The buccal buckle: the functional morphology of venom spitting in cobras
Department of Biology, Lafayette College, Easton, PA 18042, USA
* Author for correspondence at present address: School of Biological Sciences, PO Box 664236, Washington State University, Pullman, WA 99164-4236, USA (e-mail: youngb{at}wsu.edu)
Accepted 5 July 2004
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Summary |
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Key words: snake, reptile, fluid pressure, dentition, defensive behavior, venom
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Introduction |
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Some cobras can spit their venom as far as 3 m; the fluid pressures
required to propel venom that far were explored by Freyvogel and Honegger
(1965). Rosenberg
(1967
) described how venom
pressure could be generated by the contraction of the skeletal muscles that
contact the venom gland. This aspect of venom expulsion mechanics has been
supported by experimental studies of rattlesnakes
(Young et al., 2000
). Through
direct measurement of venom flow coupled with high-speed digital videography,
a new model for the mechanics of venom expulsion has been developed
(Young et al., 2002
). This new
model, termed the pressure-balance model, emphasizes the functional
significance of the soft-tissue structures in the distal portion of the venom
delivery system near the fang sheath.
Recent experimental work has demonstrated that displacement of the fang
sheath towards the base of the fang, as well as pressure changes in the venom
delivery system, can significantly influence venom flow (Young et al.,
2001a,
2003
). In most venomous snakes
physical displacement of the fang sheath, either by a container during milking
or by the target surface during fang penetration, is a prerequisite for venom
release. Spitting cobras appear to be unique among venomous snakes in their
ability to expel their venom without making direct physical contact with
another object or organism. The classic description of the mechanics of venom
spitting in cobras (Bogert,
1943
) emphasized the specialized exit orifice of the fang of
spitting cobras. The exit orifice (Fig.
1) of spitting cobras is directed more craniad and has a more
circular aperture than the exit orifice of non-spitting cobras. These
dentitional specializations explain how the venom stream expelled by spitting
cobras travels forward, rather than downward, but do not explain how the venom
is expulsed without physical contact. The goal of this study was to test the
hypothesis that venom spitting in cobras is dependent on deformation of the
fang sheath and thus is functionally convergent with the venom delivery
mechanics of crotalids.
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Materials and methods |
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High-speed videography and photography
Multiple spitting episodes (at least five episodes each for two N.
nigricollis, and one each of N. pallida and N.
siamensis) were recorded using a MotionScope 1000S (Redlake Instruments)
at 500 frames s1 with a 1/2000 s shutter speed. The digital
record was then streamed to a G4 computer (Apple) and saved using Premiere 6.5
(Adobe). Subsequent quantification of the temporal pattern of the spit and jaw
angles were performed using N.I.H. Image 1.6.3. Every spitting and
non-spitting cobra was photographed during different forms of venom expulsion
(milking, striking and spitting).
Observations
Our analyses of live specimens were augmented by standard video recordings
of over 700 spitting episodes (detailed in
Rasmussen et al., 1995), which
include footage of N. nigricollis, N. mossambica, N. pallida, N.
siamensis and Hemachatus haemachatus Lacepede. As part of an
earlier study of spitting cobra venom
(Cascardi et al., 1999
), B.A.Y.
maintained seven adult Naja pallida, which were regularly induced to
spit. The prey ingestion and transport sequence of these snakes was videotaped
by Alexandra Deufel (Deufel and Cundall,
2004
) who was kind enough to provide us with a copy of the video
footage.
Anatomy
To understand the anatomical basis of spitting in cobras, we examined the
gross and microscopic morphology of the venom apparatus of a number of
species. We examined prepared skulls of N. naja (Museum of
Comparative Zoology, MCZ 4038); N. nigricollis (MCZ 53505, 53740;
Field Museum of Natural History, FMNH 98910, and United States National
Museum, USNM 320722); N. melanoleuca (FMNH 31364, USNM 320711), as
well as one skull of N. pallida from the private collection of B.A.Y.
Gross dissection was performed on the venom delivery systems of preserved
adult specimens of N. nigricollis (MCZ 18477, USNM 40991), N.
melanoleuca (FMNH 191427, MCZ 49688, USNM 49013), as well as the
following species from the private collection of B.A.Y. (Naja kaouthia, N.
naja, N. nigricollis, N. nivea, N. pallida and H.
haemachatus).
As part of an earlier study (Young et
al., 2001b) of the comparative microscopic anatomy of the distal
venom delivery system in snakes, the head and several anterior vertebrae were
removed from previously preserved elapid specimens (Boulengerina annulata,
Hemachatus haemachatus, Naja nigricollis and N. sputatrix) and
placed in decalcifying solution (Cal-Ex, Fisher, Pittsburgh, USA) for
72168 h. Following decalcification, each head was bisected sagitally
and tissues caudal to the midpoint of the venom gland were discarded. Each
sample was dehydrated and cleared through a progressive ethanol series and
Hemo-De (Fisher) prior to embedding in Paraplast (Fisher). Serial sections
were cut at 10 µm, with one side of the head being sectioned parasagitally
and the other sectioned frontally. Sections were stained using either Van
Gieson's stain or Masson's Trichrome stain (following
Luna, 1968
;
Presnell and Schreibman,
1997
), which provide clear distinction between connective tissue,
muscle and epithelium, and then were examined and photographed using an E800M
compound microscope (Nikon, Melville, USA). Additional observations were made
on serial sections through the head of Walterinnesia aegyptia kindly
provided by Elazar Kochva.
Fully ankylosed, functional fangs were removed from preserved adult specimens of N. kaouthia and N. pallida from the private collection of B.A.Y. The fangs were air-dried prior to being critical-point-dried (Polaron, Watford, UK), and coated with 300 Å of gold (PS-2, International Scientific Instruments, Prahan, Australia). The fangs were examined and photographed at 15 kV using a Super-3A scanning electron microscope (International Scientific Instruments).
Stimulation and strain gauges
To explore the mechanical role of the M. protractor pterygoideus in
spitting, we anesthetized (via exposure to isoflurane and an
intramuscular injection of 65 mg kg1 ketamine
hydrochloride:acepromazine in a 9:1 ratio) an adult specimen of N.
siamensis and surgically exposed this muscle unilaterally. A bipolar
stimulating probe was applied to the surface of the muscle and a range of
electrical stimulations were presented using an S88 Stimulator (GRASS, West
Warwick, USA). To better document the resultant changes, these stimulations
were repeated following the attachment (via Vetbond; Sarasota, USA)
of a uniaxial strain gauge (EA-13-062AK-120, Measurements Group, Raleigh, USA)
on the oral mucosa of the roof of the mouth immediately below, and parallel to
the long axes of, the maxilloectopterygoid and palatopterygoid joints. The
strain gauge was coupled to a P122 amplifier (GRASS) and the signal from the
amplifier, along with a synch pulse from the stimulator, was transferred to a
G4 computer (Apple) at 20 kHz sampling rate using the Instrunet data
acquisition system (G.W. Instruments, Somerville, USA) and quantified using
SuperScope (G.W. Instruments).
A second uniaxial strain gauge was attached (with Vetbond) to the dorsal scales over, and parallel to the long axis of, the nasofrontal joint of an adult N. nigricollis that had been lightly anesthetized through inhalatory exposure of isoflurane. When the snake was fully recovered from the anesthesia it was placed unrestrained in an open container 75 cmx75 cmx50 cm tall. The strain gauge signal was amplified and recorded as above; one of the experimenters induced the cobra to spit and depressed a remote switch to generate a marker voltage, which was recorded by the computer simultaneously with the strain gauge output.
Venom pressure
Venom pressure was measured at two sites on two adult specimens of N.
nigricollis. Each snake was anesthetized as described above, then placed
on a heated surgical table (VSSI) and maintained on isoflurane using a
low-flow ventilator (Anesco, Waukesha, USA). A small rotary tool was used to
remove the end of the fang proximal to the exit orifice, then a 60 cm length
of polyethylene (PE) tubing was placed over the fang. The inner diameter of
the PE tubing was such that a tight fit was achieved with the outer surface of
the fang. The free end of the PE tubing was attached to a PT300 pressure
transducer (GRASS), and both were filled with Ringer's solution. The M.
protractor pterygoideus and M. adductor mandibulae externus superficialis were
then surgically exposed. Using a dual bipolar probe and the S88 Stimulator
(GRASS), each muscle was stimulated individually, then the two muscles were
stimulated simultaneously. During the stimulations the exit port of the
pressure transducer was sealed so that the transducer formed a closed system
that would not dissipate venom when pressurized. The pressure transducer was
coupled to a P122 amplifier (GRASS), and the final signal, along with a
synchronized pulse from the stimulator, was captured by the data acquisition
system as described above.
In an effort to determine the influence of the soft tissues of the distal end of the venom delivery system, venom pressures were also recorded from the proximal portion of the venom duct. For this preparation, a portion of the venom duct was surgically isolated from the surrounding connective tissue, vasculature and supralabial gland. A small incision was made on the lateral surface of the venom duct and a PE tubing catheter was inserted into the lumen of the duct. Silk suture was used to anchor the catheter in place and to prevent venom leakage around the catheter. In both preparations the muscles received at least 5 twitch stimuli (1 stimulus every 2 s, 15 ms duration, 8 V) as well as one or two train stimuli (35 p.p.s., 27 ms duration, 8 V).
Electromyography
Two adult specimens of N. nigricollis were used for the EMG
experiments. The animals were anesthetized with isoflurane and two small
incisions made in the dorsal scales of the head. Bipolar EMG electrodes 1 m in
length were fashioned from 0.05 mm diameter stainless steel wire with nylon
insulation (California Fine Wire, Grover Beach, USA), and inserted into either
the M. protractor pterygoideus or M. adductor mandibulae externus
superficialis using hypodermic needles. The incisions were closed with silk
suture and Vetbond, and the EMG leads were glued to one another and to a
tether of suture anchored to the dorsal midline of the snake's neck.
With the leads in place, and the cobra recovering from anesthesia, the snake was placed in a clear acrylic tube 50 cm long such that the anterior and posterior ends of the snake projected beyond the tube. This tube allowed us to safely restrain the snake (by holding the posterior body); though the snake had full movement of its head and neck, it had reduced opportunities to damage the EMG leads. With the snake restrained in this fashion, the EMG leads were coupled to two P511 amplifiers (GRASS), which were coupled to the data acquisition system as described above and sampled at 33 kHz. We built a spit detector to generate a marker on our computer records. This detector was constructed from a 15 cm2 plate of Plexiglas onto which we laid intermeshed strips of copper tape with only 1 mm gap between them. Alternate strips were wired to either the anode or cathode of a 6 V battery, with the termini connected to the data acquisition system. The cobra was induced to spit by one of the experimenters; if the spit detector was held in front of the experimenter's face (which the cobra targets) the venom striking the detector would form a complete circuit, sending a pulse to the data acquisition system.
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Results |
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The spit was always released with the mouth slightly open, typically around 25° (mean ± S.E.M. = 22.6±2.5°, N=16); as the venom was released the gape would decrease slightly then increase again. Immediately prior to the onset of venom expulsion, four concurrent displacements were observed in the skull. First, the snout complex rotated in the sagittal plane such that the tip of the snout was elevated relative to the resting position. Second, the caudal end of the maxilla was displaced laterally causing a bulge or deformation of the overlying supralabial scales (Fig. 2A). Third, the fang sheath, the drape of connective tissue and epithelium surrounding the fang, elevated to expose the fang tip. Lastly, two ventrally directed projections appeared in the oral mucosa of the roof of the mouth. These projections occurred side by side, with a slight gap between them, at a level slightly caudal to the scale bulge associated with the maxilla. The palatal bulges were particularly evident if the cobra was filmed from a slightly ventral perspective (Fig. 2B).
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With the cobras anesthetized prior to the surgical procedures, force was applied manually to the quadratopterygoid articulation in an attempt to protract the palato-maxillary arch. This manipulation produced the same suite of displacements observed within the skull during spitting, though the projections of the palatal mucosa were not as pronounced.
Observations
The slower frame rate of the standard video recordings of spitting made it
more difficult to discern all of the kinematic features. Nevertheless, the
displacements described above appeared to be a consistent feature of the
spitting behavior of every species. These same displacements were absent from
the video recordings of other venom expulsion events (such as milking or prey
capture) involving both spitting and non-spitting cobras. The palato-maxillary
displacement associated with prey ingestion, the well-known `pterygoid walk'
of snakes (see Boltt and Ewer,
1964; Deufel and Cundall,
2004
), was evident in video sequences of N. pallida;
however, these displacements were distinct from those observed during
spitting. The suite of displacements that was consistently observed during
spitting was never observed in non-spitting cobras, nor was it recorded from
spitting cobras engaged in any other behavior, including other forms of venom
expulsion.
Anatomy
A detailed description of the cephalic morphology of Naja is
beyond the scope of this contribution (see
Radovanovic, 1928; Haas,
1930
,
1973
;
Deufel and Cundall, 2004
), the
following is intended only as a general orientation. The upper jaw, or
palato-maxillary arch, of cobras consists of four bones, the pterygoid,
ectopterygoid, palatine and maxilla. The dentiferous pterygoid, the caudal
element in the series, is a horizontal bar of bone, the caudal end of which
deflects laterad and becomes more spatulate
(Fig. 3). The proximal end of
the non-dentiferous ectopterygoid has an elongate but poorly defined
articulation on the dorsal surface of the pterygoid. The ectopterygoid extends
craniolaterally with a slight dorsal deflection; the distal end of the
ectopterygoid is a broad, horizontal plate of bone
(Fig. 3). The caudal end of the
dentiferous palatine abuts the cranial tip of the pterygoid; from here the
palatine extends craniolaterally to approach the cranial tip of the maxilla
(Fig. 3). The cranial end of
the palatine supports a large medial process that has a connective tissue link
to the frontals, and a lateral process that is bound (via connective
tissue) to the maxilla and prefrontal; the cranial tip of the palatine has a
connective tissue attachment to the overlying ventral portions of the snout
complex (vomer, septomaxilla and nasal). The caudal end of the maxilla rests
under the distal end of the ectopterygoid
(Fig. 3); the medial portion of
this contact is heavily imbued with dense connective tissue while the lateral
portion has a more complex articulation and less connective tissue. Distally
the maxilla supports a medial process that contacts the palatine, and the
dorsal surface of the maxilla has an extensive articulation with the distal
end of the prefrontal (Fig.
3).
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Four skeletal muscles contact the palato-maxillary arch. The M. protractor pterygoideus originates from the caudolateral surface of a prominent ridge, which forms the caudolateral portion of the suture between the parietal and basisphenoid bones, and inserts on the caudal two-thirds of the pterygoid (Fig. 3). The M. levator pterygoideus originates from the lateral surface of the skull and the postorbital fossa (at a point cranial and dorsal to that of the protractor) and inserts on the pterygoid near the pterygoidoectopterygoid joint (Fig. 3). The M. retractor pterygoideus originates immediately cranial to the protractor, from the craniolateral surface of the parietobasisphenoid ridge, and inserts on the cranial tip of the pterygoid and the caudal palatine (Fig. 3). The largest of these four muscles, the M. pterygoideus, originates from the caudal end of the compound bone of the lower jaw and the adjacent caudolateral surface of the pterygoid, and inserts onto the distal end of the ectopterygoid and the connective tissue surrounding the maxilloectopterygoid joint (Fig. 3).
The fang and replacement fang are surrounded by a drape of connective tissue and epithelium termed the fang sheath (Fig. 4). The fang sheath is devoid of smooth and skeletal muscle tissue. The dorsal margin of the fang sheath is attached to the lateral and cranial surfaces of the maxilla (Fig. 4) as well as the adjacent oral mucosa. The venom duct is closely attached to the lateral surface of the maxilla. The caudolateral surface of the fang sheath is penetrated by the venom duct, which continues to course craniad within the fang sheath. At the cranial surface of the maxilla the venom duct expands in width and arches medially to form the venom vestibule (Fig. 4). The caudal margin of the venom vestibule supports two distinct foramina. These foramina extend caudally to a venom chamber, which surrounds each fang (Fig. 4). The epithelial linings of the venom chambers, the foramina and the venom vestibule are continuous with that of the venom duct (Fig. 4). This distal portion of the venom delivery system is devoid of skeletal muscle; these soft tissue chambers are contained entirely within the fang sheath.
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A comparison of the gross and histologic morphology of the palato-maxillary arch among spitting (mainly H. haemachatus, N. nigricollis and N. pallida) and non-spitting (mainly N. melanoleuca, N. naja and Walterinessia aegyptia) cobras revealed only minor differences beyond those at the fang's exit orifice (Fig. 1). The maxilla of non-spitting cobras was more horizontal at rest, the connective tissue asymmetry of the maxilloectopterygoid joint was more pronounced in spitting cobras, and the M. protractor pterygoideus appeared to be proportionately larger in spitting cobras (we lack the necessary series to quantify the latter observation). Neither these, nor the other subtle morphological differences we observed, appear to be a key morphological feature associated with the ability to spit venom.
Stimulation and strain gauges
In N. siamensis the contraction of the M. protractor pterygoideus,
triggered by electric stimulation, produced displacements of the
palato-maxillary arch that were clearly visible on the roof of the mouth
(Fig. 5). The maxilla was
protracted and rotated in the frontal plane such that the caudal end of the
maxilla moved laterally. The palatine is protracted, though seemingly not as
much as the maxilla, and appears to rotate in the frontal and sagittal plane
(the caudal palatine teeth become exposed while the anteriormost are not).
There is a ventral buckling of the palato-maxillary arch in the region of the
palatopterygoid joint and the maxilloectopterygoid joint. This buckling was
manifest as two adjacent ventral protrusions from the palatal mucosa.
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The displacement of the palatopterygoid and maxilloectopterygoid joints is evident in the signal obtained from the strain gauge attached to the oral mucosa under these joints (Fig. 6A). Twelve electrical stimulations of the M. protractor pterygoideus resulted in consistent voltage spikes from the strain gauge amplifier. The strain gauge attached to the dorsal scales over the nasofrontal joint of an unanesthetized N. nigricollis produced a clear pattern of deformation prior to each of 18 spits recorded (Fig. 6B). In this experiment the spit signal was triggered by the experimenter, and thus is delayed both by the time it takes the spit to reach the experimenter and the reaction time between spit contact and switch depression. Because no skeletal muscle contacts the snout complex of cobras, the only motive force for the rotary displacement of the snout is the palato-maxillary arch, and more specifically the palatine and maxilla.
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Venom pressure
In N. nigricollis, when identical stimulation is applied to the
same surface area of the M. protractor pterygoideus (PP) and the M. adductor
mandibulae externus superficialis (AMES) individually or simultaneously, a
clear pattern of changes in venom pressure is produced. Though the AMES is the
only muscle directly contacting the venom gland, contractions of this muscle
in isolation produce little venom pressure when measured from the fang
(Fig. 7A). The PP does not
contact the venom gland or duct, but does produce displacement of the
palato-maxillary arch and the soft tissues around the fang; stimulation of
this muscle results in modest venom pressures at the fang tip, which are
greater than those produced by the AMES
(Fig. 7A). When the two muscles
are stimulated together the resultant venom pressure at the fang tip is
greater than the sum of the two individual stimulations, with combined venom
pressures roughly double those produced by the PP alone
(Fig. 7A). The 17 stimulation
episodes produced the same pattern of relative contribution to venom pressure
whether the muscle was exposed to a single twitch stimulus, or train stimuli
(Fig. 7B).
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When the pressure catheter is inserted into the proximal venom duct, thereby avoiding the soft tissue structures of the fang sheath, a markedly different pattern of venom pressures emerges. It is important to note that the twitch stimulations and the stimulated muscle areas used during the proximal pressure recordings were the same as those used for the distal recordings. Stimulation of the AMES produces a prominent spike of venom pressure (Fig. 8), which is roughly 200 times greater than that recorded from the fang tip. The high venom pressure produced by this stimulation, coupled with the closed recording system employed, resulted in long recovery times. Stimulation of the PP produced a pulse of venom pressure that was less than 10% that of the AMES and had a more rapid recovery time (Fig. 8). The venom pressure spike produced by stimulating the two muscles simultaneously was nearly identical to that produced by stimulating the AMES alone (Fig. 8). The additive effect that characterized the venom pressures recorded at the fang tip was not observed in the pressure tracings recorded more proximally.
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Electromyography
Every spitting episode decreased the volume of venom in the venom gland,
and thus could alter the forces acting on this system. To minimize this
complication, we only recorded the first 15 spits produced by the cobras (some
species can spit over 50 times; Rasmussen
et al., 1995). The electrical activity of the muscles in N.
nigricollis revealed a consistent pattern in every spit
(Fig. 9). Nearly synchronous
electrical activity was recorded from the M. protractor pterygoideus (PP) and
the M. adductor mandibulae externus superficialis (AMES) immediately prior to
the spit. The signals from the spit detector are delayed due, among other
factors, to the time it takes the venom to travel from the fang to the spit
detector. In one-third of the spits, a second low-level burst of activity was
observed in the PP following the spit while the mouth was still open
(Fig. 9), which we believe to
be associated with a more subtle repositioning of the palato-maxillary arch
following the spit.
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The pattern of EMG activity during spitting was consistent in the two specimens of N. nigricollis. A series of spitting episodes from one of these specimens had been previously captured using high-speed videography. Quantitative analyses of these two data sets (using NIH Image for the video and SuperScope for the EMG signals) were performed to look for temporal patterns (Table 1). The quantitative features of the EMG signals presented in Table 1 are similar to those obtained from the second specimen of N. nigricollis; similarly, the spit durations presented below are similar to those observed in other specimens. Electrical activity in the PP precedes the activity in the AMES by 37 ms, and continues for 10 msbeyond the cessation of activity in the AMES (Table 1). The AMES, the compressor of the venom gland, is active for 96 ms during a spitting episode. Quantification of the high-speed digital records indicated a mean spit duration of 66 ms; this temporal measure of venom discharge includes the initial pressurization and terminal depressurization phases (Table 1). The onset of the palatal projections was clearly visible in 7 of the 12 spitting sequences captured from this specimen, and in these 7 sequences the palatal projections appeared 3 ms prior to the onset of spitting (Table 1).
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Discussion |
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The results of our investigation support both components of this model for venom spitting. During the electromyography experiments, electrical activity was consistently recorded from the M. adductor mandibulae externus superficialis (AMES) immediately prior to the discharge of the spit (Fig. 9).Electrical stimulation of the AMES of anesthetized cobras produced an increase in venom pressure (Fig. 8). The other component of this model for spitting, the displacement of the palato-maxillary arch leading to a functional release of venom, was also supported by our experimental results. High-speed digital videography and photography of spitting cobras documented the displacement of the palato-maxillary arch immediately prior to the spit (Fig. 2). The displacement of the palato-maxillary arch was also evident in the results of the strain gauge experiments. The first experiment documented the direct displacement of the palatopterygoid and maxilloectopterygoid joints; the second experiment revealed the protraction and sagittal rotation of the palatine and maxilla by documenting the ensuing rotation of the snout complex (Fig. 6). The hypothesis that the M. protractor pterygoideus (PP) is responsible for the displacement of the palato-maxillary arch is supported by the findings that electrical stimulation of this muscle produce displacements similar to those observed during the venom spitting behaviors (Fig. 5), and that electromyographic signals were consistently recorded from this muscle immediately prior to the discharge of the venom stream (Fig. 9). One consequence of this palato-maxillary displacement was evident from the manipulation of anesthetized snakes, in which manual protraction of the palato-maxillary arch led to a dorsad translation of the fang sheath. The presence of a barrier to venom flow within the fang sheath is evident in the results of the pressure recordings. Venom pressures recorded distal to the venom barrier (at the fang tip) were approximately 1/200th of those recorded proximal to the venom barrier (from the venom duct) when the AMES was stimulated independently (Figs 7, 8). The PP, which produces deformation of the fang sheath, influences venom pressures recorded distal to the fang sheath (from the fang tip), but not those recorded proximal to the fang sheath. Other muscles in this region could also potentially influence venom expulsion; however, the spatial position, size and lever arms of the AMES and PP are such that they would have the greatest influence on venom flow.
This model for the functional basis of spitting does not directly speak to
the relative timing of the two component parts. When sequential spits are
considered (multiple spits are quite common in cobras; Rassmusen et al.,
1995), three obvious temporal patterns are possible
(Fig. 11). The M. adductor
mandibulae externus superficialis (AMES) may have extended activity periods,
thus keeping the venom gland pressurized, with periodic releases of venom
associated with contraction of the M. protractor pterygroideus (PP). A second
possible pattern is a close temporal coupling between the activity of the AMES
and the PP, while the third involves asymmetrical activity of the PP extending
beyond the termination of contraction of the AMES
(Fig. 11). Cascardi et al.
(1999) reported that N.
pallida spat a rather consistent volume of venom in each spit, which is
difficult to reconcile with protracted activity in the AMES. The high-speed
videography of the venom stream revealed an initial pressurization phase,
which also seems contrary to protracted contraction of the AMES. The results
of the EMG experiments (Table
1) clearly support a close temporal coupling of the two muscles.
The longer activation period of the PP presumably reflects the greater time
and force needed to achieve displacement of the palato-maxillary complex, when
compared to compression of the venom gland by the AMES. Though recorded
separately, the temporal agreement between the duration of the spit and the
duration of electrical activity in these muscles suggests a regular temporal
coupling between these two muscles. In this view, multiple spits could be
achieved in virtually any temporal pattern with dual activation of the
muscles. Lastly, we saw no evidence of maintained displacement of the
palato-maxillary arch in any of our cobras; prolonged displacement of the fang
sheath, and the barriers to venom flow, would leave the cobra vulnerable to
accidental discharge of venom during any movement of the lower jaw.
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Rosenberg (1967) detailed
the intra-glandular pressure model for venom injection in snakes, which
described the functional relationship between contraction of the extrinsic
venom gland musculature and venom expulsion. Key aspects of this model were
supported by experimental studies of venom expulsion in Crotalus
(Young et al., 2000
), and by
the results of the present study, where contraction of the extrinsic venom
gland muscle (the AMES), either voluntarily by the cobra or through artificial
stimulation, led to increases in venom pressure. Recently, Young et al.
(2002
) developed a refined and
expanded model for venom expulsion, termed the pressure-balance model. This
model emphasized the presence of passive barriers to venom flow, the
morphology of the distal venom delivery system, and the role of the fang
sheath in influencing venom flow. Simplistically, the pressure-balance model
argues that venom pressure will build within the venom chambers of the fang
sheath until physical displacement of the fang sheath, as during fang
penetration, leads to venom flow through the fang
(Young et al., 2002
). A recent
experiment has supported the pressure-balance model by showing that
alterations of the peripheral resistance to venom flow have a significant
impact on venom expulsion (Young et al.,
2003
). Spitting cobras were a natural test of the pressure-balance
model in that, unlike apparently all other venomous snakes, they do not
require direct physical contact with a target to release venom. The results of
this study suggest that spitting cobras still utilize a pressure-balance
system for venom expulsion; however, unlike all other venomous snakes where
displacement of the fang sheath is passive, in spitting cobras the
displacement is actively produced by the contraction of the M. protractor
pterygoideus and the ensuing displacement of the palato-maxillary arch.
The results of the present study, when compared to earlier experimental
analyses of the venom delivery mechanics in rattlesnakes, suggest that the
venom delivery system of these two taxa (the Elapidae and Viperidae) have
evolved independently and functionally converged. Nearly every aspect of the
venom delivery system of elapids and viperids is morphologically distinct. The
extrinsic venom gland musculature (see
Haas, 1973;
Jackson, 2003
), venom gland
and venom duct (see Gabe and Saint Girons,
1967
; Kochva,
1978
,
1987
), maxilla and fangs (see
Duvernoy, 1832
;
Knight and Mindell, 1994
) and
the venom chambers (Young et al.,
2001b
), all show marked morphological differences between elapids
and viperids. Nevertheless, in both clades the extrinsic venom gland muscles
function to raise intraglandular pressure, the venom chambers of the distal
venom delivery system appear to pool venom and act as a barrier to venom flow,
and displacement of the fang sheath appears to be a prerequisite to venom
flow. Several scenarios (e.g. Savitzky,
1980
; Kardong,
1982
) have been proposed for the evolution of the venom delivery
system in snakes; most commonly with a rear-fanged (opisthoglyphous) colubroid
posited as the ancestral form of either viperids or elapids. Though the
phylogeny of snakes remains uncertain, many recent morphological and/or
molecular phylogenies have placed the viperids as basal within colubroid
snakes (e.g. Pough et al.,
1998
). While the exact relationship of the different lineages
within the colubroid snakes remains uncertain, there is general agreement that
venom delivery systems evolved independently multiple times within this
radiation (see Jackson, 2003
).
The similarity between our findings and the earlier experimental study of the
viperid Crotalus (principally Young et al.,
2000
,
2001a
) suggests functional
convergence between these two lineages.
The palato-maxillary arches of snakes exhibit varying kinematic patterns
during prey capture and transport (Cundall
and Greene, 2000). In viperids, protraction of the
palato-maxillary arch produces rotation of the maxilla in both the sagittal
and transverse planes, so that the width between the fangs is increased
(Mitchell, 1861
;
Zamudio et al., 2000
). Though
more modest, the maxillae of some elapids have also been described as rotating
in the transverse plane prior to envenomation (e.g.
Fairley, 1929
). The maxillary
displacements we observed in Naja during spitting differ from
previously described pre-envenomation motions in being composed of rotations
in the frontal and sagittal planes, and in being most pronounced on the
posterior surface of the maxilla. The displacement of the palato-maxillary
arch that we observed during spitting resembles the unilateral motions of the
palato-maxillary arch observed in most colubroids during prey transport (the
`pterygoid walk' of Boltt and Ewer,
1964
). The early advance phase of medial jaw transport (to use the
terminology for the pterygoid walk of
Cundall and Greene, 2000
) is
characterized by protraction of the palato-maxillary arch coupled with a
lateral rotation of the maxilla and palatine in the frontal plane.
Furthermore, experimental evidence (e.g.
Cundall, 1983
;
Kardong et al., 1986
) has
demonstrated that the M. protractor pterygoideus plays a key role in producing
these palato-maxillary displacements during ingestion. Haas
(1930
) described how the
palatine and maxilla rotate in the sagittal plane during medial jaw transport
(the pterygoid walk) in Naja; his summary figure (fig. 21 in
Haas, 1930
) depicts
displacements very similar to what we observed during spitting. The rotation
of the palatine and maxilla is a common feature in one clade of elapids (the
palatine erectors of McDowell,
1970
), and was recently explored in some detail
(Deufel and Cundall, 2004
). In
all their key features the displacements observed during spitting appear to be
pronounced, bilateral, transport mechanics.
Observations of ingestion in our spitting cobras and analysis of video
recordings of prey ingestion in N. pallida yield consistent evidence
of medial jaw transport, but not displacements of the magnitude evident during
spitting. It may be that concurrent bilateral activation of the M. protractor
pterygoideus, rather than sequential unilateral activation as seen during
ingestion, allows for greater displacement relative to the braincase. The
present study examined only the M. protractor pterygoideus from among the
palato-maxillary arch musculature. Previous studies of ingestion have
suggested that the M. protractor pterygoideus, M. levator pterygoideus, M.
retractor pterygoideus and the M. pterygoideus may all be active during the
initial (protractive) displacements of the palato-maxillary arch
(Cundall and Greene, 2000). Two
of these muscles, the M. retractor pterygoideus and the M. pterygoideus, are
antagonists of the M. protractor pterygoideus. The M. pterygoideus inserts on
the distal ectopterygoid and the ectopterygoid/maxillary joint; activation of
this muscle would limit rotation of the maxilla in the frontal plane. The more
pronounced palato-maxillary displacements we observed during spitting may be
produced by the M. protractor pterygoideus functioning independently of its
normal (ingestive) antagonists.
With the exception of the exit orifice of the fang
(Fig. 1), only subtle
anatomical differences were found among the palato-maxillary arches of
spitting and non-spitting cobras. No obvious morphological feature essential
to the venom release during spitting was found in the palato-maxillary arch.
It appears that rather than evolving a suite of morphological specializations
for spitting, cobras have instead coopted and modified a motor action pattern
employed for ingestion. The independent evolution of this behavior among
multiple independent lineages, combined with reports of occasional specimens
of non-spitting cobras that spit (e.g.
Carpenter and Ferguson, 1977;
Wüster and Thorpe, 1992
)
suggest that the palato-maxillary complex of the Najini is well suited to
achieve the displacement of the fang sheath requisite for spitting.
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References |
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