Venom Expulsion

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Mechanisms Controlling Venom Expulsion in the

Western Diamondback Rattlesnake, Crotalus atroxBRUCE A. YOUNG ANDKENNETH V. KARDONGSchool of Biological Sciences, Washington State University, Pullman,Washington 99164-4236

ABSTRACT Although many studies have documented variation in the amount of venomexpended during bites of venomous snakes, the mechanistic source of this variation remainsuncertain. This study used experimental techniques to examine how two different features of thevenom delivery system, the muscle surrounding the venom gland (the Compressor Glandulae in therattlesnake) and the fang sheath, could influence venom flow in the western diamondbackrattlesnake, Crotalus atrox. Differential contraction of the Compressor Glandulae explained only

approximately 30% of the variation in venom flow. Lifting (compression) of the fang sheath as occursduring a normal strike produced marked increases in venom flow; these changes were closelycorrelated and exceed in magnitude by almost 10 those recorded from the Compressor Glandulaealone. These results suggest that variation in these two aspects of the venom delivery systembothin terms of magnitude and temporal patterningexplain most of the observed variation in venominjection. The lack of functional or mechanical links between the Compressor Glandulae and thefang sheath, and the lack of skeletal or smooth muscle within the fang sheath, make it unlikely thatvariation in venom flow is under direct neural control. Instead, differential venom injection resultsfrom differences in the pressurization by the Compressor Glandulae, the gate keeping effects of thefang sheath and enclosed soft-tissue chambers, and by differences in the pressure returned byperipheral resistance of the target tissue. J. Exp. Zool. 307A:1827, 2007. r 2006 Wiley-Liss, Inc.

How to cite this article: Young BA, Kardong KV. 2007. Mechanisms controlling venomexpulsion in the western diamondback rattlesnake, Crotalus atrox. J. Exp. Zool.307A:1827.

Venom expulsion is any pressurized dischargeor flow of venom from the venom delivery systemof the snake. Venom injection represents onespecial form of venom expulsion in which thevenom is discharged while the fang is imbedded(typically in the tissue of the target organism).Venom injection, like other forms of venomexpulsionsuch as venom milking or venomspittingis characterized by variation in boththe volume and pressure of the venom, that is to

say, by differential venom flow. Two contrastinghypotheses have been advanced to account fordifferential venom flow in snakes. One hypothesis,termed thevenom metering hypothesis, postulatesthat the venom delivery system is under strongneural regulation and that snakes apportionvenom so as to optimize the amount of venominjected into prey. In support, experimental trialsreport that small prey receive less venom thanlarge prey (e.g., Hayes, 95). Further, this appor-tionment of venom is proposed to occur through

conscious decision making (see Hayes et al., 2002;Hayes, 2004). The second hypothesis, termed thepressure-balance hypothesis, postulates that theamount of venom injected is determined largely bythe mechanical interactions between a snakesvenom apparatus and the prey, with little neuralregulation of the venom delivery system (seeYoung et al., 2002, 2003). The venom meteringhypothesis emphasizes active regulation beforethe snake makes contact with the prey, whereas

the pressure-balance hypothesis emphasizespassive regulation that occurs during contact.

The differences between these two hypothesescan be mapped onto the structural features of thevenom delivery system. The venom gland of all

Published online 8 November 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jez.a.341.

Received 8 November 2004; Accepted 25 July 2006

Correspondence to: Dr. B.A. Young, Department of Biology,Washburn University, Topeka, KS 66621.E-mail: [email protected]

2006 WILEY-LISS, INC.

JOURNAL OF EXPERIMENTAL ZOOLOGY 307A:1827 (2007)


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viperid, elapid, and atractaspid snakes is sur-rounded by skeletal muscle (see Haas, 73). Thepresence of striated voluntary muscle abutting, ifnot attaching to, the venom gland clearly estab-lishes that muscle contraction plays a role in

pressurizing the venom gland and thereby drivingvenom through the duct system (Rosenberg, 67;Young et al., 2000). What has not been establishedto date is the nature of the relationship betweendifferential muscle contraction and differentialvenom injection. The distal portion of the venomdelivery system consists of the fang sheath, anenvelope of connective tissue and epithelium,which drapes down from the roof of the mouthto surround the fang (Young et al., 2006). Withinthe fang sheath are several soft-tissue chambersthrough which venom must flow in order to reachthe fang (Young et al., 2006). The fang sheath is

devoid of skeletal or smooth muscle, and thuscannot actively regulate venom flow. However,physiological studies have suggested that deforma-tion of the fang sheath can influence venom flow,presumably, at least in part, by altering the flow ofvenom through the soft-tissue chambers (Younget al., 2001a).

The present study was undertaken to experi-mentally explore the relative contributions ofthese two structural featuresfang sheath andthe compressor muscleto venom release in thewestern diamondback rattlesnake, Crotalus atrox.

This is a way to test the relative importance ofneural regulation on venom injection, since thefang sheath is not an active effector organ, andtherefore not under direct control by the centralnervous system. This study will examine therelationship between differential contraction ofthe skeletal muscle surrounding the venom glandand differential venom expulsion, as well as theimpact of fang sheath position on venomexpulsion. In this way, we experimentally comparethe predictions of the venom metering andpressure-balance hypotheses.

MATERIALS AND METHODS

Live animals

This study utilized seven live specimens ofwestern diamondback rattlesnake, C. atrox, withsnout lengths (svl) ranging from 66 to 135 cm. Fourof the specimens were obtained commercially(Glades Herp, Bushnell, FL) while the remainingthree were wild-caught in western Texas. Thesnakes were maintained at 27311C, with a 12:12light cycle, water ad libitum, and a diet of pre-killed

rodents. To ensure a normal venom supply, thesnakes were not fed within 2 weeks of anyexperimental procedure. Maintenance and use ofthese animals followed guidelines for reptiles andparticularly venomous snakes, and all experimental

protocols were approved by the Institutional AnimalCare and Use Committee of Washington StateUniversity.

Non-pressurized fang sheath compression

Two of the C. atrox (svls of 87 and 95 cm) wereanesthetized through exposure to Isoflurane, thenintubated and maintained on a low-level flow ofIsoflurane. Once the animals were fully anesthe-tized, the head was positioned in a clamp that heldone side of the head static while allowing manip-ulation of the contralateral side. A micromanipu-

lator was positioned behind the quadratopterygoidjoint and used to mechanically protract the upperjaw until the fang was erected to at least 701(measured from the ventral margin of the supra-labials to the leading edge of the fang).

The fang sheath was gently lifted to expose thefang tip and a rotary disk used to truncate thefang just proximal to the exit orifice; doing thisexposed the venom canal at the excised surface ofthe fang. A length of polyethylene (PE) tubing wasslipped over the fang; the inner diameter of the PEtubing was such that a tight fit was achieved withthe outer surface of the fang. The free end of the

PE tubing was attached to a Statham P23AApressure transducer (Gould, Valley View, OH),and both tubing and transducer were filled withRingers solution allowing no air within thechannel. The pressure transducer was connectedto a P511 AC/DC Amplifier (GRASS, West War-wick, RI) the output of which was converted to adigital record using a PCI-PCM12 H A/D converter(SuperLogics), recorded (at a sampling rate of10 kHz) using WINview (SuperLogics), and ana-lyzed with WINcalc (SuperLogics, Waltham, MA).

In experiments to test the effects of the fang

sheath, the fang sheath was manually lifteddorsal to simulate the elevation and compressionthat occurs normally during the strikeusingeither forceps or pads of foam rubber. The rateand extent of the fang sheath elevation varied. Atleast five sheath elevations were recorded fromeach side of the specimens.

Fatigue of the Compressor Glandulae

To access the Compressor Glandulae, the scala-tion over the venom gland was removed, exposing

VENOM EXPULSION IN RATTLESNAKES 19

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the Compressor Glandulae while leaving itsvascular supply and innervation intact. A clampwas applied to the lower jaw to stabilize theinsertion of the Compressor Glandulae. Silksuture was used to attach the origin of the

Compressor Glandulae to a force transducer(UFI model 1030). The force transducer wasconnected to the P511 amplifier and the dataacquisition system as described above.

A micromanipulator was used to position abipolar stimulating probe against the motor nerveto the Compressor Glandulae. The stimulatingprobe was connected to a S88 dual channelstimulator (GRASS), which was used to stimulate(7 V for 50 msec) the Compresssor Glandulae onceevery 5 sec for 5 min. Fatigue was calculated as thepercentage decline in twitch force over time.

Twitch venom pressure

Five specimens ofC. atrox(svls from 66135 cm)were anesthetized and prepared as describedabove to expose the Compressor Glandulae.Bipolar EMG electrodes were constructed using0.05 mm diameter stainless-steel wire with nyloninsulation (California Fine Wire), and implantedinto the Compressor Glandulae via hypodermicneedles. The electrodes were attached to a custom-built EMG amplifier which was coupled to the dataacquisition system.

A bipolar stimulating probe was used to stimu-late the surface of the Compressor Glandulae;while each stimulus was applied individually, effortwas made to standardize the manual pressureapplied to the stimulator, and to stimulate thesame general region (the anterolateral quadrant)of the muscle. Using the S88 stimulator (GRASS),the stimulus applied to the Compressor Glandulaewas varied both in terms of voltage (510 V) andduration (40120 msec). We attempted to apply1012 stimuli to both the right and left venomdelivery system of each specimen. Stimuli were notapplied to one side of two different specimens; onespecimen had shed its fang and the otherevidenced physical trauma to the fang sheath.During data analysis (see below) stimuli wereexcluded if there was evidence of cross-talk orpoor signal quality. Ultimately we quantified 68twitches, total from both sides, which were roughlyevenly divided among the five specimens.

The data tracings for this experiment (Fig. 1)consisted of the synchronized marker from thestimulator, the voltage output from the EMGamplifier, and the voltage output from the pres-

sure transducer. Using the WINcalc software(SuperLogics), the EMG voltage was rectified andthe area under the curve quantified. The peakvenom pressure was quantified, as was the areaunder the pressure curve. A curve was fit to thepooled data points for each specimen (using

maximal r2 value as the criterion for curve fit).Subsequently, the data points from all thespecimens were standardizedby adjusting themean venom pressure value for a narrow range ofEMG areato minimize intraspecific variationand the data sets combined for all specimens.

Pressurized sheath elevation

Following the twitch venom pressure experi-ments, a magnetoresistive sensor (Philips KMZ10)was positioned, using a micromanipulator, dorso-lateral to the fang sheath. A train stimulus (7 V,50 msec duration, 19.5 pps) was applied to thesurface of the Compressor Glandulae; this stimu-lus produced nearly constant pressurization of thevenom gland but did not induce a tetanic contrac-tion of the muscle (individual pulses were stillevident in the pressure tracing).

Although variable in duration, the CompressorGlandulae was always stimulated prior to thelifting of the fang sheath (Fig. 2). The fang sheathwas lifted manually by sliding a small pad of foamrubber over the PE tubing and dorsally up the

Fig. 1. Data tracing from the twitch venom pressureexperiment. The EMG waves were rectified then the areaunder the curve calculated. The peak venom pressure wasmeasured, as was the area under the venom pressure curve.

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fang. A 3.2 mm diameter Neodymium Iron Boronceramic magnet (ForceField, Inc., Fort Collins,CO) was located within the foam pad in such a waythat lifting the fang sheath moved the magnetlinearly in relation to the magnetoresistive chip.The magnetoresistive chip was connected to anAccudata 218 bridge amplifier (Honeywell, Morris-town, NJ) and then to the data acquisition system.

Only the period from the onset of magnetdisplacement to maximal fang sheath compressionwas analyzed, due mainly to the fact that the fangsheath did not rebound to its starting positionimmediately after mechanical lifting of the sheath

was discontinued. The voltage range of the datatracings from the pressure transducer andmagnetoresistive chip were balanced mathemati-cally so that the two curves (voltage over time)could be directly compared. Subtracting one curvefrom the other provided useful information aboutthe mechanics of the fang sheath. The peak venompressure was also quantified. The compression ofthe fang sheath typically led to the discharge ofvenom, thereby altering the fluid mechanics of thesystem. Due to this alteration, we only performed

one or two fang sheath manipulations on each sideof the venom delivery system.

Pressurized fang retraction

This adjunct to the preceding experiment was

performed on three of the specimens (svls of 66,85, and 115 cm). Following the termination of thepressurized fang sheath experiment, theCompresssor Glandulae was subjected to the samestimulation regime. This time the stimulatingprobe was placed on the medial surface of theCompressor Glandulae. In this position, thestimulus produced contraction not only in theCompressor Glandulae but also in the adjacentmusculature of the pterygoid arch. Stimulatingthe pterygoid arch musculature produced a retrac-tion of the fang, and with it a displacement of thefang sheath. We had no independent marker forthe displacements of either the fang or the fangsheath, but we could record the changes in venompressure that occurred when the fang sheath wasdisplaced without any direct physical contact.

RESULTS

Non-pressurized fang sheath elevation

Lifting of the fang sheath, in the absence of fluidpressure within the venom delivery system,resulted in retrograde venom flow. This retro-grade venom flow was manifest as a decrease invenom pressure (Fig. 3). Release of the sheathallows it to drop back over the fang, and producedan increase in venom pressure that was roughlyequal in magnitude to the pressure decreaserecorded during compression (Fig. 3).

Fatigue of the Compressor Glandulae

The two fatigue tests yielded similar results. Theforce output began to decrease after approxi-mately 13 stimuli (Fig. 4). The muscle thereafterdeclined to slightly less than 50% by the 35thstimulus (Fig. 4). Additional stimuli resulted in

continued force decline, although at a slower rate.

Twitch venom pressure

In every specimen, the relationship betweentwitch EMG area and venom pressure area wasbest described by a power curve with an exponentof approximately 0.70. Although these curvesrepresented the best fit to the data points, theyalways explained less than 40% of the variationin the data, as indicated by the r2 values (Fig. 5).When the data from all five specimens were

Fig. 2. Data tracing from the pressurized fang sheathelevation. In this, the first run, the gain was too high on themagnetoresistive chip, producing a supramaximal voltageoutput. Note that the Compressor Glandulae was active forover 500 msec prior to elevation of the fang sheath, and thatduring this time there was little change in venom pressure.The pulsatility within the venom pressure tracing indicatesthat the Compressor Glandulae was not in tetanic contraction.




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combined, and the venom pressures standardized,a power curve with similar exponent still repre-sented the best fit, although the amount of

variation explained decreases to less than 25%(Fig. 6).

Pressurized venom sheath

With the venom delivery system pressurized(stimulation of the Compressor Glandulae),manual lifting of the fang sheath produced asharp and distinctive rise in venom pressure(Fig. 7). If the range of venom output is standar-dized and the pressure curve subtracted from themagnetoresistive curve, two prominent deviations

from linearity are observed (Fig. 8). The largerdeviation (Fig. 8B) is at the end of curve and iscaused by the withdrawal of the magnet (and foampad) combined with the failure of the fang sheathto naturally recoil. The smaller deviation occursat the onset of fang sheath compression (Fig. 8A).This deviation is produced by a slight decrease in

Fig. 3. Data tracing from the non-pressurized fang sheathelevation showing the retrograde venom flow (arrow) typicalof those experiments (A) in contrast to twitch pressures (B)taken from the same preparation approximately 60 sec later inwhich the Compressor Glandulae is contracted.

10

20

30

40

50

60

70

80

90

100

110

PercentofMuscleForceOutput

Stimulus Number

0 10 20 30 40 50 60

Fig. 4. Fatigue curve for the Compressor Glandulae. Notethat with the exception of a short physiological rally early inthe fatigue test, the muscles has a relatively constant declinein force output over the course of the 5-min trial.

0.01

0.1

1

10

VenomP

ressureArea(Torr*Seconds)

0.1 1 10

EMG Area (Volts*Seconds)

y = 0.384x0.722 r2= 0.339

Fig. 5. Twitch venom pressure data from the 135 cm svl

specimen of Crotalus atrox. Note the range in contractileactivity of the Compressor Glandulae. The best-fit curve hasthe formulay50.384x0.722, and an r2 value of 0.339.

1

10

100

1000

10000

VenomPressure

(Torr*Seconds)

0.1 1 10

EMG Area (Volts*Seconds)

y = 59.892x0.730 r2= 0.247

Fig. 6. Combined twitch venom pressure data set from allfive specimens of Crotalus atrox after standardization of a

mean venom pressure value. Note the marked variation invenom pressure for any given level of contractile activity. Thebest-fit curve has the formulay559.892x0.73, and an r2 valueof 0.247.




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venom pressure at the onset of fang sheathelevation, and by a temporal delay between fangsheath elevation and the rise in venom pressure(Fig. 9).

In each snake, the peak venom pressuresproduced following the lifting of the fang sheathwere approximately 10 the mean peak pressuresproduced by the single twitch stimuli that werenot accompanied by lifting the venom sheath(Fig. 10).

Pressurized fang retraction

Retraction of the fang, and correspondingdisplacement of the fang sheath produced a

marked increase in venom pressure (Fig. 11). Asfang retraction progressed, with stimulation con-stant, the venom pressure subsided but did not

Fig. 7. Data tracing showing the elevation of the fangsheath (bottom tracing) and the associated change in venompressure (top trace). The vertical line represents peak fangsheath elevation after which the magnet was withdrawn. Notethe close relationship between the two curves during fangsheath elevation.

Fig. 8. Subtraction of the venom pressure curve from themagnetoresistive curve from Figure 7. Note the two promi-nent deviations from linearity. The first deviation (A) occursat the onset of fang sheath elevation, while the latter, andlarger deviation (B) occurs when the magnet is withdrawn butvenom pressure does not rebound.

Fig. 9. Synchronized recordings of venom pressure (uppercurve) and fang sheath elevation (lower curve) from the sametrial episode. In this expanded section from the start of thetrial the brief pulse drop in venom pressure (arrow) is evident,as is the slight temporal offset between fang sheath elevationand the rise in venom pressure. These two features combine toproduce the initial deviation evident in Figure 8.

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PeakVenomPressure(Torr)

66 73 85 115 135

Specimen Snout-Vent Length (cm)

Sheath Compression

Twitch

Fig. 10. Histogram of the peak pressures recorded duringpressurized fang sheath elevation (solid) and the mean venompeaks produced by the muscle twitch experiments (open) forall five specimens. Note that the impact of fang sheathelevation is roughly 10 that of the muscle compression.




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return to the baseline level. When the stimulationwas terminated, and the fang at least partiallyrebounded to the protracted position, no clearpattern of change in venom pressure was observed(Fig. 11).

DISCUSSION

In both trials, the Compressor Glandulaeshowed rapid and sustained fatigue when exposedto repeated stimulation (Fig. 4). Although thereappeared to be a brief physiological recovery in theearly part of the fatigue test, the force output fellto approximately 50% of the initial value within 35stimuli (Fig. 4). This fatigue profile is consistentwith what was observed in a previous study(Young et al., 2000). The fatigue of the CompressorGlandulae informed and dictated the experimentalprocedures used in this study. The numbers oftwitch stimuli applied were kept to a relatively lownumber and were temporally spread out in orderto lessen the fatigue. To minimize fatigue effects,only one or two fang sheath elevations per trialwere performed on each venom delivery system.

This study found only a weak relationshipbetween differential muscle force (as induced bytwitches in the Compressor Glandulae) and venompressure. This weak relationship was presentin the data obtained from each snake (Fig. 5),and in the combined data set (Fig. 6). The best fitbetween EMG and Pressure was a power curve

with an exponent of 0.722, indicating a relativedecrease in venom output with increasing muscleforce. The underlying cause of this relationshipcould not be determined from this study.

The venom gland of Crotalus consists of

extensive parenchyma supporting the venomsecreting cells (e.g., Kochva, 78; Mackessy, 91).By using spatially localized twitch stimuli, pro-duced by stimulating the surface of the muscle andnot the motor nerve, our methodology would havepreferentially expelled venom from a localizedregion of venom gland parenchyma. There maybe a limit to how much localized venom could beextruded in this fashion, which could result in arelative decrease in venom output with muscleforce. While this spatial localization might explainthe exponent of the power curve, it would notaccount for the low level of venom pressure

variation (r2 values of under 0.25) explained byvariation in muscle force.

This study used three experimental treatmentsto examine the effects of the fang sheath displace-ment. The results of each treatment revealedchanges in venom flow with fang sheath displace-ment. Lifting of the fang sheath without pressur-izing the venom gland (no muscle contraction)produced a decrease in venom pressure (Fig. 3).Lifting the fang sheath presumably altered thesoft-tissue chambers within the fang sheath; thisvolumetric displacement moved retrograde

(toward the venom gland) producing the drop invenom pressure at the tip of the fang.When the venom gland was pressurized (Com-

pressor Glandulae stimulated), manually liftingthe fang sheath resulted in an increase in venompressure (Fig. 7). A brief episode of negativepressure was also seen in these experiments asthe fang sheath first started to compress (Figs. 8and 9). The internal architecture of the fangsheath (Young et al., 2001a) including the internalfang membrane (a portion of the soft-tissuechambers) could physically obscure the entranceorifice of the fang. Lifting the fang sheath wouldthen fully expose the entrance orifice to flow of thepressurized venom. This would explain both thesharp rise in venom pressure during manuallifting of the fang sheath and the rise in venompressure during fang retraction (Fig. 11).

The difference in the directionality of the venompressure change evident in the data tracings mayreflect, at least in part, our experimental method.We connected the venom canal of the fang to afluid-filled channel (tubing and pressure transdu-cer). To minimize the loss of venom we kept the

Fig. 11. Data tracing showing the change in venompressure during pressurized fang retraction. Note that initialretraction of the fang causes an increase in venom pressure(arrow) but that with continued retraction the venompressure decreases, even though the Compressor Glandulaeis still contracting.




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exit port of the fluid transducer closed therebycreating a closed system. This meant that therewas always a pressure head acting on the end ofthe fang tip. If the pressure within the venomgland was lower than the pressure head created by

the pressure transducer, then venom flow wouldbe retrograde, as it was during the non-pressur-ized fang sheath liftings. If the pressure within thevenom gland exceeded the pressure head of thetransducer, then changes in the soft-tissuechambers within the fang sheath would havecreated positive, not negative, venom flow.Although clearly the pressure head created bythe transducer is artifactual, during a normalstrike the fang is imbedded in the target tissueand thus encounters peripheral resistance. In fact,an early study of the kinematics of venom flowthrough the venom duct documented a pulse of

retrograde flow at the termination of each venominjection episode (Young and Zahn, 2001).

The peak venom pressures recorded during fangsheath elevation were approximately 10 times themean peak values obtained during the muscletwitch trials (with no fang sheath displacement)(Fig. 10). The methodology we employed ensuredthat this pressure difference was not the result ofthe muscle stimulation and/or venom gland pres-surization (Fig. 2). Instead, the pressure differ-ences are attributable to the alterations of thesoft-tissue chambers within the fang sheath, and

the internal architecture of the fang sheath.Previous studies have documented a wide varia-tion in the amount of venom injected, even duringbites at the same target (e.g., Hokama, 78; Tun-Pe and Khin-Aung-Cho, 86). Other studies haveshown differences in the amount of venominjected associated with differences in prey items(Hayes et al., 92; Hayes, 95). The results of thisstudy suggest that two factors have substantialeffects on the amount of venom injected (Fig. 12).The differences in the amount of venom injectedcould arise either through differential contractionof the Compressor Glandulae or through differ-ential positioning of the fang sheath (therebydifferentially affecting the enclosed soft-tissuechambers), or a combination of both. Althoughthe explanatory power is low, there is a significantrelationship between muscle force and venompressure (Fig. 5). However, there is a strongerrelationship between fang sheath elevation andincreasing venom flow (Fig. 7). Since these twofactors are both mechanically and functionallydisconnected, there is no reason to conclude thata certain level of muscle activation would be

associated a priori with a certain level of fangsheath displacement.

This study concentrated on the relative magni-tudes of the contraction of the CompressorGlandulae and positional changes of the fangsheath, and how these could influence venom flow.But we also recognize that the temporal patternbetween these twomuscle contraction and fangsheath positioncould affect venom flow. If theCompressor Glandulae contracted before fang

sheath displacement, then the expelled venomcould fill the soft-tissue chambers. The relativepressure within the soft-tissue chambers wouldreduce the effect of fang sheath displacement onvenom pressure. In theory, these venom chamberscould become so turgid with pressurized venomthat lifting the fang sheath would affect neitherthe soft-tissue chambers nor venom pressure.Alternatively, lifting of the fang sheath prior tothe contraction of the Compressor Glandulaewould encounter minimal resistance in the venom

Fig. 12. Schematic of the venom delivery system. Thecontraction of the Compressor Glandulae would apply ex-ternal force (arrows) to the venom gland which wouldtranslate into fluid pressure within the venom duct (arrows).Compression of the fang sheath caused by physical interactionwith the target (arrows) would alter the volume of the soft-tissue chambers within the fang sheath. Differences in themagnitude and/or timing of these two factors would result in

variation in venom expulsion.




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chambers, and thus maximal chamber deforma-tion. A reduction in the volume of the soft-tissuechambers should maximize the volume of venomexpelled. If this unimpeded deformationreached the extent that the chambers closed down,

this kind of temporal imbalance could be a possiblemechanism behind the phenomenon of dry bites(e.g., Silveira and Nishioka, 95). It is important tonote that, as with the relative magnitude of theforces involved, there does not appear to be anymechanical or functional coupling of the Com-pressor Glandulae contraction and fang sheathelevation.

The results of this study suggest that venominjection in Crotalus is based on a two-stagemechanism, the contraction of the CompressorGlandulae and the displacement of the fangsheath. Recent experimental analyses of the

functional morphology of venom spitting incobras (Young et al., 2004) have demonstratedthis same two-stage mechanism. Though differentexperimental analyses were applied, and themechanics of venom expulsion are slightly differ-ent, the present study and the analysis of Naja(Young et al., 2004) both demonstrated that thecombination of compressor stimulation and fangsheath displacement has a supra-additive affectcompared to either factor alone. This commonalityof basic venom mechanics, between two lineagesgenerally taken to have independently evolved

their venom delivery systems (see Jackson, 2003),suggests a high degree of functional convergencewithin this system.

This study was undertaken, in part, to comparethe predictions of the two hypotheses for differ-ential venom flowvenom metering and thepressure-balance hypothesis. The venom meteringhypothesis emphasizes neural regulation andpreparation of venom volume prior to impact withthe target (Hayes et al., 2002). The results of thisstudy suggest that neural regulation of theCompressor Glandulae is not strongly correlatedwith the amount of venom injected; musclecontractile force was a poor predictor of venompressure, and the relative influence of musclecontraction on venom flow was roughly 1/10th thatof the fang sheath. The lack of active regulation ofthe fang sheath is not surprising, as it is devoid ofsmooth or skeletal muscle.

It could be argued that since the snake strike is avoluntary action, the snake is regulating theforces acting on the penetrating fang (and thus,indirectly, on fang sheath position) as well as onthe relative timing of fang sheath displacement

and coordinated contraction of the CompressorGlandulae. However, quantitative studies of thesnake strike have consistently revealed high levelsof kinematic variation (e.g., Kardong and Bels, 98;Young et al., 2001b; LaDuc, 2002) and errant

strikes (Kardong, 86); the strikes of most snakes,particularly the well-studied vipers, use more of aballistic lunge than a carefully controlled position-ing of the fangs. For a rattlesnake to activelyregulate the timing of fang penetration and thecontraction of the Compressor Glandulae, it wouldrequire that the snake could exactly determinethe distance to the target, precisely control thevelocity of the strike, and be able to factor inmovements of the target and fang penetrationangles. There is no evidence for this level ofcontrol in the snake strike.

The results of the present study are in general

agreement with the predictions of the pressure-balance hypothesis (Young et al., 2002). Thishypothesis posited that differential venom injec-tion could be achieved through differences in theCompressor Glandulae, the fang sheath andenclosed soft-tissue chambers, and by differencesin the peripheral resistance of the target tissue.The present study examined the first two possiblesources of venom variation, which should beshared by all forms of venom expulsion. Amongthe different forms of venom expulsion, onlyvenom injection incorporates peripheral resis-

tance. Earlier laboratory (Young et al., 2003) andfield (Young and OShea, 2004) studies haveexplored the influence of peripheral resistance ondifferential venom flow.

ACKNOWLEDGMENTS

The authors wish to thank J. Mellyn who kindlyprovided some of the animals used for this study,and R. Parker, who assisted during portions of theexperiments.

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Hayes W. 1995. Venom metering by juvenile prairie rattle-snakes (Crotalus v. viridis): effects of prey size andexperience. Anim Behav 50:3340.

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Venom Expulsion

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