Science of the Total Environment 666 (2019) 759–765

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Science of the Total Environment

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Evaluation of the effects of titanium dioxide and aluminum oxide

nanoparticles through tarsal contact exposure in the model insect
Oncopeltus fasciatus
Daniel López-Muñoz, Mª. Amparo Ochoa-Zapater, Amparo Torreblanca, Mª. Dolores Garcerá ⁎
Department of Cellular Biology, Functional Biology and Physical Anthropology, Universitat de València, Doctor Moliner 50, 46100, Burjassot, Valencia, Spain

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Oncopeltus fasciatus acquired Ti and Al

metal oxide nanoparticles by contact.
• TiO2 nanoparticles increased the days to
reach adulthood from first instar
nymph.
• Biochemical composition was altered in
parents and/or offspring.
• Oxidative stress evidences were not
found but for bulk Al2O3 in the offspring.
• Trans-generational effects have been
demonstrated.

a r t i c l e i n f o a b s t r a c t

Article history: Despite the increasing presence of metal nanoparticles in the biosphere as a consequence of their widespread
Received 30 December 2018 use, knowledge about the impact of these nanoparticles on fauna, ecosystems and human health is far from com-
Received in revised form 11 February 2019 pletion. This is especially true for terrestrial invertebrates. Insects are environmentally exposed to nanoparticles
Accepted 14 February 2019
by several ways, the ectopic contact being one of the most probable. The model insect Oncopeltus fasciatus, has
Available online 17 February 2019
been used in the present work for testing toxicity of nanoparticles present in a surface. Adverse effects of TiO2
Editor: Julian Blasco nanoparticles and Al2O3 in nanoparticulated or bulk form on mortality, reproductive and embryonic develop-
mental parameters have been analyzed after tarsal contact of adult individuals of O. fasciatus. Effects were mon-
Keywords: itored in the unexposed filial generation from control and exposed adults. In order to know the effect of the
Metal oxide nanoparticles nanoparticles on the insect composition, measurements of protein and lipid content as well as lipid peroxidation
Insects were also performed. The results obtained indicate that the ectopic exposure to nanoparticles at 1 mg/cm2 (TiO2)
Toxicity and 0.5 mg/cm2 (Al2O3) did not induce lethal toxicity in O. fasciatus, nor did it modify any of the reproductive pa-
Development rameters. However, NPs-TiO2 and Al2O3 produced an increase in nymphal life span. In the parental generation
Reproduction
NPs-TiO2 increased protein content whereas NPs-Al2O3 decreased it. Several effects were detected in the filial
Oxidative stress
generation as consequence of parental exposure. NPs-Al2O3 decreased protein content, NPs-TiO2 decreased
lipid content and Al2O3 in bulk form diminished protein content and increased lipid peroxidation. Responses ob-
served in the individuals of the filial generation demonstrate the existence of trans-generational effects of NPs-
Al2O3 and NPs-TiO2.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding author. Among the existing nanomaterials, metal and metal oxide nanopar-
E-mail address: garcera@uv.es (M.ªD. Garcerá). ticles were identified in N30% of the total products containing

https://doi.org/10.1016/j.scitotenv.2019.02.218
0048-9697/© 2019 Elsevier B.V. All rights reserved.
760 D. López-Muñoz et al. / Science of the Total Environment 666 (2019) 759–765
nanoparticles due to their wide variety of applications (Kumar et al.,
2018). The nanoparticles of TiO2 (NPs-TiO2) are very stable, non-toxic,
inexpensive and their properties allow them to be suitable for UV pro-
tection applications (Popov et al., 2005; Giokas et al., 2007; Ochoa
et al., 2013; Kumar et al., 2018) and in the development of new coatings
and paint systems for most surfaces (Keller et al., 2013). Due to its pres-
ence in a wide variety of products, its toxicology has attracted increasing
interest in recent years (Bernier et al., 2012). Nowadays, reports on the
toxicity of NPs-TiO2 remain conflicting. Some studies show that they do
not have toxicity (Pujalté et al., 2011); on the contrary, many others in-
dicate that they do exhibit cytotoxicity in various biological models (Li
et al., 2014; Wang et al., 2009).
Nanoparticles of Al2O3 (NPs-Al2O3) are a homogeneous powder
of high degree of purity with uniform characteristics and specific
physical and chemical properties, generally used in polymers and
tires (Stadler et al., 2010) and have also industrial, agricultural and
medical applications (Willhite et al., 2014). Results from in vivo
and in vitro studies do not indicate a clear pattern of toxicological be-
havior of these nanoparticles. In vivo experiments showed, in some
cases, nanoparticle-size-dependent toxicity, while in others no mor-
tality or clinical signs or treatment-related changes in body weight
were observed.
Research on nanoparticle toxicity in insects is of great interest. In-
sects are a very diverse group in terms of eating habits and life cycles,
and contribute in great extent to biodiversity. In other context, some
species are directly beneficial to the human activities while other are
pests. There is a lot of interest for the development of new pesticides
due to the development of resistance to them and the increasingly
strict regulation in their use. In the last decade, with the develop-
ment of nanotechnology some nanoparticles have been proposed
as potential pesticides (Sahayaraj, 2017). Therefore, insects can be
exposed to nanoparticles by two different ways: after a deliberated
exposure (when used as a pesticide) or accidentally as a conse-
quence of the increasing presence of these particles in the environ-
ment. In both cases ectopic exposure seems the most likely. The
mechanisms of action of nanoparticles on insects, reviewed by
Benelli (2018), are not fully known.
In the studies so far performed, NPs-TiO2 does not exert lethal toxic-
ity in insects but effects on the length of development stages in insects
belonging to the Lepidoptera order (Li et al., 2014; Zorlu et al., 2018)
and effects on the number of the progeny in insects from the Dipteran
order (Philbrook et al., 2011) have been reported. Recently, Al2O3
dusts (nano- and micron scale particles) have been proposed for seed
protection against a coleopteran pest (Lazarevic et al., 2018) and NPs-
Al2O3 induced mortality has been reported in different insect species
(Stadler et al., 2017). One of the most common reported mechanism
for cellular toxicity caused by metal nanoparticles in animal species is
oxidative stress (Yang et al., 2004). Lipid peroxidation is especially
harmful in insects (Ahmad, 1995).
The Hemipteran species Oncolpeltus fasciatus, have been successfully
used in bioassays to test xenobiotics and bioactive substances by ectopic
exposure (Garcerá et al., 1989; Maymó et al., 1999). The biology and re-
production of this insect species is well known (Johansson, 1958) and it
is considered a model for biological and physiological studies due to
their short life cycle, their easy rearing and handling and their key phy-
logenetic position (Chipman, 2017).
Our hypothesis is that metal oxide particles present on a surface
may interact with insects and exert cellular toxicity through the in-
crease of reactive oxygen species and other mechanisms producing
changes in the energy reserves of the individuals that can affect sur-
vival, reproduction, and development of the offspring. To test this
hypothesis, we have evaluated the lethal toxicity of metal oxide
nanoparticles in O. fasciatus after an acute exposure through tarsal
contact. Effects on different reproductive and developmental param-
eters, as well as content of protein, lipids and lipid peroxidation have
been analyzed.
2. Materials and methods
2.1. Insects
Oncopeltus fasciatus (Dallas), was maintained in a room with con-
trolled conditions of temperature (28 ± 2 °C), relative humidity (75
± 5%) and photoperiod (16:8 h light-darkness cycle). Sunflower seeds
and water were provided ad libitum.
2.2. Nanoparticles
The materials used were: titanium dioxide nanoparticles (NPs-TiO2,
21 nm in size) and aluminum oxide, both in bulk (Al2O3) and in
nanoparticulate form (NPs-Al2O3, 30–60 nm in size). Nanoparticles
were purchased from Sigma-Aldrich (NPs-TiO2 reference: 718467;
NPs-Al2O3 reference: 642991). Al2O3 were obtained from Fluka (refer-
ence: 06320).
2.3. Experimental design of bioassays
Metal oxide substances were tested at 1 mg per cm2 of petri dish sur-
face (NPs-TiO2) or 0.5 mg/cm2 for NPs-Al2O3 and Al2O3 (bulk form). In
order to obtain a homogeneous distribution of the nanoparticles when
applied on a petri dish, 5 mL of a solution of 28.6 mg/mL in ethanol of
NPs-TiO2 or 14.3 mg/mL in water of NPs-Al2O3 or Al2O3 (bulk form)
were used. The dishes were placed on a stirring plate until complete
evaporation of the solvent.
Newly molted adults (0–24 h old) were placed for 5 days in 13.5 cm
diameter glass petri dishes previously impregnated with the solution to
be tested. In this way the insects are directly in contact with the sub-
stances when walking on the impregnated dish. Peeled sunflower
seeds and water ad libitum were provided. The dishes were maintained
at the same conditions of temperature, humidity and photoperiod as the
rest of the colony.
To determine the effects of the compounds on the insects, five
groups were established, three of which were placed into dishes
where solutions of the tested compounds had been let to dry (NPs-
TiO2, NPs-Al2O3 and Al2O3) and others two into dishes where the same
procedure had been performed using only the solvents (ethanol for tita-
nium oxide and water for aluminum oxide). Each group consisted of 20
individuals, in a sex ratio of 1:1. Three replications of each experimental
and control groups were made. The insects of each replication and
group made up the parental generation.
On the fifth day of the experiment the insects of each experimental
and control group, in couples, were transferred to glass jars provided
with food and water. The eggs laid on each jar were counted daily and
separated in clean petri dishes during 11 days. After that, surviving
adults from the three replications were frozen at −80 °C for biochemical
analysis.
In order to obtain the filial generation, newly first instar nymphs
hatched from the eggs laid by females of the first replica were trans-
ferred to glass jars provided with food and water, and maintained in
the same conditions as the rest of the colony. The jars were checked
daily to calculate the reproductive and postembryonic developmental
parameters analyzed (see Section 2.5). When nymphs reached the
adult stage, they were frozen at −80 °C for subsequent biochemical de-
terminations. These adults constituted the filial generation.
2.4. Dry and wet weight
The individuals of both generations were weighed (fresh weight)
and were dried in an oven at 60 °C until constant weight (dry
weight).
 D. López-Muñoz et al. / Science of the Total Environment 666 (2019) 759–765 761

2.5. Reproductive and post-embryonic developmental parameters
The reproductive parameters considered were: pre-reproductive
period (days until the first egg laying), fecundity (total mean number
of eggs laid per female), embryonic developmental period (days from
egg laying to hatching), and fertility (mean number of nymphs hatched
per female).
In the case of post-embryonic development, the analyzed parame-
ters were: nymphal life span (days from eclosion to adulthood) and sur-
vival of nymphs (average number of nymphs reaching the fifth instar
and adulthood).
2.6. Biochemical analysis
The biochemical parameters determined were protein content, total
lipid content and lipid peroxidation.
Frozen insects were homogenized in 50 mM phosphate buffer
pH 7.4, at a rate of 2 mL for a pool of 6 insects. The homogenates were
filtered to eliminate the cuticle residues. Three homogenates of each ex-
perimental and control group of the parental generation were analyzed.
In the filial generation, four homogenates of each experimental group
were used to quantify the different biochemical parameters.
The quantification of all the biochemical parameters were per-
formed using a spectrophotometer (TECAN Spectra Fluor) equipped
with a UV-VIS microplate reader.
and a quality Merck Suprapur mixture HNO3/HCl/HF was used in diges-
tion with microwave oven, optimized in a sequence of successive stages
in a soft ramp up to 190 °C. Three insects were included in each sample
and each experimental group was measured in triplicate. The quantifi-
cation of the aluminum and titanium content has been carried out by
optical emission spectrometry with inductively coupled plasma (ICP-
OES).
2.8. Statistical analysis
The statistical analysis of the data was done with IBM SPSS Statistics
24 for Windows. Prior to the study, homogeneity of variance and nor-
mality of the data were analyzed. The comparison of parameters was
carried out using generalized linear models.
3. Results
3.1. Mortality, dry weight and fresh weight
The statistical analysis of the experimental data showed no differ-
ences among the experimental groups (Table 1). No significant differ-
ences were obtained in dry weight and fresh weight among
experimental groups or between generations (Table 1).
3.2. Reproductive parameters

2.6.1. Total proteins Results of pre-reproductive period, fecundity, embryonic develop-

The total protein content of the samples was determined in filtered mental period and fertility parameters did not show statistical differ-
crude homogenates following the method of Bradford (1976), using bo- ences between experimental groups and their controls nor between
vine serum albumin (BSA) as standard. generations (Table 1).

2.6.2. Total lipids 3.3. Parameters of post-embryonic development

For the quantitative determination of lipids (TL), the colorimetric
method of sulfo-phospho-vanillin from Spinreact was used. Previously,
the filtered crude homogenate was centrifuged at 2500g and 4 °C for
10 min. The obtained supernatant was filtered and used for the quanti-
fication of lipids content.
2.6.3. Lipid peroxidation (TBARS)
Lipid peroxidation was determined in the filtered supernatant (see
Section 2.6.2) through the quantification of species reactive to thiobar-
bituric acid (TBARS), using the method modified by Cervera et al.
(2003).
2.7. Titanium and aluminum content
The quantification of the titanium and aluminum content was made
in the Geological Techniques Unit of Universidad Complutense de
Madrid. Previous to digestion, insects were lyophilized during 24 h,
Fig. 1 shows the time it takes to reach the adult stage for individuals
belonging to each experimental group. Treatments with NPs-TiO2 and
Al2O3 increase the number of days that hatched nymphs need to reach
the adult stage. The other parameters of post-embryonic development
did not show differences between the experimental groups (Table 1).
3.4. Biochemical parameters
3.4.1. Protein content
The protein content per individual for the different groups of the pa-
rental and filial generations is represented in Fig. 2. As can be seen, there
is no pattern in the protein content, with very variable results depend-
ing on the experimental group. The statistical analysis showed a signif-
icant increase in the parental generation between individuals exposed
to NPs-TiO2 and its control, and a significant decrease in the group ex-
posed to NPs-Al2O3 and its control. In the filial generation, there were

Table 1
Summary of the results from the different parameters studied. Fresh and dry weight data belong to the parental generation.

Experimental Parameter
group
Mortality Fresh Dry Pre-reproductive Fecundity Embrionary Fertility Nymphal Survival of
(%) weight weight (g) period (days) (eggs/female) developmental (nymphs life nymphs
(g) period hatched/female) span (days) (1st-5th stage)
(days) (%)

Control 33.3 ± 0.044 ± 0.017 ± 0.001 6.92 ± 0.77 201.30 ± 88.59 4.87 ± 0.09 128.03 ± 98.51 16.63 ± 96.71 ± 4.37
ethanol 11.5 0.001 0.46
NPs-TiO2 21.7 ± 0.044 ± 0.017 ± 3.3 × 6.58 ± 0.19 208.85 ± 4.92 ± 0.13 154.58 ± 92.05 17.19 ± 92.93 ± 5.22
17.6 0.003 10−4 108.13 0.76
Control water 23.3 ± 2.9 0.042 ± 0.017 ± 0.001 7.03 ± 0.18 189.15 ± 87.22 4.80 ± 0.29 144.43 ± 86.93 16.59 ± 90.44 ± 6.19
0.003 0.44
NPs-Al2O3 35.0 ± 0.039 ± 0.015 ± 0.002 7.31 ± 0.34 167.10 ± 74.53 4.72 ± 0.12 109.12 ± 67.19 16.59 ± 84.89 ± 11.19
13.2 0.005 0.59
Al2O3 33.3 ± 0.042 ± 0.016 ± 0.002 6.98 ± 0.76 188.42 ± 80.88 4.68 ± 0.02 130.92 ± 75.38 16.85 ± 92.85 ± 7.15
15.3 0.005 0.44
762 D. López-Muñoz et al. / Science of the Total Environment 666 (2019) 759–765

24
23.5 10
Days from birth to adult

a
23 * 9

mg total lipids/insect
* 8
22.5 *
7 b *
22
6
21.5 5 Parental

21 4 Filial
3
20.5
2
20
Ethanol NPs-TiO2 Water NPs-Al2O3 Al2O3 1
0
Ethanol NPs-TiO2 Water NPs-Al2O3 Al2O3

Fig. 1. Time elapsed from the first nymphal stage to adult stage individuals of O. fasciatus
coming from parents exposed to different experimental conditions. * indicate significant
differences (p b 0.05) with the respective control group.
statistical differences between the group exposed to Al2O3 in bulk form
Fig. 3. Total lipid levels per individual of O. fasciatus corresponding to the parental and filial
generation for the different experimental groups. Different letters indicate statistical
differences between experimental groups. An asterisk indicates significant differences
between generations of the same experimental group (p b 0.05).

respect the control group. In addition, differences were also found be-
tween generations for the NPs-Al2O3 and Al2O3 (bulk form) groups. group is detected this metal. A small content of Al is observed in the
water (control) group, but the amount of this metal in alumina treated
3.4.2. Total lipids groups (both bulk form or nanoparticulate) is 17–30 times higher than
Mean content of lipids per insect in the different groups assayed is in controls.
reported in Fig. 3. As can be observed, only significant differences
were found between the group exposed to NPs-TiO2 and its control 4. Discussion
(ethanol) for the filial generation.
When comparing between generations, filial generation of insects The fact that none of the metal-oxide treatments assayed in our
exposed to ethanol or NPs-Al2O3 presented higher lipid content than study produced mortality above of that observed in their respective
the parental ones. controls allows us to affirm that the experiment has been carried out
under sublethal conditions. Xie et al. (2014) and Jovanović et al.
3.4.3. Lipid peroxidation (TBARS) (2016) also reported absence of mortality in B. mori (Lepidoptera) or
Fig. 4 shows MDA levels per milligram of protein for both genera- D. melanogaster (Diptera), respectively, after treatment of larval stages
tions in each experimental group. Considering the parental generation, with NPs-TiO2 whereas G. pyloalis (Lepidoptera) responds contrarily,
not statistical differences were observed among the experimental with a significant increase in mortality (Memarizadeh et al., 2014). Re-
groups. garding Al2O3 toxicity, either in bulk or nanoparticulate form, it caused a
On the other hand, the filial generation showed a net increase in significant increase in mortality in S. oryzae (Coleoptera) (Stadler et al.,
MDA content in the experimental groups when compared with their 2017; López-García et al., 2018) and A. lobicornis (Hymenoptera)
own control, but these results only were significant in Al2O3 (bulk (Buteler et al., 2018). Discrepancy in the responses may be due to differ-
form) group respect the NPs-Al2O3 group and its control. ences in nanoparticle characteristics (size, shape, surface, etc.), to the
specific sensitivity of the insects, to the administration route or to the
3.5. Metal content stage of development used in each test.
The content of Ti in O. fasciatus individuals is greater than that
Table 2 shows the content (μg/g) of Al or Ti in insects of the different observed in its corresponding control (ethanol), indicating that the
experimental groups. As can be observed, the amount of Ti in the water
(control) group are below the detection limit, whereas in the NPs-TiO2
0.007
b
4.5 0.006
a
MDA (mM/mg protein)

4 B' A
3.5
* * 0.005
A' A,B
mg protein/insect

B
3 0.004
2.5
b Parental a Parental
2 0.003 a
Filial Filial
1.5
0.002
1
0.5
* * *
0.001
0
Ethanol NPs-TiO2 Water NPs-Al2O3 Al2O3 0.000
Ethanol NPs-TiO2 Water NPs-Al2O3 Al2O3

Fig. 2. Protein levels per individual of O. fasciatus for the different groups of the parental Fig. 4. mM MDA/mg protein in O. fasciatus corresponding to the parental and filial
and filial generation. Different lowercase letters indicate significant differences between generation for the different experimental groups. Different letters indicate significant
experimental groups of the filial generation, capital letters among the parental differences between experimental groups. An asterisk indicates significant differences
generation. An asterisk indicates significant differences between generations (p b 0.05). between generations of the same experimental group (p b 0.05).
 D. López-Muñoz et al. / Science of the Total Environment 666 (2019) 759–765
Table 2
Mean values of aluminum or titanium content in insects from the different experimental
groups.
Experimental Mean content of Mean content of
group titanium aluminum
(μg/g) (μg/g)
Ethanol b2 – because the amount of Ti in the insect is not sufficient to induce the pro-
NPs-TiO2 47.33 ± 12.22 –
Water – 18.67 ± 3.79
NPs-Al2O3 – 581.67 ± 341.54
Al2O3 – 316.67 ± 118.09
tarsal contact treatment allows the incorporation of NPs-TiO2, either by
its adhesion on the cuticle of the insect or by its introduction through it,
as has been observed with other nanoparticles and organisms (Pandey
et al., 2013). The quantities of Ti measured in O. fasciatus are different
from those observed in Ch. tentans after the administration of TiO2 in
the sediment (Savic-Zdravkovic et al., 2018), but the differences ob-
served may be due to the method of application and the amounts used.
Regarding Al content in the different experimental groups, the small
amount observed in the control (water) could be explained by its pres-
ence in the drinking water or in the food supplied during the experi-
ments. On the other hand, the amounts of Al accumulated by insects
of the NPs-Al2O3 and Al2O3 groups are approximately 31 and 17 times
higher, respectively, than those of their control. It has been indicated
that alumina has sorptive properties, so it adheres strongly to the cuticle
(Stadler et al., 2017, 2018), increasing the rate of water loss from the in-
sect and its dehydration. Also, NPs-Al2O3 could penetrate into the insect,
as discussed above, thus contributing to the alterations in some of the
parameters studied.
Reproductive parameters were not altered by Al2O3 or the oxide-
metal NP ectopic exposure. While lack of response in the same parame-
ters here studied was also found by other authors when testing other
types of nanoparticles in other insect species (Small et al., 2016;
Philbrook et al., 2011), some studies reported that NPs-TiO2 and NPs-
Al2O3 affected the fecundity in D. melanogaster or G. mellonella
(Philbrook et al., 2011; Zorlu et al., 2018), as well as in the nematode
model C. elegans (Wang et al., 2009).
One of the most interesting post-embryonic developmental results
is the prolongation of the time that it takes for the nymphs to reach
adulthood after exposure to NPs-TiO2 in comparison to their ethanol
control. This result indicates that NPs-TiO2 affect development in in-
sects, independently of the species investigated and the administration
type, as is demonstrated by Small et al. (2016) in Blatella germanica in-
dividuals after exposure to gold nanoparticles, or by Zorlu et al. (2018)
after ingestion of NPs-TiO2 by Galleria mellonella. On the other hand, di-
atomaceous earth is absorbed by insect's cuticular waxes causing water
loss (Stadler et al., 2017) which could affect postembryonic develop-
ment as in the case of O. fasciatus exposed to Al2O3 (bulk form).
Analyzing the results of the exposure to nanoparticles on the differ-
ent biochemical parameters studied, it could be said that the composi-
tion of Oncopeltus fasciatus itself is affected, since differences were
found between some exposed groups and their controls.
NPs-TiO2 exposure did not produce any adverse effects on parame-
ters related to the health status (wet and dry weight, lipid content,
lipid peroxidation) of adult Oncopeltus of the parental generation but in-
creased protein content, probably by promoting protein synthesis (Tian
et al., 2016). However, their offspring had a longer post-embryonic de-
velopment and presented lower lipid content. The mechanism of action
of titanium nanoparticles is very little known. Tian et al. (2016) found
an increase in triglyceride and lipase activity in fat body of the silkworm
after ingestion of NPs-TiO2, indicating that NPs can enhance the metab-
olism of lipids. This is not the case in O. fasciatus. Li et al. (2014) found an
increase in 20-hydroxyecdisone biosynthesis when administered to
B. mori, which reduced the duration of molting. In the present study,
the duration of the nymphal period was longer, what is contrary to
763
that pointed by Li et al. (2014). On the other hand, TBARS content has
not been affected by NPs-TiO2. It has been reported that B. mori treated
with these nanoparticles at 30 °C reduce oxidative stress by promoting
expression of antioxidant genes (Li et al., 2018) or decreased ROS accu-
mulation (Xie et al., 2014). The fact that, according to the results pre-
sented here, the NPs-TiO2 do not induce oxidative stress may be
duction of ROS, or O. fasciatus has low sensitivity to this metal.
NPs-Al2O3 exposure produced a decrease in protein content of the
parental generation as the sole significant effect and Al2O3 did not
cause any effect on the parental generation but increases the days to
reach adulthood and caused a decrease in protein content and an in-
crease in lipid peroxidation in the filial generation. It has been pointed
out that charged alumina NPs could be absorbed by the insect cuticle
and dehydrate it (Stadler et al., 2017, 2018) which may influence
some of the effects observed after these exposures.
We have not found any work that evidences a decrease in the total
protein content due to exposure to alumina nanoparticles. But, if we
consider that body weight is directly related with total protein content,
Stadler et al. (2017) found a reduction of 51,6% in body weight of
Sitophylus oryzae after exposure to nanostructured alumina. In our
case the reduction is similar for the bulk form of alumina.
The finding of an increase in lipid peroxidation in the filial genera-
tion after Al2O3 exposure is in line with the effects found by Prabhakar
et al. (2012) in kidney cells from rats exposed to this compound. It is
known that the oxidative damage caused by the nanoparticulate form
of various compounds is greater than those produced by their bulk
form (Wang et al., 2009). Therefore, we see that the group of the filial
generation exposed to Al2O3, in addition to the decrease in their protein
levels, also presented higher levels of lipid peroxidation, suggesting that
a higher protein content leads to less oxidative damage. Insects, to com-
bat the effects of ROS, develop enzymatic mechanisms of antioxidant
defense (Felton and Summers, 1995; Kodrik et al., 2015; Zorlu et al.,
2018), so it can be assumed that the higher the protein content, the
greater the possibility of defense against oxidative stress and reduces
the damage produced.
We have observed differences between generations in all the ana-
lyzed biochemical parameters, with the filial generation presenting, in
most cases, higher levels of these parameters than the parental one.
Usually, older insects tend to present greater oxidative damage, since
this increases exponentially with age in various organisms (Sohal and
Weindruch, 1996; Rodríguez and Céspedes, 1999). However, in our
study, the parental generation, composed of older adult individuals
(16 days of age when analyzed) than the filial one (1 day old), showed
less oxidative damage.
Some authors (McCahon and Pascoe, 1991; Wu et al., 2011) indicate
that during the early stages of growth insects are more sensitive to ox-
idative stress, and any alteration in the insect's environment can in-
crease it. In our case eggs hatched were kept in containers with a high
population density, which could cause such oxidative damages due to
stress generated by competition for food, water or simply due to
crowded conditions (Feir, 1974).
Also, it is known that the adipokinetic hormone acts as an anti-stress
hormone (Kodrik et al., 2015; Zemanová et al., 2016) stimulating the
catabolic reactions that mobilize the energetic stores and inhibiting
the synthesis reactions, which allows the mobilized energy to be used
to eliminate stress situations. These anti-stress reactions trigger physio-
logical responses to combat it, such as, for example, the activation of an-
tioxidant mechanisms (Krishnan and Kodrík, 2012; Kodrik et al., 2015).
The fact that the individuals of the filial generation only had one day of
age makes us suppose that the level of this hormone in hemolymph has
not reached the right level, as it happens, for example, with the juvenile
hormone. Therefore, an insufficient concentration of adipokinetic hor-
mone could suppose a greater oxidative stress, which is reflected in
our results by higher TBARS values in the filial generation with respect
to the parental one.
764 D. López-Muñoz et al. / Science of the Total Environment 666 (2019) 759–765
5. Conclusions
The presence of measurable amounts of Ti or Al in individuals of each
experimental group corroborates the incorporation (external or inter-
nal) of NP in the animal body through the tarsal contact assay, assuring
the existence of an effective interaction.
Topic administration of NPs-TiO2 to adult individuals of O. fasciatus
caused an increase in the total protein content and interferes with de-
velopmental processes in their non-exposed offspring by increasing
the days elapsed from birth to adulthood and resulting in adults with
a lower lipid content than those coming from parents of the control
group.
Tarsal contact of NPs-Al2O3 decreased total protein content of the
parental generation and enhanced lipid content of adults of the filial
generation. Administration of Al2O3 affects post-embryonic develop-
mental period as well as protein and TBARS in the filial generation.
All compounds tested in the present study, when present at suble-
thal amounts in surfaces transitted by insects, may be incorporated by
them and produce trans-generational effects that are not quickly de-
tected and that may have long-term effects in non-detrimental insect
species.
Conflict of interest
The authors declare that they have no conflict of interest.
CRediT authorship contribution statement
Daniel López-Muñoz: Investigation, Methodology, Software, Writing -
original draft. Mª. Amparo Ochoa-Zapater: Formal analysis, Visualiza-
tion, Methodology, Resources. Amparo Torreblanca: Conceptualization,
Writing - review & editing, Funding acquisition. Mª. Dolores Garcerá:
Conceptualization, Project administration, Supervision, Funding acquisi-
tion, Writing - review & editing.
Acknowledgments
This work was supported by grant from the Ministerio de Economía
y Competitividad (project AGL2010-21555). The authors acknowledge
M.A. Silvestre for the statistical support.
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