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First published online January 31, 2006
Journal of Experimental Biology 209, 702-710 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02067
Hagfish slime ecomechanics: testing the gill-clogging hypothesis
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 27 December 2005
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Summary |
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Key words: biomechanics, slime, hagfish, gill resistance, predator defence, Eptatretus stoutii
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Strahan, 1959;
Downing et al., 1981a;
Martini, 1998). In a previous
paper (Fudge et al., 2005), we
demonstrated that hagfish slime is an extremely dilute assemblage of mucins
and seawater held together by a network of fine protein threads. Measurements
of water egress from hagfish slime indicated that it is not a coherent
material that immobilizes water, but instead a fine sieve that slows water
down via viscous entrainment. These experiments, along with the many
papers on hagfish slime by the late Elizabeth Koch and other researchers
(Downing et al., 1981b;
Fernholm, 1981;
Koch et al., 1991;
Fudge et al., 2003), answer
several questions about slime morphology and mechanics. However, fundamental
questions about the function of the slime persist.
The list of common hagfish predators includes certain species of seabirds,
pinnipeds and cetaceans but exhibits a conspicuous lack of fishes
(Martini, 1998;
Fudge, 2001). This fact has led
researchers to speculate that the slime functions as a defence against
gill-breathing predators by clogging the gills
(Fernholm, 1981;
Martini, 1998). The mechanical
data we report in Fudge et al.
(2005) on slime formed in
vitro do not contradict this hypothesis. We found that the threads within
hagfish slime are extremely effective at catching on projections and making
continuous connections across substantial distances. While the slime does not
possess the coherence of a solid material, it is capable of trapping large
volumes of water via viscous entrainment. From these data it is not
difficult to imagine that the slime would attach easily to gills and seriously
impair respiratory flow across them. Here, we test the gill-clogging
hypothesis by measuring the effect of hagfish slime on water flow through an
artificial gill analogue and real gills in isolated fish heads and demonstrate
that the slime has dramatic effects on flow at physiological water pressures.
We also provide information from high-speed video trials on the details of
slime release and formation by free-swimming hagfish.
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A series of tubing formed the rest of both versions of the slime vacuum. Each gill setup was connected to polyvinyl tubing (1.52 m long, 8 mm inner diameter) followed by a short segment of rubber tubing (225 mm long, 6.6 mm inner diameter), which could be clamped to restrict water flow. Screw adapters joined the consecutive pieces. Experiments were held in a 20-litre aquarium of cold artificial seawater (8-10°C, 32%thou). For artificial gill trials, the gill setup was attached to a plastic rod and held in position underwater by clamping the rod to the rim of the aquarium. In fish head trials, the apparatus was kept in place at one end of the aquarium with bricks. A bucket on a top-loading balance placed below the aquarium collected the siphoned water. The free end of the rubber tubing rested in a small overflowing beaker positioned directly above the bucket, reducing the incidence of air bubbles within the tubing. All trials had a starting pressure head of 3.48 kPa, which was determined from the vertical distance between the water level in the aquarium and the top of the overflowing beaker.
A live hagfish was gently placed in the aquarium, and 40-90 s after the
start of the siphon the hagfish was pinched on the tail with padded forceps to
induce sliming (Fudge et al.,
2005). A video camera and VCR recorded the display on the
top-loading balance for later review. Outputs from an external timer and a
second camera filming a view of the aquarium were recorded simultaneously on
to the same tape so that data from the balance could be correlated with events
in the tank and time-stamped. Recording was stopped after the balance reached
its upper limit (3000 g).
Water flow rates were determined from the change in mass of water in the bucket and the time interval between mass measurements. To adjust for the decreasing pressure head as water flowed from the aquarium, we calculated standardized water flow rates (ml s-1 kPa-1) over time by dividing each flow rate measurement by the pressure head at the time of the measurement. All subsequent calculations involving flow rates used these standardized values. The siphon system consisted of two components in series that contributed to the total resistance (R) that the system presented to the flow of water: the gills (test tube brush or fish head gills) and the narrow tubing connected to the gills. That is, Rsystem=Rgills+Rtube. Measurements of flow rates with and without the gills present were used to calculate gill resistance relative to the rest of the siphon, and the pressure drop across the gills. For the artificial gill setup, the test tube brush was removed from its thick polyvinyl tube housing to achieve a gill-less condition. In the fish head setup, the gills were removed by pulling off the dental dam holding the fish head and removing the entire head from the PVC pipe.
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Convective mixing effects and slime collapse
In the high-speed video trials using constrained hagfish, we observed that
exudate released by the hagfish did not hydrate fully, as indicated by it
remaining opaque and sinking to the bottom of the aquarium. This observation
led us to test the hypothesis that some convective mixing is required for
proper slime hydration and formation. To test this hypothesis, we conducted
two additional kinds of video trials in which we filmed the introduction of
freshly collected slime exudate into still seawater either using a spatula or
via injection with a syringe fitted with a shortened 18-gauge needle.
The capture rate for these trials was 60 frames s-1.
We also assessed the effect of mixing on slime formation using a `removable
mass' assay modified from Koch et al.
(1991). A small volume (0.12
ml) of slime exudate stabilized in a high osmotic strength buffer
(Downing et al., 1984) was
injected into 50 ml of artificial seawater on a shaker table set at 200 revs
min-1. After shaking for a precise amount of time (0, 10, 20, 40,
80, 160, 320, or 640 s), a custom hook, which was placed in the beaker before
the addition of slime, was removed (Fig.
3). Removable mass was quantified by weighing the hook and
adherent slime and subtracting the mass of the hook.
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 | (1) |
where 
P is the pressure head,
is the flow rate, and R is
the resistance. Standardized water flow rates, which we will call
p, are given by
/P. Consequently, Eqn
1 can be written in terms of
p, and then rearranged to
give:
 | (2) |
where Rgills is the resistance of the gills and
Rtube is the resistance of the tubing. Using measurements
of water flow rates with and without the gills present, we determined the
relative magnitudes of Rgills and
Rtube. The relative resistance of the gills is given by:
 | (3) |
and the relative resistance of the tubing is simply:
 | (4) |
For the artificial gills, the mean flow rate without gills was 8.3 ml
s-1 kPa-1, while the mean rate with gills present was
7.9 ml s-1 kPa-1; thus, Rgills,rel
is 0.044±0.0037 (mean ± s.d.; N=3), and
Rtube,rel is 0.956. That is, the tube resistance is
approximately 20 times greater than the artificial gill resistance, which
accounts for only 4% of the total resistance in an unslimed system. Because
the rockfish heads used in the fish head trials varied in size, the relative
resistance of the real fish gills was more variable, ranging from 0.061 to
0.15 (mean ± s.d., 0.11±0.047; N=3). The pressure drop
across the gills was found by multiplying the relative gill resistance by the
mean pressure head in the trial. Mean pressures (± s.d.) across the
artificial gills (0.17±0.014 kPa; N=3) and real fish gills
(0.35±0.16 kPa; N=3) were comparable to pressures found during
normal ventilation in other fishes (e.g. white sucker Catostomus
commersoni, 0.2 kPa; carp Cyprinus carpio, 0.5 kPa)
(Saunders, 1961).
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 | (5) |
We know Rtube,rel (Eqn 4), and, because we assume that
Rtube remains constant, we can calculate its absolute
magnitude using data on the pre-slime conditions in the system. Rearranging
Eqn 2 (Ohm's Law) and indicating initial conditions before the gills are
exposed to slime (denoted by the zero subscript) gives:
 | (6) |
We define Rtube as the constant C, and
substitute Eqn 6 into Eqn 5 to get the constant value:
 | (7) |
Equation 2 can now be written in terms of the flow rate and the gill
resistance as functions of time (t):
 | (8) |
Rearranging gives:
 | (9) |
which we can use to calculate gill resistance during the experiment from the flow rate data.
All trials showed slowed water flow and an increase in gill resistance
following slime release (Table
1). Flow rate and resistance data are presented as normalized
values, p,norm and
Rgills,norm, obtained by dividing
p(t) and
Rgills(t) by their mean pre-slime values. The
start of slime suction, as observed from video recordings of the aquarium,
corresponded well with abrupt changes in flow and resistance. Slime uptake
into the artificial gill corresponded with a decrease in flow rate by a factor
of 70-80 (Fig. 5A) and an
increase in resistance of approximately three orders of magnitude
(Fig. 5B). Two trials with the
fish head setup were usable for data analysis. In these trials, slime caused
the flow rate to decrease by a factor of 4-8
(Fig. 6A) and the gill
resistance to increase by one to two orders of magnitude
(Fig. 6B).
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). The slime's greater
effect on the artificial gill model is likely to be due to the multi-layered
and densely packed bristles of the test tube brush, which would catch more
slime than the single layer of wider-spaced gill rakers in the fish head.
Also, the smaller area of the model gill's opening compared with the open area of the fish mouth makes the model gills easier to block with a given amount of slime; the slime is more concentrated in this small area, and water flow is impaired to a greater extent.
For a live fish predator, sustained low water flow over the gills might
lead to insufficient oxygen delivery and reduced gas exchange. Furthermore,
the increase in diffusion distance across the gills caused by a slime coating
should also decrease gas exchange, as diffusion rates are inversely
proportional to distance (Fick's Law)
(Vogel, 2003). This hypothesis
will be tested in future experiments using respirometry of live fish exposed
to hagfish slime. The potential for suffocation through one or both of these
mechanisms might discourage gill-breathing predators from preying on
hagfish.
High-speed video of free-swimming hagfish revealed that they do not
generally release slime and then hide within it
(Fig. 12). The local release
of exudate supports this idea; simultaneous slime release from all of the
glands would likely be more effective at producing a mass of slime for an
instant refuge. Hagfish have an ingenious behaviour, however, that implies
that they do occasionally have to free themselves from their own slime.
Covered in slime and facing eventual suffocation, a hagfish will tie its body
in a knot and pass the knot toward its head to slough off the slime
(Strahan, 1963;
Martini, 1998). While not a
protective shroud, the behaviour of slime release suggests that it may have a
more active role in defending hagfish against predators. When pinched, slime
glands near the region of contact respond by forcefully ejecting exudate as a
coherent jet. It is possible that the combination of local and forceful
release of slime is functionally important in `targeting' the gills of an
attacking fish predator.
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 | (10) |
where 
sphere is the wall stress, p is the
pressure, r is the radius and d is the wall thickness. Using
a typical muscle stress of 200 kPa, a gland radius of 0.65 mm and a wall
thickness of 45 mm (Lametschwandtner et
al., 1986), we get a pressure inside the gland of 28 kPa, or about
double the blood pressure of a mammal.
To calculate the velocity of the exudate as it exits the gland, we use the
Hagen-Poiseuille equation for flow through a pipe:
 | (11) |
where is flow, a is the
radius of the pipe (45 µm), p is the pressure head, µ is
the dynamic viscosity and l is the duct length. Since we already know
the jet velocity (0.175 m s-1) from high-speed video, we can use
this equation to calculate the viscosity of the exudate. If it gives us a
reasonable value, then we know that the muscular gland capsule is capable of
ejecting the slime without help from the surrounding myotomal muscle.
Rearranging the equation above, we get:
 | (12) |
), we get a viscosity of 0.08 Pa s, which is about
60 times the viscosity of seawater at 10°C and not an unreasonable value.
If the calculation predicted a viscosity considerably less than water, then
clearly we would need to invoke another source of pressure. Our estimates
indicate, however, that the gland capsule can eject a fluid with a viscosity
60 times that of seawater at the velocities we have measured; thus, another
mechanism, such as compression of the gland via contraction of nearby
myotomal muscle, is not required to explain our data.
After exudate is discharged into seawater, convective mixing is essential
for rapid hydration and full expansion of the slime. The Reynolds number
(Re) of the exudate jet is informative on this point. Using the
values for exudate viscosity and jet velocity that we calculated above, and
the gland duct diameter, Re within the duct is 
0.1. Because flow
immediately outside the duct is unlikely to differ much from the flow inside
the duct, the Re indicates that the exudate jet is laminar. As a
result, the exudate experiences very little mixing from inherent turbulence in
the jet despite its seemingly forceful ejection. Also, given the relatively
large size-scale of the slime, diffusion alone is insufficient to cause
formation once the exudate is in seawater. In nature, convective mixing is
likely fulfilled by the hagfish itself, as escape behaviours often include
vigorous thrashing after slime release. While this requisite mixing appears at
first to be a limitation, it may serve an important function: if expansion
were faster, the slime would form closer to the slime gland pore. This could
decrease the distance that the slime is shot and potentially even clog the
gland pore. The laminar character of the exudate jet and the full formation of
slime some time after release from the gland also support the idea that the
jet is more important in the targeting of predator gills than other functions,
such as mixing.
Removable mass trials showing the non-linear relationship between the
amount of final slime product and stirring time underscore the convective
mixing result. They also indicate, however, that mixing past a certain point
decreases the mass of slime produced. This agrees with previous studies that
have demonstrated that the slime collapses when it is disturbed
(Ferry, 1941;
Fudge et al., 2005). In a
future study, we will explore in more detail the mechanism by which the mucins
and fibrous threads interact with seawater and each other to form fully
hydrated slime.
The sieve model of hagfish slime in which water is loosely bound is
consistent with the anti-predator role of the slime when one considers the
functional trade-offs between a slime that binds water loosely versus
a gel that binds it tightly. Hydration is slower in a loosely binding slime,
meaning the exudate jet can travel farther than it would if it had a greater
affinity for water. In addition, the resulting slime has a greater volume and
is less mechanically coherent. Such slime may have more opportunity to
initially stick to the gills of a predator and tangle between the gill rakers
compared with a more coherent and smaller slime mass. Prolonged agitation of
the slime from any subsequent thrashing will also cause the slime to collapse
more completely on the gills, as the results of our removable mass experiments
imply. At one extreme, slime with little coherence might be more likely to
catch on the gills but may not interfere much with respiratory flow. At the
other extreme, coherent slime might effectively block water flow but may be
ineffective at lodging in the gills in the first place. In addition, a tight
plug of slime would be easier for a fish to dislodge via `coughing'.
Thus, the strength of the interaction between the slime and seawater may be a
compromise among several requirements for effective anti-predator activity.
While the focus of the present study has been the anti-predator function of
hagfish slime, the slime should be equally effective at endangering
gill-breathing competitors. Hagfish also release slime during feeding
(Martini, 1998) and this could
serve to deter competitors from imposing themselves on a hagfish's meal.
Conclusions
We demonstrate here that hagfish slime can clog fish gills, which increases
gill resistance and slows water flow through them. The potential for entrapped
slime to interfere with gill respiration suggests that the slime may have
evolved to deter gill-breathing animals from preying on hagfish. We have shown
that the release of slime exudate is local and that its forceful ejection from
the slime gland can be accomplished by contraction of the gland capsule muscle
alone. Once slime is released into the water, the extent of its hydration and
expansion depends on the amount of convective mixing in the water. The
mechanical consequences arising from different models of how tightly water is
bound to the slime imply that hagfish slime's loose water binding is
functionally important in defending hagfish against gill-breathing
predators.
p
p,no gills
p,norm
p,0
p,with gills
p
P
sphere
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Downing, S. W., Salo, W. L., Spitzer, R. H. and Koch, E. A.
(1981a). The hagfish slime gland: a model system for studying the
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214,1143
-1145.
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.
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