Characterization of Applicable

Bio-activities in Octopus
vulgaris Saliva
Nirit Abras, Tali Almon, Yael Goldberg, Morani Landau, Tom Salman
Supervisors: Dr. Nir Nesher, Dr. Tal Shomrat

School of Marine Sciences, Ruppin Academic

Center
2015/16

Objective
To conduct a broad survey revealing octopus saliva properties and
bio-activities with applicable potential

Table of Contents
Abstract.................................................................................................... 3
Introduction.............................................................................................. 4
Materials and methods.............................................................................6
Results.................................................................................................... 10
Conclusions............................................................................................ 18
Bibliography........................................................................................... 23

Abstract
Octopus vulgaris is a well-developed, opportunistic predator that
feeds on an eclectic pool of prey including crabs, bivalves,
gastropods and various species of bony fish (Anderson and Mather,
2007; Wood, 1963). These organisms display defense mechanisms
which present the octopus with a number of challenges, whether
these require overpowering the prey, external digestion of prey due
to its size, or a form of self-defense. Furthermore, proceeding prey
capture, the octopus immediately attaches it to its mouth. From this
observation we deduced that the saliva plays an important role in
the hunting and feeding process, and may hold beneficial biological
uses (Grisley and Boyle, 1987). In this project we conducted a broad
survey of the saliva's properties, while using a variety of bio-assays
in order to characterize potentially useful bioactivities including
neuroactive, degenerative and antibacterial activities. Paralytic
properties of the saliva were tested and found to be effective by
injection to Blowfly larvae (ED50, 0.676g/(ml*mg larvae)). We
strengthened this result by testing the salivas effect on a
vertebrate model of Zebrafish larvae. This test too, showed positive
anesthetic like effects, which may hint at a potentially effective
anesthetic. Proteolytic properties were examined and the saliva was
found to degrade proteins. The saliva comprises of both soluble and
insoluble substances, while the insoluble fraction comprises mostly
of a gel like substance. We compared the effect of the saliva's
insoluble fraction on human erythrocytes to that of the soluble
fraction. The insoluble fraction demonstrated protective properties
of the cells by adsorbing them, and consequently reducing
hemolytic activity.
Saliva anti-fouling activity was tested on different surfaces and
found to be effective through two different mechanisms; the soluble
fraction prevented fouling accumulation from the beginning, while
the insoluble fraction initially adsorbed the fouling, and later when
3

peeled, it left a clean surface. Finally, saliva antibacterial properties

were tested on 12 Escherichia coli mutant bacteria and the marine
bacteria Photobacterium leiognthi. The saliva was found to be
effective on some of the bacterial strains, implying a potentially
effective antibiotic activity. Through our experiments, we discovered
an array of saliva functionalities which present great applicable
potential.

Introduction
As humans, we are constantly seeking possible solutions to improve
our quality of life. These include the search for medical and
pharmaceutical agents, such as antibiotics, anesthetics and useful
enzymes. It is a known fact that there is a growing need for
discovering new antibiotics, as bacteria are developing resistance to
the existing ones, and new epidemics are being introduced to the
human

population

(Livermore,

2004).

Another

aspect

for

improvement is agricultural yield, including the discovery of possible

new natural pesticide compounds which are not harmful to humans
and are environmentally friendly. Marine form of transport has
grown in the last few decades, and with it the demand for new
antifouling agents that are nontoxic for the environment. Marine
biofouling is an undesirable accumulation of microorganisms, algae
and other organisms, which can lead to bio-deterioration in marine
vessels and other equipment. The existing antifouling agents have
proven to contaminate the food chain, to be highly toxic for many
aquatic

organisms,

environmentally

and

friendly

are

nontoxic

nondegradable.
agents

are

highly

Therefore
required

(Marechal and Hellio, 2009).

The oceans conceal many undiscovered and potentially beneficial
compounds within them. It is in our interest to explore this
untouched natural source, specifically through the O.vulgaris saliva

which may encompass many properties of interest and are evident

through distinct behavior and physical attributes.
The octopus is a cephalopod mollusk composed of soft tissue. The
esophagus of the octopus, as in many other invertebrates, is a thin
tube passing through the brain, limiting the expansion of the former
(see Fig. 1). The large paired salivary glands in the octopus lie within
the body cavity posterior to the head, but deliver their secretion
forward through a single duct which terminates within the buccal
cavity in a muscular salivary papilla (Nixon, 1984).

Fig. 1. Diagram of the octopuss gut from a dorsal viewpoint. The organs
inside the octopuss gut are shown with a focus on the brain (orange), posterior
salivary glands (pink), esophagus (green) and crop (blue). (Wells, 1978). Note the
esophagus- a thin tube passing through the brain.

O.vulgaris is an opportunistic superior predator, with an immense

prey diversity that requires many hunting tactics and methods. The
octopus preys on organisms of diverse nature, some being relatively

large, tough-shelled, or bonny. For example: fish, crabs and other

mollusks which consist of bones, carapaces, shells, etc.
Due to the physiology and diet described above, the octopus is
faced with a number of challenges while feeding: The exposure to
possible injuries which may occur while hunting (such as arm loss),
the inaccessibility of the prey's meat, since it's often attached to
inedible tissue (i.e. carapace and shells), and the passage of the
feed through the octopus's size limited esophagus.
While feeding, the Octopus is known to externally digest the
arthrodial membrane of its prey and detach the muscle from the
skeleton of the crab, by disintegrating its joints and dissolving the
muscles (Nixon, 1984). This allows the feed to pass through the
narrow space of the esophagus (personal communication with N.
Nesher and T. Shomrat). This idea was also contemplated by (Nixon,
1984) as to the saliva's important role in external octopus's
digestion. This behavior points to the option that the saliva contains
digestive substances such as proteolytic enzyme.
While hunting, the octopus ensures its safety initially by attaching
the prey to its mouth which, in turn, leads to prey neutralization and
to the beginning of the feeding process. This behavior suggests that
the octopus uses its saliva in order to paralyze or tranquilize the
prey (Cariello and Zanetti, 1977; Grisley and Boyle, 1987).
The octopus observed tendency to insert its arm into its mouth
following amputation, along with the knowledge we have about antiseptic characteristics of the saliva in other species (Dawes et al,
2015), lead us to the assumption that the objective here is an
attempt at fighting and preventing infections by using its saliva.
The saliva was shown to contain a gel like substance in addition to
the liquid, while excreted into a shell through a particular form of
milking (personal communication with N. Nesher and T. Shomrat).
According to Gennaro et al, 1965, it was concluded that the gel
property of saliva plays a role in enhancing toxicity of the venomous
6

saliva, although in and of itself is not toxic. Based on the above we

assume the gel like component of the octopus saliva to enhance
existing activity.
The octopus anatomy along with its' characteristic behavior,
suggest that the saliva contains unique properties of which are
potentially applicable for human use. Therefore our goal is to
conduct a broad survey to reveal the different octopus saliva
properties and bio-activities. These include neurotoxic, enzymatic
and degenerative, and antibacterial properties.

Materials and methods

Based on octopus behavior, we decided to examine properties of the
octopus saliva while focusing on three main characteristics. For each
characteristic, we conducted bioassays which allowed us to
determine the presence or the absence of properties of interest.
We began our research by scanning a number of potentially
applicable saliva properties and conducting various assays in
accordance. Following preliminary examination of the various
properties, we decided to focus on three main properties which were
chosen in accordance with the given time limitations and initial
relatively promising results. This paper reviews the effectiveness of
these three properties of the octopus saliva (fig.2).
First, we created an initial saliva stock by milking live octopi. The
octopi were presented with Lined-top shells which were sealed with
hypoxic glue. The octopi then bore a hole in the sealed shells into
which they excreted their saliva in an attempt at feeding (Wodinsky,
1969). The saliva was then collected, lyophilized and stored as a
powder in freezing conditions. When ready to be used, the
powdered saliva was fluidized with a hydrophilic solvent.
The three main saliva features/characteristics that we focused on
are described in the flowchart below:
7

Fig.

2.

Project

flowchart.
research

The

plan

is

presented in this
diagram. In red is
the

enzymatic

and degenerative
branch, in green
the
branch,
purple

neurotoxic
and
the

in

anti-

bacterial branch.

Enzymatic

and

degenerative

activity

was

divided

into

Proteolytic activity and Hemolytic activity. Proteolytic activity was

examined by analyzing the effect of octopus saliva on BSA (standard
protein)

band

pattern,

by

running

different

samples

on

an

acrylamide gel electrophoresis. BSA samples were incubated for one

hour in the presence of the saliva's soluble fraction (which was prediluted with sea water). These samples, as well as BSA samples
without any treatment, were profiled on Acrylamide gel (using
running buffer with DTT) alongside the soluble fraction alone as
control. Band patterns of the different samples were compared to
locate any differences.
For the Hemolytic assay, human blood cells were separated and
washed with PBS from the serum. Samples of the saliva's soluble
fractions

were

diluted

with

PBS

and

added

in

different

concentrations (0.2g/ml, 0.4g/ml, 0.6g/ml and 0.8g/ml) to the

washed human erythrocytes. Additional samples of the saliva's
soluble fraction together with the insoluble fraction were added to
the washed human erythrocytes.

All samples were incubated in 37C for one hour, and then
centrifuged. OD (optical density) of the samples was measured at
450nm wavelengths on an OD plate reader. In addition, erythrocytes
were incubated in the presence of DDW or PBS as positive and
negative controls (respectively).

Neurotoxic activity (paralytic effect) was examined through three

different assays. The first one was a Zebrafish larvae escape
response

assay,

which

focused

on

determining

the

saliva's

neurotoxic effect on vertebrae.

Zebrafish larvae were placed in a 24 well plate, while each well
contained 1ml E3 buffer and one of four different protein
concentrations of the saliva's soluble fraction (636.65, 318.32,
159.16, 79.58g/ml). The larvae were then poked with a tip at
different times (5, 10, 30, 60 minutes). The response behavior was
monitored in order to measure the effect of the saliva's soluble
fraction. Larvae in the presence of E3 buffer alone served as a
control group. ED50 values were calculated in order to determine
toxic effect.
The objective for the second assay was to determine the saliva's
neurotoxic effect on arthropods. Blowfly larvae were injected with
one of four different protein concentrations (0.336, 0.6744, 1.34936,
2.7g/ml) of the saliva's soluble fraction. The larvae's behavior and
viability were then monitored and recorded over time. The control
group was injected with PBS alone. ED50 values were calculated in
order to determine the neurotoxic effect.
Protein concentrations in the saliva's insoluble fraction were
determined through Bradford reagent assays. The Bradford reagent
changes its color from brown to blue in accordance to the protein
content in the tested sample. The change in color was detected by
UV absorption at 450nm (blue) and 595nm (brown). Protein

concentrations in the samples were determined by using a

calibration curve of standard protein (BSA).

Antibacterial activity was examined in several assays, all in

sterile conditions. Twelve E.coli mutant strains and an additional
marine bacteria Photobacterium leiognathi (which naturally emits
luminesce when not under stress) were used for these assays. Each
E. coli mutant contains a specific bio reporter gene insertion (Table
1, Results), which triggers luminescence in response to stress. Each
bacterial

strain

was

placed

in

prepared

LB

substrates

and

distributed into four wells in a 96 well plate. Saliva samples of one of

three concentrations (11.83, 23.66, 47.32g/ml) were added to each
well, as well as a control well which did not contain any saliva
extract. The plate was incubated at 37C and read every 10 minutes
for the next 24 hours. The plate was read in a plate reader both for
OD,

monitoring

bacterial

growth

and

for

Luminescence,

for

determination of the saliva's mechanism of action on the E.coli

mutant

bacteria.

All

data

was

registered

and

scanned

for

antibacterial activity.
For The Antifouling assay we used newly milked saliva. The saliva
was centrifuged and separated into the soluble and insoluble (gel)
fractions, while a portion was left untouched. The three forms of
saliva were applied to three types of surfaces: wood, glass and
fiberglass. These surfaces, along with clean surfaces serving as
controls, were then connected to weights and placed in a natural
environment of shallow sea water. The surfaces were monitored for
antifouling activity following 24, 48, 72 hours and one week later.

10

Results
Different circumstances cause the octopus to use its saliva whilst
performing unique behaviors. A number of these behaviors, which
have been studied and are described in the introduction unit, give
us

reason

to

believe

the

saliva

contains

specific

bioactive

compounds that may be of great potential and interest to us as

humans.
As a starting point we determined the extent of the soluble and
insoluble fractions that compose the full octopus saliva, as well as
the protein content (Fig. 3).

Fig. 3. Octopus saliva composition. The saliva comprises of both water

soluble and insoluble substances. The insoluble fraction comprises mostly of a gel
like substance and the soluble fraction, which contains most of the activity, was
further analyzed to determine its protein content.

Enzymatic and degenerative activity

Saliva secretion directed at prey allows the meat to be more
accessible for feeding and seems to be an initial step toward
external digestion. Indeed, we observed proteolytic enzymatic
activity in the saliva through comparison of standard protein BSA vs.
the BSA post one hour incubation in the presence of octopus saliva.
11

Both samples were run and profiled on a gel acrylamide as

presented in Fig 4. Distinct differences in the band patterns were
observed, indicating the presence of proteolytic activity in the
saliva.

Fig. 4. Saliva proteolytic activity as shown on gel acrylamide profiling of

standard BSA samples. BSA protein samples of different concentrations: 1g/ml
(1:10) and 2g/ml (1:5) were incubated with octopus saliva and run on gel
acrylamide. Control samples of saliva alone, BSA alone and filtered sea water
were run as well. Presented on the right is a copy of wells No. 8 and 9 for
emphasis of the proteolytic effect of the saliva on BSA. Effected bands are
indicated with arrows. Well No.2 is loaded with marker PageRuler Plus Prestained
Protein Ladder and well No.6 is loaded with Spectra Multicolor Low Range
Protein Ladder. The Running conditions include Running Buffer+ SDS for 120 min
in 130V. The gel was stained with Forte Seaband.

In his study, Gennaro (1965) suggested that the saliva's gel fraction
plays a role in enhancing toxic activity, although it is not toxic in and
of itself. In order to explore this possibility we conducted hemolytic
assays (Fig. 5), while comparing the soluble fraction hemolytic
performance to that of the insoluble fraction. These assays showed
12

that the soluble fracture in the saliva displayed hemolytic activity

while the gel like substance not only did not enhance the activity
but also reduced it. Furthermore, we noticed that the gel substance
interacted with the erythrocytes in a way which the erythrocytes
seemed to be captured in the gel substance, and therefore
inaccessible to the hemolytic compounds.

Fig. 5. Hemolytic Activity. (A) Hemolysis as a function of the protein

concentration in octopus saliva. The soluble fraction showed greater hemolytic
activity than the insoluble fraction. Percentage was calculated from DDW activity.
(B) Erythrocytes were found to interact with the insoluble fraction.

Neurotoxic activity
In correlation with the hypothesis, we were able to verify existence
of paralytic activity in the octopus saliva. The saliva was shown to
affect both vertebrates and arthropods, as shown through the effect
on Zebrafish larvae (Fig. 6A) and on Blowfly larvae (Fig. 6B). The
ED50 value (of 0.676g/(ml*mg larvae)) was determined for Blowfly
larvae directly injected with octopus saliva, indicating saliva
potency.
The effect on Zebrafish larvae was tested through an escape
response assay in which the larvae were placed in a medium
containing saliva. The larvae were touched at four different times
13

over a period of one hour, and their behavioral response was

monitored. Four ED50 values were determined according to
behavior at each inspection. Interestingly, a transient effect was
observed, as ED50 values increased over time (fig. 6B).

Fig.

6.

Paralytic

activity. (A) Paralytic

median

effective

concentration of the
octopus

saliva

as

determined by effect
on

Blowfly

larvae

through injection. The

graph

indicates

activity

in

concentration
dependent
(B)

manner.
Neurotoxic

activity

as

determined by effect
on Zebrafish larvae.
Zebrafish

larvae

escape response was

tested at four time
points (5, 10, 30 and
60

minutes)

from

exposure to saliva. At
each time point ED50
value was determined
according to observed behavior at that time. Error bars are presented.

Anti-bacterial
Following limb amputation, it has been observed that the octopus
immediately inserts the remaining stump into its mouth. This
14

observation in addition to the fact that no known immune system

was characterized in octopi may point to the possibility that the
saliva actually has antibacterial properties and provides immunity
(Dawes et al, 2015). These assumptions were confirmed through
antibacterial experiments (Fig. 7 and 8). The octopus saliva inhibited
bacteria cultural growth, as indicated by reduction of OD levels in a
concentration dependent manner in 12 mutants of E.coli and the
marine bacteria P.leiognathi (Fig. 7).

Fig. 7. Antibacterial properties: Saliva effect on growth of E.coli mutants

(orange) and a wild type P.leiognathi (red), indicated by average optical density
(OD600nm). (*) Indicates a statistically significant difference between effected
bacteria and control. Two-tails unpaired student T-Test, p<0.05. n=3, Error bars
are presented.

Following

the

conformation

of

the

presence

of

antibacterial

properties in the octopus saliva, we investigated the saliva

mechanism of action by performing antibacterial assays (Fig. 8). The
mechanism was postulated by using stress indicator bacterial
strains (Table. 1).

15

Fig. 8. Presumed saliva mechanism of action against bacterial growth:

Saliva's effect on bacterial growth of 12 E.coli mutants and the wild type marine
bacteria P.leiognathi. (A) OD (Optical Density) values indicate bacterial growth,
and RLU (Relative Luminescence Unit) values indicate the salivas targeting
mechanism. Strains with significantly higher luminescence in comparison to
control (threshold in dotted blue line) demonstrate a specific saliva target. (B)
Example for one experiment result of the effect of the saliva on E.coli mutant grpE
which emits luminescence in response to stress. (C) Example of one experiment
result of the effect of the saliva on wild type P.leiognathi which naturally emits
luminescence in positive relation to the culture density (quorum sensing).
Luminescence is represented by Relative Luminescence Units (RLU), which are
simply the luminescence values relative to the OD values (LU/OD).

16

By

comparing

luminescence intensity of
each bacterial strain to
control, we were able to
determine
was

which

activated

saliva

by

extract.

stress
the
The

activated stressors can be

seen in Fig. 8A, where the
bacterial
surpassed

mutants
the

which

threshold

are considered affected.

The induced mutants and
their mechanism appear
in detail in Table 1 below.

Table 1. 12 E.coli mutant

promoters and their stress
mechanism. Description of the
mutant promotors used for the
antibacterial assay. Mutants
.affected by the saliva (surpassed the threshold in Fig. 8A) are highlighted in grey

17

In succession to verifying antibacterial properties in the octopus

saliva, we decided to test for antifouling properties, since biofouling
initiates through bacterial accumulation on surfaces (Banerjee et al,
2011). Antifouling assays were conducted by applying different
saliva samples (soluble, insoluble, and full saliva) to wood surfaces
which were then incubated in shallow sea water. The saliva's soluble
fraction was shown to prevent fouling accumulation during the first
few days, while the insoluble fraction was shown to accumulate
18

excess fouling during the first few days, and later peeled away
leaving a clean spot. The full saliva showed a combination of both
mechanisms.

Fig. 9. Saliva Anti-Fouling as exemplified by the effect on wood surfaces:

relative fouling level over the course of one week (A). Representation of the saliva
effect by comparing the soluble fraction surface to control at day 3 (B) and by
comparing the full saliva surface at two time points (C).

19

Conclusions
In this project the O.vulgaris saliva's properties were examined
through various bio-assays in order to identify potentially beneficial
products which are nature based.
The saliva comprises of a soluble fracture and an insoluble (gel like
substance) fracture. The protein content in the saliva's soluble
fraction was determined through Bradford assays to be ~0.8% (Fig.
3).
The saliva demonstrated its proteolytic potential by altering the
band pattern of the BSA standard protein (Fig. 4). Considering the
fact that the soluble fraction contains proteins, it is possible that
enzymes found in this fraction are responsible for the proteolytic
activity (Cariello and Zanetti, 1977; Grisley and Boyle, 1987). In
nature, the octopus has been observed to use its saliva in order to
detach the muscle tissues of its prey as part of the digestion
process. We assume that this activity is of proteolytic nature. The
saliva can potentially be used to develop new enzymes and expand
the available protein cleaving capabilities.
Following the proteolytic bio-assay we examined the hemolytic
properties of the saliva. The existence of hemolytic activity was
confirmed in a previous project and academic paper (Key et al,
2002), however, we conducted the bio-assay in order to determine
how the different saliva fractions effect the activity. The initial
hypothesis was that the insoluble fraction may increase existing
activity in the saliva (Gennaro et al, 1965). While the soluble
fraction caused an increasing hemolytic response relative to the
saliva's concentration, the full saliva, containing both the soluble
and insoluble fractions, demonstrated relatively low hemolytic
activity (Fig. 5). In the sample containing full saliva, we observed an
interaction between the gel like substance and the erythrocytes (Fig.
5B). It seems that the insoluble fraction adsorbed the erythrocytes,
20

acting as a physical barrier between the hemolytic agents found in

the insoluble fracture and the erythrocytes. The adsorbing property
suggests a possible use for the gel like substance as a substrate for
cell culturing.
Through this assay, it became clear that the gel like substance does
not increase activity in this case, in contrast to our initial hypothesis.
We believe that the gel like substance may act as an isolator for the
active compounds found in the soluble fraction, preventing them
from diffusing in the ocean's water before reaching their target.
The hemolytic process may benefit the octopus while feeding by
contributing to the digestion process. The hemolytic agents target
the erythrocytes by deteriorating their membranes, thus lowering
the amount of oxygen transferred in the blood. Tissues suffering
from hypoxia may be easier to dismantle and digest.
Contrary to the proteolytic and hemolytic properties that contribute
to digestion, the neurotoxic properties serve in an earlier stage in
the feeding process. The neurotoxic assay was performed on Blowfly
larvae and on Zebrafish larvae (Fig. 6), which represent arthropods
and vertebrates (respectively). While the Blowfly larvae received a
direct injection of saliva, the Zebrafish larvae came in contact with
the saliva through the water medium they were in. This can possibly
explain why the effective concentrations for the Blowfly larvae were
considerably lower than the effective concentrations on the
zebrafish larvae (Fig. 6). Furthermore, the Blowfly larvae showed
signs of complete yet temporary paralysis following the injection,
while the Zebrafish larvae demonstrated behavioral changes, mainly
by slower response time to probing and a lowered movement
activity. In the case of the Zebrafish larvae, higher concentrations of
saliva were needed for a longer effect, suggesting of a transient
effect.
Neurotoxins affect the function of neurons, either by altering the
ionic concentrations across the neuron cell membrane which
21

induces their function, or by disrupting the communication between

neurons. The paralytic effect of the saliva may target the muscles
which are responsible for movement. The precise affected muscles
are unknown, however if the toxin effect the respiratory muscles, a
sufficient concentration may prove lethal.
The saliva showed great potential in neurotoxic activity, calling for
further research. The neurotoxic activity shown on vertebrates
suggests a possible anesthetic to be developed for human use as
well as for other species. The results obtained from the neurotoxic
assays on arthropods may imply a possible pesticide to be
developed against pests of this phylum.
Looking into much smaller organisms, we explored the saliva's antibacterial properties. Bacteria that came in contact with the saliva
showed lowered OD values (Fig. 7) in comparison with the control
group, suggesting that the saliva restricted bacterial growth.
The mechanisms of action of the saliva were then examined by
using 12 E.coli strains inserted with specific promotor genes, which
are

activated

in

response

to

stress

and

are

indicated

by

luminescence emittance (Fig. 8A). The genes katG, micF, fabA, grpE,
sodA and soxS were shown to be affected by the saliva. The mutants
differ in their action inducing factors as mentioned in the results unit
(Table 1). However, we were able to detect two main action
mechanisms responsible for the anti-bacterial activity: Oxidative
stress and hyperosmosis.
Oxidative stress is caused by an increase in free radicals within the
cell which the organism struggles to neutralize. Free radicals are a
by-product of the energy production process taking place in the
mitochondria of every cell. Most commonly they are made of oxygen
molecules that lack an electron, making them unstable and harmful.
In order to stabilize, the free radicals alter molecules such as
proteins, enzymes, and DNA in their vicinity while retrieving their
missing electron. DNA damage ranges from few mutations to
22

complete dysfunction. When the body enters a state of stress, as

can occur as the result of the invading saliva toxins, it boosts its
energy

producing

mechanisms

in

order

to

maintain

regular

homeostasis. However, increase in energy production results in the

increase of free radicals (Sies, 1985).
Hyperosmosis, the second antibacterial activity mechanism, may
lead to cell apoptosis, DNA damage and cell cycle arrest. Alterations
of solute concentrations in cells may be the result of a damaged
membrane. Maybe there a relation between the hemolytic activity of
the saliva and the induction of hyperosmosis. This relation is
apparent, as hemolytic agents penetrate the lipid membranes of the
cells and lead to their destruction.
The saliva mechanism of action was determined by monitoring
stress levels of the various E.coli mutants and P.leiognathi bacteria.
The E.coli mutant grpE as presented in Fig. 8B, exemplifies the
saliva's stress effect. Stress levels were clearly raised in response to
saliva presence, as seen by the increase in RLU/OD values over time
followed by a decrease. Possible explanations for this decrease are
that the damage to the bacteria by this stage was too severe and
caused dysfunction to the luminescence mechanism, or decreased
luminescence due to bacterial death. This was not the case with
P.leiognathi, naturally emitting luminescence bacteria, triggered by
quorum sensing, resulting in a correlation between concentration
and luminescence.
In continuation to the found antibacterial properties in the saliva,
antifouling properties were examined. The different saliva fractions
were shown to effect fouling accumulation through two different
mechanisms (Fig. 9). The saliva's soluble fraction prevented settling
of fouling during the first 3 days, losing its effect over time. It seems
that the soluble fraction contains the chemically active compounds,
which are responsible for prevention of fouling accumulation.

23

The active compounds prevented settlement of microorganisms to

the

different

surfaces,

implying

antibacterial

activity.

Micro-

organisms are the first step in the succession of fouling, producing

bio-films which serve as a substrate for larger organisms and abiotic substances.
In contrast, the insoluble fraction enhanced fouling accumulation
during the first 3 days. Later, the fouling was removed with the
peeling of the fraction. Although the gel adsorbed fouling, it non-the
less served as a physical barrier between the surface and the
environment, thus preventing direct contact between fouling
components and the surfaces. The fraction was eventually washed
away with the substances attached, leaving a clean surface, that
was now exposed to fouling.
It appears that the full saliva was more effective than the insoluble
fraction, though their action mechanism seems to be similar (Fig.
9A). The full saliva encompasses both fractions, and it seems that it
operated in a combination of both described mechanisms. The
insoluble fraction may have extended the duration of the active
compound present in the soluble fraction, creating a longer lasting
shield of both chemical and physical properties, enabling the effect
to last longer. The active compounds in the soluble saliva prevented
fouling from settling, even after the insoluble fraction peeled off.

Through the antifouling assay, the octopus saliva was shown to have
potential as an antifouling agent. That said, more research is
needed in order to confirm these preliminary findings, and to
deepen our understanding of its potential and mechanism.
It is likely the saliva might be effective for a longer period of time in
an environment of lower kinetics (less waves and currents).
However, since antifouling compounds are designed to be used in
an active marine environment, it is important to extend its lasting
effectiveness.
24

Awareness of the damage caused to our world in response to

excessive use of toxic and harmful chemicals has increased in
recent years (Birnbaum, 2008). Environmentally friendly products of
natural origin are highly sought after for the purpose of replacing
environmentally harmful ones (Shu, 1998). The octopus's saliva is
an example of a natural compound containing interesting traits that
may fulfill this demand in different areas, benefiting humans as well
as the environment. In this project we opened a hatch to its
potential, and showed some of its abilities including proteolytic,
neurotoxic and antibacterial potential.
Through this research project, we were able to expose, though
preliminarily, a variety of potentially applicable properties of the
O.vulgaris saliva although the obtained results call for further
research. For future projects we would recommend to focus
separately on each property, while thoroughly examining them and
chemically characterizing their active compounds.

Acknowledgements: our thanks to Ori Koshet from the S. Belkin

lab at Hebrew University for the providence of bacterial strains.

25

Bibliography
Anderson RC. and Mather JA. (2007). The packaging problem:
bivalve prey selection and prey entry techniques of the octopus
Enteroctopus dofleini. Journal of Comparative Psychology. 121(3):
.300305
Banerjee I, Pangule RC. And Kane RS. (2011). Antifouling coatings:
recent developments in the design of surfaces that prevent fouling
by
proteins,
bacteria,
and
marine
organisms. Advanced
.Materials. 23(6): 690-718
Birnbaum LS. (2008). The effect of environmental chemicals on
.human health. Fertility and sterility. 89(2): e31
Burg MB, Ferraris JD. and Dmitrieva NI. (2007). Cellular response to
.hyperosmotic stresses. Physiological reviews. 87(4): 1441-1474
Cariello L. and Zanetti L. (1977). - and -cephalotoxin: Two
paralyzing proteins from posterior salivary glands of Octopus
vulgaris. Comparative Biochemistry and Physiology Part C:
.Comparative Pharmacology. 57(2): 169173
Dawes C, Pedersen AML, Villa A, et al. (2015). The functions of
human saliva: A review sponsored by the World Workshop on Oral
.Medicine VI. Archives of oral biology. 60(6): 863-874
Gennaro JF, Lorincz Jr AE. and Brewster HB. (1965). The anterior
salivary gland of the octopus (Octopus vulgaris) and its mucous
secretion. Annals of the New York Academy of Sciences. 118: 1021
.1025
Grisley MS. and Boyle PR. (1987). Bioassay and proteolytic activity
of digestive enzymes from octopus saliva. Comparative
Biochemistry and Physiology Part B: Comparative Biochemistry.
.88(4): 11171123
Key LN, Boyle PR. and Jaspars M. (2002). Novel activities of saliva
from
the
octopus
Eledone
cirrhosa
(Mollusca;
.Cephalopoda). Toxicon. 40(6): 677-683
Livermore DM. (2004). The need for new antibiotics. Clinical
.Microbiology and Infection. 10(s4): 1-9

26

Marechal JP. and Hellio C. (2009). Challenges for the development of

new non-toxic antifouling solutions. International Journal of
.Molecular Sciences. 10(11): 4623-4637
Nixon M. (1984). Is there external digestion by Octopus?. J. Zool.
.202(3): 441447
Shu YZ. (1998). Recent natural products based drug development: a
pharmaceutical
industry
perspective. Journal
of
natural
products. 61(8): 1053-1071.
Sies HE. (1985). Oxidative stress: introductory remarks. Oxidative
.stress. pp 1-8
Wells M. (1978). Octopus physiology and behavioral responses of an
.advanced invertebrate. London: Chapman Hall. pp 1-417
Wodinsky J. (1969). Penetration of the shell and feeding on
gastropods by octopus. Integrative and Comparative Biology. 9(3):
.9971010
Wood FG. (1963). Observations on the behavior of octopus.
.Int.Congr.Zool. 16(1): 73

27