NEUROBIOLOGY, PHYSIOLOGY, BIOCHEMISTRY

Isolation and Partial Characterization of Proteins with Vasodegenerative

and Proinflammatory Properties from the Egg-Nests of
Hylesia metabus (Lepidoptera: Saturniidae)
ULF LUNDBERG,1,2 VICTOR SALAZAR,3 MARIA TOVAR,4 AND JESSICA RODRIGUEZ4

J. Med. Entomol. 44(3): 440Ð449 (2007)

ABSTRACT The Hylesia genus comprises a group of Neotropical moths ubiquitous in the Americas from
Arizona to Argentina. One of the species of the Hylesia genus in Venezuela, French Guyana, and Trinidad
has been identiÞed as Hylesia metabus (Cramer 1775) (Lepidoptera: Saturniidae). In Venezuela, these
moths are found in abundance in the mangrove (Avicennia spp.) swamps surrounding the Gulf of Paria
and the Orinoco Delta in the eastern part of the country. During the mating season, the female adults shed
copious amounts of urticating setae in the air, producing a severe papulovesicular dermatitis among the
population in the affected areas. The females also use their urticating setae to protect the eggs during the
hatching period. In the current study, we have isolated and partially characterized proteins with proin-
ßammatory properties from the urticating setae in the egg-nests by using sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis and anionic exchange-high-performance liquid chromatography (HPLC).
We also have studied the biological response of the egg-nest extract and the HPLC puriÞed fractions by
inoculation in guinea pigs; and, analyzing the tissue samples by means of histopathological methods. The
results of this study show that the extracted venom and HPLC puriÞed subcomponents give rise to an
intense inßammatory reaction characterized by massive inÞltration of inßammatory cells, echymoses, and
vascular degeneration. Chromatographic separation showed that the venom was made up of proteins
having selectively vasodegenerative-Þbrinolytic or proinßammatory-quimotactic properties.

RESUMEN El género “Hylesia” está conformado por un grupo de polillas neotropicales que abundan
desde el estado de Arizona de los Estados Unidos hasta el extremo sur del subcontinente. Una de las
especies de las polillas “Hylesia” encontados en Venezuela, la Guayana Francesa y Trinidad ha sido
identiÞcado como Hylesia metabus (Cramer, 1775) (Lepidoptera, Saturniidae). En Venezuela estas
polillas se encuentran en gran abundancia dentro de los manglares (Avicennia spp.) del Golfo Triste
(Golfo de Paria) y los caños adyacentes del Delta de Orinoco en el oriente del paṍs. Durante las épocas
de empareamiento las hembras adultas de este especie libera cantidades enormes de una minúscula
pelusa urticante (setas) en el medio ambiente, produciendo una dermatitis papulo-vesicular severo
entre los pobladores de las zonas adyacentes a los manglares. En el presente estudio hemos logrado
aislar y caracterizar sustancias protéicas con propiedades proinßamatorias de las setas urticantes de
las posturas de huevos de este especie usando electroforesis en poliacrilamida y cromatografṍa de
intercambio aniónico lṍquida de alta resolución (HPLC). Hemos estudiado la respuesta inßamatoria
del extracto de las posturas de huevos y sus componentes aislados en un modelo animal utilizando el
pabellón auricular del Acure (Cavia porcellus) mediante métodos de histopatologṍa. Los resultados del
presente estudio muestran que el veneno extraṍdo, y sus subcomponentes, dan origen a una reacción
inßamatoria intensa caracterizado por inÞltración masiva de células inßamatorias, echimosis, y de-
generación vascular. La separación cromatográÞca mostró que el veneno contiene proteṍnas con
propiedades selectivamente Þbrinolṍtico-vasodegenerativos o quṍmotactico-proinßamatorios.
KEY WORDS Hylesia metabus, erucism, proteolytic enzymes, inßammation

Caterpillars from !12 families of Lepidoptera species
worldwide can inßict serious injuries such as urticarial
dermatitis, atopic asthma, osteochondritis, consump-
1 Departamento de Biologṍa Estructural, Instituto Venezolano de
Investigaciones CientṍÞcas, Carretera Panamericana. Km 11. Estado
Miranda, Venezuela, Apdo. 21827, Caracas 1020A, Venezuela.
2 Corresponding author, e-mail: ulundber4412@cantv.net.
3 Centro de Biofṍsica y Bioquṍmica, Instituto Venezolano de Inves-
tigaciones CientṍÞcas, Carretera Panamericana. Km 11. Estado
Miranda, Venezuela, Apdo. 21827, Caracas 1020A, Venezuela.
4 Instituto de Investigaciones en Biomedicina y Ciencias Aplicadas,
Universidad de Oriente, Cumana, Venezuela.
tion coagulopathy, renal failure, and intracerebral
hemorrhage (Diaz 2005). The term erucism is used to
describe the pruritic maculopapular to bulbous con-
tact dermatitis caused by exposure to urticating setae
from the larvae or the adults (Benaim-Pinto et al. 1991,
Diaz 2005). In most cases, the urticating setae are
present in the larval stage, and it is only in a few species
that they are found in adults. For example, in the
genera Acyphas and Euproctis in the Lymantridae and
in the genus Hylesia of the Saturniidae (Rodriguez et
al. 2004). Approximately 115 species of the Hylesia
genus have been described in the Americas, 25 of

0022-2585/07/0440Ð0449$04.00/0 ! 2007 Entomological Society of America

May 2007 LUNDBERG ET AL.: ISOLATION OF PROINFLAMMATORY PROTEINS FROM H. metabus
which have been identiÞed in Venezuela (The Insect
Company 2006). Most species identiÞed in Venezuela
do not seem to cause any discomfort with the notable
exception of Hylesia metabus (Cramer 1775). This
moth is considered a major public health problem for
the populations living in the vicinity of the mangrove
(Avicennia spp.) swamps of the coast off the Gulf of
Paria (Golfo Triste) and the Delta Orinoco in north-
eastern Venezuela (Fornéz and Hernandez 2001).
The biological cycle of H. metabus lasts !101 d,
producing 3.65 successive generations each year
(Boyé 1932, Benaim-Pinto et al. 1991). Adult females
shed copious amounts of urticating arrow-like setae
during mating and oviposition at the end of each cycle.
The females also use their urticating abdominal setae
to cover the egg masses to protect them from preda-
tors and parasites (the males do not possess urticating
setae) (Rodriguez et al. 2004). Adults exhibit a posi-
tive phototropism, and they are strongly attracted by
the streetlights and other light sources at nighttime.
Thus, during the ßight periods, all lights must be
turned off after sunset. Most activities of daily life are
restricted, e.g., agricultural work, Þshing, commerce,
and educational activities.
Contact with the urticating abdominal setae of the
females, or accidental contact with the egg-nests of H.
metabus, gives rise to an intense pruritic papuloery-
thematous dermatitis with discrete vesicular and vas-
cular degeneration occurring without previous expo-
sure (Benaim-Pinto et al. 1992). The condition often
requires systemic administration of potent steroids
and antihistamines to alleviate symptoms. The dermal
reaction lasts typically from 5 to 10 d. However, in
some cases the dermatitis may persist for considerably
longer periods (months).
Several studies have demonstrated the presence of
proteins with proinßammatory properties in the urti-
cating setae (Benaim-Pinto et al. 1992, Lundberg et al.
2002). These studies also reported the presence of a
proteolytic enzyme with a molecular mass of 32 kDa
as determined by gel Þltration chromatography with a
pH optimum of 8.5Ð9.0. The determination of K3/Km
values in different chromogenic substrates showed a
preference for the plasma kallikrein substrate S-2302
(H-D-Pro-Phe-Arg-pNA) followed by the broad-spec-
trum serine protease substrate S-2288 (H-D-Ile-Pro-
Arg-pNA). In addition, the enzyme showed a marked
Þbrinolytic activity in vitro (Lundberg et al. 2002).
Substances with serine-protease activities also have
been demonstrated from Euproctis spp. caterpillars
(Bleumink et al. 1982). A protein with a molecular
mass of 28 kDa extracted from the setae of Thaumeto-
poea pityocampa (Schiff) (Lepidoptera: Thaumeto-
poeidae) caused similar dermal reactions as the eru-
cism produced by exposure to setae from H. metabus
(Lamy et al. 1986). In the current study, we have
explored the biochemical properties of the compo-
nents obtained from the extracts of adult males, fe-
males, and egg-nests from H. metabus, and we also
implemented a sensitive in vivo assay to investigate the
biological activities of the puriÞed proteins.
441
Materials and Methods
Reagents. All reagents used were of the highest
degree of purity available and obtained from Sigma-
Aldrich (St. Louis, MO) unless otherwise speciÞed.
Insect Material. H. metabus samples were obtained
by recollection of adults and egg-nests in various sites
of the mangrove swamps in the Gulf of Paria and
rapidly transferred to the Venezuelan Institute for
ScientiÞc Investigation (Instituto Venezolano de In-
vestigaciones CientṍÞcas, Caracas, Venezuela) where
the material was stored at "80#C until use.
Venom Extraction. Five grams of setae (equivalent
to !170 adults or 120 egg-nests) were extracted in a 50
mM Tris buffer adjusted to pH 8.5 supplemented with
0.5 M NaCl at a proportion of 10 ml of buffer per gram
of raw material for 24 h. The resulting material was
Þltered through Þve layers of gauze cloth and clariÞed
by centrifugation for 30 min at 10,000 rpm in a RC-5B
centrifuge (Sorvall, Newton, CT) by using an HB4
rotor (DuPont, Wilmington, DE). ClariÞed extracts
were concentrated by the addition of solid ammonium
sulfate to reach a Þnal saturation of 60%. Precipitated
proteins were harvested by centrifugation at 20,000
rpm for 120 min in a Sorvall RC-5B centrifuge using an
HB4 rotor. The pellets were redissolved in 50 mM
Tris-buffer, pH 8.5, supplemented with 0.05 M NaCl at
a ratio of 0.5 ml/g raw material and dialyzed exten-
sively against the same buffer. For the chromato-
graphic separation and subsequent histopathological
studies, we used the venom extracted from the egg-
nests, because of the relative lack of contaminating
proteins and lipids. The Bradford protein assay was
used for the determination of total protein content
(Bradford 1976). Qualitative analysis of proteins was
done by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) in 10 and 12% gels (Lae-
mmli 1970). Protein bands were visualized by the
Coomassie Brilliant Blue R-250 method (Andrews
1986) or by the periodic acid-Schiff reagent for glu-
coprotein detection (Jay et al. 1990). The amidolytical
activity was determined using the chromogenic sub-
strates S-2288 and S-2302 (Instrumentation Labora-
tory, Milan, Italy). Initial p-nitroaniline (p-NA) for-
mation rate was monitored by absorbance at 410 nm.
in a PerkinElmer Lambda Bio 20 spectrophotometer
(PerkinElmer Life and Analytical Sciences, Boston,
MA). The concentration of chromogenic substrates
was 80 !M. Fibrinolytic activity also was evaluated by
the Þbrin lysis plate method (Marsh and Arocha-
Pinango 1972) and expressed in square millimeters of
Þbrin lysis area.
Purification of Egg-Nest Extract. Analytical and pre-
parative separation of venom components was per-
formed by anion exchange AE-HPLC on a Waters 626
LC system using a Protein-Pak Q 8HR anion exchange
column (Waters, Milford, MA). Separated fractions
from several high-performance liquid chromatogra-
phy (HPLC) runs were combined, concentrated by
lyophilization, and dissolved in a Þnal volume of 1 ml
by the addition of 50 mM Tris, pH 8.5, supplemented
with 0.05 M NaCl.
442 JOURNAL OF MEDICAL ENTOMOLOGY
Histamine Determination. Determination of hista-
mine in extracts and chromatographic fractions were
done using the histamine enzyme-linked immunosor-
bent assay (ELISA) kit (catalog no. RE59221, IBL
Immuno-Biological Laboratories, Hamburg, Germany)
according to the instructions provided by the manufac-
turer.
Histopathological Studies. The proinßammatory ef-
fect of the 60% ammonium sulfate precipitate or
HPLC-puriÞed fractions was evaluated in an in vivo
bioassay by subcutaneous injection in the central dor-
sal part of the auricle of guinea pigs, Cavia porcellus,
weighing between 300 and 350 g. The animals were
inÞltrated with either whole extract equivalent to 25
!g of total proteins or 5 !g of HPLC-puriÞed proteins.
The total volume injected was 25 !l with proteins
dissolved in sterile normal saline solution adjusted to
pH 7.4 with 10 mM phosphate buffer. Control animals
received only 25 !l of sterile normal saline solution.
After 4 h, the animals were sacriÞced, and the auricles
were removed, Þxed in 4% formaldehyde in phos-
phate-buffered saline, pH 7.4, for 12 h, washed exten-
sively with deionized water, and preserved in 70%
ethanol. The 4-h inoculation time was chosen as op-
timal based on previous observations of the skin re-
actions.
Auricles were sectioned, and tissue samples were
taken from the site of inÞltration, noninÞltrated
sites of the auricle and from the auricular adnexa of
the cranial insertion of the auricle (distal zone).
Sample sections were stained with the following
methods: hematoxylin and eosin, periodic acid-Shiff
for carbohydrate (Cook 1990), and GomoriÕs
trichrome for connective tissue (Bradbury and Gor-
don 1990). Samples were observed in a Nikon E600
light microscope (Nippon Kogahu KK, Tokyo, Ja-
pan). Images were obtained with a Nikon Coolpix
5000 digital camera. The ImageJ (version 1.34n)
public domain Java image processing package (Na-
tional Institute of Health, Bethesda, MD) was used
to calculate the areas and count particles (Johans-
son et al. 2001, Gao et al., 2002). The brightness and
contrast of the images were increased to separate
the hematoxylin-stained nuclei from the back-
ground in the connective tissue, and the color range
threshold was set to exclusively encompass the nu-
clei. The area percentage occupied by nuclei was
calculated and taken as an indicator of the number
of nucleated cells and is referred to as cellularity in
this study.
Statistical Analysis. Values for determination of total
protein concentration, speciÞc activity in synthetic
substrates, and Þbrinolytic activity were done with
samples from Þve separate reproductive cycles. Cell
count values in the histopathological studies of sam-
ples from animals treated with either whole precipi-
tate or chromatographic fractions were analyzed by
StudentÕs t-test. The data analysis package Microsoft
Excel for Windows (Microsoft, Redmond, WA) was
used for descriptive statistic analysis of the data.
Vol. 44, no. 3
Fig. 1. SDS-PAGE analysis of extracts from adults and
egg-nests of H. metabus. Lanes 1 and 10, molecular mass
markers (kilodaltons). Lanes 2, 3, 8, and 9, extract from adult
male setae. Lanes 4 and 5, extract from adult female setae.
Lanes 6 and 7, extract from egg-nests.
Results
Characterization of Proteins. The SDS-PAGE pro-
Þle of whole extracts from adult males, females, and
egg-nests are shown in Fig. 1. A band of !36.8 kDa was
present in all samples. Adult males (lanes 2, 3, 8, and
9) showed a double band of !41Ð 42 kDa, which also
was seen in the adult females (lanes 4 and 5) but not
in the egg-nest extract (lanes 6 and 7). However, a
band of 38.5 kDa was observed in the adult females,
and also more prominently in the egg-nest extract, but
not in males. Faint bands of higher molecular masses
(50 and 66 kDa.) were observed from all extracts.
There was no difference when gels were run under
dissociating or nondissociating conditions.
The separation of the proteins present in the 60%
ammonium sulfate precipitate of the egg-nest whole
extract by AE-HPLC is shown in Fig. 2. Peaks are
numbered 1 to 6. Separate chromatographic fractions
from several runs were pooled. The SDS-PAGE anal-
ysis of the protein fractions obtained from the IE-
HPLC runs are shown in Fig. 3. Peak 1 (lane 3) showed
prominent bands at 30.5, 34.0, and 44.0 kDa and fainter
bands at 27.5 and 29 kDa. Peak 2 (lane 4) shows very
faint bands at 27.5, 31.2, and 32.2 kDa. Peak 3 (lane 5)
shows three bands at 29.0, 33.0, and 34.0 kDa. Peak 4
(lane 6) shows faint bands with the same molecular
masses as peak 3 and also a very prominent band at 38.5
kDa. The only band that stained positive for carbo-
hydrate using the periodic acid-Schiff glycoprotein
detection method was the 38.5-kDa band (data not
shown). Peak 5 (lane 7) contained a single band at 40
kDa, whereas peak 6 (lane 8) showed no bands what-
soever.
The protein concentration, speciÞc activity in chro-
mogenic substrate S-2288 and in vitro Þbrinolytic ac-
tivity of the whole egg-nest extract, 60% ammonium
sulfate, and HPLC fractions are shown in Table 1.
Histamine Determinations. No histamine could be
detected in the extracts and chromatographic frac-
tions used in this study.
Histopathological Studies of Whole Extract Inocu-
lation. Figs. 4 and 5 show the biological response after
inoculation with 25 !g of proteins from the 60% am-
May 2007 LUNDBERG ET AL.: ISOLATION OF PROINFLAMMATORY PROTEINS FROM H. metabus 443

Fig. 3. SDS-PAGE analysis on a 10% gel under reducing

conditions of AE-HPLC puriÞed fractions of 60% ammonium
sulfate precipitate of crude egg-nest extract. Fraction num-
bers refer to chromatogram shown in Fig. 2. Lane 1, molec-
ular mass markers. Lane 2, 60% crude ammonium precipitate
of egg-nest extract. Lane 3, HPLC fraction 1. Lane 4, HPLC
fraction 2. Lane 5, HPLC fraction 3. Lane 6, HPLC fraction
4. Lane 7, HPLC fraction 5. Lane 8, HPLC fraction 6.

leukocytes (PMNLs) and mononuclear cells with pre-

Fig. 2. HPLC chromatogram of the 60% ammonium sul-
fate precipitate obtained from the egg-nest extracts. Chro-
matography was performed on a Waters 626 LC system using
a Protein-Pak Q 8HR anion exchange column operated at a
ßow-rate of 0.4 ml/min in the anionic exchange mode. The
solvent system consisted of 10 mM Tris, pH 8.5, supple-
mented with either 0.05 M NaCl (solvent A) or 0.6 M NaCl
(solvent B). Left side ordinate shows absorbance at 280 nm,
whereas the right side ordinate shows the percentage of
solvent B at any given instant. Analysis by high-performance
liquid chromatography was carried out using a Waters 626 LC
system. Sample volumes of up to 1 ml were applied to the
column. Elution was carried out using a NaCl gradient from
0.05 to 0.5 M.
monium sulfate precipitate of the egg-nest extract in
the dorsal part of the auricle of the experimental
guinea pigs as explained under Materials and Methods.
Control specimens receiving normal saline solution
are shown in Fig. 4A. There was massive migration,
inÞltration, and aggregation of polymorphonuclear
dominance of PMNLs in the extracellular matrix,
veins, and venules at the site of inÞltration (Fig. 4BÐ
D). In some areas petechiae with erythrocytes scat-
tered around the vessels (Fig. 5C) and vasculitis (Fig.
5D) could be observed. Diapedisis of PMNLs through
the vascular wall also could be seen (Fig. 5B). The
intensity of the inßammatory response decreased with
distance from the site of inoculation. The cellularity in
the inÞltrated area was signiÞcantly higher than in
noninÞltrated areas of the ear, such as the tissue of the
ventral side of the intra-auricular cartilage or at sites
distant to the site of inoculation on the dorsal side (P $
0.05, P % 2.8319 " & 10"9) (Fig. 5A and B; Table 2). The
extract used was highly active in the in vitro Þbrin clot
lysis assay (Table 1).
Histopathological Studies of Inoculation with
HPLC Fractions. Fraction 1. This fraction did not
cause any inßammatory reaction after inoculation
with 5.0 !g of protein. The observed tissue samples did
not show any difference compared with tissue samples

Table 1. Protein concentration, specific amidolytic acticity, and fibrinolytic activity of crude 60% ammonium sulfate extract and
HPLC-purified fractions of H. metabus egg-nest extract

Sample Protein content (mg/ml) SpeciÞc activity in S-2288a Fibrin lysis area (mm2)b
"2 "3
Crude extract 1.309 ' 0.21 7.34 & 10 ' 2.49 & 10 45 ' 2.5
60% (NH4)2SO4 precipitate 1.569 ' 0.17 1.51 & 10"1 ' 3.3 & 10"3 58 ' 4.5
HPLC fraction 1 0.66 ' 0.033 0 0
HPLC fraction 2 0.07 ' 0.0073 1.51 & 10"3 ' " 4.5 & 10"5 24 ' 3.0
HPLC fraction 3 0.28 ' 0.0023 1.52 & 10 ' " 2.7 & 10"4
"2
24 ' 20.8
HPLC fraction 4 0.36 ' 0.001 8.4 & 10"3 ' 1.0 & 10"4 0
HPLC fraction 5 0.386 ' 0.002 3.14 & 10"2 ' " 3.5 & 10"4 0

Data are presented as mean ' SD from Þve cycles of puriÞcation of egg-nest extract.
a
SpeciÞc activity is expressed as moles of p-NA released from chromogenic substrate S-2288 (broad-spectrum serine protease substrate)
per mole of protein per minute.
b
For in vitro Þbrin lysis assay, 5 ! l of sample was loaded onto a preformed Þbrin gel.
444 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 3

Fig. 4. Histopathological studies showing the inßammatory response of crude 60% ammonium sulfate precipitate of H. metabus
egg-nest extract. (A) Response of control animals in receiving sterile isotonic saline solution in 10 mM phosphate buffer, pH 7.4.
(BÐD) Massive inÞltration of PMNLs and mononuclear cells in the extracellular matrix and within the venules after receiving
egg-nest extract as described under Materials and Methods. Arrows shown in B and C indicate intravascular aggregates of mainly
PMNLs. D shows massive tissue cellular inÞltration. MagniÞcation, 100& (A) and 200& (BÐD).

from control animals with respect to cellularity (P ( Table 2. Cellularity determined from tissue samples taken
from guinea pigs auricles infiltrated with sterile normal saline (con-
0.05, P % 0.576008) and morphology. There was no trol), egg-nest extract precipitated with 60% ammonium sulphate
lysis of preformed Þbrin (Table 1). and HPLC-purified fractions of precipitate
Fraction 2. Inoculation of 5 !g of HPLC fraction 2
proteins (Fig. 2) on the dorsum of the auricle of the Fraction Cellularity (mean ' SD)a
guinea pig ear caused prolonged bleeding ((20 min.) Control 3.353 ' 0.418
and formation of macroscopic hematomas. The mi- Extract, inÞltrated zone 27.207 ' 9.780
croscopic study of the tissue showed no evidence of Extract, noninÞltrated zone 9.780 ' 3.040
mechanical damage to the vascular structures. There Extract, distal zone 6.233 ' 2.322
HPLC fraction 1 3.580 ' 0.322
was a marked degeneration of the structure of the HPLC fraction 2 13.080 ' 3.825
vessel walls as shown by the lobulation and engorge- HPLC fraction 3 11.887 ' 4.695
ment of venules and extensive extravasation of large HPLC fraction 4 10.380 ' 5.697
quantities of erythrocytes (Fig. 6A, arrow). Impor- HPLC fraction 5 15.580 ' 3.896
tantly, there was no evidence of Þbrin clot formation a
The area percentage occupied by nuclei as an indicator of the
in association with the massive erythrocyte accumu- number of nucleated cells, calculated as described under Materials
lation. There was a slight inßammatory reaction show- and Method. Values are average from 10 determinations.
May 2007 LUNDBERG ET AL.: ISOLATION OF PROINFLAMMATORY PROTEINS FROM H. metabus 445

Fig. 5. Histopathological studies showing the inßammatory response of crude 60% ammonium sulfate precipitate of
H. metabus egg-nest extract. (A and B) Massive intravascular aggregates and tissue inÞltration of leukocytes in the
connective tissue of the dorsal part of the auricle. Petechiae were a common Þnding in these samples. (C) Petechial
bleeding of the dorsal part of the auricle in vicinity to the lesion produced by the venom (arrow). Frank vasculitis was
also a common Þnding as shown in D (arrow). MagniÞcation, 100& (A) and 200& (BÐD).

ing PMNLs scattered in the tissue surrounding the
inÞltrated area. The cellularity value for inÞltrating
leukocytes was statistically different from the control
tissue (P $ 0.05, P % 2.3269 " & 10"9). The fraction 2
proteins did produce a signiÞcant lysis of preformed
Þbrin in vitro (Tables 1 and 2).
Fraction 3. The lesions caused by inÞltration with
fraction three proteins showed a moderate leuko-
cyte inÞltration, statistically different from control
animals with regard to cellularity in connective tis-
sue (P $ 0.05, P % 0.0003) but no statistically dif-
ferent from fractions 2 (P ( 0.05, P % 0.186189) and
4 (P ( 0 0.05, P % 0.152094) (Fig. 6B; Table 2). The
animals inoculated with fraction 3 protein showed
prolonged bleeding at the injection site and also a
moderate number of areas with erythrocyte extrav-
asation. However, the amount and severity of the
bleeding were far less than that seen from fraction
2 inoculation. Intravascular degradation of clots was
observed. Fraction 3 proteins produced an in vitro
Þbrin lysis of the same magnitude as fraction 2
proteins (Table 1).
Fraction 4. Inoculation with fraction 4 proteins pro-
duced a moderate but statistically signiÞcant increase
of intravascular leukocyte aggregation and tissue in-
Þltration compared with the control group (P $ 0.05,
P % 0.0003) (Fig. 6C; Table 2). A marked inÞltration
of leukocytes into the surrounding extravascular con-
nective tissue with predominance of PMNLs could be
seen adhering to the endothelium and in the process
of diapedesis. No hemorrhage or damage to blood
vessels was observed. Fraction four proteins did not
cause any in vitro lysis of preformed Þbrin (Table 1).
Fraction 5. Fraction 5 protein caused a marked
tissue leukocyte inÞltration (Fig. 6D) compared
with the control group (P $ 0.05, P % 0.0004). This
446 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 44, no. 3

Fig. 6. Histopathological studies showing the inßammatory response of HPLC-puriÞed fractions of H. metabus egg-nest
extract. (A) Structural derangement with lobulation and engorgement of the vessel with erythrocytes is clearly visible
(arrow), caused by inoculation of 5 !g of HPLC fraction 2 proteins (see Fig. 2). Note the absence of Þbrin clot formation
in association with the erythrocyte masses. (B) Effect of HPLC fraction 3 on the dorsal tissues of the auricle showing moderate
leukocyte inÞltration and erythrocyte extravasation. Note the absence of Þbrin clot formation associated with erythrocytes
(arrow). (C) Tissue response to HPLC fraction 4 proteins. A marked inÞltration of leukocytes with a predominance of PMNLs
in the perivascular connective tissue was evident. In addition, leukocytes adhering to the vascular endothelium and actively
migrating toward the perivascular tissue are clearly seen (arrow). (D) Effect of HPLC fraction 5 protein on the auricular
tissue. There was a high degree of tissue cellular inÞltration (signiÞcantly higher than HPLC fraction 4 proteins). SigniÞcant
aggregation of intravascular leukocytes was clearly observed (arrow). MagniÞcation, 200&.

cellular inÞltration was signiÞcantly higher than
fraction four proteins (P $ 0.05, P % 0.009). The high
degree of tissue leukocyte inÞltration was the chief
characteristic of this fraction. Fraction 5 did not
cause any lysis of preformed Þbrin in in vitro assays
(Tables 1 and 2).
Fraction 6. Fraction 6 did not produce any signs of
inßammatory reaction or edema. There was no lysis of
preformed Þbrin in vitro assays (Table 1).
Discussion
Dermatitis caused by exposure to the urticating
setae of H. metabus gives rise to an intense pruritic
dermatitis. There is a consensus that it is the femalesÕ
abdominal setae (ßéchettes) that are responsible
for the dermal lesions by carrying and introducing
highly irritant substances into the dermisÐ epider-
mis (Hill et al. 1948, Vicente 1952, Gusmao et al.
1961, Dinehart et al. 1985). These dart-like ßéch-
ettes, 50 Ð100 !m in length and !1.5 !m in diameter,
have been seen incrusted deeply in the dermal le-
sions (Dinehart et al. 1985). During oviposition, the
female sheds large amounts of setae, including the
ßéchettes originating from the last abdominal seg-
ments (!50,000 per mm2) to cover and protect the
eggs. These egg-nests have very pronounced urti-
cating properties (Tisseuil 1935, Benaim-Pinto et al.
May 2007 LUNDBERG ET AL.: ISOLATION OF PROINFLAMMATORY PROTEINS FROM H. metabus
1991). Adult females and egg-nests both have a
prominent band at !38.5 kDa in the SDS-PAGE
analysis of extracts, which is absent in adult males
(Fig. 1). Because the adult males do not have any
urticating properties, the 38.5-kDa band may be
associated with the proinßammatory activity ob-
served in this study.
The papuloerythematous dermatitis produced by
exposure to the urticating setae of H. metabus varies
in size from a few millimeters to several centimeters,
being solitary, or occurring in large numbers in
exposed areas. In the latter case, papules are usually
conßuent. The onset of the dermal reaction is typ-
ically of a retarded nature starting from 1 to 6 h after
exposure to urticating material in humans. The der-
mal lesions give rise to an intense pruritus that
worsens with scratching (Benaim-Pinto et al. 1991,
1992). Ocular complications are fortunately rare,
but they require careful treatment when present
(Vicente 1952, Ducombs et al. 1983, Dinehart et al.
1985). General malaise and arthralgia also have been
reported (Dinehart et al. 1985). Respiratory symp-
toms are rare and usually seen only in predisposed
persons or in cases of direct exposure to the upper
airways (Ducombs et al. 1983, Dinehart et al. 1985).
Histopathological examination of skin biopsies of
skin lesions resulting from exposure to Hylesia
paulex (a Hylesia species reported exclusively from
Brazil) revealed rounded areas of patchy necrosis of
the dermis and epidermis with an inßammatory in-
Þltrate consisting of disintegrating PMNLs. Small
dermal vessels exhibited Þbrinoid necrosis. Ne-
crotic vessels were surrounded by an inßammatory
inÞltrate composed mainly of PMNLs and lympho-
cytes presenting nuclear pyknosis and karyorrhexis.
On the basis of these Þndings, the lesions were
characterized as a cutaneous leukocytoclastic vas-
culitis (Benvenuti et al. 1998). The lesions produced
by adult females of Hylesia metabus (Cramer) (east-
ern Venezuela) have been described clinically as an
urticarial vesicular or papuloerythematous dermatitis
with discrete vesicular degeneration and necrosis. Ec-
chymotic spots also have been reported. Histopatho-
logical studies have shown hydropic degeneration of
epidermal cells, spongiosis and cellular inÞltration by
lymphocytes, histocytes, and neutrophils. (Zaias et al.
1969, Benaim-Pinto et al. 1991). The results of the
current study using an animal model are in clear ac-
cordance with these Þndings. Thus, inoculation with
egg-nest extract caused a massive migration, inÞl-
tration, and aggregation of PMNLs and mononu-
clear cells with predominance of PMNLs in the
extracellular matrix, veins, and venules. PMNLs
could be observed in the process of diapedesis
through the vessel walls. Petechiaes and frank vas-
culitis also was observed frequently.
The nature of the urticating substances and their
mechanism of action in the etiology of these lesions
have been a matter of controversy. The presence of
histamine in the urticating hairs has been reported
previously and has been implicated as a mediator
responsible for the dermal lesions (Dinehart et al.
447
1985). However, the effect of histamine is of very
short duration. Therefore, it seems unlikely that
histamine alone would be of importance in the long-
lasting inßammatory reactions that are typically
seen after exposure to the urticating setae of H.
metabus. In addition, the extracts and chromato-
graphic fractions used in this study were all negative
when assayed for the presence of histamine by using
a sensitive histamine ELISA test. Other studies also
have failed to detect histamine in extracts from H.
metabus urticating setae (Benaim-Pinto et al. 1992).
Therefore, we do not think that histamine present
in the urticating setae has any signiÞcant role in the
extended inßammatory response.
It has been suggested that there must be other
inßammatory mediators present in the setae (Dine-
hart et al. 1985). Several studies have noted the
presence of substances with highly antigenic and
enzymatic properties (Benaim-Pinto et al. 1992,
Lundberg et al. 2002). In the current study, we have
shown in an animal model that the aqueous extracts
from the egg-nests of H. metabus are capable of
reproducing the histopathological alterations de-
scribed previously. In addition, we have shown that
this venom may be separated by anionic exchange
chromatography into separate components having
different biological activities. Fractions 2 and 3 from
the chromatographic separation caused vascular
damage and petechial bleedings. There was clear
evidence of marked degradation of the vessel walls
evidenced by lobulation, engorgement of venules,
and extensive extravasation of large quantities of
erythrocytes. Both of these fractions also showed an
in vitro Þbrinolytic activity. There was no signiÞcant
increase of inÞltration of inßammatory cells in these
fractions. Fractions 4 and 5, however, induced a
marked inÞltration of inßammatory cells with no
signs of vascular damage or Þbrinolytic activity. It
should be noted that all fractions contained proteins
with a molecular mass between 31 and 41 kDa with
an amidolytic activity in synthetic peptide substrate
S-2288 speciÞc for a broad range of serine proteases.
The molecular mechanisms by which the puriÞed
fractions of the venom bring about their respective
biological effects are at present not well understood.
The results of the studies of cutaneous leukocytoclas-
tic vasculitis resulting from exposure to H. paulex sug-
gests an antibody-mediated vascular inßammation
with either deposition or formation in situ of immune
complexes in the vessel wall and complement activa-
tion (Benvenuti et al. 1998). However, a wide range of
stimuli, e.g., cytokines, bioactive lipids, and even
agents such as monosodium urate crystals and serum-
activated zymosan particles, are efÞcient activators of
PMNLs (Claesson et al. 1981; Serhan et al. 1984a,
1984b; Štvrtinová et al. 1995). In addition, a trypsin-
like activity has been described previously with ex-
tracts of setae from adult females that may be related
to the vasodegenerative and necrotic traits of the
whole extract as well as fractions 2 and 3 (Benaim-
Pinto et al., 1992). Apart from the direct toxic effect of
the components of the moth venom, the possibility of
448 JOURNAL OF MEDICAL ENTOMOLOGY
allergic reactions toward the moth must also be eval-
uated. Studies are presently being undertaken to de-
termine what immunological mechanisms and medi-
ators may be involved in the pathophysiology of the
cutaneous lesions caused by exposure to the urticating
setae of H. metabus.
Acknowledgments
We thank Frances Osborn of the “Instituto de Investiga-
ciones en Biomedicina y Ciencias Aplicadas, Universidad de
Oriente, Cumana, Venezuela” and also the Þeld technicians
of the H. metabus project for invaluable support in making
this study possible. We deeply acknowledge the patience and
spirit of collaboration of the people living in the affected
areas (municipalities of Benitez, Libertador, Cajigal, Mariño,
and Valdéz of the state of Sucre). We also are indebted to the
staff of FUNDACITE of the state of Sucre, Venezuela. We
acknowledge the support provided by the Department of
Structural Biology of the Venezuelan Institute for ScientiÞc
Investigation (Departamento de Biologṍa Estructural, Insti-
tuto Venezolano de Investigaciones CientṍÞcas, IVIC). We
also thank Antonio Aguado for assistance in the animal ex-
periments. Finally, we acknowledge the skillful help of
Rebeka Powell de Vaz in the preparation of this manuscript.
This project, identiÞed as “Proyecto Reto Hylesia metabus,
subproject Caracterización de la sustancia urticante de la
mariposa Hylesia metabus (”Palometa Peluda“) y identiÞca-
ción de inhibidores de la misma”, (Project Hylesia metabus,
sub-project: Characterization of the Urticating Substance of
the butterßy Hylesia metabus and identiÞcation of its inhib-
itors) has been Þnanced by the Ministerio de Ciencia y
Tecnologṍa de la República Bolivariana de Venezuela (“Min-
istry of Science and Technology of the Boliviarian Republic
of Venezuela”).
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Received 3 November 2006; accepted 1 February 2007.