Partial characterization of lethal and neuroactive components of the brown recluse spider (Loxosceles reclusa) venom

0041-010117910701-0347502.00/0

Toxtron, Vol. 17, pp . 347-334. ® PerQamon Pte~e Ltd. 1979. Printed in Great Britain.

PARTIAL CHARAC"PERIZATIOti OF LE"hI-~,~L A~II~ 'NEU?20ACTIVE CUI~IPONENTS OF TI?E B?G~~dtT R~ECLLTSE SPIDER (LO.YOSCELES I ECLUSA) ~7~`,jl,?i1~i'>< L. D. Foll., J. L. FRAZIER and B. R. NoxMENT

Department of Entomology, Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, Mississippi State, MS 39762, U.S .A . (Acceptedjor publication 30 October 1978) L. D. Fo1L, J. L. Fnnzll:a and B. R. NORMENT. Partial characterization of lethal and neluoactive wmponents of the brown recluse spider (Loxosceles reclusa) venom. Toxicon 17, 347-354, 1979.-Loxosceles reclusa venom was separated by Sephadex G-50 gel filtration into three 280 nm absorbing peaks, two of which contained components lethal to insects. The heavy molecular weight (mol . wt) protein found in peak 1 caused knockdown and mortality in Musca domestics 20-24 hr after injection. The low mol, wt (less than 10,000) polypeptide in peak 2 was heat stable at 90°C and caused knockdown within 30 min and mortality within four hr after injection. Both whole venom and peak 2 contained components which caused a dramatic increase in the amplitude and frequency of the endogenous activity of the ventral nerve cord of Periplaneta americana. The neuroactive factor stimulated this activity to 680-fold and with 5 min produced a block which was reversible by saline irrigation . Gel filtration elutions of the low mol. wt lethal factor and the neuruactive factor overlapped but did not coincide . 1NTRODUC I70N

Tlm sROwrr recluse spider, Loxosceles reclusa Gertsch and Mulaik, is a medically important

arachnid found in the southern two-thirds of the Mississippi-Missouri-0hio River Basin (ArmStsoN, 1973). Envenomation by this spider often results in a spreading, necrotic lesion (DILLAHA et al., 1963) and can produce systemic manifestations including hemolytic anemia (DExrtY et al., 1964). Although the effects of L. reclusa venom have been studied on at least five vertebrate species (ATxINS et al., 1958 ; GxoTxnus, 1968 ; SMrrli and Mlexs, 1968), no chemotherapeutic agent which will specifically inhibit formation of the pathological lesion has been found. ELGI?ItT et al. (1974) established that antiserum has to be administered within 30 min after the bite to inhibit local necrosis. Antivenin therapy would, therefore, normally be of little practical use in preventing dermonecrosis, since individuals may be unaware that envenomation by L. reclusa has occurred until 6-8 hr later. The effects of L. reclusa venom on insects, the natural prey ofthe spider, have also been studied . NoRMENT and SMITH (1968) observed reduced hemocyte counts in the common house cricket, Acheta domesticus, following venom injection. Insect tissues were also found to undergo pathological changes following either experimental venom injection (NoItM$NT and VINSON, 1969) or envenomation by the spider (ESICAFI and NOI2MENT, 1976) . The purpose of this study was to investigate the toxicities and pathophysiological effects of L. reclusa venom upon insects . MATERIALS AND METHODS Adult L. reclusa were collected in north and central Mississippi, maintained in the laboratory at room temperature, and fed greater wax moth, Galleria mellonella, larvae every two weeks. Venom was extracted 'Publication No . 3986 of Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, Mississippi State, MS 39762. This research was supported by NIH grant No . ROlES00651 . 347

348

L. D. FOIL, J. L. FRA7TFR and B, R. NORMENT

from adult spiders by a mild electrical stimulus (Noxnuavr and Snarlr, 1968) and collected in 1 ul Drummond® capillary tubes. The venom contained 215 ug protein per pl as determined by the Folin spectra photometric method (LowxY et al., 1951). Adult house flies, Musca domestica, were injected intrathoracically with 066 pl of either whole venom or gel filtration fractions using 025 ml syringes with 30 gauge needles mounted on an Isco Model M microapplicator. Five venom concentrations with 25 flies per concentration were tested in each of three trials . Mortality was determined 24 hr after injection and flies not responding to prodding were considered dead . Peroent mortality was corrected by Abbott's method (AHH01T, 1925) and transformed into log-probit values for linear regression analysis . Whole venom samples between 0~5 and 0~7 mg were fractionated by gel filtration on Sephadex G-50 columns (0~9 cm x 30 cm) which were equilibrated and eluted with either 0~1 M NaCI or a phosphate buffered saline (Romar, 1948) at 4°C. Venom elution was continuously monitored by an Isco Model UA-5 absorbance monitor at 280 nm and collected in lml fractions in an Isco Model 1200 fraction colleJtor. The absorption spectrum of peak fractions betwcen 310 and 240nm was also scannedusing a Beckman DB-GT spectrophotometer. The R values were calculated for fractions from each column using the formula 1~, = Y, - V,lYt - V, in which V, is the elution volume of the fraction, Y, (void volume) is the elution volume for Blue Deztran 2000, and Vt is the total bed volume . Conventional electrophysiological techniques (ROEDHR and Rosnar, 1939) were used to record the activity in ventral nerve cords of the American cockroach, Periplaneta amertcana. Ventral nerve cords with either the second to the sixth or the second to the fifth abdominal ganglia were extirpated and placed in a plexiglass chamber containing a 200 pl saline bath . Electircal activity of the isolated ventral nerve cords was monitored by a suction electrode (FLORY and 1{xn:BFi, 1966), amplified with a Grass 7 P3 B preamp, and recorded on magnetic tape with a Hewlett Packard 3960A instrumentation tape recorder . Nerve spike frequency was calculated by passing the amplified signals through an Ortec 730 single channel analyzer, which excluded all signals below 33 pV. Pulses from the Ortec were then counted by a Beckman 4325 LR counter and converted to spike frequency. Whole venom applications were made by draining the fluid level of the saline bath and replenishing with saline containing venom or by adding venom to the existing saline bath with a microcapillary pipette. Additionally, gel filtration fractions from the same columns which were used to establish the lethal factor elution were tested for neuroactivity. These fractions were tested in both ascending and descending order to prevent any bias due to potentiating or desensitizing effects of exposure to previous fractions. Saline irrigation was continued between column fraction applications until the control level of endogenous activity returned, and each cord was treated with all column fractions unless wntrol level was not attained by saline irrigation . RESULTS AND DISCUSSION

The LD 60 of L. reclusa venom in house flies was 026 ltg per g making it 18-fold more toxic to house flies than to mice (LD6o 4~6 p.g per g ; GExErr et al., 1975). House flies injected with sub - LD6u doses developed uncoordinated leg movements and lost wing movements within 1 hr. Within 4 hr, total paralysis, frequently accompanied by excessive salivation, marked the final stages of toxicosis. House flies injected with doses above the LDSO were often knocked down within 5 min and died within 1 hr. Because house flies are extremely sensitive, ng quantities of L. reclusa venom are sufficient for investigations of the properties of the venom responsible for its lethal activity . Upon electrical stimulation, adult female spiders normally yield about 701íg of venom protein. Thus, the venom from a single spider is enough to kill 50 ~ of 13,460 injected house flies. Trypsin digestion for 1 hr at 32°C completely eliminated the lethal activity of whole venom (Table 1), indicating that peptide bonds are essential to the venom's toxicity. When whole venom was heated to 90°C for 1 hr, the Ln bu increased from 026 !ag per g to 036 Ftg per g (Table 1) indicating a loss of approximately 25 ~ of the venom's toxicity . The toxicity of L. reclusa venom for insects, therefore, cannot be attributed to one heat stable compound . Whole venom was separated into three 280 nm absorbing peaks by Sephadex G-50 gel filtration (Fig. 1). Peak 1 contained venom components excluded by the gel pore size. Peaks 2 and 3 were those components retained by the gel, indicating that they were less than 10,000 daltons. The absorbance spectra between 310 and 240 nm of the three separated peaks were also scanned. The 280/260 nm ratio of peak 1 was 224. This ratio was 179 in peak 2 (within the range of polypeptides which have only amino acid residues) and 067 in

Loxosceles reclusa Venom Neurotoxicity TAHLB

1.

349

THB EFFECTS OF HEAT TREATMENT AND TRYPSIN DIOFSrION UPON THE LErHAL ACTIVITY OF WHOLE VENOM AND ßEL FILTRATION FRACTIONS IN Musca domestics

1 hr

Sample Whole venom Whole venom Whole venom HMW fraction+ HMW fraction HMW fraction LMW fractionj' LMW fraction LMW fraction

treatment

L. reclusa

Lethality

4°C

LD,p

90°C

LD

= 026 Pg/g§ = 0~361Ig/g~~

No mortality~j 75~ mortality~j No mortality~j No mortality~j 90% mortality~j 95~ mortalityQ No mortality~j

Trypsin digestion$ 4°C

90°C

Trypsin digestion 4 °C

90 °C

Trypsin digestion

+HMW fraction = peak 1 fraction K 029 (see Fig. l) (066 ul of final gel filtration dilution) . tLMW fraction = peak 2 fraction K 080 (see Fig. l) (066 l11 of Seal gel filtration dilution) . $Trypsin digestion = 1 hr incubation at 32°C with 0~1 mglml trypsin in pH 72 buffer . §Three trials-450 flies. ~jTwo trials-200 flies. ~jT'wo trials-100 flies.

peak 3 (closer to the expected ratio for nucleotides) (WHrra et al., 1973). L. reclusa venom has previously been separated into one low and one high mol. wt peak by Sephadex G-25 gel filtration (GE1tEN et al., 1975). The low mol. wt fraction (less than 5000) was reported to contain at least two ninhydrin positive components and multiple nucleotides when further separated by ion exchange and thin-layer chromatography.

0.11

I

~I~Unllu na .L050~s/ul/mq aqulv.

O N

40

ó



O n O

d AS u c O a

ó N a

20

a

_ _ 0 .2

0 .4

0 .6

_ 0 .6

_ K a~

_ _ I .0

_ 1 .2

1 .4

1 .6

L8

FIG. 1 . LLV. ABSORPTION SCAN OF SEPHADEX G-Stl GEL FILTRATION OF L. reclusa vENOM . The lethal factors contained in peaks 1 and 2 are presented as the number Of LD's for Musca domestlca per l11 of each fraction adjusted to a one mg venom application .

Gel filtration fractions from three different columns were injected into house flies, and the number of Ln s o's contained in one ltl were expressed as the number per mg whole venom applied to the column (Table 2). Venom components in peak 1 and peak 2 produced above control mortality (Fig. 1). The high mol. wt lethal components in peak 1 lost their lethal activity during either heat treatment or trypsin digestion (Table 1). Injection of the high mol. wt fractions resulted in house fly knockdown rapidly followed by death

330 TABLE

L. D. FOIL, J. L. FRAZIER and B. R. NORMENT 2.

THE DISTRIBUTION OF THE HIGH AND LOW MOLECULAR WEIGHT LETHAL FRACTION FOR MUSCLZ CIOItt¢S11C0 IN A G-SO GEL PII.TRATION ELUTION OF L. r¢CilISli VENOM

Peak no . 1 1 2 2 2 2 2 2 3§

Ka,* 0'19 0'29 0'35-0'64 0'65-0'74 0"73-0'84 0'85-0'94 0'95-1 "04 l'03-1 "l4 1"13-1 "23

Replicationst 1 2 3 3 4 4 3 3 3

Mean no . LDb p~l mg equiv$ ~ S.E, 3'37 4'42 f 0'38 3'43 f l'72 T88 f 0"42 13'18 ~ 1"74 15'95 f 0 7"2 ~ l'67 3'97 f 0'48 3'04 f 0'03

Initial knockdown time (hr) 20 20 0'S 0'S 0'S 0'S 0'3 0'3 0"3

V~ - Vo~ Vt - Vo (~ Fig . l). tEach replicate represents an LD6p calculation of S dilutions with 23 Hies per dilution . $Mgequiv .-all LD 6p values were adjusted to mgproteinappliedto the column . §A possible tailing of peak 2. *Kav =

20-24 hr after injection (Table 2). Previously, two L. reclusa venom components, each approximately 34,000 mol. wt, have been demonstrated to be toxic to vertebrates (GER>rx et al., 1976). Due to their molecular size, these two toxins should be in peak 1 of the G-50 gel filtration . The low mol. wt lethal fractions associated with peak 2 did not lose toxicity following heat treatment for one hour at 90°C. These fractions, however, like the high mol. wt fractions, lost all lethal activity during trypsin digestion . The heat stability (similar to some scorpion neurotoxins) and rapid knockdown of the low mol. wt lethal factor (Table 3) suggested that the venom possessed neurotoxic properties . TABLE

3.

THB DISTRIBUTION OF L, r¢ciusa VENOM G-SO GEL FILTRATION FRACTIONS WHICH EVOKH ABOVE CONTROL LEVELS IN THE ENDOGENOUS ACTMTY OF ISOLATED P. al)i¢TfCQlia

Sample Pre-void or saline Peak It Peak 2§ Peak 3$ Peak 2 Ka, 0'55-0'64 Ke, 0'65-0'74 Ka, 0'73-0"84 Ko, 0"83-0'94 Kdo 0'93-1'04

Number Mean no . spikes per observations min f S.E! 19 80 ~ 22 l9 80 f 27 33 9120 f 1G63 23 93 ~ 28 ~ 6 9 6 6 8~

6218 14,140 17,210 7174 863

f 3392 f 3314 f 3120 f 1840 f 292

Range 3-382 0-467 102-32,093 0-630 481-21,681 800-32,026 8277-32,093 2386-13,193 102-2381

'Spike counts were adjusted to 0"5 mg column application and all spikes counted were greater than 33 uV . tK,~ 0'09-0'54. §K8o 0'53-1'04. $Ke, 1'05-1'40.

Application of whole venom to isolated P. americana ventral nerve cords evoked a dramatic increase in both amplitude and frequency of endogenous nervous activity (Fig. 2). Analysis of the frequency of nerve spikes larger than 33 uV revealed increases to 680-fold above control levels (Fig. 3). This increase was approximately six times greater than that caused by Latrodectus mactans venom in P. Americana ventral nerve cord preparations (N>nu et al., 1965). Endogenous activity was then blocked in 2-5 min by venom applica-

Loxosceles reclusa

35 1

Venom Neurotoxicity

~~~/~;h 7~~e

F " /,

FIO . T . A POLYGRAPH TRACE OF THE EFFECT OP lO FIg L. reclusa WHOLE VENOM ON THE ENDOGENOUS ACTIVITY OF THE VENTRAL NERVE CORD OF Periplanela an;erlCpna.

tions (Fig. 3) and addition of more whole venom did not evoke further stimulation ; however, the venom block was reversible by saline washing. Up to 70 min of saline irrigation was often required for recovery to control level. During the irrigation, rhythmic bursts of spikes often occurred (Fig. 4) with the rhythmicity lasting over 30 min. When maximum increase in frequency was plotted as a function of venom concentration the relationship shown in Fig . 5 was found. Thus, whole venom demonstrated both time and concentration dependent stimulation of endogenous activity in P. americana ventral nerve cords .

6 5 ~0

~OSug/ul ____CONTROL

4

X

~3 w N O_

\Z M W Y

V1 1

Z

1 2 3 MINUTES AFTER APPLICATION

vENOM oN P. americana VENTRAL NERVE coRD ENDOGENOUS ACTIVITY. Each point represents the number of spikes greater than 33 tIV during a ten second sampling period. The block of activity at 2-3 min was not affected by a second venom addition but was reversed by saline irrigation . FIG . 3 . THE ePFECr oP

0~5 kg/ul L . reclusa

3S2

L. D. FOIL, J. L. FRAZIER and B. R. NORMENT

FIG . 4. THE RHYTHMIC HURS'TING AGTMTY OBSERVED DURING SALINE IRRIGATION OF P . QIIlCrICgRR VENTRAL NERVE CORD$ PREVIOUSLY TRHATED WITH L . rECIUSü VENOM .

This activity was recorded after 35 min of saline irrigation . A, B and C are polygraph traces . D and E are oscilloscope records taken with a kymograph camera. (A) Time reference in S second intervals. (B) Integrated spike activity with a time constant of 0'2 sec. (C) Spike frequency without integration (pen frequency response prevents the true display of burst spike amplitude). (D) 25 Hertz signal for time reference. (E) Two expanded bursts of B and C revealing a single spike amplitude class.

When gel filtration fractions were tested on ventral nerve cords, peak 2 aliquots were the only ones which caused increased frequency of endogenous activity . Neuroactive fractions were found between Ke 060 and K, 1~0 with the maximum activity at 080. Frequency analysis, presented in Table 3, indicated that Kao 080 applications increased the endogenous

N XS U W N y

w 3 Y

ay

MG/ML VENOM FIG . S . THE EFFECT OF L . rCCiüSa WHOLE VENOM CONCENTRATION ON THE FREQUENCY OF SPIKES GREATER THAN 33 lIV RECORDED IN THE SPONTANEOUS ACTIVITY OF P. QIfIENCQ11a NERVE CORDS .

Each point represents a 10 sec sampling period at maximum stimulation of different cords. (Line fitted by linear regression R = 082.)

Loxosceles reclusa Venom Neurotoxicity

35 3

activity to 215 times that of control level. Additionally, an increased amplitude of the endogenous activity coincided with the increased frequency responses (Fig. ~. This increase in amplitude suggests that there may be either axonal changes in venom stimulated nerve cells or that additional classes of nerve cells are stimulated at high concentrations of the low mol. wt component. Neuroexcitatory effects of whole venom and gel filtration fractions were, like the low mol. wt lethal factor, heat stable and susceptible to trypsin digestion. When the low mol. wt lethal factor and the neuroactive elutions were compared, the two activities overlapped but did not coincide . GmaN et al. (1976) reported that L. reclusa venom contained two polypeptides of mol. wt less than 5000. Therefore, the differences in elution patterns of the neuroactive and low mol. wt lethal factors could be attributed to the elution of two venom components . Further biochemical separation should reveal whether the low mol. wt lethal factor and the neuroexcitatory factor are the same or two different polypeptides .

Fia. 6. OSCIU.OORAFHIC RECORDS OF L . reclusa SEPSADEX aEZ . FaTRATtoN FRACnoNS' sT~arvuTION OF THE ENDOGENOUS ACITVITY OF TFIE P. Q117erJCaIJa VENTRAL NERVE CORD . (A) The top trace is a SO Hertz signal for time reference and the insert on the right is a f ]00 pV calibration pulse both applicable to all traces . The lower trace is activity following a saline application (one min count-33 spikes) . (B) Fraction K 1~0 (one min count-1117 spikes) . (~ Fraction K,~ 0~6 (one min count-3980 spikes). (D) Fraction K 0~7 (one min count-13,190 spikes) . (E) Fraction K 0~8 (one min count-32,080 spikes) .

354

L. D. FOiL, J. L. FRA 7IF.R and B. R. NORMENT

In addition to its excitatory effects upon endogenous activity, L. reclusa venom also blocks transmission in cereal nerve-giant fiber synapses in the sixth abdominal ganglion of P. americana; previously, these have been shown to be cholinergic (SHANKLANI) et a1.,1969). This study represents the first report of neuroactive components in L. reclusa venom, Since this venom has been considered primarily a cytotoxin in vertebrates, some of the subtleties ofthe pathology associated with envenomation could have previously been overlooked. For instance, changes in the rate of heartbeat (which is partly under neuronal control) have been recorded in patients bitten by L. reclusa (NANCS, 1961). These changes in the rate of heartbeat were reported as secondary complications in patients suffering from systemic manifestations following envenomation . Additionally, the spike potentials evoked by L. reclusa venom represent changes in nerve cell membrane permeability to selected canons, but these changes in permeability are not necessarily confined to nerve cells. A shift in ionic composition of red blood cells could be involved in intravascular hemolysis, a fatal syndrome in victims of loxoscelism. Acknowledgements-Thanks to LnxxY HAa~zn for technical assistance and Dr. Howaxo C1iAMBfiR$ and Dr. Huax KEecex for review of the manuscript . A special thanks to Dr. Dnx Sxwxxi nxn for supply of equipment and advice concerning experimentation and manuscript preparation. REFERENCES AHao~rr, W. S. (1923) A method for computing the effectiveness of an insecticide. J. Fcon . Entomol. ]8, 265. ANDERSON, P. C. (1973) What's new in Ioxoscelism7 Missouri Med.70, 711 . ArxtNS, J. A., WiNCO, C. W. and SoDEHtaN, W. A. (1938) Necrotic arachnidism . Am . J. Trop . Med. Hyg. 7, 165 . DENNY, W. F., Du tnse, C. J. and MDRaeN, P. N. (1964) Hemotoxic effect of Loxosceles reclusa venom: in vrvo and in vitro studies. J. Lab. clip. Med. 64 : 291 . Drr .i.~w~ , C. J., JerrsEN, G. T., HoNEVturr, W. M. and HAYDEN, C. R. (1963) The gangrenous bite of the brown recluse spider in Arkansas. J. Arkansas Med. Soc. 60, 91 . EtaERT, K. D., Ross, M. A., C.~sE.r., B. J. and BARNE'IT, J. T. (1974) Immunological studies of brown recluse spider venom. IrIJect. Immun. 10, 1412 . Esruxi, F. M . and NORMErrr, B. R. (1976) Physiological action of Loxosceles reclusa (G & M) venom no insect larvae . Toxkon 14, 7. FLORY, E. and KRIE9EL, M. E. (1966) A new suction electrode system . Comp . Brochem. Physiol. l8, 173 . GExEN, C. R., CanN, T. K., HowEia ., D. E. and ODELL, G. V. (1975) Partial characterization of the low molecular weight fractions of the extract of the venom apparatus of the brown recluse spider and its hemolymph. Toxkon 13, 233. GEREN, C. R., Cxnrr, T. K., HowEi t, D. E. and ODEtL, G. V. (1976) Isolation and characterization of toxins from brown recluse venom (Laxosceles reclusa) . Archs Brochem. Brophys. 174, 90 . GROranus, R. H. (1968) The toxicity of spider venoms . Tech . Monogr . U.S. Navy Dis. Vector Ctr. l, 63 . LOWRY, O. H., RosEeROUax, N. J., FnRR, A. L. and RANDELL, R. J. (1931) Protein measurement with the Folin phenol reagent. J. biol. Chem. ]93, 265. NaNtE, W. E. (1961) Hemolytic anemia of necrotic arachnidism . Am. J. Med.31, 801 . NERr,L .,BErrnvr, S. and FR~Nx, M . (1963) The effect of Latrodectus madams tredecrrr~guttatus venom of the endogenous activity of Periplamta americana nerve cord . Toxkon 3, 93 . NoRMENr, B. R, and SMrrx, O. E. (1968) EB~ect of Loxosceles reclusa Gertsch and Mulaik venom against hemocytes of Acheta domesticus (Linnaeus) . Toxrcon 6, 141 . NoRMENr, B. R. and VrNSON, S. B. (1969) Effect of Loxosceles reclusa Gertsch and Mulaik venom on Heltothrs vrrescens (F .) larvae . Toxkon 7, 99. ROEDER, K. D. (1948) The effect of potassium and calcium on the nervous system of the cockroach Periplaruta Americana. J. cell comp . Physiol. 31, 327. ROEDER, K. D. and ROBDER, S. (1939) Electrical activity in the isolated nerve cord of the cockroach . J. cell comp . Physiol. 14, 1 . Sxwxx~rrD, D. L., Rose, J. A. and DoxNiNCER, C. (1969) The cholinergic nature of the cereal nerve-giant fiber synapse in the sixth abdominal ganglion of the American cockroach, Periplaneta amerkana (I-.). J. Neurobrol. 2, 247. $Ail'1Ti, C. W. and Mitxs, D. W. (1968) A rnmparative study of the venom and other components of three species of Loxosceles . Am. J. trop. Med. Hyg. 17, 631. Wz-nrE, A., HnNDr.ER, P. and SIKITH, E. L. (1973) Principles of Biochemistry, p. 133. New York : McGrawHill .