raia

1. Toxicon 50 (2007) 676–687
Comparative study on extracts from the tissue covering the
stingers of freshwater (Potamotrygon falkneri) and marine
(Dasyatis guttata) stingrays
Katia C. Barbaroa,Ã, Marcela S. Liraa
, Marı´lia B. Maltaa
,
Sabrina L. Soaresa
, Domingos Garrone Netob
, Joa˜ o L.C. Cardosoc
,
Marcelo L. Santorod
, Vidal Haddad Juniorc,e
a
Laboratory of Immunopathology, Butantan Institute, Av. Vital Brazil 1500, 05503-900 Sa˜o Paulo, SP, Brazil
b
Department of Zoology, UNESP, 18618-000 Botucatu, SP, Brazil
c
Hospital Vital Brazil, Butantan Institute, Av. Vital Brazil 1500, 05503-900 Sa˜o Paulo, SP, Brazil
d
Laboratory of Pathophysiology, Butantan Institute, Av. Vital Brazil 1500, 05503-900 Sa˜o Paulo, SP, Brazil
e
Dermatology and Radiotherapy, UNESP, 18618-000 Botucatu, SP, Brazil
Received 3 April 2007; received in revised form 1 June 2007; accepted 4 June 2007
Available online 22 June 2007
Abstract
Stingrays are elasmobranchs found along the seacoast and in some rivers of Brazil. Pain is the most conspicuous
symptom observed in patients wounded by the bilaterally retroserrate stingers located in the tail, which are covered by
glandular and integument tissues. In addition, cutaneous necrosis is commonly observed in injuries caused by freshwater
stingrays. The aim of this work was to characterize and compare certain properties of tissue extracts obtained from the
glandular tissues covering the stinger apparatus of Potamotrygon falkneri and Dasyatis guttata stingrays. By sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), tissue extracts have similar bands above 80 kDa, but
most differences were observed below this molecular mass. Lethal, dermonecrotic and myotoxic activities were detected
only in P. falkneri tissue extract. Edematogenic activity was similar and dose dependent in both tissue extracts. Nociceptive
activity was verified in both tissue extracts, but P. falkneri presented a two-fold higher activity than D. guttata tissue
extract. No direct hemolysis, phospholipase A2 and coagulant activities were observed in both tissue extracts. Antigenic
cross-reactivity was noticed by ELISA and Western blotting, using antisera raised in rabbits. Species-specific sera reacted
with several components of both tissue extracts, noticeably above 22 kDa. Both tissue extracts presented gelatinolytic,
caseinolytic and fibrinogenolytic activities, which were not caused by the action of metalloproteinases. Hyaluronidase
activity was detected only in P. falkneri tissue extract. Our experimental observations suggest that P. falkneri tissue extract
is more toxic than D. guttata tissue extract. These results may explain why injuries caused by freshwater stingrays are more
severe in human accidents.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Stingrays; Potamotrygon; Dasyatis; Venom; Toxin
ARTICLE IN PRESS
www.elsevier.com/locate/toxicon
0041-0101/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2007.06.002
ÃCorresponding author. Tel.: +55 11 37267222x2278/2134; fax: +55 11 37261505.
E-mail addresses: kbarbaro@butantan.gov.br, kbarbaro@usp.br (K.C. Barbaro).

2. 1. Introduction
In Brazil, freshwater stingrays are very common
in northern, central-western and southeastern rivers,
and marine stingrays are distributed throughout
Atlantic Ocean coast. The family Potamotrygonidae
comprises the only group of rays totally restricted to
fluvial systems (Rosa, 1985; Compagno and Cook,
1995), occurring only in the major river basins of
South America; they are distributed in three genera:
the monotypic genera Plesiotrygon (Plesiotrygon
iwamae), Paratrygon (Paratrygon aiereba), and the
Potamotrygon genus, which has 16–18 valid species
(Rosa, 1985; Charvet-Almeida et al., 2002; Carval-
ho et al., 2003; Garrone Neto et al., 2007). Stingrays
of the Dasyatidae family are marine, encompassing
six genera and about 50 species that are widely
distributed in tropical and temperate waters of
Australia, Asia, Africa and America (Taniuchi,
1979; Roberts and Karnasuta, 1987; Taniuchi
et al., 1991). Similar to other elasmobranchs, certain
species of dasyatids are euryhaline, invade rivers
and live in estuaries for extended periods of time
(Thorson, 1983; Thorson et al., 1983). On account
of that, some authors, based on physiological/
ecological findings, suggest that potamotrygonids
might be nested within dasyatids in a unique
family—Dasyatidae, with two subfamilies: Dasya-
tinae and Potamotrygoninae (Nelson, 1994; Nishi-
da, 1990). However, systematic studies with
parasites suggested that potamotrygonids are most
closely related to Pacific coast members of the genus
Urolophus (Urolophidae) than dasiatids (Brooks
et al., 1981; Lovejoy, 1996). Taxonomists consid-
ered urolophids to be the sister group to the
neotropical freshwater rays, and used them as the
outgroup to assess intra-potamotrygonid relation-
ships (Rosa, 1985; Rosa et al., 1987).
Stingrays are commonly found in superficial
waters, where they lay on the sandy or muddy
bottom, or remain partially hidden beneath it
(Halstead, 1966; Halstead, 1970). These animals
have one or more retroserrated stingers on the tail,
covered by integumentary and glandular tissues,
where the venom is produced (Charvet-Almeida
et al., 2002; Carvalho et al., 2003; Haddad et al.,
2004). Such habits increase the possibility of
accidents, which occur when humans accidentally
step on the dorsal part of stingrays, and animals
whip the tail to the stimulated site to defend
themselves. Almost all patients present pain at the
injury site, followed by edema and erythema. Skin
necrosis and ulcers can occur, mainly if injuries are
caused by freshwater stingrays, and they may take
up to 3 months to heal (Haddad, 2000; Haddad
et al., 2004). The most inflicted anatomic region is
the lower limbs (Haddad et al., 2004; Brisset et al.,
2006; Lim and Kumarasinghe, 2007).
Lethal injuries are very rare and usually occur
when the stinger reaches vital organs (Isbister,
2001). In Brazil, injuries provoked by freshwater
stingrays are common in the northern and middle-
western regions, where they are considered as an
important public health problem. In other regions,
such as the southwestern one, due to the construc-
tion of hydroelectric power plants, stingrays have
gradually occupied larger areas in the last years,
causing alterations in the epidemiological aspects of
accidents caused by venomous animals. Thirty years
ago, stingrays have been observed only in the Lower
and Medium Parana´ River, in the boundaries with
Paraguay and Argentina countries. Today, stingrays
occur in areas located upstream, such as the Upper
Parana´ River and tributaries, in Sa˜ o Paulo and
Mato Grosso do Sul States, Brazil. The risk of
accidents increases when sand ‘‘beaches’’ are formed
along the river, providing a touristic appeal to the
region and increasing the possibility of an encounter
between fishermen or swimmers and stingrays
(Garrone Neto et al., 2007).
Several toxic activities have already been reported
in extracts obtained from the tissue covering the
stingers, and therefore many authors have named
them as ‘‘venom’’. Neurotoxic, cardiotoxic and
lethal activities were observed by Vellard (1931,
1932). Nociception, dyspnea, agitation and convul-
sion were also noted when experiments with the
intact stinger of Dasyatis pastinaca were carried out
in guinea pigs (Fleury, 1950). Cardiac and circula-
tory disturbances observed in cats were likely due to
the direct action of Urolophus halleri venom in
myocardium cells (Russell and Van Harreveld,
1954; Russell et al., 1957). Recently, Magalha˜ es
et al. (2006) compared Potamotrygon cf. scobina and
Potamotrygon gr. orbignyi venoms and showed that
they could induce nociception, edema, necrosis and
disturbances in murine microcirculation. These
authors also showed that the mucus dispersed
through the stingers could potentiate necrosis.
Many enzymes were detected in marine and
freshwater stingray venoms: phosphodiesterase
and 50
-nucleotidase activities in U. halleri venom
(Russell et al., 1958); 50
-nucleotidase, phospholi-
pase, hyaluronidase and protease activities in
ARTICLE IN PRESS
K.C. Barbaro et al. / Toxicon 50 (2007) 676–687 677

3. Potamotrygon motoro (Magalha˜ es, 2001), and hya-
luronidase, caseinolytic, gelatinolytic and fibrino-
genolytic activities in Potamotrygon falkneri venom
(Haddad et al., 2004). A peptide with vasoconstric-
tive activity was isolated from Potamotrygon
orbignyi venom by Conceic-a˜ o et al. (2006).
Aiming to obtain detailed information about
stingray tissue extracts, a comparative study of
some biochemical, biological, enzymatic and im-
munological features of freshwater (P. falkneri) and
marine (Dasyatis guttata) stingray tissue extracts
was carried out herein.
2. Materials and methods
2.1. Animals and tissue extracts
Swiss male mice (18–20 g) and adult rabbits
(3–4 kg) were provided by Butantan Institute
Animal House. Animals received food and water
ad libitum. Specimens of P. falkneri and D. guttata
were collected in Sa˜ o Paulo and Mato Grosso do
Sul States. Tissue extracts were obtained from the
integumentary tissue covering the stinger as pre-
viously described (Haddad et al., 2004). The protein
content of tissue extract pools was determined by
bicinchoninic acid method (Smith et al., 1985),
using bovine serum albumin (BSA) as a standard.
The procedures involving animals were conducted
in conformity with national laws and policies
controlled by Butantan Institute Animal Investiga-
tion Ethical Committee (protocol no. 115/2002).
2.2. Production of sera against P. falkneri and
D. guttata tissue extracts
Sera against P. falkneri and D. guttata tissue extracts
were obtained by immunization of two rabbits, one for
each type of tissue extract. Tissue extracts (200mg) were
diluted in 500ml of PBS and added to 500ml of
complete Freund’s adjuvant, and these mixtures were
injected i.m. into rabbits. After 1 month, animals
received five additional boosters of antigen, suspended
in incomplete Freund’s adjuvant, at fortnight intervals.
Blood was collected and sera was separated and stored
at À201C until used.
2.3. ELISA
Rabbit species-specific sera were titrated by
ELISA, using P. falkneri and D. guttata tissue
extracts (10 mg/ml) to coat the microplates (Nunc,
USA), according to Theakston et al. (1977). The
reaction was read using an ELISA reader (Multis-
kan EX) and the titer determined as the reciprocal
of the highest dilution that causes an absorbance
greater than 0.050 at 492 nm, since non-specific
reactions were observed below this value.
2.4. Sodium dodecyl sulfate– polyacrylamide gel
electrophoresis (SDS– PAGE)
Proteins of P. falkneri and D. guttata tissue extracts
(5mg) were analyzed by SDS–PAGE (4–20%, Pierce,
USA) under non-reducing conditions (Laemmli, 1970),
and then proteins were silver stained (Blum et al.,
1987). Myosin, b-galactosidase, BSA, carbonic anhy-
drase, soybean trypsin inhibitor, lysozyme and aproti-
nin were used as the molecular mass markers
(Kaleidoscope prestained standards; BioRad, USA).
2.5. Western blotting
Proteins of P. falkneri and D. guttata tissue
extracts (20 mg) were first fractionated by
SDS–PAGE as described above, and then electro-
blotting was performed as described by Towbin
et al. (1979). Nitrocellulose membranes were in-
cubated with species-specific rabbit antisera, diluted
at 1/250. Immunoreactive proteins were detected
using peroxidase-labeled anti-rabbit IgG and the
blot was developed with 0.05% (w/v) 4-chloro-1-
naphtol in 15% (v/v) methanol, in the presence of
0.03% (v/v) H2O2. Non-immunized rabbit serum
was used as a control. Pre-stained molecular mass
markers (BioRad, USA) were used.
2.6. Proteolytic and hyaluronidase assays
Zymography was used to assay protease and
hyaluronidase activities. Casein (Merck, Darmstadt,
Germany), gelatin and fibrinogen (Sigma, St. Louis,
MO) were used as substrates to assay proteolytic
activity (Heussen and Dowdle, 1980; Barbaro et al.,
2005), and hyaluronic acid from rooster comb (Sigma,
St. Louis, MO) to determine hyaluronidase activity
(Miura et al., 1995; Barbaro et al., 2005). Samples of
P. falkneri and D. guttata tissue extracts in non-
reducing sample buffer were loaded into gels and run at
20mA/gel. Clear areas in the gel indicated regions
of enzyme activity. When required, Na2-EDTA or
1,10-phenanthroline (Sigma, St. Louis, MO) was added
in a final concentration of 5mM to gel washing
and incubation buffers, and then gel was stained as
ARTICLE IN PRESS
K.C. Barbaro et al. / Toxicon 50 (2007) 676–687678

4. usual. Pre-stained molecular mass markers (BioRad,
Hercules, CA) were used.
2.7. Lethal activity
Mice (n ¼ 4) were injected i.p. with 100, 200, 400
or 800 mg of P. falkneri and D. guttata tissue extracts
diluted in 200 ml of PBS. Lethality was observed 24
and 48 h after i.p. tissue extracts injections.
2.8. Local reaction and dermonecrotic activity
induced by P. falkneri and D. guttata tissue extracts
Local reaction (edema/erythema and paleness/
ecchymosis areas) and dermonecrotic activity were
determined by i.d. injection of 100, 200 or 400 mg of
P. falkneri or D. guttata tissue extracts, dissolved in
0.1 ml of PBS, into the mouse dorsum skin (n ¼ 4).
Animals were sacrificed by CO2 inhalation and the
inner dorsum skin was observed. Areas of edema/
erythema, paleness/ecchymosis and dermonecrosis
were inspected 72 h after injection and reported as
the mean of four different areas (mm2
) for each
parameter studied. Animals injected only with PBS
were used as negative controls.
2.9. Nociceptive and edematogenic activities of
P. falkneri and D. guttata tissue extracts
To detect nociceptive activity, mice (n ¼ 8) were
injected with different doses (1, 4, 16 and 64 mg) of
P. falkneri and D. guttata tissue extracts dissolved in
30 ml of PBS into the right hind paw. Animals were
individually placed under glass funnels on a mirror.
Afterwards, the reactivity of animals to lick or bite
the injected paw was measured, in seconds, during
30 min of experimental evaluation (Hunskaar et al.,
1985). Animals injected only with PBS were used as
negative controls.
Edema-forming activity was evaluated at differ-
ent time intervals (15 min, 1, 2, 24 and 48 h) as the
difference of thickness (mm) measured with a
caliper between the right hind paw—injected with
different doses (1, 4, 16 and 64 mg) of P. falkneri and
D. guttata tissue extracts diluted in PBS, or PBS
alone (negative control)—and the left hind paw of
mice, which received no injection.
2.10. Estimation of myotoxic activity
Mice (n ¼ 8) were injected i.m. into the right
gastrocnemius muscle with 100, 200 or 300 mg of
P. falkneri and D. guttata tissue extracts dissolved in
50 ml of PBS. The control group was injected with
PBS alone. After 3 h, blood was collected from the
ophthalmic plexus. Sera of mice were separated and
was immediately assayed for creatine kinase (CK)
activity (CK-NAC Liquiform, LABTEST, Brazil).
One unit corresponds to the amount of enzyme that
hydrolyzes 1 mmol of creatine per min at 25 1C.
Myotoxic activity was expressed as U/mg of tissue
extract of three independent experiments. Bothrops
jararacussu snake venom (150 mg) was used as a
positive control.
2.11. Coagulant activity
Clotting time was performed according to
Santoro and Sano-Martins (1993). P. falkneri and
D. guttata tissue extracts (1, 4, 16 and 64 mg) diluted
in 50 ml of PBS were added to 200 ml human plasma.
Samples (duplicate) were observed for 5 min at
37 1C to determine the clotting time. Thereafter, to
verify fibrinogen hydrolysis, 50 ml of bovine throm-
bin (30 U/ml) (Sigma, USA) was added to the
mixture. As a positive control, 50 ml samples of two-
fold serially diluted B. jararaca snake venom
(1.56–200.0 mg) were used to determine the minimal
coagulant dose (MCD). Experiments were carried
out twice.
2.12. Direct hemolytic activity
Human blood (type O, Rh+) was collected in the
presence of 0.15 M sodium citrate (9:1) and cen-
trifuged at 1900g for 15 min at 10 1C, as described
by Boman and Ralleta (1957). Red blood cells were
obtained and washed three times with PBS. Samples
(50 ml) of PBS containing 3% of red blood cells were
mixed with 100 ml of different doses (1, 4, 16 and
64 mg) of P. falkneri and D. guttata tissue extracts.
Each sample (50 ml) was placed (duplicates) in
microplates. As controls distilled water (100%
hemolysis) and PBS (0% hemolysis) were used.
Microplates were kept at room temperature for 3 h.
The absorbance was read using an ELISA reader
(Multiskan EX) at 595 nm.
2.13. Phospholipase A2 activity
Phospholipase activity was determined as de-
scribed elsewhere (Santoro et al., 1999). P. falkneri
and D. guttata (15, 30, 60 and 120 mg) tissue extracts
diluted in 15 ml of PBS, pH 7.4 were added to 1.5 ml
ARTICLE IN PRESS
K.C. Barbaro et al. / Toxicon 50 (2007) 676–687 679

5. of the reaction solution (100 mM NaCl, 10 mM
CaCl2, 7 mM Triton X-100, 0.265% soybean
lecithin, 98.8 mM phenol red, pH 7.6) in a spectro-
photometer cuvette. The solution was immediately
homogenized and read at 556 nm. The definition of
1 U of phospholipase A2 activity was taken as the
amount of toxin (mg of protein/assay) producing a
decrease of 0.001 absorbance units per min under
the conditions described. Crotalus durissus terrificus
snake venom (6 mg) was used as a positive control.
Phospholipase activity was expressed as U/mg of
two independent experiments.
2.14. Statistical analysis
Results were expressed as mean7SD. Two-way
ANOVA followed by Bonferroni’s test was used to
analyze data, using SigmaStat 3.0 software. Values with
po0.05 were considered statistically significant.
3. Results
3.1. Analyses of tissue extracts by SDS– PAGE
Fig. 1 shows the electrophoretic pattern of
P. falkneri and D. guttata tissue extracts. After
SDS–PAGE, under non-reducing conditions, many
components with similar molecular masses were
noticed in both tissue extracts, mainly above
84 kDa, which were difficult to be separated. Some
bands around 22 kDa were observed exclusively in
P. falkneri tissue extract. A strong and diffuse band
was observed between 43 and 65 kDa in P. falkneri
tissue extract. Gels also showed major components
below 18 kDa in both tissue extracts. At least 16
bands were distributed along the gel in both tissue
extracts (Fig. 1).
3.2. Cross-reactivity determined by ELISA and
Western blotting
Table 1 shows the comparison of antibody titers
obtained by ELISA between the two antisera
assayed against homologous and heterologous
antigens. Both tissue extracts were immunogenic
and could induce high levels of antibodies in
rabbits. Intense cross-reactivity between both tissue
extracts was detected, and no significant differences
on titers were noticed (only variations higher
than two-fold dilutions were considered significant).
Fig. 2 shows immunoblots of D. guttata and
P. falkneri tissue extracts after incubation with
species-specific sera produced in rabbits. Many
components of both tissue extracts, mainly above
23 kDa, reacted with both antisera, likely due to the
presence of many common proteins in the integu-
mentary tissue. However, when compared with
homologous antisera, heterologous antisera failed
to react or faintly reacted with some components
below 30 kDa, demonstrating the presence of
unique components showing no immunological
identity between each tissue extract.
3.3. Enzymatic activities of tissue extracts
Casein, gelatin, fibrinogen or hyaluronic acid were
incorporated as substrates in 12.5% acrylamide gels,
ARTICLE IN PRESS
Pf
83.91
133.08
205.73
41.56
31.35
17.25
7.01
Dg
Fig. 1. Electrophoretic profiles of P. falkneri (Pf) and D. guttata
(Dg) tissue extracts (5 mg) after 4–20% SDS–PAGE under non-
reducing conditions. Gel was silver stained. Numbers on the right
correspond to the position of molecular mass markers.
Table 1
Antigenic cross-reactivity between D. guttata and P. falkneri
tissue extracts using rabbit antisera by ELISA
Antisera Tissue extracts
D. guttata P. falkneri
Anti-D. guttata 1,024,000a
1,024,000
Anti-P. falkneri 512,000 1,024,000
Normal o200b
o200b
a
ELISA titers. Microplates were coated with each tissue
extract, and then incubated with homologous or heterologous
rabbit antisera.
b
Initial dilution 1/200.
K.C. Barbaro et al. / Toxicon 50 (2007) 676–687680

6. and used to assay enzymatic activities of tissue extracts.
Several components with molecular mass above 83kDa
showed similar profiles for caseinolytic, gelatinolytic
and fibrinogenolytic activities (Fig. 3). However, some
components below 60kDa showed these activities only
in D. guttata tissue extract. Incubation with Na2-
EDTA or 1,10-phenanthroline, inhibitors of metallo-
proteinases, could not abolish the activity of most
components (data not shown). No hyaluronidase
activity was observed in D. guttata tissue extract
(Fig. 3). A diffuse band between 41 and 65kDa was
observed in P. falkneri tissue extract. Minimal phos-
pholipase A2 activity was detected in both tissue
extracts (2267U/mg to P. falkneri and 5200U/mg to
D. guttata tissue extracts) even when high doses of
tissue extract (120mg) were used. C. d. terrificus snake
venom (114,500U/mg) and PBS were used as a positive
and negative control, respectively.
3.4. Toxic activities
Only P. falkneri tissue extract could evoke a dose-
dependent local reaction—areas of edema/erythema
and paleness/ecchymosis, and necrosis—showing an
intense inflammatory reaction at the site of injection
(Table 2). No signs of inflammatory reactions were
observed in animals injected with D. guttata tissue
extract, even when higher doses were administered
ARTICLE IN PRESS
Fig. 2. (A) Electrophoretic profiles of P. falkneri (Pf) and
D. guttata (Dg) tissue extracts (25 mg) after 12.5% SDS–PAGE
under non-reducing conditions. Gel was silver stained. (B)
Antigenic cross-reactivity of P. falkneri (Pf) and D. guttata
(Dg) tissue extracts was determined by Western blotting, using
anti-D. guttata and anti-P. falkneri rabbit sera. Numbers on the
left correspond to the position of molecular mass markers.
Pf Dg
210-
131-
89-
41.3-
31.8-
18.1-
Casein
Pf Dg
210-
131-
89-
41.3-
31.8-
18.1-
Gelatin
Pf Dg
210-
131-
89-
41.3-
31.8-
18.1-
Fibrinogen Hyaluronic Acid
Pf Dg
17.26-
7.01-
31.35-
41.56-
83.91-
133.08-
205.73-
Fig. 3. Caseinolytic, gelatinolytic, fibrinogenolytic (20 mg) and hyaluronidase (60 mg) activities of P. falkneri (Pf) and D. guttata (Dg) tissue
extracts were determined using the technique of substrate SDS–PAGE 12.5%. Numbers on the left correspond to the position of molecular
mass markers. Clear areas in the gel indicate regions of enzymatic activity.
K.C. Barbaro et al. / Toxicon 50 (2007) 676–687 681

7. (400 mg). Deaths could be observed only in animals
injected with P. falkneri tissue extract. All animals
injected with 800 mg of tissue extract died within 24 h
after the tissue extract administration (Table 3).
Both tissue extracts induced nociceptive activity
(Fig. 4), which was dose dependent. However, only
the highest dose of D. guttata tissue extract (64 mg)
showed a statistically significant difference with the
control, whereas low doses of P. falkneri could
induce pain. The edematogenic activity of both
tissue extracts peaked at 15 min after the tissue
extract injection. Edema decreased gradually until
24 h, when statistically significant edema was no
more observed (Fig. 5).
No direct hemolytic activity was detected in both
tissue extracts, even when 64 mg of each tissue
extract was used. P. falkneri and D. guttata tissue
extracts showed no coagulant activity. The MCD of
B. jararaca snake venom (positive control) was
10.56 mg/ml. P. falkneri and D. guttata tissue
extracts could not clot human plasma. When bovine
thrombin (30 U/mg, final concentration) was added
to plasma previously incubated with all the tested
doses of tissue extracts, clotting times were compar-
able to those of plasma incubated with saline
(10–15 s).
Fig. 6 shows that higher myotoxic activity
was present in P. falkneri tissue extract than in
D. guttata tissue extract. The latter showed CK
levels similar to the control group (PBS). P. falkneri
tissue extract, even in a low dose (100 mg), could
induce a remarkable CK release, and this activity
was dose dependent. Animals injected with PBS
(7997367 U/l) or B. jararacussu (100 mg) snake
venom (435071596 U/l) were used as negative and
positive controls, respectively.
4. Discussion
Stingrays have one or more retroserrated stingers
adhered to their tail, which are covered by an
integument composed of glandular tissues contain-
ing toxic activities (Halstead, 1970). When intro-
duced into the victim, the stinger causes a traumatic
injury that may worsen if the integument is retained
in the wound. Herein, we present a comparative
study between tissue extracts of freshwater
(P. falkneri) and marine (D. guttata) stingrays.
By SDS–PAGE, we observed several components
of high molecular masses in P. falkneri and
D. guttata tissue extracts. We suppose that many
of those components belong to the tissue that
recovered the stinger, once the stingrays do not
have a venom-individualized gland. A great number
of different components are observed below 84 kDa
in both tissue extracts. Morphological studies of the
stinger structure showed that more specialized cells
with high protein content are observed in P. falkneri
than in D. guttata venom-secreting tissue (Pedroso
et al., submitted). This structural difference could
explain the variation observed in the electrophoretic
profile noticed between both tissue extracts.
Both tissue extracts were immunogenic and could
induce large amounts of antibodies. No significant
difference was observed between the titers of each
species-specific antiserum, even when heterologous
ARTICLE IN PRESS
Table 2
Local reaction and necrosis induced by P. falkneri and D. guttata
tissue extracts
Tissue extracts Necrosis (mm2
) Local reaction (mm2
)
D. guttata
100 mg 0 0
200 mg 0 0
400 mg 0 0
P. falkneri
100 mg 0 7.5711.4
200 mg 21.0718.5 31.5714.8
400 mg 13.3710.8 191.5716.5
Mice were injected i.d. with different doses of P. falkneri or
D. guttata tissue extracts diluted in 0.1 ml of PBS. Local reaction
(edema/erythema and paleness/ecchymosis) and necrosis were
evaluated after 72 h. Data are expressed as mean7SD.
Table 3
Lethal activity of P. falkneri and D. guttata tissue extracts
Tissue extracts Lethality (h)
24 48
D. guttata
100 mg 0/4a
0/4
200 mg 0/4 0/4
400 mg 0/4 0/4
800 mg 0/4 0/4
P. falkneri
100 mg 0/4 0/4
200 mg 0/4 0/4
400 mg 2/4 3/4
800 mg 4/4 4/4
Mice (n ¼ 4) were injected i.p. with different doses of P. falkneri
or D. guttata tissue extracts, diluted in 0.2 ml of PBS. Deaths were
observed 24 and 48 h after tissue extract injection.
a
Dead/injected.
K.C. Barbaro et al. / Toxicon 50 (2007) 676–687682

8. tissue extract was used to coat the microplates.
Antigenic cross-reactivity was detected by ELISA
and Western blotting, indicating the presence of
many common epitopes among tissue extract
components, especially above 41 kDa. This was
expected since many common housekeeping pro-
teins are shared by the constitutive tissue covering
both stingers. Both antisera weakly reacted with
components below 17 kDa, indicating that they are
weakly immunogenic, since they are present in high
quantities, as detected by electrophoresis.
The intense inflammation reaction observed in
human injuries is suggestive of disturbances in
the extracellular matrix. As previously reported
ARTICLE IN PRESS
Time (hours) Time (hours)
Thickness(cm)
0.00
0.05
0.10
0.15
0.20
0.25 1 2 24 48
∗
∗
∗∗
∗
PBS
1 μg
4 μg
16 μg
64 μg
#
Thickness(cm)
0.00
0.05
0.10
0.15
0.20
0.25
0.25 1 2 24
∗
∗
∗
∗
∗
PBS
1 μg
4 μg
16 μg
64 μg
∗
#
#
48
Fig. 5. Edematogenic activity of P. falkneri (A) and D. guttata (B) tissue extract. To investigate edematogenic activity, different doses of
tissue extract were diluted in 30 ml of PBS solution and injected intraplantarly, using vehicle as a negative control. Edema evaluation was
carried out by measuring the difference in thickness (cm) between the injected and non-injected hind paws at different time intervals.
*po0.001 and #po0.05—statistically significant difference between the experimental and control (PBS) groups.
PBS 1 μg 4 μg 16 μg 64 μg
Reactivity(seconds)
0
20
40
60
80
100
120
140
160
PBS
P. falkneri
D. guttata
∗
∗
∗
∗
Fig. 4. Nociceptive activity of P. falkneri and D. guttata tissue extracts. To evaluate nociceptive activity, different doses of each tissue
extract were diluted in 30 ml of PBS and injected intraplantarly. Vehicle was used a negative control. Reactivity was expressed as the time
(in s) to animals to lick and/or bite the injected paw during a period of 30 min. *Statistically significant (po0.05) difference between the
experimental and control (PBS) groups.
K.C. Barbaro et al. / Toxicon 50 (2007) 676–687 683

9. (Haddad et al., 2004), Zymographic analyses
showed that P. falkneri tissue extract contains
enzymes that can degrade distinct proteins such as
casein, gelatin and fibrinogen. In fact, enzymatic
activity was more intense in D. guttata than in
P. falkneri tissue extract, and enzymes with mole-
cular mass below 83 kDa could hydrolyze all
substrates. In both tissue extracts, the profile of
enzymatic degradation was similar using distinct
substrates, indicating the presence of proteases with
broad substrate specificity. These results suggest
that such proteases could contribute to degradation
of proteins and components present in the extra-
cellular matrix, favoring the establishment of local
injury. On the other hand, most of those enzymes
are not metalloproteinases, once the incubation
with Na2-EDTA or 1,10-ortho-phenantroline could
not inhibit their enzymatic activity (data not
shown). Under our experimental conditions, we
only observed hyaluronidase activity in P. falkneri
tissue extract, corroborating results described pre-
viously (Magalha˜ es, 2001; Haddad et al., 2004). The
presence of hyaluronidases in P. falkneri tissue
extract could amplify the local damage caused by
toxins as well as the injury caused by the stinger.
Several components with enzymatic activity have
been described in venomous animals (Tan and
Ponnudurai, 1992; Birkedal-Hansen et al., 1993;
Veiga et al., 2000; Haddad et al., 2004; Lira et al.,
2007). These enzymes could degrade components of
extracellular matrix and function as diffusion
factors, or they can act directly in the degradation
of proteins, likely contributing to tissue injury.
Biological activities were investigated according
to the major symptoms described in human
accidents. We observed that P. falkneri and
D. guttata tissue extracts could induce a dose-
dependent nociceptive activity. However, P. falkneri
tissue extract was two times more active than
D. guttata tissue extract. In human envenomation,
pain is worsened by the mechanical damage caused
by the stinger, which lacerates the local tissues,
potentiating the action of toxins. Nociceptive
activity might be associated with the direct action
of toxic components, since it was observed imme-
diately after tissue extract injection. Severe pain is
also reported in human injuries (Isbister, 2001;
Haddad et al., 2004). Besides, components with
enzymatic activity could also promote a tissular
injury, inducing an inflammatory reaction with the
release of mediators involved in nociception. Our
results showed that both tissue extracts evoked a
similar dose-dependent edema, which is more
intense within 15 min after injection, returning to
ARTICLE IN PRESS
CreatinekinaseU/L
0
1000
2000
3000
4000
5000
6000
7000
PBS Bjssu Dg Pf Dg Pf Dg Pf
100 μg 200 μg 300 μg
∗
∗
∗
∗
#
#
#
Fig. 6. Myotoxic activity of P. falkneri and D. guttata tissue extracts (100, 200 and 300 mg). *Statistically significant difference between the
experimental and negative control groups (PBS) (po0.05). #
Statistically significant difference between the experimental and the positive
control group (Bjssu, Bothrops jararacussu snake venom, 150 mg) (po0.05).
K.C. Barbaro et al. / Toxicon 50 (2007) 676–687684

10. basal levels within 24 h. Edema is also observed in
the limbs of patients afflicted by the stinger
(Haddad et al., 2004).
Under our experimental conditions, we verified
that only the tissue extract of P. falkneri could
induce necrosis and an intense inflammatory reac-
tion at the site of injection. These data are in
agreement with reports of accidents in humans that
demonstrate that necrosis and local inflammation
are much more prominent in injuries caused by
freshwater stingrays (Haddad, 2000; Haddad et al.,
2004). Our results agree with those reported by
Castex et al. (1964), who stung or injected the mixed
tegument, which covered the stinger of Potamotry-
gonidae freshwater stingrays, i.m. or i.p. into guinea
pigs. Magalha˜ es et al. (2006) also observed edema,
nociception and necrosis in Potamotrygon cf.
scobina and P. gr. orbignyi venoms, and that the
mucus covering the animal could augment this
necrotic activity. The mechanism of the pain
induction, edema and necrosis in the accidents for
stingrays is still uncertain, but mucus certainly
contributes to the injury caused by stingrays (Castex
et al., 1964; Magalha˜ es et al., 2006).
Myotoxicity was found only in P. falkneri tissue
extract. CK release could be due to the action of
myotoxins, as it occurs in B. jararacussu envenoma-
tion (Gutie´ rrez and Lomonte, 2003), or caused by
the intense inflammatory reaction, which can induce
muscular damage, as observed in Loxosceles spider
envenomation (Franc-a et al., 2002). In humans, the
local damage caused by stingers could also con-
tribute to expose muscular tissue to noxious
enzymes and toxins.
Lethal activity was detected only in P. falkneri
tissue extract. High amounts of samples were
necessary to cause the death of mice, probably
because the sample used was a mixture of toxic
components and integument constitutive tissue.
Russell et al. (1957) reported that the LD50 of
Urolophus stingray tissue extract was 28 mg/kg.
That value is similar to that obtained in our
experiments, since mortality started to be detected
from 20 mg/kg. Other studies observed neurotoxic
symptoms in animals experimentally injected with
stingray tissue extracts, and i.v. injection could
cause the death of mice (30 g) in 10–20 min (Vellard
1931, 1932).
We verified that D. guttata and P. falkneri tissue
extracts did not induce direct hemolysis, confirming
data published by Vellard (1931, 1932) using
Potamotrygon sp. venom. Besides, tissue extracts
could not prolong the clotting time of plasma nor
consume plasma fibrinogen under our experimental
conditions, indicating that these tissue extracts do
not act directly in the coagulation cascade, as
observed in many animal venoms, such as Bothrops
snakes or Lonomia caterpillars (Sano-Martins and
Santoro, 2003).
The results presented herein enhance the informa-
tion available about Brazilian stingray envenoma-
tion. We verified that stingray tissue extract is a
complex mixture of components, inducing different
toxic activities depending on the species being
studied. The morphological differences observed in
the tegument that covers the stinger can also
contribute to explain the different toxicity between
tissue extracts (Pedroso et al., submitted). Our
results suggest that P. falkneri and D. guttata tissue
extracts have peculiar characteristics that can
influence their toxic activity, and consequently the
clinical picture manifested by patients after stingray
accidents. Since a specific treatment does not exist
to stingray envenomation, and the therapeutical
approach is symptomatic (use of anti-inflammatory,
analgesic drugs and antimicrobials to prevent
infection), our data may contribute to understand
the mechanisms of action of these tissue extracts.
Acknowledgments
This work was supported by FAPESP (03/06873-4).
The authors thank Danieli M. Rangel, Guilherme
C. Rocha, Thais A. Oliveira and Letı´cia
M. P. Martins for technical assistance and Miss
Ottilie Carolina Forster and Dr. Maria Jose´ Alencar
Vilela, who provided some conditions to develop
this work. The authors also thank the fishermen in
Ubatuba (Zeca, Bideco, Adalto, Quim, Elias,
Major, Cebolinha, Paco, Nando, Santana, Amorim,
Natalı´cio, Rafael, among others) and Treˆ s Lagoas
(Marquinhos and Edmilson) cities for helping in the
capture of stingrays. We also thank CNPq for the
Grants of Katia C. Barbaro (306158/2004-3) and
Domingos Garrone Neto (142985/2005-8). IBAMA
provided animal collection permits (02027002992/
2004-79) and CGEN provided the license for genetic
patrimony access (041/05).
References
Barbaro, K.C., Knysak, I., Martins, R., Hogan, C., Winkel, K.,
2005. Enzymatic characterization antigenic cross-reactivity
and neutralization of dermonecrotic activity of five Loxosceles
ARTICLE IN PRESS
K.C. Barbaro et al. / Toxicon 50 (2007) 676–687 685

11. spider venoms of medical importance in the Americas.
Toxicon 45, 489–499.
Birkedal-Hansen, H., Moore, W.G., Bodden, M.K., Windsor,
L.J., Birkedal-Hansen, B., DeCarlo, A., Engler, J.A., 1993.
Matrix metalloproteinases: a review. Crit. Rev. Oral Biol.
Med. 4, 197–250.
Blum, H., Beier, H., Gross, H.J., 1987. Improved silver staining
of plant proteins, RNA and DNA in polyacrylamide gels.
Electrophoresis 8, 93–95.
Boman, H.G., Ralleta, U., 1957. Chromatography of rattlesnake
venom—a separation of 3 phosphodiesterases. Biochim.
Biophys. Acta 24, 619–631.
Brisset, I.B., Schaper, A., Pommier, P., Haro, L., 2006.
Envenomation by amazoniam freshwater stingray Potamo-
trygon motoro: 2 cases reported in Europe. Toxicon 47, 32–34.
Brooks, D.R., Thorson, T.B., Mayes, M.A., 1981. Fresh-water
stingrays (Potamotrygonidae) and their helminth parasites:
testing hypotheses of evolution and coevolution. In: Funk,
V.A., Brooks, D.R. (Eds.), Advances in Cladistics. New York
Botanical Garden, New York, pp. 147–175.
Carvalho, M.R., Lovejoy, N.R., Rosa, R.S., 2003. Family
Potamotrygonidae (river stingray). In: Reis, R.E., Kullander,
S.O., Ferraris, Jr., C.J. (Eds.), Check List f the Freshwater
Fishes of South and Central Ame´ rica. Edipucrs, Porto Alegre,
pp. 22–28.
Castex, M.N., Pedace, E., Maciel, I., Meyer, S.J., Murphy, M.,
Remonda, G., Alewaerts, F., 1964. La enfermedad para-
trygo´ nica: cuadros clı´nicos experimentales y estudio anato-
mopatolo´ gico. Prensa Med. Arg. 51, 1085–1095.
Charvet-Almeida, P., Arau´ jo, M.L.G., Rosa, R.S., Rinco´ n, G.,
2002. Neotropical freshwater stingrays: diversity and con-
servation status. Shark News 14, 47–51.
Compagno, L.J.V., Cook, S.F., 1995. The exploitation and
conservation of freshwater elasmobranchs: status of taxa and
prospects for the future. J. Aquaric. Aquat. Sci. 7, 62–90.
Conceic-a˜ o, K., Konno, K., Melo, R.L., Marques, E.E., Hiruma-
Lima, C.A., Lima, C., Richardson, M., Pimenta, D.C.,
Lopes-Ferreira, M., 2006. Orpotrin: a novel vasoconstrictor
peptide from this venom of the Brazilian stingray Potamo-
trygon gr. orbignyi. Peptides 27, 3039–3046.
Fleury, R., 1950. L’appareil venimeux des se´ laciens trygoni-
formes. Mem. Soc. Zool. France 30, 1–37.
Franc-a, F.O., Barbaro, K.C., Abdulkader, R.C., 2002. Rhabdo-
myolysis in presumed viscero-cutaneous loxoscelism: report
of two cases. Trans. R. Soc. Trop. Med. Hyg. 96, 287–290.
Garrone Neto, D., Haddad Jr., V., Vilela, M.J.A., Uieda,
V.S., 2007. Registro de ocorreˆ ncia de duas espe´ cies de
Potamotrigonı´deos na regia˜ o do Alto do Rio Parana´ e
algumas considerac-o˜ es sobre biologia. Biota Neotrop. Jan/
Abr 2007 7, 1. http://www.biotaneotropica.org.br/v7n1/pt/
abstract?shortcommunication+bn00707012007/http://www.
biotaneotropica.org.br/v7n1/pt/abstract?short communication
+bn00707012007S ISSN:1676-0603.
Gutie´ rrez, J.M., Lomonte, B., 2003. Efectos locales en el
envenenamiento ofı´dico en Ame´ rica Latina. In: Cardoso,
J.L.C., Franc-a, F.O.S., Wen, F.H., Ma´ laque, C.M.S.,
Haddad, Jr., V. (Eds.), Animais Pec-onhentos no Brasil.
Biologia, Clı´nica e terapeˆ utica dos Acidentes, Sarvier,
pp. 310–323.
Haddad Jr., V., 2000. Atlas de animais aqua´ ticos perigosos do
Brasil: guia me´ dico de identificac-a˜ o e tratamento. Editora
Rocca, Sa˜ o Paulo, pp. 123–128.
Haddad Jr., V., Garrone, N.D., de Paula, N.J.B., Marques,
F.P.L., Barbaro, K.C., 2004. Freshwater stingrays: study of
epidemiologic, clinic and therapeutic aspects based on 84
envenomings in humans and some enzymatic activities of the
venom. Toxicon 43, 287–294.
Halstead, B.W., 1966. Venomous marine animals of Brazil. Mem.
Inst. Butantan 33, 1–26.
Halstead, B.W., 1970. Poisonous and Venomous Marine Animals
of the World. Gov Print Office: 3, Washington, DC, USA,
pp. 29–69.
Heussen, C., Dowdle, E.B., 1980. Electrophoretic analysis of
plasminogen in polyacrylamide gels containing sodium
dodecyl sulfate and copolymerized substrates. Anal. Biochem.
102, 196–202.
Hunskaar, S., Fasmer, O.B., Hole, K., 1985. Formalin test
in mice, a useful technique for evaluating mild analgesics.
J. Neurosci. Methods 14, 69–76.
Isbister, G., 2001. Venomous fish stings in tropical northern
Austra´ lia. Am. J. Emerg. Med. 19, 561–565.
Laemmli, U.K., 1970. Cleavage of structural proteins during
assembly of the head of bacteriophage T4. Nature 227,
680–685.
Lim, Y.L., Kumarasinghe, S.P.W., 2007. Cutaneous injuries from
marine animals. Singapore Med. J. 48, 25–28.
Lira, M.S., Furtado, M.F., Martins, L.M.P., Lopes-Ferreira, M.,
Santoro, M.L., Barbaro, K.C., 2007. Enzymatic and im-
munochemical characterization of bothrops insularis venom
and its neutralization by polyspecific Bothrops antivenom.
Toxicon 49, 982–994.
Lovejoy, N.R., 1996. Systematics of myliobatoid elasmobranchs:
with emphasis on the phylogeny and historical biogeography
of neotropical freshwater stingrays (Potamotrygonidae:
Rajiformes). Zool. J. Linnean Soc. 117, 207–257.
Magalha˜ es, K.W., Lima, C., Piran-Soares, A.A., Marques, E.E.,
Hiruma-Lima, C.A., Lopes-Ferreira, M., 2006. Biological and
biochemical properties of the Brazilian Potamotrygon stin-
grays: Potamotrygon cf. scobina and Potamotrygon gr.
orbignyi. Toxicon 47, 575–583.
Magalha˜ es, M.R., 2001. Estudos bioquı´micos do veneno de raias
Potamotrygon motoro (Chondrichthyes: Dasyatidae, Potamo-
trygonidae)—Purificac-a˜ o e caracterizac-a˜ o de uma hialuroni-
dase. Dissertac-a˜ o de Mestrado, Instituto de Cieˆ ncias
Biolo´ gicas, UFG.
Miura, R.O., Yamagata, S., Miura, Y., Harada, T., Yamagata, T.,
1995. Analysis of glycosaminoglycan-degrading enzymes by
substrate gel electrophoresis (Zymography). Anal. Biochem.
225, 333–340.
Nelson, J., 1994. Fishes of the World, third ed. Wiley, New York,
NY, 416pp.
Nishida, K., 1990. Phylogeny of the suborder Myliobatidoidei.
Mem. Fac. Fish. Hokkaido Univ. 37, 1–108.
Pedroso, C.M., Jared, C., Charvet-Almeida, P., Almeida, M.P.,
Garrone-Neto, D., Lira, M.S., Haddad Jr., V., Barbaro,
K.C., Antoniazzi, M.M., 2007. Morphological characteriza-
tion of the venom secretory epidermal cells in the stinger of
marine and freshwater stingrays. Toxicon 50, 688–697.
Roberts, T.R., Karnasuta, J., 1987. Dasyatis laosensis, a
new whiptailed stingray (family Dasyatidae), from the
Mekong River of Laos and Thailand. Environ. Biol. Fish. 20,
161–167.
Rosa, R.S., 1985. A systematic revision of the South American
freshwater stingrays (Chondrichthyes: Potamotrygonidae).
ARTICLE IN PRESS
K.C. Barbaro et al. / Toxicon 50 (2007) 676–687686

12. Unpublished D. Phil. Thesis, College of William and Mary,
Williamsburg, Virginia.
Rosa, R.S., Castello, H.P., Thorson, T.B., 1987. Plesiotrygon
iwamae, a new genus and species of neotropical freshwater
stingray (Chondrichthyes: Potamotrygonidae). Copeia 2,
447–458.
Russell, F.E., Van Harreveld, A., 1954. Cardiovascular effects of
the venom of the round stingray, Urobatis halleri. Arch.
Intern. Physiol. 62, 332–333.
Russell, F.E., Barrit, W.C., Fairchield, M.D., 1957. Electro-
cardiographic patterns evoked by venom of the stingray.
Proc. Soc. Exp. Biol. Med. 96, 634–635.
Russell, F.E., Fairchield, M.D., Michaelson, J., 1958. Some proper-
ties of the venom of the stingray. Med. Arts Sci. 12, 78–86.
Sano-Martins, I.D., Santoro, M.L., 2003. In: Cardoso, J.L.C.,
Franc-a, F.O.S., Wen, F.H., Ma´ laque, C.M.S., Haddad, Jr., V.
(Eds.), Animais Pec-onhentos no Brasil. Biologia, Clı´nica e
terapeˆ utica dos Acidentes, Sarvier, pp. 289–309.
Santoro, M.L., Sano-Martins, I.S., 1993. Different clotting
mechanisms of Bothrops jararaca snake venom on human
and rabbit plasmas. Toxicon 31, 733–742.
Santoro, M.L., Sousa-e-Silva, M.C., Gonc-alves, L.R., Almeida-
Santos, S.M., Cardoso, D.F., Laporta-Ferreira, I.L., Saiki, M.,
Peres, C.A., Sano-Martins, I.S., 1999. Comparison of the
biological activities in venoms from three subspecies of the South
American rattlesnake (C. durissus terrificus, C. durissus cascavella
and C. durissus collilineatus). Comp. Biochem. Physiol. C
Pharmacol. Toxicol. Endocrinol. 122, 61–73.
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K.,
Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke,
N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of
protein using bicinchoninic acid. Anal. Biochem. 150, 76–85.
Tan, N.H., Ponnudurai, G., 1992. Comparative study of the
enzymatic, hemorrhagic, procoagulant and anticoagulant
activities of some animal venoms. Comp. Biochem. Physiol.
C 103, 299–302.
Taniuchi, T., 1979. Freshwater elasmobranchs from Lake
Naujan, Perak River, and Indragiri River, Southeast. Asia
Jpn. J. Ichthyol. 25, 273–277.
Taniuchi, T., Shimizu, M., Sano, M., Baba, O., Last, P.R., 1991.
Descriptions of freshwater elasmobranchs collected from
three rivers in northern Australia. Univ. Museum Univ.
Tokyo Nat. Culture 3, 11–26.
Theakston, R.D.G., Lloyd-Jones, M.J., King, L.E., 1977. Micro-
ELISA for detecting and assaying snake venom and anti-
venom antibody. Lancet 2, 639–641.
Thorson, T.B., 1983. Observations on the morphology, ecology,
and life history of the euryhaline stingray, Dasyatis guttata
(Bloch and Schneider) 1801. Acta Biol. Venez. 11, 92–125.
Thorson, T.B., Brooks, D.R., Mayes, M.A., 1983. The evolution
of freshwater adaptation in stingrays. Nat. Geol. Soc. Res.
Rep. 15, 663–694.
Towbin, H., Staehelin, T., Gordon, J., 1979. Eletrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose
sheets: procedure and some applications. Proc. Natl. Acad.
Sci. USA 76, 4350–4354.
Veiga, S.S., da Silveira, R.B., Dreyfuss, J.L., Haoach, J., Pereira,
A.M., Mangili, O.C., Gremski, W., 2000. Identification of
high molecular weight serine proteases in Loxosceles inter-
media (brown spider) venom. Toxicon 38, 825–839.
Vellard, J., 1931. Venin des rais (Taeniura) du Rio Araguaya
(Bre´ sil). C. R. Acad. Sci. 192, 1279–1281.
Vellard, J., 1932. Mission scientifique au Goyaz et au Rio
Araguaya. Me´ m. Soc. Zool. France 29, 513–539.
ARTICLE IN PRESS
K.C. Barbaro et al. / Toxicon 50 (2007) 676–687 687