831

Toxicon. Vol. 19, No. 6. pp. 831-839, 1981.

Printed in Great Britain.

0041-0101/81/06083I-08 $02.00/0

1981 Pergamon Press Ltd.

THE TOXIC DUVERNOY'S SECRETION OF THE WANDERING

GARTER SNAKE, THAMNOPH IS E LEGANS VAGRANS

DARWIN K. VEST

Department of Zoology, Washington State University, Pullman, WA 99164, U.S.A.

(Accepted for publication 10 June 1981)

DARWIN K. VEST. The toxic Duvernoy's secretion of the wandering garter snake (Thamnophis elegans vagrans). Toxicon 19, 831-839, 1981.The Duvernoy's secretion of the wandering garter snake (Thamnophis elegans vagrans) is highly toxic to mice, causing marked hemorrhaging in the lungs, diaphragm, mesentery and stomach lining, as well as mild local hemorrhaging. Systemic hemorrhaging was most pronounced in mice receiving doses approximating the p. LD50, while doses two times the LD50 or greater produced massive hermorrhaging in the lungs and diaphragm only. Local extravasations were directly proportional to dose. Oral secretions other than Duvernoy's secretion failed to produce lethal effects in mice challenged with doses up to 7 times the LD50 of Duvernoy's secretion. A micro-aspiration techniques for the collection of Duvernoy's secretion from colubrid snakes is described, and liquid as well as dried secretion yields for Thamnophis elegans vagrans are presented.

INTRODUCTION

Toxic oral secretions associated with the saliva of some colubrid snakes have occasionally been

demonstrated (ALCOCK and ROGERS, 1902; COWLES and BOGERT, 1935; CECCALDI and TRINQUIER, 1948;

MEBS, 1968; WILLARD, 1967; DOMERGUE and RICHAUD, 1971). Additionally, a number of envenomations

by opisthoglyphic Colubridae have been reported (CRIMMINS, 1937; BROWN, 1939; FITZSIMONS and SMITH,

1958; POPE, 1958). More recently, cases of poisoning involving aglyphous serpents have been documented

(HEATWOLE and BANUCHI, 1966; MITTLEMAN and GORIS, 1974; NICKERSON and HENDERSON, 1976;

MATHER et al., 1978; SEIB, 1980). NAHAS et al., (1976) presented studies of the Duvernoy's secretions of

the aglyphous Japanese yamakagashi (Rhabdophis tigrinus), while THEAKSTON et al. (1979) investigated

toxic properties of its Asiatic relative, the red-neck keelback (Rhabdophis subminiatus).

Toxicity of the oral secretions of North American garter snakes (Thamnophis) has been suspected

(MCKINSTRY, 1978), but not confirmed. An instance of human poisoning following a bite by a western

aquatic garter snake (Thamnophis couchi) has been reported in the popular literature (MINTON, 1978), and

recently VEST (1981) described a case of envenomation following a prolonged bite by a wandering garter

snake (Thamnophis elegans vagrans). The study herein reported confirms the presence of toxic moieties in

the Duvernoy's secretion of Thamnophis elegans vagrans, a common serpent of the western United States

and adjacent southwestern Canada.

MATERIALS AND METHODS

Two hundred and twenty adult specimens of Thamnophis elegans vagrans were collected mainly from agricultural areas of Whitman County, Washington and the adjacent Latah County, Idaho, U.S.A. Most' specimens were

DARWIN K. VEST

collected in the spring (March 15-June 30) and were subjected to initial extraction procedures as soon as possible following collection.

Extraction and yield of Duvernoy's secretion Each snake was measured, sexed and then grasped gently behind the head with the left hand of the operator and

brought to eye level. The teeth of the right maxilla were exposed by either sliding the lower jaw to the left or by manually forcing the mouth open with a blunt probe. The tip of a 5 1 disposable micropipet (Van Waters and Rogers 353432-706), attached to a 15 inch micropipet aspirator tube, was then carefully placed in contact with the tip of the posterior-most maxillary tooth and held in place. Great care was taken to prevent oral mucosa contact with the tip of the micropipet, otherwise liquid flow rate was impeded (Fig. 1). In each extraction a stopwatch was started the instant the tooth-tip touched the micropipet, and when liquid flow was noted the elapsed time was recorded. A vacuum was then applied to the system by very gentle oral suction applied at the mouthpiece of the micropipet holder. When Duvernoy's secretion no longer flowed, extraction was discontinued and the total volume of collected secretion was recorded. The micropipet was then evacuated into a 5 ml test tube packed in ice. The snake was then grasped in the operator's right hand and the procedure repeated for the left posterior maxillary tooth. Periodically, the micropipet lumen was washed with distilled-deionized water, which was then added to the collected secretion in the 5 ml test tube. The secretion was frozen, lyophilized and weighed. Protein content of the secretion was determined via BioRad Protein Assay (BioRad Laboratories, Richmond, CA, U.S.A.), according to the method of BRADFORD (1976). Absorbance was plotted at 595 nm, using a bovine serum albumin standard.

Non-Duvernoy's oral secretions

Other oral secretions were also collected from each snake. A 20 l disposable micropipet was attached to a 70 cm length of polyethylene tubing (Clay Adams No. 7420,1.D. 0.86 mm), the opposing end terminating in an ice-packed 5 ml test lube enclosed in a 125 ml Erlenmeyer flask equipped with a vacuum spigot. A small vacuum pump was attached to the flask establishing a vacuum aspiration system. The oral cavity, exclusive of the posterior maxillary region, was then subjected to aspiration. The micropipet tip was passed slowly along the entire inner margin of the lower jaw, collecting secretions present at the base of the dentary teeth. The micropipet was then passed over the mucosa between the maxillary and palatine teeth, anterior to the maxillary diastema. Finally, the micropipet tip was drawn along the inner bases of the pterygoid and then the palatine teeth. Secretions thus obtained were deposited in the collection receptacle. The entire aspirating system was flushed with distilled-deionized water every 3 - 5 aspirations and the product added to the collection vessel. The aspirate was then immediately frozen, lyophilized and weighed.

Toxicity determination Lyophilized, first extraction Duvernoy's secretion was pooled and reconstituted in 0.9% physiological saline solution

to a working concentration of 1 mg/ml. Healthy, young male Swiss-Webster laboratory mice weighing 10.5 0.5g each were used. Six mice were challenged via the i.p. route with graduated doses of 10-30mg/kg and fatalities were recorded 24 hr post-injection. The dose range was then narrowed, with two subsequent groups of 6 mice each, and an estimated LD50 calculated. This final estimate was confirmed with a group of 8 mice, each receiving the calculated LD50 dose. All mice were autopsied immediately after death and gross examination of all major organs was performed. Mice surviving challenges (over 24 hr) were killed by cervical dislocation and autopsied. Possible toxicity of the non-Duvemoy's oral aspirate was investigated similarly.

RESULTS

Extraction and yield of Duvernoy's secretion

Following insertion of the micropipet on the posterior maxillary tooth, a lag period elapsed (2-154sec; x

= 40sec) in which no release of secretion occurred. The lag period ended when a clear, somewhat viscous

fluid was first observed accumulating in the orifice of the micropipet tip. A front of liquid could then be

observed migrating down the lumen of the pipet. The flow rate of the front could be increased by gentle

teasing of the tooth with the micropipet. Circular massage of the Duvernoy's gland produced an increased

flow rate in some snakes, but appeared to have no effect in others. Neither teasing nor massage appeared to

affect the ultimate collectable volume, but excessive application of either technique produced artifacts

(blood, mucus), rendering the sample unusable. Protein content of freshly extracted secretion was 55.5

mg/ml.

Liquid yields of secretion were highly variable (Table 1). Forty-five per cent of specimens in our total

series did not yield measurable secretion. The other 55% produced an average of 0.71 l of secretion per

session, although exceptional yields (2.04.5 1) were obtained from

FIG. 1. MICRO-ASPIRATION TECHNIQUE USED FOR EXTRACTION OF DUVERNOY'S SECRETION FROM COLUBRID SNAKES.

At right : disposable micropipet (dm) in collection position : differing sizes may be desirable, depending upon posterior maxillary tooth size and anticipated liquid volume. Micropipet holder (mh), aspiration tubing (at) and mouth piece (mp) complete the aspiration system. At left : micrograph showing the posterior region of the maxilla of Thamnophis elegans vagrans with 5 l micropipet in place on the tip of the posterior maxillary tooth.

FIG. 2. SYSTEMIC HEMORRHAGE IN LUNGS FOLLOWING INJECTION OF SECRETION FROM DUVERNOY'S

GLAND OF Thamnophis elegans vagrans. (A) Lungs of mouse challenged with 30 mg/kg of T.e. vagrans Duvernoy's secretion. Mice challenged with doses of this magnitude die relatively quickly and often may not exhibit significant hemorrhaging in other internal organs. Mice lethally challenged with lower doses may show prolonged survival times, but exhibit more extensive systemic hemorrhaging. (B) Lungs of control mouse, no injection.

Toxic Duvernoy's Secretion

835

some individual snakes. One 51.2 cm female delivered a total of 6.5 l during one of the extractions.

Individual specimens gave similar yields at each extraction.

The average yield of dried secretion was estimated to be approximately 57.7 g per snake, with some

specimens producing up to 528.1 g. The lyophilized secretion appeared as a flocculant white powder

and had an approximate protein content of 46%. This powder was soluble in distilled-deionized water and

in 0.9% saline.

Non-Duvernoy's secretions

The aspirate of non-Duvernoy's oral secretions appeared mucoid in nature. The lyophilized preparation

was only partially soluble in distilled-deionized water or in 0.9% saline.

Toxicity of Duvernoy's secretion

The i.p. LD50 of Thamnophis elegans vagrans Duvernoy's secretion in mice was 13.85 mg/kg. All mice

receiving challenges of 15.0mg/kg or more died. Fatalities were not observed in mice challenged with 11.8

mg/kg or less. Mice receiving lethal challenges of T. e. vagrans Duvernoy's secretion exhibited no

immediate manifestations of pain, but within 30min became torpid and assumed a "hunched" posture.

Those receiving 15.0 mg/kg or more became progressively more lethargic, refused food and water and

could be stimulated to move only by direct physical contact. Within 90min all animals at this dose level

developed respiratory difficulties, which in turn evolved into a rapid breathing pattern accompanied by

gasping. Once these latter signs developed, death followed within 10 min. All mice challenged at this dose

level died within 150 min of injection. Mice lethally challenged with doses less than 15.0 mg/kg followed

a similar evolution of signs, but occasional individuals experienced a temporary remission of torpor just

prior to the anticipated respiratory distress. These remissions were short-lived (5-10min) and were

followed by l-4hr of torpor, then apnea and death. Mice receiving sub-lethal doses also became torpid and

periodically exhibited transitory patterns of rapid breathing, but these episodes did not persist. Prior to

torpidity sub-lethally challenged animals exhibited signs of pruritis, as evidenced by compulsive

scratching of extremities, face and ears. Following several hours of intermittent periods of restlessness and

lethargy the mice resume normal behavioral patterns.

Post-mortem examination of lethally challenged mice revealed localized hemorrhaging at the point of

injection, restricted to the peritoneum and the capillaries of the adjacent dermal layer. The extent of local

hemorrhaging appeared directly related to dosage: 30 mg/kg doses typically produced extravasations

measuring 3 cm2 or greater; 12-20 mg/kg elicited lesions totalling 1cm2 or less; sub-lethal quantities (less

than 11.8mg/kg) did not produce local extravasations in either the dermal layer or the peritoneum.

Systemic hemorrhaging was the most significant post-mortem finding. Massive pulmonary hemorrhage

was present in all mice receiving lethal challenges (Fig. 2). Other viscera also exhibited hemorrhage, the

extent of which appeared dependent upon post-injection survival time as well as the dose administered.

Mice challenged with 30 mg/kg exhibited extrapulmonary hemorrhaging only in the diaphragm, while

mice lethally challenged with doses of 15 mg/kg or less typically showed extensive hemorrhage in the

diaphragm, mesentery, stomach lining and, occasionally, the liver. No challenge produced significant

bleeding in the brain, intestines, kidneys or spleen. Mice receiving non-lethal challenges approaching the

LD50 level exhibited some evidence of extravasation in the diaphragm and the stomach lining, but no

evidence of hemorrhage was detected in subjects receiving less than 11 mg/kg.

Yields* liquid: l/snake (dry: g/snake)

Lag time* (sec. per tooth) Size range

(cm)

No. snakes: yielders (non-

yielders)

Total no. of

extractions Max Min Mean S.D. Max Min Mean S.D.

19.2-30.7 10(5) 15 3.7

(300.6)

0.1

(8.1)

0.8 + 1.1 (65 89)

140 6 35 33

33.3-43.5 7(5) 10 2.5 (203.1)

0.1 (8.1)

0.9 0.9 (77 71)

85 4 30 + 26

46.1-58.9 21(15) 29 6.5 (528.1)

0.1

(8.1)

1.2 1.5 (94 120)

118 2 41 33

61.5-71.7 1(7) 1 0.4 (32.4)

0.4 (32.4)

113 113

Table 1. Yields of Duvernoy's secretion from one initial extraction session on a series of 71 Thamnophis elegans vagrans snakes

* Calculated for yielding snakes only.

Toxic Duvernoy's Secretion

837

Non-Duvernoy's secretion

Non-Duvernoy's secretion failed to produce lethal effects in mice at doses up to 100 mg/kg. Diffuse

minor local hemorrhaging (but not systemic) did occur following administration of doses exceeding 85

mg/kg. Mice challenged with non-Duvernoy's secretion exhibited no abnormal behavioral syndromes.

DISCUSSION

The wandering garter snake (Thamnophis elegans vagrans) harbors a toxic Duvernoy's secretion

capable of producing extensive systemic hemorrhaging as well as local extravasations in mice. Systemic

hemorrhage first appeared in the lungs of envenomated mice, then progressed to the diaphragm,

mesentery, stomach lining and liver. Death of lethally envenomated mice appeared to be related to

massive pulmonary hemorrhage, a consistent finding in post-mortem subjects. Hemorrhage in other

internal organs may have contributed to fatality, but was not a consistent finding in mice receiving doses

high enough to kill within 2.5 hr. Systemic hemorrhage caused by T. e. vagrans secretion was similar to

that reported for the venoms of some Crotalidae (TU and HOMMA, 1970; TU 1971). Local extravasations

produced by T. e. vagrans Duvernoy's secretion, however, were far less remarkable than those seen

following crotalid poisoning. Relatively large quantities were required to elicit notable local responses and

these were restricted to capillaries in the vicinity of secretion deposition. It was notable that the threshold

for hemorrhagic activity, whether systemic or local, is approximately 11.0mg/kg, suggesting that a single

moiety may be responsible for both actions.

Collection of Thamnophis and other colubrid Duvernoy's secretions in a form suitably homogeneous for

qualitative and quantitative studies can be accomplished utilizing the "micro-aspiration" technique

described herein. While slight contamination with non-Duvernoy's secretions may sometimes occur, and a

steady hand is required for execution, micro-aspiration is far preferable to macerated gland preparations

(MCALISTER, 1963) or techniques utilizing washable absorbents (THEAKSTON et al, 1979): the former

procedure yields a product considerably different from uncontaminated Duvernoy's secretion, while the

latter does not allow for accurate estimation of secretion yields. Micro-aspiration eliminates such obstacles

and offers an alternative methodology with many of the advantages of standard venom extraction

procedures.

Although mechanical vacuum devices may be successfully implemented for micro-aspiration, oral

suction provided a more controllable means of regulating vacuum. The lag time observed between

micropipet placement and flow initiation appeared to be associated with the viscous nature of T. e.

vagrans Duvernoy's secretion. TAUH (1967) found that Thamnophis elegans possessed a "mixed"

Duvernoy's gland, e.g., one containing both serous and mucous secretory epithelia. This condition

probably contributed to the viscous character of the Duvernoy's secretion of Thamnophis elegans vagrans.

Yields reflected in this study indicate that considerable variation exists among individual specimens in

regard to secretion yield (Table 1).

No clear explanation has emerged as to what function the inherent toxicity of Duvernoy's secretion in

Thamnophis elegans vagrans may serve. The temptation to ascribe this toxicity to promotion of rapid prey

death should be resited in the light of certain physical and behavior al limitations of T. e. vagrans

(KARDONG, 1979). The enlarged posteior maxillary teeth of T.e. vagrans lack a secretion groove or its

equivalent, but have a sharp, prominent edge on the posterior surface of these same teeth which may

promote entrance of oral secretions into the tissues of prey items (TAUB, 1967; WRIGHT et al, 1979).

Northwest populations of T. e.

838 DARWIN K. VEST

vagrans are known to feed quite heavily on small mammals, and laboratory mice have occasionally been

observed to die following prolonged bites by this species which caused only minor mechanical damage

(PETERSON, personal communication). Nevertheless, T. e. vagrans and other populations of T. elegans

generally control prey by coiling-like manuevers, including constriction (PETERSON, 1978; GREGORY et al,

1980). The quantities of available secretion in many T. e. vagrans are probably insufficient to provide a

rapid dispatch of mammalian prey, although a small percentage of specimens harbor enough secretion to

easily kill one or more mice. It is feasible, therefore, that one role of Thamnophis elegans vagrans

Duvernoy's secretion may be to serve as an alternative or supplemental means of subduing struggling prey

(for additional interpretations see KARDONG, 1979).

AcknowledgementsThe author expresses utmost appreciation to KENNETH V. KARDONG for his sustained support of this project, use of laboratory facilities, advice and comments on the manuscript and numerous other kindnesses. Special thanks is also given REBECCA J. VEST for preparation of line drawings and general assistance. For the procurement and use of live specimens thanks are due DICK R. HIGHFILL, RODNEY A. MEAD and DAVID J ANSEN. I am pleased to acknowledge RAYMOND REEVES for the use of lyophilization equipment and Vic VINSON and DEBRA L WRIGHT for photography. This investigation was supported in part by NSF Grant No. 79-16568 to K. V. KARDONG.

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