J. Exp. Biol. (1965), 43. 79-106
fVith 1 plate and 10 text-figures
Printed in Great Britain
jg
THE OCCURRENCE AND FUNCTIONAL CHARACTERISTICS
OF HETEROMORPH ANTENNULES IN AN EXPERIMENTAL
POPULATION 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 eyestalks is a well-documented phenomenon among the higher Crustacea, and there are
extensive 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 demonstrate functional connexions between sensory elements on the heteromorph and the
central nervous system. In some cases, the behaviour and neuromuscular activity
elicited 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 the
findings therefore suffered because one has been unable to say how representative the
individuals studied may have been, or whether the responses described represented
stable or transient patterns in the individual. The need to have some answer to such
questions became particularly evident in the course of a detailed analysis of the
neurophysiology of a ' naturally occurring' heteromorph in a spiny lobster, Panulirus
argus (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 an
experimental population of P. argus whose eyestalks were removed unilaterally.
Functional connexions between heteromorph and brain were found in some form in
every instance tested. They often appeared to be quite specific, and, in at least some
individuals, relatively stable for periods of 12 months or more.
METHODS
Eyestalks were removed unilaterally from three series of adult or near-adult P. argus
Latreille. In Series O eyestalks were removed from eighteen lobsters on 17 March
1962. In six of these eighteen, only eyestalks were ablated; but in the remaining
twelve, the distal segment and flagella of an antennule were also removed, six ipsilateral, six contralateral. In addition, six more lobsters with only antennules removed
• Contribution number 365 from the Bermuda Biological Station, St George's West, Bermuda. The
first modern report of the occurrence of a heteromorph antennule in a spiny lobster was made 24 October
1864 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 oq
8 August 1962. In Series Q, eyestalks were removed from twenty-five lobsters on
16 March 1963. After operation the lobsters were maintained in running sea water in
large cement tanks at the Bermuda Biological Station. Water temperatures ranged
from below 150 C. during February and March to over 300 C. during August and
September. The lobsters were fed periodically on fresh or frozen fish.
Eyestalks were removed by cutting across the eyestalk with heavy scissors between
the proximal and distal chitinous segments. This removed the medulla terminalis and
all distal optic ganglia (see Maynard & Dingle, 1963). The stump was crushed with
haemostats to prevent excessive bleeding.
Mortality at the operation was nil, but subsequently a number of lobsters were lost
in the holding tanks, often at ecdysis. Animals were also killed periodically for histological study and in the course of acute experiments. One animal from Series O was
maintained 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, glassfronted aquaria. For electrophysiological observations lobsters were removed from
the tanks and immobilized by tying to a partially submerged board. The anterior
carapace and appendages remained out of water. When moistened periodically the
animal remained in good condition for several hours and at the end of the experiment
could be returned to the tanks without apparent damage. Conventional amplifiers,
oscilloscopes, cameras and stimulators were used. Initially, recording electrodes for
electromyography were connected, via cuts in the antennular cuticle, to the muscles
involved, but in subsequent experiments surface electrodes placed on undamaged
cuticle 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 previous
publication (Maynard, i960).
RESULTS
Ecdysis and growth
Thirty-seven of the forty-three lobsters of Series O and Q remained alive to the
first moult. Fig. 1 gives the size distribution of the population at the time of the
operation together with the size distributions of the subpopulations moulting within
the first 3 months of the operation (before 18 June); during the fourth month (18 June
to 18 July); and during or after the fifth month. The smaller animals tended to moult
earlier 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 few
animals. In one lobster with a cape length of 11*35 cm., the interval between the
first 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., the
interval between thefirstin August and the third post-operative moults in June ranged
from 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 per
year (Sutcliffe, 1952). Slightly smaller (8-O-8-9 cm cape length) immature lobsters
kept in the Bermuda laboratory moulted 3-4 times per year with intervals varying
according 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 effects
on the intermoult interval in these lobsters.
The incremental increases in carapace length are plotted for fifty-two moults in
thirty-two lobsters in Fig. 2. Although a few individuals showed very little or no
8
9 10 11 12 13 14
Cape length (cm.)
Mean
2 -
6
7
8
9
10 11 12 13 14
-
1
0
1
2 3 4 5 6 7
Cape increase (mm.)
Fig. 2
Fig. 1. A. Size distribution, in premoult cape lengths, of experimental population alive after
first moult. B. Size distribution, in premoult cape lengths, according to interval between
operation and first moult (O-E interval), see text. The open histograms represent animals in
which no heteromorph appeared at the first moult; the hatched histograms represent animals
which produced a heteromorph at the first moult.
Fig. 2. Distribution of growth increments, in millimetres, at moult of experimental population; 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 comparable 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 of
factors: 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 at
6
Exp. BioL 43, 1
82
D. M . MAYNARD
ecdysis (Travis, 1954), the above observations suggest that the lobsters used in these
experiments were in reasonably good health. There was no significant difference in
growth at ecdysis between lobsters which did not regenerate a heteromorph at first
moult (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 which
were alive at the first post-operative moult. Five lobsters from Series P which lacked
Table 1. Moult interval and heteromorph growth
ist moult
mobster
Sex
Q-4
O-i*
M
F
F
M
M
F
F
F
F
M
F
M
M
M
M
M
F
F
M
F
F
M
F
F
F
M
F
M
M
M
F
F
F
F
F
F
M
M
O-21 •
O-2
O-io*
O-6*
O-i9 #
O-i7»
O-18
Q-2
Q-24
O-i 3 »
Q-7
O-5 *
Q-I3
Q-15
Q-20
Q-6
O-23»
O-3*
Q-5
Q-i
Q-21
Q-8
O-9»
Q-19
Q-12
Q-23
Q-14
Q-17
O-15
Q-25
Q-22
O-7
Q-16
Q-10
P-i
Initial Days Het.
size post-op. size
9-4 cm. 88
0
0
94
< 93
0
8-2
< 93
76
0
< 93
70
0
< 93
0
< 93
94
0
7'5
< 93
0
89
< 93
69
99
11-35
95
io-6
97
97
99
100
104
104
145
145
0
0
152
0
0
154
170-190
0
108
24
925
io-8
0
95
112
112
116
IO-2
120
2-6
no
121
i-5
104
120
127
127
128
29
3-4
IOI
129
27
II-45
130
130
0
514
3:8
.
.
152-321
2-1
152-321
2-7
30
I-I
321
452-456 3"5
36 +
g
152-321
0
469
0
3"5
I-I
131
2-9
152-321
40
n-45
139
0
152-321
0
II-I
23
II-2
140
141
io-6
142
0
290-321
152-321
41
0
469
0
IO-O
142
2-5
152-321
0
458
0
91
an heteromorph length (cm.)—
41
33
131
144
> 152
< 202
514-685
.
3'4
1005
1185
137
Het.
size
I-I
107
121
Days
post-op.
0
i°5
123
Het. Days Het.
size post-op. size
4th moult
a-6
99
975
n-35
Days
post-op.
3rd moult
065
29
I-I
0
IOI
103
2nd moult
3'5
0
21
30
24
'306
3-8
34
355-524
49
4-1
—
Heteromorph length is given in cm.
• Animals of Series O in which an antennule was removed along with an eyestalk. Mean heteromorph 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 the
eyestalk and appeared in place of the ablated eyestalk in twenty-two lobsters at the
first moult.
Heteromorph occurrence and the operation-ecdysis (O-E) interval
Heteromorphs were completely lacking in the eight animals moulting within less
than 3 months after eyestalk ablation, but they occurred in over two-thirds of the
lobsters moulting during the fourth and fifth months after the operation. Heteromorph
failures were evenly distributed over these latter two months. Presumably there was
a critical period in the intermoult interval—apparently just preceding ecdysis—in which
eyestalk removal did not initiate heteromorph regeneration. When the operation
occurred at any time outside this period, however, the probability of heteromorph
regeneration seemed to remain essentially constant.
Nine lobsters lacking heteromorphs after the first moult were followed through the
second moult; only one developed a heteromorph. Three of these animals were then
followed through the third moult, 15 months post-operative; none developed heteromorphs. If only O-E intervals of more than 3 months are considered, twenty-two out
of thirty first moults produced heteromorphs, while only one out of twelve second or
third moults produced new heteromorphs. Apparently heteromorph flagella must
develop 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 not
examined.
Regeneration of heteromorph and normal antennule
In eleven of the fifteen Series O lobsters of Table 1 (indicated by (•)), the distal
portion of either the ipsilateral or the contralateral antennule was also removed at the
time of eyestalk ablation. The normal antennule regenerated within the stump in all
of these animals and appeared at first moult. It even occurred in those moulting
within 3 months of the operation and in which heteromorphs failed to appear.
Such simultaneous regeneration of normal antennules, however, did not obviously
affect either the probability of heteromorph occurrence or the length of the heteromorph in the fourth and fifth months.
Certain features of normal antennule regeneration differed from heteromorph
formation. Although one individual of a control series with antennule ablation only
failed to regenerate on the first moult it did so on the second, and in most cases the
critical period for antennule regeneration was obviously less than that required for
heteromorph development. Regeneration of the normal antennule was also more
extensive 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. and
ranged 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 experiments
was 4-9 cm.
These values and those of Table 1, however, are not completely reliable as measures
of 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 minimal
and 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 progressive
increases with longer O-E intervals. In lobsters of Series Q heteromorphs appearing
during the 4th post-operational month averaged 1 -7 cm.; those appearing during the
5th month averaged significantly longer, 2-6 cm.
Normal
eye-stalk
Interocular
yoke (cut)
Normal outer flagellum
Fig. 3. Eyestalk, heteromorph flagellum, regenerated outer flagellum (distal portion only) of
normal antennule, and original outer flagellum (distal portion only) of normal antennule on
opposite side: all from same lobster. Animal killed after first post-operative moult and tracing
made from photograph of preserved specimens. See text for further discussion.
Heteromorph form
As in other Palinuridae (Herbst, 1900; Milne Edwards, 1864) the heteromorph
antennule of Panulirus argus resembled the outer flagellum of a normal antennule.
It characteristically lacked structures analogous to either basal segments or inner
flagellum.
The heteromorph arose as a ringed flagellum directly from the remnants of the
heavy chitinous basal segment of the eyestalk (Fig. 3). There was some variation in
the direction taken by the flagellum, but in most cases it was oriented horizontally or
at a slight upward angle and pointed straight ahead (Figs. 4, 5, PI. 1). There was no
independent movement of the heteromorph. However, since it was attached rigidly
to 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 the
heteromorph varied considerably. There were occasional protuberances or sculpturings
but no true segmentation.
The flagellum itself consisted essentially of two parts. The proximal portion was
made up of wider annuli that bore a scattered assortment of relatively short sensory
hairs (see Laverack, 1964). In the distal portion the annuli tended to be narrower, and
in addition to the sensory hairs mentioned above bore guard, companion and aesthetasc
hairs on the ventral side. The aesthetasc hairs were situated in two transverse rows at
the forward and rear edge of each annulus, and were bordered laterally on each side
by one large guard hair and one or two companion hairs. This gave a toothbrush-like
appearance to the flagellum.
The aesthetasc hairs are apparently chemoreceptors (Laverack, 1964), while many
of the other hairs mediate various kinds of mechanical stimuli. The brush of aesthetasc
hairs and associated companion and guard hairs distinguished the outer from the inner
flagellum in the normal antennule. In all major respects, therefore, except number of
annuli and proportion of individual annuli, the heteromorph flagellum was like the
outer flagellum of the normal antennule.
The essential structure of the heteromorphflagellumas described above was present
in all twenty-two heteromorphs observed in this study. There was individual variation, however, with respect to length-breadth proportions of the flagellum, and with
respect to the relative number of proximal and distal rings. There were also changes
with time. After the first moult, the flagellum tended to retain bends and folds which
had 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 were
lost on the second moult (Fig. 5, PL 1). In the shorter heteromorphs the distal annuli
were 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 characteristic
flagellum measuring 2-9 cm., there were twenty-four proximal and twenty-eight distal
annuli. Increases in flagellar length, both during primary growth and between
moults, 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 thirtyeight in one moult, while the proximal annuli increased by one, from twenty-four to
twenty-five. In none of the animals observed, however, did the heteromorph flagellum
approach the normal flagellum in length or number of segments. The ' oldest' animal,
examined 27 months and four moults after operation, had twenty-six proximal and
sixty distal annuli in the heteromorph compared with sixty-seven proximal and over
116 distal annuli in the normal outer flagellum. Moreover, in lobsters simultaneously
regenerating normal flagella and heteromorphs the normal regenerate formed more
distal 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 regenerate
at first moult, but only twenty-five in the heteromorph.
When the heteromorph flagellum failed to appear the eyestalk scar generally healed
over 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 second
moult, 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 of
response (Herbst, 1910; Lissman & Wolsky, 1933; Maynard & Cohen, 1965): (1)
general escape activity and bodily withdrawal from the stimulus; (2) cleaning of the
stimulated heteromorph with ipsilateral walking legs—these motions are analogous to
those employed in cleaning the normal eyestalk; (3) movements of the ipsilateral
antennule. The latter have been of particular interest because they often resemble
responses normally specific to stimulation of the outer flagellum of the normal
antennule (Maynard & Dingle, 1963; Maynard & Cohen, 1965).
Table 2. Behavioural responses to heteromorph stimulation
Stimulus
Mechanical
Lobster
#
Heteromorph
length
(cm.)
Manipu-
Chemotactile
lat
Response
Days
O-E
cl.
gen.
ant.
cl.
X
X
X
X
X
X
X
X
X
0
X
X
X
X
X
X
gen.
ant.
127
127
X
X
X
0
202
X
X
X
an
Q-i
Q-2I*
2-9
P-I
3-8
Q-I4*
Q-io*
Q-5
Q-7
Q-8
O-12
29
131
X
X
0
X
X
2-1
152 +
X
0
i'5
121
-
X
—
—
X
—
?
—
I-I
104
X
0
X
0
3'4
128
X
X
X
0
X
X
0
0
3'5
065
2-4
130
X
?
0
0
X
X
0
X
—
0
97
X
—
-
0
108
X
0
X
X
?
X
0
i'i
X
X
?
X
X
0
3'5
131
141
?
0
X
?
34
112
X
0
0
X
0
0
i'i
100
X
0
0
—
—
—
0
34
170—190
X
0
?
0
0
0
0
8
3
3
12
6
7
6
0
1
0
0
0
0
4
11
I
3
4
6
10
Q-2
Q-15
Q-23
Q-25*
Q-6
Q-24
O-i7 #
i'i
<
Total no. lobsters responding
Total no. questionable 1•esponses
Total no. not responding
15
0
-
X
0
X
X
X
0
0
0
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 of
stimulated 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 specified
stimulus.
Table 2 gives the responses to various kinds of heteromorph stimulation as observed
in sixteen lobsters—fourteen first-moult heteromorphs from Series Q, one secondmoult 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 heteromorph 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 the
lobster is held by hand out of water. In Table 2 the first two stimulus types are
combined under ' mechanical stimulation', while the third and fourth types are labelled
'chemotactile' and 'manipulate' respectively. Because of the complex nature of the
stimuli used and the variability in heteromorph anatomy, stimuli to different animals,
or to the same animal at different times, cannot be considered quantitatively equivalent. None the less, the range of stimulus intensities used was usually sufficient to
assure responses if any could be evoked by non-injurious stimuli (see however
O-17 below), and the failure of a response could not generally be ascribed to abnormally weak stimuli. Responses were variable, but fell roughly into the three classes
mentioned above: (1) non-specific response, usually escape activity and bodily
withdrawal; (2) specific cleaning of stimulated heteromorph with 4th ipsilateral walking
leg; (3) specific movement of ipsilateral antennule; the response to manipulation in
the hand-held animal generally involved depression of the outer flagellum (Maynard &
Cohen, 1965). Within any one response class individual lobsters differed both in
threshold strength of the effective stimulus and in the exact form and intensity of the
evoked motor activity. Some of these variations will be detailed for selected individuals 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 were
present in all lobsters of this population.
B. Specific connexions between heteromorph sensory neurons and ipsilateral
antennular motor neurons are common but not universal. On the basis of clear
behavioural 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 the
population.
D. Chemoreceptors sending afferents from the heteromorph often formed functional connexions in the brain. This is implied by the increase of heteromorph cleaning
activity following chemotactile stimulation over that following mechanical stimulation
alone.
E. Functional effectiveness does not obviously correlate with the length of the
heteromorph.
Specific responses
Several lobsters (indicated by (•) in Table 2) were re-examined in detail in June
1964 (see Fig. 5, PI. 1). Particular attention was given to the ipsilateral antennulai
responses, and to the comparison of these with responses evoked by stimulation of the
normal antennule. Over a period of 4 days there were three or four sessions with each
of the four kinds of stimulation: glass rod, cloth swab, hand manipulation, and fish
extract on cloth swab. Responses to the glass rod and cloth swab, both mechanical
stimuli, were similar and are considered together (Table 3). Antennular responses to
chemotactile stimulation of the heteromorph with the fish extract swab were generally
Jike 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 extraci
while mechanical stimulation alone never evoked heteromorph cleaning in this series
of observations (see, however, Table 2). Responses to heteromorph manipulation in
the lobsters held out of water differed slightly from other responses to mechanical
stimuli, and are treated separately in Table 4. The distal portion of the heteromorph
flagellum was generally more effective than the proximal portion in eliciting this
response.
Mechanical stimulation. The major responses of an unrestrained lobster to mechanical
stimulation of the normal antennule were described by Maynard & Dingle (1963).
The fast withdrawal reflex of the antennule (antennular reflex) and the bodily withdrawal responses are relevant here. The antennular reflex following stimulation of the
outer flagellum involves an inward and downward twitch of the first antennular segment, 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)
*—
Segmental 1novements
,
Non-specific
(% stimulus sessions1
responding:
A
Basal
n == 6-8)
Outer
Any ant. (inSecond Distal flagellum
A
Startle Crouch response down) (straight) (down) (down)
0-17
Q-25
0
0
0
0
38
0
0
0
0
0
0
62
87
38
Q-21
0
100
57
29
0
43
29
Q-i
Q-10
29
67
71
86
100
43
83
29
16
57
83
50
57
83
Q-14
33
67
100
100
67
5°
50
Comment
Basal twitch very
slight
Slight, slow movements
Basal twitch
Slow (single basal
twitch)
Basal twitch, other
movements slow
Average response to normal outer flagellum stimulation
— In-down Lateral Down Down All twitches
(usually)
depression of the outer flagellum of the antennule stimulated (Table 3). The contractions are fast and twitch-like. Bodily withdrawal may have two or more components: an initial startle reaction characterized by a synchronized twitch-like contraction or extension of most of the appendages, and subsequent tonic crouch or
stepping withdrawal from the locus of the stimulus.
Table 3 compares responses to normal outerflagellarstimulation with those observed
following heteromorph stimulation. Response frequencies were calculated from the
number of sessions—out of a total of six to eight—in which at least one response of
the kind indicated occurred; they are expressed in percentages. Usually several presentations were made in any one session. Individual differences are apparent. For
example, in this particular series of tests, O-17 gave no obvious non-specific response
of any kind—its reaction to normal antennular stimulation, however, was prompt and
appropriate—while Q-25 and Q-10 usually showed both startle and withdrawal
reflexes. 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 particularly, always responded with strong reflexes. The pattern of response also varied
so that, although all responding lobsters included an inward and downward movement
of the basal segment in some of their responses, this appeared as a rapid, twitch-like
contraction in some individuals (Q-25, Q-i, Q-14), or as a slower, more prolonged
movement in others (Q-21, Q-10). The relative frequency of movements of the more
distal antennular segments also differed among individuals. In general all these
responses resembled the normal antennule reflex, but in addition to individual variation seemed to have two consistent discrepancies: (1) most of the antennular contractions elicited by heteromorph stimulation, and particularly those involving the distal
segments and outer flagellum, tended to be slower and more prolonged than the fast
withdrawal reflex; and (2) where movement of the second segment occurred it commonly took the form of forward extension rather than the lateral flexion more usual
with normal outer flagellar stimulation. Elevation of the distal segment and outer
flagellum, which characterized the reflex evoked by innerflagellarstimulation, was not
obvious in these animals.
Table 4. Behavioural responses to flagellar manipulation
Specific antennular response
(% stimulus sessions responding: n = 3)
Segmental movements
-obster
Second
Basal
Any ant.
response (in-down) (straight)
O-i7
0
Q-25
Q-21
0
Q-i
Q-10
Q-14
33
0
0
0
Distal
(down)
Outer
flagellum
(down)
0
0
0
0
0
0
0
33
67
33
67
Comment
No movement of basal or
second segment
0
0
Weak response
33
33
33
100
100
100
100
Strong response, = normal
67
antennular response
Average response to manipulation of normal outer flagellum
In-down, Lateral
Down
Down
and
or up
and down straight
67
0
0
Manipulation. Maynard & Cohen (1965) found that flagellar depression in a normal
hand-held lobster was specifically induced by manipulation of the outerflagellumof the
antennule. Similar stimulation of the heteromorph discovered on a lobster' in nature'
evoked the same response. Table 4 compares the response of the antennule to such
stimulation of its outer flagellum with responses resulting from stimulation of experimentally induced heteromorphs. The slow depression of distal segment and outer
flagellum found in the response to normalflagellarstimulation was often associated with
movements of the basal segments in these animals. An inward and downward movement
of the basal segment and lateral flexion of the second segment were common, but in
many cases an up-down oscillation of the basal segment and an extension of the second
segment 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 flagellar
manipulation, and that, if strong stimulation was continued after depression, elevation
and other avoiding movements often followed.
Only one of the six experimental lobsters (Q-14) gaveflagellardepression following
heteromorph stimulation during each of the three stimulus sessions. The detailed
form of the response was very similar to that evoked by stimulation of the normal
uterflagellum, 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 outer
flagellum were involved; in this series of experiments the basal and second segments
remained motionless.
Tables 3 and 4, together with Table 2, seem to suggest that, although heteromorphs
do occur whose stimulation evokes several responses essentially like those caused by
stimulation of the outer flagellum of the normal antennule, animals bearing them are
not very common. Moreover, there may be significant variations in certain components 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 necessarily unorganized. Most lobsters gave some indication of specific responses, and
these generally seemed to resemble components or incomplete sequences of activity
normally elicited by the normal outer flagellum more closely than any other kind of
behaviour observed thus far. Inappropriate responses such as those specific for the
inner 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 of
withdrawal activity upon mechanical stimulation of the stump, and one demonstrated
' place sense' with appropriate cleaning motions by the fourth ipsilateral walking leg
directed at the stump following strong mechanical stimulation. In none of these
animals, however, was the response potentiated by chemotactile stimuli, nor were
specific antennular responses evident.
Stability of behavioural responses over time
In an attempt to determine whether progressive changes in heteromorph function
occur with time or ecdysis, the behaviour of the six lobsters of Tables 3 and 4, together 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-August
1963, February, April, June 1964). O-17 was followed over a longer period of
20 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 behavioural responses evoked by heteromorph stimulation. The population is somewhat
small, however, and discrimination of small differences by the behavioural methods
used is difficult so I cannot exclude the possibility of subtle changes with time in some
or all of the lobsters tested, or indeed, the possibility of major changes in the occasional
individual. 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 behaviours
included—from non-responsive to very responsive individuals—does suggest that
heteromorph-induced behaviour is reasonably stable, and is consistent with the
tentative hypothesis that once connexions are established in the course of initial
development further modification of connexions is normally insignificant or very slow.
Fig. 6. Compound action potentials recorded in vivo from flagellar nerves. A, Normal nerve
from antennular innerflagellum(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 of
major fast component; slow components are not visible. C. As in B, but with slow trace to show
later slow components; only component'c' is obvious; see Table 5 and Fig. 10. D-I, Nerves
from 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 baseline represents the first component of the compound action potential. Calibration, 60 eye./sec.
Table 5. Conduction velocities in hcteromorph and flagellar nerves
Flagellar nerve
Ipsilateral
Heteromorph nerve
Lobster
Temp.
(° C.)
O-17
Q-25
Q-21
26-7-27-5
26-4-27-2
260-26-7
Q-i
270
Q-10 26-8-27
Q-14 27-3
Mean (m/sec)
Outer flagellum
Contralateral
Inner
flagellum
a
b
c
d
a
b
c
a
b
5'4
19
—
26
19
—
076
0-41
56
o-86
047
058
76
7-4
7'4
o-33
0-41
0-33
39
4-4
—
7-0
049
038
—
—
56
5'5
6-8
69
8-3
67
054
0-38
41
6-2
37
37
39
45
i-5
—
048
042
032
028
037
2 0
0-72
0-37
—
—
o-6i
5'3
O-54
°5 5
0-56
3-8
5'5
7-0
51
67
4-5
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 the
flagellum and on toward the cerebral ganglia along the course of the former optic tract.
Direct evidence for conducting pathways was obtained by recording compound action
potentials in vivo in the proximal part of the flagellum while stimulating the distal
region. Conduction velocities were calculated from response latencies which remained 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 under
sections 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 are
particularly prominent in O-17 and Q-10. In view of the unresponsiveness of O-17
to natural heteromorph stimulation the demonstration of functional conducting
elements in the heteromorph nerve is of particular interest.
Fig. 6 and Table 5 also illustrate the conduction velocity spectra of nerves in the
inner flagellum and outer flagellum of normal antennules, and in one regenerated
antennule. The innerflagellarnerve 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 slightly
slower elements, but apparently lacks a major slow component. The outer flagellum
is characterized by a major fast component, 'a', often with several slower subcomponents, 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 in
Q-10 and Q-14 might be ascribed to the position of recording electrodes. In the
record from the single regenerate outer flagellum both a slow and a fast component
were present, but both were slower than any recorded from normal outer flagella. In
general, the conduction velocity spectrum of the outer flagellum resembles that of the
heteromorph 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 muscle
activity during heteromorph and normal antennular stimulation were made from the six
lobsters of Fig. 5 (PI. 1). External electrodes clipped to the intact cuticle of an antennular segment recorded action potentials in the muscles of that segment only. Simultaneous recordings (Fig. 7) illustrate independent activity in muscles of the contiguous second and distal segments. In the second segment functional interpretation
is 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 depresses
the outer flagellum; flagellar elevation is passive. In the following experiments
simultaneous recordings were made from the distal segments of both antennules
(Fig. 7C). Two kinds of action potential were usually evident in recordings from
m. 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 flick
of the outer flagellum (Maynard & Dingle, 1963). It appeared in partially contracted
as well as in completely relaxed muscle. The second pattern was simply a maintained
barrage of potentials at varying frequencies; the higher the frequency, the greater the
depression of the outer flagellum. During strong contractions instantaneous frequencies occasionally approached 200/sec.
1.3
0-2
Fig. 7. Action potentials recorded in vivo from antennular muscles. A, Lobster Q—21. Upper
trace recorded from second segment of antennule, lower trace recorded from third or distal
segment 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 third
or distal segment of antennule. Lifting inner flagellum of the same antennule—indicated by
marker trace—produced increased activity in muscles of the second segment, but inhibited
on-going discharges from the muscle of the third segment. Note that electrodes only recorded
activity tvithin one segment; no 'cross-talk' was evident. C, Lobster Q—10. Upper trace
recorded from third segment of left antennule. Note on-going activity in muscle of right
antennule, action potentials of two amplitudes and apparently independent discharge frequencies are present. In simultaneous recording from muscle of left antennule background
activity 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. Antennular
flagella, eyestalks and heteromorphs remaining out of water were moistened periodically. For stimulation, flagella were lifted or bent with a glass rod, or were pinched
between rubber-tipped forceps. Stimuli were obviously imprecise, and varied with
time and individual; the general excitability of the animal also varied. Analyses of
fine detail in the responses are therefore inappropriate. Nevertheless, in individual
lobsters re-examined after an interval of days, responses were usually consistent, and
some general statements are possible. As a basis for comparison normal responses are
considered first.
Stimulation of the normal flagellum. Maynard & Cohen (1965) reported that
manipulation 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 slightly
different stimuli used several complications appeared.
In all six lobsters mechanical elevation of the inner flagellum always caused a reduction
in action-potential frequency in m. reductor4, and, sometimes, nearly complete
inhibition. A typical response appears in Fig. 8C, G. In some animals this was
associated with contralateral excitation and with augmented frequencies as the
flagellum dropped at the end of stimulation. In most, but not all lobsters, mechanical
depression orpinching of the innerflagellum caused excitation, not inhibition; when inhibition 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 the
ipsilateral muscle; sometimes there were contralateral effects (Fig. 8A, 8F). In two
of the six lobsters, however, responses to outer flagellar stimulation, particularly
flagellar depression, were complicated by inhibition following an initial excitation, or
occasionally by inhibition only (Fig. 8B).
These observations demonstrate that both excitatory and inhibitory reflexes can be
elicited from both antennular flagella. Complex sequences of inhibition and excitation
can also occur with proper stimulation of either flagellum. Thus far, however, the
evidence seems to confirm the initial impression that net excitation is usually characteristic of the responses to gross stimulation of the outerflagellum,while net inhibition
is more prevalent upon stimulation of the inner flagellum. This also is in accordance
with behavioural observation (Maynard & Dingle, 1963).
Stimulation of the heteromorph. Responses of the antennular muscle to heteromorph
stimulation were observed in all six lobsters, even those which were behaviourally
unresponsive, lobster O-17 (Fig. 9F, G) and lobster Q-25 (Fig. 9J). The form of the
response, however, differed between individuals, and often within one individual
according to the kind of stimulus. The latter is particularly well illustrated in lobster
Q-14 (Fig. 9D, E), where an inward bend of the heteromorph caused ipsilateral, and
to a much lesser extent, contralateral excitation, while an outward bend caused ipsilateral inhibition. Lobster Q-10 (Fig. 9H, I) is another example in which an upward
bend again caused bilateral, asymmetrical excitation, while pinching the distal portion
of the heteromorph caused ipsilateral inhibition and contralateral excitation. In fact,
in all lobsters tested except lobster Q-21 (Fig. 9C), certain kinds of mechanical
stimulation tended to produce inhibition while others favoured excitation. In many
cases (see lobsters Q-i, Q-25 and O-17 in Fig. 9 for examples) both excitation and
inhibition were apparent in single response sequences, excitation commonly being
followed by inhibition. In at least one lobster, O-I7, relatively strong ipsilateral
excitation was associated with contralateral inhibition, while in another, Q-i, bilateral
inhibition occasionally occurred.
Individual variation was difficult to evaluate. Heteromorph responses were not
specific enough to be identified with either 'inner flagellar responses' or 'outer
flagellar responses', and direct intra-lobster comparison of heteromorph and normal
responses showed almost no consistent pattern of difference. The muscle responses
of lobsters O-17 and Q-25, lobsters with little or no apparent heteromorph-induced behaviour, were somewhat weaker than others, but further correlation between individual
behaviour and electromyography was not obvious. Nevertheless, two generalizations
Heteromorph antennules in Panulirus argus
.JJ.^J.^.I „..]
„..]
1J . I II I
..I., i
I J
i j i j
95
I I I I I
j .
11 i
i 111
i
i i
iy^myy^|oLjlJ A-il.JJ J JJl J .1 .J LJ J. J. I. I.
i i
.III.
LJ
j< I i ii.liiihi.il lil
J kJUl Llillllll Hal Illl III II II 111 11 i I
ilLl I
Mli
0-5
Fig. 8. Responses of m. reductor4 in third antennular segment to mechanical stimulation of
antennular flagella. In each record the upper trace is taken from the right antennule, the
lower trace from the left antenule, simultaneous recording. The marker signal above the upper
trace in each case indicates the approximate duration of the stimulus, but is displaced to the
right 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 flagellum
lifted; 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 flagellum
depressed. Note that responses are largely, although not completely, unilateral. R and L
indicate antennule stimulated. Calibration, C5 sec.
1
A
, 1
,LJ
B
IllJUliJ ±,JU
III
iu,,iili
l i l l i l 1 lit k Ii I 1
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llill
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-Uil
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25
kkUllklhl
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ii Iki
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i
It h i l l
1
l l r i l L l l I
L. ,.,.„„„„,,
I t
1
i l
0-5
Fig. 9. Responses of m. reductorj in third antennular segment to mechanical stimulation of
heteromorph flagellum. Upper trace, right antennule; lower trace, left antennule; simultaneous 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 heteromorph
inward, B, lobster Q-i, pinch left heteromorph; C, lobster Q-21, manipulate left heteromorph; D, lobster Q—14, bend right heteromorph inward; E, lobster Q—14, bend right heteromorph outward; F, lobster O-17, bend left heteromorph backward; G, lobster O-17, pinch
left heteromorph; H, lobster Q-10, bend left heteromorph upward; I, lobster Q-10, pinch
left hetermorph; J, lobster Q—25, bend left heteromorph downward. R and L indicate side of
stimulated heteromorph. Calibration, 0-5 sec.
Heteromorph antemtules in Panulirus argus
97
seem possible for the responses of these particular six lobsters: (1) Although contralateral effects occurred the primary excitatory response to heteromorph stimulation was ipsilateral. (2) None of the excitatory responses was as vigorous as those
elicited by normal antennular stimulation. In this respect these six lobsters contrast
with the lobster described by Maynard & Cohen (1965) in which heteromorph
stimulation produced very strong excitation. The extent to which gross and uncontrolled 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. Single
electrical stimulus given to distal flagellum at artifact. Lobster Q— i. A, Stimulus and upper
trace from outer flagellum, stimulus subthreshold for slow components (6 and c) of outer
flagellar nerve. B, As in A, but stimulus suprathreshold for slow components. Early response in
muscle (i) is more apparent. C, Stimulus and upper trace from inner flagellum. Stimulus supra
threshold, 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 heteromorphs, Q-16 and Q-22, were examined. Responses to stimulation of the normal
antennular flagellum resembled those described above. Antennular responses to
manipulation 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 particularly
strong and were somewhat variable. They were usually bilateral, and in at least half
the cases were inhibitory. There were also excitatory responses however (see Maynard
& Cohen, 1965) and occasionally potentiation of burst frequency associated with
antennular flicks occurred. In lobster Q-14 ipsilateral excitation was associated with
contralateral inhibition. In general, responses to eyestalk manipulation did not closely
resemble responses to antennular or heteromorph manipulation.
Responses to electrical stimulation
Stimulation of outer flagellum. A single stimulus applied to an outer flagellum just
proximal to the distal brush of sensory hairs characteristically elicited a four-part
response in the ipsilateral m. reductor^. An initial burst of action potentials (1) was
followed first by inhibition (2), then by a subsequent second burst (3) and, often, by
a final prolonged discharge at increased frequency (4). Contralateral responses were
not observed. The afferent fibres mediating this response apparently belong to the
fast component of the antennular nerve because the response occurred before the
arrival of the slow component at the brain, and also occurred at stimulus strengths
subthreshold 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
A
l i l i l l ' i n I I l i I I n Li i l l
l u i l l n i 1 1 1 l i d u i l i l l n 11 1 1 1 1 1 I I 1 1 1 1 1 1
I'Hjrkiii
\
\
2
3
JJUJjUu-JUUUoUUJUUJJl
I. J L . i l . i l . I . I L I L L . , I . J . . L . I . J . I . J . I . I ,
1
LJ1.J.L.I.I...1..L.
l
Fig. 11. Responses of m. reductor4 in third antennular segment to electrical stimulation—single
stimulus—of the ipsilateral antennular inner flagellum (if), the ipsilateral antennular outer
flagellum (of), and the heteromorph flagellum (het). Upper trace, right antennule; lower
trace, left antennule, simultaneous recording. Dots (•) indicate stimulus. R and L indicate
side 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 nagella
and left heteromorph stimulated. C, Lobster Q—21, left nagella and left heteromorph stimulated. 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 heteromorph stimulated. G, Lobster Q-25, left nagella and left heteromorph stimulated. Components of reflex response: i, initial burst; 2, following inhibition; 3, second burst; 4, prolonged
discharge, only beginning visible on record. Calibration, 05 sec.
Heteromorph antennules in Panulirus argus
99
Maynard & Cohen (1965) and was relatively consistent within the population examined
here (Fig. 11, of). There is some individual variation, however, in the relative
prominence of the four parts of the response. Such variation seemed characteristic of
the individual, not the specific antennule, because it was reflected to varying degrees
in 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 stimulation occurred. Although the second, inhibitory response continued to appear and to
interrupt briefly the discharge after each stimulus the net effect was of strong
excitation.
Stimulation of inner flagellum. Fig. 11 compares the two-part responses to single
stimuli of inner flagella with outer flagellar responses. The initial burst of the latter
seems to be absent and the first obvious component of the inner flagellum response is
an inhibition of spontaneous discharge. This in turn is followed by the second component, moderate excitation. The inhibition tends to be stronger and the excitation
weaker 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 was
apparent. 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 well
defined within individual lobsters and were consistent with repeated stimuli. Fig. 11
compares responses to single stimuli applied to heteromorphs with responses to
single stimuli applied to normal antennular flagella and Fig. 12 illustrates responses to
repetitive stimulation. There was considerable variation among lobsters. The most
typical 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 inhibition
while in another, lobster Q-25 (Fig. 11G), the initial burst was absent or nearly so but
was replaced by strong inhibition. Repetitive stimulation produced complete ipsilateral inhibition in lobster Q-25 (Fig. 12G). In the other four lobsters, repetitive
stimulation produced net excitation similar to that resulting from normal outer
flagellar stimulation. A contralateral inhibitory response was observed in two lobsters.
In lobster Q-i (Fig. 12B) it appeared only with repetitive stimulation, but in lobster
O-17 (Fig. 11E) it was also very prominent following single stimuli.
The latency of the excitatory burst following heteromorph stimulation ranged
between 40 and 80 msec. Although weaker and briefer, the response appears
analogous to that reported in the lobster described by Maynard & Cohen (1965). If
so, it may well correspond to the second excitatory burst of the normal antennular
response. It is clear that there are major differences between the total response to
stimulation of the normal outer flagellum and to stimulation of the heteromorph, but
it remains easier to correlate the heteromorph response with portions of the outer
flagellar response than with any other reflex seen so far.
Like responses to mechanical stimulation, responses to electrical stimulation were
often 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 able
to 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 and
electrical stimulation, however. For example, three animals showing clear ipsilateral
inhibition to certain mechanical stimuli gave inhibitory (lobster Q-25), weak excitatory (lobster Q-14), or combined excitatory-inhibitory (lobster Q-10) responses to
electrical stimuli. And the two lobsters displaying contralateral inhibition with
mechanical stimulation also showed it with electrical stimuli (lobsters Q-i and O-17).
1
r
i-
• 11111 III III lull III Illll
T
Ullll 11
I
"
.
I
T
I
•
•
'
-
t
•
I
AlU --
1 Jit -\]\ WS, l J|M,i J l l flttifc|-III U I I I l l l I t J ll A 1 . J l Mi 111 _k 1
illJlllllll JlllilUiili
L
ill.
1 1 1 1
1 1
k III 1
1 1 1 1
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, left
antennule, simultaneous recording. Dot (.) indicates beginning of repetitive stimuli at
10/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 indicate side stimulated. A, Lobster Q-22, right eyestalk stump; B, lobster Q-i, left heteromorph
stimulated; C, lobster Q-21, left heteromorph; D, lobster Q-14, right heteromorph; E, lobster
O-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 the
ablated eystalk in one lobster lacking a heteromorph regenerate, Q-22. No responses
were observed in m. reductor4 (Figs. 11A and 12 A).
Stimulation of eyestalk. No obvious or consistent responses to single stimuli applied
to eyestalks were recorded in m. reductor4. Stimuli were applied across the peduncle
and 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 unilateral
or excitatory responses occurred.
Table 6. Responses to electrical stimulation of heteromorph
Single stimuli
Repetitive stimuli
Lobster
Ipsilateral
Contralateral
Ipsilateral
O-17
Q-a5
Q-ai
Q-i
Q-10
Q-14
+
(+)+
+
H—
+?
-
+
0
0
o
o
o
+
H—
+?
Contralateral
0
+ 0
o
o
+ , excitatory burst; —, inhibition; H—, excitation followed by inhibition; ( + ), very weak excitation; ?, response questionable.
DISCUSSION
Heteromorph occurrence. The high proportion of heteromorphs observed in this
experimental 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 been
higher if all eyestalk ablations had been made shortly after ecdysis, for, among those
lobsters with long operation-ecdysis intervals, nearly 75%, twenty-two out of thirty,
produced heteromorphs. The population ranged from sexually immature to fully
mature animals (Sutcliffe, 1952) and included both sexes so the above proportions
seem sufficient reason to accept heteromorph development as a normal consequence
of eyestalk ablation in adult Panulirus argus.
The causes of heteromorph failure in the remaining 46 % of the population are not
clear. Since, however the first thirteen lobsters moulting after eyestalk ablation
failed to develop heteromorphs, the time at which ablation occurs in the moult cycle
is presumed important. Heteromorph failure in such animals must involve more than
simply lack of sufficient time for development before ecdysis. If this were so, heteromorph primordia should appear as found in normal limb regeneration (see Nouvel,
1939; Bliss, i960) and these should develop into full heteromorphs on the second
moult. 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 the
first moult, they rarely did at all. One may suppose that after some critical period in
the intermoult preceding ecdysis potential heteromorph tissue either lost its competence to respond to 'heteromorph-inducing mechanism', or the hypothetical
102
D. M .
MAYNARD
mechanism itself became inactive. In either event, the regenerating tissue presumablf
began differentiation as non-specific epithelium, and, once begun, this process was
apparently irreversible. Heteromorph regeneration thus differs from normal flagellum regeneration in its failure to continue differentiation after an early moult and
in the longer time required for heteromorph differentiation before moult.
Heteromorph failure in lobsters moulting at longer intervals (4 or 5 months) after
eyestalk ablation did not obviously correlate with the moult cycle, and probably
resulted 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 flagellum
of the antennule. There was no evidence of development of basal segments or of a
second flagellum with successive moults (up to four moults). In this respect Panulirus
heteromorphs resemble those reported in Palinurus vulgaris (Herbst, 1910), and
contrasted with those reported in Palaemon. In the latter genus the heteromorph first
appeared as a non-articulated nubbin with sensory hairs, but with subsequent
moults often developed both inner and outer ' antennular' flagella.
Just as the external heteromorph morphology resembled the outer flagellum of the
normal antennule, so did many of its functional properties. Laverack (1964), for
example, demonstrated functional mechano- and chemoreceptors in the heteromorph
by direct electrical recording from two lobsters of the Q series. The properties of these
were similar to receptors found in the outer flagellum. Likewise, the compound action
potential of the heteromorph nerve resembled that of the normal outer flagellum in
having major fast and slow components. Some differences such as slower conduction
velocities and additional conduction components were apparent and may have had
functional significance (see below and Maynard & Cohen, 1965), but in general the
concept of the heteromorph as a misplaced outer antennular flagellum seems reasonable as a first approximation.
Growth and temporal change. There are two aspects of heteromorph growth or
temporal change important in the present context. The first is the peripheral aspect,
and concerns change in number, size, distribution, or functional properties of sense
organs and nerve fibres in the heteromorph itself. Most of these changes would be
expected to occur stepwise, becoming evident only at each successive moult. The
second aspect is the central aspect, and concerns growth and differentiation within the
brain of afferent fibres from the heteromorph nerve. Although obviously related to
peripheral events and structures the central changes could presumably be continuous,
and in some respects rather independent of peripheral growth. At the early stages they
may also be complicated by degenerative reorganization occasioned by the loss of eyestalk
ganglia. Whereas the first aspect of growth might be revealed by changes in heteromorph morphology, the second would be detected primarily by behavioural changes.
Unfortunately, evidence available on either aspect of heteromorph temporal change
is limited. The lobsters maintained for more than one moult were selected for wide
variation in both morphological and functional characteristics, but the sample was
small, and none was kept for longer than four moults. Furthermore, the behavioural
tests 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 illustrated 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 tentative conclusions are possible.
The peripheral, external signs of growth are straightforward. The heteromorph
increases in length and in number of annuli with each moult. The distal annuli
supplied with chemosensory and associated sense hairs increase disproportionately
over the less well-endowed basal annuli, so periodic increases in the afferent neural
supply from the heteromorph seem inevitable. Presumably this would lead to continued invasion of the central ganglia with new heteromorph afferents. Functional
evidence for such an increase in total input strength from the heteromorph has not
yet been found.
Evidence on central changes with time is more ambiguous. Some lobsters with
small or poorly differentiated heteromorphs (e.g. lobsters Q-i, Q-10) responded with
strong, 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 periods
after heteromorph appearance for initial differentiation. Individual behavioural
variants were also remarkably stable, even over two moults, indicating that such
variations probably represent adventitious differences in central connectivity patterns
rather than different stages in a common sequence of progressive, functional differentiation. Although it is premature to claim that patterns of functional connexion,
once established, never develop further, it does seem likely that an initial stage of
central fibre differentiation, possibly terminated at the first moult, is followed by a
more prolonged functionally inert period. Further observations over a longer period
and utilizing more precise measures of functional connexion are necessary.
Patterns of central connexion. From the behavioural observations it is apparent that
at least some of the fibres from the heteromorph always make effective contact with
a general alarm or avoidance system in the cerebral ganglia, and that often a rather
discrete 'place sense' may occur (see also Herbst, 1910; Lissman & Wolsky, 1933;
Maynard & Cohen, 1965). Although some central specificity was undoubtedly required for the establishment of these connexions, they are less suitable for analysis
than connexions associated with the specific antennular responses, and will not be
considered further here.
The principal question concerning heteromorph connexions is simply whether the
new, incoming afferent fibres from antennule-like sensory receptors in the heteromorph flagellum form patterns of central connexion like those normally formed by
original antennular afferents. In a previous specimen of Panulirus an extensive analysis
involving behavioural tests, electromyography of antennular muscles and intracerebral
recording from single neurons did indicate great similarity (Maynard & Cohen, 1965).
In the present population the situation is less clear. Behavioural tests and electromyography indicate that in the majority of lobsters specific movement of the ipsilateral antennule can be elicited by some form of heteromorph stimulation. They also
demonstrate that according to the kind of sensory structures stimulated either
excitation or inhibition of an antennular muscle may occur. In both of these respects
heteromorph and outer fiagellar stimulation are similar. Upon closer analysis of
patterns of movement of antennular segments, or of the pattern of motor impulses
reaching 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, witH
that described by Maynard & Cohen, or with responses elicited by similar stimulation
of the normal antennular outer flagellum. Furthermore, in one or two instances,
contralateral effects not normally observed with outer flagellar stimulation were seen
following heteromorph stimulation (lobsters Q-i, O-17). Clearly, in total effect the
pattern of afferent functional connexion connectivity of the heteromorph differs from
that of the normal flagellum.
The inferences about patterns of central connexion which may be legitimately
drawn from such behavioural differences are difficult to assess. The divergences
observed do not seem sufficiently critical necessarily to disprove the hypothesis that
afferents from similar kinds of sensory receptors in the two flagella make specific
connexions with similar motor neurons and interneurons in the cerebral ganglia. The
responses of the system are obviously complex, requiring co-ordinated activity in a
number of elements, and one might propose several factors unrelated to specificity
of individual afferent connexions which could produce variations in response. Three
can be indicated: (1) Since the stimuli used presumably activated peripheral fibres
producing both central inhibition and excitation, the sequence in which inhibitorinducing and excitor-inducing impulses arrive should be important. Consequently
differences in relative conduction velocities of these two components in the two
flagella, normal and heteromorph, might produce significantly different responses. As
illustrated in Fig. 6 and Table 5, differences in conduction velocity are entirely
possible. (2) Differences in sensitivity of peripheral receptors, or in ratios of fibre
types between heteromorph and normal flagella, should result in different responses
to similar stimuli. One extreme example of this seemed to occur in lobster O-17, in
which electrical stimulation of the heteromorph was necessary to evoke flagellar
depression, presumably because insufficient peripheral receptors were stimulated by
the usual mechanical stimulation. (3) The degree of specificity need not be identical for
all afferent fibres—perhaps being greatest for the aesthetascs and associated guard and
companion hair fibres, which are normally found only on the outer flagellum, and less
for 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 beneath
extensive individual variation. Unfortunately none of these factors, although plausible
and supported by indirect evidence, have been demonstrated to act in the manner
postulated. Consequently, although general similarities are apparent, the question of
the preciseness of neuron-neuron specificity in this population of lobsters remains
incompletely resolved.
In the final analysis, however, the present observations must be considered in
relation to the lobster described in detail by Maynard & Cohen (1965). In that
individual, similarities between the excitatory responses of the heteromorph and of the
outer 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 experiments to exclude the possibility of the earlier animal being a unique or completely
fortuitous occurrence. Rather, it appears to have represented one possible condition
within the potential range of specific heteromorph responses suggested in this experiment. If this interpretation is accepted the conclusions derived from the intracellular
analysis 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 seems
indicated; and variations in heteromorph response may be supposed to result, as
proposed for individual motor neurons, from relative differences in the intensity of
the 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 thus
become accountable in terms of quantitative rather than qualitative differences.
Supported with evidence of specific movements at other antennular joints the variations remain compatible with the argument for specificity of central connexions of at
least some of the heteromorph afferents, and with the initial impression from behavioural observation of some degree of identity between the connexion patterns of
heteromorph and of flagellum.
It must be emphasized, however, that peripheral observations cannot reveal the
extent of such central identities, nor the immediate causes of individual variation which
do exist. Accordingly, all behavioural observations, and more particularly those
reported here, must ultimately remain ambiguous with respect to inferred patterns of
central connexion until more direct evidence from the systems involved have been
obtained.
SUMMARY
1. Eyestalks were removed unilaterally from forty-nine Panulirus argus. Twentythree of the forty-three animals remaining alive to the first moult regenerated a
heteromorph antennular outer flagellum in place of the amputated eye.
2. None of the lobsters moulting within less than 3 months of eyestalk ablation
regenerated a heteromorph; 70% of those moulting after more than three months
recovery regenerated a heteromorph. If the heteromorph did not appear upon the
first moult it usually continued to be missing after the second or third moult.
3. Mechanical stimulation of the heteromorph produced a general withdrawal
reaction in all lobsters tested, and in most either a specific cleaning reflex directed
toward the stimulated heteromorph, or a specific movement of the normal ipsilateral
antennule, 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 periods
ranging from one to nearly two years, and over one or two moults. No significant
changes in behavioural responses to heteromorph stimulation were detected within
that time.
5. The conduction-velocity spectrum of fibres in the heteromorph nerve resembled
that in the outer flagellar branch of the normal antennule nerve in having both a fast
and a slow component. However, the velocity of the fast component of the heteromorph nerve was often less than normal for theflagellarbranch 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 heteromorph and normal flagella elicited inhibitory and excitatory responses in the nerve
to the depressor muscle of the outer flagellum of the normal ipsilateral antennule.
7. Responses of theflagellardepressor muscle of the ipsilateral antennule to electrical
stimulation of the heteromorph varied among individuals, but in general resembled
later components of the response to similar stimulation of the normal antennular
flagellum.
106
D . M . MAYNARD
8. It is tentatively concluded that at least some of the heteromorph afferents make
specific central connexions on neuron pools normally associated with antennular
flagellum afferents. The difficulties of drawing such inferences from indirect observation are indicated.
The hospitality and co-operation of the Director and staff of the Bermuda Biological
Station permitted the maintenance of a healthy experimental lobster population for
nearly two and one-half years with minimal natural attrition. I should like to express
my 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 and
E. 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 abgeschnittenen
Augen. 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 ausgelost
werden. 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 von
Augen. 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 Antennule
bei 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 regenerated
nubbin. Compare contorted and bent form of heteromorph after first moult with heteromorph
form after second moult (Fig. 5). Calibration rectangle in F is 1 cm. long.
Fig. 5, Regenerated heteromorphflagellaafter one to four post-operative moults. A, Lobster
O-17, fourth moult; B, lobster Q-i, third moult; C, lobster Q-25, second moult; D, lobster
Q-10,firstmoult; E, lobster Q-21, second moult; F, lobster Q-14, second moult. Behavioural
responses 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
D M. MAYNARD
Plate 1
{Facing p. 106)