Ontogenetic changes in Phoneutria nigriventer (Araneae, Ctenidae) spider venom

Toxicon 44 (2004) 635–640 www.elsevier.com/locate/toxicon

Ontogenetic changes in Phoneutria nigriventer (Araneae, Ctenidae) spider venom Volker Herziga, Richard John Wardb, Wagner Ferreira dos Santosc,* a

Department of Neuropharmacology, Zoological Institute, Faculty of Biology, University of Tu¨bingen, Germany b Departamento de Quimica, FFCLRP-USP, Ribeira˜o Preto, Brazil c Laboratorio de Neurobiologia e Pec¸onhas, Departamento de Biologia, FFCLRP-USP, Ribeira˜o Preto, Av. Bandeirantes, 3900, 140140-901, Brazil Received 21 January 2004; revised 28 April 2004; accepted 26 July 2004 Available online 30 September 2004

Abstract Venom-yield and composition of differently sized individuals of the medically most important Brazilian spider Phoneutria nigriventer (Keyserling, 1891) was analysed. During growth the venom-mass increases according to a fourth order function of the prosoma size, which mainly reflects an increase of the venom gland volume. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed increasing percentages of proteins %17 kDa from 4.1% in the smallest analysed spiders (2–3 monthsold) to 79.1% in adult female venom. Additionally, high-pressure liquid-chromatography showed an increase of a single (‘main’) peak from 4.6 to 64.9%, while the overall number of other major-peaks decreased. Venom from young instars completely lacked lethality in mice up to a dose of 3.28 mg/kg i.v. as compared to a LD50 of 0.63 mg/kg for adult female or 1.57 mg/kg for adult male venom that we reported previously. In conclusion, ontogenetic changes in venom proteincomposition of growing P. nigriventer are suggested to produce increasing lethality in vertebrates. q 2004 Elsevier Ltd. All rights reserved. Keywords: Phoneutria nigriventer; Ontogenetic changes; Venom yield; Protein composition; Vertebrate-lethality (LD50)

1. Introduction In Brazil the genus Phoneutria Perty, 1833 (family: Ctenidae) is responsible for about 60% of all spiderbiteaccidents (Lucas, 1988). According to the latest revision (Simo´ and Brescovit, 2001), only five valid species of Phoneutria have been described (fera, nigriventer, boliviensis, reidyi, bahiensis) and in Brazil P. nigriventer (Keyserling, 1891) accounts for the majority of accidents (Bu¨cherl, 1969). The length (prosomaCopisthosoma) of adult P. nigriventer female can reach 42 mm (Vital-Brazil and Vellard, 1925). The mating season is from April to July

* Corresponding author. Tel.: C55 16 6023657; fax: C55 16 6331758. E-mail address: [email protected] (W.F. dos Santos). 0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2004.07.020

(Ramos et al., 1998) and females with egg-sacs appear at the middle of July. After copulation females build up to four white oval egg-sacs, and from the time the young spiders leave the egg-sac, they are able to move and capture prey. Because spiders have an exoskeleton they have to shed it at regular intervals (moult) during growth. Depending on the availability of food, young Phoneutria moult 5–10 times in the first, 3–7 times in the second and 1–3 times in the third year (Lucas, 1969, 1988; Bu¨cherl, 1969). They become adult in the third year and the maximum age that can be reached is 6 years (Bu¨cherl, 1969). More than 25 neurotoxic peptides and proteins in the range from 2–15 kDa have been purified from the venom of P. nigriventer, and to date the complete amino acid sequence of 15 of these polypeptides is known (Diniz et al., 1993; Cordeiro et al., 1995). For intravenous (i.v.) injections in mice, the median lethal dose (LD50)

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was determined as 0.33 mg/kg mice (Bu¨cherl, 1956; Schenberg and Pereira-Lima, 1966). The venom causes dyspnea, prostration, paralysis of hind legs and tail and finally death by paralysis of the respiratory system. However, individual venom quantity varies considerably, ranging from 0.3–8.0 mg dried weight per spider (Lucas, 1988). Furthermore, P. nigriventer venom quantity can vary seasonally in summer (with 1.8 mg average) and winter (2.5 mg) (Schenberg and Pereira-Lima, 1966) and intersexual differences can also account for variations (Herzig et al., 2002). Variations in venom quantity, caused by different factors, have also been documented in other spider species. In electrically extracted venom from Loxosceles reclusa, smaller spiders yielded less venom than larger ones (Morgan, 1969). Kuhn-Nentwig and Nentwig (1997) reported that venom production in adult Cupiennius salei (Ctenidae) can depend on age, gender, degree of hunger and probably on the reproductive status. Periods of high or low temperature may also influence the venom yield (Jong et al., 1979). Therefore, variations in the venom quantity can be an important aspect regarding the danger of a spiderbite. Individual P. nigriventer with high venom quantities may be dangerous for physically weak or diseased adult humans. Not only the venom quantity, but also its composition, can show intraspecific variations. In the brown recluse spider L. intermedia, the ontogenetic development of a single 35 kDa protein (denominated F35) was demonstrated (De Andrade et al., 1999). F35 has dermonecrotic and haemolytic activity and is responsible for the main toxic effects of the venom. Eggs and spiderlings of the first and second instar contained no F35. It was first detected in instars of the third stage and its amount increased until adulthood. The authors conclude that F35 is an important factor for the spiders’ survival, because it precisely occurred at the point of development when the spider is able to hunt, and defend itself. For the ctenid spider Cupiennius salei, Malli et al. (1993) showed that the insect-lethality is highest in the sixth instar (the first instar stage that was tested) and in adult spiders (without intersexual differences). The present study was designed to determine if and how ontogenetic development affects venom yield or venom composition in the ctenid spider P. nigriventer. Furthermore, the effect of such developmental changes in venom composition on the lethality was examined in mice.

2. Materials and methods 2.1. Spiders The spiders for the present study were all collected between March and August 1999 on the campus of the University of Sa˜o Paulo (USP) in Ribeira˜o Preto (Brazil). The spiders were identified as P. nigriventer by Antonio Brescovit (Butantan Institute, Sa˜o Paulo, Brazil).

After capturing the spiders were maintained in the laboratory for a maximum of 8 months, where they were fed on crickets and cockroaches (see also Herzig et al., 2002). During this period, the spiders were allowed to reproduce in order to have enough young spiders for venom extraction. However, venom-extraction from neonate spiders was not possible therefore they were kept until they reached a size that allowed venom-extraction (after 2–3 months). The size and (if possible) sex of all spiders was determined before venom extraction and the length of the prosoma was measured. Since P. nigriventer requires 3 years from leaving the egg-sac to adulthood and since all spiders were collected only over a period of 6 months, a correlation of the spiders sizes with the instar-stages (as done by Malli et al., 1993) was not possible. Thus, all spiders were organized into seven size-classes according to their prosoma-length, i.e. size-class 1 (adult females); size-class 2 (sub-adult females) and size-classes 3–7 (instars in order of decreasing size). The size-classes 1–3 contain only female P. nigriventer, whereas the size-classes 4–7 include spiders of both sexes. 2.2. Method of venom extraction and treatment The spiders were anesthetized with CO2 and venom was extracted by applying a few times electric shocks of 13–18 V (depending on the size of the spider) that lasted about 1 s each. To avoid contamination with saliva, venom was only collected from the tips of the fangs. Venom was extracted about once per month over a period of 8 months. Other procedures are described in detail by Herzig et al. (2002). 2.3. Gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a running gel with a linear gradient of 5–20% acrylamide at a voltage of 120 V and a current of 20 mA (Laemmli, 1970). Venom samples were dissolved in 20 ml of water (milli-Q) and 5 ml sample buffer (25% b-mercaptoethanol and 75% of 0.313 M Tris–HCl; 10% glycerol and 0.001% bromphenol blue, pH 6.8) was added. Samples were boiled for 2 min, vortexed for 30 s and applied to the gel. After electrophoresis, gels were stained in a 0.25% Coomassie Brilliant Blue R250 solution and destained by using a solution containing 10% methanol and 10% acetic acid. The molecular weights of the venom proteins were estimated by using known molecular weight standards (Sigma 6H standard). The gel was scanned and analysed using the program Easy Win 32 (Herolab). The total intensity of the protein bands in each size-class was set to 100% and the intensities of the individual protein bands were normalized to this value. 2.4. Reverse-phase liquid chromatography High-pressure liquid-chromatography (HPLC) was performed using a dual pump solvent delivery system

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(LC-10Ai, Shimadzu, Japan), coupled to a fraction collector (FRC-10A, Shimadzu, Japan). The flow rate was 1 ml/min and a linear gradient (2.5%/min) of solvent A (0.1% aqueous solution of trifluoracetic acid (TFA, Nuclear)) and solvent B [60% acetonitrile (chrom HR HPLC, Mallinckroft)C40% solution A] was used according to Rego et al. (1996). The lyophilized venom samples were dissolved in 120 ml water and applied to a reversephase C-18 column (Waters Spherisorb ODS2 5 mm, 250!4.6 mm), previously equilibrated with a 0.1% aqueous TFA. The absorbance of the eluate was monitored continuously at 220 nm. Based on the total peak area per run, a percentage value for each peak area was calculated (using the program CLASS-LC10, version 1.63, Shimadzu) and compared with peaks from different size-classes that showed the same retention times. 2.5. Lethality test in mice The lethality test in mice was performed to determine any potential dangers for vertebrates caused by venom from young P. nigriventer. Assuming varying proportions of different toxins in different size-classes, a bioassay using invertebrates could therefore not be used, since different toxins in the venom are responsible for effects in vertebrates and invertebrates. Venom from size-class 6 (about 6–7 month-old spiders) was dissolved in 100 ml of 0.9% NaCl solution and injected into the tail-vein (i.v.) of male Swiss white mice. The mice were supplied from the main animal house, USP Ribeira˜o Preto, Sa˜o Paulo, Brazil and had a body-weight of 22–26 g. Three increasing venom concentrations were injected into 5 mice at each concentration and 15 mice were used in total. All animal experiments comply with the guidelines of the Brazilian Society of Neurosciences that follow the guidelines for animal care of the Committee on Care and Use of Laboratory Animal Resources, National Research Council (USA).

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3. Results The spiders of size-class 7 (Table 1) were the smallest spiders that yielded sufficient venom for analysis, however the quantity was to low for weight-determination. Instead, the venom was used for a single SDS-PAGE run and no HPLC analysis was performed. The next size-class analysed (size-class 6) was approximately 6–7 months old and yielded enough venom for both HPLC and SDS-PAGE analysis. Data for size-classes 1–3 have already been published (Herzig et al., 2002) and are included for reasons of comparison. Table 1 further shows an increase of the venom-mass yielded per spider during growth, with the exception that size-class 6 yielded more venom than sizeclass 5. The dried venom yield (y, in mg) increased exponentially during the spider’s growth according to the equation yZ0.01 * x4.21, where x is the prosoma length in millimeter. This equation shows a r2 of 0.998 and was iterated by using the ‘solver’ tool in Microsoft Excel 2002. According to Fig. 1, each of the size-classes 1–5 showed nine protein bands of the same molecular weights or weightranges (i.e. 7–12, 13, 16, 19, 20, 22–27, 32, 35 and 41–47 kDa), however their percentages varied among different size-classes. In size-class 6, 10 bands were detected, which is due to an additional protein of 17 kDa. The venom of the youngest spiders (size-class 7) consisted mainly (82%) of a 45 kDa protein and very low quantities of some of the proteins that have been detected in size-classes 1–6. By delineating two molecular-weight classes (Table 1), a general variation in the venom protein composition was revealed. The quantity of proteins with more than 17 kDa molecular weight decreased during the spiders’ growth from about 96% in size-class 7 to about 21% in adult females. On the other hand, the quantity of proteins %17 kDa increased from 4% in size-class 7 to 79% in size-class 1. By HPLC, a total number of 31 different peaks was detected in the venoms of all size-classes. The number of peaks varied between 25 in size-class 2 and 20 in sizeclass 4 (Table 2). A decrease in the number of ‘major-peaks’

Table 1 Ontogenetic changes in prosoma-length, venom yield and venom protein sizes: venom data of different size-classes of Phoneutria nigriventer are shown by decreasing size Size-class

Sex of spiders

Length of prosoma (mm)GSD

Number of spiders

Venom-yield per spider (mg)

Protein content (%) with MW O17 kDa

Protein content (%) with MW %17 kDa

1 2 3 4 5 6 7

Adult female Subadult female Instar female Instars, sex not det.

15.8G0.6 13.5G0.6 11.5G0.7 9.5G0.6 7.5G0.5 5.5G1.7 1.9G0.4

21 17 16 7 33 36 9

1079 548 296 117 36 50 not det.

20.9 not det. 21.6 29.9 37.2 44.1 95.9

79.1 not det. 78.4 70.1 62.8 55.9 4.1

The data of size-classes 1–3 (in italics), have already been published (Herzig et al., 2002) and are only provided for ease of comparison. SDS-PAGE revealed increasing percentages of proteins %17 kDa and decreasing percentages of proteins O17 kDa during the spiders growth (MW, molecular weight; not det., not determined).

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of size-classes 2–5 are not presented, since they are very similar to the chromatogram of size-class 1. The venom of small spiders (size-class 6) showed no lethality in Swiss white mice (i.e. no mice died) even after injection of the highest dose (3.28 mg/kg) and also no signs of toxicity (e.g. disturbance of respiration or paralysis of the extremities) were observed.

4. Discussion The dried venom yield (y) is a fourth order function (x4) of the prosoma-length (x). A third order function would have been easily explained by the increase of the venom gland volume (and therefore its venom content) within three dimensions, whereas the length of the prosoma increases only in one dimension. The observation of a fourth order function is difficult to interpret but it is likely that the increase in the venom gland volume accounts for the main increase in the venom yield. However, other (unknown) factors also play an additional role in this phenomenon (e.g. state of nutrition, time since the last feeding or ecdysis, etc.). Interestingly, the observed exponential increase in venom yield is consistent with the results obtained by Malli et al. (1993) on electrically extracted venom from instars of Cupiennius salei. In summary, the observed increase of the dried venom yield mainly reflects the increase of the venom gland volume during growth of the spiders. The observed increase in the relative amounts of proteins %17 kDa (up to 79%) during the development of the spiders could indicate that in young spiders the venom gland and especially the venom secreting cells are limited in their capacity to synthesize these proteins. Another possible explanation could be that young spiders already have the ability to synthesize these proteins, but are limited in their ability to posttranslationally process the prepropeptide precursors of the mature toxins. Similar changes in venom composition during ontogenetic development have been detected by HPLC, where an increase of the ‘main’-peak

Fig. 1. SDS-PAGE gel. Coomassie-blue stained SDS-PAGE gel of venom from six different size-classes (for details about the sizeclasses see Table 1) of Phoneutria nigriventer (with exception of size-class 2 that was not analysed). The data of size-classes 1 and 3 were already published (comply with venom pools 3 and 4 in Herzig et al., 2002) and are only provided for ease of comparison. The dried venom quantities applied to the gel are indicated below. The standard marker proteins (‘M’Z6H, Sigma) and the two molecular weight classes (! or R17 kDa, corresponding to Table 1) are also indicated.

(i.e. peaks with more than 5% of the total peak area) was observed during the spiders growth (Table 2). Venom of small spiders from size-class 6 showed nine major-peaks, while only two major-peaks were observed in venom of sizeclass 1. A comparison of HPLC data from size-class 1 and size-class 6 venoms (Fig. 2) showed various differences in the venom composition of both size-classes, but a clear tendency towards an increase of a single peak (eluting at 19.2 min). In venom of size-class 1, this peak (the ‘main peak’) accounted for about 65% the total protein content, while in size-class 6 the content of this peak was only 4.6%. The chromatograms

Table 2 Ontogenetic changes in P. nigriventer venom protein composition: percentage content of ‘major’ protein peaks (peaks with more than 5% peak area) and their retention times according to HPLC of all analysed size-classes HPLC major protein peaks Retention time (min) Size-class 1 2 3 4 5 6

2.3

2.8

3.0

3.2

3.5

3.7

4.3

8.1

11.1

11.5

16.9

19.2 ’main’ peak

25.6

29.3

Total number of peaks

% of total protein content 5.6 6.1

8.5 5.4 5.8

8.0 11.5

8.3

7.5

7.1

6.1

6.5

8.9

6.2 6.2

5.5

64.9 56.3 65.4 38.5 43.1 4.6

12.0 6.4 10.3

8.2

21 25 22 20 23 23

The total number of all peaks (incl. peaks !5% peak area) is also presented. The percentage of the ‘main’ peak increased with the size of the spiders. Venom of size-class 7 was not analysed due to a lack of sufficient venom quantity.

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Fig. 2. HPLC results. HPLC venom chromatograms of size-class 1 (adult females, 700 mg venom applied) compared to size-class 6 (instars, about 6–7 months-old, 741 mg venom applied). The absorbance of the eluates was monitored at 220 nm for 30 min. The peak showing the largest percental variation during ontogenetic development was termed as ‘main-peak’. The chromatograms of size-classes 2–5 are not presented, since they are very similar to the chromatogram of size-class 1. Due to the low quantity, venom of size-class 7 was not analysed by HPLC.

(up to 65%) was observed. Thus, it would be of interest to further analyse this ‘main-peak’ to confirm if it contains proteins %17 kDa. However, only based on the results obtained in the present study and due to different physicochemical bases of the separation techniques, it is not possible to establish a correlation of HPLC and SDS-PAGE data. This could be achieved in future experiments, using LC-MS techniques. The only other report considering the distribution of a specific toxin during ontogenetic development showed that even instars of the third stage of Loxosceles intermedia (exactly the stage when young spiders leave their egg-sac) contain F35, which is responsible for the main toxic effects of the venom (De Andrade et al., 1999). However, no report about the distribution of other venom components of L. intermedia was given. In line with these findings, quantitative but not qualitative differences in the venom components have been reported from adults of both sexes and immature juveniles of the theraphosid spider Poecilotheria rufilata (Escoubas et al., 2002). Another study failed to correlate the lethality for insects with the total protein content in different growth-stages of the ctenid spider C. salei (Malli et al., 1993). Thus it remains unclear whether venom secreting cells generally undergo ontogenetic development in young spiders, if this phenomenon can only be observed in some spider species (e.g. P. nigriventer), or if it’s restricted to the synthesis of some toxins. Based on the observed increase in the proportion of proteins %17 kDa during growth, it could be assumed that

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venom from larger specimens of P. nigriventer has a higher lethality in mice, since toxins with lethal effects in vertebrates (PhTx 1, PhTx 2–5 and PhTx 2–6) also show molecular weights in that range (Diniz et al., 1990; Cordeiro et al., 1992). This hypothesis is strongly supported by the lack of lethality in venom from 6–7 month-old spiders (up to 3.28 mg/kg), whereas adult spiders showed lethal effects (LD50 of 0.63 mg/kg for female venom and 1.57 mg/kg for male venom according to Herzig et al., 2002). It furthermore strengthens the assumption that venom-secreting cells undergo ontogenetic development, showing full capacity for toxin-synthesis only in larger instars. On the other hand, the insect-lethality of venom from several stages of C. salei was highest in young instars (sixth instar) and in adults, suggesting a high content of invertebrate-specific toxins especially in young spiders (Malli et al., 1993). The present findings do not exclude the possibility that venom from young P. nigriventer also has a higher invertebrate-lethality compared to venom from larger instars. Biologically it would even make sense that young spiders show a higher invertebrate-lethality, since this forms their main prey-type. Furthermore, our study was not designed to identify specific proteins and due to the lack of sufficient venom quantity especially from the smallest instars, the isolation and characterisation of single venom proteins was not possible. Therefore more detailed studies are necessary to confirm our suggestion that the lack of lethality of venom from instars of P. nigriventer is due to the absence of exactly these known vertebrate-specific toxins (see Diniz et al., 1990; Cordeiro et al., 1992) and to account for alterations in the distribution of invertebrate-specific toxins within differently sized spiders. The ‘male specific’ venom components, that have been detected in our previous study of intersexual differences in P. nigriventer venom (Herzig et al., 2002), were completely absent in size-classes 1–6. This is interesting, since the sizeclasses 4–6 include both male and female spiders. Therefore we suggest that these male specific components develop fairly late, i.e. after their last moult. The function of these components is unknown, yet the late development could indicate that it may be for sexual purposes. Because adult males roam around in search of females, another possible explanation for these components may be defensive purposes, however, the lower vertebrate lethality of male P. nigriventer venom (Herzig et al., 2002) argues against this hypothesis. A partial loss of the protein-producing or processing capacity in male P. nigriventer is therefore more likely. This explanation is supported by the fact that traces of some ‘male specific’ components were detected by SDS-PAGE (2.9% of the 66 kDa protein and 0.7% of the 80 kDa protein) in venom from the smallest instars (2–3 months-old, size-class 7) that are presumably also limited in their protein-producing or processing capacity. A partial loss of toxicity of male P. nigriventer venom fits to their reduced need of venom to overcome potential prey, because they rarely eat and mainly roam in search of females.

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Acknowledgements Volker Herzig was the recipient of a DAAD grant within the exchange program University Tu¨bingen (Germany)/University of Sa˜o Paulo (USP), campus Ribeira˜o Preto (Brazil). We wish to thank Profs. Norberto Cysne Coimbra, Roy Larson, Antunes-Rodriguez, Lewis Greene, Jarbas Georgini and Ronaldo Zucchi (USP, Ribeira˜o PretoSP) for use of their laboratories; Renato Guizzo (USP, Ribeira˜o Preto-SP) for assisting in the mouse-assay; Johannes Mu¨ller and Birgitt Scho¨nfisch (University of Tu¨bingen, Germany) for statistical advice and Antonio Brescovit (Butantan Institute, Brazil) for identification of the spiders. Furthermore, we wish to thank all the anonymous reviewers for their helpful and constructive comments on the manuscript.

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