The Buccal Buckle: The The Buccal Buckle: The Functional Morphology of Functional Morphology of Venom Spitting In CobrasVenom Spitting In Cobras Presented by: Qudirat JamiuPresented by: Qudirat Jamiu

Young, B., Dunlap, K., Koenig, K., and Singer, M. “Young, B., Dunlap, K., Koenig, K., and Singer, M. “The buccal The buccal buckle: the functional morphology of venom spitting in buckle: the functional morphology of venom spitting in cobras”cobras” The Journal of ExperimentalThe Journal of Experimental Biology Biology Volume 207 (2004) Volume 207 (2004)

Pages 3483-3494. Pages 3483-3494.

BackgroundBackground

Asiatic and African cobras independently evolved Asiatic and African cobras independently evolved ability to expel venom as pressurized horizontal ability to expel venom as pressurized horizontal stream stream Known as spittingKnown as spitting Form of active defensive behaviorForm of active defensive behavior

Some cobras can spit venom as far as 3 metersSome cobras can spit venom as far as 3 meters Pressure requiredPressure required

Most venomous snakes have to make direct Most venomous snakes have to make direct physical contact with an object/organism to physical contact with an object/organism to propel venom propel venom Spitting cobras have the ability to expel venom without Spitting cobras have the ability to expel venom without

making direct physical contactmaking direct physical contact

Have specialized exit orifice of the Have specialized exit orifice of the fangfang Exit orifice is directed more craniad and Exit orifice is directed more craniad and

has a more circular opening than the exit has a more circular opening than the exit orifice of non-spitting cobrasorifice of non-spitting cobras

This explains why venom is propelled forward, This explains why venom is propelled forward, rather than downward.rather than downward.

Fangs surrounded by CT and Fangs surrounded by CT and epitheliumepithelium Attached to lateral and cranial surface of Attached to lateral and cranial surface of

the maxillathe maxilla No smooth or skeletal muscleNo smooth or skeletal muscle Venom duct is also attached to lateral surface Venom duct is also attached to lateral surface

of maxillaof maxilla

The upper jaw/ palato-maxillary arch The upper jaw/ palato-maxillary arch of cobras consist of four bonesof cobras consist of four bones Pterygoid, ectopterygoid, palatine and Pterygoid, ectopterygoid, palatine and

maxillamaxilla Four skeletal muscles contact the Four skeletal muscles contact the

palato-maxillarypalato-maxillary M. Protractor pterygoideus, M. levator M. Protractor pterygoideus, M. levator

pterygoideus, M. Retractor pterygoideus, M. Retractor pterygoideus, and M. pterygoideuspterygoideus, and M. pterygoideus

Jaw Anatomy Jaw Anatomy

Illustration of the lateral view of the skull, and ventral view of the palato-maxillary arch, of Naja nigricollis. Muscle attachment sites are shown as solid colors (lateral surface) or broken colors (medial surface) for the following muscles: M. adductor mandibular externus superficialis (yellow); M. levator pterygoideus (purple); M. protractor pterygoideus (red); M. retractor pterygoideus (blue); M. pterygoideus (green). e, ectopterygoid; m, maxilla; pa, palatine; pf, prefrontal; pt, pterygoid.

ObjectiveObjective

To determine the functional bases of To determine the functional bases of venom spitting venom spitting Muscle involvementMuscle involvement Bone Displacement Bone Displacement

Materials and MethodsMaterials and Methods

5 Black-necked sitting cobras 5 Black-necked sitting cobras Naja Naja nigricollis, nigricollis, 1 Red spitting cobra 1 Red spitting cobra Naja Naja pallida, pallida, 1 Indochinese spitiing cobra 1 Indochinese spitiing cobra Naja Naja siamensissiamensis, 4 Egyptian cobras , 4 Egyptian cobras Naja haje, Naja haje, and 3 Forest cobras and 3 Forest cobras Naja melanoleuca.Naja melanoleuca.

Venom expulsion measured using high Venom expulsion measured using high speed videography and photographyspeed videography and photography Multiple spitting episode recorded using Multiple spitting episode recorded using

MotionScope 1000S at 500 frames sMotionScope 1000S at 500 frames s-1-1

Recorded venom expulsions of spitting and Recorded venom expulsions of spitting and non-spitting cobrasnon-spitting cobras

Sagital bisections of the heads of Sagital bisections of the heads of various species were examined to various species were examined to understand anatomy of spittingunderstand anatomy of spitting

Analysis of mechanical role of the M. Analysis of mechanical role of the M. protractor pterygoideus (PP) in spittingprotractor pterygoideus (PP) in spitting Exposure of muscleExposure of muscle Applied bipolar stimulating probe to Applied bipolar stimulating probe to

surface of mucslesurface of mucsle Electrical stimulationsElectrical stimulations Repeated stimulations with uniaxial strain Repeated stimulations with uniaxial strain

guageguage

Venom pressure measurementsVenom pressure measurements Fang removedFang removed Polyethylene (PE) tubing placed over Polyethylene (PE) tubing placed over

fang and attached to a pressure fang and attached to a pressure transducertransducer

M. protractor pterygoideus (PP) and M. M. protractor pterygoideus (PP) and M. adductor mandibulae externus adductor mandibulae externus superficialis (AMES) exposedsuperficialis (AMES) exposed

AMES directly contacts venom gland, PP AMES directly contacts venom gland, PP does notdoes not

Muscles were stimulated individually Muscles were stimulated individually and simultaneouslyand simultaneously

Electromyography Electromyography (Instrument that produces an visual (Instrument that produces an visual

record of the electrical activity of a record of the electrical activity of a skeletal muscle by electrode inserted skeletal muscle by electrode inserted into the muscle or placed on the skin )into the muscle or placed on the skin )

Inserted into PP and AMES through Inserted into PP and AMES through incisions in headincisions in head

Cobras were induced to spit by Cobras were induced to spit by experimenterexperimenter

Spit detector held in front of experimenters Spit detector held in front of experimenters faceface

Striking sent signal to data system Striking sent signal to data system

VGVG =AMES =AMESPGPG = PP = PP

ResultsResults The high speed digital videography revealed that The high speed digital videography revealed that

the spitting behavior had consistent patterns of the spitting behavior had consistent patterns of motionmotion

Four displacements occurred immediately prior to Four displacements occurred immediately prior to venom expulsion venom expulsion Snout complex rotated in sagital plane elevating snout Snout complex rotated in sagital plane elevating snout Lateral displacement of caudal maxilla causing Lateral displacement of caudal maxilla causing

bulge/deformation of supralabial scales bulge/deformation of supralabial scales Elevation of CT and epithelium surrounding fang to Elevation of CT and epithelium surrounding fang to

expose fang tipexpose fang tip Spit released with mouth slightly open at ~ 25Spit released with mouth slightly open at ~ 25oo

Deformations of the Deformations of the palato-maxillary arch palato-maxillary arch during spitting. (A) during spitting. (A) Naja nigricollisNaja nigricollis immediately prior to immediately prior to spitting; deformation spitting; deformation of the supralabial of the supralabial scales (arrow) (B) a scales (arrow) (B) a high-speed digital high-speed digital videograph recording videograph recording of of N. nigricollisN. nigricollis spitting spitting

The displacement of the palato-The displacement of the palato-maxillary arch observed during maxillary arch observed during spitting resembled the unilateral spitting resembled the unilateral motions of the palato-maxillary arch motions of the palato-maxillary arch associated with prey ingestion known associated with prey ingestion known as the ‘pterygoid walk’as the ‘pterygoid walk’

Displacements observed during Displacements observed during spitting was never observed in non-spitting was never observed in non-spitting cobras, nor was it observed spitting cobras, nor was it observed from spitting cobras engaged in any from spitting cobras engaged in any other behavior, including other forms other behavior, including other forms of venom expulsionof venom expulsion

Gross histological morphology of Gross histological morphology of palato-maxillary arch of spitting and palato-maxillary arch of spitting and non-spitting cobras revealed minor non-spitting cobras revealed minor differences except for the exit orificesdifferences except for the exit orifices

Contractions generated by electrical Contractions generated by electrical stimulation produced stimulation produced displacements of palato maxillary arch displacements of palato maxillary arch Protracted maxillaProtracted maxilla Protracted palatine Protracted palatine Buckling of palato maxillary arch and Buckling of palato maxillary arch and

maxilloectopterygoid joint maxilloectopterygoid joint

Ventral view of the palate of an Indochinese spitting cobra Naja siamensis before (A) and after (B) stimulation of the M. protractor pterygoideus. Both A and B are photos of the same side of the same animal Note the protraction and rotations of the palato-maxillary arch. f, fang; pa, palatine; pt, pterygoid.

Strain gauges placed on the palatal mucosa (A) or scales over the nasofrontal joint (B) of two separate spitting cobras Naja nigricollis. In both experiments contraction of the M. protractor pterygoideus, either artificially (A) or during spitting (B), resulted in deformations of the palato-maxillary arch evident in the strain gauge tracings.

12 electrical stimulations measured

Displacement of palatoperygoid and

maxilloectopterygoid can be seen by the signals obtained from the strain guage

Clear pattern of deformation observed prior to spits recorded

Venom PressureVenom Pressure Individual stimulations of PP and AMES Individual stimulations of PP and AMES

produced clear patterns in pressure produced clear patterns in pressure changeschanges

AMES produced little pressureAMES produced little pressure PP produced displacement of palato-PP produced displacement of palato-

maxillary arch and tissue surrounding fang maxillary arch and tissue surrounding fang and produced pressure greater than that of and produced pressure greater than that of the AMESthe AMES

Two muscles stimulated together Two muscles stimulated together produced pressure greater than the sum produced pressure greater than the sum of the individual muscle stimulationsof the individual muscle stimulations

Venom pressure recorded from the fang tip of Naja nigricollis. Note that stimulation of the M. adductor mandibulae externus superficialis (AMES) simultaneously with the M. protractor pterygoideus (PP) produces greater venom pressure than when either is stimulated alone. This pattern holds whether the muscles are given twitch (A) or train (B) stimuli.

Stimulation trials Stimulation trials exposed to single exposed to single twitch and train twitch and train stimulistimuli

EMG activityEMG activity Electrical activity during spitting in the Electrical activity during spitting in the

PP lasted 37 ms and in AMES 96msPP lasted 37 ms and in AMES 96ms Mean spit duration 66msMean spit duration 66ms Palatal projections appeared 3 ms prior Palatal projections appeared 3 ms prior

to onset of spitting to onset of spitting

ConclusionConclusion Analyses suggest that a two-component Analyses suggest that a two-component

mechanism form the functional basis of venom mechanism form the functional basis of venom spitting in cobras. spitting in cobras. displacement of the palato-maxillary arch displacement of the palato-maxillary arch increase in pressure caused by contraction of the M. increase in pressure caused by contraction of the M.

adductor mandibulae externus superficialis adductor mandibulae externus superficialis Spitting cobras utilize a pressure-balance system Spitting cobras utilize a pressure-balance system

for venom expulsion; however, unlike all other for venom expulsion; however, unlike all other venomous snakes where displacement of the fang venomous snakes where displacement of the fang sheath is passive, in spitting cobras the sheath is passive, in spitting cobras the displacement is actively produced by the displacement is actively produced by the contraction of the M. protractor pterygoideus and contraction of the M. protractor pterygoideus and the displacement of the palato-maxillary arch the displacement of the palato-maxillary arch

Phylogeny of snakes remains Phylogeny of snakes remains uncertain, however functional uncertain, however functional convergence between Elapidae convergence between Elapidae (rattlesnakes) and Viperidae (rattlesnakes) and Viperidae (spitting cobras) has been suggested.(spitting cobras) has been suggested.

Venom delivery system of Elapidae Venom delivery system of Elapidae and Viperidaeand Viperidae Nearly every aspect of the venom Nearly every aspect of the venom

delivery system of elapids and viperids delivery system of elapids and viperids is morphologically distinct is morphologically distinct

It appears that rather than evolving a It appears that rather than evolving a suite of morphological specializations suite of morphological specializations for spitting, cobras have instead for spitting, cobras have instead modified a motor action pattern modified a motor action pattern employed for ingestionemployed for ingestion

SummarySummary

Prior to spit discharge AMES involved in Prior to spit discharge AMES involved in increasing venom pressureincreasing venom pressure AMES displaces the palato-maxillary archAMES displaces the palato-maxillary arch Contractions of the PP causes displacement of Contractions of the PP causes displacement of

the palato-maxillary arch and fang sheath the palato-maxillary arch and fang sheath which also influences venom pressurewhich also influences venom pressure

AMES and PP are activated simultaneouslyAMES and PP are activated simultaneously This increases venom pressure within the This increases venom pressure within the

venom gland, propelling stream of venom venom gland, propelling stream of venom through the venom duct and out the fangthrough the venom duct and out the fang