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Geographical variation in Crotalus scutulatus scutulatus (Mojave rattlesnake) venom properties
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GEOGRAPHICAL VARIATION IN CROTALUS SCUTULATUS SCUTULATUS (MOJAVE RATTLESNAKE) VENOM PROPERTIES JAMES
L.
GLSxrr', RICIiARD C. STRAICI~r,' MARTIiA DAVID L. HARDY'
C. WOLFE' and
'Venom Research Laboratory, Veterans Administration Medical Center, Sah Lare City, UT 84148 and 'Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ, 85724, U .S .A. (Amepted for publirntion 10 August 1982) J . L . Ot .eww, R. C. S~afrr, M. C . wot .ae and D. L . hL+anv . Oeographical variation in Crotahcs srutulatus scutulotus (Mojave rattlesnake) venom properda. Taxirnn 21, 119-130, 1983 . - Individual venom samples were analyzed from 12 specimens of Crota+ss scutulatus srutulatus, from north of Tucson to the extreme southeastern region of Arizona . Six of the specimens, from north of Tucson, produced venan lethal toxicity C.p. t.n ) values in mice of 2 .0-6.0 mg/kg. These coincided with the values previously reported for C, s. srutulatus in the Phoenix, Arizona, region and designated as type B venom (OtBtav and Snwatir, 1978) . i:n contrast, the venom t.n of six individuals from extreme southeastern Arizona, including one individual near Tucson, ranged from 0.22-0 .46 mg/kg. This corresponds to the values for C. s. scutulatus venom previously reported and designated as type A venom (Ot .amv and Snutattr, 1978) . Specimens with type A venom have been collected in California, Nevada, Utah and regions of Arizona. In addition to differences in lethal toxicity, the type B venom consistently exhibits a different protein profile, greater proteolytic activity, greater hemorrhagic activity and contains little or none of the m$jor lethal toxin, Mojave toxin, compared to the type A venom. No external morphological characteristic could be found differentiating the type A venom specimens from the type B venom specimens . These findings further confum the geographical variation of C. s. scurulatus venom in Arizona.
INTRODUCTION
and STRAIGI-IT (1978) reported a significant geographical difference in the lethal toxicity of Mojave rattlesnake (Crotales scetulatus scutulatus Kennicott) venom in laboratory mice . The more toxic venom from California, Utah and Southwestern Arizona, with i.p. LD,o values of 0.13 - 0.54 mg/kg, was designated venom A. The less toxic venom from the Phoenix, Arizona, region, with i.p. LDss values of 2.3 - 3.8 mg/kg, was designated venom B. Also, commercial antivenin was found to be more effective against venom H than venom A. These findings suggested that both venom research and the clinical events following C. s. scetulatus envenomation could be affected, depending on the type of venom involved. It has been reported that C. s. scutulatus envenotnations produce very few local symptoms and signs, but marked neurotoxic effects (RUSSELL, 1969; RUSSELL and PUFFER, 1970; RUSSELL et al., 1975; CASTILONIA et al., 1981). However, HARDY (1982) has reported that the clinical symptoms and signs of patients bitten by C. s. scutulatus in the Tuaon and Phoenix, Arizona regions often include significant local effects, with few neurotoxic signs. This suggests that the composition of C. s. scutulates venom may differ GLENN
"To whom correspondence should be addressed.
120
J. L. GLENN, R. C. STRAIGHT, M. C. WOLFF and D. L. HARDY
regionally, and may be related to the regional differences in lethal toxicity of this species reported previously (GLENN and STRAIGHT, 1978) . The purpose of this report is to: (1) compare some biochemical properties of C. s. scutulal`es venom from specimens from various regions of the southwestern U.S.A . ; (2) further define the geographical limits of venom A and venom B from C. s. scutulatus . MATERIALS AND METHODS Specimens and venom samples Five male and seven female Crotales scetelates scutelatus were collected near Tucson, Arizona and in the extreme southeastern corner of Arizona, as illustrated in Fig. 2. The spximens ranged from 40-110 cm in total length. The mouse i.p . LDN results were determined under single blind study conditions for each spedmen and related to its identification number. Additional C. s. scwtulatus venom samples, used forcomparative analysis in this study, were from apedmens previously reported (GLernv and STRAIGHT, 1978) and were from locations in Arizona, California, Nevada and Utah (as illustrated in Fig. 2) . Snakes utilized in this studyare catalogued in the Brigham Young University, Life Silence Muscum, Provo, Utah . Three adult specimens of the H~~nmgn tlan rattlesnake (C. scutulates salvine) were from Vera Cruz, Mexico . Western diamondback rattlesnakes (Crotales atrox) were from Oklahoma and Texas. Lyophilized "Mojave toxin" used in this report, is the add -basic protein complex (Mil-9V) isolated by CATs and Bmame (1978) and was gradously supplied by Allan Bieber, Arizona State University, Tempe, Arizona . Venom rnUection, processing and lethal tazldty testing Veaoms were collected from individual specimens by manual venom extraction tahniques and centrifuged at 1000 a for 15 min. The supernatant fhdd was frozen, lyophilized to dryness and stored at 4°C. All testing of venoms from specimens of the Tucson and Southestern Arizona region was accomplished within four months or less of venom eollecdon. The protein content of the venoms was determined by the biuret atethod of GORNALL et al. (1949). Venom lethal toxidty was determined for each individual sample by raonatituting the lyophilized venom in normal saline at 1 mg/ml and igjecting the venom i.p . into 18-22 g Swiss-Webster mice (female), using four serial doses and a minimum of six mice per dose . The reauhing LDN values were calculated from the survival ratios at 48 hr by the method of KARHBR (1931) . Proteolytic acrd esttmae actlvlty assays Venom proteolytic activity was assayed for each individual spatimen of C. s. scutulates and C. s. satvint and pooled C. atroz venom, using a modification of the method of RnvnBRnvscr et al. (1968), with Remazolbrilliant bhre hide-powder azure (Cal-Biochem) as substrate. Venom (100 ~, in 0.1 ml of 0.1 M sodium phosphate buffer, pH 7.5, was added to S ml of hide powder suspension (5 mg/ml in 0.01 M sodium phosphate buffer, pH 7.3). The solution was continuously stirred for 13 min in a water bath at 37°C, filtered through a No. 1 Whatman filter and the proteolytic activity determined at 320 nm . Esterase activity was measured using abenzoyl-L-arginine ethyl ester (BASE) substrate, as outlined by ScHweRT and TAIO:NAKA (1955), using 20 Ng of venom. Xemorrhagic assay The hemorrhagic activity of the venoms was determined by modifying the method of ICo~wo et al. (1960) . Venom (1-SO ~, in 0.1 ml of 0.911 saline, was igjected intradermally into the shaven back of rabbits, marked into grid squares. The rabbits were sacrificed by ether inhalation 24 hr after igjection and the dorsal skins removed and spread on a glass plate. The hemorrhegic spots were measured and photographed from the dorsal side ova a fluorescent light sotu~oe. h;jections of 25 I+g of C. s. scvtulates (venom A and B) and C. s. salvinl venom were selected for oomparatIve analysis. Crotales adaneantees (1 pg) and C. atrox (1 ~ venoms were utilized as reference venoms . Calelectrophorrsis Polyacrylamide gel electrophoresis (PAGE) was run in a Bio-Rad vertical slab dauophoresis cell Mode1220. The gd was 1 .5 mm thick. Sample wells were formed with Canaloostacking gel (pH 6.6 - 8.6)and were 10 mm in length . The 71i acrylamide separating gel (pH 8.9-9 .0) was 80 mm in length . The sample was electrophoreaed toward the anode (+) at 2 mA constant current per sample and was stopped 10 mm from the bottom of the gel. Blectrophoreaia buffer was 0.025 M Tris/0.192 M glycine, pH 7.83. Hromophmol blue was used as tracking dye. The gel was fixed in 10% acetic add/40sfir isopropanoV3019 water for 60 min, stained with Coomassie Blue (O.OSy.) in 101Y acetic add/10% isopropanol/80% water far 60 min and deatained in 1011 acetic add/10~K iaopropanoV801i water, using 3 -4 dungea of solution over a 24 hr period . The stained gels with their protein patterns were photographed and each gd was sliced into strips 7.S mm wide and the protein patterns scanned at 280 nm oa s Gilford gel scanner Mode12520.
Crorahrs scetuiates sewtelatus venom
121
RESU1.Ts
The lethal toxicities of six of the C. s. scututatus specimens collected north of Tucson, Arizona, were similar to the C. s. scutulatus venom B values previously reported (GLENN and $TRAIaI-IT, 1978) for the Phoenix region (Table 1). The mouse i.p. LD variation of 2.0- 6.0 mg/kg for venoms from the six individuals was similar to the variation within multiple venom samples from a single specimen for venom collected at three monthly intervals (i.p. LD 2.6-S.S mg/kg) . All five specimens collected from the southeastern corner of Arizona and one specimen from just north of Tucson (see Fig. 2, nearest Tucson) produced type A venom l,n values (0.22-0.46 mg/kg) (Table 1). The differences in lethal toxicity between type A and B venoms were not linked to sex or age of the specimens. Death times were noticeably different for venoms A and B. Calculating deaths at 48 hr, rather than 24 hr, proved critical for venom B LDeo values, since a significant number of deaths occurred between 24 and 48 hr. Conversely, deaths from venom A occurred within 24 hr; usually within 12 hr. Crotales s. salvini is the southernmost form of the species, C. scutulatus. The i.p. LD,o values from three specimens were similar to type A venom from C. s. scutulatus (Table 1), and are in agreement with that previously reported from a single individual (GLENN and STRAIGIiT, 1978) . TABLE
1.
SLLpliareS
COMPARISON
Crorarus Croraha sruruiQres
OF VENOM LETHAL TOXICITY OF
sC7lrLlQle4 VENOM FROM ARIZONA AND
salvini vENOM
FROM VERA CRUZ, MEXICO
Number of "Venom i.p. LD (dry wt) Venom species specimens mg/kg - mean (range) C. s. sretuiarus Venom At 6 0.28 (0 .22-0.4~ Venom B ~ 6 3.33 (2 .00-6.00) C. s. salvini 3 0.30 (0 .22-0.40) "LD Was determined foréach specimen by injecting venom i.p. into 18-22 g Swiss Webater mice, as described in teat . fi Five specimens from extreme southeastern Arizona, near Portal, Dos Cabezas and Apache, and one spaimen from near the northern city limits of Tucson, Arizona (see Fig. 2). # Six specimens from north of Tucson, Arizona, SO hm N.W . of Tucson near Red Racr, N.W . Tucson 17 lnn, San Manue147 km N.E. Tucson (see Fig. 2) .
Enzyme activity Another major difference between C. s. scetulates venoms A and B was in the hide powder proteolytic activity . All of the individual venom A samples were low or lacking in proteolytic activity using this substrate. Crotales s. salvini venom, which was similar in i.p . LD value to venom A, was also similarly low or lacking in hide powder protoolytic activity . In contrast, the venom B samples were strongly proteolytic and approximately oqual to pooled C. atrax venom (Table 2). The variation of proteolytic activity in venom samples from 13 specimens of C. s. scetulates producing venom B (Table 2) was similar to the variation in venom samples from a single specimen collected by four monthly venom extractions. The variation between individual specimens in venom proteolytic activity may reflect the normal variation in venom protein synthesis in an individual snake.
12 2
J. L. GLENN, R. C. STRAIGHT, M. C. WOLFF and D. L. HARDY
Other Crotales species with relatively low or no hide powder proteolytic activity are C. viridis concolor (Wyoming), C. mitchellü mitchellü (Baja Sur), C. horridus atricaudatus (Texas), C. durissus durissus (unknown locale) and C. adamanteus (Florida). All of the above species exhibited i.p. LD6, values of 0.6 mg/kg or lower, except C. adamanteus (2.1 ~kg)~ Both A and B type venoms from C. s. scutulates had similar esterase (BASE) activity (Table 2) and the activity was highly variable among individual specimens. TABLE 2. COMPARATIVE ANALYSIS OF VENOM PROTEOLYTIC AND ESTERASE ACTIVITIES FROM INDIVIDUAL SPECIMENS OF CrOtaIiL4 sCIfrHiQtYS SCIIhIlQttfS (VENOM A AND VENOM B) AND Crotalus srutulatus sialvini
venom species
Number Df specimens
Proteolytic activity" eonnoo,~ls min mean (range)
Esterase activity fi eoDi2o,$min mean (range)
C. s. scutulatus Venom A $ Venom B $ C. s. snivini C. atrax
23 13 3 Pool
0.005 (0 .000-0.013) 0.192 (0 .114-0.246) 0.001 (0 .000-0.003) 0.201
0.90 (0.37-1 .78) 0.88 (0.35-1.59) 1.04 (0.92-1 .18) 0.31
"Remazol-brilliant blue hide-powder azure substrate (at 520 nm). fi a-Benzoyl-L-arginine ethyl ester (BASE) substrate (at 253 nm). $ Venom type determined by i.p . Ln in mice previous to enzyme assays .
Hemorrhagic activity The comparison of the hemorrhagc activities of C. s. scutulatus venoms A and B and C. s. salvini venom are illustrated in Fig. 1, using 25 pg of each venom. Eight individual venom A samples and two individual C. s. salvini venom samples produced no detectable hemorrhagc lesion . These venom samples were also tested at s0 ~g and no hemorrhage resulted. Conversely, four individual venom B sâmples produced hemorrhagc spots, 10 mm in cross-diameter, at 25 )tg. When using as little as 5 ltg of type B venom, small hemorrhagc lesions were noted. Comparatively, only 1 ~g of venom from C. adamanteus and C. atrax was required to produce hemorrhaic lesions of equal size. In both venom A and B, the level of hemorrhagc activity correlated with the level of proteolytic activity . Our results with venom A were consistent with the results of GIROUX and LACHMANN (1981), who found little or no hide powder proteolytic activity and no hemorrhaic activity in mice with C. s. seutulatus venom. Geographical distribution Of the eight specimens of C. s. scutulatus with venom B previously reported from Arizona (GLENN and STRAIGHT, 1978), only six collection locations were known. Three were from east of Phoenix, two from south of Phoenix and one north of Phoenix (Fig. 2). In this report, 6 additional venom A and 6 venom B specimens were collected with accurate locality data. The results indicate that specimens with type B venom extend from the Phoenix region to the Tucson region of Arizona. Venom protein patterns by gel electrophoresis The Mojave toxin complex (MD-9~ (LATE and BIEBER, 1978), the major lethal toxin in C. s. scutulatus venom, disassociates into two major subunits when fractionated in a
123
FIO . 1 . Col~wwsox OF Tl~ tI~NOAR~iAOIC ACTIVITY OF Crotales scetelatus srlrtelates (varloM A wrm vlavol~t B) t~ Crotales sretetatus siohint vsrlo~t. All igjections was intradamal in 0 .1 ml volume, in the shaven back of a 2 .5 kg rabbit . A, Bight individual venom samples from C. s. scutulates venom A, 25 I+g in each grid square ; B, fow individual venom samples from C. s. scutelatus venom B, 25 Pg in each grid square ; CA, 1 kg of pooled C adamantees venom ; CAA, 1 Kg of pooled Crotales atrox venom ; SALV, 2S kg of individual C. s . sohlni venom samples . The hemorrhaic lesions of venom B, CA and CAA are appro~matdy 10 mm in cross-diameter . Venons A and SALV produced no hemorrhage . The darkening in the SALV grid squares is due to diffusion of hemorrhage from the lateral venom B lemons .
124
FIO . 3 . POLYACRYLAAl1DE OEL BLHCIROPHORHSIS OP V8NOM3.
Elaxrophoreaia buffo 0.025 M Tria/0.192 M Blycine, pH 7 .gS . MT, Mojave toxin, 100 ~ of protein; A, C. s. scvtulahtf venom A, 200 Ng of pooled venom protein from four apaimena ; H, C. s. snrtulatus venom B, 200 Pg of pooled venom protein from four apecimena; SALV, C. s. ,~alvlni 200Pg of pooled venom protein from three apedmena ; 1, Mojave toxin ; 2, acidic aubunit of Mojave taatin. (Stained with Coomesaie brilliant blue.)
Crotales srutulates sretu/attrs Venom
125
F~a, 2. DDIYBVI'ION OF Crotales scutelatus scutelates veraM A ,uvn vFxoM B sracu~ns. ~, Closed circles designate venom A specimens ; o, open circles designate venom B specimens.
basic buffer (pH 7.85) by gel electrophoresis (Figs. 3 and 4). Repeated freezing and thawing of the protein complex resulted in degradation of the toxin. The acidic subunit migrates with the electrophoresic front and serves as an easily identifiable marker for the presence or absence of the toxin in venom samples. Since variations occurred in venom electrophoresic profiles between individual snakes, venom samples of equal portions of venom protein from four individual C. s. st utulatus venom A and venom B and three individual C. s. salvini were each pooled, respectively, and analyzed (Figs. 3 and 4). C. s. scutulatus venom A samples consistently showed protein bands (280 nm) corresponding to the acidic component of Mojave toxin (Figs. 3 and 4), although considerable quantitative variation occurred among six individual samples examined . Peaks corresponding to the Mojave toxin complex were also present in the C. s. salvini venom. Estimates were made of the amount of dissociated acidic subunit contained in venom samples by cutting out and weighing the areas of graph paper corresponding to the gel scan tracings of protein peaks. Six individual venom A samples ranged from 6 to 18% in dissociated acidic subunit content and the content of the pooled venom (four snakes) represented 9% of the total protein recorded (Fig. 4). Mojave toxin was low or lacking in the pooled venom B sample . Interestingly, two of the six venom B samples from the Tucson region exhibited faintly stained protein bands similar to the acidic subunit and Mojave toxin. This may represent an intergradal situation between specimens of the two types of venom in the Tucson region . This regional variation in Mojave toxin content in Arizona specimens appears similar to the regional variation in C. d. tet~cus venom toxins, crotamine and
126
J . L . GLENN, R . C . STRAIGHT, M. C. WOLFF and D. L . HARDY
Addic Subunh
Begin Bei
Verwm A
_ / "v \
Fta . 4 . Gal, scwrt (280 nm) of Mo~wvs ~roxnr, C. s. scutulahas vatvoM A, C. s. scutLlatus vt~voM B wrro C. s. salvirtt vt~oAf u.~usrsw~n nv sta. 3 . The peak corresponding to the acidic subunit of Mojave toxin is illustrated in C. s. scumlatrrs venom A and C. s. salvirt! venom but is absent (or extrentdy low) in C. s. scutulatus venom B .
convulxin, in South America (GONCALVES, 1956 ; GONCALVES and DEUTSCH, 1956; HOGS, 1965 ; PRADO-FYtANCESCHI et al., 1981). Morphological examination GLENN and STRAIGHT (1978) proposed that the differences in the venom lethal toxicity of C. s. scutulatus might correlate with squamation differences, especially if the
Crotales scetulates scetelatus Venom
127
geographical limits of venom B specimens could be further defined. However, in the examination of 13 venom H specimens and 18 venom A specimens, no significant differences in external morphology could be established . All of the features of the specimens examined were similar and overlapped in numerical range. Characters examined included color; body/tail ratios ; rattle width total body length ratios ; the number of body blotches, tail bands, dorsal scale rows, mid-dorsal caudal rows, ventrals, subcaudals, supralabials, infralabials, intersupraoculars; the organization of crown scales ; size and shape of canthals, loreals and preoculars ; posterior connections of dorsal - lateral blotches ; width of preocular and postocular stripes. DISCUSSION Despite earlier confusion with other rattlesnakes, ICLAUBER (1930) provided adequate evidence that C. s. scetulatus was a valid species. By 1939, there was also evidence that the venom of this species was exceptionally toxic (lethal) as compared to other North American rattlesnake forms (GITHENS and WOLFFE, 1939). GLOYD (1940) subsequently concurred with Klauber's speciation of C. scutulates and further nominated C. scutulatus salvini Gunther, as a valid subspecies . Conflicting clinical reports involving envenomation by C. s. scutulatus were often thought to be due to misidentification of the species involved, e.g . C. atrax confused with C. s. scutulatus. Although envenomation by C. s. scetulatus has been characterized as predominantly producing neurotoxic effects (including respiratory paralysis) with little or no local effects, a recent clinical study in Arizona indicates that some C. s. scutulatus envenomations do, in fact, produce local tissue effects, sometimes severe (HaxDY, 1982 ; HARDY, 1983). In the majority of the Arizona clinical cases (14 of 1S), neurotoxic signs were notably absent . The discovery of a major lethal toxin in C. s. scutelatus venom (BIEBER and TU, 1974 ; B>EBER et al ., 1975 ; PATTABHIRAMANN and RUSSELL, 1975 ; HENDON, 1975) that produces neurotoxic effects (CASrILONIA et al., 1979 ; GOPALAKRISNAKONE et al., 1980 ; CASTILONIA et al., 1981 ; Ho and LEE, 1981) corroborates the neurotoxic effects previously reported in human clinical cases. The geographical difference in venom lethal toxicity in Arizona C. s. scetulatus reported previously (GLENN and S'rxnlal-l'r, 1978) is further confirmed in this report. The data also suggests that venom B may produce more severe local tissue effects than venom A. Interestingly, Hardy (1983) noted clinical findings of swelling, bleb formation, ecchymosis and/or necrosis in patients bitten by snakes from the venom B region . The only patient exhibiting a neurotoxic effect (eyelid ptosis) was bitten by a snake from the venom A region . The venom B geographical distribution appears limited to the region north of Tucson to north of Phoenix (Fig . 2) . However, many more specimens are required to determine the exact distribution limits of venom B and to determine if intergradation occurs between snake populations of the two venom types. MIN~roN (1958) reported on C. s. scutulatus venom lethal toxicity from the Big Bend Region of western Texas, indicating that type A venom also exists there. No venom data is available for populations of C. s. scetulates throughout its range in Mexico. A total of four specimens of C. s. salvini have been tested and all produced type A venom lethality and were similarly low in proteolytic and hemorrhagic activities . The C. s. salvini venom also cross-reacts immunochemically (Ouchterlony agar double-diffusion)
128
J . L. GLENN, R. C. STRAIGHT, M. C . WOLFF and D. L. HARDY
with antibodies to the basic phospholipase subunit of Mojave toxin from C. s. scutulatus venom A, further indicating a possible similarity in the lethal toxins from the two subspecies (Allan Bieber, personal wmmunication) . Other crotalid venoms and toxins which also cross-react immunochemically with Mojave toxin are C. horridus atricaudatus, C. viridis concolor, C. durissus te~cus, C. durisrus durissus, crotoxin from C. d. ter~cus and concolor toxin from C. v. concolor (HENDOx and BISSER, 1982). C. d. terr~cus and C. s. scutulatus have received considerable attention by toxicologists due to their venoms' high lethal toxicity and potent neurotoxic components . However, other rattlesnake venoms are now known to exhibit LD6o values comparable to C. s. scutulatus, such as C. v. concolor (GLENN slid STRAIGHT, 1977; Pool et al., 1981 ; GLENN and STRAIGHT, 1982; HENDON and BIEBER, 1982), C. h. atricaudatus (MINTON, 1967 ; GLENN and STRAIGHT, 1982), C. m. mitchellü (GITHENS and WOLFFS, 1939 ; RUSSELL et al ., 1960 ; GLENN and STRAIGHT, 1982), C. s. salvini (GLSNx and STRAIGHT, 1978), C. vegrandis (VILLALAZ, 1966 ; SCANNONE et al., 1978) and Sistrurus c. catenatus (MINTON, 1956). Therefore, no longer can C. d. terr~cus and C. s. scutulatus be considered as unique in rattlesnake venom lethality, as has been presented in the past . The importance of geographical differences occurring in venom from a given species/subspecies is becoming quite evident. At the binomial species level, extreme geographical differences in venom composition, including lethal toxicity, venom toxins and/or enzyme activity, have been reported in Crotalus durissus, C. lepidus, C. horridus, C. mitchellü, C. viridis (see review by GLENN and STRAIGHT, 1982), Bothrops asper (ARAGOx et al., 1981), Bothrops numm ;fer (JIMINEZ-PORRAS, 1964), Ngja hgje (IRWIN, et al., 1970), Ngja nigricollis (IRWIN et al., 1970) and others . In this regard, a number of venom research reports continue to use only bionomenclature taxonomy and give little concern to the regional origin of the venom species. The differences in venom lethal toxicity and proteolytic and hemorrhagic activities between the type A and B venoms of C. s. scutulatus are too significant and consistent to be disregarded . They give reason to suspect that dramatic differences might occur in the clinical picture of human envenomation . Considerable variation even occurs between individual snakes in venom protease activity, esterase activity and Mojave toxin content, possibly influencing clinical symptoms and signs following envenomation . These differences in venom quality, combined with other unknowns in envenomation, such as the dose of venom received, further complicate the clinical aspects of C. s. scutulatus envenomations in Arizona. However, in all cases, the early therapeutic use of antivenin is important if significant envenomation is suspected. Due to the relatively poor efficacy of existing commercial antivenin against the type A venom (STRAIGHT et al., 1976; GLENN and STRAIGHT, 1977 ; GLENN and STRAIGHT, 1978), an antivenin prepared against rattlesnake venoms that included venom A or Mojave toxin would be useful in medical ~PY~ The distribution data in this report provide a beginning for determining the range limitation of C. s. scutulatus type B specimens. The lack of recognizable e~zternal characters between venom A and B specimens is unfortunate and thereby places more importance on the establishment of well-defined geographical limits on each type of C. s. scutylatus venom. Presently, the only consistent criteria for distinguishing the two varieties are their venons, with some evidence that venom B spocimen distribution is limited and contained within a certain geographical region of Arizona.
Crotales srutulatus scvtulates Venom
129
Acknowkdgttr~nts -
We thank the Arizona Game and Fish Department and the Utah Division of Wildlife Raout+ces for their cooperation . We also thank Dr . At.LArt Bmeap, Arizona State University, Tempe, Arizona, for supplying the Mojave toxin and immunodiffudon tests utilized is this report . We are grateful to the following persons and institutions for Weir assistance : ToM RArmAZZO, RoaattT LApsorr, Mt~ COFr~N, Lane PowtAS, JAwtes JApcct~ttow, MAROAAEr HApux, HowAttu LwwLen, Joe CApAr~wp, Pttn. Ovetawae, YAp PtTtxxs~zxty and the Animal Research Facllity staff and the Medical Media Production Servis staff, V .A . Medical Center, Salt Lake City, Utah . This research wan supported by We Veterans Administradon Medical Itexarch Program, Veterans Admi~nistratian Medical Center, Sah Lake City, Utah and Hogle Zoological Gardens, Salt Lake City, Utah .
REFERENCES ARAGON, F . and Guaerrsr;x, F. (1981) Bothropts riper venom from We Atlantic and Pacific zones of Costa Rica . Taxkon 19, 797 . Htt~p, A. L . and Tu, A . T . (1974) Purification of an acidic toxic protein from We venom of We Mojave rattlesnake (Crotales scutelatus) . Fedn Proe. Fedn Am. Socs exp. Blol. 33, 1564 . Btuawrt, A . L ., Tu, T . and Tu, A . T. (1975) Studies of an acidic cardiotoxin isolated from We venom of We Mojave rattlesnake (Crotales scutulatus) . Biochim. biophy. Acts 400, 178 . CAS171ANIA, R. R ., PA7TAHFIIRAMAN, T. R . and RussEr.t., F . E. (1979) Neuromuscular effects of fractions of Crotalesscutulates scetulatus venom. Proc. wrst, pharmat. Soc. 22, 203 . CASI'ILANrA, R . R ., PATTABHIRAMAN, T. R., RUSSELL, F. E . and Gon~zA~ac, H . (1981) Electrophysiological studies on a protein fraction (K' ) fran Mojave rattlesnake (Crotahcs scutulatusscutulates) venom . Taxirnn 19, 473 . CxrE, R . L . and Bn?eER, A . L . (1978) Purification and cheracteri7stion of Mojave (Crotales scutulatus scutulatus) toxin and its subunits. Archs Biochem. Blophys. 189, 397 . GtROUx, E . and L.ACFiMANN, P . J . (1981) In vivo diminution by chelators of snake venom-provoked hemorrhage and to vitro inhibition of proteolytic activity . Tazicon l9, 481 . Grttmrrs, T . S. and WoLrrE, N. O'C . (1939) The polyvalency of crotalidic antivenins . III . Mice as test animals for study of antivenins. J. Immun. 37, 47 . Gr.Ervrr, J . L. and $TRAIOHT, R. (1977) The midget faded rattlesnake (Crotales viridis contolor) venom : lethal toxicity and individual variability. Taxiton 1S, 129 . Gt .Ervrv, J . L . and $TRA1OHr, R . (1978) Mojave rattlesnake Crotales scutulatus stutulatus venom: variation in toxicity wiW geographical origin. Taxirnn 16, 81 . GtBrav, J . L . and SraNOFrr, R. L . (1982) The rattlesnakes and Weir venom yield and lethal toxicity . ln: Rattlesnake Venons: Their Action acrd TiaQtr»ent, pp. 3 -119 (Tu, A . T., Ed.) New York : Marcel Dekker Inc . GLOYD, H . K . (1940) The Rattlesnokes, Genera Sistrurus and Crotales. Chicago Academy of Sciences Special publication No . 4 . Chicago : Chicago Academy of Sciences (Reprinted in 1978 by We Society for We Study of Amphibians and Reptiles) . GorvcALVES, J . M. (1956) Purification and properties of Crotamine. In : Versons, pp. 261-274 (BUCKLEY E. E. and PopaES, N ., Eds,) Washington DC : American Association for We Advancement of Science . GoNCALVES, J. M. and Detrrsctt, H. F . (1956) Ultracentrifugal and zone electrophoresis studies of some Crotaltdae venons . Archs Biochen . Btophys . 60, 402 . GOPwi .wrQrc yNAitONE, p HAwooon, B. J ., HoLapoor~, S. E ., SAN'l'ANA, DE S . S . and Tu, A. T . (1980) Sites of action of Mojave toxin isolated from the venom of the Mojave rattlesnake. Br. J. Pharnrac. 69, 421 . GorwALL, A. G ., BARDAWrLL, G . J . end DAVID, M. M . (1949) Determination of serum proteins by means of the biuret reaction. J. biol. Chem. 177, 731 . HARnx, D. L. (1982) Envenomation by We Mojave rattlesnake (Crotales scutulatus srutulatus) in SouWern Arizona, U .S .A . (abstract) . Toxkon 20, 338. HARDY, D. L. (1983) Envenomation by We Mojave rattlesnake (Crotales stutulatus stutulatus) in SouWern Arizona, U .S .A . Taxirnn 21, 111 . HEImON, R. A . (1975) Preliminary studies on We neurotoxin in We venom of Crotahrs scetulatus (Mojave rattlesnake) . Tazirnn 13, 477 . HENDON, R . A . and B>~ax, A. L . (1982) Presynaptic toxins from rattlesnake venons . In : Rattlesnake Venons: Their Action and Treatment, pp, 211-246 (nT, A . T ., Ed .) . New York: Marcel Dekker Inc . Ho, C . L . and Lam, C . Y . (1981) Presynaptic actions of Mojave toxin isolated from Mojave rattlesnake (Crotales stutulatus scvtulatus) venom . Taxirnn 19, 889. HOOF, R . A. (1%S) Preliminary aceount on neotropical Crotalinae (Serpentes, Viperidae) . Mem. Inst. Butantan 32, 109. IRwEV, R. L ., OLivER, K. L ., MOi~iAr~mn, A . H. and HAASr, W . E . (1970) Toxicity of Elapidae venons and an observation in relation to geographical location . Tazicon 8, S 1 . Jn~~PoiueAS, J . M . (1964) Intraspecific variations in composition of venom of We jumping viper, Bothrops nummjfero. Taxiton 2, 187 .
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J. L. GLENN, R. C. STRAIGHT, M. C. WOLFE and D. L. HARDY
KARaBR, G. (1931) Beitrag zur kollektiven behand lung pharmakologischer reihenversuche .Arcb. exp. Path. Pharmak. 1f2, 480. Kt .wuamt, L. M. (1930) Differtntial characteristics of Southwestern rattlesnakes allied to Crotahcs atrax. Bull. zool. Soc. S Diego 6, 1. Kormo, H., Kotvno, S., h~zwww, H. and MunwTw, R. (1960) Studies on the quantitative method for determination of hemorrhagic activity of baba snake venom. Jap. J. med. Scl. Biol. 13, 43 . Mtxrotv, S. A., Je . (1956) Some properties of North American pit viper venoms and their correlation with phylogeny. In: Ytnoms, pp . 145 -151 (BuctQ.ev, E. E. and POaaES N., Eds.). Washington, DC : American Association for the Advancement of Science . Mtxrort, S. A., JR . (1958) Observations on the amphibians and reptiles of Big Bend region of Texas . Surest. Nat. 3, 28 . Mtxrort, S. A., Ja . (1967) Observations of toxicity and antigenic makeup of venoms from juvenile snakes. In : Anima! Toxins, pp. 211-222 (Russt3LL F. E. and $wUNDHRS, P. R., Eds .) . New York: Pergamon Press. PATTABHIAM4AN, T. R. and RUSSaLt F. E. (1975) Isolation and purification of the toxic fractions of Mojave rattlesnake venom. Taxiton 13, 291. Poot., W. R. and Bmaea, A. L. (1981) Fractionation of midget faded rattlesnake (Crotalus viridis cortcolor) venom: lethal fractions and enzymatic activities . Taurirnn 19, 517. Pawno-Fawt~Fn, J. and Bßwzu., O. V. (1981) Convulxin, a new toxin from the venom of the South American rattlesnake Crotahas durLsstrs terr(fuws. Tazicon 19, 875 . Rtrmeatan3cFrr, H., Geotcws, M. C., Su.vsteMwtv, P. and Hwvcaawcx, B. J. (1968) A new ultrasensitive method for the determinadon of proteolytic activity. Clinica chin. Acta 21, 197. Russeu ., F. E. (1968) Clinical aspects of snake venom poisoning in North America. Taunton 7, 33 . Russtn.t ., F. E. and Puts, H. W. (1970) Pharmacology of snake venons. Clan . Taxk. 3, 433 . RUSSELL, F. E., Et~ttY, J. A. and Lotva, T. E. (1960) Some properties of rattlesnake venom following 26 years storage. Proc. Sot. arp. Blo. Med. 103, 737. RtlSSBLt F. E., CwteLSON, R. W., WwurscEmL, J. and Osaoaro:, A. H. (1975) Snake venom poisoning in the United States. J. Am. mad. Ass. 223, 341. Scwravora:, H. R., RonRtauEZ, O. G. and Lwivctrn, A. R. (1978) Enzymatic activities and other characteristics of Crotahcs vegrondïs snake venom. In : Toxins: Animal, Plant and Microbial, pp . 223 -229 (RosEtvEExa, P., Ed .) New York: Pergamon Press. ScewEnr, G. W. and Twt~tvwtcw, Y. (1955) A spectrophotometric determination of trypsin and chymotrypsin . Blochlm . biophys . Acto 16, 570. Srawtotrr, R. and GLENN, J. L. (1976) Antivenom activity of rattlesnake blood plasma . Nature 261, 259. Vn ~ "~ ", A. R. L. (1966) Crotahas vegrandis Klauber: redescripcion y distribution . Men. Inst . Butantan 33, 725.
r .va.n,rb .~,oa.~i9-~30.~sx+ . t+rintea m orat a~n~.
GEOGRAPHICAL VARIATION IN CROTALUS SCUTULATUS SCUTULATUS (MOJAVE RATTLESNAKE) VENOM PROPERTIES JAMES
L.
GLSxrr', RICIiARD C. STRAICI~r,' MARTIiA DAVID L. HARDY'
C. WOLFE' and
'Venom Research Laboratory, Veterans Administration Medical Center, Sah Lare City, UT 84148 and 'Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ, 85724, U .S .A. (Amepted for publirntion 10 August 1982) J . L . Ot .eww, R. C. S~afrr, M. C . wot .ae and D. L . hL+anv . Oeographical variation in Crotahcs srutulatus scutulotus (Mojave rattlesnake) venom properda. Taxirnn 21, 119-130, 1983 . - Individual venom samples were analyzed from 12 specimens of Crota+ss scutulatus srutulatus, from north of Tucson to the extreme southeastern region of Arizona . Six of the specimens, from north of Tucson, produced venan lethal toxicity C.p. t.n ) values in mice of 2 .0-6.0 mg/kg. These coincided with the values previously reported for C, s. srutulatus in the Phoenix, Arizona, region and designated as type B venom (OtBtav and Snwatir, 1978) . i:n contrast, the venom t.n of six individuals from extreme southeastern Arizona, including one individual near Tucson, ranged from 0.22-0 .46 mg/kg. This corresponds to the values for C. s. scutulatus venom previously reported and designated as type A venom (Ot .amv and Snutattr, 1978) . Specimens with type A venom have been collected in California, Nevada, Utah and regions of Arizona. In addition to differences in lethal toxicity, the type B venom consistently exhibits a different protein profile, greater proteolytic activity, greater hemorrhagic activity and contains little or none of the m$jor lethal toxin, Mojave toxin, compared to the type A venom. No external morphological characteristic could be found differentiating the type A venom specimens from the type B venom specimens . These findings further confum the geographical variation of C. s. scurulatus venom in Arizona.
INTRODUCTION
and STRAIGI-IT (1978) reported a significant geographical difference in the lethal toxicity of Mojave rattlesnake (Crotales scetulatus scutulatus Kennicott) venom in laboratory mice . The more toxic venom from California, Utah and Southwestern Arizona, with i.p. LD,o values of 0.13 - 0.54 mg/kg, was designated venom A. The less toxic venom from the Phoenix, Arizona, region, with i.p. LDss values of 2.3 - 3.8 mg/kg, was designated venom B. Also, commercial antivenin was found to be more effective against venom H than venom A. These findings suggested that both venom research and the clinical events following C. s. scetulatus envenomation could be affected, depending on the type of venom involved. It has been reported that C. s. scutulatus envenotnations produce very few local symptoms and signs, but marked neurotoxic effects (RUSSELL, 1969; RUSSELL and PUFFER, 1970; RUSSELL et al., 1975; CASTILONIA et al., 1981). However, HARDY (1982) has reported that the clinical symptoms and signs of patients bitten by C. s. scutulatus in the Tuaon and Phoenix, Arizona regions often include significant local effects, with few neurotoxic signs. This suggests that the composition of C. s. scutulates venom may differ GLENN
"To whom correspondence should be addressed.
120
J. L. GLENN, R. C. STRAIGHT, M. C. WOLFF and D. L. HARDY
regionally, and may be related to the regional differences in lethal toxicity of this species reported previously (GLENN and STRAIGHT, 1978) . The purpose of this report is to: (1) compare some biochemical properties of C. s. scutulal`es venom from specimens from various regions of the southwestern U.S.A . ; (2) further define the geographical limits of venom A and venom B from C. s. scutulatus . MATERIALS AND METHODS Specimens and venom samples Five male and seven female Crotales scetelates scutelatus were collected near Tucson, Arizona and in the extreme southeastern corner of Arizona, as illustrated in Fig. 2. The spximens ranged from 40-110 cm in total length. The mouse i.p . LDN results were determined under single blind study conditions for each spedmen and related to its identification number. Additional C. s. scwtulatus venom samples, used forcomparative analysis in this study, were from apedmens previously reported (GLernv and STRAIGHT, 1978) and were from locations in Arizona, California, Nevada and Utah (as illustrated in Fig. 2) . Snakes utilized in this studyare catalogued in the Brigham Young University, Life Silence Muscum, Provo, Utah . Three adult specimens of the H~~nmgn tlan rattlesnake (C. scutulates salvine) were from Vera Cruz, Mexico . Western diamondback rattlesnakes (Crotales atrox) were from Oklahoma and Texas. Lyophilized "Mojave toxin" used in this report, is the add -basic protein complex (Mil-9V) isolated by CATs and Bmame (1978) and was gradously supplied by Allan Bieber, Arizona State University, Tempe, Arizona . Venom rnUection, processing and lethal tazldty testing Veaoms were collected from individual specimens by manual venom extraction tahniques and centrifuged at 1000 a for 15 min. The supernatant fhdd was frozen, lyophilized to dryness and stored at 4°C. All testing of venoms from specimens of the Tucson and Southestern Arizona region was accomplished within four months or less of venom eollecdon. The protein content of the venoms was determined by the biuret atethod of GORNALL et al. (1949). Venom lethal toxidty was determined for each individual sample by raonatituting the lyophilized venom in normal saline at 1 mg/ml and igjecting the venom i.p . into 18-22 g Swiss-Webster mice (female), using four serial doses and a minimum of six mice per dose . The reauhing LDN values were calculated from the survival ratios at 48 hr by the method of KARHBR (1931) . Proteolytic acrd esttmae actlvlty assays Venom proteolytic activity was assayed for each individual spatimen of C. s. scutulates and C. s. satvint and pooled C. atroz venom, using a modification of the method of RnvnBRnvscr et al. (1968), with Remazolbrilliant bhre hide-powder azure (Cal-Biochem) as substrate. Venom (100 ~, in 0.1 ml of 0.1 M sodium phosphate buffer, pH 7.5, was added to S ml of hide powder suspension (5 mg/ml in 0.01 M sodium phosphate buffer, pH 7.3). The solution was continuously stirred for 13 min in a water bath at 37°C, filtered through a No. 1 Whatman filter and the proteolytic activity determined at 320 nm . Esterase activity was measured using abenzoyl-L-arginine ethyl ester (BASE) substrate, as outlined by ScHweRT and TAIO:NAKA (1955), using 20 Ng of venom. Xemorrhagic assay The hemorrhagic activity of the venoms was determined by modifying the method of ICo~wo et al. (1960) . Venom (1-SO ~, in 0.1 ml of 0.911 saline, was igjected intradermally into the shaven back of rabbits, marked into grid squares. The rabbits were sacrificed by ether inhalation 24 hr after igjection and the dorsal skins removed and spread on a glass plate. The hemorrhegic spots were measured and photographed from the dorsal side ova a fluorescent light sotu~oe. h;jections of 25 I+g of C. s. scvtulates (venom A and B) and C. s. salvinl venom were selected for oomparatIve analysis. Crotales adaneantees (1 pg) and C. atrox (1 ~ venoms were utilized as reference venoms . Calelectrophorrsis Polyacrylamide gel electrophoresis (PAGE) was run in a Bio-Rad vertical slab dauophoresis cell Mode1220. The gd was 1 .5 mm thick. Sample wells were formed with Canaloostacking gel (pH 6.6 - 8.6)and were 10 mm in length . The 71i acrylamide separating gel (pH 8.9-9 .0) was 80 mm in length . The sample was electrophoreaed toward the anode (+) at 2 mA constant current per sample and was stopped 10 mm from the bottom of the gel. Blectrophoreaia buffer was 0.025 M Tris/0.192 M glycine, pH 7.83. Hromophmol blue was used as tracking dye. The gel was fixed in 10% acetic add/40sfir isopropanoV3019 water for 60 min, stained with Coomassie Blue (O.OSy.) in 101Y acetic add/10% isopropanol/80% water far 60 min and deatained in 1011 acetic add/10~K iaopropanoV801i water, using 3 -4 dungea of solution over a 24 hr period . The stained gels with their protein patterns were photographed and each gd was sliced into strips 7.S mm wide and the protein patterns scanned at 280 nm oa s Gilford gel scanner Mode12520.
Crorahrs scetuiates sewtelatus venom
121
RESU1.Ts
The lethal toxicities of six of the C. s. scututatus specimens collected north of Tucson, Arizona, were similar to the C. s. scutulatus venom B values previously reported (GLENN and $TRAIaI-IT, 1978) for the Phoenix region (Table 1). The mouse i.p. LD variation of 2.0- 6.0 mg/kg for venoms from the six individuals was similar to the variation within multiple venom samples from a single specimen for venom collected at three monthly intervals (i.p. LD 2.6-S.S mg/kg) . All five specimens collected from the southeastern corner of Arizona and one specimen from just north of Tucson (see Fig. 2, nearest Tucson) produced type A venom l,n values (0.22-0.46 mg/kg) (Table 1). The differences in lethal toxicity between type A and B venoms were not linked to sex or age of the specimens. Death times were noticeably different for venoms A and B. Calculating deaths at 48 hr, rather than 24 hr, proved critical for venom B LDeo values, since a significant number of deaths occurred between 24 and 48 hr. Conversely, deaths from venom A occurred within 24 hr; usually within 12 hr. Crotales s. salvini is the southernmost form of the species, C. scutulatus. The i.p. LD,o values from three specimens were similar to type A venom from C. s. scutulatus (Table 1), and are in agreement with that previously reported from a single individual (GLENN and STRAIGIiT, 1978) . TABLE
1.
SLLpliareS
COMPARISON
Crorarus Croraha sruruiQres
OF VENOM LETHAL TOXICITY OF
sC7lrLlQle4 VENOM FROM ARIZONA AND
salvini vENOM
FROM VERA CRUZ, MEXICO
Number of "Venom i.p. LD (dry wt) Venom species specimens mg/kg - mean (range) C. s. sretuiarus Venom At 6 0.28 (0 .22-0.4~ Venom B ~ 6 3.33 (2 .00-6.00) C. s. salvini 3 0.30 (0 .22-0.40) "LD Was determined foréach specimen by injecting venom i.p. into 18-22 g Swiss Webater mice, as described in teat . fi Five specimens from extreme southeastern Arizona, near Portal, Dos Cabezas and Apache, and one spaimen from near the northern city limits of Tucson, Arizona (see Fig. 2). # Six specimens from north of Tucson, Arizona, SO hm N.W . of Tucson near Red Racr, N.W . Tucson 17 lnn, San Manue147 km N.E. Tucson (see Fig. 2) .
Enzyme activity Another major difference between C. s. scetulates venoms A and B was in the hide powder proteolytic activity . All of the individual venom A samples were low or lacking in proteolytic activity using this substrate. Crotales s. salvini venom, which was similar in i.p . LD value to venom A, was also similarly low or lacking in hide powder protoolytic activity . In contrast, the venom B samples were strongly proteolytic and approximately oqual to pooled C. atrax venom (Table 2). The variation of proteolytic activity in venom samples from 13 specimens of C. s. scetulates producing venom B (Table 2) was similar to the variation in venom samples from a single specimen collected by four monthly venom extractions. The variation between individual specimens in venom proteolytic activity may reflect the normal variation in venom protein synthesis in an individual snake.
12 2
J. L. GLENN, R. C. STRAIGHT, M. C. WOLFF and D. L. HARDY
Other Crotales species with relatively low or no hide powder proteolytic activity are C. viridis concolor (Wyoming), C. mitchellü mitchellü (Baja Sur), C. horridus atricaudatus (Texas), C. durissus durissus (unknown locale) and C. adamanteus (Florida). All of the above species exhibited i.p. LD6, values of 0.6 mg/kg or lower, except C. adamanteus (2.1 ~kg)~ Both A and B type venoms from C. s. scutulates had similar esterase (BASE) activity (Table 2) and the activity was highly variable among individual specimens. TABLE 2. COMPARATIVE ANALYSIS OF VENOM PROTEOLYTIC AND ESTERASE ACTIVITIES FROM INDIVIDUAL SPECIMENS OF CrOtaIiL4 sCIfrHiQtYS SCIIhIlQttfS (VENOM A AND VENOM B) AND Crotalus srutulatus sialvini
venom species
Number Df specimens
Proteolytic activity" eonnoo,~ls min mean (range)
Esterase activity fi eoDi2o,$min mean (range)
C. s. scutulatus Venom A $ Venom B $ C. s. snivini C. atrax
23 13 3 Pool
0.005 (0 .000-0.013) 0.192 (0 .114-0.246) 0.001 (0 .000-0.003) 0.201
0.90 (0.37-1 .78) 0.88 (0.35-1.59) 1.04 (0.92-1 .18) 0.31
"Remazol-brilliant blue hide-powder azure substrate (at 520 nm). fi a-Benzoyl-L-arginine ethyl ester (BASE) substrate (at 253 nm). $ Venom type determined by i.p . Ln in mice previous to enzyme assays .
Hemorrhagic activity The comparison of the hemorrhagc activities of C. s. scutulatus venoms A and B and C. s. salvini venom are illustrated in Fig. 1, using 25 pg of each venom. Eight individual venom A samples and two individual C. s. salvini venom samples produced no detectable hemorrhagc lesion . These venom samples were also tested at s0 ~g and no hemorrhage resulted. Conversely, four individual venom B sâmples produced hemorrhagc spots, 10 mm in cross-diameter, at 25 )tg. When using as little as 5 ltg of type B venom, small hemorrhagc lesions were noted. Comparatively, only 1 ~g of venom from C. adamanteus and C. atrax was required to produce hemorrhaic lesions of equal size. In both venom A and B, the level of hemorrhagc activity correlated with the level of proteolytic activity . Our results with venom A were consistent with the results of GIROUX and LACHMANN (1981), who found little or no hide powder proteolytic activity and no hemorrhaic activity in mice with C. s. seutulatus venom. Geographical distribution Of the eight specimens of C. s. scutulatus with venom B previously reported from Arizona (GLENN and STRAIGHT, 1978), only six collection locations were known. Three were from east of Phoenix, two from south of Phoenix and one north of Phoenix (Fig. 2). In this report, 6 additional venom A and 6 venom B specimens were collected with accurate locality data. The results indicate that specimens with type B venom extend from the Phoenix region to the Tucson region of Arizona. Venom protein patterns by gel electrophoresis The Mojave toxin complex (MD-9~ (LATE and BIEBER, 1978), the major lethal toxin in C. s. scutulatus venom, disassociates into two major subunits when fractionated in a
123
FIO . 1 . Col~wwsox OF Tl~ tI~NOAR~iAOIC ACTIVITY OF Crotales scetelatus srlrtelates (varloM A wrm vlavol~t B) t~ Crotales sretetatus siohint vsrlo~t. All igjections was intradamal in 0 .1 ml volume, in the shaven back of a 2 .5 kg rabbit . A, Bight individual venom samples from C. s. scutulates venom A, 25 I+g in each grid square ; B, fow individual venom samples from C. s. scutelatus venom B, 25 Pg in each grid square ; CA, 1 kg of pooled C adamantees venom ; CAA, 1 Kg of pooled Crotales atrox venom ; SALV, 2S kg of individual C. s . sohlni venom samples . The hemorrhaic lesions of venom B, CA and CAA are appro~matdy 10 mm in cross-diameter . Venons A and SALV produced no hemorrhage . The darkening in the SALV grid squares is due to diffusion of hemorrhage from the lateral venom B lemons .
124
FIO . 3 . POLYACRYLAAl1DE OEL BLHCIROPHORHSIS OP V8NOM3.
Elaxrophoreaia buffo 0.025 M Tria/0.192 M Blycine, pH 7 .gS . MT, Mojave toxin, 100 ~ of protein; A, C. s. scvtulahtf venom A, 200 Ng of pooled venom protein from four apaimena ; H, C. s. snrtulatus venom B, 200 Pg of pooled venom protein from four apecimena; SALV, C. s. ,~alvlni 200Pg of pooled venom protein from three apedmena ; 1, Mojave toxin ; 2, acidic aubunit of Mojave taatin. (Stained with Coomesaie brilliant blue.)
Crotales srutulates sretu/attrs Venom
125
F~a, 2. DDIYBVI'ION OF Crotales scutelatus scutelates veraM A ,uvn vFxoM B sracu~ns. ~, Closed circles designate venom A specimens ; o, open circles designate venom B specimens.
basic buffer (pH 7.85) by gel electrophoresis (Figs. 3 and 4). Repeated freezing and thawing of the protein complex resulted in degradation of the toxin. The acidic subunit migrates with the electrophoresic front and serves as an easily identifiable marker for the presence or absence of the toxin in venom samples. Since variations occurred in venom electrophoresic profiles between individual snakes, venom samples of equal portions of venom protein from four individual C. s. st utulatus venom A and venom B and three individual C. s. salvini were each pooled, respectively, and analyzed (Figs. 3 and 4). C. s. scutulatus venom A samples consistently showed protein bands (280 nm) corresponding to the acidic component of Mojave toxin (Figs. 3 and 4), although considerable quantitative variation occurred among six individual samples examined . Peaks corresponding to the Mojave toxin complex were also present in the C. s. salvini venom. Estimates were made of the amount of dissociated acidic subunit contained in venom samples by cutting out and weighing the areas of graph paper corresponding to the gel scan tracings of protein peaks. Six individual venom A samples ranged from 6 to 18% in dissociated acidic subunit content and the content of the pooled venom (four snakes) represented 9% of the total protein recorded (Fig. 4). Mojave toxin was low or lacking in the pooled venom B sample . Interestingly, two of the six venom B samples from the Tucson region exhibited faintly stained protein bands similar to the acidic subunit and Mojave toxin. This may represent an intergradal situation between specimens of the two types of venom in the Tucson region . This regional variation in Mojave toxin content in Arizona specimens appears similar to the regional variation in C. d. tet~cus venom toxins, crotamine and
126
J . L . GLENN, R . C . STRAIGHT, M. C. WOLFF and D. L . HARDY
Addic Subunh
Begin Bei
Verwm A
_ / "v \
Fta . 4 . Gal, scwrt (280 nm) of Mo~wvs ~roxnr, C. s. scutulahas vatvoM A, C. s. scutLlatus vt~voM B wrro C. s. salvirtt vt~oAf u.~usrsw~n nv sta. 3 . The peak corresponding to the acidic subunit of Mojave toxin is illustrated in C. s. scumlatrrs venom A and C. s. salvirt! venom but is absent (or extrentdy low) in C. s. scutulatus venom B .
convulxin, in South America (GONCALVES, 1956 ; GONCALVES and DEUTSCH, 1956; HOGS, 1965 ; PRADO-FYtANCESCHI et al., 1981). Morphological examination GLENN and STRAIGHT (1978) proposed that the differences in the venom lethal toxicity of C. s. scutulatus might correlate with squamation differences, especially if the
Crotales scetulates scetelatus Venom
127
geographical limits of venom B specimens could be further defined. However, in the examination of 13 venom H specimens and 18 venom A specimens, no significant differences in external morphology could be established . All of the features of the specimens examined were similar and overlapped in numerical range. Characters examined included color; body/tail ratios ; rattle width total body length ratios ; the number of body blotches, tail bands, dorsal scale rows, mid-dorsal caudal rows, ventrals, subcaudals, supralabials, infralabials, intersupraoculars; the organization of crown scales ; size and shape of canthals, loreals and preoculars ; posterior connections of dorsal - lateral blotches ; width of preocular and postocular stripes. DISCUSSION Despite earlier confusion with other rattlesnakes, ICLAUBER (1930) provided adequate evidence that C. s. scetulatus was a valid species. By 1939, there was also evidence that the venom of this species was exceptionally toxic (lethal) as compared to other North American rattlesnake forms (GITHENS and WOLFFE, 1939). GLOYD (1940) subsequently concurred with Klauber's speciation of C. scutulates and further nominated C. scutulatus salvini Gunther, as a valid subspecies . Conflicting clinical reports involving envenomation by C. s. scutulatus were often thought to be due to misidentification of the species involved, e.g . C. atrax confused with C. s. scutulatus. Although envenomation by C. s. scetulatus has been characterized as predominantly producing neurotoxic effects (including respiratory paralysis) with little or no local effects, a recent clinical study in Arizona indicates that some C. s. scutulatus envenomations do, in fact, produce local tissue effects, sometimes severe (HaxDY, 1982 ; HARDY, 1983). In the majority of the Arizona clinical cases (14 of 1S), neurotoxic signs were notably absent . The discovery of a major lethal toxin in C. s. scutelatus venom (BIEBER and TU, 1974 ; B>EBER et al ., 1975 ; PATTABHIRAMANN and RUSSELL, 1975 ; HENDON, 1975) that produces neurotoxic effects (CASrILONIA et al., 1979 ; GOPALAKRISNAKONE et al., 1980 ; CASTILONIA et al., 1981 ; Ho and LEE, 1981) corroborates the neurotoxic effects previously reported in human clinical cases. The geographical difference in venom lethal toxicity in Arizona C. s. scetulatus reported previously (GLENN and S'rxnlal-l'r, 1978) is further confirmed in this report. The data also suggests that venom B may produce more severe local tissue effects than venom A. Interestingly, Hardy (1983) noted clinical findings of swelling, bleb formation, ecchymosis and/or necrosis in patients bitten by snakes from the venom B region . The only patient exhibiting a neurotoxic effect (eyelid ptosis) was bitten by a snake from the venom A region . The venom B geographical distribution appears limited to the region north of Tucson to north of Phoenix (Fig . 2) . However, many more specimens are required to determine the exact distribution limits of venom B and to determine if intergradation occurs between snake populations of the two venom types. MIN~roN (1958) reported on C. s. scutulatus venom lethal toxicity from the Big Bend Region of western Texas, indicating that type A venom also exists there. No venom data is available for populations of C. s. scetulates throughout its range in Mexico. A total of four specimens of C. s. salvini have been tested and all produced type A venom lethality and were similarly low in proteolytic and hemorrhagic activities . The C. s. salvini venom also cross-reacts immunochemically (Ouchterlony agar double-diffusion)
128
J . L. GLENN, R. C. STRAIGHT, M. C . WOLFF and D. L. HARDY
with antibodies to the basic phospholipase subunit of Mojave toxin from C. s. scutulatus venom A, further indicating a possible similarity in the lethal toxins from the two subspecies (Allan Bieber, personal wmmunication) . Other crotalid venoms and toxins which also cross-react immunochemically with Mojave toxin are C. horridus atricaudatus, C. viridis concolor, C. durissus te~cus, C. durisrus durissus, crotoxin from C. d. ter~cus and concolor toxin from C. v. concolor (HENDOx and BISSER, 1982). C. d. terr~cus and C. s. scutulatus have received considerable attention by toxicologists due to their venoms' high lethal toxicity and potent neurotoxic components . However, other rattlesnake venoms are now known to exhibit LD6o values comparable to C. s. scutulatus, such as C. v. concolor (GLENN slid STRAIGHT, 1977; Pool et al., 1981 ; GLENN and STRAIGHT, 1982; HENDON and BIEBER, 1982), C. h. atricaudatus (MINTON, 1967 ; GLENN and STRAIGHT, 1982), C. m. mitchellü (GITHENS and WOLFFS, 1939 ; RUSSELL et al ., 1960 ; GLENN and STRAIGHT, 1982), C. s. salvini (GLSNx and STRAIGHT, 1978), C. vegrandis (VILLALAZ, 1966 ; SCANNONE et al., 1978) and Sistrurus c. catenatus (MINTON, 1956). Therefore, no longer can C. d. terr~cus and C. s. scutulatus be considered as unique in rattlesnake venom lethality, as has been presented in the past . The importance of geographical differences occurring in venom from a given species/subspecies is becoming quite evident. At the binomial species level, extreme geographical differences in venom composition, including lethal toxicity, venom toxins and/or enzyme activity, have been reported in Crotalus durissus, C. lepidus, C. horridus, C. mitchellü, C. viridis (see review by GLENN and STRAIGHT, 1982), Bothrops asper (ARAGOx et al., 1981), Bothrops numm ;fer (JIMINEZ-PORRAS, 1964), Ngja hgje (IRWIN, et al., 1970), Ngja nigricollis (IRWIN et al., 1970) and others . In this regard, a number of venom research reports continue to use only bionomenclature taxonomy and give little concern to the regional origin of the venom species. The differences in venom lethal toxicity and proteolytic and hemorrhagic activities between the type A and B venoms of C. s. scutulatus are too significant and consistent to be disregarded . They give reason to suspect that dramatic differences might occur in the clinical picture of human envenomation . Considerable variation even occurs between individual snakes in venom protease activity, esterase activity and Mojave toxin content, possibly influencing clinical symptoms and signs following envenomation . These differences in venom quality, combined with other unknowns in envenomation, such as the dose of venom received, further complicate the clinical aspects of C. s. scutulatus envenomations in Arizona. However, in all cases, the early therapeutic use of antivenin is important if significant envenomation is suspected. Due to the relatively poor efficacy of existing commercial antivenin against the type A venom (STRAIGHT et al., 1976; GLENN and STRAIGHT, 1977 ; GLENN and STRAIGHT, 1978), an antivenin prepared against rattlesnake venoms that included venom A or Mojave toxin would be useful in medical ~PY~ The distribution data in this report provide a beginning for determining the range limitation of C. s. scutulatus type B specimens. The lack of recognizable e~zternal characters between venom A and B specimens is unfortunate and thereby places more importance on the establishment of well-defined geographical limits on each type of C. s. scutylatus venom. Presently, the only consistent criteria for distinguishing the two varieties are their venons, with some evidence that venom B spocimen distribution is limited and contained within a certain geographical region of Arizona.
Crotales srutulatus scvtulates Venom
129
Acknowkdgttr~nts -
We thank the Arizona Game and Fish Department and the Utah Division of Wildlife Raout+ces for their cooperation . We also thank Dr . At.LArt Bmeap, Arizona State University, Tempe, Arizona, for supplying the Mojave toxin and immunodiffudon tests utilized is this report . We are grateful to the following persons and institutions for Weir assistance : ToM RArmAZZO, RoaattT LApsorr, Mt~ COFr~N, Lane PowtAS, JAwtes JApcct~ttow, MAROAAEr HApux, HowAttu LwwLen, Joe CApAr~wp, Pttn. Ovetawae, YAp PtTtxxs~zxty and the Animal Research Facllity staff and the Medical Media Production Servis staff, V .A . Medical Center, Salt Lake City, Utah . This research wan supported by We Veterans Administradon Medical Itexarch Program, Veterans Admi~nistratian Medical Center, Sah Lake City, Utah and Hogle Zoological Gardens, Salt Lake City, Utah .
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