Composition, morphology and mechanics of hagfish slime
Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada
* Author for correspondence at present address: Department of Integrative Biology, University of Guelph, Guelph, ON N1G-2W1, Canada (e-mail: dfudge{at}uoguelph.ca)
Accepted 26 October 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: biomechanics, slime, mucus, hagfish, fibre-reinforced composite
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
To answer this question we measured the mechanical properties of hagfish slime mucins and whole slime. We also made a variety of measurements required for a complete understanding of hagfish slime form and function, such as slime thread length and diameter, the concentration of mucins and slime threads, and the amount of slime exudate stored by hagfishes as a function of body mass. From our results we conclude that the mucin component of the slime is not capable of transferring significant forces between slime threads as in a typical fibre composite. Furthermore, experiments with whole slime demonstrate that slime threads indeed dominate the mechanical behavior, while the mucin component imparts viscosity and aids in the rapid deployment of the slime into its mature and fully hydrated state. Although the precise ecological function of hagfish slime is not known, our results are consistent with the hypothesis that hagfish release the slime in order to thwart attacks by gill-breathing predators.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Concentrations of threads and mucins in native slime
Hagfish were induced to produce a single mass of slime in their 200 l
aquarium by pinching them on the tail with forceps. Care was taken to insure
that the slime was produced by hagfish swimming in the middle of the water
column rather than close to the bottom or surface, which could have
constrained slime hydration. The slime was gently collected by scooping it
into a plastic kitchen colander lined with 53 µm nylon mesh. As the
colander was lifted out of the aquarium, free water was allowed to run out of
the bottom through the mesh, and the colander was tipped slightly to allow
free water sitting on top of the slime to spill out. The contents of the
colander were transferred to a bucket for subsequent measurement of slime
volume. Threads were removed from the slime by twirling them onto a glass rod
until they collapsed and squeezed out most of the entrapped mucins and water.
This technique was very effective at collecting the vast majority of the
threads in the slime, and concentrating them into a fibrous ring that could be
easily handled for subsequent purification and drying. The ring of threads was
placed back into the original slime solution to which a pinch of
dithiothreitol (DTT) was added. Along with gentle heating of the slime (to
about 60°C), DTT helped in the removal of mucins that were still bound to
the threads. The threads were treated in this way until they were no longer
slippery to the touch (about 30 min), at which point they were removed, rinsed
with several changes of distilled water, and dried in an oven at 80°C for
the determination of dry mass. Thread concentration was calculated by dividing
the dry mass of threads by the total volume of the slime collected.
Mucin concentration was measured by dialysis of 50 ml of the remaining slime solution using Spectra/Por dialysis tubing with a 1214 kDa cut-off (Spectrum Laboratories Inc., Rancho Dominguez, CA, USA). Mucin samples were dialysed four times against 5 l of distilled water in a cold-room (4°C) for 8 h. Preliminary trials demonstrated that the above procedure was adequate to lower the concentration of salts to a negligible amount. Mucin dry mass was obtained by drying 25 ml of the dialyzed solution in a drying oven at 80°C. Mucin concentration was obtained by dividing the mass of mucins by the volume of solution dried. Mucin concentrations were adjusted by subtracting out the concentration of material in the distilled water, which was measured by drying down 25 ml of the distilled water used for the dialyses. Mucin and thread concentrations were measured in this way for five independent slime masses.
The high ratio of salts to mucins in the slime made it technically
challenging to accurately measure the mucin concentration. In contrast,
because the slime threads are insoluble, they were easily separated from the
slime and therefore their concentration could be measured more accurately. To
confirm the mucin concentrations obtained via dialysis, we also
measured the mucin concentration via a centrifugation technique we
will refer to as `slimatocrit', due to its similarity to the measurement of
hematocrit. The premise of this technique is that the volume ratio of mucin
vesicles to threads can be measured by centrifuging slime exudate in
hematocrit tubes. From these data, the concentration of mucins in the slime
can be calculated from the thread concentration data. Slime exudate was
collected from anaesthetized hagfish and transferred into an aqueous
stabilization buffer (Downing et al.,
1984) containing 0.5% Toluidine Blue. Stained and stabilized slime
exudate was drawn up into 75 mm microhematocrit capillary tubes, and spun for
10 min in a hematocrit centrifuge. Centrifugation resulted in two distinct
layers, a slime thread skein layer, and a Toluidine Blue stained mucin vesicle
layer topped by stabilization buffer (Fig.
3). The relative volume of the layers was calculated from their
dimensions, which were measured under a dissecting scope with a Filar eyepiece
micrometer. Sixteen slimatocrit measurements were made in total.
|
Slime storage vs hagfish mass
Measurement of the amount of exudate stored in slime glands was performed
on hagfish that were anaesthetized as described in Fudge et al.
(2003). Hagfish that released
slime in the anesthesia bucket were rejected and placed back in the aquarium.
Immediately after losing touch sensitivity, hagfish were removed from the
anesthesia bucket, blotted dry and weighed. The animals were then placed on a
chilled dissection tray where they were kept hydrated and cool, except for the
area from which slime exudate was collected, which was rinsed with distilled
water and blotted dry. Rather than trying to measure the mass of exudate
expressed from every slime gland, five glands were chosen as representatives.
Exudate was expressed from the glands via mild electrical stimulation
as described in Fudge et al.
(2003
). Exudate from all five
glands was collected with a spatula and transferred to a pre-weighed
microcentrifuge tube. Glands were stimulated until exhausted of exudate. Wet
and dry mass were measured to the nearest 0.1 mg using a Mettler H31 balance
(Mettler Instruments, Zurich, Switzerland). Exudate samples were dried in a
drying oven at 80°C until the mass was stable over time. The total mass of
stored exudate was calculated by multiplying the pooled exudate mass by the
total number of glands and dividing by five.
Slime thread length
When ejected from the slime glands into seawater, slime thread skeins
quickly unravel. When they are collected into stabilisation buffer, however,
they retain their original ellipsoid shape and coiling. Thread length was
measured by transferring single stabilized skeins to a seawater-filled test
chamber, allowing it to partially unravel, and attaching its respective ends
to two glass rods using techniques described in Fudge et al.
(2003). The original intent of
this setup was to slide one rod away from the other until the thread was just
taut; the distance between the two rods would then reveal the length of the
thread. This plan was confounded by the tendency of the coiled thread to
unravel in stages, with the thread going taut at times when there were clearly
large sections that had not yet unraveled. Because it was not possible to
observe the glass rod under the microscope and simultaneously observe the
entire length of the thread for clusters of thread loops, using tautness as an
endpoint was abandoned. Instead, thread failure was used as an endpoint and
the resting length calculated from the average failure strain of slime threads
as reported in Fudge et al.
(2003
)
(
max=2.2). While the popping open of clusters of loops caused
deflections of the glass rod, these were always transient and easily
distinguished from the long steady deflection that occurred before failure of
the thread. In essence, these length measurements were extensibility tests of
entire slime threads.
Slime thread taper
Diameter measurements were made at eleven equispaced positions along the
length of intact slime thread skeins in distilled water. While most slime
thread skeins rupture and lose their coiled structure in seawater, some remain
mostly intact, and it was these that were used for diameter measurements.
Slime thread skeins were visualized under high power (100x interference
contrast oil immersion objective) on a Leitz polarizing microscope (Ernst
Leitz Canada, Midland, ON, Canada) fitted with a Panasonic WV-BL600 video
camera. Thread diameter was measured on captured images using Scion Image
release v. 3b software (Scion Corp., Frederick, MD, USA).
To isolate skeins and prepare them for scanning electron microscopy (SEM) imaging, slime exudate was collected and stirred into stabilization buffer. Stabilized slime was filtered through 53 µm nylon mesh, which retained the skeins and allowed the mucin vesicles to pass through. After washing the filter disk with excess buffer, skeins were removed from the disk by gentle shaking with 10 ml of buffer in a capped vial. Skeins were fixed for 2 h in 4% glutaraldehyde in stabilization buffer, rinsed with fresh buffer, and then rinsed again with 0.2 mol l1 cacodylate buffer (pH 7.1). Skeins were transferred onto a Nucleopore Track-Etch membrane (13 mm diameter, 6 µm pore size, Corning, Acton, MA, USA) in-line with a 5 ml syringe. The cells were dehydrated with an ethanol series consisting of 5 ml of the following ethanol solutions: 30%, 50%, 70%, 80%, 90%, 95%, 100%, 100%. While still wet with 100% ethanol, the filter disk was transferred into a Balzer CPD 020 critical point drying apparatus (Bal-Tec, Manchester, NH, USA). Before critical-point drying, the ethanol was replaced by ten rinses with liquid carbon dioxide. Dried skeins were immediately transferred into a Nanotech Semprep 2 gold sputter coater, and coated under vacuum for 3.2 min. Images were collected using a Hitachi S4700 scanning electron microscope.
Mucin mechanics
Our original intent was to characterize the viscoelastic properties of
hagfish mucins, but after isolating mucins from the slime, it became clear
that mucins in seawater at native concentrations possess no elastic
properties, and if they have any effect on the properties of seawater at all,
it is only to raise the viscosity. We therefore measured mucin mechanics using
a simple Ostwald viscometer (Fisher Scientific, Hampton, NH, USA). Slime
masses from five different animals were isolated and viscosity measured on
three different 7 ml subsamples from each. Transit times through the
viscometer were measured with a stopwatch to the nearest 0.01 s and averaged.
The viscometer was mounted in a water bath maintained at 9°C. After
introduction into the viscometer, mucin samples were allowed to equilibrate
for 20 min before testing. Between trials, the viscometer was flushed with the
following solvents: 10 ml distilled water (dH2O; 3x), 10 ml 1
mol l1 HCl, 10 ml dH2O, 5 ml acetone, 5 ml
acetone, and then flushed with filtered air until dry. Mucin samples were
obtained by collecting slime masses from the 200 l hagfish tank using a
mesh-lined colander. Slime threads were removed by twirling them out onto a
glass rod. Mucins bound to the threads on the glass rod were removed by gently
massaging the threads until they were no longer slippery to the touch. Before
testing, mucin samples were filtered twice through 53 µm mesh to remove
particulates that might have interfered with the viscosity measurements.
The concentration-dependence of mucin viscosity was measured by preparing concentrated slime solutions in distilled water and diluting them to attain a concentration series that spanned two orders of magnitude. Concentrated stock solutions were prepared by stirring slime exudate from anaesthetised hagfish directly into a beaker of chilled distilled water. This resulted in a thick mixture containing both slime mucins and slime threads. Threads were removed by inserting a glass rod into the mixture and spinning the threads onto the rod. The remaining mucin solution was then vacuum-filtered on ice through two layers of 53 mm nylon mesh to remove any remaining slime threads or other particulates. Mucin stock solutions were prepared from four different hagfish and had an average mucin concentration of 470±67 mg l1 (mean ± S.E.M.), which was measured gravimetrically by drying down 2x 25 ml samples of the stock solution at 80°C. Mucin solutions in seawater were prepared by diluting mucin stocks with an equal volume of double strength artificial seawater (Coralife, Carson, CA USA) to obtain a solution half as concentrated as the distilled water stock. Further dilutions were made with full strength seawater. Viscosity measurements were made as described above.
Whole slime mechanics
The extreme heterogeneity of the slime required a mechanical testing
apparatus that could simultaneously quantify the viscous and elastic
components of its mechanics but not destroy its delicate structure in the
process. Ideally, the slime would be formed from exudate and seawater within
the testing apparatus itself. In addition, the apparatus had to be large
enough to allow for the complete unraveling of the slime threads. The
apparatus shown in Fig. 4
fulfils all these criteria. We used an Instron model 5500 (Norwood, MA, USA)
universal testing machine to move a 2 l beaker of hagfish slime up and down
while forces were measured by a 100 g load cell from which hung a stationary
plunger. The plunger consisted of a 40.5 mm nylon disk fitted with eight
radial spikes that protruded 12 mm from the edge of the disk. The plunger was
attached to the load cell via a 0.36 m stainless steel rod. The nylon
disk was sandwiched between 3 mm thick lead disks that gave the plunger a
total mass of 104 g and kept sufficient tension on the attachment rod so that
compression forces could be measured without the rod lifting off from the load
cell. The inside surface of the beaker was modified by inserting a 62 mm wide
plastic collar studded with 72 1.3 mm diameter nails that protruded 12 mm
toward the centre of the beaker (Fig.
4). We found that after the slime formed within this setup, it
became attached to both the plunger and the spiked collar. For each trial, the
plunger was oscillated up and down in 2 l of 9°C seawater at a rate of
11.7 mm s1 over an amplitude of 100 mm and force and
displacement data were collected at 100 Hz. After 50 s of data collection in
seawater only, 100 mg of fresh slime exudate from an anaesthetised hagfish was
added via a spatula to the top of the beaker. As the slime
concentration measurements demonstrate later in this paper, this amount of
exudate is enough to produce about 1 l of slime. Some trials were performed in
the presence of 5 mmol l1 DTT, which reduces disulfide bonds
within and between mucin molecules and has been shown to decrease the
viscosity of mucus (Bell et al.,
1985). Stressrelaxation trials were performed on slime that
had been allowed to develop for 500 s of plunger oscillations. As the
stainless steel rod oscillated up and down in the beaker, the length of rod
immersed in the water changed over time. This resulted in a highly predictable
buoyant force that in some trials represented a considerable amount of the
force variation. We therefore measured the change in force on the plunger as a
function of the degree of (static) immersion, which allowed us to remove the
contribution of the buoyant force from our data. The relationship between rod
immersion (x) in mm and static force on the load cell (y) in
mN was y=0.026x in seawater and y=0.024x
in distilled water. Preliminary trials revealed a predictable 30 Hz source of
noise in the data that presumably arose from resonant oscillations of the
force transducer. This noise was digitally filtered using a second order
Butterworth recursive algorithm (Winter,
1979
) with a 10 Hz cut-off. Filtering the data in this way had no
effect on the low frequency events resulting from deformation of the slime.
Raw data were additionally processed to remove a force spike that occurred at
the extremes of the crosshead excursion. This spike arose from the rapid
deceleration of the beaker of water at the turnaround point and had nothing to
do with the material properties of the slime. About 0.1 s worth of data points
were deleted from each half cycle of the force traces to remove these
spikes.
|
Water egress from the slime
Our mucin and thread concentration measurements suggested that hagfish
slime is able to organize heroic amounts of water with very little material.
In making these measurements, we collected the slime by lifting it out of an
aquarium using a colander. This procedure took only a few seconds to perform.
To test whether the slime could organize water on longer timescales, we
measured water egress from slime formed in vitro. Slime was formed by
stirring about 100 mg of fresh slime exudate into a 1 l plastic cylinder
(diameter=88 mm, length=148 mm) that was nested in a 2 l beaker of seawater.
The cylinder hung from a 5 kg load cell and was covered on the bottom with
plastic 4 mm mesh. At the start of the experiment, the bridle connecting the
cylinder to the load cell was slack. The beaker of water was quickly lowered
well below the cylinder and the mass of the cylinder, plus water and slime was
recorded at 10 Hz by the load cell. These measurements were performed with
four different slime samples.
Congo Red staining
In a previous study, we established that Congo Red (CR) staining can be
used to detect an ß transition in intermediate filament
proteins from mechanically strained slime threads. Here we used CR as a way of
evaluating the strains induced in slime threads as a result of mechanical
perturbation of the whole slime in seawater. Hagfish were coaxed into
producing a mass of slime in their aquarium as described above. `Unperturbed'
slime was gently collected from the aquarium in a shallow, mesh-lined colander
with a glass slide on the bottom. The slime was allowed to drain and adhere to
the mesh before being rinsed with a very gentle, continuous flow of tapwater
for 15 min. The slime was then rinsed with distilled water, and allowed to
completely dry onto the glass slide and the mesh. When dry, the glass slide
was freed from the mesh by trimming the slime threads with a razor blade. The
slime was stained in a 1% CR, 10% ethanol solution for 1 h, after which it was
de-stained in distilled water for 5 min, followed by a gentle distilled water
rinse. `Perturbed' samples were prepared in the manner described above, except
a ruler was pushed into the slime and moved back and forth 20x at an
approximate frequency of 1 Hz and an amplitude of 10 cm before the sample was
collected into the colander.
Values are reported as means ± S.E.M.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Stored slime represents 34% of hagfish body mass
Collection of slime exudate from eleven anaesthetized hagfish revealed a
significant positive linear relationship with body size (P=0.014,
r2=0.50), with a regression equation of
S=0.036Mb, where S is the total mass of
stored slime (g) and Mb is the hagfish mass (g). From this
equation, one can conclude that slime exudate makes up about 34% of
hagfish body mass (Fig. 5).
Slime threads break at 34 cm and have a resting length of 1017 cm
The average length at failure for the ten slime threads tested was
34±1 cm. Because a small length of thread at either end was used to
attach the slime thread to mounting rods, this value is a slight underestimate
of failure length. If we assume that the extensibility of entire slime threads
is the same as the extensibility as the thread segments tested in Fudge et al.
(2003) (i.e.
max=220%), then the resting length of slime threads is
1011 cm. However, because whole slime threads are tapered, it is more
likely that they will break at their narrowest point, with the thicker regions
never reaching their maximum extensibility. Slime threads exhibit a threefold
difference in diameter from their thickest to thinnest points
(Fig. 6), and therefore a
ninefold difference in cross-sectional area. This means that when the thinnest
part of the thread is at the breaking stress (180 MPa), the stress in the
thickest part will be only 1/9th of that, or 20 MPa, which corresponds to a
strain of only 1.0. This means that the overall breaking strain of an entire
thread should fall somewhere between 100% and 220%, giving a possible range of
resting lengths of 1017 cm. These values are consistent with Fernholm's
estimate of 611 cm, and a bit lower than the estimate of 2460 cm
by Downing et al. (1981b
).
|
Slime threads are bi-directionally tapered
Measurement of thread diameter within intact slime thread skeins using
light microscopy revealed a distinct bi-directional taper
(Fig. 6), in contrast to the
uni-directional taper proposed by Downing et al.
(1981b). Hydrated threads
within skeins exhibited a maximum diameter of 3.0±0.4 µm, which
occurred in the middle of the skeins. Thread diameter was 1.5±0.2 µm
at the pointed end, and 1.0±0.2 µm at the blunt end. Inspection of
thread diameter under SEM confirms this result
(Fig. 6B).
Mucin viscosity is low, even at high concentrations
The average dynamic viscosity of the mucin solutions collected from slime
masses produced in aquaria was indistinguishable from the viscosity of
seawater controls (1.44x103 Pa s
±0.003x103 Pa s). Mucin solutions prepared in
distilled water exhibited a linear, but weak concentration dependence, with
the viscosity of the stock solutions exceeding that of their hundredfold
dilutions by an average factor of only 3.2
(Fig. 7). The viscosity of
mucin solutions prepared in seawater exhibited even less concentration
dependence, with a linear slope that was over 14x lower than the slope
for mucins in distilled water. The viscosity of stock solutions in seawater
exceeded that of their 50-fold dilutions by an average factor of only 10%
(Fig. 7).
|
|
Slime threads dominate the material properties of hagfish slime
Close inspection of the slime maturation process revealed that the increase
in force generation by the slime over time corresponded with the unravelling
of skeins and the subsequent entanglement of slime threads on the projections
of the plunger and the inside of the beaker. Focusing in on a single
oscillation for each treatment reveals almost identical force traces
(Fig. 9). To test whether the
forces measured were indeed a result of the straining of solid slime threads
and not just a viscous phenomenon, stressrelaxation trials were
performed after the initial 500 s period of slime maturation and oscillations.
The three treatments (seawater, distilled water and 5 mmol
l1 DTT in seawater) each exhibited distinct stress
relaxation properties (Fig.
10). The forces generated by slime produced in seawater relaxed on
average by about 74% after 500 s of relaxation. Although such a large
relaxation suggests a significant viscous component to the peak forces
generated by the slime, the fact that it still held significant force after
500 s confirms that a solid structure is involved in the forces measured.
Slime formed in seawater in the presence of 5 mmol l1 DTT
relaxed significantly less, losing only about 52% of the peak force after 500
s of relaxation. DTT cleaves disulfide bonds and has been shown to be an
effective means of disrupting networks of mucins
(Bell et al., 1985). In
contrast, the proteins that make up the slime threads have been shown to be
almost completely devoid of cysteine (Koch et al.,
1995
,
1994
), thus one would expect
DTT to have no effect on the thread mechanics. The slower force decay with DTT
is consistent with our assertion that it is indeed the slime threads that are
responsible for the majority of the forces generated by the slime in these
oscillation experiments. This effect of DTT also suggests that some of the
force generated by the slime in seawater can be attributed to the mucin
component of the slime. It also suggests that the presence of cross-linked
mucins allows the threads to slip more easily past each other and off the
projections of the apparatus.
|
|
Hagfish slime is known to collapse when it is mechanically disturbed, with
a massive loss of mucins and water (Ferry,
1941). We expected to see evidence of this process in our force
traces over time. Interestingly, we saw no degradation of slime properties
over the 500 s of data collection, and even extended trials of plunger
oscillations (up to 3000 s) had no effect on the magnitude or shape of the
force traces (data not shown). This was likely due to the limited volume of
seawater used for these measurements as well as the fact that slime threads
that became entangled on the projections of the plunger and the walls of the
beaker had little opportunity to collapse onto each other.
From the four trials performed in seawater, the average peak forces in the
positive and negative directions were 24.6 mN and 12.6 mN, respectively, for a
total peak force (± S.E.M.) of
37.2±7.6 mN. Because the mucin component exhibited no elastic
properties and only marginally elevated viscosity, it is reasonable to
conclude that these forces arose mostly from the straining of slime threads.
The stability of the force traces over time suggests that the slime threads
were able to recover between cycles, and this suggests that the strain they
experienced was less than their yield strain of 35%
(Fudge et al., 2003). If we
assume that attached slime threads are extended to their yield strain (about
35%) during each cycle, then each thread will exert about 7 µN of force.
Assuming that each thread contributes to the force in one direction only
(which admittedly will not hold for threads whose minimum strain occurs close
to the middle of the plunger excursion), then this corresponds to about 5300
slime threads. A typical slime mass contains about 24 000 slime threads, so
this suggests either that the threads are strained less than 35% on average,
and/or a majority of the threads were not ever brought into tension due to the
orientation of their attachment. It is unlikely that many of the threads were
strained to the breaking point, since a similar calculation predicts that only
about 150 threads strained maximally could account for the forces measured.
This is clearly much lower than the number of threads that could be seen with
the naked eye and would require that the vast majority of threads were never
put into tension.
Hagfish slime binds water loosely
Measuring the flow of water out of hagfish slime formed within a 1 l
cylinder revealed that the slime is not able to organize large volumes of
seawater over timescales of more than a few seconds. When the 2 l beaker was
dropped away from the hanging mesh-bottomed cylinder containing the slime, an
average of 1100±110 g of slime was trapped in the cylinder. The rate of
water egress from the slime was rapid at first (340±80 ml
s1 over the first 0.5 s) and declined rapidly over time
(Fig. 11). The decrease in
egress rate resulted from the decrease in the pressure head as the water level
dropped in the cylinder, and presumably the collapse of the slime and
obstruction of the mesh by the threads. Curiously, the rate of egress was not
linear when plotted on a semi-log scale, showing a distinct bump where the
flow rate increases and then decreases again
(Fig. 11). After 250 s of
hanging in air, the average mass left in the cylinder for the four trials was
only 62±13 g or about 5.6% of the initial mass.
|
Slime perturbation induces an ß transition in slime thread proteins
Slime threads from both unperturbed and perturbed slime bound CR, but only
threads from perturbed slime exhibited strong and extensive CR metachromasia
(Fig. 12). The few threads
that did exhibit CR metachromasia in unperturbed samples were likely strained
during collection or rinsing. Threads within perturbed slime also showed a
tendency to form parallel bundles, whereas unperturbed samples did not. The
presence of bundles is consistent with previous observations of bundles in
perturbed slime and in the slime found near hagfish eggs
(Koch et al., 1991a).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hagfish slime is 1000x more dilute than other mucus secretions
Gravimetric and `slimatocrit' measurements indicate that the mucins in
hagfish slime are present at only 15 mg l1, which is over
1000x more dilute than typical mammalian mucus secretions
(Table 1). Along with the slime
storage data reported in Fig.
5, the mucin concentration data can be used to calculate the
maximum volume of slime that a typical hagfish can produce. A typical E.
stoutii weighs about 60 g, so according to
Fig. 5, it should have about
2.2 g of slime exudate in its arsenal. Of these 2.2 g, about 66% of the mass
is water, leaving about 0.73 g of dry mucins and threads. The concentration
data show that mucins and threads make up approximately equal amounts of the
dry mass of the slime, which implies that about 0.36 g of the stored exudate
is dry mucins. At a final mucin concentration of 15 mg l1,
this amount of mucin could be used to make about 24 l of slime, or about
400x the hagfish's own volume. In contrast, if the mucins in hagfish
slime were as concentrated as they are in typical mammalian mucus (about 30 mg
ml), the hagfish would be able to produce only 12 ml of slime.
Our estimate of 24 l is far higher than Strahan's measurement of 0.5 l, but
closer to the estimates of Koch et al.
(1991b) and Goode and Bean
(1895
) (about 78 l).
Strahan's estimate came from placing hagfish in only 1 l of water. Because the
vast majority of slime volume is seawater, holding the animal in such a low
volume imposes an artificial ceiling on the maximum volume that can be
measured. Indeed, the average slime volume we measured in our 200 l aquarium
from a single pinch on the tail was 0.91 l, and this surely was only a
fraction of the animal's maximum capacity.
Why is the mucin concentration in hagfish slime so low? From an
evolutionary standpoint, one would expect selection to favour hagfish that can
produce functionally competent slime with as little energetic investment as
possible. Because it is mostly water, mucus may not seem like an energetically
expensive material to make, but for many marine organisms, mucus production
represents a large portion of their energy budget. From 1380% of the
energy intake of gastropods and chitons is used in the production of mucus
(Denny, 1989). In light of
these facts and the fact that 34% of a hagfish's wet mass is slime, it
is not surprising that selection has favoured hagfish that can produce slime
as cheaply as possible. The question of how hagfishes make such dilute slime
will be addressed later in the Discussion.
Hagfish slime is not a fibre-reinforced composite
The bi-directional taper of the slime threads depicted in
Fig. 6 is reminiscent of the
taper exhibited by collagen fibrils in tendon, which is generally attributed
to collagen's role as fibrous reinforcement in these structures
(Trotter and Koob, 1989).
Although they are quite long, collagen fibrils do not span an entire tendon,
and so stress must be transferred to adjacent fibrils via shear of
the proteoglycan matrix. Composite theory predicts that the tensile forces
borne by the fibrils decrease toward the fibril ends and are highest in the
middle. Thus, the most economical use of collagen protein is to make fibrils
that are tapered at both ends. Could this explain the taper of hagfish slime
threads?
If hagfish slime behaves as a fibre-reinforced composite-like tendon, then
shear transfer between adjacent slime threads must be significant. The
critical fibril aspect ratio (sc=fibril length divided by
radius) at which reinforcement is effective in a fibre composite is determined
by the ultimate stress (fu) of the fibril divided by the
yield stress of the matrix in shear (
my) or:
![]() | (1) |
CR staining demonstrates that during modest deformations of the slime, as
might occur while a hagfish or predator thrashes within it, most of the
threads are loaded to stresses higher than the yield stress
(Fig. 12), effecting an
ß transition in the thread intermediate filament (IF)
proteins. How do the threads get stretched past their yield point if the
mucins do not link them together? One possibility is that the slime threads
are so long that pulling one through water generates drag forces that
accumulate as significant tension in the thread. The drag force D on
a long cylinder pulled through seawater can be calculated as:
![]() | (2) |
A model of hagfish slime structure and mechanics
What is the function of the mucins if not to link the threads? Koch et al.
(1991b) demonstrated that
isolated slime thread skeins stirred into seawater in the absence of mucin
vesicles fail to produce anything resembling naturally formed slime. Instead,
the slime threads collapse into a small fibrous clot that binds relatively
little water. In addition, whole slime exudate containing both skeins and
mucin vesicles fails to produce viable slime in the presence of the disulfide
breaker DTT (Koch et al.,
1991b
). Here we demonstrated that DTT also delays slime
development and affects the viscoelastic properties of the slime. These
results suggest that the presence of cross-linked mucins is important to the
function of the slime. And yet, the extremely low concentration of mucins and
their low viscosity indicate that they can't possibly exist as a cross-linked
network that permeates the entire volume of slime. The only way to reconcile
these facts is if the distribution of mucins in the slime is heterogeneous,
with discrete networks of mucins at relatively high concentrations dispersed
in seawater with relatively few mucins. If this is indeed the case, then to
understand how the slime works, it is important to know the size of these
mucin networks.
A natural answer to the question of network size comes from looking at the
vesicles in which the mucins are packaged. Ejected slime exudate consists of
slime threads skeins and mucin vesicles from ruptured gland mucus cells. Mucin
vesicles are ellipsoids with a major axis length of about 7 µm
(Luchtel et al., 1991) and it
is not difficult to imagine that the mucins within a vesicle are cross-linked
into a discrete networks that would be dispersed by DTT. If mucin vesicles
swell but remain as intact networks of mucins, how are they distributed in the
slime? Mucin vesicles are known to bind readily to slime threads
(Koch et al., 1991b
), and it
is possible that the ratio and dimensions of slime threads and vesicles has
evolved to optimize the interaction between the two. The ratio of slime thread
surface area to the number of vesicles supports this idea. We estimate the
ratio of mucin vesicles to slime thread skeins to be about 5700:1. Given that
the threads are about 150 mm long and are tapered with a middle diameter of
3.0 µm and end diameters of 1.0 µm and 1.5 µm, the surface area of a
single thread is about 700 000 µm2. Each mucin vesicle should
therefore have about 120 µm2 of slime thread surface area on
average on which to bind. This means that all of the condensed mucin vesicles
(each with a projected surface area of about 21 µm2 assuming
major and minor axes of 7, 3 and 3 µm) could bind to a thread with room to
spare. If the vesicles swell to a degree that is typical of mucus granules
(i.e. 200% increase in radius; Verdugo,
1991
), they will cover more of the thread surface. This process is
depicted in Fig. 13.
|
If most of the slime is bulk seawater, how does the slime manage to be as
coherent as it is? The simplest answer is that the water is confined to
channels between the slime threads, and the flow through these channels is
slow enough to make the slime act as a coherent structure over short
timescales. Indeed, one of the things one notices when the slime is lifted out
of water is that water streams out of it, reducing its volume substantially.
Ferry (1941) noted this
phenomenon, and estimated that the slime collapses to 1/50th of its original
volume when it is handled. The results of our water egress experiment
(Fig. 11) support the idea
that the slime contains channels of bulk seawater. The take-home message from
these trials is that in its most expanded state, the slime does not immobilize
water like a gel, but only slows it down. A few calculations demonstrate that
our egress data are consistent with the structure of slime proposed above.
If we assume, as we did above, that a slime mass consists of equally spaced
and parallel mucin-coated slime threads that are 150 mm long, then we can
estimate the average distance between slime threads to be about 500 µm. The
rate of water flow through a pipe Q can be calculated using the
HagenPoiseuille equation (Vogel,
1994):
![]() | (3) |
According to this new model of hagfish slime structure, the slime is not as
much a coherent material as a very fine three-dimensional sieve that can trap
water over short timescales, but over longer timescales simply slows it down.
Thinking about the slime in this way is remarkably consistent with the
hypothesis that the slime functions as a defence against gill-breathing
predators (Fernholm, 1981;
Martini, 1998
). If the slime
indeed evolved as something that could bind to gills and disrupt respiratory
flow, one would not necessarily expect it to bind water irreversibly; slowing
it down would be sufficient. Furthermore, the tendency of the slime to
contract over time will decrease the distance between threads, and
dramatically reduce the flow rate, which varies according to the fourth power
of pipe radius.
Conclusions
In this paper we demonstrate that hagfish slime is remarkably dilute and
consists mostly of bulk seawater entrained between mucin-coated threads.
Although the threads that permeate the slime exhibit a bidirectional taper, we
have shown that the mucin component does not provide shear linkage between
threads as in a fibre-reinforced composite. The slime owes its coherence
mainly to the thousands of slime threads that are long enough to span the
entire structure. We propose that the main functions of the mucins are to bind
to the slime threads and aid in its rapid deployment, although the exact
mechanism of the latter is unknown. The slime is unlike a gel in that it does
not bind water over long timescales, and instead appears to function as a fine
three-dimensional sieve that may have evolved as a deterrent to gill-breathing
predators and competitors.
![]() |
List of abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bell, A. E., Sellers, L. A., Allen, A., Cunliffe, W. J., Morris, E. R. and Rossmurphy, S. B. (1985). Properties of gastric and duodenal mucus effect of proteolysis, disulfide reduction, bile, acid, ethanol, and hypertonicity on mucus gel structure. Gastroenterology 88,269 -280.[Medline]
Denny, M. (1979). The role of mucus in the locomotion and adhesion of the pulmonate slug, Ariolomax columbianus, pp. 298. PhD thesis, University of British Columbia, Vancouver, Canada.
Denny, M. (1989). Invertebrate mucus secretions: functional alternatives to vertebrate paradigms. In Mucus and Related Topics (ed. E. Chantler and N. A. Ratcliffe), pp. 337-366. Cambridge: Company of Biologists Limited.
Downing, S. W., Salo, W. L., Spitzer, R. H. and Koch, E. A. (1981a). The hagfish slime gland: a model system for studying the biology of mucus. Science 214,1143 -1145.[Medline]
Downing, S. W., Spitzer, R. H., Salo, W. L., Downing, S. D., Saidel, L. J. and Koch, E. A. (1981b). Hagfish slime gland thread cells: organization, biochemical features, and length. Science 212,326 -327.
Downing, S. W., Spitzer, R. H., Koch, E. A. and Salo, W. L. (1984). The hagfish slime gland thread cell. I. A unique cellular system for the study of intermediate filaments and intermediate filament-microtubule interactions. J. Cell Biol. 98,653 -669.[Abstract]
Fernholm, B. (1981). Thread cells from the slime glands of hagfish (Myxinidae). Acta Zool. 62,137 -145.
Ferry, J. D. (1941). A fibrous protein from the slime of the hagfish. J. Biol. Chem. 138,263 -268.
Fudge, D. S., Gardner, K. H., Forsyth, V. T., Riekel, C. and
Gosline, J. M. (2003). The mechanical properties of
hydrated intermediate filaments: Insights from hagfish slime threads.
Biophys. J. 85,2015
-2027.
Goode, G. B. and Bean, T. H. (1895). Oceanic Ichthyology: A Treatise on the Deep-Sea and Pelagic Fishes of the World, based chiefly upon the collections made by the steamers Blake, Albatross and Fish Hawk in the Northwestern Atlantic. Washington: Government Printing Office.
Koch, E. A., Spitzer, R. H. and Pithawalla, R. B. (1991a). Structural forms and possible roles of alinged cytoskeletal biopolymers in hagfish (slime eel) mucus. J. Struct. Biol. 106,205 -210.[CrossRef]
Koch, E. A., Spitzer, R. H., Pithawalla, R. B. and Downing, S. W. (1991b). Keratin-like components of gland thread cells modulate the properties of mucus from hagfish (Eptatretus stoutii). Cell Tissue Res. 264,79 -86.[CrossRef][Medline]
Koch, E. A., Spitzer, R. H., Pithawalla, R. B. and Parry, D.
A. (1994). An unusual intermediate filament subunit from the
cytoskeletal biopolymer released extracellularly into seawater by the
primitive hagfish (Eptatretus stoutii). J. Cell
Sci. 107,3133
-3144.
Koch, E. A., Spitzer, R. H., Pithawalla, R. B., Castillos, F. A., 3rd and Parry, D. A. (1995). Hagfish biopolymer: a type I/type II homologue of epidermal keratin intermediate filaments. Int. J. Biol. Macromol. 17,283 -292.[CrossRef][Medline]
Luchtel, D. L., Martin, A. W. and Deyrup-Olson, I. (1991). Ultrastructure and permeability characteristics of the membranes of mucous granules of the hagfish. Tissue Cell 23,939 -948.[CrossRef]
Majima, Y., Sakakura, Y., Matsubara, T., Murai, S. and Miyoshi, Y. (1983). Mucociliary clearance in chronic sinusitis-related human nasal clearance and in vitro bullfrog palate clearance. Biorheology 20,251 -262.[Medline]
Martini, F. H. (1998). The ecology of hagfishes. In The Biology of Hagfishes (ed. J. M. Jorgensen, J. P. Lomholt, R. E. Weber and H. Malte), pp.57 -77. New York: Chapman and Hall.
Pain, R. H. (1980). Gastric mucus gel: a challenge for biophysics. In Biomolecular Structure, Conformation, Function and Evolution (ed. R. Srinivasan). London: Pergamon.
Sellers, L. A. and Allen, A. (1989). Gastrointestinal mucus gel rheology. In Mucus and Related Topics (ed. E. Chantler and N. A. Ratcliffe), pp.65 -71. Cambridge: Company of Biologists Limited.
Spitzer, R. H., Downing, S. W., Koch, E. A., Salo, W. L. and Saidel, L. J. (1984). Hagfish slime gland thread cells. II. Isolation and characterization of intermediate filament components associated with the thread. J. Cell Biol. 98,670 -677.[Abstract]
Strahan, R. (1959). Slime production in Myxine glutinosa Linnaeus. Copeia 2, 165-166.
Trotter, J. A. and Koob, T. J. (1989). Collagen and proteoglycan in a sea-urchin ligament with mutable mechanical properties. Cell Tissue Res. 258,527 -539.[Medline]
Veerman, E. C., Valentijn-Benz, M. and Nieuw Amerongen, A. V. (1989). Viscosity of human salivary mucins: effect of pH and ionic strength and role of sialic acid. J. Biol. Buccale 17,297 -306.[Medline]
Verdugo, P. (1991). Mucin exocytosis. Am. Rev. Respir. Dis. 144,S33 -S37.[Medline]
Vogel, S. (1994). Life in Moving Fluids, p. 467. Princeton NJ: Princeton University Press.
Winter, D. A. (1979). Biomechanics of Human Movement. New York: John Wiley Press.