Autophagy in Octopus

Abstract

Automutilation, specifically of the arms, is well known in some octopod species. It occurs in two forms, autotomy and autophagy. Autotomy of an arm is achieved by breaking off at a predetermined site, or by biting off by the animal itself. Biologically, autotomy is a meaningful behaviour. It is well known, e.g. in male Argonauta during reproduction; it has also been described in several octopod species as a survival strategy. Autophagy, in contrast, is more puzzling; it is distinct from cannibalism because the animals eat (parts of) their own arms. This paper is based on 161 cases of autophagy in Octopus vulgaris. Although the data are still limited, they indicate that autophagy is not caused by hunger or stress, but is an infectious, deadly disease. Incubation time is between one and two weeks; death occurs 1–2 days after autophagy starts. Some data suggest that autophagy is caused either by a (so far unknown) substance released by the animal itself or, more likely, by viruses or bacteria; these, in turn, seem to affect the nervous system. Stress (often thought to be the reason for autophagy) may contribute to it but it is not its primary cause.

Full text

Cephalopod Biodiversity, Ecology and Evolution
Payne,
AI.
L., Lipinski, M.
R,
Clarke, M.
R
and M.
A
C. Roeleveld (Eds). S.
Afr.l
mar. Sci.
20: 101-108
1998
AUTOPHAGY IN OCTOPUS
B. U. BUDELMANN*
Automutilation, specifically of the arms, is well known in some octopod species. It occurs in two forms,
autotomy and autophagy. Autotomy of an arm is achieved by breaking off at a predetermined site, or by biting
off by the animal itself. Biologically, autotomy is a meaningful behaviour. It is well known, e.g. in male Arg-
onauta during reproduction; it has also been described in several octopod species as a survival strategy. Au-
tophagy, in contrast, is more puzzling; it is distinct from cannibalism because the animals eat (parts of) their
own arms. This paper is based on 161 cases of autophagy in Octopus vulgaris. Although the data are stilllirnited,
they indicate that autophagy is not caused by hunger or stress, but is an infectious, deadly disease. Incubation
time is between one and two weeks; death occurs 1-2 days after autophagy starts. Some data suggest that
autophagy is caused either by a (so far unknown) substance released by the animal itself or, more likely, by
viruses or bacteria; these, in turn, seem to affect the nervous system. Stress (often thought to be the reason for
autophagy) may contribute to it but it is not its primary cause.
Autophagy ("self-eating") and autotomy (voluntary
amputation
=
breaking off body parts) are well known
forms of automutilation (self-destruction). They occur
in a variety of animal species, from invertebrates, such
as echinoderms, molluscs and crustaceans (e.g.
Riggenbach 1901) to vertebrates, including man (e.g.
Meyer-Holzapfel 1968, Allyn et at. 1976, Levitt 1985,
Brown et at. 1987, Fisch 1987, Wiesenfeld-Hallin et
at. 1987, Dodman et at. 1988).
In cephalopods, automutilation, specifically of the
arms, is well known in some octopod species, and
animals with a missing, or a regenerating, much smaller,
arm are not uncommon. Whereas some of those cases
are certainly attributable to the action of predators, others
might well be due to automutilation. Automutilation of
the arms includes both autotomy and autophagy.
Autotomy of an arm is achieved either by a direct
break, which starts internally (below the skin) at a pre-
determined site, or by the animal biting off the arm
itself. Biologically, autotomy is a meaningful behaviour.
It is well known in sexually mature male Argonauta,
Tremoctopus and Ocythoe in the process of reproduction
and is frequent in Octopus defilippi. In addition, it has
been described in many other species at the end of their
larval stages, during sexual maturation and spawning,
during fights, and as a form of survival strategy when
an arm of an animal is trapped, ulcerated or otherwise
damaged (Jatta 1896, Riggenbach 1901, Lo Bianco
1909, Nesis 1987, Hanlon and Wolterding 1989, Nor-
man 1992).
In contrast, autophagy of an arm is more puzzling. It
is distinct from cannibalism because the animals eat
(parts of) their own arms. It has been reported in several
octopod species but, with one exception (Taki 1936),
only as a matter of secondary importance or in anecdotal
form: in Octopus vulgaris (Lo Bianco 1909, Taki 1936,
Boycott 1954, I. G. Gleadall, Tohoku University, Japan,
pers. comm.,
A
Mauro, Rockefeller University, New
York, pers. comm., M. Nixon, Broughton, England,
pers. comm.); O. variabilis (Taki 1936, 1941); 0. ocel-
latus (Taki 1936); O. dofleini (Reimschuessel and
Stoskopf 1990); O. bimaculoides and O. joubini
(J. B. Wood, Dalhousie University, Canada, pers.
comm.); and Eledone moschata (Lo Bianco 1909).
Remarkably, there is no report of autophagy in any of
the decapod (cuttlefish and squid) species.
Quantitative data on autophagy in Octopus vulgaris
are presented for the first time in this paper. Although
the data are still rudimentary, they allow some
comments on the behaviour itself, the number and type
of arms affected, whether autophagy is infectious, the
incubation time, the time between start of autophagy
and death, the agent that may cause autophagy, and a
possible strategy for prevention.
MATERIAL AND METHODS
The data were collected between June 1982 and
January 1985. They stem from 161 Octopus vulgaris
(males and females, 30-450 g), obtained from the
Gulf of Naples, Italy. The animals were caught by
local fisherman and brought to the Zoological Station
*
Marine Biomedical Institute and Department of Otolaryngology, University of Texas Medical Branch, Galveston, Texas 77555-1163,
USA. Email: bubudelm@utrnb.edu
Manuscript received: September 1997
101
~--

102 Cephalopod Biodiversity, Ecology and Evolution
South African Journal of Marine Science 20 1998
Fig. 1: Octopus vulgaris male (280 g) with the first right arm bitten off at its base, close
to the mouth
"Anton Dohrn" in large buckets, with several animals
sitting together in one bucket. In the laboratory, they
were housed individually in either buckets (20
£)
or
tanks (120
£)
connected to a large system of natural
seawater. Unfortunately, there is no record on whether
the seawater system ran as an "open" or a "closed"
circulation during those times. When it recirculated,
its turnaround time was between I and 2 days, with
no mechanical or other filters involved. Owing to the
design of the tanks and the connection of the buckets
to the system, the water of individual tanks/buckets
ran through
2-4
other tanks before it was either
disposed of into the Gulf, or collected in huge reservoirs
for recirculation. The animals were fed live Carcinus
every day until two days before they were transported
to Germany.
The animals were kept between
2
and
24
days in
Naples. During transport to the University of Regens-
burg, Germany, they were kept individually in small
buckets (5 £), filled with about 4
£
of seawater and
connected to an oxygen supply; the animals had neither
visual nor water contact. After arrival in Regensburg,
they were acclimatized for up to 20 h to the tempera-
ture and salinity of the local seawater system, then
transferred into individual buckets (20
£)
that were part
of a single closed system (approx. 4 000
£,
for a maxi-
mum of 80 animals) of recirculating artificial seawater.
From that moment on, the animals were in permanent
"water contact" via the recirculating water, but they
still had neither visual nor body contact. However, the
water contact was only after the water had first passed
through the entire circulation. In brief, the overflowing
surface water of each bucket passed through a small
protein skimmer, then through a biological [liter system
that included many different species of algae and
invertebrates (from protozoans to tunicates), then
through a bacterial/sand filter, and finally through a
large (2 m high, 30 ern diameter) protein skimmer.

1998
Budelmann: Autophagy in Octopus
Thereafter, it was collected in a 700
£
collection reser-
yoir, from where it was pumped (about every 30 minutes)
mto a 700
£
storage reservoir for recirculation via gravity
(for a diagram of the system, see Boletzky 1989). The
animals were kept at a water temperature of l5-18°C.
"Yater quality was monitored regularly for salinity, pH,
dissolved oxygen, ammonia nitrogen, and nitrite- and
nitrate-nitrogen. In each bucket, oxygen supply was
from a pressurized air system via wooden air stones
~.d, in addition, via the fine jet of recirculating water
injected from above the water surface. The animals
were initially fed once per day with live or frozen
(thawed) Carcinus, but later more sparsely (2-3 times
per week). Whenever animals stopped feeding, food
was offered again once a day.
As octopuses in captivity often have small skin
lesions at the tips of or along their arms (and mantle),
autophagy was recorded as such only when an arm
was actually bitten off and at least parts of it eaten,
or wh~n an arm had a large wound in its proximal
one-third to as deep as the nerve cord (i.e. the arm's
proximal part became non-functional because of the
damage to the nerve cord).
Although 161 cases of autophagy are on record,
the data for each topic discussed do not stem from all
161 animals but only from various fractions thereof
for two reasons. First, the relevant data were not
recorded for all animals. Second, many animals were
used before they died (i .e. right after they showed the
first signs of autophagy) for research on statocyst
transmitters (Auerbach and Budelmann 1986) and
neuroanatomical tracing of brain pathways (Plan
1987).
RESULTS AND DISCUSSION
The data are based on records of 161 Octopus vul-
garis from two large populations of 56 (June 1982)
and 65 animals (May 1983), and several smaller ones
(4 animals - June 1983; 5 animals - November 1983'
12 animals - May 1984; 19 animals - November
1984/January 1985). Over a period of 20 years
(1967-1987), during which the author regularly (twice
per year) transported animals from Italy to Germany
and kept them under (almost) identical conditions in
artificial seawater for several months, these were the
only Octopus populations that ever showed autophagy.
Behaviour
Some animals fed regularly until the day before
103
(f)
60 ,--
-I
-c
~
z
-c
40
u..
o
a:
w
co
~ 20
~
z
II II
2 34' 5 6 7 8
NUMBER OF AFFECTED ARMS
Fig. 2: Distribution of the number of affected arms in 84
Octopus vulgaris that displayed autophagy
autophagy started, whereas other animals stopped
feeding two days before. This, plus the fact that quite
often larger parts of the arm bitten off were not eaten
indicates that hunger does not seem to be the primary
cause of autophagy, as stated already by Lo Bianco
(1909) and Taki (1936). However, regular controls of
crop and stomach contents were not performed.
The clearest early symptom that an animal was
going to bite off one (or more) of its arms (usually
the following day) was uncoordinated, unstable and,
to various degrees, trembling arm movements. This
finding may indicate that those parts of the central
nervous system and/or of the arm nerve cords that
control arm movements are in some way affected.
When an animal actually bites off an arm, it shows
a characteristic posture. Several centimetres of the
arm bases are held close together in a stiff and stalky
way, whereas the distal ends of the arms are often
curled and do not adhere to the substratum. The arm
bitten off is so heavily bent inwards at its very base
that the site of bite off is as close to the mouth as the
anatomy allows (Fig. 1). This is different from Taki's
(1936) description that autophagy begins at the tip of
the arm and ends at its base. After being bitten off,
the arm's wound on both sides is slimy. Further, no
obvious contraction of the muscular skin can be seen
that may at least partly close the wound (as is known
in healthy animals after surgical procedures).
Taki (1936). reports that no bleeding occurs during
autophagy. This, however, seems doubtful, because in
all those cases where autophagy was in progress, or
had taken place, large quantities of protein foam were

104 Cephalopod Biodiversity, Ecology and Evolution
South African Journal of Marine Science 20
1998
4
-
-
r--
I-
.--
r:--
r-- r--
r-- r--
-
I"'"
'L
I:>L'1)
24
20
(J)
....J
-c
~ 16
Z
-c
u,
o
12
a:
UJ
CD
~
~ 8
4
3
2
2
3
4
Fig. 3: Distribution by left or right affected arm (n
=
115) of the same 84 Octopus vulgaris from which Figure 2
was constructed
found accumulated on the water surface of the respec-
tive tanks as a result of the very tine air bubbles of the
water jet inflow and of the air stone. At the same time,
no foam was seen in any of the other tanks (with animals
not undergoing autophagy) connected to the same sea-
water system. Therefore, unexpected foam bubbles on
the water surface of single tanks are another indicator,
besides the animal's posture, that autophagy might be
in progress, or that an arm has already been bitten off.
Whether the animals finally die from blood loss, how-
ever, is not known.
Number and type of arms affected
For 84 animals, a record was kept of the number
of and which arms were affected by autophagy. In
most cases (62
=
74%) it was only one arm, in some
cases (16
=
19%) it was two, and only occasionally it
was three (3
=
3.5%) or four (3
=
3.5%). There were
no cases with more than four arms affected (Fig. 2).
However, Wood (pers. comm.) had an animal
(0.
bi-
maculoides) in his laboratory that had bitten off six
arms.

1998
Budelmann: Autophagy in Octopus 105
30 a)
n-=
108
Octopus vulgaris
26
D
n
1
=
57
(June
1982)
22
•n2
=
51 (May 1983)
18
C/)
...J
14
«
~
z
10
«
LL
0
6
a::
w
co
2
~
~
z
b)
D
n
=
5 (7
months)
3
•n
=
8 (6
months)
2
14 16 18 20
Fig. 4: Incubation time of autophagy in Octopus vulgaris - (a) number of days between the date the animals
were transferred into a closed system of recirculating seawater and the start of autophagy; (b) number
of days between the arrival of fresh animals in the closed system of recirculating seawater and the start
of autophagy in animals that already had been in that seawater system for 6 or 7 months
2
46
8 10 12
NUMBER OF DAYS
As mentioned by Taki (1936), there is no clear pref-
erence as to whether left or right arms were affected
(Fig. 3). The first left arm (22 cases
=
26%) might be
more often involved than any of the other seven (Fig. 3),
but the number of cases is still too few to make this a
significant statement
(X
2
goodness-of-fit test;
X2
=
7.65,
df= 7,p
>
0.30).
Is autophagy infectious?
On the first two occasions when autophagy occurred
(June 1982 and May 1983), the entire populations
(57 and 51 animals respectively) were affected and all
ultimately died. At the same time, two small popula-
tions that had been in the Regensburg seawater system
already for respectively six (5 animals) and seven
(8 animals) months prior to the arrival of the "new"
animals were affected and died. These cases indicate
that autophagy is able to spread from one animal to the
next and, therefore, is infectious. At the times when
autophagy happened, the physico-chemical properties
of the seawater did not change, neither prior to auto-
phagy nor when it actually occurred. This, and in
particular the fact that the two established populations
became infected, makes it very likely that an agent was
introduced into the seawater circulation by one, or
several, of the new arrivals. This would then have been
distributed by the recirculating water throughout the
system, infecting all the other octopuses, including
those already established in the system for several
months.
During the four later observations of autophagy
(June 1983-November 1984/January 1985), not all the

106 1998 •Cephalopod Biodiversity, Ecology and Evolution
South
African
Journal of Marine Science 20
40
-
en
...J
-c
~
30
z
-c
u..
0
20
a:
w
CD
~
:::J
10
z
234
NUMBER OF DAYS
5
Fig. 5: Number of days between the onset of autophagy
and death of 50 Octopus vulgaris
octopuses were affected, presumably because of the
preventive measures that were taken at those times (see
below).
Incubation time
In each of the two large populations (June 1982,
57 animals; May 1983,51 animals), the first cases of
autophagy were noted six days after the animals
were transferred into the closed seawater system in
Regensburg (Fig. 4a). Thereafter, the number of
cases increased steadily to a maximum of 30 cases at
Day 9 before decreasing again, with the last case at
Day 17 (Fig. 4a).
A similar time-frame was observed for the two
established populations (8 and 5 animals) that had
already been in the Regensburg seawater system for
respectively six and seven months. In those two popu-
lations, the first cases of autophagy were noted
respectively 5 and 7 days after arrival of (and possible
infection by) the new animals from Naples; the last
case was again at Day 17 (Fig. 4b).
From the observations that autophagy took between
5-6 and 17 days in a closed seawater system to manifest
itself (Fig. 4), it may be concluded that the disease has
an incubation time of approximately 1- 2 weeks. How-
ever, there were some exceptions from this timing. In
the other four arriving populations (40 animals
between June 1983 and November
1984/January
1985), 11 cases of autophagy were recorded 1-5 days
after transfer of the animals into the closed seawater
system in Regensburg (1 day, 1 case; 2 days, 2 cases;
3 days, 2 cases; 4 days, 3 cases; 5 days, 3 cases). Nev-
ertheless, all these cases can still be explained in terms
of a 1-2 week incubation time because 6-16 days be-
fore autophagy started in Regensburg these
animals were in physical contact with other animals
when they were brought into the Zoological Station in
Naples. In the same four arriving populations, there
were an additional six cases of autophagy 18-26 days
after arrival, and another five cases 57 - 84 days after
arrival and transfer into the closed seawater system
in Regensburg. These extended incubation times are
more difficult to explain. One possibility is that the
agent that caused autophagy survived in the seawater
system for those extended periods of time (because
of the preventive actions [see below] that were taken
during those times) and only later became infectious.
Another possibility is that the infection took place
but was initially "subthreshold" with regard to the
symptom, i.e. biting off an arm.
Time between start of autophagy and death
Of the 50 cases recorded, all animals died within
1-5 days after the onset of autophagy. Of those, 80%
died within one day, 10% after two days, and the
remaining 10% within 3-5 days (Fig. 5). This agrees
with Taki's (1936) note that the animals die "soon after
autophagy occurs" and the two cases reported by
Reimschuessel and Stoskopf (1990) where the animals
died within 1-2 days.
What is the agent that causes autophagy?
At present, the data do not allow a clear answer to
that question. The data show, however, that the agent
can be transmitted through a closed seawater system
and is able to bypass biological and sand filters. Possi-
ble candidates for the agent are a chemical substance
that is released by the animals themselves or, more
likely, because of the 1-2 week incubation time,
mi-
croorganisms such as viruses or bacteria.
Are there morphological changes of the arm nerve
cord and/or the central nervous system associated
with autophagy?
The brains of several of the infected animals were
processed for a neuroanatomical study of brain path-
ways (Plan 1987). At gross morphological and light
microscopical levels, no obvious changes or anomalies
in the structure of any of the brain lobes were seen
(bearing in mind the different focus of that study). Un-

.1998
107Budelmann: Autophagy in Octopus
fortunately, no records exist on the histopathology of
the arm nerve cords, e.g. on inflammations and oede-
mas, as have been described by Reimschuessel and
Stoskopf (1990) as what they called "automutilation
syndrome of the arms" (without proof that lesions of
1
the skin and the missing suckers and arm tips were due
to a self-mutilation and not simply to mechanical or
other damage associated with secondary infection). To
, research autophagy more fully, a more thorough mor-
phological investigation and bacterial culture and viral
testing are certainly needed.
Strategy for prevention
After having lost two entire populations of octopuses
in the Regensburg facility (June 1982 and May 1983),
two preventive measures were taken:
(i) before transfer into the closed seawater system,
as many animals as possible (for technical reasons
usually not all) were kept in quarantine for
between two and three weeks;
(ii) the protein skimmers were set at high perfor-
mance for the first 2-3 weeks; that way, about
5% (200
£)
of the seawater was eliminated from
the 4 000
£
circulation as "wet foam" and
replaced by fresh artificial seawater every day.
Although there were no detailed studies on the ef-
fectiveness of those measures, what was clear was that
there were fewer cases of autophagy after the preven-
tive measures and on no occasion were all animals af-
fected. It is also possible that some animals were, or
became, immune against the disease.
CONCLUSIONS
Although some data are still rudimentary and not
yet conclusive, they indicate that:
• autophagy is a disease that can cause death of an
entire population of animals in a closed seawater
system, i.e. it can be infectious;
there is no preference as to which arm is affected;
the agent that causes the disease might either be a
substance that is released by the animals them-
selves or, more likely, a microorganism (e.g. a
bacterium or virus);
the agent can be transmitted through a closed system
of recirculating seawater and is able to bypass
biological and sand filters;
the incubation time is 1-2 weeks;
the agent seems to affect the nervous system that
controls movement of the arms;
the infected animal usually dies within 1 or 2 days;
high-performance protein skimming might be a
way to impede, or-possibly prevent, the agent's
transmission in a closed seawater system.
Stress and hunger, which are often assumed to
cause autophagy, are obviously not its primary cause.
Whether they contribute to it, e.g. by lowering the
threshold for the infection, or the expression of the
symptoms, needs to be researched further.
ACKNOWLEDGEMENTS
The data were collected during the years
1982-1985, when I was affiliated to the Zoological In-
stitute of the University of Regensburg, Germany. I
therefore acknowledge grant support of the Deutsche
Forschungsgemeinschaft (Bu 404/1- 3 and SFB
41
A2),
and thank the Director and Staff of the Zoological
Station in Naples for their hospitality and help with the
animal supply. I also thank Mr
1.
B. Wood and two
anonymous reviewers for critical comments on some
topics of the manuscript and Dr I. G. Gleadall for helpful
comments on some literature sources. My participation
at the CIAC '97 conference was made possible by the
Marine Medicine Budget of The Marine Biomedical
Institute of The University of Texas Medical Branch at
Galveston.
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