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ORIGINAL ARTICLE /
ARTÍCULO ORIGINAL
FIRST ELECTRON PROBE X-RAY MICROANALYSIS OF THE ELEMENTAL
COMPOSITION OF THE LYOPHILIZED LATEX OF
EUPHORBIA MILII
VAR.
HISLOPII
AND ITS IMPACT IN THE MALE
SCHISTOSOMA MANSONI
LA PRIMERA SONDA DE ELECTRONES DE MICROANÁLISIS DE RAYOS X DE LA
COMPOSICIÓN ELEMENTAL DEL PRODUCTO LIOFILIZADO DE LATEX DE
EUPHORBIA MILII
VAR.
HISLOPII
Y SU IMPACTO EN EL MACHO DE
SCHISTOSOMA
MANSONI
1,211
Ronaldo de Carvalho Augusto; Clélia Christina Mello-Silva; Mario Jorge Gatti;
31
*
Claudio L. Mafra & Claudia Portes Santos
1
Laboratório de Avaliação e Promoção da Saúde Ambiental, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Av. Brasil
4365, Manguinhos, Rio de Janeiro, RJ, 21040-361, Brasil.
2
Programa de Pós-graduação em Biologia Parasitária, Instituto Oswaldo Cruz, Fiocruz.
3
Laboratório de Parasitologia e Epidemiologia Molecular, Universidade Federal de Viçosa, Minas Gerais, MG, Brasil.
* Corresponding author to Ronaldo de Carvalho Augusto (Augusto, RC) Tel.: +55 21 25621526
E-mail address
: augustorc@ioc.fiocruz.br (Ronaldo de Carvalho Augusto).
Neotropical Helminthology, 2016, 10(1), ene-jun: 13-22.
ABSTRACT
Keywords
: Cercariae - Chemical elements -
Euphorbia milii -
Schistosomiasis - Tegument
-
X-ray microanalysis
The aim of this work was to study the elemental composition of the lyophilized latex obtained
from
Euphorbia milii var. hislopii
(N.E.Br.) Ursch & Leandri) and the tegument of adult male of
Schistosoma mansoni
Sambon, 1907 originated from cercariae previously exposed to the latex.
The composition and concentrations of 15 elements were characterized using scanning electron
microscopy (SEM) and energy dispersive spectroscopy (EDS). The
Magnesium, Potassium and
Rhodium were found in high
weight percent
in the lyophilized latex. High levels of Sodium,
Nitrogen and Magnesium elements were detected respectively in control adult male
S. mansoni
.
The cercariae exposition of
S. mansoni
to the
E. milii
latex changed the elemental composition of
adult parasite, which showed higher level of Nitrogen instead of Sodium as observed in normal
parasites. Furthermore the comparison of elements of the control group and the adult worms from
cercariae exposed to the latex showed a significant decrease in Nitrogen and increase in Sodium,
Potassiumand Magnesium. The present work represents the first analysis of the elemental
composition of the lyophilized latex of
E. milii var. hislopii
and its impact in the male
S. mansoni
by Electron Probe X-Ray Microanalysis.
13
ISSN Versión impresa 2218-6425
ISSN Versión Electrónica 1995-1043
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14
RESUMEN
Palabras-clave
: cercarias - Elementos químicos -
Euphorbia milii
- Esquistosomiasis - Tegumento
microanálisis de rayos X -
El objetivo de este trabajo fue estudiar la composición elemental del látex liofilizado obtenido de
Euphorbia milii
var.
hislopii
(N.E.Br.) Ursch & Leandri) y el tegumento del macho adulto de
Schistosoma mansoni
Sambon, 1907 originado a partir cercarias previamente expuesta al látex.
La composición y las concentraciones de 15 elementos se caracterizaron mediante microscopía
electrónica de barrido (SEM) y la espectroscopía de dispersión de energía (EDS). El magnesio,
potasio y sodio se encontraron en un alto porcentaje en peso en el látex liofilizado. Altos niveles
de los elementos sodio, magnesio y nitrógeno se detectaron, respectivamente, en el control de los
machos adultos de
S. mansoni
. La exposición de cercarias de
S. mansoni
al látex de
E. milii
cambió la composición elemental del parásito adulto, que mostró un mayor nivel de nitrógeno en
lugar de sodio como se observó en los parásitos normales. Además, se compararon los elementos
del grupo de control y de los gusanos adultos de las cercarias expuestas al látex y mostraron una
disminución significativa de nitrógeno y un aumento de sodio, potasio y magnesio. El presente
trabajo representa el primer análisis de la composición elemental del látex liofilizado de
E. milii
var.
hislopii
y su impacto en el macho de
S. mansoni
por la Sonda de microanálisis de Electrones
de Rayos X.
Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016
INTRODUCTION
On Schistosomiasis control studies, despite the
efficacy of chemotherapy in reducing
morbidity, a decrease in the transmission of the
parasite was not achieved (Bockarie
et al.,
2013). Among these, many studies using
bioactive plant compounds have been
undertaken (Yadav & Jagannadham, 2008;
Singh
et al.,
2010; Hamed, 2010; Mello-Silva
et al.,
2011; Lima
et al
., 2012). In these,
Euphorbia milii
var.
hislopii
has been
indicated as the most promising for use in
official WHO programs for schistosomiasis
control (WHO 2002). The biocidal activity of
the latex has been tested for the control of
snail's intermediate hosts of different parasites
(Vasconcellos & Schall, 1986; Giovanelli
et
al.
, 2001; Vasconcellos & Amorim, 2003;
Mello-Silva
et al.,
2010; Crignis
et al.,
2012).
One of the main practices to evaluate the effect
of control actions in
S. mansoni
is to evaluate
the tegument of the parasite, because this
structure is important to the survival,
performing vital functions such as the
absorption of organic and inorganic
compounds and the evasion of the host
immune system (Braschi
et al.
, 2006). In
addition, the male worm tegument is especially
important because it is also responsible for the
development and maturation of female worms
(Ribeiro-Paes & Rodrigues, 1997). The
tegumental surface of
S. mansoni
exposed to
anthelmintic drugs, exhibits deformations
such as scaling, erosion, spikes and bubbles
(Xiao
et al.
, 2000; Lorsuwannarat
et al.,
2013).
Despite the extensive accumulated knowledge
of tegumental functions and biochemistry,
little is known of the inorganic composition of
this structure under the pressure of
anthelmintic drugs. In recent years, the
elemental microanalysis method, based on
scanning or transmission electron
microscopy/energy dispersive X-ray
spectrometry (SEM/EDS or TEM/EDS), has
been applied to the detection and semi-
quantification of chemical elements across a
broad spectrum of physical and biological
samples, including parasites (Goldstein
et al.
,
Carvalho
et al.
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15
2003; Buron
et al
., 2009; Morais & Malta,
2016).
The aim of this work was study the elemental
composition of the lyophilized latex obtained
from
Euphorbia milii
var.
hislopii
and the
tegument of adult male of
S. mansoni
originated from cercariae previously exposed
to the latex.
Euphorbia milii
var.
hislopii
samples
The
E. milii
var.
hislopii
latex was collected in
the Ilha do Governador district
(22°48´09´´S/43°12´35´´W), Rio de Janeiro,
Brazil (Vasconcellos & Amorin, 2003). The
latex sample collected was pre-frozen in dry
ice and absolute ethanol and subsequently
-1
lyophilized at -52°C on 8 x10mBar during
three cycles of 12 h in a Modulo 4K Freeze
Dryer with an acrilic chamber (Edwards High
Vacuum Int., UK). The lyophilized pellet
obtained was diluted in distilled water and
homogenized by sonication for 20 min.
Schistosoma mansoni
adult's samples
The
B. glabrata
(Belo Horizonte-lineage BH)
used in the experiments had shell diameters
between 8-10 mm and they were individually
infected with 7±1 miracidia of
S. mansoni
(lineage BH) at the Laboratório de
Esquistossomose Experimental (LEE),
Instituto Oswaldo Cruz. After thirty days, the
snails were individually exposed to light (60-
watt incandescent bulb for 1 h) in becker (5ml)
with distilled water to look for the release of
cercariae. After this period, the snails were
removed and the cercariae were separated into
two groups.
The first cercarial group was kept in distilled
water for one hour. The second group was
exposed for the same period (one hour) to a
sublethal concentration of the powdered
lyophilized latex of
E. milii
(LC=1.4 mg/L) as
50
described by Schall
et al.
(1992) for
Biomphalaria glabrata
snails.
After this, fifteen female Swiss-Webster mice
were infected with exposed cercariae and
another fifteen with unexposed cercariae
(control group). At 65 days post-infection, part
of
S. mansoni
males, both the exposed and
control groups, were collected and fixed in
70% ethanol.
The worms were dehydrated in an ethanol
series (80%, 90% and three changes of 100%)
prior to being critical point dried using CO.
2
Powdered lyophilized latex, four specimens of
the control group and four of the exposed
group of S. mansoni were mounted in stubs,
sputter-coated with carbon and observed in a
Scanning Electron Microscope Leo 1430VP
coupled to a probe X-ray (EDS) at the Núcleo
de Microscopia e Microanálise of Centro de
Ciências Biológicas e da Saúde, Universidade
Federal de Viçosa, Minas Gerais. We selected
for SEM-EDS analysis the following
elements: N (Nitrogen), Na (Sodium), Mg
(Magnesium), Al (Aluminum), K (Potassium),
Cr (Chromium), Fe (Iron), Cu (Copper), Rh
(Rhodium), Pd (Palladium), Ag (Silver), Cd
(Cadmium), Pt (Platinum), Hg (Mercury) and
Pb (Lead). The qualitative and semi-
quantitative composition of these elements
were determined using the X-ray spectrum
performed on a manually selected area and
took into consideration 100% of the total
amount of weight percent of elements
measured (wt.%).
The results were expressed for each sample as
the mean ± standard deviation and submitted to
analysis of variance (ANOVA) and Student's t
tests
(α = 5%
, p
≤
0,05) (GraphPad, V.4.00,
Prism, GraphPad, vol. 3.02 Prism Inc.).
Ethics
This research was approved by the Animal
Ethics Committee of the Oswaldo Cruz
MATERIALS AND METHODS
Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016
X-ray microanalysis
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16
Foundation (CEUA-FIOCRUZ LW-07/13), in
accordance with the guidelines of the Brazilian
College for Animal Experiments (COBEA).
The ultrastructure of the lyophilized latex
varied in the size and shape of the particles.
Even after lyophilization, the latex formed
pellets, clustered and arranged its particles of
different sizes and shapes in the form of a resin
(Fig. 1A).
The energy dispersive spectroscopy (SEM-
EDS) of the lyophilized latex sample showed
the
weight percent
(wt.%)
of
K (45.14), Rh
(31.39), Mg (11.45), Hg (2.58), Pt (2.24), Al
(2.14), Na (2.03), Pb (1.85) Cu (0.47), Cd
(0.42), Cr (0.25) e Fe (0.13). The elements N,
Pd and Ag were absent from the sample of
lyophilized latex.
Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016
RESULTS
Figure. 1
. Structural feature of powered lyophilized latex of
Euphorbia milii
var.
hislopii
and morphology of adult
Schistosoma
mansoni
tegument came from cercariae previously exposed to this latex, by
S
canning electron microscopy: (A) Po
wdered
lyophilized latex. (B) Control tegument of adult male of S. mansoni. (C-D) Tegument of adult male of S. mansoni
exposed to the
latex at the cercarial stage showing intense damage to the external surface.
Carvalho
et al.
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17
Table 1.
Chemicals concentration on tegument of four
adult males of
Schistosoma mansoni
.
Control group (C) and group exposed (E) for one hour to 1.4 mg/L
Euphorbia milii var. hislopii
. Data are expressed as mean ± standard deviation
(α = 5%
. p
≤
0.05). N (Nitrogen). Na (Sodium). Mg (Magnesium). Al (Aluminum).
K (Potassium). Cr (Chromium). Fe (Iron). Cu (Copper). Rh (Rhodium). Pd (Palladium). Ag (Silver). Cd (Cadmium). Pt (Platinum). Hg (Mercury) and Pb (Lead).
Elements
N
Na
Mg
Al
K
Cr
Fe
Cu
Parasites
C
E
C
E
C
E
C
E
C
E
C
E
C
E
C
E
1
6.9
35.0
23.2
17.0
14.6
10.8
13.5
9.2
1.6
0.8
0.2
0.2
0.9
0.8
13.0
12.2
2
15.2
39.5
20.8
14.9
14.3
11.6
14.4
10.4
1.2
0.5
0.2
0.1
0.6
0.5
12.6
8.8
3
23.4
31.5
20.4
13.3
13.33
8.2
10.0
5.6
0.9
0.9
0.1
0.3
0.8
1.1
17.8
21.4
4
3
0.1
41.8
17.4
12.7
11.1
9.9
10.3
9.0
0.9
0.6
0.1
0.1
0.7
0.9
11.8
10.7
Mean
18.9±10.0
37.0±4.6
20.4±2.3
14.5±1.9
13.3±1.6
10.1±1.4
12.1±2.2
8.5±2.0
1.1±0.3
0.7±0.1
0.2±0.0
0.2±0.0
0.7±0.1
0.8±0.2
13.8±2.6
13.3±5.5
P
–
value
0.017
0.008
0.025
0.060
0.037
0
.
479
0.624
0.878
Elements
Rh
Pd
Ag
Cd
Pt
Hg
Pb
Parasites
C
E
C
E
C
E
C
E
C
E
C
E
C
E
1
11.3
3.6
0.6
0.0
0.8
0.000
0.614
0.000
3.423
3.674
3.472
2.680
5.322
3.59
2
9.78
6.3
0.7
0.0
1.1
0.213
0.000
0.000
3.077
1.876
2.845
2.634
2.806
2.09
3
1.3
1.6
0.0
0.0
0.0
0.000
0.000
0.242
3.768
4.794
4.146
5.428
3.606
5.15
4
7.4
5.2
0.0
0.0
0.3
0.108
0.000
0.000
3.137
2.581
3.553
2.783
2.664
3.07
Mean
7.4±4.4
4.2±2.0
0.3±0.4
0.0±0.0
0.5±0.5
0.08±0.1
0.15±0.3
0.06±0.12
3.35±0.3
3.2±1.2
3.5±0.5
3.3±1.3
3.6±1.2
3.4±1.2
P
-
value
0.227
-
0.091
0.593
0.861
0.872
0.898
Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016
X-ray microanalysis
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18
The SEM-EDS analyses of adult males of
S.
mansoni
in control and exposed groups were
conducted in the mid-dorsal section of the
tegument (Figs 1B-1D). Each of the 15
elements analyzed in each worm and their
weight as a percentage (wt.%) are presented in
Table 1. As determined by EDS, all elements
were present in both groups, but Pd was absent
in the exposed group. The levels of
alkali
metals Na (p<
0,008), K
(p<
0,037) and of
alkali earth metal Mg (
p<
0,025) w
ere
significantly higher in the tegument of the
control group
and the non-metal N
was
significantly higher in the exposed
group. The
percentages of Cr, Fe, Cu, Pt, H and Pb did not
vary significantly between the control and the
exposed groups.
The ultrastructure of the normal tegument in
the mid-dorsal part of males of
S. mansoni
showed a pattern of a well organized
distribution of tubercles and spines (Fig. 1B).
The tegument of adult parasites originating
from
cercariae exposed to the latex of
E. milii
var.
hislopii
exhibited pronounced changes in
the mid-dorsal surface. The most significant
morphological changes observed were (i) a
reduction in the number and size of the
tubercles and spines, (ii) a disorganized
distribution of the tubercles and (iii) damage to
the tegument in the form of hollows and
wrinkles (Figs 1C-1D).
The bioaccumulation of metals by helminths is
a well acknowledged phenomenon; this is a
process which, as pointed out in the overview
by Buron
et al.
(2009), can be influenced by
factors such as the nature of the metal itself,
host age and motility, and the age, stage of
development, sex and site of the parasite. The
case, where cercariae of
S. mansoni
, which had
been exposed to the latex of
E. milii
var.
hislopii
, produced effects in adult worms,
showed that bioaccumulation can pass through
different life stages from the environment to
the definitive host.
The
E. milii
var.
hislopii
is a widely distributed
plant that occurs in almost all habitats and
occupies a wide range of climes and soils
(Zahra
et al.
2014). The products of their
secondary metabolites have been described as
rich in diterpenes, triterpenes, flavonoids,
saponins, tannins, alkaloids, esters, ricin and
phenols (Mwine & Damme, 2011). These
compounds have proved to be useful as anti-
plasmodial, anti-malarial and molluscicidal
products (Mwine & Damme, 2011). Previous
tests with cercariae of
S. mansoni
exposed to
this latex showed a slightly toxic effect, but
without death of cercariae (De-Carvalho
et al.,
1998). At present, this is the first description of
the chemical elements of the lyophilized latex
of
E. milii
var.
hislopii
.
Toxic metals were found in the latex, as Al
(2.14%), Pt (2.24%), Hg (2.58%) and Rh
(31.39 %). The occurrence of Rh is rare, being
reported, in the environment as a discharge of
anti-cancer medications or emitted by catalytic
converters for automobile (Sures
et al
., 2003;
Singer
et al
., 2005). In our study, the Rh even in
high concentrations in the latex did not
accumulate significantly in the exposed group
(4.2%) when compared to the control group
(7.4%). In the latex the weight percent of Al,
Cu (0.47) and Pb (1.85) were low in relation to
the control (12.1, 13.8 and 3.6 respectively)
and exposed groups (8.5, 13.3 and 3.4). Morais
& Malta (2016) also observed Cu, Pb and Al,
among other elements, in different digenean
metacercariae from fish caught in Manaus
polluted streams (Brazil). Although they
correlated the presence of these metals with
toxic effluents originating from Manaus
Industrial District factory sewers, it is
interesting to note that Cu, Pb and Al were
present in both our control and exposed
groups, probably indicating that these
elements may occur in different digenean
species.
DISCUSSION
Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016
Carvalho
et al.
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19
Other elements, such as Cd, Ag, and Pd were
found in low concentrations in the tegument of
the worms; the latter was only detected in the
control group. Even though the biological
functions of these elements are understudied, it
is known that they are toxic in high
concentrations, but essential for life in small
quantities (Morley
et al.,
2002). According to
Sigel
et al.
(2009), there is a metallothionein-
protein (MT) responsible for cell protection
against reactive oxidants, including nitric
oxide which has the capacity to bind to heavy
metals such as Cu, Cd and Ag. González-Mejia
et al.
(2014) commented that the nitric oxide
radical has a biological activity limiting the
local inflammation caused by parasites. The
reduction of Cd, Al, Rh and Ag in our exposed
group in relation to MT protection against
schistosomiasis is still to be evaluated.
Shaw & Erasmus (1984) evaluated the
tegument of male of
S. mansoni
by electron
probe X-Ray microanalysis using eight
inorganic compounds. These authors observed
high concentrations of K and low
concentrations of Na, Fe and Mg, respectively.
In our experiment, on the contrary, control
group showed highest values of Na and Mg and
small values of K and Fe. Shaw & Erasmus
(1984) suggested that wt% values can change
according to factors such as the strain of the
parasite and host, fixation method and sample
preparation and the metabolic/physiology
status
of the parasites.
The comparison of elemental composition of
surface tegument between the control group of
male of
S. mansoni
with the parasites formed
from cercariae previously exposed to the latex,
showed significant differences with higher
levels of Na, Mg and K in the control group. In
the exposed group, only N was significantly
higher than in control group.
The tegument of
S. mansoni
seems to be
++
primarily dependent on K and Na gradients
that are responsible for a partial
depolarization
of the surface membranes that leads to
muscular contractions of the parasite (Smyth
& Halton 1983). Comparing the elemental
composition present in the latex with both
male
S. mansoni
groups, we observed a large
amount of K in the lyophilized latex (45.14), a
low percentage in control group (1.1) and the
exposed group had an even lower one (0.7).
With regard to Na present in the latex (2.03)
there was a significant difference (p=0.008)
between the control (20.4) and the exposed
groups (14.5). We observed that the exposure
of cercariae to the latex did not change the
muscle contraction of live adult parasites, in
+
accordance with the proportion of ions of K
+
and Nathat remained regular. Therefore, the
++
latex had no influence in the K and Nabomb.
Galaktionov & Dobrovolskij (2003) described
that the contents in the excretory vesicle of
trematodes also include Calcium (Ca), Mg and
Phosphorous (P). The Mg was found in the
control (13.3) and exposed groups (10.1) with
a significant decrease in the latter. The
alteration of Mg levels may be related to
osmoregulation and also play an important role
in muscle contraction together with Na and K
(Galaktionov & Dobrovolskij 2003).
The tegument of
S. mansoni
constantly renews
the outer layer of its syncytium, being adapted
for ion transport, osmoregulation, absorption
and the secretion of substances (Faghiri
et al.,
2010). Besides, the parasite presents the
mimetism capacity to scape to the host's
immune system. The changes in wt% of the
elemental composition of
S. mansoni
tegument
may be involved in the morphological changes
observed in tegument of the exposed group
such as: reduction of tubercles and spines.
Similar results were reported in the male
tegument of
S. mansoni
exposed to
praziquantel, artemether and the latex of allicin
(Liang
et al.
, 2001; Xiao
et al.,
2002; Lima
et
al.,
2012). These morphological changes may
weaken the tegument, interfering with
exchanges through the syncytium and thus
Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016
X-ray microanalysis
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20
affecting the physiology of
S. mansoni.
We conclude that when the cercariae of
S.
mansoni
are exposed to
E. milii
latex, the
subsequent adult male presents alterations in
morphological and elemental composition of
the tegument. It is possible that these changes
may indirectly affect the ontogeny of the
parasite.
We are grateful to David I. Gibson from The
Natural History Museum, London, UK, for his
valuable suggestions, Francisco Paumgartten
from Escola Nacional de Saúde Pública
(ENSP/Fiocruz) for the use of equipament and
Patricia Pinto Machado from Laboratório de
Esquistossomose Experimental (IOC/Fiocruz)
for the technical and scientific support. This
study was funded by the
Fundação Carlos
Chagas Filho de Amparo à Pesquisa no Rio de
Janeiro
(FAPERJ–BIOTA 2012), the
Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior
(CAPES Parasitologia
Básica/2011), PAEF/IOC/Fiocruz and
Conselho Nacional de Pesquisa
(CNPq).
C.P.Santos was sponsored by fellowships from
the
Conselho Nacional de Pesquisa
(CNPq)
and R.C. Augusto from CAPES.
2006.
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Ringwood, AH, Rolando, E &
Richardson, D. 2009.
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Silva, AG & Cruz ZMA .2012.
De-Carvalho, RR, Maldonado, Jr A, Oliveira-
Filho, EC, Ribeiro, AC, Paumgartten,
FJR, & Rey, L. 1998.
Faghiri, Z, Camargo, SMR, Huggel, K,
Forster, IC, Ndegwa, D, Verrey, F &
Skelly, PJ. 2010.
Galaktionov, A & Dobrovolskij, AA. 2003.
Giovanelli, A, Silva, CLPAC, Medeiros, L &
Vasconcellos, MC. 2001.
The tegument surface membranes
of the human blood parasite
Schistosoma mansoni:
A proteomic
analysis after differential extraction
.
Proteomics, vol. 6, pp.1471–1482.
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