A comparative study of properties and toxic constituents of funnel web spider (Atrax) venoms

Camp. Biochem. Phvsiol. Vol. 18C. No. I, pp. 55-68. 1984

Printed in Great Bhtain 0306~4492/84 $3.00 +O.OO

0 1984 Pergamon Press Ltd

A COMPARATIVE STUDY OF PROPERTIES AND TOXIC CONSTITUENTS OF FUNNEL WEB

SPIDER (A T&4X) VENOMS

D. D. SHEUMACK,* B. A. BALDO,~ P. R. CARROLL,~ F. HAMPSON,* M. E. H. HOWDEN* and A. SKORULIS*

*School of Chemistry, Macquarie University, North Ryde, N.S.W. 2113, Australia. Telephone: 888-8000; tKolling Institute, Royal North Shore Hospital, St. Leonards, 2065, Australia; and

fRiker Laboratories Australia Pty. Ltd, Chilvers Road, Thornleigh, N.S.W. 2120, Australia

(Received 5 September 1983)

Abstract-l. The crude venoms of male and female Sydney funnel web spiders, Afrux robustus, were compared by cation-exchange and high-performance liquid chromatography, lethality to new-born mice, polyacrylamide gel isoelectric focusing, immunoelectrophoresis, phospholipase A analysis, effects on the mouse phrenic nerve hemidiaphragm and passive paw oedema in the rat and, except in the case of rat paw oedema, were found to exhibit quite different properties.

2. Polyacrylamide gel isoelectric focusing and high-performance liquid chromatography proved to be suitable for gender and species determination when applied to the venoms of A. formidubilis, A. infensus, A. robustus and A. versutus. These venoms were also compared by lethality, promotion of muscle fasciculation and phospholipase A activity.

INTRODUCTION

Funnel web spiders of the genus Atrax are a group of arachnids which are indigenous to Australia. The existence of 33 species ranging from Southern Queensland to Tasmania has been established (Gray, 1981). At least 14 human deaths have been attributed to the bite of the Sydney funnel web spider, Atrax robustus. Only male A. robustus have been implicated in these fatalities (Gray and Sutherland, 1978). A lethal polypeptide neurotoxin, named robustoxin, has been isolated by us from the venom of male A. robustus spiders (Carroll et al., 1982; Sheumack et al., 1983) and partly characterized. Gender and species determination amongst members of the genus Atrax is difficult (Gray, 1981), yet may be important for emergency treatment of bites by these spiders. This study was designed to compare the properties and toxic constituents of venoms from male and female representatives of A. robustus, A. formidabilis, A. infensus and A. versutus as a means of distinguishing between them. The distribution of A. robustus is centred on the Sydney metropolitan area, whereas A. formidabilis is a tree-dwelling species from the North Coast of New South Wales, A. infensus is found in Northern New South Wales and Southern Queensland and A. versutus is located in hilly areas West of Sydney.

MATERIALS AND METHODS

Collection of venom

Colonies of Atrax species were set up by providing a collection service in the Sydney region. Spiders were identified by Mr M. R. Gray of the Australian Museum. Venom was collected by aspiration of the venom from the tips of the live spiders’ fangs into washed and silylated pipettes. The crude venom was washed from the pipette with 1.5% aqueous acetic acid into polypropylene vials, freeze-

dried and stored at -20°C until required. Care was taken to ensure that there was no confusion of juvenile male A. robustus with the females, by selecting only large mature females which had been kept in captivity from the summer to spring in order to allow time for observation of the appearance of any tibia1 spur (characteristic of the male) after moulting.

Lethality assay

The LD,, of crude venom and various purified fractions was measured using new-born mice (Sutherland, 1980) weighing 1.5-1.8 g. Freeze-dried fractions were dissolved in 1.5% aqueous acetic acid to 10 mgcmm3 and serially diluted. Samples (total volume each 50 ~1) were injected subcuta- neously into the dorsum of the mouse parallel to the spine. Each fraction was injected into a group of four mice. Each group was kept under conditions which minimized temperature fluctuations. The number of deaths was measured every 3 hr for 24 hr.

The average yield of crude dry venom from male and female A. robustus was calculated from the total dry weight of venom obtained from each “milking” of the colony. These yields were measured over a period of 6 months, during the summer, autumn and early winter. To compare variations in lethality of A. robustus venom, 20 individual male and female spiders’ “milkings” were tested in new- born mice. As no accurate measure of the dry weight of an individual “milking” can be obtained, each “milking” was dissolved in 1 cm3 of 1% v/v aqueous acetic acid, and the number of mouse lethal doses in that volume was deter- mined. These values are thus expressed as mouse units per milking. The lethal dose for half the number of mice in a group (LD,,,) for batches of Atrux venoms was also deter- mined in new-born mice.

Extraction of venom apparatus

The venom apparatus (fangs and venom glands) from male and female A. robustus were extracted essentially according to the procedure of Gregson and Spence (1983). The supernatants after centrifugation were not diafiltered, but were freeze-dried and then tested for lethality and effects on the mouse diaphragm.

55

56 D.D. SHEUMACK et al.

Ion-exchange chromatography

Ion-exchange chromatography was performed using CM Sephadex C-25 (Pharrnacia South Seas) that had previously been converted to the ammonium form by washing 100 g of the gel on a Biichner funnel successiveiy with 5 dm’ each of 1 M acetic acid, deionized water, 1.0 M ammonium acetate solution and distilled water. The gel was then suspended in 0.1 M ammonium acetate solution at pH 7.0 and a 1.6 x 30 cm column of the gel was packed. The column was equilibrated overnight with 2dm3 of 0.1 M ammonium acetate solution at pH 7.0 before use. Samples of venom were dissolved in distilled water to a concentration of 0.17; w/v and the pH adjusted to 7.0 with concentrated ammonia solution. The sample was then absorbed onto the column and elution was performed by a linear ionic strength gradient of 0.1-1.1 M ammonium acetate solution at pH 7.0. At completion of the gradient elution, 100 cm3 of 1.5 M acetic acid was passed through the column to remove any remaining adsorbed material, The column effluent was monitored at 280nm using a Paton CM 2 absorbance monitor and equivalent volume fractions were collected using a Paton LTDM Fraction Collector. All fractions were bulkid, freeze-dried and stored at -20°C. All fractions were screened for lethality towards new-born mice.

Polyacrylamide gel isoelectric focusing

Polyacrylamide gel isoelectric focusing was carried out on OSmm plates according to the LKB appli~tion note, “High-Performance Thin Layer Isoelectric Focussing”. Gels were polymerized to give concentrations of 10% polyacrylamide and 4% w/v pH 3.5-10 ampholines. Tetra- methylethylenediamine was added to the gel to afford a concentration of 0.035’~ v/v, in order to extend the alkaline end of the pH gradient (Yao-Jun and Bishop, 1982). Crude venom was dissolved in water to give a concentration of lOpg/pl and 1OOpg samples were loaded onto the plates. Isoelectric focusing was performed at 10” with an initial setting of 30mA and 200V. A maximum power level of 15 W was set. Focusing was complete after 80min. The focused plate was fixed in 15% w/v aqueous trichloroacetic acid containing 7.5% w/v of sulphosalicylic acid for 30 min. Staining was performed with 0.03% w/v Coomassie Blue G-250 in 3%v/v aqueous perchloric acid. destaining was performed with 5% v/v aqueous acetic acid containing 5% v/v glycerol.

High-performance liquid chromatography

High-performance liquid chromatography was carried out with a Waters Associates C-8 reverse phase Radial Pat column. The column was eluted during 25 min with a linear gradient of Ift35%v/v acetonitrile in an aqueous buffer which was 5 mM in sodium dihydrogen phosphate pH 3.0, 5 mM in lysine and 100 mM in sodium sulphate.

Preparation of antisera

Antisera for male and female A. robustus venom were prepared for immunological examination of the venom. Adult male Californian white rabbits were immunized as follows: for antiserum against male A. robustus venom, 0.5mg of crude venom was mixed with 0.5cm3 of 1% v/v acetic acid and 0.5 cm3 of Freund’s complete adjuvant (Commonwealth Serum Laboratories) until a homogeneous emulsion was obtained. The emulsion was injected intra- muscularly into the front and hind limbs. A second injection was made two weeks later. This consisted of 0.5mg of venom in 1 cm3 of 1% v/v acetic acid, which was administered as ten individual subcutaneous injections along the flank of the rabbit. These injections continued in an alterna- ting fashion fortnightly. The regimen for preparation of antiserum to female A. robustus venom was the same as that for male venom, except that 1 mg of female venom was used per injection. At the end of each month, 40cm’ of whole blood was removed from the immunized rabbit by bleeding

from a marginal ear vein. After allowing the blood to stand at room temperature for 60 min, the clot was centrifuged at 1OOOg for 2Omin and the antiserum was removed by pipette. The antiserum was transferred to sterile 1 cm3 sample tubes and stored at -20°C.

Grabar- Williams immunoelectrophoresis

Immunoelectrophoresis was performed with a 1% w/v agarose (Sigma Chemical Co.) matrix and a pH 8.4 sodium barbital buffer. The buffer was prepared by dissolving 8.42 g of sodium diethylbarbitu~c acid and 1.6 g of diethyl- barb&uric acid in 2 dm3 of distilled water and diluting 1: 1 with water before use. Samples were dissolved to a concentration of lOpg/pl in l%v/v acetic acid and 50pg were applied to the agarose in a punched well. Electrophoresis was performed at IOmA for 45 min at 5” using bro- mophenol blue as a tracking dye. Whole rabbit antiserum (0.2 cm’) was placed in each trough and allowed to diffuse for 18 hr. The plate was then pressed and washed with five successive changes of 0.860/, w/v saline containing 0.01% w/v sodium azide for a total of 48 hr. The plates were then dried and stained with Coomassie R-250 as a 0.5% w/v solution in ethanol-acetic acid-water 4.5 : 1: 4.5. Destaining was performed in ethanol-acetic acid-water 4.5: 1 :4.5 until a clear background was obtained.

Rat passive paw oedema

Rat paw thickness was assayed with a thickness- measuring transducer manufactured by Hoffman La Roche, Base! (Bald0 et al., 1980). Following the measurement of the thicknesses of both hind paws, male Oxford Hooded [HO(PVG/C)] rats of approximately 200 g body weight were given a subplantar injection of SOnI of a suitable saline dilution of material to be tested in the right hind paw and 50 ~1 of 0.86% w/v saline in the left paw. Injections were administered via a 27 gauge needle. At the desired times after challenge, the ensuing swelling in the paw was measured by the thickness-measuring transducer. Paw swelling in each rat due to injection of venom was obtained by deducting the thickness of the control (left) paw from the thickness of the right paw. Four rats were used in each experiment. The first was injected with a IOpglnl stock solution of sample in 0.86”/, w/v saline; the second with a 1: 2 dilution of this stock solution; the third with a I:4 dilution; and the fourth with a 1: 8 dilution. Fractions obtained from the ion-exchange chromatography of crude male and female A. robustus venom were also tested at l~~g;50~1 of 0.86 w/v saline for oedema-pr~ucing ac- tion. Those fractions causing oedema were analyzed by proton nuclear magnetic resonance (‘H n.m.r.) and mass spectrometty. N.m.r. spectra were obtained from 1 mg/0.5ml solutions in D,O on a Varian XL-200 instru- ment. Mass spectra were measured on an AEI MS-12 electron impact spectrometer at 14O”C, 70eV and 100 PA.

Mouse diaphragm muscle fasciculation

Crude venoms from four species of Atrax and robustoxin were compared for muscle fasciculation activity using a mouse phrenic nerve diaphragm muscle preparation according to Gage and Spence (1977). The organ preparation was pi&red ina 5 cm3 organ bath made of silastic (Dow Chem- ical Co.) and bathed in modified Kreb’s solution which was bubbled with 5% v/v CO2 in oxygen. Venoms were diluted in distilled water and added to the organ bath to give concentrations of 1 pg/cm3 for venoms from male spiders, 20 ~g/crn’ for female venoms and 0.1 yg/cm3 for robustoxin. Muscle stimulation and twitch responses were applied and measured respectively by a Grass Stimulator and a Grass Force Transducer.

A comparative study of funnel web spider venoms 57

Phospholipase A activity

Phospholipase A activity was measured in crude An-ax venoms by the method of Salach et al. (1968) in a 1Ocm’ bath. The reaction buffer was 0.02 M calcium chloride, 0.7 mM ethylenediaminetetracetic acid, 0.5 mM 2-amino-2- hydroxymethylpropane-1,3-dial, 0.05 M NaCl and 0.5% w/v Triton X-100 at pH 8.0. The substrate was egg lecithin. The reaction mixture was magnetically stirred at 25°C under nitrogen. When the pH was stable after addition of the substrate, samples of crude Atrax venom (100 pg in 100 ~1 water) were added in distilled water, and the change in pH measured with time. Crude Pseudechis australis venom was used as a standard for a potent, phospholipase A-containing venom (Sharp, Howden and Spence, unpublished results).

RESULTS

Venom yield and lethality

The average yields of crude dry venom from male and female A. robustus are summarised in Table 1. The variations in lethality of male and female A. robustus venom are reported in Table 2. Table 3 summarises the LD,, values obtained for crude male and female venoms of the various Atrax species.

Extracts of venom apparatus

Venom apparatus from 15 male (total wet wt of fangs and venom glands, 3.8 g) and 15 female (4.7 g) A. robustus yielded 0.18 and 0.38 g respectively of dry extracts. The LD,, of these extracts were 33 and 200 mg/kg mouse, respectively. These values were ten and four times less potent, respectively, than average figures for crude male and female A. robustus venom (Table 3). The minimum concentrations of these extracts causing detectable muscle fasciculation in the mouse diaphragm were 30 and 250 pg/cm3 respectively. These concentrations were also markedly higher than those for the corresponding venoms (Fig. 16). The dry extracts were not soluble in 1.5-3.5x v/v acetic acid and had to be sonicated for 30 min to achieve an even suspension for analysis. The acetic acid in such suspensions was found to inhibit muscle twitch. When 100 mg of dry extract from male venom apparatus was subjected to cation-exchange chromatography, no lethal fractions were obtained.

Table 1. Average yield (p g) of dry crude venom per “milking” per A. robustus spider over a 6 month period

Month male female

January (sumner) 179

February 132

March 180

April 182

May 186

JUW 152

July (winter) 130

Average * 163

*Average of monthly values.

340

310

350

302

311

318

304

319

Ion-exchange chromatography of crude A. robustus venoms

Samples of crude male and female A. robustus venoms were subjected to cation-exchange chromatography. The elution profiles for crude male and female venom are shown in Figs 1 and 2 respectively. Bulked fractions were freeze-dried and tested for lethality in new born mice. The last eluting peak (Fraction 1A) from the chromatography of 70 mg of male venom (Fig. 1) had an LD,, of 0.16 mg kg- ’ and weighed 7mg; no other fractions from male venom were lethal to new-born mice. When 70 mg of crude

Table 2. Lethality (mouse units per spider) of single “milkings” of individual male and female A. robustus spiders. The lethality was for subcutaneous injection into new-born mice. Each “milking” was dissolved in 1 cm’ of 1% v/v acetic acid and serial dilutions were injected until a dose was reached at which three out of four mice died within 24 hr. All spiders were collected in February and March and had moulted twice before this assay was applied. Thus there is no possibility of any of the female spiders being mis-

taken for juveniles males (Gray, 1981).

Spider number

male felMle

1 20 5

2 25 7

3 50 a

4 52 10

5 60 10

6 68 10

7 70 11

8 100 12

9 100 14

10 133 20

Table 3. LD, values for crude venoms from male and female Arrax species and for robustoxin

*tra.z species Gender LO,mg kg-' of nwuse

robustus male 3.3

robustus female 50

verSUtu(S male 0.7

"‘XVUtUO female 0.5

infensus male 0.7

fcxT+:utiiis fefiale 16

,S,rri<:abiiis male 50*

,? bi<rt,x;,! male 0.16.

*This value was obtained from a single “milking” and is expressed as mouse units per “milking”.

tsheumack et al. (1983).

D. D. SHEUMACK et al.

1 50 100 150 200 250 300 350 400 450

VOLUME OF EFFLUENT b13i

Fig. 1. Ion exchange chromatogram of 70 mg of crude male A. robustus venom chromatographed on a 1.6 x 30 cm column of Sephadex CMC25 at pH 7.0. Elution was carried out with a linear ionic strength

gradient of 0.1-1.1 M ammonium acetate solution at pH 7.0.

female A. robustus venom were subjected to cation- for crude male and female A. roburtus venoms run exchange chromatography under identical condi- against male venom rabbit antiserum and female tions, the fractions obtained (Fig. 2) were not lethai venom antiserum respectively. Complex mixtures of to new-born mice at doses up to 1 mg per mouse. antigens were revealed for both venoms. As can be

Immunoelectrophoresis of A. robustus venoms seen, there was a high degree of apparent cross- antigenicity between male venom and female anti-

Figure 3 shows the immunoelectrophoretograms serum and female venom and male antiserum. Addi-

1

100 200 300 400

VOLUME OF EFFLUENT (CM3)

Fig. 2. Ion exchange chromatogram of 70 mg of crude female A. robustus venom chromatographed on a 1.6 x 30 cm column of Sephadex CMC25 at pH 7.0. Elution was carried out with a linear ionic strength

gradient of 0.1-1.1 M ammonium acetate solution at pH 7.0


Fig. 3. Immunoelectrophoretograms of (A) 50 pg of crude male A. robustus venom in well numbers l-3 and 50 pg of crude female venom in wells 4 and 5, against 0.2 cm3 of crude male venom rabbit antiserum, in the troughs at pH 8.4; and of (B) 50 ng of crude male A. robustus venom in wells 1-3 and 50 pg of crude female venom in wells 4 and 5 against 0.2 cm’ of crude female venom rabbit antiserum at pH 8.4.

Wells are numbered l-5 from the top downwards. The anodes are on the right.

tional precipitin lines occurred at the cathode for male venom. One of these presumably corresponded to robustoxin (since it is cationic at pH 8.6). These bands were not observed when female A. robustus venom was incubated with either male (Sheumack and Howden, unpublished results) or female antiserum. These results are consistent with the apparent lack of robustoxin in female venom (Sheumack et al., 1983).

Polyacrylamide gel isoelectric focusing

Figure 4 shows the isoelectric focusing patterns for crude venoms from seven different species and gen- ders of Atrax spiders, together with robustoxin and protein standards of known PI. A band at or near the position for robustoxin was found in the patterns for crude venoms from male A. robustus and A. infensus and male and female A. versutus, but was absent from those of female A. robustus and A. formidabilis. Only one “milking” was obtained from one specimen of male A. formidabilis and the loading on the plate was insufficient to give any detectable bands. Robustoxin

and bovine cytochrome c focused at the same p1. Thus the pI of robustoxin was 10.2.

High-performance liquid chromatography of Atrax venoms

The high-performance liquid chromatograms of the crude venoms of male and female specimens of five known Atrax species and of two unidentified variants are given in Figs 5-14. The crude venom of each species and gender had its own distinctive elution profile, and these were reproducible from batch to batch of venom. Figure 15 is the chromatogram for robustoxin. A peak corresponding to robustoxin was present in the chromatogram for crude male A. robustus venom (Fig. 5) at an elution time of 19 min. A similar peak appeared at 17 min in crude male and female A. uersutus venoms (Figs 7 and 8). The venoms of the two unidentified Atrax variants (Figs 12 and 14) showed profiles similar to A. robustus and A. versutus.

Mouse diaphragm muscle fasciculation

The effects of four Atrax crude venoms and of

60 D. D. SHEUMACK et al.

Fig. 4. Polyacrylamide gel isoelectric focusing patterns for (A) a mixture of standards (Pharmacia) consisting of /?-lactoglobulin A (PI 5.2), bovine carbonic anhydrase B (5.9), human carbonic anhydrase B (6.6), horse myoglobin (7.4), lentil lectin (8.1), lentil lectin (8.5), lentil lectin (8.7), trypsinogen (9.3) and bovine cytochrome c (10.2); (B) crude male A. robustus; (C) female robustus; (D) male uersutus; (E) female aersutus; (F) male formidabilis; (G) female~ormidabilis; (H) male infensus venoms; and (I) a mixture of robustoxin and cytochrome c (p1 10.2). Polyacrylamide (10% w/v), 4% w/v pH 3.SlO ampholines and tetramethylethylenediamine (0.035% v/v) were used. Initial settings were 30 mA and 200 V. The band at p1 10.2 in (B) was identified as robustoxin* by comparison with the pure toxin. The anode was at the

top of the figure.

5 IO 15 20 25

Retention time (mini

Fig. 5. High-performance liquid chromatogram of 0.15 mg of crude male A. robustus venom on a C-8 Radial-Pat column eluted by 10-35x aqueous acetonitrile which was 5mM in sodium dihydrogen phosphate, 5 mM in lysine and 100 mM in sodium sulphate at pH 3.0. The flow rate was 1.5 cm3 mini.

Aufs denotes absorbance units full scale.


5 IO 15 20 25

Retention time i m!n I

Fig. 6. High-performance liquid chromatogram of 0.15 mg of crude female A. robustus venom under the same conditions as Fig. 5.

robustoxin on the mouse diaphragm are summarised female A. robustus and A. f~rm~dubi~is produced only in Fig. 16. The venoms from the male Atrux spiders slight muscle fasciculation at a concentration 20 times provoked intense muscle twitching within 30 set to that for male A. robustus venom. Robustoxin pro- 2 min. Female A. versutus venom also caused marked duced intense twitching at a concentration one-tenth fasciculation and, in addition, the muscle tension that of male A. robustus crude venom. increased quite dramatically. The latter result may be attribu~ble to the presence of phosphoiipase A in ~~rnp~ris5~ of yearns-producing properties of venoms

this venom (see below). The crude venoms from Crude venom from male and female A. robustus

5 10 I5 20 25

Retention time (mini

Fig. 7. High-performance liquid chromatogram of 0.15 mg of crude male A. uersufus venom under the same conditions as Fig. 5.

62 D. D. SHEUMACK et al

5 IO 15 20 25

Retention time (rwnl

Fig. 8. High-performance liquid chromatogram of 0.15 mg of crude female A. uersutus venom under the same conditions as Fig. 5.

produced maximum swelling 1&20min after injection into the hind paw of a rat (Figs 17 and 18 respectively). The swelling produced by female venom was slightly greater than that for male venom. Robustoxin at a concentration of 1 mgcm-’ produced no significant oedema. The fractions obtained from the cation-exchange chromatography of crude male and female venom (Figs 1 and 2) were screened for oedemic activity. Only the ultraviolet-absorbing fractions in each venom and which eluted at 3 10 cm3 (Figs 1 and 2) produced oedema, and the swelling reached a maximum in 30min. This material gave

mass and n.m.r. spectra which were identical to those of authentic Smethoxytryptamine (Sigma Chemical Co.). Authentic 5_methoxytryptamine, at a concentration of 1 mgcme3, gave rise to oedema in the rat paw comparable to that due to the same concentration of the above fraction. Duffield et al. (1979) detected this compound in the venom of female A. robustus.

Phospholipase A activity

It was found that, of the Atrax venoms studied in this report, only crude female A. uersutus venom

Retention trne (mlnl

Fig. 9. High-performance liquid chromatogram of one-fifth of a single “milking” of crude male A. formidabilis venom under the same conditions as Fig. 5.

A comparative study of funnel web spider venoms

5 IO 15 20 25

Retenbon twne (m!n)

Fig. IO. High-performance liquid chromatogram of 0.15 mg of crude female A. formidabifis venom under the same conditions as Fig. 5.

5 10 15 20 25

Retention time I mm)

Fig. I f . High-performance liquid chromatogram of 0.15 mg of crude male A. injk.ws venom under the same conditions as Fig. 5.

63

64 D. D. SHEUMACK ei al.

v\i \--R,-- 5 IO i5 ------75 20

Retention time i mm i

Fig. 12. High-performance liquid chromatogram of 0.15 mg of crude venom from a female Atrux variant collected at Bombala, New South Wales, run under the same conditions as Fig. 5.

showed phospholipase A activity. Robustoxin at units/l~~g venom/min. Crude A. uersutus venom IO fig in 100~1 water did not possess such activity. gave an almost identical value of 1.8 pH units/100 pg Crude P. australis venom had an activity of 2.0pH venom/min.

Retention time Imin)

Fig. 13. High-performance liquid chromatogram of 0.15 mg of crude male A. cerberea venom under the same conditions as Fig. 5.


5 IO I5 20 25

Retention time (mln)

Fig. 14. High-performance liquid chromatogram of 0.15 mg of crude venom from a female Atrax variant collected at Jervis Bay, New South Wales, run under the same conditions as Fig. 5.

DISCUSSION

The individual lethality values (Table 2) covered a wide range for both male and female A. robustus spiders. Such a broad spectrum of lethality would arise from variations in potency and/or output (Table 1) of venom for individual spiders and between spiders, and could be due to many causes; such as the size, health and diet of the spiders, how recently they have used their venom, etc. Such variations in lethality may partly explain the numerous non-fatal or asymptomatic bites by A. robustus (Sutherland, 1972). The LD,, values in Table 3 indicate that genuine crude female A. robustus venom is 19 times

; ; 6 ;I lb 1; 1111 ’ 16 ;6 ;O

RETENTION TIME ( min I

Fig. 15. High-performance liquid chromatogram of 0.0 I mg of robustoxin under the same conditions as Fig. 5.

less potent towards new-born mice than the crude male venom. This finding is consistent with the relatively mild symptoms observed in humans en- venomated by female A. robustus (Sutherland, 1972), with the presence of the lethal neurotoxin, robustoxin, in the crude male venom (Figs 3, 4 and 5) and the lack of significant quantities of it in crude female venom (Figs 3, 4 and 6). In contrast, extracts of venom glands from male Latrodectus (black widow) spiders were non-toxic to rats, whereas venom from female spiders was lethal (Bettini and Maroli, 1978).

Crude male A. infensus and female A. uersutus venoms were the most lethal of any of the venoms tested, being approximately three times as potent as crude male A. robustus venom. Based on these figures, all of the spiders listed in Table 3 should be regarded as dangerous to humans, except female A. robustus and perhaps female A. formidabilis. Sutherland (1983) reported that the venom of female A. formidabilis was at least as toxic to new-born mice as the venom of male A. robustus. This conclusion is not in accord with the results in Table 3. It is difficult to distinguish between female A. robustus and juvenile male spiders since both lack the tibia1 spur possessed by the mature male (Gray, 1981). Until they are positively identified, it is therefore necessary to treat all of these Atrax species as potentially lethal.

The high-performance liquid chromatograms (Figs 5-14) and polyacrylamide gel isoelectric focusing patterns (Fig. 4) of the crude Atrax venoms consis- tently and reproducibly showed marked differences between them. Such differences could be exploited in using these techniques to determine gender and species for Atrax spiders. All of the Atrax spiders studied, except perhaps A. formidabilis because of its larger size, are difficult to identify. As to the methods

66 D. D. SHEUMACK ef al.

Fig. 16. The effects of Atrax crude venoms and robustoxin on the murine diaphragm muscle in z&o. The muscle was constantly stimulated ria the phrenic nerve. The techniques of Gage and Spence (1977) were employed. The organ preparation was pinned in a 5 cm3 bath and bathed in Krebs’ solution which was bubbled with 57; v/v CO, in oxygen. Venoms or toxin were diluted in distilled water and added to the bath to give the following concentrations (pg/cm’ top to bottom traces) (a) robustoxin 0.1, (b) crude male A. robustus 1.0, (c) female robustus 20, (d) male versutus 1.0, (e) female versufus 20, (f) male in&sus 1.0 pg/cm’. (g) male formidabitis 0.2 of a single “milking”

and (h) female ,formidabilis 20.

of analysis, isoelectric focusing requires 100 pg of crude venom per analysis, while high-performance liquid chromatography may be easily applied using only lpg of crude venom. Thus, the latter is the method of choice for this analysis.

The oedema-promoting properties observed following the subplantar injection of A. robustus venoms and venom fractions into rat hind paws demonstrates in vivo another potent biological activity of the venoms from this species. The paw thickness increases observed were large, being greater than the swelling generally found with commonly-used inflammatory agents such as carrageenan, turpentine or rat homocytotropic antibody (Bald0 et al., 1980).

When this study was almost completed, Gregson

and Spence (1983) published a report describing the isolation and characterization of a semi-purified neurotoxin, named “atraxin”, from the venom glands of male or female A. robustus. This neurotoxin appeared to have quite different structure and properties to robustoxin (Sheumack et al., 1983) and we are unable to reconcile our results with theirs. “Atraxin” was reported to have been isolated from macerated venom apparatus of both male and female spiders whereas robustoxin was separated by us from “milked” venom of male spiders only (Carroll et al., 1982; Sheumack et al., 1983). The isolation of “atraxin” was monitored by mouse phrenic nerve-hemidiaphragm instead of mouse lethality. We repeated the extraction of venom apparatuses from authentic groups of male and female A. robustus by the procedure of Gregson and Spence (1983) and obtained extracts which were only sparingly soluble in the dilute acetic acid solvent favoured by them. These extracts had low lethality towards new-born mice compared to crude “milked” venom. The extract from male spiders did not yield any lethal eluate upon ion-exchange chromatography under the same conditions as those used to isolate robustoxin from crude venom.

Acknowledgements-The authors are grateful to many peo- ple in New South Wales and Queensland including Mr P. Walker, Mr A. Gill and Mr R. Swift for the donation of spiders; to the Councils of Baulkham Hills, Blacktown, Ku-ring-gai, Ryde and Warringah, the Housewives Associ- ation of New South Wales, the National Health and Med- ical Research Council and many other individuals and organisations for financial support; and to Dr I. Spence and Mr M. R. Gray for special assistance.

REFERENCES

Baldo B. A. (1982) Inflammation, counter irritation and rat serum acute phase a,-macroglobulin levels. Agents and Actions 12, 333-339.

Baldo B. A., Thomas P. G., Nelson C. L. and Taylor K. M. (1980) Passive paw anaphylaxis in the rat. Optimum conditions for use in studies on immediate hyper- sensitivity. Allergy 35, 3999408.

Bettini S. and Maroli M. (1978) Venoms of Theridiidae, genus Latrodectus. In Handbook of Experimental Phar- macology (Edited by Bettini S. and Maroli M.), Vol. 48, p. 156. Springer-Verlag, New York.

Carroll P. R., Hampson F., Howden M. E. H., Inglis A. S., Roxburgh C. M., Sheumack D. D., Skorulis A. and Strike P. M. (1982) The isolation and primary structure of the lethal neurotoxin from the venom of the male Sydney funnel web spider, Atrax robustus. Toxicon 20, Suppl. 1, 9.

Duffield P. H., Duffield A. M., Carroll P. R. and Morgans D. (1979) Analysis of the venom of the Sydney funnel web spider Atrax robustus using gas chromatography mass- spectrometry. Biomed. Mass Spectrom. 6, 105-108.

Fisher M. McD., Carr G. A., McGuinness R. and Warden J. C. (1980) Atrax robustus envenomation. Anaesth. In- tens. Care 8, 410-420.

Gage P. W. and Spence 1. (1977) The origin of muscle fasciculation caused by funnel-web spider venom. Aust. .I. exp. Biol. med. Sci. 55, 453-461.

Gray M. R. (1981) Getting to know funnel-webs. Aust. Nat. H&t. 20, 256270.

Gray M. R. and Sutherland S. K. (1978) Venoms of Dipluridae. In Arthropod Venoms (Edited by Bettini S.), pp. 121-148. Springer-Verlag, Berlin.


5.0 -

‘i‘ r 4.0-

D s u’ 5 3.0

I; ;! g 2.0

I

0 5 10 15 20 25 30 35 40 45 50 55 60 120

TIME (mini

Fig. 17. Rat paw thickness increases caused by crude male A. robustus venom. The right hand paws of male Oxford Hooded [HO(PVG/C)] rats were injected with 50 ~1 of solution containing 1.25, 2.5, 5 or 1Omg of dry venom/cm--‘. Left hand rear paws were injected with physiological saline. Oedema (as swelling) was assessed by measuring paw thicknesses at 5 min intervals up to 1 hr and then at 2 hr. The solid line corresponds to a concentration of 10 mg crne3 of crude venom; (--) line to 5 mg cm-) of crude

venom; line (--------) to 2.5mgcm-3; and line (-.-.) to 1.25mgcm-3.

0 5 10 15 20 25 30 35 40 45 50 55 60 120

TIME (mint

Fig. 18. Rat paw thickness increases caused by crude female A. robustus venom. The right hand paws of female Oxford Hooded [HO(PVG/C)] rats were injected with 50 ~1 of solution containing I .25, 2.5, 5 or 10 mg of dry venom/cml. Left hand rear paws were injected with physiological saline. Oedema (as swelling) was assessed by measuring paw thicknesses at 5 min intervals up to 1 hr and then at 2 hr. The solid line corresponds to a concentration of IO mg crnm3 of crude venom; (--) line to S mg cmm3 of crude venom;

line (--------) to 2.5mgcme3; and line (-.-,) to 1.25mgcm-‘.

Gregson R. P. and Spence I. (1983) Isolation and characterization of a protein neurotoxin from the venom glands of the funnel-web spider (Atrax robustus). Comp. Biochem. Physiol. 74C, 125-132.

Salach J. I., Turini P., Hauber J., Seng R., Tisdale H. and Singer T. P. (1968) Isolation of phospholipase A iso- enzymes from Naja naja venom. Biochem. biophys. Res. Commun. 33, 936-940.

Sheumack D. D., Carroll P. R., Hampson F., Howden M.

E. H., Inglis A. S., Roxburgh C. M., Skorulis A. and Strike P. M. (1983) The isolation and N-terminal se- quence of the lethal neurotoxin from the venom of the male Sydney funnel web spider, Atrax robustus. Toxicon Suppl. 3, 397400.

Sutherland S. K. (1972) The Sydney funnel-web spider. A review of some clinical records of human envenomation. Med. J. Aust. 2, 643-646.

Sutherland S. K. (1980) Antivenom to the venom of the

68 D. D. SHEUMACK et al.

male Sydney funnel-web spider Atrax robustus. Med. J. Yao-Jun G. and Bishop R. (1982) Extension of the alkaline Aust. 2, 437-441. end of a pH gradient in thin-layer polyacrylamide electro-

Sutherland S. K. (1983) Australian Animal Toxins, p. 291. focusing gels by addition of tetramethylethylene diamine. Oxford University Press, Melbourne. J. Chromat. 234, 459462.

Documents

A comparative study of properties and toxic constituents of funnel web spider (Atrax) venoms