ABSTRACT
Free-swimming Sepioteuthis lessoniana hatchlings were
videotaped during both feeding and strobe flash sessions. Analysis
showed that while feeding the peak instantaneous velocity obtained
by the hatchling is twice as great and the peak acceleration is 50%
greater than during an escape response to a strobe flash in which
only the giant axon is used. Behavioral measurements of mantle
radius made with video were used to compute jet thrust and compare
performance with varying patterns of motor neuron activity in the
restrained hatchling. The coordinated, adultlike pattern of giant and
small fiber use resulted in mantle contractions and jet thrusts that
were always greater than contractions and thrusts produced by
giant-only or small-only responses. Thus, evidence strongly
suggests that hatchling Sepioteuthis use the giant fiber in feeding
attacks.
INTRODUCTION
The squid neural system is comprised of two motor pathways,
one slow and one fast, that can interact in complex ways. The fast
pathway originates in the brain from two first order giant cells
which make contact with seven second order giant neuons. One
second-order axon projects to the stellate ganglion and synapses
there with the third order giant axons that innervate mantle muscles
associated with rapid escape responses. The slow pathway runs
basically in parallel, starting from the median basal lobe of the
brain, connecting with the pallio-visceral lobe, and then continuing
on to the stellate ganglion via the pallial nerve. From the stellate
ganglion, the slow motor axons emerge, with the third-order giant
axons, in the stellar nerves and synapse on circular mantle muscles
that are slow to fatigue (Mackie 1990; Otis and Gilly 1990).
A major advantage of such a complicated system lies in the
the ability to use the two pathways separately or in concert. The
giant axons, with much larger diameters (280-718 um in Loligo
forbesi ) than the small axons (40-50 um), also have greater
conduction velocities (15.0-22.3 m/s vs 3.1-5.6 m/s) (Pumphrey and
Young 1938).
The two pathways can work together in a coordinated manner,
and two electrophysiological escape response patterns have been
identified, one for adults and one for embryos. The adult, or
delayed-type, pattern shows multiple cycles of small motor activity
at long latency (300-500 ms) with the giant spike(s) present, if
occurring at all, in the middle of bursts of small motor axon spikes
(Otis and Gilly, 1990). The embryonic, or fast-start, pattern
characteristically shows a single giant spike preceding any small
motor activity in the same cycle, and occurs at short latency (-50
ms) (Gilly et al. 1991). The embryonic pattern is retained by the
hatchling, and within a few weeks, the adult pattern develops. Äfter
the change to the adult pattern, the fast-start pattern of giant axon
firing is seen only following sudden visual stimulation (Otis and
Gilly, 1990).
The factors responsible for the progressive development from
one system to the other are currently unknown. However, one
function that may require the use of the adultlike motor patterns is
prey capturing. Sepioteuthis hatchlings develop the ability to feed
on live prey as early as 4 days posthatching (Hanlon, 1990), and
precise timing of a strike would be advantageous in capturing a
rapidly moving prey item, such as a copepod or larval fish. Thus, it
is of interest to compare in more detail the time course of evolution
of the adult-like useage of the giant fiber system with the
development of feeding behavior. Additionally, it is possible that
the attacks made on live prey may positively reinforce the adult
pattern of giant fiber utilization or even be necessary for the
changeover. As a first step in exploring these ideas, it must be
ascertained whether or not the giant axons are used in feeding by
hatchling squid.
MATERIALS AND METHODS
Animals
Late-stage Sepioteuthis lessoniana eggs were obtained from
the Marine Biomedical Institute, University of Texas Medical Branch,
Galveston, and kept in circular plastic tubs (28 cm in diameter, 15
cm deep) in an aquatron (100 gallon tank with 50 gallon reserve)
with gently circulating seawater (22-23 °0). The tubs had two
Nytex (0.4 x 0.4 mm pores) panels in the sides and one in the bottom
to keep water circulating in the tubs. Each morning, hatchlings born
overnight were put in one group and transferred by beaker to either
another plastic tub or the open aquatron, so that animals of
different ages were separated from each other. Groups of animals
were arbitrarily chosen to be "fed" or "unfed". Fed animals were
kept in the open portion of the aquatron and were given many small
mysid or Gambusia fry immediately after transfer and then at
intervals of approximately twelve hours; unfed animals were
maintained in the plastic tubs.
Experiments
Free-swimming escape responses to strobe flashes and feeding
sessions of "fed" hatchlings were videotaped from the side of the
tank using a Canon Al videocamera set at 1/250 shutter speed.
Strobe flashes were made with a Nikon Speedlight SB-15 camera
flash. Data were analyzed with a Megavision image analysis system
that can store 28 real-time frames (33 ms per frame) and replay
them in digitized form.
For electrophysiological recordings, an adapted version of a
procedure developed by Ötis and Gilly (1990) was used. Hatchlings
were first anaesthetized in 0.5% urethane, and the dorsal side of the
mantle was glued, with cyanoacrylate cement, to a 30-gauge needle
fitted with Tygon tubing. The animals were suspended in a small
aquarium with seawater and gently bubbling Ö2. The skin and muscle
overlying the left stellate ganglion were surgically removed, and a
polyethylene suction electrode was placed on the interface between
the stellate ganglion and the second-hindmost stellar nerve.
Electric stimulation was applied by two separate electrodes, one to
the pallial nerve and one to the head region of the hatchling.
Behavioral data were recorded by vidoetaping the profile of the
restrained animal with a Canon A1 or LI Hi-8 Camcorder on 1/250
exposure, and data were analyzed by the Megavision. Electrical data
were either digitized and stored on a videotape or were stored on
the audio track of the behavioral videotape.
Calculations
In the videotapes of the free-swimming animal, the Megavision
system was used to collect the x,y coordinates of the eye in
sequential frames. The calculated distance that the squid's eye
moved in sequential frames was used to measure the instantaneous
velocity. Acceleration is the derivative of the velocity: dy/dt.
Experiments were normalized for comparison by dividing all values
of velocity and acceleration by the body length of the squid, which
was the average measured distance from the squid's eye to its tail
in the frames used for data collection.
Videotapes of the restrained animal were used to measure the
change in radius (r) of the mantle over sequential frames. Volume
(Vol) is proportional to r2 for a cylindrical or conical object. The
thrust of the water jet produced by the mantle contraction can be
calculated based on equations provided by Ö'Dor, Foy, and Helm
(1986), and is proportional to (dVol/dt)2.
RESULTS
Instantanoeus velocity and acceleration during feeding and free¬
swimming escape responses to strobe flashes
Strobe-driven escape responses in free-swimming
Sepioteuthis hatchlings are very stereotyped, like the case in adult
or hatchling Loligo (Otis and Gilly, 1990; Gilly, et al, 1991). In an
experiment with an 11 day-old animal that had been actively
feeding, the maximum instantaneous velocity reliably occurred
between the between the second and third frame (33 ms per frame)
after the strobe flash and had a value ranging from 0.45-0.56 body
lengths/frame and corresponding accelerations of 0.37-0.44 body
legths/ framee. Examples of such responses are indicated by the
open symbols in Figs. 1A, B, respectively. Similar responses were
also obtained in younger squid (3-8 days; data not illustrated).
During an attack on a small fish, the same squid displayed a
maximum velocity of 1.1 body lengths/frame and a peak acceleration
of 0.57 body lengths/frame2. Thus, the feeding attack of a hatchling
squid can be substantially more rapid than an escape resoponse to a
strobe flash. Based on the published work on Loligo (Otis and Gilly,
1990; Gilly et al, 1991) and on the data on Sepioteuthis described
below, it is assumed that these escape responses are largely, or
entirely, diven by a single giant axon spike and that the small motor
axon system plays a negligible role. The significantly greater values
of both peak velocity and acceleration during the feeding episode
suggest that the giant axon may also be utilized during this non¬
escape behavior. In order to pursue this idea, the relationship
between motor activity and jetting performance was investigated in
restrained hatchlings.
Electrophysiological recordings in conjunction with behavior in the
restrained hatchling
Electrophysiological recordings in restrained animals have
shown that escape responses to strobe flashes give a distinctive
motor discharge in the stellar nerves in both adult, hatchling, and
late-stage embryonic Loligo opalescens (Otis and Gilly, 1990; Gilly
et al, 1991). In "simple" escape responses of this type, the giant
axon fires first at brief latency (25-50 ms) and may be followed by
small unit activity of unidentified origin. Firing of the giant is
always accompanied by a sudden jetting reaction. When the giant
does not fire in response to the strobe stimulus, no mantle response
is detectable with video observations, even though some small
motor activity may occur. Sepioteuthis lessoniana hatchlings
displayed similar behavior in the present study.
Just supra-threshold electrical stimulation of the pallial
nerve in a restrained 11 day-old squid (same animal as in behavioral
experiment of Fig. 1) results in the fairly selective firing of the
giant fiber with a delay of approximately 1 ms, as shown in Fig. 2. A
strobe flash stimulus (Fig. 3) also triggers a giant axon spike but at
a longer delay (20 ms). The recordings in both Figs. 2 and 3 also
show some small unit activity of uncertain origin following the
giant spike. If strobe stimulation fails to trigger a giant spike,
there is no observable mantle response. In the case of pallial nerve
stimulation, proper positioning of the stimulating electrode can
yield a similar pattern, but often weak mantle responses
(determined by simple visual observation) can be elicited without
activation of the giant axons. In either case, pallial nerve
stimulation was a reliable means of providing a "template" for the
waveform and amplitude of the giant spike to aid in its
identification during more complex motor discharges and in
obtaining an indication of the mantle response due to preferential
activation of the giant axon.
Recordings of mantle dynamics and the underlying motor
patterns in the stellar nerve were also obtained from a 4 day-old
restrained hatchling that had not been fed (Fig. 4). A 5 V shock to
the pallial nerve was delivered, and this resulted in a single, abrupt
jet response. Firing of the giant axon within one ms after the shock
(Fig. 4A) leads to the onset of mantle contraction within one frame
(Fig. 4B), and the mantle contraction reaches peak response (the
smallest diameter) by 2-3 frames. The rapid onset of this mantle
response and its temporal relationship to the giant spike are thus
very similar to the results discussed above in conjunction with
strobe flash stimuli. Pallial nerve stimulation appears to produce a
mantle reponse that is longer lasting than that due to a strobe
stimulation (compare Figs. 4B and 1A). However, this may be due to
secondary small unit activity that accurs with a sizeable delay only
after the nerve shock. Such late activity is apparent in Fig. 2A.
Shocks applied to the surface of the head of the same 4 day-
old squid give behavioral and neural responses that are more
complex than either pallial nerve shock or strobe stimulation. A
single 20 V shock to the mouth during frame zero results in the
behavioral data shown in Fig. 5A, and the corresponding stellar nerve
activity is shown in Fig. 5B. Expanded traces for each of the 3
jetting cycles are shown in Fig. 50. The short latency for the first
cycle of neural activity (30 ms) is characteristic of hatchling and
embryonic squid (Gilly et al, 1991) and is not seen with adult
animals (Otis and Gilly, 1990).
From examination of records like those in Fig. 5, it appears
that the most powerful jetting responses are those that involve the
coordinated ("adult-like") use of the giant and non-giant motor
pathways. Thus, contractions associated with small-unit activity
alone (Fig 5, cycles 1 and 2) are not as powerful as cycle 3, although
they are definitely stronger than the contraction due to a normal
breathing cycle (prior to frame zero in Fig. 5A). Finally, a
contraction due to the firing of a short-latency, embryonic-type
giant spike acting more or less alone (e.g., due to the pallial nerve
stimulation) is at best equal to the contraction due to small motor
axon activity alone. This can be seen by comparing behavioal data
from Fig. 4B with cycle 1 in Fig. 5 (filled symbols in Fig. 5A). Thus,
a giant axon spike does not necessarily lead to a marked increase in
jetting performance over that provided by the non-giant system. The
boosting effect of the giant axon only comes about when its
activation is properly timed in relation to the burst of non-giant
activity.
Differences in the level of behavioral performances achieved
in jetting cycles invoking coordinated discharge of giant and non¬
giant motor units and in those cycles generated by small activity
alone can also be more subtle than indicated by the data in Fig. 5.
Fig. 6 shows the time course of the mantle radius and the
corresponding stellar nerve recording of the same 4 day-old unfed
squid (as in Fig. 5) in response to a 30 V shock to the mouth. Again,
the motor discharge displays a combination of embryonic and adult
patterns: An embryonic, giant-type occurs at short latency before
the burst of small unit activity in the first cycle; small activity
only is present in the second cycle; the adultlike-coordinated
giant/non-giant discharge exists in cycles 3 and 4. In Fig. 6A, the
corresponding measurements of the mantle radius do not show
marked differences in speed or degree of contraction. However,
when the corresponding thrust of the water jet (calculated as
(dVol/dt)2, where volume is proportional to the mantle radiuse
(O'Dor, Foy, and Helm, 1986)) produced by each mantle contraction is
compared in Fig. 7, there is a notable correlation between the type
of motor activity and the strength of the jet thrust for each cycle:
the thrust produced by the embryonic giant (cycle 1) is on the same
order as, but slightly greater than, the thrust from the small motor
activity alone (cycle 2); adult-like cycles 3 and 4, where the giants
fall in the middle of small unit firing, are associated with jet
thrusts considerably larger than those of cycles 1 and 2.
DISCUSSION
The higher values of instantaneous velocity and acceleration
in the feeding hatchlings compared with the giant-only escape
response produced by a strobe flash seem to indicate that, in order
to produce the extra burst of speed, the giant and the small motor
systems must be working together to produce an added behavioral
effect. In this experiment, behavioral and electrophysiological work
on restrained hatchlings shows that the degree of mantle
contraction and jet thrust produced are related to the motor
patterns of escape response elicited by the hatchling.
In considering mantle radii, all escape responses produce
stronger contractions than those made during a normal breathing
cycle. Small motor activity can produce a contraction as strong, if
not stronger than the giant acting alone (overlay, Figure 5A). Small
motor activity also gives weaker contractions than when both the
small and the giant systems are being used in conjunction (Figures
5A and 6A). While Figure 6A shows that the contraction caused by
small activity only is almost equivalent to other cycles in which the
giants are involved, it is worth noting that the lowest point in cycle
2 is indicated by a single point, whereas the other cycles have
multiple points defining the full contraction. The difference in the
position of this low point and the points clustered at a higher level
in this contraction could easily be due to noise in sampling
measurements.
While embryonic and adultlike giant spikes show almost no
difference in the degree of mantle contraction, the effect of each
response pattern is made apparent when jet thrust is calculated.
The jet thrust resulting from the embryonic giant is only about half
of the thrust produced by a single adultlike giant spike. Thus, the
presence of a giant acting with small motor activity does not
necessarily imply that an added behavioral effect will be produced.
Instead, the position of the giant spike seems to be the factor that
determines if the effects from the giant and the small systems will
add or not: a giant fired at long latency, in the middle of a burst of
small motor activity will give a summed effect, while a short
latency giant that preceeds small motor activity produces behavior
that is similar to small motor activity acting alone. Also, the
summed effect of the adultlike pattern of giant fiber use
consistently produces mantle contractions that are larger than that
of the giant alone or the small fiber alone. This would seem to
indicate that, in order to attain the high velocity and acceleration
seen in feeding, the hatchlings must be using the giant in an
adultlike manner.
The effect of feeding vs. not feeding on the degree of mantle
contraction and jet thrust is yet to be determined. Both fed and
unfed squid showed combinations of the adult and embryonic
patterns at the age of 4 days. If animals were in the "fed" category,
the adultlike response would predominate by the age of 8 days; if
"unfed", the hatchling exhibited only embryonic behavior by as early
as 6 days old, though they did not survive or were not kept alive
beyond 7 days. This might indicate that adultlike-use of the giant
fiber must be exercised through an activity such as feeding,
otherwise the ability to use the adult pattern is lost once the
hatchling is past 4-5 days old. This also supports the idea that the
giant fiber is used in an adultlike manner for feeding.
Future work may focus on obtaining more video data on
hatchling feeding. Due to the difficulty in videotaping the feeding
attacks of the Sepioteuthis hatchlings, only a small number of
sequences were of use for analysis. The feeding attack/capture
sequence of an 11 day-old Sepioteuthis hatchling seemed to be
representative of the feeding attacks made by other 7-8 day-old
hatchlings, but this is not certain. At the present time, electrical
recordings from free-swimming hatchlings are not possible.
Therefore, in order to assess whether the adultlike giant is actually
being used in feeding, the behavioral data from the restrained
animals must be converted to values of free-swimming acceleration
and velocity via a mathematical model. Once it is known whether or
not the giant fiber is used in an adultlike manner during feeding, it
may be determined whether the the development of prey-catching
ability is actually responsible for the establishment of the adult
pattern of escape response.
ACKNOWLEDGEMENTS
Many thanks go to Professor Gilly, whose patience, advice,
wisdom, and humor were much appreciated; without his help this
project would never have been accomplished. I am also indebted to
the following people: Professor Baxter for providing the video
equipment, Roger Hanlon and the University of Texas for supplying
the squid eggs, Bruce Hopkins for advice and help with setting up
equipment for experiments, Professor Denny for his infinite
knowledge of energetics, and Professor Thompson, director of
Megavision Studios. I would also like to thank the 1991 Biology
175H dudes for their support and friendship.
LITERATURE CITED
Gilly, W. F Hopkins, Bruce, and Mackie, G. O. 1991. Development of
giant motor axons and neural control of escape responses in
squid embryos and hatchlings. Bio. Bull. 180: 209-220.
Hanlon, Roger T. 1990. Maintenance, Rearing, and Culture of
Teuthoid and Sepioid Squids. Squid as Experimental Animals.
Plenum Press, New York. 35-62.
Mackie, G.O. 1988. Giant axons and control of jetting in the squid
Loligo and the jellyfish Aglantha . Can. J. Zool. 68: 799-805.
O'Dor, R. K., Foy, E. A., and Helm, P.L. 1986. The locomotion and
energetics of hatchling squid, Illex illecebrosus. Amer. Malac.
Bull. 4: 55-60.
Otis,
Thomas S. and Gilly, W. F. 1990. Jet-propelled escape in the
squid Loligo opalescens: concerted control by giant and non¬
giant motor axon pathways. Proc. Natl. Acad. Sci. USA. 87:
2911-2915.
Pumphrey, R. J. and Young, J. Z. 1938. The rates of conduction of
nerve fibres of various diameters in cephalopods. J. Exp. Biol.
84: 303-318.
FIGURE LEGEND
Figure 1A: Instantaneous velocity of an 11 day-old hatchling during
a feeding attack and during an escape to a strobe flash
Figure 1B: Acceleration of the feeding attack and escape to the
strobe
Figure 2A: Electrical recording of an 11 day old, "fed" hatchling in
response to a 5 V pallial nerve shock
Figure 2B: Same as 2A on an expanded timescale
Figure 3:
Electrical recording of the restrained, 11 day-old, "fed'
hatchling escape to a strobe flash
Figure 4A: Oscilliscope trace of a 5V shock to the pallial nerve of a
4 day-old unfed hatchling
Figure 4B: Corresponding mantle radius, before, during, and after the
shock (frame zero) described in Figure 44
Figure 5A: Mantle radius of 4 day, "unfed" hatchling before, during
and after a 20 V shock to the mouth at frame zero
Figure 5B:
Electrical recording of the hatchling's reaction to the
shock
Figure 50: Cycles 1, 2, and 3 from figure 5B shown on an expanded
timescale
Figure 6A: Mantle radius and electrical recording of same 4
day, "unfed" hatchling before, during, and after a 30 V
shock to the mouth at frame zero. This shock was given
approximately 1/2 hour after the shock corresponding
with Fig. 5
Figure 6B: Expanded timescale of cycles 1-4 in Figure 6A
Figure 7:
Calculated thrust of the water jet corresponding with Fig.
6A,B
INSTANTANEOUS VELOCITY DURING
FEEDING AND ESCAPE
121
0
08
—0— Strobe
06
— — Feeding
0.4
0.2


og
0.0
-10
Frame
Figure 1A
ACCELERATION DURING FEEDING AND ESCAPE
0.6
0.4
0.2
00
—0— Strobe


— —— Feeding
-0.2
-0.4
-0.6 +
Frame
Figure 1B
50
—
Pallial Stimulation

10 ms
Figure 2A

4 ms
Figure 28
Light Flash
10 ma
Figure 3
60 -
50
40
Figure 4A
Radius of hatchling during
pallial nerve stimulation

20 40
60 80
-20 0
Frame
Figure 48
500
RADIUS OF HATCHLING DURING ESCAPE JETTING
60 -

50 -
Figure 5A

40

-30 -20 -100 10 20 30 40 50 60 70
Frame

Figure 5B
3

30 ms
Figure 5c
2 effrp
THRUST OF WATER JET
8o0000
600000
0000
ooo00
RR R
-20
60 80
Frame
Figure 7
RADIUS OF HATCHLING DURING ESCAPE JETTING
60





50

40

30 +
—

-20
40
20
60
80
Frame
Figure 6A
—
o
3
30 ms
2  ygukpe
4 —r
Figure 6B