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Ecological Indicators 95 (2018) 663–672
Contents lists available at ScienceDirect
Ecological Indicators
journal homepage: www.elsevier.com/locate/ecolind
Original Articles
Ariadna spiders as bioindicator of heavy elements contamination in the
Central Namib Desert
T
⁎
E. Contia, , G. Costaa, G. Liberatorib, M.L. Vannuccinib, G. Protanob, F. Nannonib, I. Corsib
a
b
Department of Biological, Geological and Environmental Sciences, University of Catania, Italy
Department of Physical, Earth and Environmental Sciences, University of Siena, Italy
A R T I C LE I N FO
A B S T R A C T
Keywords:
Biomonitoring
Heavy elements
Oxidative and cholinergic responses
Central Namib Desert
Spiders
Ariadna sp.
The present study represents the first attempt to promote Ariadna spider as bioindicator of heavy elements
contamination in the central region of the Namib Desert. Various spider populations belonging to undescribed
Ariadna species have been described in Central Namib gravel plains and identified as sit-and-wait predators. By
spending their life in individual tunnels, Ariadna spiders resemble the behaviour of ground-dwelling spiders that
are well known as bioaccumulators of heavy elements. In the present study, 60 individuals of Ariadna spiders
were collected from three populations located at various distances from major mining areas of Namib Desert.
Contents of fifteen heavy elements were measured in sand samples taken around spider burrows as well as in
spider bodies. Several oxidative stress responses as CAT and GST, MDA levels, aside cholinergic function
(Cholinesterases), were assayed. Body burden of Ag, Cd, Cu and Zn resulted significantly higher than levels
measured in sand, while Co, Cr, Ni, Pb, U and V levels were one or two order of magnitude lower in spider body
than in sand samples. All enzymes activities (CAT, GST, ChE) as well as MDA levels were detectable in Ariadna
spiders despite lower values than other species. Though we cannot affirm that all sampled spiders belong to the
same species, their enzymatic activities reflect their ability to accumulate heavy elements regardless the specific
habitat features, confirming so the value of this genus as bioindicator of heavy elements in the Central Namib
Desert.
1. Introduction
Mining activities, including mine exploitation, ore processing/
smelting and waste disposal in dump, represent a source of toxic heavy
elements that may cause adverse impact on the environment by contaminating air, soil and natural waters (Asensio et al., 2013; Bussinow
et al., 2012; Dudka and Adriano, 1997; Heath et al., 1993; Maxwell and
Strager, 2013; Mudrak et al., 2012).
Namibia is very rich in natural resources such as ore deposits of
copper, zinc, gold, tin, lead, uranium and other heavy elements.
Therefore, an intense mining industry has been long developed over the
past 50 years in the territory of Namibia, including the Namib Desert
and large parks and protected areas (e.g., Dorob and Namib Naukluft
coastal parks; Schneider, 2000).
However, any potential impact associated with mining activities in
terms of ecological processes in super arid areas like the Namib Desert
(Mansfeld, 2006; Sims et al., 2011; Taylor and Kesterton, 2002;
Wassenaar et al., 2013) has not yet been investigated, as well as potential synergism with documented climate changes (Ford et al., 2011;
⁎
Norgate and Haque, 2010; Pearce et al., 2011). Therefore, it is urgent to
understand ecological disturbance caused by the increasing mining
activities in Namibia in order to devise better restoration techniques
and to inform decision makers about suitable management options
(Wassenaar et al., 2013).
The use of invertebrate species as bioindicator of heavy element
contamination has been largely adopted by monitoring their distribution, levels and effects in mining areas (Migula et al., 2013; Yang et al.,
2016). Biomonitoring programs using resident, widespread and
adapted species facilitate an efficient and early intervention, and represent a fundamental contribution to ecological risk assessment and
environmental management. The most suitable bioindicator species are
undoubtedly the K-selected ones as they are very sedentary, by binding
tightly to very stable environments, and respond to changes with high
specificity and sensitivity. For this reason, predators such as spiders
have already been used as ecological indicators of environmental pollution (Clausen, 1986; Maelfait, 1996; Wilczek et al., 2013).
From an ecotoxicological point of view, spiders have been also increasingly used as non-conventional terrestrial bioindicators (Wilczek,
Corresponding author.
E-mail address: econti@unict.it (E. Conti).
https://doi.org/10.1016/j.ecolind.2018.08.014
Received 5 April 2018; Received in revised form 23 July 2018; Accepted 9 August 2018
1470-160X/ © 2018 Published by Elsevier Ltd.
Ecological Indicators 95 (2018) 663–672
E. Conti et al.
three sites.
2017). Their ability to survive under extreme conditions has allowed
them to colonize several ecosystems; there are no terrestrial zones
where they are not present, consequently their ecological role cannot be
underestimated (Babczynska et al., 2006; Nyffeler and Birkhofer,
2017).
Spiders are often used also as bioaccumulators due to their ability to
concentrate heavy elements present in the surrounding environment
(Wilczek and Babczyńska, 2000; Hendrickx et al., 2004; Jung and Lee,
2012; Yang et al., 2016). According to Dallinger (1993), spiders can be
recognized among macroconcentrators of heavy elements sharing this
peculiar property with other terrestrial invertebrates. In fact, spiders
are able to store heavy elements such as Zn, Pb, Cu, Hg, Cd, Fe, in
temporal spherites (Ludwig and Alberti, 1988) or mineral granules
(Brown, 1982; Hopkin, 1989) inside midgut gland cells which are involved in metal digestion, storage and detoxification (Wilczek and
Babczyńska, 2000; Wilczek, 2017).
The bioaccumulation of heavy elements and the response of antioxidative enzymes have been suggested to be species-specific in spiders.
They largely depend on animal sex and physiological status, but also on
element properties, concentration and behavior in the environment in
which the spider is living (Wilczek and Babczyńska, 2000; Wilczek
et al., 2013; Stalmach et al., 2015a,b). In this study, we considered
psammophilous spiders that, living in burrows in the sand (where the
temperature is much more tolerable than the surface), are enable to
retain a certain degree of humidity avoiding the dryness of the organism itself (Seely and Mitchell, 1987; Costa, 1995). They are not
active hunters, but they wait the prey in their burrows, identifying it
through sand vibrations (Costa et al., 1993; Costa and Conti, 2013).
Spiders can accumulate more than 80% of their prey, which are usually
more mobile, while they spend all life in the burrows; therefore they
may reflect a contamination from a larger area (Wilczek and Migula,
1996). Furthermore, these spiders are generally polyphagous and many
of them can tolerate long periods of starvation due to their low metabolism, lower than other spiders living in other deserts of the World
(Henschel, 1994; Lubin and Henschel, 1996).
The existence on the gravel plains of the Namib Desert of numerous
and large tube-dwelling spider populations belonging to undescribed
Ariadna species, has been discovered long time ago (Costa et al., 1993).
In spite of different samplings carried out at various seasons and years,
no adult males have been so far found inside Ariadna burrows, but only
spiderling, immature and adult females. However, the existence of
males has been indirectly demonstrated, both considering the high
value of genetic variability and finding spermatozoa in female spermathecae (Dallai et al., 2006; Conti et al., 2007).
The individual burrow of these spiders is very particular, as it is dug
vertically into the ground, covered internally with silk, and has a circular entrance surrounded by a stone ring, with sometimes also lichen
bits (Costa et al., 1995). The features of the burrow rings vary according
to population and habitat (Costa et al., 2000).
As stated above, Ariadna spiders could be very sensitive towards
habitat features and changes in mining areas of Namibia (Costa and
Conti, 2013; Conti et al., 2015).
The present study is the first attempt to use Ariadna spider as
bioindicator of heavy element contamination in the Central region of
the Namib Desert by using an integrated approach based on ecological,
geochemical and biochemical analyses. Spider burrows were measured
by recording their entrance diameter and depth. Concentrations of fifteen heavy elements were determined in spider whole body as well as in
sand samples collected around their burrows. Several oxidative stress
responses as Catalase (CAT) and Glutathione-s-transferase (GST),
Malondialdeide
(MDA)
levels
and
cholinergic
function
(Cholinesterases) were assayed for the first time in this species using
whole body due to small size of the animal.
Through a detailed statistical analysis, the levels of heavy elements
in sand and spider body were compared, as well as the biometric
parameters and biological responses in spider populations from the
2. Materials and methods
2.1. Geological and climatic features of the Central Namib Desert
The territory of Namibia experienced a long and complex geologic
history resulting in a great variety of rocks and tectonic structures. The
geology of western sector consists of metamorphic, magmatic and sedimentary rocks formed from 2600 to 132 million years ago (Miller,
1983; Vollgger et al., 2015). In the eastern sector of Namibia younger
and terrestrial sediments of the Kalahari Group (70 Ma-present) crop
out. The geology of Namibia is also characterized by several ore deposits that are mainly found in the western sector, associated mostly to
the rocks of the Namaqua metamorphic complex and Damara Supergroup. Ore formation processes, geological setting, host rocks and ore
mineralogy distinguished various types of base, precious and rare metal
mineralizations (e.g., Cu, Pb, Zn, Sn, W, Au and U).
The geological and geomorphological framework is completed by
the very old coastal Namib Desert (Ward et al., 1983; Ward and Corbett,
1990), which stretches for more than 1900 km along the southwestern
Atlantic coast. It is featured giant sandy orange dunes, extensive unique
gravel plains and scattered rock outcrops. The climate of the coastal
areas of Namibia is closely dependent on the cold Antarctic upwelling
Benguela current (Van Zinderen Bakker, 1975; Wefer et al., 1996),
which frequently causes the formation of a dense advective morning fog
due to air thermal inversion (Lancaster et al., 1984; Robertson et al.,
2012). Driven by the wind, this fog can penetrate up to 60 km inland
(Robertson et al., 2012) and even cause rainfall (Olivier, 2004). In the
Central Namib Desert, the northwestern winds are dominant, though
there are both diurnal and seasonal inversions on wind prevailing direction (Lancaster et al., 1984; Lindesay and Tyson, 1990; Tyson and
Seely, 1980). The presence of another type of fog in the Central Namib
Desert has recently been ascertained, a radiation fog that forms by the
radiative cooling of stagnant air close to the surface, and is blown by
the land wind to westward (Kimura, 2005).
The geographic and climatic characteristics of this desert, and above
all the availability of water supplied by the fog, have shaped the flora
and fauna not only by welcoming a large number of endemic species
(e.g., Cloudsley-Thompson, 1977; Louw and Seely, 1980; Mulder and
Ellis, 2000; Seely and Pallett, 2008; Simmons et al., 1998; WilkinsEllert, 2004; Wirth, 2010), but also by stimulating extraordinary
adaptive responses to the environment that hosts them (e.g., CloudsleyThompson, 1990; Costa, 1995; Hamilton and Seely, 1976; Holm and
Scholtz, 1980; Louw and Seely, 1982; Polis and Seely, 1990; Mulroy
and Rundel, 1977; Withers et al., 1980).
2.2. Sampling sites
Ariadna spider specimens and sand samples were collected in 3 sites
located in gravel plains of the Central Namib Desert. These sampling
sites, named R, G and W, are within the Namib Naukluft Park at different distance from some major mining areas of Namibia: Navachab
Gold mine (21°58′56″S, 15°45′51″E), Rössing Uranium mine
(22°29′3″S, 15°2′56″E), Husab Uranium mine (22°37′0.12″S,
14°52′60″E), Langer Heinrich Uranium mine (22°48′53″S,
15°20′1.68″E), Gorob Copper mine (23°32′55″S, 15°25′13″E), Hope
Copper-Gold-Silver mine (23°34′0″S, 15°16′0″E), Matchless Copper
mine (22°41′30″S, 16°50′47″E) (Fig. 1).
The R site (23°0.0′32.7″S, 14°43.0′38.0″E) is about 20 km east of
Walvis Bay, an important town on the Atlantic coast, which has
100,000 inhabitants and a commercially strategic, natural deep-water
harbor, and lies a few kilometers from the Rooikop International
Airport. This sampling site is about 70 km away from some large open
pit mines of uranium, 60 km from the Khan Copper-Zinc-Nickel mine
and 90 km from the abandoned Gorob Copper mine. The R site is in a
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Ecological Indicators 95 (2018) 663–672
E. Conti et al.
Fig. 1. Map of the Central Namib Desert showing the sampling sites and the main mining areas.
Fig. 2. Ariadna burrow features from R, G and W sites; in the lower right a spider specimen.
site, there are a large number of Ariadna burrows, whose rings include
6–15 quartz stones mixed with pieces of lichens and arranged in one to
four strata to shape a typical turret (Fig. 2).
The G site (23°19.0′38.4″S, 15°2.0′23.3″E) is in the central part of
the Namib Desert, 56 km from the Atlantic coast and 35 to 90 km from
several mines (e.g., Hope Copper mine and Larger Heinrich Uranium
mine). It lies within a very wide gravel plain characterized by a sediment with a prevalent sandy component (≈77%), but without lichens
gravel plain consisting of a gravelly-sandy sediment with small quartz
pebbles, very rich in lichens. Based on the grain size, sand (≈80%) is
the principal component of sediment. As in other coastal gravel plains
in Namib Desert, the sediment has high amount of gypsum, which,
according to various authors (e.g., Eckardt and Spiro, 1999; Ward,
2009), mainly comes from the ocean transported inward from fog and
wind. The site receives thick fog daily and is beaten by wind that can
reach up to 80 km/h, often causing sandstorms (Seely, 1987). In this
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Ecological Indicators 95 (2018) 663–672
E. Conti et al.
From 7 to 20 January 2017 we located and marked 35 spider burrows with spoons at the R, G and W sites and collected 20 specimens of
adult female Ariadna spiders per site. Moreover, depth and diameter of
entrance burrows of each spider were measured with a Borletti caliper
(measurement error of 0.02 mm). Live spiders were individually placed
in Falcon tubes (50 mL) filled by half with sand collected from the
burrow, and kept at temperature of 20–22 °C, approximately 67–69%
RH (relative humidity), and photoperiod corresponding to that of the
sampling area (i.e., 13 h15’L:10 h45’D) until their shipping to Italy for
laboratory analyses.
All spider specimens arrived alive at the Laboratory of
Ecotoxicology of emerging contaminants and nanomaterials of the
University of Siena (Italy), where they were immediately removed from
sand and rinsed with MilliQ water to remove any sand particles from
the body of the animals. Then, spiders were weighed and individually
transferred in Eppendorf tubes and stored at -80 °C. Half of spiders
(n = 10) were used for the analysis of heavy elements and the rest
(n = 10) for the biochemical assays.
In each site, seven sand samples were collected up to 20 cm depth
around spider burrows and stored at −20 °C until heavy element analysis.
The activity was expressed as µmol H2O2 min−1 mg protein−1.
GST activity was carried out in S9 fraction and measured according
to the spectrophotometric method of Habig et al. (1974) and using a
Victor 3 1420 Multilabel Counter (Wallak) spectrophotometer. GST
activity was performed in a microplate were we placed 1.5 mM 1chloro-2,4-dinitrobenzene
(CDNB,
extinction
coefficient
9.6 mM−1 cm−1) in 0.1 M NaH2PO4/Na2HPO4 buffer pH 7.42, 1.5 mM
reduced glutathione (GSH) in 0.1 M NaH2PO4/Na2HPO4 buffer and
20 µL of S9 fractions diluted 1:3 in homogenization buffer. The increase
in absorbance of the reaction was measured at 340 nm, before and after
3 min of incubation at 20 °C. The activity was expressed in µmol min–1
mg prot−1.
The alteration of membrane phospholipids was evaluated through
lipid peroxidation by measuring the concentration of a secondary metabolite, the malondialdeide (MDA). 200 µL of sample was added to
150 µL 0.1 M phosphate buffer (pH 7.4), 250 µL of 20% TCA-0.01%BHT
solution and centrifuge at 7300 rpm for 10 min at 4 °C. 400 µL of resulting supernatant was mixed with 80 µL 0.6 M HCl and 320 µL of
25 mM TRIS-120 mM TBA solution (pH 7.9) and incubated at 80 °C for
15 min. The supernatant was used to spectrophotometrically quantify
the MDA content at 530 nm using a Victor 3 1420 Multilabel Counter
(Wallak) spectrophotometer. Levels of MDA were expressed as µmol
MDA mg protein−1.
Cholinergic function was measured using Cholinesterases (ChE)
activity assay from composite pool of 3 individual spiders using 20 mM
Tris-HCl, 5 mM MgCl2, 0.1 mg mL−1 bacitracin, 8 × 10−3 TIU mL−1
aprotinin and 1% Triton X-100 buffer (pH 7.4). Tissue was homogenized in TissueLyser (Quiagen, Retsch®, Germany) and then centrifuged at 8400 rpm for 20 min at 4 °C. After centrifugation, the supernatants were stored at −80 °C. ChE activity was measured on
microplates according to Ellman et al. (1961). Assays were performed
at 20 °C in 0.1 M Na2PO4, pH = 7.2, and 1 mM DTNB, using 1 mM of
Acethythiocholine (ASCh) as ChE substrates. ASCh was selected as
specific substrates for AChE. Selective ChE inhibitor, BW284c51 for
AChE was tested at concentration of 3 mM in order to characterize ChE
specificity towards ASCh substrate. The inhibitor was incubated with
the homogenates for 15 min at 20 °C before substrate kinetic study. The
increase in absorbance at 405 nm was followed for 5 min using a 550
Model microplate reader (Bio-Rad). ChE activity was at last expressed
as nmol min−1 mg protein−1. Total proteins in spider homogenates
were measured as described above.
2.4. Biochemical assays
2.5. Sample treatment and heavy elements analysis
Activities of antioxidants enzymes as Catalase (CAT), Glutathione-stransferase (GST) and the determination of lipid peroxidation levels
(MDA by TBARs method) were carried out in composite pool of 5 individual spiders. Due to small size of spiders, all body was used for
biochemical assays.
Biological samples were homogenized (w:v 1:8) using buffer of
50 mM K2HPO4, 0.75 M sucrose, 1 mM EDTA, 0.5 mM DTT, 400 µM
PMSF, 10 µM leupeptin, 1 µM pepstatin A, 1 mg/L aprotein, pH 7.5,
with a glass Potter-Elvehjem and centrifuged at 12,000 rpm for 20 min
at 4 °C. The supernatants (fraction S9) were used to analyze total proteins, CAT and GST activity and level of MDA.
Total proteins in S9 homogenates were measured according to
Bradford (1976) using a Shimadzu UV-160A visible recording spectrometer and bovine serum albumin as standard, reading at 595 nm. The
activity of the catalase enzyme (CAT, EC 1.11.1.6) was observed
through its peroxidase function, using the spectrophotometric method
according to Regoli et al. (2004) for estimating the 240 nm absorbance
decrease due to the reaction with H2O2. Aliquot of 40 µL of S9 fraction,
diluted 1:5 in homogenization buffer, were added to 100 mM phosphate
buffer (pH = 7) and the reaction started by addiction of the H2O2
(150 mM in 100 mM phosphate buffer) at 25 °C. Kinetic was recorded
for 1 min using a Shimadzu UV-160A visible recording spectrometer.
In the laboratory, sand samples were air-dried and sieved with a
metal-free 2 mm sieve. The fraction < 2 mm was homogenized by
quartering and an aliquot of about 100 g was powdered in an agate
mortar using a mechanical pulveriser.
The sand samples were solubilised by acid digestion adding 1 mL
HF, 2 mL HNO3, 2 mL HCl, 1 mL HClO4 (ultrapure reagents) to 250 mg
of powdered sample. Solubilisation was performed in Teflon bombs
placed in a Milestone Ethos 900 microwave lab station. The solution
was filtered and diluted with ultra-pure water to a final volume of
100 mL.
The specimens of Ariadna spider, dehydrated and weighed before
their utilization, were grouped forming a pool of 10 individuals per
each sampling site. Three samples of each pool weighing about 150 mg
were digested with 3 mL HNO3 and 1 mL H2O2 (ultrapure reagents) in
Teflon bombs in a Milestone Ethos 900 microwave lab station. The
solution was filtered and diluted with ultra-pure water to a final volume
of 25 mL.
The concentrations of 15 heavy elements, 13 metals (Ag, Cd, Co, Cr,
Cu, Fe, Mo, Ni, Pb, Tl, U, V, Zn) and two metalloids (As, Sb), were
determined in sand samples and spider specimens by inductively coupled plasma-mass spectrometry (ICP-MS) using the Perkin Elmer
NexION 350 spectrometer. The accuracy of ICP-MS analyses was
(Conti et al., 2015). Fog is less frequent and humidity is lower than in
the areas closer to the coast; wind can blow very strong. Spider burrow
rings include most commonly seven quartz stones, similar in size, shape
and color, arranged in only one layer (Fig. 2).
The W site (23°36.0′32.9″S, 15° 10.0′2.7″E), characterized by the
presence of some specimens of the dwarf gymnosperm Welwitschia
mirabilis, is located 72 km from the Atlantic coast. This sampling site is
only a few kilometers from the Gobabeb Desert Research and Training
Center, an internationally recognized research station for ecological
study of arid environments, as well as 11 and 27 km from the Hope
Copper-Gold-Silver mine and Gorob Copper mine, respectively. The W
site includes a 3–20 m wide ravine with a sandy channel (Henschel and
Seely, 2000), that is a dry tributary of the Kuiseb River flowing from the
Khomas Highlands to Walvis Bay. The sediment is sandy (≈54%) and
gravelly (≈41%) (Conti et al., 2015). The spider burrows are dug on
the slopes of the tributary and adjacent areas; the ring stones are numerous, irregular and arranged in up to four strata (Fig. 2).
2.3. Spider and sand sampling
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Ecological Indicators 95 (2018) 663–672
E. Conti et al.
checked using the following certified analytical standards: NIST
(National Institute of Standards and Technology) SMR 2709 (San
Joaquin soil) for sand samples, and NIST SRM 2977 (Mussel Tissue) for
spider specimens. The analytical precision was determined by five replicate analyses of each soil sample and spider specimen.
2.6. Statistical analysis
The mean and standard deviation of heavy element concentrations
in sand samples and spider specimens collected in each sampling site
were calculated. The bioaccumulation of each element in the sampled
spiders was investigated by comparing sand and animal concentrations
by means of the Wilcoxon rank test.
All data were ln-transformed (natural logarithms) to improve their
fit to normal distribution for parametric statistical analysis. The normality of distribution was tested by the Shapiro-Wilk test and the
homogeneity of variance by the Levene’s test. One-way ANOVA was
employed to test for differences in the heavy element concentrations in
sand and spiders from the sampling sites.
One-way ANOVA was also performed to compare biometric parameters as well as enzyme activity. Post-hoc comparisons were conducted using Tukey HSD method (Sokal and Rohlf, 1981).
Principal component analysis (PCA) (Hair et al., 1998) was conducted to assess how sand samples and spider specimens were distributed in the Euclidean space when all heavy elements were considered. The Pearson correlation coefficient between enzyme activities
and heavy element concentrations was also calculated. The significance
limit for the statistical analyses was P < 0.05. All analyses were performed using the SPSS package for Windows (version 22.0) and XLSTAT
2017 for Windows.
Fig. 3. Biometric parameters expressed as mm for burrow diameter (DIA), cm
for burrow depth (DEPTH) and cg for spider weight (WEIGHT). Different
symbols (Roman letters for DIA; Greek letters for DEPTH and Roman numerals
for WEIGHT) indicate difference at P < 0.05 level according to post-hoc
comparisons for each site.
total variance of the first component is mainly due to Co, Cr, Cu, Fe, Ni,
V and Zn (λ ≈ 0.3) and Pb (λ = −0.29); the main contribution to the
second component is instead provided by As and Cd (λ = 0.49), Mo
(λ = 0.41) and U (λ = 0.37).
Significant differences were found for the heavy element concentrations in spider specimens among the R, G and W sites, except for
Cd and Co (P < 0.006 for Zn, P < 0.001 for the other elements). Posthoc comparisons revealed that such differences are mainly due to the
highest concentrations of Ag, As, Cu, Ni and U at R site, Mo at G site, V
at W site. The levels of Ag, Cd, Cu and Zn in spiders were generally
higher than those in sand samples (P = 0.033 for Ag; P = 0.008 for Cd;
P = 0.028 for Cu; P = 0.008 for Zn). In contrast, the concentrations of
the other analyzed heavy elements were higher in sand than in spiders
(P = 0.008 for all elements).
Concerning the biological responses of spiders, all tested enzymes
(CAT, GST, ChE) as well as MDA levels resulted detectable in whole
body samples. Except for MDA, significant differences were observed
among specimens from the three sites (Fig. 5). Lowest levels of ChE
activity expressed as nmol min−1 mg protein−1, were recorded in
spiders from G site (9.21 ± 1.39) and W site (11.20 ± 1.21) compared to those from spiders collected in R site (23.68 ± 1.13;
P < 0.001). A similar trend was observed also for antioxidant enzymes
which showed slight but still significant differences in spiders from the
three sites: CAT expressed as µmol H2O2 min−1 mg prot−1, show levels
of 7.92 ± 0.38 in spiders from R site, 9.44 ± 1.76 from G site,
9.12 ± 1.62 from W site, and GST expressed as µmol min−1 mg
prot−1, activities of 16.84 ± 2.14 in spiders from R site, 18.79 ± 2.72
from G site, 21.09 ± 3.71 from W site (P < 0.01 for CAT; P < 0.002
for GST).
The PCA based on the concentrations of heavy elements and biochemical assays in spider body burden (Fig. 4), further demonstrated a
clear distribution among the sites, although different from that showed
by the PCA based on heavy element concentrations in sand samples. In
fact, spiders collected in R site had higher levels of heavy elements
compared to those from the other two sites. The first two principal
components (PC1 and PC2) accounted for 35.30% and 20.90% of total
3. Results
The burrow diameter measurements of the spiders captured in the
three sites gave the following results: 5.47 ± 0.49 mm in R site,
6.26 ± 0.74 mm in G site, 6.09 ± 0.89 mm in W site; the burrow
depths were: 6.73 ± 1.62 cm in R site, 8.20 ± 1.47 cm in G site,
12.30 ± 2.25 cm in W site. The analysis of these burrow parameters
showed significant differences among the sites (Fig. 3). Burrow depth
(F = 30.613, P < 0.001) showed the lowest values in R site and the
highest ones in W site. The comparison of diameter measurements
displayed significant difference (F = 4.842, P = 0.013) highlighting the
burrow diameter in R site as the lowest one; no difference arose for
burrows in W and G sites.
The following outcomes emerged for the weight of collected spider
specimens (0.032 ± 0.008 g in R site, 0.053 ± 0.02 g in G site,
0.069 ± 0.025 g in W site); the post-hoc comparison indicated that
spiders in R site have the smallest body weight (F = 14.602,
P < 0.001).
The concentrations of heavy elements in sand samples and spider
body burden are shown in Table 1. Sb and Tl were not considered in
statistical analyses due to their low levels in spider specimens, normally
below the respective detection limit.
The sand samples collected at R, G and W sites clearly differ in the
concentration of heavy elements. With the exclusion of Ag and Mo, the
levels of the heavy elements were found to be significantly different
among the three sites (P < 0.001). In particular, Cr, Co, Cu, Fe, Ni, V
and Zn were significantly higher in sand from W site, while As, Pb and
U in that from G site. Cd, Cr, Cu and V showed the lowest levels in sand
from R site.
The PCA based on the concentrations of the heavy elements in sand
samples showed a clear distribution among the R, G and W sites (Fig. 4).
The highest levels of heavy elements were found at W site, the lowest
ones at R site. The first two principal components (PC1 and PC2) accounted for 59.27% and 20.94% of total variance, respectively. Considering the λ value of the eigenvectors, the main contribution to the
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Table 1
Mean ± SD of heavy element concentrations (expressed in mg/kg dry weight) in sand and Ariadna spiders from R, G and W sites. Five replicate analyses of each soil
sample and spider specimen were performed.
Element
Ag
As
Cd
Co
Cr
Cu
Fe
Mo
Ni
Pb
Sb
Tl
U
V
Zn
R site
G site
W site
Sand
Spiders
Sand
Spiders
Sand
Spiders
0.18 ± 0.02
2.44 ± 0.17
0.07 ± 0.01
7.08 ± 0.18
46.97 ± 1.85
17.90 ± 0.38
9770 ± 180
0.68 ± 0.07
15.38 ± 0.54
16.91 ± 0.35
0.11 ± 0.01
0.39 ± 0.02
2.34 ± 0.11
73.56 ± 3.50
29.36 ± 1.41
0.74 ± 0.14
1.44 ± 0.32
1.57 ± 0.85
0.15 ± 0.03
1.53 ± 0.36
55.72 ± 14.48
341.10 ± 80.05
0.41 ± 0.05
5.86 ± 2.77
0.62 ± 0.26
0.03 ± 0.015
< 0.01
0.24 ± 0.03
1.08 ± 0.24
212.63 ± 42.0
0.20 ± 0.02
3.02 ± 0.03
0.19 ± 0.01
7.02 ± 0.50
56.68 ± 2.07
20.05 ± 0.98
11440 ± 750
0.86 ± 0.06
14.84 ± 0.56
18.75 ± 0.45
0.18 ± 0.01
0.30 ± 0.01
2.99 ± 0.07
83.19 ± 2.04
23.61 ± 0.74
0.31 ± 0.03
0.69 ± 0.06
1.60 ± 0.83
0.14 ± 0.03
1.45 ± 0.21
33.62 ± 13.90
227.40 ± 18.01
0.63 ± 0.06
3.32 ± 0.98
0.35 ± 0.05
0.01 ± 0.004
< 0.01
0.12 ± 0.02
0.68 ± 0.02
180.00 ± 32.07
0.16 ± 0.01
2.68 ± 0.07
0.18 ± 0.02
12.53 ± 0.69
82.39 ± 2.60
25.36 ± 0.88
17290 ± 1240
0.81 ± 0.06
29.35 ± 1.09
14.77 ± 0.15
0.09 ± 0.01
0.45 ± 0.02
2.15 ± 0.07
165.28 ± 3.71
59.63 ± 1.39
0.14 ± 0.03
0.26 ± 0.04
1.31 ± 0.71
0.14 ± 0.02
1.04 ± 0.17
29.12 ± 8.54
337.03 ± 43.81
0.28 ± 0.06
2.93 ± 1.16
0.56 ± 0.38
0.01 ± 0.003
< 0.01
0.08 ± 0.02
1.64 ± 0.49
206.63 ± 20.41
a similar significant negative correlation with Ag, Cr, Ni and U and, in
addition, with Fe and Pb for CAT and As for GST. ChE had significant
positive correlation with Ag, As, Cu, Fe, Ni, Pb, U and Zn. It is also to be
noted that Cd, Mo and V showed no correlation with the analyzed enzymatic activities; on the contrary, Ni is correlated with all enzymes
and lipid peroxidation, negatively with CAT and GST and positively
with ChE and MDA.
4. Discussion
In order to investigate Ariadna spider as potential bioindicator of
heavy elements in the Central Namib Desert, specimens of populations
inhabiting three sites characterized by peculiarities in habitats, were
collected. The differences in sites are reflected in the behavior of
Ariadna spiders, which adopt various strategies for building their homes
(Fig. 2). Besides, burrows and spiders in the selected areas have biometrical parameters depending on local climatic factors, such as the
presence or absence of wind and the consequent overheating level of
surface.
Heavy elements in sand samples collected from the three sitesshowed levels comparable with those reported for clastic sediments,
rocks and soils (Reimann and de Caritat, 1998). Nevertheless, heavy
element concentrations resulted significantly different in the three sites
with higher levels in W site compared to R and G sites. The highest
concentrations of several heavy elements recorded in the sand from W
site, are likely due to the geolithological and geochemical features of
the local rocks as well as the provenance of the tributary of the Kuiseb
River. Iron and V are abundant in the schists bordering the dry canal
(Phillips et al., 1989; Iilende, 2012). Other heavy elements probably
come from the mining area present in the Khomas Highlands, associated to the particles dragged by the Kuiseb River and its tributaries.
The sand from G site is slightly enriched in U, As and Pb probably
due to particle dispersion caused by the extraction activity of the mines
located northeast of the site. The possible spreading of heavy elements
in sand due to mining activities has already been reported (e.g., Ashraf
et al., 2012; Rodriguez et al., 2009; Salomons, 1995; Wuana and
Okieimen, 2011).
The R site deserves special attention since sand samples showed the
lowest levels of several heavy elements compared to W and G sites.
Despite being located in the proximity of the Rössing and Husab
Uranium mines, U levels detected in sand samples resulted not high
(Fig. 1). Such mines are located northeast the R site and therefore the
prevailing northwestern winds, which can transport mine-derived airborne particles, may not directly affect the site. Nevertheless, spiders
living in this site have the highest body concentrations of Ag, As, Cu, Ni
Fig. 4. PCA diagrams (1st and 2nd principal components) based on heavy
element concentrations in sand samples (top) and spider specimens (bottom)
from R, G and W sites.
variance, respectively. The maximum contribution on PC1 is given by
Ag, As and U (0.36 ≤ λ ≤ 0.39), ChE activities (0.31 ≤ λ ≤ 0.33) and
Cr (λ = −0.26), and on PC2 by Pb, Zn, Fe and V (0.33 ≤ λ ≤ 0.50) and
Mo (λ = −0.42).
The correlation analysis (Table 2) highlighted that MDA has significant positive correlation with Co, Cr, Ni and Pb, and negative correlation with Zn. Regarding antioxidant enzymes, CAT and GST shared
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Ecological Indicators 95 (2018) 663–672
E. Conti et al.
Fig. 5. CAT and GST activities, MDA levels and AChE vs ASCh activities in spider whole body of specimens collected from R, G and W sites. Different letters are
indicative of significant differences (P < 0.05) among sites. All biochemical assays were run in replicates of three (n = 5 per group).
westward. Lichens covering the gravel plain at R site could easily sequester the chemicals present in the water vapor. In fact, given the lack
of cuticle, lichens can absorb nutrients as well as atmospheric contaminants as both gas and particle-bound solution (Tuominen and
Jaakkola, 1973; Brown and Beckett, 1985). Therefore, lichens have
been used as bioindicators in air pollution assessments in various settings and countries (e.g., Andersen et al., 1978; Branquinho et al., 1999;
Conti, 2008; Herzig et al., 1989; Ng et al., 2005; Pignata et al., 2004).
The ability of lichens to uptake U particles is well known (e.g., Beckett
et al., 1982; Richardson et al., 1985). Recently, Boryło et al. (2017)
stated that lichens and mosses have a high efficiency in capturing U
from atmospheric fallout. So they concluded that these cryptogams can
be good indicators of U contamination in the environment. Uranium
and other chemicals absorbed by lichens can thus enter the body of
spiders that feed on lichenophagous prey.
The hypothesis that in an area there may be atmospheric pollution
rather than soil pollution has already been postulated (e.g., Rossini
Oliva and Fernández Espinosa, 2007). Schemenauer and Cereceda
(1992) have demonstrated that in the foggy Atacama Desert aerosols
originating primarily from evaporated cloud droplets on the Pacific
Ocean, contain As, Cd, Cu, Fe, Ni, Pb, V and other heavy elements. It is
likely that analogous phenomenon can occur in the Namib Desert. Indeed, high concentrations of several heavy elements, such as Cu, Cd,
Pb, Zn, Ni, Cr, and Fe, were found in the atmosphere over the Atlantic
Ocean from the Antarctic to the European coasts (Rädlein and
Heumann, 1992). Moreover, a recent assessment of trace metal pollution along the central Namibian coastline (Vellemu, 2014) has shown
that the coastline of Walvis Bay recorded more Cu, Fe, Zn and other
metals than all the other studied areas. Therefore, aerosols coming from
Table 2
Pearson’ correlation analysis between enzymes and heavy elements in spiders.
The statistical significance is expressed with asterisks (* = P < 0.05;
** = P < 0.01; *** = P < 0.001; n.s. = not significant). All activities of antioxidants enzymes were performed in replicates of three (n = 5 per group).
CAT
Ag
As
Cd
Co
Cr
Cu
Fe
Mo
Ni
Pb
U
V
Zn
GST
−0.35
−0.28 n.s.
0.02 n.s.
−0.05 n.s.
−0.30*
−0.22 n.s.
−0.30*
0.04 n.s.
−0.43**
−0.33*
−0.41**
−0.26 n.s.
−0.09 n.s
*
−0.41
−0.48**
−0.03 n.s.
−0.02 n.s.
−0.45**
−0.25 n.s.
−0.04 n.s.
−0.13 n.s.
−0.37*
0.06 n.s.
−0.39**
0.16 n.s.
−0.07 n.s
**
MDA
ChE
0.08 n.s.
0.20 n.s.
0.27 n.s.
0.42**
0.38**
−0.22 n.s.
0.14 n.s.
0.13 n.s.
0.35*
0.58***
−0.06 n.s.
−0.14 n.s.
−0.39**
0.72***
0.65***
0.12 n.s.
0.21 n.s.
0.25 n.s.
0.65***
0.50***
−0.17 n.s.
0.53***
0.40**
0.81***
0.19 n.s.
0.32*
and U. Copper and Ni could come from the Atlantic coastal area, and As
and Ag from the large town of Walvis Bay by the morning advective fog
moved inward by the sea breeze, but U deserves a separate discussion.
Even if U levels in the sand of R site are quite comparable with those in
the other two sites, those detected in spider tissues resulted two to three
times higher than those in specimens from G and W sites. We cannot
rule out the possibility that the U-containing particles coming from the
mines and transported to the interior of the Central Namib Desert from
the prevailing winds, are intercepted by the night radiation fog, which,
according to Kimura (2005), is driven by the land wind to the
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Ecological Indicators 95 (2018) 663–672
E. Conti et al.
et al., 2006).
Regarding the Malondialdehyde (MDA), the absence of variations
among sites further supports the hypothesis of some limitations of using
whole body instead of midgut glands.
Our study was the first attempt to identify ChE activities in Ariadna
spider from Namib Desert and it was done by using an AChE specific
inhibitor (1,5-bis (4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW284c51)). According to Meng et al. (2016), different types
of AChEs have been identified in Arachnida with high ASCh substrate
specificity and sensitivity to specific AChE inhibitor. Our findings further support both the presence of AChE enzyme in Ariadna spiders and
its selectivity towards ASCh as preferential substrate. Significant differences have been observed also in the ChE vs. ASCh activity in spiders
collected in the three sites, suggesting a potential role of this enzyme as
biomarker of exposure to chemicals with inhibitory or inductive effect.
Moreover, ChE vs. ASCh activities measured in Ariadna spiders are similar to those reported in wolf and funnel-web spiders chronically exposed to heavy elements and organophosphorus insecticides
(Babczynska et al., 2006). Concerning specific site responses, the differences observed between the spiders from R, G and W sites and stimulate the need for a further characterization of enzyme functionality
in terms of age, gender as well as adaptive responses of specimens to
local habitats. In fact, in the absence of a clear correlation with spider
heavy element body burden, the observed differences in ChE vs. ASCh
activities among specimens from the three sites motivate a deeper
knowledge in terms of their sensitivity to chemicals or environmental
parameters for a proper use as a biomarkers of exposure. In particular,
spiders from R site, which had the highest levels of several heavy elements, show higher ChE vs. ASCh activities compared to spiders from G
and W sites, whose activities had very similar values even if statistically
different. Despite the inhibition of this enzyme is widely considered as a
biomarker of organophosphorus and carbamate insecticides exposure,
few contributions documented both inhibition as well as induction
upon heavy element exposure in invertebrate species (Payne et al.,
1996; Frasco et al., 2005).
In particular, Devi and Fingerman (1995) first described a strong
AChE activity inhibition (up to 48%) in crayfish short-term exposed
(24–48 h) to heavy metals such as Hg, Cd and Pb, but also controversial
data concerning different sensitivities towards specific heavy metals as
well as time of exposure (short vs. long-term) in the AChE responses.
For instance, Thaker and Haritos (1989) hypothesized that Cd exposure
could activate multiple molecular forms of esterases from which a doseresponse increases of esterase activities are reported upon Cd exposure
(0.1–0.8 ppm). Although in other studies significant inhibition of AChE
has been also documented in both terrestrial and aquatic organisms
upon exposure to Pb, U, Cd and Zn, a clear hypothesis on mechanism of
AChE inhibition of heavy elements has not been provided (Labrot et al.,
1996; Antonio et al., 2003; Richetti et al., 2011). In spiders, inhibition
of AChE has been reported to cause hyperactivity, locomotors disturbances and perturbations in the body, often leading the organism
death (Tietjen and Cady, 2007; Behrend and Rypstra, 2018). Therefore,
further knowledge is needed in order to better interpret the ChE activities detected in Ariadna spiders from the three sites, although ChE
vs. ASCh activities are clearly recognizable as responsible for the cholinergic function in these spiders from Namib Desert.
both the Atlantic Ocean and the Walvis Bay town can be transferred by
fog inward transporting the associated chemicals.
The higher levels of Cd, with Cu and Zn, in spider whole body than
in sand, confirm the ability of these arachnids to be the best Cd accumulators among all investigated terrestrial invertebrates (Hendrickx
et al., 2004; Jung et al., 2005). The typical feeding behavior of spiders
as well as pronounced assimilation efficiencies are considered the main
drivers of large amount of Cd assimilated in their body (Heikens et al.,
2001; Jung and Lee, 2012; Li et al., 2016). Moreover, the midgut gland
diverticulae are considered the main storage organs also involved in the
excretion of Cd in some spiders species (Hopkin, 1989). Therefore,
based on such similarity in Cd accumulation, Ariadna seems a suitable
candidate as bioindicator of Cd in Namib Desert.
By comparing body burden levels of heavy elements in Ariadna
spiders with those reported from other spider species, they result
comparable to those detected in population inhabiting unpolluted sites
(Migula et al., 2013; Yang et al., 2016). Specie-specific factors have
been hypothesized to account for 70% of the variety of element contents in spider body (Wilczek and Babczyńska, 2000).
Concerning the biological responses investigated in spider whole
body, both antioxidant enzymes as CAT and GST, cholinergic ones as
ChE as well as MDA levels resulted detectable. Such results, reported for
the first time in Ariadna species from Namib Desert, confirm also the
suitability of the proposed sampling protocol which allows to keep
specimens alive during shipping to Europe and more important prevent
any degradation of enzymes.
The absence of significant correlations in Ariadna spiders between
heavy element body burden and antioxidant enzymes CAT and GST,
known to be induced for instance by Cd in other spider species, could be
related to the fact that due to small dimension of Ariadna specimens,
whole body has been used for enzyme assays instead of specific metabolizing organs as midgut glands. In fact, by comparing CAT activities
measured in Ariadna spiders with those reported for other spider species, they resulted two orders of magnitude lower probably as a consequence of dilution of enzymes content caused by whole body subcellular fraction extraction (Wilczek, 2005; Wilczek et al., 2013). CAT
for instance is commonly assayed in midgut gland in invertebrates but
also in hemolymph showing very often a high sensitivity towards heavy
element exposure and in particular to Cd (Wilczek and Migula, 1996;
Wilczek, 2005; Wilczek et al., 2013; Stalmach et al., 2015a,b).
Similarly, GST has been reported to be significant induced in spiders
exposed to Cd (Wilczek and Migula, 1996; Wilczek et al., 2004). GST is
a family of isoenzymes that catalyze the conjugation of various toxic
molecules with reduced glutathione making them less reactive and
more easily eliminated by the organism that can be sensitive also to
other contaminants exposure (Jakoby, 1978; Mannervik and Danielson,
1988).
GST activities in Ariadna spiders are in line with those reported in
other spiders species in field studies using the families Lycosidae or
Agelenidae, chronically exposed to metals and insecticides (Wilczek
et al., 2004; Babczynska et al., 2006). It is known that spiders that live
in chronically contaminated environments including those by heavy
elements, cope very well with stress, activating antioxidant defense
mechanisms, which however depend heavily on the state, sex and
species, as well as their specific behavior and physiology. In particular,
in males glutathione plays a fundamental role in protecting from high
concentrations of xenobiotics, while in females it very often shows a
higher activity, since their main objective is to protect their genetic
material and maintain a reproductive fitness (Foelix, 1996; Wilczek
et al., 2008). Some spiders oppose this problem by limiting the activity
of different enzymes as it might have occurred in Ariadna specimens
during the travel to Europe without any food and water, except for
moisture present in the sand. The activation of compensation mechanisms, very useful in case of high stress and limited energy reserves,
might have been occurred during this travel and explain the low responses observed in antioxidant enzymes in Namib spiders (Babczynska
5. Conclusions
Interesting and promising aspects for further studies rise up from
this research.
Ariadna spiders are able to differently concentrate heavy elements
according to the position and characteristics of their living site.
Vanadium prevails only in spider specimens from W site, and Mo in
those from G site. In the R site, there were the highest values of Fe, Ni,
Cu, Zn, As, Ag and U. Ariadna spiders bioaccumulated Cu, Zn and Cd in
all three sites, as well as Ag in R and G sites.
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Ecological Indicators 95 (2018) 663–672
E. Conti et al.
The case of the R site is particularly interesting, as the Ariadna
spiders inhabiting it have uranium concentrations higher than that
found in the other sites, without there being an enrichment of this metal
in the sand; conversely, the air seems to be influenced by the transport
of uranium particles from the combined action of fog and wind. In the
transfer of this metal to the Ariadna spiders, an important involvement
of lichens covering the sand at the R site and the ingested lichenophagous prey is hypothesized. For a subsequent validation of this hypothesis, the analysis of uranium concentration in lichens will be decisive.
Regarding the results obtained with biochemical assays, for the first
time carried out in this spider genus, oxidative stress responses as well
as cholinergic function averaged their sensitivity to heavy elements as
well as other environmental pollutants. Future studies should better
characterize their sensitivity towards exposure to environmental pollutants as well as the use of spider midgut glands instead whole body.
Despite we cannot affirm that all our spiders belong to the same species,
their enzymatic activities reflect their ability to accumulate heavy
elements regardless the specific habitat features, confirming so the
value of this genus as bioindicator of heavy elements in the Central
Namib Desert.
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Acknowledgements
We would like to thank the Ministry of Environment and Tourism,
Namibia, for a permit (number 2245/2016) to conduct research work in
the Namib-Naukluft Park and to collect some specimens of Ariadna. We
also thank Gobabeb Research and Training Centre for hosting during
samplings. Furthermore, we are very grateful to Prof. Christian Mulder
for his precious comments and suggestions on the final draft of the
manuscript. Finally, we would also like to express our thanks to the
anonymous reviewers for providing constructive critiques of the
manuscript.
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