, left nagellaand left heteromorph stimulated. C, Lobster Q—21, left nagella and left heteromorph stimu-lated. D, Lobster Q—14, right nagella and right heteromorph stimulated. E, Lobster O—17,left nagella and left heteromorph stimulated. F, Lobster Q-10, left flagella and left hetero-morph stimulated. G, Lobster Q-25, left nagella and left heteromorph stimulated. Com-ponents of reflex response: i, initial burst; 2, following inhibition; 3, second burst; 4, prolongeddischarge, only beginning visible on record. Calibration, 05 sec.

Heteromorph antennules in Panulirus argus 99

Maynard & Cohen (1965) and was relatively consistent within the population examinedhere (Fig. 11, of). There is some individual variation, however, in the relativeprominence of the four parts of the response. Such variation seemed characteristic ofthe individual, not the specific antennule, because it was reflected to varying degreesin responses to both ipsi- and contralateral flagella.

With repetitive stimulation at 10/sec. the ipsilateral excitatory effects summated,and a high-frequency discharge that continued somewhat beyond the end of stimula-tion occurred. Although the second, inhibitory response continued to appear and tointerrupt briefly the discharge after each stimulus the net effect was of strongexcitation.

Stimulation of inner flagellum. Fig. 11 compares the two-part responses to singlestimuli of inner flagella with outer flagellar responses. The initial burst of the latterseems to be absent and the first obvious component of the inner flagellum response isan inhibition of spontaneous discharge. This in turn is followed by the second com-ponent, moderate excitation. The inhibition tends to be stronger and the excitationweaker than in comparable portions of the outer flagellar response. In several lobsters,Q-i (Fig. 11B), Q-14 (Fig. 11D), O-17 (Fig. 11E), contralateral excitation wasapparent. With repetitive stimulation, inhibition rather than excitation summates,and the net effect during stimulation is one of inhibition.

Stimulation of heteromorph. Responses to heteromorph stimulation were welldefined within individual lobsters and were consistent with repeated stimuli. Fig. 11compares responses to single stimuli applied to heteromorphs with responses tosingle stimuli applied to normal antennular flagella and Fig. 12 illustrates responses torepetitive stimulation. There was considerable variation among lobsters. The mosttypical response to single stimuli was a simple, excitatory ipsilateral burst (see Table 6).In one individual, lobster Q-10 (Fig. 11F), this appeared to be followed by inhibitionwhile in another, lobster Q-25 (Fig. 11G), the initial burst was absent or nearly so butwas replaced by strong inhibition. Repetitive stimulation produced complete ipsi-lateral inhibition in lobster Q-25 (Fig. 12G). In the other four lobsters, repetitivestimulation produced net excitation similar to that resulting from normal outerflagellar stimulation. A contralateral inhibitory response was observed in two lobsters.In lobster Q-i (Fig. 12B) it appeared only with repetitive stimulation, but in lobsterO-17 (Fig. 11E) it was also very prominent following single stimuli.

The latency of the excitatory burst following heteromorph stimulation rangedbetween 40 and 80 msec. Although weaker and briefer, the response appearsanalogous to that reported in the lobster described by Maynard & Cohen (1965). Ifso, it may well correspond to the second excitatory burst of the normal antennularresponse. It is clear that there are major differences between the total response tostimulation of the normal outer flagellum and to stimulation of the heteromorph, butit remains easier to correlate the heteromorph response with portions of the outerflagellar response than with any other reflex seen so far.

Like responses to mechanical stimulation, responses to electrical stimulation wereoften difficult to correlate with behavioural responses. In some cases, such as O-17,this failure must be considered to reflect peripheral rather than central differences,for when stimulated by repetitive electrical shocks the heteromorph was perfectly ableto elicit good flagellar depression, although this response was normally absent with

7-2

ioo D. M. MAYNARD

mechanical stimulation. There were similarities between responses to mechanical andelectrical stimulation, however. For example, three animals showing clear ipsilateralinhibition to certain mechanical stimuli gave inhibitory (lobster Q-25), weak excita-tory (lobster Q-14), or combined excitatory-inhibitory (lobster Q-10) responses toelectrical stimuli. And the two lobsters displaying contralateral inhibition withmechanical stimulation also showed it with electrical stimuli (lobsters Q-i and O-17).

1i-

r

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i l l J l l l l l l l J l l l i lU i i l i i l l . 1 1 1 1 1 1 1 1 1 1L 0-5

Fig. I I . Responses of m. teductor4 in third antennular segment to electrical stimulation—repetitive—of the heteromorph flagellum. Upper trace, right antennule; lower trace, leftantennule, simultaneous recording. Dot (.) indicates beginning of repetitive stimuli at10/sec. which continue until end of record. In some instances—B, D, E, and G particularly—stimulus artifacts are apparent and should not be confused with action potentials. R and L in-dicate side stimulated. A, Lobster Q-22, right eyestalk stump; B, lobster Q-i, left heteromorphstimulated; C, lobster Q-21, left heteromorph; D, lobster Q-14, right heteromorph; E, lobsterO-i 7, left heteromorph; F, lobster Q-10, left heteromorph; G, lobster Q-25, left heteromorph.Calibration, 05 sec.

Heteromorph antenmdes in Panulirus argus 101

Stimulation of eyestalk stump. Electrical stimuli were applied to the stump of theablated eystalk in one lobster lacking a heteromorph regenerate, Q-22. No responseswere observed in m. reductor4 (Figs. 11A and 12 A).

Stimulation of eyestalk. No obvious or consistent responses to single stimuli appliedto eyestalks were recorded in m. reductor4. Stimuli were applied across the peduncleand were strong enough to cause eyestalk twitches and occasional systemic reactions.With repetitive stimuli summation occurred and variable mild responses appeared.These were usually , but not always, bilateral and inhibitory. Occasionally unilateralor excitatory responses occurred.

Table 6. Responses to electrical stimulation of heteromorph

Single stimuli Repetitive stimuli

Lobster Ipsilateral Contralateral Ipsilateral Contralateral

O-17 + - + -Q-a5 ( + ) - 0 - 0Q-ai + 0 + 0Q-i + o +Q-10 H— o H— oQ-14 +? o +? o

+ , excitatory burst; —, inhibition; H—, excitation followed by inhibition; ( + ), very weak excita-tion; ?, response questionable.

DISCUSSION

Heteromorph occurrence. The high proportion of heteromorphs observed in thisexperimental population of Panulirus argus—54% of those surviving the first moult—compares favourably with observations on other forms; Herbst (1900), for example,obtained heteromorph development in 27 % of about 370 shrimps, Palaemon spp.Undoubtedly the proportion of heteromorphs among the lobsters would have beenhigher if all eyestalk ablations had been made shortly after ecdysis, for, among thoselobsters with long operation-ecdysis intervals, nearly 75%, twenty-two out of thirty,produced heteromorphs. The population ranged from sexually immature to fullymature animals (Sutcliffe, 1952) and included both sexes so the above proportionsseem sufficient reason to accept heteromorph development as a normal consequenceof eyestalk ablation in adult Panulirus argus.

The causes of heteromorph failure in the remaining 46 % of the population are notclear. Since, however the first thirteen lobsters moulting after eyestalk ablationfailed to develop heteromorphs, the time at which ablation occurs in the moult cycleis presumed important. Heteromorph failure in such animals must involve more thansimply lack of sufficient time for development before ecdysis. If this were so, hetero-morph primordia should appear as found in normal limb regeneration (see Nouvel,1939; Bliss, i960) and these should develop into full heteromorphs on the secondmoult. With one or two exceptions, however (see Fig. 4, PI. 1, and lobster O-17),neither of these effects occurred in this population. There are two problems therefore:(1) why heteromorphs did not occur; and (2) why, if they did not develop before thefirst moult, they rarely did at all. One may suppose that after some critical period inthe intermoult preceding ecdysis potential heteromorph tissue either lost its com-petence to respond to 'heteromorph-inducing mechanism', or the hypothetical

102 D. M. MAYNARD

mechanism itself became inactive. In either event, the regenerating tissue presumablfbegan differentiation as non-specific epithelium, and, once begun, this process wasapparently irreversible. Heteromorph regeneration thus differs from normal flag-ellum regeneration in its failure to continue differentiation after an early moult andin the longer time required for heteromorph differentiation before moult.

Heteromorph failure in lobsters moulting at longer intervals (4 or 5 months) aftereyestalk ablation did not obviously correlate with the moult cycle, and probablyresulted from other factors preventing initial differentiation.

Heteromorph form. The morphology of the heteromorph was remarkably constant,a single ftagellum bearing sensory armament like that of the normal outer flagellumof the antennule. There was no evidence of development of basal segments or of asecond flagellum with successive moults (up to four moults). In this respect Panulirusheteromorphs resemble those reported in Palinurus vulgaris (Herbst, 1910), andcontrasted with those reported in Palaemon. In the latter genus the heteromorph firstappeared as a non-articulated nubbin with sensory hairs, but with subsequentmoults often developed both inner and outer ' antennular' flagella.

Just as the external heteromorph morphology resembled the outer flagellum of thenormal antennule, so did many of its functional properties. Laverack (1964), forexample, demonstrated functional mechano- and chemoreceptors in the heteromorphby direct electrical recording from two lobsters of the Q series. The properties of thesewere similar to receptors found in the outer flagellum. Likewise, the compound actionpotential of the heteromorph nerve resembled that of the normal outer flagellum inhaving major fast and slow components. Some differences such as slower conductionvelocities and additional conduction components were apparent and may have hadfunctional significance (see below and Maynard & Cohen, 1965), but in general theconcept of the heteromorph as a misplaced outer antennular flagellum seems reason-able as a first approximation.

Growth and temporal change. There are two aspects of heteromorph growth ortemporal change important in the present context. The first is the peripheral aspect,and concerns change in number, size, distribution, or functional properties of senseorgans and nerve fibres in the heteromorph itself. Most of these changes would beexpected to occur stepwise, becoming evident only at each successive moult. Thesecond aspect is the central aspect, and concerns growth and differentiation within thebrain of afferent fibres from the heteromorph nerve. Although obviously related toperipheral events and structures the central changes could presumably be continuous,and in some respects rather independent of peripheral growth. At the early stages theymay also be complicated by degenerative reorganization occasioned by the loss of eyestalkganglia. Whereas the first aspect of growth might be revealed by changes in hetero-morph morphology, the second would be detected primarily by behavioural changes.

Unfortunately, evidence available on either aspect of heteromorph temporal changeis limited. The lobsters maintained for more than one moult were selected for widevariation in both morphological and functional characteristics, but the sample wassmall, and none was kept for longer than four moults. Furthermore, the behaviouraltests were not always identical over the extended observation periods (up to 20 months)and were therefore sufficient to detect only the grossest variations. Finally, as illu-strated with lobster O-17, behavioural response did not always necessarily reflect aU

Heteromorph antetmules in Panulirus argus 103

Punctional connexions. Nevertheless, if these restrictions are recognized some tenta-tive conclusions are possible.

The peripheral, external signs of growth are straightforward. The heteromorphincreases in length and in number of annuli with each moult. The distal annulisupplied with chemosensory and associated sense hairs increase disproportionatelyover the less well-endowed basal annuli, so periodic increases in the afferent neuralsupply from the heteromorph seem inevitable. Presumably this would lead to con-tinued invasion of the central ganglia with new heteromorph afferents. Functionalevidence for such an increase in total input strength from the heteromorph has notyet been found.

Evidence on central changes with time is more ambiguous. Some lobsters withsmall or poorly differentiated heteromorphs (e.g. lobsters Q-i, Q-10) responded withstrong, specific responses within a few days of first appearance of the heteromorph,and these remained essentially unchanged throughout the months of observation.Obviously, specific central connexions do not necessarily require prolonged periodsafter heteromorph appearance for initial differentiation. Individual behaviouralvariants were also remarkably stable, even over two moults, indicating that suchvariations probably represent adventitious differences in central connectivity patternsrather than different stages in a common sequence of progressive, functional dif-ferentiation. Although it is premature to claim that patterns of functional connexion,once established, never develop further, it does seem likely that an initial stage ofcentral fibre differentiation, possibly terminated at the first moult, is followed by amore prolonged functionally inert period. Further observations over a longer periodand utilizing more precise measures of functional connexion are necessary.

Patterns of central connexion. From the behavioural observations it is apparent thatat least some of the fibres from the heteromorph always make effective contact witha general alarm or avoidance system in the cerebral ganglia, and that often a ratherdiscrete 'place sense' may occur (see also Herbst, 1910; Lissman & Wolsky, 1933;Maynard & Cohen, 1965). Although some central specificity was undoubtedly re-quired for the establishment of these connexions, they are less suitable for analysisthan connexions associated with the specific antennular responses, and will not beconsidered further here.

The principal question concerning heteromorph connexions is simply whether thenew, incoming afferent fibres from antennule-like sensory receptors in the hetero-morph flagellum form patterns of central connexion like those normally formed byoriginal antennular afferents. In a previous specimen of Panulirus an extensive analysisinvolving behavioural tests, electromyography of antennular muscles and intracerebralrecording from single neurons did indicate great similarity (Maynard & Cohen, 1965).In the present population the situation is less clear. Behavioural tests and electro-myography indicate that in the majority of lobsters specific movement of the ipsi-lateral antennule can be elicited by some form of heteromorph stimulation. They alsodemonstrate that according to the kind of sensory structures stimulated eitherexcitation or inhibition of an antennular muscle may occur. In both of these respectsheteromorph and outer fiagellar stimulation are similar. Upon closer analysis ofpatterns of movement of antennular segments, or of the pattern of motor impulsesreaching the most distal antennular muscle, m. reductor4, however, individual

104 D. M. MAYNARD

variations in detail appear, and none are identical at all points with one another, witHthat described by Maynard & Cohen, or with responses elicited by similar stimulationof the normal antennular outer flagellum. Furthermore, in one or two instances,contralateral effects not normally observed with outer flagellar stimulation were seenfollowing heteromorph stimulation (lobsters Q-i, O-17). Clearly, in total effect thepattern of afferent functional connexion connectivity of the heteromorph differs fromthat of the normal flagellum.

The inferences about patterns of central connexion which may be legitimatelydrawn from such behavioural differences are difficult to assess. The divergencesobserved do not seem sufficiently critical necessarily to disprove the hypothesis thatafferents from similar kinds of sensory receptors in the two flagella make specificconnexions with similar motor neurons and interneurons in the cerebral ganglia. Theresponses of the system are obviously complex, requiring co-ordinated activity in anumber of elements, and one might propose several factors unrelated to specificityof individual afferent connexions which could produce variations in response. Threecan be indicated: (1) Since the stimuli used presumably activated peripheral fibresproducing both central inhibition and excitation, the sequence in which inhibitor-inducing and excitor-inducing impulses arrive should be important. Consequentlydifferences in relative conduction velocities of these two components in the twoflagella, normal and heteromorph, might produce significantly different responses. Asillustrated in Fig. 6 and Table 5, differences in conduction velocity are entirelypossible. (2) Differences in sensitivity of peripheral receptors, or in ratios of fibretypes between heteromorph and normal flagella, should result in different responsesto similar stimuli. One extreme example of this seemed to occur in lobster O-17, inwhich electrical stimulation of the heteromorph was necessary to evoke flagellardepression, presumably because insufficient peripheral receptors were stimulated bythe usual mechanical stimulation. (3) The degree of specificity need not be identical forall afferent fibres—perhaps being greatest for the aesthetascs and associated guard andcompanion hair fibres, which are normally found only on the outer flagellum, and lessfor other antennular receptors, which seem to occur on both outer and inner flagella.If this were so, then a core of constant, specific patterns might be concealed beneathextensive individual variation. Unfortunately none of these factors, although plausibleand supported by indirect evidence, have been demonstrated to act in the mannerpostulated. Consequently, although general similarities are apparent, the question ofthe preciseness of neuron-neuron specificity in this population of lobsters remainsincompletely resolved.

In the final analysis, however, the present observations must be considered inrelation to the lobster described in detail by Maynard & Cohen (1965). In thatindividual, similarities between the excitatory responses of the heteromorph and of theouter flagellum were more apparent than in any reported here. There were, moreover,enough basic similarities between it and such lobsters as Q-14 of the present experi-ments to exclude the possibility of the earlier animal being a unique or completelyfortuitous occurrence. Rather, it appears to have represented one possible conditionwithin the potential range of specific heteromorph responses suggested in this experi-ment. If this interpretation is accepted the conclusions derived from the intracellularanalysis of that animal are relevant for the present discussion. In particular, thfl

Heteromorph antennules in Panulirus argus 105

common absence of a functional fast afferent component in the heteromorph seemsindicated; and variations in heteromorph response may be supposed to result, asproposed for individual motor neurons, from relative differences in the intensity ofthe output of common interneuron pools. The observed responses of m. reductor4

(see Fig. 11) could be caused by such a mechanism, and the individual variations thusbecome accountable in terms of quantitative rather than qualitative differences.Supported with evidence of specific movements at other antennular joints the varia-tions remain compatible with the argument for specificity of central connexions of atleast some of the heteromorph afferents, and with the initial impression from be-havioural observation of some degree of identity between the connexion patterns ofheteromorph and of flagellum.

It must be emphasized, however, that peripheral observations cannot reveal theextent of such central identities, nor the immediate causes of individual variation whichdo exist. Accordingly, all behavioural observations, and more particularly thosereported here, must ultimately remain ambiguous with respect to inferred patterns ofcentral connexion until more direct evidence from the systems involved have beenobtained.

SUMMARY

1. Eyestalks were removed unilaterally from forty-nine Panulirus argus. Twenty-three of the forty-three animals remaining alive to the first moult regenerated aheteromorph antennular outer flagellum in place of the amputated eye.

2. None of the lobsters moulting within less than 3 months of eyestalk ablationregenerated a heteromorph; 70% of those moulting after more than three monthsrecovery regenerated a heteromorph. If the heteromorph did not appear upon thefirst moult it usually continued to be missing after the second or third moult.

3. Mechanical stimulation of the heteromorph produced a general withdrawalreaction in all lobsters tested, and in most either a specific cleaning reflex directedtoward the stimulated heteromorph, or a specific movement of the normal ipsilateralantennule, or both. The withdrawal and antennular movement, when it occurred,resembled responses elicited via stimulation of the normal antennular flagellum.

4. Behavioural responses were followed in a selected subpopulation for periodsranging from one to nearly two years, and over one or two moults. No significantchanges in behavioural responses to heteromorph stimulation were detected withinthat time.

5. The conduction-velocity spectrum of fibres in the heteromorph nerve resembledthat in the outer flagellar branch of the normal antennule nerve in having both a fastand a slow component. However, the velocity of the fast component of the hetero-morph nerve was often less than normal for the flagellar branch of the antennule nerve,and additional components of intermediate velocity occurred in some animals.

6. According to the direction or nature of the mechanical stimulus both hetero-morph and normal flagella elicited inhibitory and excitatory responses in the nerveto the depressor muscle of the outer flagellum of the normal ipsilateral antennule.

7. Responses of the flagellar depressor muscle of the ipsilateral antennule to electricalstimulation of the heteromorph varied among individuals, but in general resembledlater components of the response to similar stimulation of the normal antennularflagellum.

106 D. M. MAYNARD

8. It is tentatively concluded that at least some of the heteromorph afferents makespecific central connexions on neuron pools normally associated with antennularflagellum afferents. The difficulties of drawing such inferences from indirect observa-tion are indicated.

The hospitality and co-operation of the Director and staff of the Bermuda BiologicalStation permitted the maintenance of a healthy experimental lobster population fornearly two and one-half years with minimal natural attrition. I should like to expressmy particular appreciation to Drs Sutcliffe and Beers and Messrs Burgess and Spurling.I should also like to thank Dr M. Laverack, Miss F. Yen, and Messrs G. Wyse andE. Bendit for assisting in various aspects of the study.

This work was supported in part by USPHS Grant NB-03271.

REFERENCES

BLISS, D. E. (i960). Autotomy and regeneration. Ch. 17 in The Physiology of Crustacea, Vol. I.(ed. T. H. Waterman), pp. 561-89. New York and London: Academic Press.

HERBST, C. (1896). (Jber die Regeneration von antennenShnlichen Organen an Stelle von Augen.I. Mittheilung. Arch. EntxeMech. Org. a, 544-58.

HERBST, C. (1900). tJber die Regeneration von antennenahnlichen Organen an Stelle von Augen.III. Weitere Versuch mit total exstirpirten Augen. IV. Versuche mit theilweise abgeschnittenenAugen. Arch. EntwMech. Org. 9, 315-92.

HERBST, C. (1910). {Jber die Regeneration von antennenahnlichen Organen an Stelle von Augen.VI. Die Bewegungsreaktionen, welche durch Reizung der heteromorphen Antennulfl ausgelostwerden. Arch. EntwMech. Org. 30, 2, 1-14.

HERBST, C. (1917). Ober die Regeneration von antennenahnlichen Organen an Stelle von Augen.VII. Die Anatomie der Gehirnnerven und des Gehirnes bei Krebsen mit Antennulis an Stelle vonAugen. Arch. EntwMech. Org. 43, 407-89.

HOFHR, B. (1894). Ein Krebs mit einer Extremitflt »tatt eines Stielauges. Verh. dtsch. Zool. Ges. 4,82-91.

LAVERACK, M. (1964). The antennular sense organs of Panuiirus argus. Comp. Biochem. PhysM. 13,301-321.

LISSMANN, H. W. & W0L8KY, A. (1933). Funktion der an Stelle eines Auges Regenerierten Antennulebei Potamobius leptodactyhu Eschh. Z. vergl. Physiol. 19, 555-73.

MAYNARD, D. M. (i960). Heart rate and body size in the spiny lobster. Physiol. Z06I. 33, 241-51.MAYNARD, D. M. & COHEN, M. J. (1965). The function of a heteromorph antennule in a spiny lobster,

Panuiirus argus. J. Exp. Biol., 43, 55-78.MAYNARD, D. M. & DINGLE, H. (1963). An effect of eyestalk ablation on antennular function in the

spiny lobster, Panuiirus argus. Z. vergl. Physiol. 46, 515—40.MILNE EDWARDS, A. (1864). Sur un cas de transformation du petioncle oculaire en une antenne, observe

chex une Langouste. C.R. Acad. Sci., Paris, 59, 710-12.NOUVEL, L. (1939). Sur le mode de regeneration des appendices locomoteurs chex ScyUarus arctus et

les Crustacea Decapodes en giniral. Bull. Inst. Ocean., Monaco, no. 773, 7 pp.SUTCLIFFE, W. H. Jr. (1952), Some observations of the breeding and migration of the Bermuda spiny

lobster, Panuiirus argus. Proc. Gulf Carib. Fish. Inst. 1952, pp. 64—9.TRAVIS, D. (1954). The molting cycle of the spiny lobster, Panuiirus argus Latreille. I. Molting and

growth in laboratory-maintained individuals. Biol. Bull., Woods Hole, 107, 433-49.WOLSKY, A. (1931). Natlirliche Ffllle heteromorpher Regeneration am Auge des Sumpfkrebses. Zool.

Anst. 96, 18-32.

EXPLANATION OF PLATE

Fig. 4, PI. 1. Regeneration after first post-operative moult, Series O. A, Lobster O-23; B,lobster O-3; C, lobster O-7; D, lobster O-13; E, lobster O-2, no heteromorph regeneration,note healed stump of left eyestalk; F, lobster O-6 (2nd moult), arrow points to regeneratednubbin. Compare contorted and bent form of heteromorph after first moult with heteromorphform after second moult (Fig. 5). Calibration rectangle in F is 1 cm. long.Fig. 5, Regenerated heteromorph flagella after one to four post-operative moults. A, LobsterO-17, fourth moult; B, lobster Q-i, third moult; C, lobster Q-25, second moult; D, lobsterQ-10, first moult; E, lobster Q-21, second moult; F, lobster Q-14, second moult. Behaviouralresponses and electrical recordings from these six lobsters are given in Tables 2—6 and Figs. 6—12.

Journal of Experimental Biology, Vol. 43, No, 1 Plate 1

D M. MAYNARD {Facing p. 106)