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Drosophila melanogaster as a model organism to study nanotoxicity

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 Nanotoxicology

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Drosophila melanogaster as a model organism to

study nanotoxicity

Cynthia Ong, Lin-Yue Lanry Yung, Yu Cai, Boon-Huat Bay & Gyeong-Hun Baeg

To cite this article: Cynthia Ong, Lin-Yue Lanry Yung, Yu Cai, Boon-Huat Bay & Gyeong-
Hun Baeg (2015) Drosophila melanogaster as a model organism to study nanotoxicity,
Nanotoxicology, 9:3, 396-403, DOI: 10.3109/17435390.2014.940405

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ISSN: 1743-5390 (print), 1743-5404 (electronic)

Nanotoxicology, 2015; 9(3): 396–403

! 2014 Informa UK Ltd. DOI: 10.3109/17435390.2014.940405

REVIEW ARTICLE

Drosophila melanogaster as a model organism to study nanotoxicity

Cynthia Ong1, Lin-Yue Lanry Yung2, Yu Cai3, Boon-Huat Bay1, and Gyeong-Hun Baeg1
1
Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 2Department of Chemical & Biomolecular
Engineering, Faculty of Engineering, National University of Singapore, Singapore, and 3Temasek Life Sciences Laboratory, National University of
Singapore, Singapore
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Abstract Keywords
Drosophila melanogaster has been used as an in vivo model organism for the study of genetics Drosophila melanogaster, in vivo model
and development since 100 years ago. Recently, the fruit fly Drosophila was also developed organism, nanomaterials, toxicity
as an in vivo model organism for toxicology studies, in particular, the field of nanotoxicity.
The incorporation of nanomaterials into consumer and biomedical products is a cause for History
concern as nanomaterials are often associated with toxicity in many in vitro studies. In vivo
animal studies of the toxicity of nanomaterials with rodents and other mammals are, however, Received 24 February 2014
limited due to high operational cost and ethical objections. Hence, Drosophila, a genetically Revised 18 June 2014
tractable organism with distinct developmental stages and short life cycle, serves as an ideal Accepted 19 June 2014
organism to study nanomaterial-mediated toxicity. This review discusses the basic biology Published online 22 July 2014
of Drosophila, the toxicity of nanomaterials, as well as how the Drosophila model can be used
to study the toxicity of various types of nanomaterials.

Introduction consumer and biomedical products (Eby et al., 2009; Hemmati

et al., 2009; Lin et al., 2013; Tripp et al., 2007). Despite the
Drosophila melanogaster was first introduced by Thomas Hunt
extensive use of nanomaterial today, there is still limited
Morgan as a model for research since 100 years ago (Morgan,
understanding of nanomaterial-mediated toxicity in vivo. The
1910). From 1910 to 1960s, genetic approaches directed the
Drosophila model presents an interesting alternative in the study
research carried out in Drosophila, while genetic concepts
of nanotoxicity. A recent editorial has also encouraged the use
developed during these 50 years led the way to novel discoveries
of the fruit fly Drosophila in nanotoxicity and nanomedicine
in the biological systems (Bellen et al., 2010). Subsequently, the
research in view of the possible novel scientific knowledge and
Drosophila was used to study human diseases and therapeutic
technological breakthroughs that Drosophila can bring about
strategies (Pandey & Nichols, 2011). Recently, the Drosophila
(Vecchio, 2014). In this review, we will discuss in greater depth
was also successfully developed as a model organism in
the toxicity of nanomaterials, the basic biology of Drosophila and
toxicology studies (Bhargav et al., 2008; Coulom & Birman,
how the Drosophila model was used to study nanotoxicity.
2004; Dean, 1985; Hosamani, 2013; Siddique et al., 2013), and
the new term Drosophotoxicology was proposed (Rand, 2010).
Toxicity of nanomaterials
Among other compounds, the use of the Drosophila in inorganic
mercury toxicity testing has identified toxicity effects on various Engineered nanomaterials can be classified into carbon-based,
physiological functions of Drosophila and possible signaling polymer-based, silicon, ceramic, metal or metal oxide materials.
cascades associated with inorganic mercury toxicity, providing a The minute size of nanomaterials, which are between 1 and 100
better understanding of the mechanism mediating inorganic nanometers, brings about a high surface area to volume ratio,
mercury toxicity (Paula et al., 2012). resulting in novel properties that are absent in their bulk form
In light of the recent success of the Drosophila model in (Powell & Kanarek, 2006a). The physicochemical properties
toxicology studies, there has been an enhanced interest in the use such as high conductivity, strong optical scattering properties,
of Drosophila for the understanding of nanomaterial-mediated strong absorbance and ease of functionalization give rise to
toxicity. Nanomaterial is defined as any material with the size of many nanomaterial-related consumer and biomedical products
1–100 nanometers in one or more dimensions (Powell & Kanarek, (Jang et al., 2010; Lim et al., 2011; Ong et al., 2013; Panahifar
2006b; SCENIR (Scientific Committee on Emerging and Newly et al., 2013). However, recent in vitro studies have revealed that
Identified Health Risks)). Their small size and unique properties nanomaterials could result in toxicity. Toxicity refers to the extent
have spurred the incorporation of nanomaterials in a myriad of of a substance to cause harm to an organism and it is closely
related to substance exposure, distribution, metabolism, inter-
action with macromolecules and the toxic end point (Hodgson,
2004). For instance, titanium dioxide nanoparticles (TiO2 NPs),
Correspondence: Gyeong-Hun Baeg, Department of Anatomy, Yong Loo
which are one of the most common nanomaterials used in
Lin School of Medicine, National University of Singapore, Block MD10, consumer products, were found to be cytotoxic and genotoxic in
4 Medical Drive, Singapore 117594, Singapore. Tel: +65 6516 7973. Fax: human epidermal cells (A431) after 48 h and 6 h exposure
+65 67787643. E-mail: [email protected] to 80 mg/ml of TiO2 NPs, respectively (Shukla et al., 2011).
 DOI: 10.3109/17435390.2014.940405 Toxicity study in Drosophila 397

The increase in reactive oxygen species (ROS) production and

effect on cellular calcium homeostasis were attributed to be the
cause of the nanotoxic effects observed (Shukla et al., 2011;
Simon et al., 2011). Some studies on TiO2 NPs toxicity revealed
varying toxicity profile in different crystalline structure of TiO2
NPs. TiO2 NPs can exist in the anatase form and rutile form in
a mix of 80/20, and anatase was found to result in more ROS
production and toxicity than rutile, especially after UV irradiation
(Petkovic et al., 2011; Sayes et al., 2006). Carbon nanotubes
also caused genotoxicity in human bronchial epithelial cells
and mesothelial cells. Dose-dependent DNA damage in human
bronchial epithelial cells was detected by the comet assay
(Lindberg et al., 2009, 2013). Gold nanoparticles (AuNPs),
which are inert in its bulk form, are cytotoxic as well. AuNPs
induce oxidative stress-mediated genotoxicity in MRC-5 human
lung fibroblast cells. The presence of autophagosomes, which
are concomitantly observed with AuNPs uptake under the
transmission electron microscope and proteomic analysis, indi-
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cates that autophagy is induced by nanoparticles and may be a Figure 1. The whole life cycle of the fruit fly Drosophila is relatively
cellular defense mechanism against oxidative stress (Li et al., rapid and takes only approximately 10–12 days at 25  C. The Drosophila
2008, 2010a, 2011). Alteration in micro RNA expression and development is divided into various stages: embryo, larva (first instar,
epigenetic processes was also implicated during AuNP exposure second instar and third instar), pupa and adult.
(Ng et al., 2011). Recently, more in vivo studies of nanomaterials
have been performed for a better understanding of nanomaterial- were identified, of which 95% of the genes were encoded on three
mediated toxicity. Silver nanoparticles (AgNPs) of size of the four chromosomes (Rand, 2010). A systemic analysis
7.9 ± 0.95 nm were intravenously injected (0.5 mg/kg and further revealed that 77% of distinct genes related to human
5.0 mg/kg AgNPs) at a single dose into rabbits and monitored diseases matches Drosophila sequences (Reiter et al., 2001).
for up to 28 days. AgNP accumulation was observed in liver, Studies have also shown that Drosophila proteins involved in the
kidney, spleen, lung, brain, testis and thymus, indicating that regulation of gene expression and metabolism exhibit close
AgNPs can be transported through the blood circulation. Notably, similarity to human counterparts. Furthermore, genomic analysis
excretion of silver was mainly found in the feces than urine, revealed a good correspondence of Drosophila biosynthetic
suggesting that biliary excretion may be the main mechanism networks of important biochemical pathways to human (Adams
for the removal of AgNPs from the body (Lee et al., 2013). et al., 2000).
On the other hand, anatase TiO2 NPs exposure to newborn mice Drosophila is easy to maintain, propagate and manipulate.
by intranasal instillation as a single dose (1 mg/g body weight) Flies can be kept in vials and fed on food medium consisting
on postnatal day 4 or three doses on postnatal days 4, 7 and 10 of cornmeal, glucose, agar and fungicide (Rand, 2010). The
was shown to induce inflammation and inhibit lung development. whole life cycle of Drosophila is relatively rapid and takes only
As the study endpoint is at 14 days of age, a longer study period approximately 10–12 days at room temperature. The Drosophila
is required to determine the risk of respiratory problems later development is divided into various stages: embryo, larva, pupa
in life after TiO2 NPs exposure (Ambalavanan et al., 2013). and adult (Figure 1). Eggs are laid on the food, and embryogenesis
Nanotoxicity studies using non-mammalian in vivo models takes place within the egg. In less than 24 h, the first instar larva
have also enabled a greater understanding of the toxicity effects hatches and begins feeding. This feeding and growth phase
of nanomaterials. In Caenorhabditis elegans (roundworm), will last for four days. About a 200-fold increase in weight of the
reduced growth rate, decreased lifespan, declined reproduction larva is expected during the growth phase and is largely due to
and defective embryogenesis were observed after the ingestion of the endoreplication of larval tissues. However, the larval tissues
highly soluble amide-modified single-walled carbon nanotube. will not be the part of the adult fly as these tissues are broken
The observed toxicity effects were shown to be resulted from down during metamorphosis in the pupa stage. The imaginal
defective endocytosis, decreased citrate cycle and nuclear trans- discs, which are made up of diploid cells of undifferentiated
location of DAF-16 transcription factor, indicating the molecular epithelium, will eventually contribute to the development of adult
mechanism underlying the nanotoxicity (Chen et al., 2013). fly structures. At the end of third instar stage, the larvae stop
The Danio rerio (zebrafish) model has also shown to be useful. feeding and leave the food in search of an area for pupariation.
Exposure to various concentrations of 62 nm silica nanoparticles During the pupa stage, metamorphosis occurs for four days,
to 4–96 h old embryos can pose adverse effects on the mortality, following which adult flies eclose. Adult female flies are normally
hatching rate and larval locomotor activity in a dose-dependent larger than adult male flies with females weighing 1.4 mg and
manner. Malformations of embryos such as pericardial edema, males 0.8 mg. Females are ready to mate in less than 24 h after
yolk sac edema, tail and head malformation were also observed by eclosion and can lay up to 100 eggs per day. Adult flies live about
the treatment of silica nanoparticles (Duan et al., 2013). Together, two months after eclosion (Pandey & Nichols, 2011; Stocker &
these in vitro and in vivo studies have provided important insights Gallant, 2008).
into our understanding of nanomaterial-mediated toxicity.
Drosophila as a model to study the toxicity
Biology of D. melanogaster of nanomaterials
Drosophila melanogaster is often used to study genetics due to Despite the increase in the number of in vivo nanomaterial
its simple genetic makeup consisting of only four chromosomes. toxicity studies being carried out today, it is still insufficient
The complete Drosophila genome has previously been sequenced to address important issues in the field of nanotoxicology. Key
and annotated (Adams et al., 2000). Approximately 13 600 genes questions such as what are the long-term effects of nanomaterial
 398 C. Ong et al. Nanotoxicology, 2015; 9(3): 396–403

exposure and what is the exact molecular mechanism underlying investigated at various developmental stages. When Drosophila
nanomaterial-mediated toxicity remain to be addressed. Many eggs were exposed to AgNP-food, a significant effect on
nanotoxicology studies are often limited to in vitro models as survivorship was observed during the adult stage. Many of the
in vivo models are expensive and difficult to carry out, and often resulting larvae failed to pupate, and time to pupation was
raise ethical objections. delayed. In addition, there was a decrease in the number of flies
Drosophila can live up to 40–60 days after eclosion, and thus leaving the pupa stage and emerging as adult flies compared to
nanotoxicity can be assessed at various ages of adult flies. control flies (personal communication). CdSe-ZnS quantum dots
Furthermore, due to its short lifespan, chronic nanotoxicity studies (QDs) are toxic to Drosophila. Ingestion of CdSe-ZnS QDs
can easily be carried out using specific tissues or organs of caused a strong decline in lifespan when compared to controls.
subsequent filial generations to examine the effects of nanoma- Lifespan refers to the length of time between eclosion and death.
terials on genome stability, development, reproduction and Notably, different types of coating on nanomaterials can contrib-
viability (Greenspan, 2004). The various developmental stages ute to different toxicological profile. CdSe-ZnS QDs coated with
of Drosophila are also essentially good models for toxicology poly(maleic anhydride octadecene) and polyethylene glycol were
studies. For instance, at the embryonic stage, developmental more toxic than those coated with mercaptoundecanoic acid or
studies regarding cell fate determination, neuronal development poly(maleic anhydride octadecene) alone (Galeone et al., 2012).
and organogenesis upon exposure to toxins can be carried out The lifespan of Drosophila can also be used to study how various
while the wandering third instar larva can be used for develop- metrics can affect the toxicity of nanomaterials, as exemplified
mental, physiological and behavioral studies. Furthermore, the by the finding that the total number of particles but not the
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identification of how toxins affect the imaginal discs during the total surface area of ingested citrate-capped AuNPs shorten the
late larval through the pupa stage will also be useful to understand lifespan of Drosophila (Pompa et al., 2011b). Importantly,
the adverse effects of toxins on endoreplication and morpho- however, not all nanomaterials are toxic to Drosophila, indicating
logical changes from larval to adult stage (Pandey & Nichols, the robustness and reliability of nanotoxicity study in Drosophila.
2011; Stocker & Gallant, 2008). Notably, the anatomical struc- Organically modified silica nanoparticles (20 nm) and gallium
tures such as the brain, heart, lung, kidney, liver, gut and phosphide nanowires (80 nm) have no significant effects on the
reproductive tract of the adult Drosophila are physiologically development and viability of larvae and adult Drosophila
very similar to those of humans (Pandey & Nichols, 2011). (Adolfsson et al., 2013; Barandeh et al., 2012). Likewise,
For example, the Drosophila fat body functions like the human Gellan Gum-PEI nanocomposites showed no significant effects
liver. The fat body utilizes lipoprotein particles for the transpor- on the survivorship of Drosophila (Goyal et al., 2011). Submicron
tation of lipids to peripheral tissues, while lipid metabolism in size insulin-small lipid nanoparticles, developed for insulin
Drosophila is regulated by insulin signaling (Canavoso et al., delivery, were also non-toxic to Drosophila even after chronic
2001; Diangelo & Birnbaum, 2009). The respiratory system of exposure from egg to adult, providing important preliminary
Drosophila, named Drosophila tracheal system, is a branched evidence of the suitability of insulin-small lipid nanoparticles
network of epithelial tubes that ramifies throughout the body for oral insulin delivery in patients (Fangueiro et al., 2013).
for the transport of oxygen and other gases. Considering Nanomaterials can also be introduced to Drosophila via dry
these physiological similarities of organs between Drosophila physical exposure, which is equivalent to dermal exposure to
and humans, Drosophila can serve as a suitable model for organ- human. Dry nanomaterial such as carbon black and single-walled
specific toxicology studies. nanotubes were added as a powder to the bottom of sealed glass
Recommendations by the European Centre for the Validation vials in the absence of food and water. Nanomaterials were found
of Alternative Methods indicated that D. melanogaster is an to adhere strongly to the surface of the flies, resulting in mortality
ideal model organism to study nanomaterial-mediated toxicity in all exposed flies within a few hours. As nanomaterials were
(Ahamed et al., 2010). The short life cycle, the distinct found to partially block spiracle openings in Drosophila, defects
developmental stages, the availability of various tools and in respiration were considered the primary cause of the mortality
reagents, known genome sequence and the physiological similar- during dry exposure (Lehmann, 2001; Liu et al., 2009).
ity of Drosophila with humans make Drosophila an excellent Nanotoxicity study in Drosophila can also be carried out in the
in vivo model organism to rapidly test toxicity in whole organism methodology of inhalation exposure, using a nebulizer-based
and elucidate the molecular mechanisms underlying the toxicity. method that exposes Drosophila to aerosolized nanoparticles,
which are small enough to enter the spiracle openings of
Drosophila. This study has shown that different sizes and types
Survivorship
of nanoparticles such as FluoSpheres (24, 100 and 210 nm), silver
The most direct method to evaluate nanomaterial toxicity in the (20 nm) and CdSe/ZnS (5.7 ± 0.5 nm) can be delivered to the
Drosophila model is through the determination of survivorship Drosophila respiratory system (Posgai et al., 2009). Subsequent
after nanomaterial exposure. As nanomaterials can gain entry into toxicity testing such as survivorship can then be studied. Findings
the human body by several ways such as oral, dermal and from this mode of exposure can thus be a good preliminary study
inhalation routes, it is essential that such entries are modulated to investigate nanomaterial inhalation toxicity in human.
in the Drosophila model (Li et al., 2010b). The availability of simple yet versatile ways of administration,
One possible route of nanomaterial exposure to Drosophila and the reproducibility and specificity of nanomaterial-mediated
is through ingestion. For instance, nanomaterials can be added toxicity, make Drosophila an excellent model organism to study
directly to the standard Drosophila food at various concentrations. nanotoxicity in vivo. However, it is worth to note that relevant
Newly enclosed male adult flies were transferred directly to the doses of nanomaterials should be administered to investigate the
food containing AgNPs (20 nm) after 6 h starvation, and survivors toxicity of nanomaterials in vivo. Doses that are too low or high
were accounted every 24 h for a period of 10 days. A significant may not yield meaningful conclusions regarding the toxicological
dose-dependent decrease in survival rate was observed for flies profile and mechanisms of nanomaterial as such concentrations
fed with AgNPs as compared to control flies. However, food may never be encountered by humans through occupational or
containing similar dose of AgNO3 did not affect the survival daily exposure. Furthermore, the method of administration is vital
rate of flies, suggesting that the toxicity was caused specifically to consider as different routes of entry of nanomaterials may
by AgNPs (Tian et al., 2013). Survivorship can also be induce toxicity in different ways. For instance during inhalation
 DOI: 10.3109/17435390.2014.940405 Toxicity study in Drosophila 399

exposure of AuNPs in rats, AuNPs was found to accumulate

Genotoxicity
mostly in the lungs, while intravenously injected AuNPs
were found to be accumulated more in the liver and spleen Specific damage to the genetic material (genotoxicity) is often the
(Balasubramanian et al., 2010; Yu et al., 2007). Therefore, the cause of various diseases such as cancer and hereditary genetic
differential accumulation of AuNPs due to the different routes disorders. Hence, there is an increased interest in the field of
of entry may account for varying organ specific toxicity. nanotoxicology to study the effects and interactions of nanoma-
terials with the genome of organisms. Drosophila that was
traditionally used for genomic discovery is an ideal model to
Oxidative stress
examine genotoxicity of nanomaterials in vivo, as demonstrated
Numerous in vitro studies showed that increased ROS production by the pioneering studies of Muller and Auerbach in investigating
after exposure to nanomaterials is one of the most common the genotoxic effects of X-ray and mustard gas compounds,
causes of nanomaterial-mediated toxicity (Jaeger et al., 2012; respectively (Auerbach, 1947; Muller, 1928). The known genome
Ng et al., 2013; Passagne et al., 2012; Pichardo et al., 2012). sequence, high genetic homology to humans and short lifespan
Excess ROS may cause damage to proteins, lipids and DNA are also advantageous for the use of Drosophila as a model in
in cells, eventually leading to diseases such as cardiovascular assessing acute and/or chronic genotoxicity.
diseases and neurological disorders (Turrens, 2003). Higher doses Exposure of the flies to 15 nm sodium citrate-capped AuNPs
of nanomaterials can induce greater oxidative stress. However, resulted in DNA fragmentation in the gastrointestinal (GI)
it is often challenging to investigate oxidative stress in an in vivo tissue, as demonstrated by the terminal transferase dUTP nick-
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mammalian model due to ethical issues of exposing high doses end-labeling (TUNEL) assay. A significant higher occurrence of
of nanomaterials to rodents. Drosophila, which raises less ethical DNA damage was encountered in AuNP-treated Drosophila
concern, thus can serve as a suitable in vivo model to study (8%) than the unexposed controls (51%) (Pompa et al.,
nanomaterial-induced oxidative stress. 2011a). Notably, larger AuNPs (40 and 80 nm) were found to
Oxidative stress was identified to be the primary cause of induce less genotoxic effects as compared to smaller AuNPs (5
toxicity induced by nanomaterials. Intracellular ROS level in and 15 nm), suggesting that smaller AuNPs may enter the tissue of
Drosophila after oral ingestion of various sizes of AuNPs (5, 15, the GI tract more efficiently than larger AuNPs (Vecchio et al.,
40 and 80 nm) was measured from the homogenate of the 2012b). The p53 is one of the key molecules involved in the
resulting flies using the 2,7-dichlorofluoresceindiacetate (DCF- regulation of cellular and genomic integrity, the inhibition of cell
DA) dye. There was a significant increase in ROS level in growth and apoptosis (Marcel et al., 2011). Quantitative real-time
Drosophila exposed to AuNPs as compared to unexposed control PCR expression profiling revealed an increase in p53 gene
flies. However, the different sizes of AuNPs had no effect on ROS expression in Drosophila upon AgNP treatment as compared to
production, indicating that the total surface area of AuNPs is not controls, corresponding to the increase in DNA damage observed
an important parameter in inducing oxidative stress (Vecchio in the TUNEL assay (Pompa et al., 2011a; Vecchio et al., 2012b).
et al., 2012b). Treatment of flies with high doses of amorphous Long-term mutagenic effects of AuNPs were investigated through
silica nanoparticles (10 and 100 mg/ml) also caused a time and F0, F1 and F2 generations. While F0 generations were fed with
dose-dependent increase in oxidative stress in larval midgut, AuNP-food, F1 and F2 generations were kept in untreated food.
as demonstrated by flow cytometry using DCF dye and Systematic screening of the phenotype of F1 generation identified
lipid peroxidation assay that measures malonyl dialdehyde. flies with malformed wings, eyes or thorax. The F1 flies with the
Consistently, increased antioxidant activities of superoxide malformed phenotype were then crossed with wild-type flies to
dismutase (SOD) and catalase, which are thought to be related obtain F2 generation flies. Interestingly, some F2 flies were
to the cellular defense of Drosophila against nanomaterials, were observed to have severely impaired body parts that include
detected (Pandey et al., 2013). Similar experiments with AgNPs malformed eyes or wings. Therefore, this study suggests that
also revealed the high induction of both oxidative stress and AuNPs are genotoxic and induce genetic mutations in germline
antioxidant activity in third-instar larvae. Western blotting cells, causing chronic genotoxicity on subsequent generations
analysis using the larval tissue extract showed an increase in (Vecchio et al., 2012a). Genetic damage can also be induced by
the expression of heat-shock protein (HSP) 70, which plays an AgNPs in Drosophila. The expression of p53 and p38 was found
important role in biological stress and is a valuable marker for the to be up-regulated upon AgNPs ingestion during the third-instar
evaluation of the adverse effects of AgNPs (Ahamed et al., 2010). larval stage, indicating the effects of AgNPs on DNA integrity and
Ingestion of CdSe/ZnS QDs also showed to increase the cell viability (Ahamed et al., 2010). CdSe-ZnS QDs also induce
transcription of hsp70 and hsp83 genes in the larva (Brunetti genotoxicity in Drosophila. More TUNEL-positive nuclei were
et al., 2013). observed in hemocytes exposed to the QDs than those of control
Interestingly, however, when vitamin C or vitamin C palmitate flies (Galeone et al., 2012).
was added to AgNP-food for ingestion during the larval stage, a Genotoxicity can be investigated using the wing somatic
significant increase in survivorship, development and mating mutation and recombination test (SMART). The SMART assay
success was observed. This suggests that the antioxidant vitamin enables to identify somatic recombination and other genomic
C can suppress the induction of oxidative stress caused by aberrations, such as point mutations, deletions and chromo-
nanomaterial treatment. In support of this, a short-term 24-hour somal aberrations (Graf et al., 1984). Genotoxic effects induced
exposure of flies to AgNPs with vitamin C was shown to reduce can be observed as an increase in the number of mutant spots,
SOD levels and increase Glutathione levels as compared to flies which are caused by the expression of two recessive markers,
exposed to AgNP alone (Posgai et al., 2011). Finally, alumina namely, multiple wing hairs or flare-3 on the wings. Third
nanoparticles decreased the frequency of oscillations in the local instar larvae fed with AgNP-food had an increase in occurrence
interneurons (LNs) and synchronizations in paired LNs, and these of small single mutant spots as compared to the negative
adverse effects of the nanomaterials on the central nervous system control. By contrast, balanced heterozygous flies, which have
of Drosophila were thought to be caused by oxidative stress abolished somatic recombination, had no difference in the
(Huang et al., 2013). All these studies suggest that increased number of mutant spots, indicating that the genotoxic effects
levels of oxidative stress are the primary cause of nanomaterial- are due to somatic recombination. On the other hand, silver
mediated toxicity across phyla. nitrate at various concentrations failed to induce genotoxicity,
 400 C. Ong et al. Nanotoxicology, 2015; 9(3): 396–403

suggesting that genotoxic effects are due to AgNPs and

not Ag+ (Demir et al., 2011). Dose-dependent genotoxic
effects of cobalt nanoparticles were also detected by the
SMART assay in Drosophila (Vales et al., 2013). Similarly,
not all nanomaterials are genotoxic. Gallium phosphide
nanowires, multi-walled carbon nanotubes, zirconium oxide,
aluminum oxide and TiO2 failed to induce genotoxicity in
Drosophila (Adolfsson et al., 2013; de Andrade et al., 2014;
Demir et al., 2013).

Fecundity
Drosophila also serves as a model for fecundity testing due to its
rapid life cycle and numerous offsprings (Stocker & Gallant,
2008; Tiwari et al., 2011). In particular, fecundity testing
in toxicology studies is increasingly being investigated in
Drosophila (Gupta et al., 2007; Kislukhin et al., 2012). In the
case of nanomaterials, the effect of AgNPs on fertility was
investigated for a long-term exposure of up to eight filial Figure 2. Drosophila treated with AgNPs exhibit defects in cuticle
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generations. Both males and females (parental generation) were development and melanization. Drosophila exposed to 5 mg/L of AgNPs
fed with food containing 5 mg/L of AgNP (29 ± 4 nm) for 10 via ingestion has unpigmented cuticle (right) as compared to normal
days. F1 progenies were then transferred to new AgNP-food to control Drosophila (left).
obtain the F2 generation. This was repeated till F8 generation.
While no significant changes in the number of F1 flies were Conclusion and future directions
observed, a gradual decrease in the number of eclosed flies was
The ease of synthesis and manipulation of nanomaterial today has
observed in the subsequent generations. However, fecundity was
given rise to the development of a variety of nanomaterial-related
found to improve from F4 generation and returned to normal at
products. However, many in vitro studies have pointed out that
F8 generation. Adaptability by decreasing development time and
these nanomaterials are potentially cytotoxic and capable of
thus less ingestion of AgNP-food in the larval stage were
resulting in oxidative stress, followed by genotoxicity. Several
suggested as the main reason for the improvement in fecundity
in vivo studies of nanotoxicity in mammals also provided
(Panacek et al., 2011). Similar decline in fecundity upon AgNP
supporting evidence of cytotoxicity, but the number of in vivo
or AuNP exposure was also observed in other studies
studies are still limited due to reasons such as high operational
(Armstrong et al., 2013; Pompa et al., 2011a; Posgai et al.,
cost and ethical issues.
2011; Vecchio et al., 2012b). Fecundity can also be measured
Many studies to determine the effects of nanomaterials on the
by accounting the number of eggs laid by females fed with
survivorship, oxidative stress production, genotoxicity and fec-
AuNP-food during larval stage (Vecchio et al., 2012a). Taken
undity have been carried out in Drosophila, and this Drosophila
together, these observations indicate that Drosophila can serve
model has proven to be a useful for nanotoxicological studies
as an in vivo model to study both short-term and long-term
(Figure 3). There are many advantages of using Drosophila as a
effects of nanomaterials on fecundity. In Drosophila, anatomical
model organism. The genetically tractable organism Drosophila is
structures and cellular characteristics of germline stem cells
easy to maintain in the laboratory and inexpensive to keep and has
and their stem cell microenvironments (niches) in male and
a short life cycle and high genetic similarity with humans. The
female reproductive organs have been well described, and thus
Drosophila model is advantageous over other non-mammalian
subsequent detailed studies for understanding the molecular
model organisms. In particular, compared with zebrafish and
and cellular mechanisms of fecundity can be carried out in
C. elegans, Drosophila allows nanomaterials to be administered
Drosophila (Fuller & Spradling, 2007).
through various routes including oral ingestion, direct dermal
exposure and respiratory exposure, while the route of nanomater-
Metabolic defects
ial exposure in zebrafish and C. elegans is mainly through water-
The adult Drosophila fed with AgNP-food during larval stage borne or food-exposure, respectively. Drosophila organs and
exhibits defects in cuticle development and melanization. genes are more homologous to humans than those of C. elegans.
Drosophila that ingested AgNPs has non-pigmented soft cuticle In addition, while the sexual reproducing Drosophila is more
as compared to controls (Figure 2) (Armstrong et al., 2013; Gorth appropriate for the aspect of toxicology study, the hermaphrodite
et al., 2011; Key et al., 2011; Panacek et al., 2011; Philbrook characteristic of C. elegans makes it unsuitable for the study of
et al., 2011; Posgai et al., 2011). The pigmentation defect is due mating behaviors and toxicology effects on the reproductive
to the influence of AgNPs on copper transporters. Excess Ag systems. Zebrafish is also another attractive model for nanotox-
results in competitive inhibition of copper uptake at the copper icology study, but it requires special space and equipment to
transporters, causing a depletion of copper in cells. As the copper- maintain and has a relatively long life cycle. Despite the many
dependent tyrosinase is required for melanin synthesis, its decline benefits of the Drosophila model for the study of nanotoxicology,
in activity may be the reason for AgNP-induced pigmentation there are several limitations to this model. Since insects are very
defect (Armstrong et al., 2013). Indeed, the phenotypic modifi- susceptible to stress, different raising and handling practices
cation was first reported by Rapoport in 1939, and the reduction such as temperature, humidity, day/night cycle and density may
in body pigmentation was thought to be related to treatment result in varying biological effects on flies, possibly confounding
with silver salts (Stefano, 1943). Hence, more comprehensive the toxic effects observed in nanomaterials (Matsumoto et al.,
studies are required to better evaluate if pigmentation defects 2003). In addition, the effect of nanomaterials on the feeding
are due to AgNPs or silver salts. Nonetheless, these suggest pattern and food intake of Drosophila is not well established and
that Drosophila can also be used to study metabolic disorders remains challenging to be investigated (Deshpande et al., 2014).
caused by nanomaterials. Hence, nanotoxicity effects observed through the ingestion of
 DOI: 10.3109/17435390.2014.940405 Toxicity study in Drosophila 401
Figure 3. Nanotoxicity study in Drosophila.
Drosophila can be exposed to nanomaterials
by ingestion, physical exposure or inhalation
exposure, leading to a variety of acute and
chronic effects.
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nanomaterials may be confounded by the change in the food the opportunity to design systemic functional genomic approaches
consumption pattern of the flies. The stability of various to many cell biological processes caused by nanomaterials, such
nanomaterials during the different routes of exposure to as ROS induction and genomic aberration. Finally, Drosophila
Drosophila remains to be studied. As nanomaterials are affected can be used as a rapid, reliable, robust and cost-efficient in vivo
by its local environment, the physio-chemical properties of model for fast assessing the possible adverse effects of
nanomaterials may differ in different conditions such as fly nanomaterials. Taken together, Drosophila can serve as an
food recipes and fly food temperature (Pfeiffer et al., 2014). excellent model organism for evaluating and studying nanotoxi-
Finally, even with a high structural homology and genetic city in vivo, and thus this tiny animal presents limitless
similarity between Drosophila and human, it still remains possibilities in the study of nanotoxicity.
challenging to translate the nanotoxicology findings to effects
of nanomaterial on human health. Nonetheless, nanotoxicological Acknowledgements
studies in the Drosophila model can yield important lessons
The authors would like to thank Ms Song-Lin Bay for her assistance in
for understanding nanotoxicity effects in vivo and provide preparing the artwork for the figures used in this article and Ms You Fang
paradigms for researchers working in mammalian systems. from the Department of Chemical and Biomolecular Engineering,
Fundamental new knowledge obtained about the nanotoxicity National University of Singapore, for synthesizing the AgNPs used in
effects on whole organism is expected to advance the general field this study.
of nanotoxicology.
In Drosophila, numerous genetic tools and reagents to identify Declaration of interest
genes and/or dissect their function are available. Drosophila
The authors declare that they have no conflict of interest.
transgenic RNA interference (RNAi) project has generated tens This research was funded by the National Research Foundation
of thousands transgenic animals with an RNAi hairpin under (NRF), Prime Minister’s Office, Singapore, under its Campus for
UAS/Gal4 system that allows to inhibit certain gene function in a Research Excellence and Technological Enterprise (CREATE) pro-
cell or tissue specific manner in vivo; the Drosophila RNAi gramme and the NUS start-up grant R-181-000-142-133.
Screening Center (www.flyrnai.org) has generated a set of 21 000
double-stranded RNAs that covers every annotated gene in the
Drosophila genome and has developed high-throughput RNAi References
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