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Venom characteristics as an indicator of hybridization between Crotalus viridis viridis and Crotalus scutulatus scutulatus in New Mexico
TO,I/('Oll
Vol. 28, No, 7, pp, 857-862, 1990,
Pergamon Pres" p1c
Prtnted in Great Britain,
SHORT COMMUNICATIONS VENOM CHARACTERISTICS AS AN INDICATOR OF HYBRIDIZATION BETWEEN CROTALUS VIRIDIS VIRIDIS AND CROTALUS SCUTULATUS SCUTULATUS IN NEW MEXICO JAMES 1. GLENN and RICHARD C. STRAIGHT Venom Research Laboratory, Veterans Administration Medical Center, Salt Lake City, UT 84148, U.S.A.
(Accepted/or publica/ion 7 November 1989)
J. 1. GLENN and R. C. STRAIGHT. Venom characteristics as an indicator of hybridization between Crotalus viridis viridis anjrotalus scutulatus scutulatus in New Mexico. Toxicon 28, 857-862, 1990. One hundred and thirteen venoms from 46 populations of Crotalus virid s viridis were screened by
immunodiffusion for protein toxins antigenically similar to the phospholipase A 2 (PLA~toxin 'Mojave toxin', using a polyclonal antibody to it's basic PLA subunit. enom i.p. LD so values in mice were recorded from 22 of the 46 populations. he venoms of three of 14 specimens from southwest (S.W.) New Mexico and one specimen from northern Arizona were immunologically positive by the immunodiffusion tests and produced low LD so values (0.38-0.65 mg/kg) compared to all immunologically negative venoms (0.9-5.5 mg/kg). These four specimens were morphologically typical for C. v. viridis and their venoms were the only samples of 15 southern New Mexico specimens examined by reverse phase HPLC to exhibit peaks corresponding to the acidic and basic subunits of Mojave toxin. Alkaline polyacrylamide gel electrophoresis (PAGE) analysis of the recombined subunit peaks from the C. v. viridis venom from the S.W. New Mexico specimens showed more similarity to Mojave toxin from C. s. scutulatus venom than to similar toxins in C. v. conc%r venom. The combined results of the immunodiffusion, lethal toxicity tests, HPLC profiles and PAGE analysis strongly suggest that the venoms of the three New Mexico specimens contain Mojave toxin(s), as a result of some previous hybridization with C. s. scutulatus. The northern Arizona specimen likely contains 'concolor toxin' through intergradation with C. v. concolor in its' genetic background.
THE WESTERN rattlesnake, Crotalus viridis, is a widely distributed species across the western half of the U.S.A. Among the nine subspecies of Crotalus viridis, only the venom of the subspecies C. viridis concolor (midget faded rattlesnake) has been reported (POOL and BIEBER, 198 I; HENDON and BIEBER, 1982) to contain concolor toxin (a phospholipase A 2 toxin complex with neurotoxic activity) and exhibit low LD so values similar to other rattlesnake venoms that contain similar toxins (GLENN et al., 1977; GLENN and STRAIGHT, 1982). Over the past several years we have screened by immunodiffusion the venoms of numerous populations of C. viridis viridis (prairie rattlesnake) and found no toxin 857
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antigenically similar to Mojave toxin from C. scutt/latus scutulatus (Mojave rattlesnake) venom. More recently, however, the venom of one of two specimens of C. viridis viridis collected in S.W. New Mexico reacted strongly to the antibody and had a low LD so value indicative of venoms containing the toxin. Subsequently, 12 additional specimens from S.W. New Mexico were collected and examined for the presence of the toxin. The results of the investigation are presented in this report. All venoms were collected by manual extraction, centrifuged (cold) at 1500g for 15 min. The supernatant fluid was frozen, lyophilized and stored at ca 4°C. Ouchterlony immunodiffusion tests were performed in pre-cast agarose discs (Miles Laboratories) in 4.5 cm petri dishes, using rabbit antibody provided by ALLAN BIEBER (HENDERSON and BIEBER, 1986) to the PLA 2 subunit of Mojave toxin vs individual venoms (40 mg/ml) as previously described (GLENN and STRAIGHT, 1985). A total of 113 venom samples have been examined by immunodiffusion tests. Lethal toxicity by i.p. injection was tested using 3-4 serial doses of venom in groups of 5 mice each (Swiss-Webster, female, 18-22 g). The resultant i.p. LD so values were calculated at 48 hr, following the statistical analysis method of KARBER (1931). Initial chromatographic analysis of the venoms was achieved by applying 500 pg of each venom to a reverse-phase HPLC column (LC8-DB, Supelco), using a linear gradient of 30-70% acetonitrile in 0.1 % TFA as previously reported (GLENN and STRAIGHT, 1989). For isolation and collection of the acidic and basic subunits of the toxin from C. viridis viridis venom, 4 mg of venom was repeatedly applied. The subunits were recombined in H 20 for electrophoretic analysis and lethal toxicity tests. Alkaline polyacrylamide gel electrophoresis (PAGE) analysis was performed as previously described (GLENN et al., 1983). b
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10
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50
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60
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70
Retention Time (min.) FIG. 1.
HPLC OF Crotalus scutulatlls scutulatlls. Crotalus viridis concolor AND THREE VENOM TYPES FROM Crotalus viridis viridis. Venom (500 Jig) applied to a LC8-DB column (Supelco), using a linear gradient of 30-70% acetonitrile in 0.1 % TFA. CSS "A" = Crotalus scutulatus SCUll/latus (venom A, GLENN and STRAIGHT, 1978); CVV-NM "A" = Crotalus viridis viridis. S.W. Ncw Mexico, i.p. LD," 0.38 mg/kg; CVV-NM "B", S.W. New Mexico, i.p. LD" 5.5 mg/kg; CVV-AZ "A" = Crotalus viridis viridis, near Page, AZ, i.p. LD,o 0.65 mg/kg; CVC-Pool = Crotalus viridis cOllcolor venom pool; a = acidic subunit of Mojave toxin, retention time = 15.6 min; b = basic subunit of Mojave toxin, retention time = 24.6 min. REVERSE-PHASE
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Comparative HPLC profiles of venoms from C. scutulatus scutulatus, C. viridis conc%r and C. viridis viridis are presented in Fig. 1. Three individual venoms of C. viridis viridis from Hidalgo County, NM (CVV-NM "A") and one individual from near Page, AZ (CVV-AZ "A") contained protein peaks with identical retention times as the acidic and basic subunits of Mojave toxin (CSS "A") and concolor toxin (CVC-Pool). Eleven of the 14 C. viridis viridis specimens from the same S.W. New Mexico region lacked two peaks in their venom profiles (data not shown) except that one specimen's venom exhibited a low peak in the region of the basic subunit (CVV-NM "B" venom, Fig. 1). This peak did not
(-)
I-I I I I
1_-
1
2
3
4
(+)
FIG. 2. BASiC-PAGE ANALYSIS OF THE RECOMBINED TOXIN (PEAKS a AND b, FIG. I) FROM Crowlus viridis viridis VENOM (CVV-NM "A", FIG. I). Polyacrylamide gel electrophoresis run as previously described (GLENN er al., 1983), using electrophoresis buffer 0.025 M Tris/0.192 M glycine, pH 9.0 and 251lg protein of each toxin stained with Coomassic brilliant blue. Lane I = Mojave toxin (CATE Hnd BmuER, 1978); Lane2 = recombined peaks a and b from CVV-NM "A" venom, Fig. I; Lane 3 = concolor toxin (POOL and BiEBER, 1981); Lane 4 = canebrake toxin (STRAIGHT and GLENN, 1989); a = unbound or disassociated acidic subuOIt; dashed line = recombined toxins.
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FIG. 3. DISTRIBUTION OF Crotalus viridis virielis AND POPULATIONS OF VENOMS SCREENED BY IMMUNOUIFFUSION FOR TOXINS SIMILAR TO MOJAVE TOXIN, USING ANTIBODY TO THE BASIC SUBUNIT OF MOJAVE TOXIN .
• = positive venoms;
0
= negative
venoms.
react with the antibody to the PLA subunit. The 4 CVV "A" venoms reacted strongly in the immunodiffusion tests and the 11 CVV "B" venoms were negative, using a high venom concentration of 40 mg/ml. Peaks a and b of the CVV-NM "A" venom (Fig. 1) were collected, recombined in H 20 and the toxins' electrophoretic mobility was compared to similar venom toxins of C. scutulatus (Mojave toxin), C. viridis cOl1color (concolor toxin) and C. /lOrridus atricaudatus (canebrake toxin) (Fig. 2.). Two major bands (likely isomeric
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toxins formed on recombination) were resolved with the CVV-NM "A" toxin. These were identical in electrophoretic mobility to Mojave toxin and distinct from concolor toxin and canebrake toxin which migrate faster anodally. The i.p. LD so of the recombined CVV-NM "A" toxin was O.064mg/kg. Among the Arizona and S.W. New Mexico specimens, the i.p. LO so values ranged from 0.38-0.65 mg/kg for the 4 venoms containing the toxin(s) (CVV "A") and ranged from 104-5.5 mg/kg for 11 venoms lacking the toxin(s) (CVV "B"). The latter venom "B" values were similar to the i.p. L0 50 values (0.9-4.1 mg/kg) obtained from the venoms of 20 populations of C. viridl:~ viridis throughout its range. Venoms from 46 different populations of C. viridis viridis have been assayed using immunodiffusion for the presence of the toxin and 44 were negative (Fig. 3). The only immunologically positive venoms were from regions where the distribution of C. viridis viridis overlaps with C. scutulatus or is near the range of C. viridis concalor (see KLAUBER, 1956; GLENN and STRAIGHT, 1982). The HPLC venom profile of the Page, AZ specimen (CVV-AZ "A", Fig. 1) is more similar to C. viridis concalor venom than to the New Mexico C. viridis viridis venom. Therefore, the toxins' presence is likely due to its close proximity to C. viridis concalar, through intergradation at some point in its genetic history. Further analysis of the Arizona specimens' venom could not be achieved due to lack of venom. Morphologically, the Arizona specimen appears as a typical C. viridis viridis, as do the three New Mexico specimens containing the toxin. All three of these New Mexico specimens (males) were of similar size (total length == 74-78 em) but were collected 5 and 15 miles distant from each other, t~erefore, the probability of their being from the same parentage is remote, but possible'IThe combined results of the immunodiffusion, lethal toxicity, HPLC and PAGE analyses indicate that the venoms of the three New Mexico specimens contain Mojave toxin as a result of ancestoral hybridization with C. scutulatus scutulatus in these specimens' genetic background.1
1
Ack/lowledgcmcll/s-Wcapprcciatc thc help of BARNEY TOMBERLIN and TONY SNELL, Portal, AZ, in providing collection data and in their diligent efforts to collect the S.W. New Mexico specimens for us. We thank DAVID HARDY, Phoenix, AZ for the loan of the Page, AZ specimen and HUGH QUINN, Houston Zoo, Houston, TX, for the loan of C. I'iridis I'iridis from several localities. The technical assistance of ELIZABETH ARNOLD, ROIlERT NOHAVEC, MARTHA WOLFE and TERRY WaLT is gratefully acknowledged. We are thankful to ALLAN BIEBER, Arizona State University, Tempc, AZ, for supplying the rabbit antibody used in this report. This research was supported by the Department of Veterans Affairs Medical Research Program, VA Medical Center, Salt Lake City, UT, and, in part, by Hogle Zoological Gardens, Sail Lake City, UT.
REFERENCES CATE, R. L. and BlEilER, A. L. (1978) Purification and characterization of Mojave (Crotalusscum/a/usSCUll/Ill/US) toxin and Its subUnits. Archs Biodu!fIl.Biophys. 189, 397---407. GLENN, .T. L. and STRAIGHT, R. C. (1977) Thc midget faded rattlesnake (Cro/alus viridiscOllcolor)venom: lethal tOXIcity and individual variability. To.\icoJl 15, 129-133. GLENN, .T. L. and STRAIGHT, R C. (1978) Mojave rattlesnake Cro/alus scu/lIla/llsscUlulalusvcnom: variation in toxicity with gcographical origin. Toxicoll 16, 81-84 GLENN, .T. L. and STRAIGHT, R. C. (1982) The rattlcsnakes and their venom yield and lethal toxicity. In: Ralllesllake Venoms: Their Actio/land Treatmell/, pp. 3~119 (Tu, A. T., Ed.). New York: Marcel Dckker. GLENN, .T. L. and STRAIGHT, R. C. (1985) Venom properties of the rattlesnakes (Cro/aills) inhabitIllg the Baja California region of Mexico. Toxicoll 23, 769-775. GLENN, .T. L. and STRAIGHT. R. C. (1989) Intergradation of two different venom populations of the Mojave rattlesnake (Crowills,lcu/llla/ussCII/II/allls) in Arizona. Toxicon 27,411-418. GLENN, .T. L., STRAIGHT, R. C, WOLFE, M. C. and HARDY, 0. L. (1983) Geographical variation in Crotaills sCllll/latusSCUll/laIUS (Mojavc rattlesnake) venom properties. To.\lcon 21, l19~ 130.
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HENDERSON, J. T. and BIEBER, A. L. (1986) Antigenic relationships between Mojave toxin subunits, Mojave toxin and some crotalid venoms. Toxicon 24, 473-479. HENDON, R. A. and BIEBER, A. L. (1982) Presynaptic toxins from rattlesnake venoms. In: Raulesnake Venoms: Their Action and Treatment, pp. 21/-246 (Tv, A. T., Ed.). New York: Marcel Dekker. KARBER, G. (1931) Beitrag Zur Kollektiven behandlung pharmakologischer reihenversuche. Arch. expo Path. Pharmak. 162, 480-489. KLAUBER, L. M. (1956) Raulesnakes: Their Habits, Life Histories and Influence on Mankind, Vol. I, p.56. Berkeley: University of California Press. (Partially revised in 1972, p. 61). POOL, W. R. and BIEBER, A. L. (1981) Fractionation of midget faded rattlesnake (Crotalus viridis conca/or) venom: lethal fractions and enzymatic activities. Toxicon 19, 517-527. STRAIGHT, R. C. and GLENN, J. L. (1989) Isolation and characterization of basic phospholipase (PLA,) and acidic subunits of canebrake toxin from Crolalus horridus alriclludolus venom using HPLC. Toxicon 27, 80 (A bstract).
Vol. 28, No, 7, pp, 857-862, 1990,
Pergamon Pres" p1c
Prtnted in Great Britain,
SHORT COMMUNICATIONS VENOM CHARACTERISTICS AS AN INDICATOR OF HYBRIDIZATION BETWEEN CROTALUS VIRIDIS VIRIDIS AND CROTALUS SCUTULATUS SCUTULATUS IN NEW MEXICO JAMES 1. GLENN and RICHARD C. STRAIGHT Venom Research Laboratory, Veterans Administration Medical Center, Salt Lake City, UT 84148, U.S.A.
(Accepted/or publica/ion 7 November 1989)
J. 1. GLENN and R. C. STRAIGHT. Venom characteristics as an indicator of hybridization between Crotalus viridis viridis anjrotalus scutulatus scutulatus in New Mexico. Toxicon 28, 857-862, 1990. One hundred and thirteen venoms from 46 populations of Crotalus virid s viridis were screened by
immunodiffusion for protein toxins antigenically similar to the phospholipase A 2 (PLA~toxin 'Mojave toxin', using a polyclonal antibody to it's basic PLA subunit. enom i.p. LD so values in mice were recorded from 22 of the 46 populations. he venoms of three of 14 specimens from southwest (S.W.) New Mexico and one specimen from northern Arizona were immunologically positive by the immunodiffusion tests and produced low LD so values (0.38-0.65 mg/kg) compared to all immunologically negative venoms (0.9-5.5 mg/kg). These four specimens were morphologically typical for C. v. viridis and their venoms were the only samples of 15 southern New Mexico specimens examined by reverse phase HPLC to exhibit peaks corresponding to the acidic and basic subunits of Mojave toxin. Alkaline polyacrylamide gel electrophoresis (PAGE) analysis of the recombined subunit peaks from the C. v. viridis venom from the S.W. New Mexico specimens showed more similarity to Mojave toxin from C. s. scutulatus venom than to similar toxins in C. v. conc%r venom. The combined results of the immunodiffusion, lethal toxicity tests, HPLC profiles and PAGE analysis strongly suggest that the venoms of the three New Mexico specimens contain Mojave toxin(s), as a result of some previous hybridization with C. s. scutulatus. The northern Arizona specimen likely contains 'concolor toxin' through intergradation with C. v. concolor in its' genetic background.
THE WESTERN rattlesnake, Crotalus viridis, is a widely distributed species across the western half of the U.S.A. Among the nine subspecies of Crotalus viridis, only the venom of the subspecies C. viridis concolor (midget faded rattlesnake) has been reported (POOL and BIEBER, 198 I; HENDON and BIEBER, 1982) to contain concolor toxin (a phospholipase A 2 toxin complex with neurotoxic activity) and exhibit low LD so values similar to other rattlesnake venoms that contain similar toxins (GLENN et al., 1977; GLENN and STRAIGHT, 1982). Over the past several years we have screened by immunodiffusion the venoms of numerous populations of C. viridis viridis (prairie rattlesnake) and found no toxin 857
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antigenically similar to Mojave toxin from C. scutt/latus scutulatus (Mojave rattlesnake) venom. More recently, however, the venom of one of two specimens of C. viridis viridis collected in S.W. New Mexico reacted strongly to the antibody and had a low LD so value indicative of venoms containing the toxin. Subsequently, 12 additional specimens from S.W. New Mexico were collected and examined for the presence of the toxin. The results of the investigation are presented in this report. All venoms were collected by manual extraction, centrifuged (cold) at 1500g for 15 min. The supernatant fluid was frozen, lyophilized and stored at ca 4°C. Ouchterlony immunodiffusion tests were performed in pre-cast agarose discs (Miles Laboratories) in 4.5 cm petri dishes, using rabbit antibody provided by ALLAN BIEBER (HENDERSON and BIEBER, 1986) to the PLA 2 subunit of Mojave toxin vs individual venoms (40 mg/ml) as previously described (GLENN and STRAIGHT, 1985). A total of 113 venom samples have been examined by immunodiffusion tests. Lethal toxicity by i.p. injection was tested using 3-4 serial doses of venom in groups of 5 mice each (Swiss-Webster, female, 18-22 g). The resultant i.p. LD so values were calculated at 48 hr, following the statistical analysis method of KARBER (1931). Initial chromatographic analysis of the venoms was achieved by applying 500 pg of each venom to a reverse-phase HPLC column (LC8-DB, Supelco), using a linear gradient of 30-70% acetonitrile in 0.1 % TFA as previously reported (GLENN and STRAIGHT, 1989). For isolation and collection of the acidic and basic subunits of the toxin from C. viridis viridis venom, 4 mg of venom was repeatedly applied. The subunits were recombined in H 20 for electrophoretic analysis and lethal toxicity tests. Alkaline polyacrylamide gel electrophoresis (PAGE) analysis was performed as previously described (GLENN et al., 1983). b
JwU~ J
J'
E
c
o
a:J N
o o
J
CW-AZ "A"
J~LJ~
o ,
j
10
i
20
•
30
I
40
I
50
I
60
I
70
Retention Time (min.) FIG. 1.
HPLC OF Crotalus scutulatlls scutulatlls. Crotalus viridis concolor AND THREE VENOM TYPES FROM Crotalus viridis viridis. Venom (500 Jig) applied to a LC8-DB column (Supelco), using a linear gradient of 30-70% acetonitrile in 0.1 % TFA. CSS "A" = Crotalus scutulatus SCUll/latus (venom A, GLENN and STRAIGHT, 1978); CVV-NM "A" = Crotalus viridis viridis. S.W. Ncw Mexico, i.p. LD," 0.38 mg/kg; CVV-NM "B", S.W. New Mexico, i.p. LD" 5.5 mg/kg; CVV-AZ "A" = Crotalus viridis viridis, near Page, AZ, i.p. LD,o 0.65 mg/kg; CVC-Pool = Crotalus viridis cOllcolor venom pool; a = acidic subunit of Mojave toxin, retention time = 15.6 min; b = basic subunit of Mojave toxin, retention time = 24.6 min. REVERSE-PHASE
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Comparative HPLC profiles of venoms from C. scutulatus scutulatus, C. viridis conc%r and C. viridis viridis are presented in Fig. 1. Three individual venoms of C. viridis viridis from Hidalgo County, NM (CVV-NM "A") and one individual from near Page, AZ (CVV-AZ "A") contained protein peaks with identical retention times as the acidic and basic subunits of Mojave toxin (CSS "A") and concolor toxin (CVC-Pool). Eleven of the 14 C. viridis viridis specimens from the same S.W. New Mexico region lacked two peaks in their venom profiles (data not shown) except that one specimen's venom exhibited a low peak in the region of the basic subunit (CVV-NM "B" venom, Fig. 1). This peak did not
(-)
I-I I I I
1_-
1
2
3
4
(+)
FIG. 2. BASiC-PAGE ANALYSIS OF THE RECOMBINED TOXIN (PEAKS a AND b, FIG. I) FROM Crowlus viridis viridis VENOM (CVV-NM "A", FIG. I). Polyacrylamide gel electrophoresis run as previously described (GLENN er al., 1983), using electrophoresis buffer 0.025 M Tris/0.192 M glycine, pH 9.0 and 251lg protein of each toxin stained with Coomassic brilliant blue. Lane I = Mojave toxin (CATE Hnd BmuER, 1978); Lane2 = recombined peaks a and b from CVV-NM "A" venom, Fig. I; Lane 3 = concolor toxin (POOL and BiEBER, 1981); Lane 4 = canebrake toxin (STRAIGHT and GLENN, 1989); a = unbound or disassociated acidic subuOIt; dashed line = recombined toxins.
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FIG. 3. DISTRIBUTION OF Crotalus viridis virielis AND POPULATIONS OF VENOMS SCREENED BY IMMUNOUIFFUSION FOR TOXINS SIMILAR TO MOJAVE TOXIN, USING ANTIBODY TO THE BASIC SUBUNIT OF MOJAVE TOXIN .
• = positive venoms;
0
= negative
venoms.
react with the antibody to the PLA subunit. The 4 CVV "A" venoms reacted strongly in the immunodiffusion tests and the 11 CVV "B" venoms were negative, using a high venom concentration of 40 mg/ml. Peaks a and b of the CVV-NM "A" venom (Fig. 1) were collected, recombined in H 20 and the toxins' electrophoretic mobility was compared to similar venom toxins of C. scutulatus (Mojave toxin), C. viridis cOl1color (concolor toxin) and C. /lOrridus atricaudatus (canebrake toxin) (Fig. 2.). Two major bands (likely isomeric
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toxins formed on recombination) were resolved with the CVV-NM "A" toxin. These were identical in electrophoretic mobility to Mojave toxin and distinct from concolor toxin and canebrake toxin which migrate faster anodally. The i.p. LD so of the recombined CVV-NM "A" toxin was O.064mg/kg. Among the Arizona and S.W. New Mexico specimens, the i.p. LO so values ranged from 0.38-0.65 mg/kg for the 4 venoms containing the toxin(s) (CVV "A") and ranged from 104-5.5 mg/kg for 11 venoms lacking the toxin(s) (CVV "B"). The latter venom "B" values were similar to the i.p. L0 50 values (0.9-4.1 mg/kg) obtained from the venoms of 20 populations of C. viridl:~ viridis throughout its range. Venoms from 46 different populations of C. viridis viridis have been assayed using immunodiffusion for the presence of the toxin and 44 were negative (Fig. 3). The only immunologically positive venoms were from regions where the distribution of C. viridis viridis overlaps with C. scutulatus or is near the range of C. viridis concalor (see KLAUBER, 1956; GLENN and STRAIGHT, 1982). The HPLC venom profile of the Page, AZ specimen (CVV-AZ "A", Fig. 1) is more similar to C. viridis concalor venom than to the New Mexico C. viridis viridis venom. Therefore, the toxins' presence is likely due to its close proximity to C. viridis concalar, through intergradation at some point in its genetic history. Further analysis of the Arizona specimens' venom could not be achieved due to lack of venom. Morphologically, the Arizona specimen appears as a typical C. viridis viridis, as do the three New Mexico specimens containing the toxin. All three of these New Mexico specimens (males) were of similar size (total length == 74-78 em) but were collected 5 and 15 miles distant from each other, t~erefore, the probability of their being from the same parentage is remote, but possible'IThe combined results of the immunodiffusion, lethal toxicity, HPLC and PAGE analyses indicate that the venoms of the three New Mexico specimens contain Mojave toxin as a result of ancestoral hybridization with C. scutulatus scutulatus in these specimens' genetic background.1
1
Ack/lowledgcmcll/s-Wcapprcciatc thc help of BARNEY TOMBERLIN and TONY SNELL, Portal, AZ, in providing collection data and in their diligent efforts to collect the S.W. New Mexico specimens for us. We thank DAVID HARDY, Phoenix, AZ for the loan of the Page, AZ specimen and HUGH QUINN, Houston Zoo, Houston, TX, for the loan of C. I'iridis I'iridis from several localities. The technical assistance of ELIZABETH ARNOLD, ROIlERT NOHAVEC, MARTHA WOLFE and TERRY WaLT is gratefully acknowledged. We are thankful to ALLAN BIEBER, Arizona State University, Tempc, AZ, for supplying the rabbit antibody used in this report. This research was supported by the Department of Veterans Affairs Medical Research Program, VA Medical Center, Salt Lake City, UT, and, in part, by Hogle Zoological Gardens, Sail Lake City, UT.
REFERENCES CATE, R. L. and BlEilER, A. L. (1978) Purification and characterization of Mojave (Crotalusscum/a/usSCUll/Ill/US) toxin and Its subUnits. Archs Biodu!fIl.Biophys. 189, 397---407. GLENN, .T. L. and STRAIGHT, R. C. (1977) Thc midget faded rattlesnake (Cro/alus viridiscOllcolor)venom: lethal tOXIcity and individual variability. To.\icoJl 15, 129-133. GLENN, .T. L. and STRAIGHT, R C. (1978) Mojave rattlesnake Cro/alus scu/lIla/llsscUlulalusvcnom: variation in toxicity with gcographical origin. Toxicoll 16, 81-84 GLENN, .T. L. and STRAIGHT, R. C. (1982) The rattlcsnakes and their venom yield and lethal toxicity. In: Ralllesllake Venoms: Their Actio/land Treatmell/, pp. 3~119 (Tu, A. T., Ed.). New York: Marcel Dckker. GLENN, .T. L. and STRAIGHT, R. C. (1985) Venom properties of the rattlesnakes (Cro/aills) inhabitIllg the Baja California region of Mexico. Toxicoll 23, 769-775. GLENN, .T. L. and STRAIGHT. R. C. (1989) Intergradation of two different venom populations of the Mojave rattlesnake (Crowills,lcu/llla/ussCII/II/allls) in Arizona. Toxicon 27,411-418. GLENN, .T. L., STRAIGHT, R. C, WOLFE, M. C. and HARDY, 0. L. (1983) Geographical variation in Crotaills sCllll/latusSCUll/laIUS (Mojavc rattlesnake) venom properties. To.\lcon 21, l19~ 130.
862
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HENDERSON, J. T. and BIEBER, A. L. (1986) Antigenic relationships between Mojave toxin subunits, Mojave toxin and some crotalid venoms. Toxicon 24, 473-479. HENDON, R. A. and BIEBER, A. L. (1982) Presynaptic toxins from rattlesnake venoms. In: Raulesnake Venoms: Their Action and Treatment, pp. 21/-246 (Tv, A. T., Ed.). New York: Marcel Dekker. KARBER, G. (1931) Beitrag Zur Kollektiven behandlung pharmakologischer reihenversuche. Arch. expo Path. Pharmak. 162, 480-489. KLAUBER, L. M. (1956) Raulesnakes: Their Habits, Life Histories and Influence on Mankind, Vol. I, p.56. Berkeley: University of California Press. (Partially revised in 1972, p. 61). POOL, W. R. and BIEBER, A. L. (1981) Fractionation of midget faded rattlesnake (Crotalus viridis conca/or) venom: lethal fractions and enzymatic activities. Toxicon 19, 517-527. STRAIGHT, R. C. and GLENN, J. L. (1989) Isolation and characterization of basic phospholipase (PLA,) and acidic subunits of canebrake toxin from Crolalus horridus alriclludolus venom using HPLC. Toxicon 27, 80 (A bstract).