Chapter 12

Effect of Teratogens on Development of Drosophila

melanogaster
Dong Li and Xiaolin Bi

Abstract
Prenatal exposure to various environmental teratogens such as drugs, toxins, and infectious etiologies
results in a wide range of developmental abnormalities, including growth deficiency, developmental delay,
structural defects, functional CNS abnormalities, and fetal death. Here, we describe the use of Drosophila
melanogaster as a model organism for investigating teratogenic effects on embryonic development.

Key words Teratogen, Drosophila, Embryonic cell culture, Development, Immunostaining

1  Introduction

A teratogen is defined as any environmental factor that can pro-

duce a permanent abnormality in the embryo or fetus. In recent
decades, people are more frequently exposed to potentially terato-
genic compounds such as commercial chemicals, industrial by-­
products, wastes, and numerous drugs that have not been properly
tested [1]. It is estimated that approximately 10–15% of congenital
malfunctions are the result of the adverse effect of environmental
factors on prenatal development [2]. Thus, establishment of effec-
tive approaches to assess teratogenic risks becomes more and more
important in the field of teratology.
Animal-based studies of developmental toxicology provide the
initial guidelines on whether a drug or chemical may present a tera-
togenic risk during pregnancy. Toxicology studies using animal
models have been used to determine the risk of toxicity from chemi-
cal exposures and the mechanism of action of drugs and chemicals.
Mammals, especially rodents, are the most commonly used species
for evaluating potential human teratogens. Traditionally, in vivo
assays were developed by exposing suspected teratogen to embryos
in utero and then abnormalities classified at the birth. These in vivo
assays proved to be expensive, time c­ onsuming and not always pre-
dictive due to the variation in species-­specific responses. As a result

Luís Félix (ed.), Teratogenicity Testing: Methods and Protocols, Methods in Molecular Biology, vol. 1797,
https://doi.org/10.1007/978-1-4939-7883-0_12, © Springer Science+Business Media, LLC, part of Springer Nature 2018

233
234 Dong Li and Xiaolin Bi

of the problems encountered with in vivo testing, alternative in vitro

assays have recently been developed. These assays range from whole
embryo, derived from invertebrates and vertebrates, to cell and
organ cultures. For instance, numerous tests involving submamma-
lian and invertebrate embryos have been described to assay for tera-
togenic potential, such as fish [3–5], frogs [6, 7], crickets [8],
Drosophila [9–11], Hydra [12], and chicks [13]. The Drosophila
shares a lot of pharmacologic and physiological similarities with
human beings [14]. Moreover, the Drosophila has many advantages
in research studies, including fast reproduction, short life cycles,
facility in maintenance, and fewer ethical problems [15]. The
Drosophila system, based upon the monitoring of cell death and pro-
liferation events, has shown a high degree of concordance not only
with in vivo animal studies but also with human epidemiological
data; furthermore, the vast and detailed knowledge of Drosophila
molecular biology could potentially be of use in the derivation of
teratogenic mechanisms. Therefore, fruit fly models have great
potential to study the teratogenic effects and then generalize to
other organisms, including mammals. In recent years, many investi-
gations have been done using the Drosophila model to determine
the effects of teratogens, such as heavy metals, ethanol, cocaine, and
nanoparticles on embryonic development [16–18].
In this chapter, detailed methods for examining teratology
using the Drosophila as the model organism are provided. This
assay can be useful not only in teratogenic screening but also in
mechanistic studies of abnormal development and genes involved
in teratogenic responses.

2  Materials

2.1  Fly Husbandry Flies are raised at 25 °C, 65% humidity, under a 12:12 h light-dark
cycle.
1. Wild-type Dm flies (such as w1118, Oregon R, Bloomington
Stock Center).
2. Fly food: 17 L of water, 93 g of agar, 1716 g of cornmeal,
310 g of Brewer’s yeast, 517 g of sucrose, 1033 g of dextrose,
200 mL of acid mix.
3. Acid mix stock solution: add 164 mL distilled water to 836 mL
of propionic acid. Add 917 mL distilled water to 83 mL of
phosphoric acid. Combine the two diluted acid solutions to
produce the acid mix.
4. Yeast paste: 700 mL of water, 25–30 g agar, 300 mL of apple
juice concentrate, 0.5 g methyl paraben, and 30 g sucrose.
5. Dissection microscope.
6. CO2 gas.
7. CO2-dispensing porous pad.
 Study of Teratogenicity in Drosophila melanogaster 235

8. Fly incubator with light and additive humidity control.

9. Food vial 28.5 mm wide × 95 mm high.
10. Round bottom bottles, 64 mm diameter × 103 mm height.

2.2  In Vitro 1. 50% (v/v) bleach.

Teratogen Assay Using 2. Apple agar plates.
Drosophila Embryonic
3. Shields and Sang M3 Insect Medium.
Cell Culture
4. 70% (v/v) ethanol.
5. Dounce homogenizer.
6. Plastic petri dish.
7. Table centrifuge (such as Hermle Labortechnique with
220.80V02 type rotor).

2.3  Testing 1. Teratogen to be tested.

the Effects 2. 1× phosphate buffered saline (PBS): NaCl 137 mM, KCl
of Teratogen 2.7 mM, Na2HPO4 10 mM, KH2PO4 2 mM, pH 7.4.
on Drosophila
3. Plastic petri dish, 5 1/2 in. diameter for dissection.
Development
4. Dissecting instruments including sharpened, fine point forceps.
5. 1× PBS with 0.1% Tween 20.
6. DMSO.
7. Glass slides.

2.4  Immunostaining 1. Fixative: 3.7% paraformaldehyde in PBS.

2. PBST: 1× PBS with 0.1% Tween 20.
3. Blocking buffer: PBS, 10% goat serum, 0.1% Tween 20 (see
Note 1).
4. Antibody which detects candidate protein (see Note 2).
5. Fluorescence-conjugated secondary antibodies.
6. Fluorescent mounting medium: 80% glycerol, diluted in PBS,
4% n-propyl-gallate (see Note 3).
7. Laser scanning confocal microscope.
8. Glass coverslips.
9. Glass slides.
10. Nail polish.

3  Methods

3.1  Fly Food 1. Boil water.

Preparation 2. Add agar, stirring constantly.
3. After agar has dissolved, add yeast, cornmeal, and sugar and
continue stirring until the mix boils again.
236 Dong Li and Xiaolin Bi

4. Shut off steam.

5. Allow the mix to cook with the mixer running for about
15 min.
6. Add acid mix and mix thoroughly.
7. Dispense.
8. Allow vials to cool for several hours before plugging.

3.2  In Vitro 1. For optimal egg collection, transfer young (<7 days), healthy
Teratogen Assay Using male and female flies to large embryo-collection cages. On the
Drosophila Embryonic bottom of the cage will be the apple juice plate with yeast paste
Cell Culture (see Note 4).
2. Move the cages into a room with a temperature of ~25 °C and
3.2.1  Preparation
humidity ~65% in a 12:12 h light–dark cycle to facilitate the
of Fly Cages
collection of synchronous embryos (see Note 5).

3.2.2  Collection 1. Prior to egg collection, flies are fed with yeast paste on fresh
of Embryos plates, changed at least twice a day for at least 2 days.
2. On the day that the embryos are collected, feed the flies with
fresh plates coated with yeast paste for a 1–2 h prelay period.
3. Collect embryos for a period according to different types of
tests.
4. Loosen the embryos from the plates with a paintbrush, and
rinse thoroughly with tap water through a stacked set of sieves,
so that the embryos collect in the finest mesh sieve at the bot-
tom. Rinse for a few min to remove as much yeast and fly
debris as possible.

3.2.3  Preparing Primary 1. Dechorionate the embryos by immersing them in 50% (v/v)
Embryonic Cells bleach for 3–5 min (see Note 6). Rinse the embryos with dis-
tilled water and then sterilize them with 70% (v/v) ethanol.
From this point on, embryos should be handled in sterile
conditions.
2. Rinse the embryos thoroughly to remove the ethanol with
autoclaved distilled water.
3. Homogenize the embryos using a Dounce homogenizer
(loosen pestle) filled with ten volumes of room temperature
M3 insect medium (see Note 7).
4. Transfer the homogenate into a sterile centrifuge tube and
centrifuge for 10 min at 30 × g at room temperature to pellet
the tissue debris. Transfer the supernatant to a clean tube and
repeat the above step once.
5. Carefully transfer the supernatant by pouring or pipetting to a
clean tube and centrifuge for 10 min at 500 × g at room tem-
perature to pellet the cells. Discard the supernatant. Resuspend
the cell in 10 mL of M3 insect medium.
 Study of Teratogenicity in Drosophila melanogaster 237

6. Count cells using a hemocytometer to estimate the cell density

and resuspend the cells to a desired concentration. To start, try
a range of 1–5 × 106 cells/mL and plate out in 35 mm tissue
culture dishes.

3.2.4  Test Teratogenic
Effect in Embryonic Cell
Cultures
1. After the cells had attached to the dish, the medium was
removed and replaced with medium containing different con-
centration of the teratogen to be tested. The control cultures
receive a change of medium lacking teratogen.
2. Culture the cells at 25 °C for 1–2 days and harvest the cells for
subsequential assays to test the teratogenic effect on embry-
onic cells.

3.3  Testing 1. Prepare fly food containing different concentrations of terato-

the Effects gens to be tested (see Note 8). Prepare five repetitions in each
of Teratogen concentration.
on Drosophila 2. Five pairs of 3-day-old fruit flies are added to the medium cul-
Development ture for mating and egg hatching at 25 °C. The parental flies
are exited after 8 h.

3.3.1  Quantitative
Morphological Studies
of Developmental Stages
in Fruit Fly
1. Record the time started from egg laying to the emergence of
the 1st instar larvae, from 1st instar larvae until the emergence
of pupae and from pupa to adult emergence, which shows the
embryonic, larval, and pupal periods, respectively.
Morphological defects of Drosophila by teratogens treatment
are shown in Fig. 1.
2. Record the number of embryos transforming into larvae, lar-
vae to pupae, and pupae to adult to calculate the surviving rate.
3. Five pairs of 3-day-old adults, who spent their fetus period in
the medium containing teratogen and emerged from pupae,
are transferred to the medium without teratogen, and removed

Fig. 1 Morphological defects of Drosophila by teratogens treatment. Third instar larva after treatment with
normal food (a) or food containing 0.1% carbamazepine (b). Two- to three-day-old pupae after treatment with
normal food (c) or food containing 0.1% carbamazepine (d). Adult flies after treatment with normal food (e) or
food containing 30 mM valproate (f). Note: abnormal wing indicated by the arrow
238 Dong Li and Xiaolin Bi
3.3.2  Morphometric
Variation in Larvae, Pupae,
and Adults
3.4  Immunostaining
after 8 h, and given the opportunity for mating and egg laying.
The number of eggs is counted by stereomicroscope. The bea-
kers are incubated and the larvae are counted in the stage of
third instar larvae. Larvae are counted and the hatching rate of
eggs is calculated according to the number of eggs.
1. The first sampling is done 24 h after the release of the adults.
At this moment, the larvae are at 1st instar stage. Afterward,
the sampling is done once every 12 h. At collection, all indi-
viduals are lifted off or dissected from the substrate. They are
then transferred to just-boiled water for 1 min to fix the larvae
and stop enzyme activity and to produce elongation compara-
ble to maximum extension of a live larva. The larvae are then
towel-­dried and preserved in 70% EtOH.
2. Adult flies are collected 3 days after eclosion and then anesthe-
tized with ether and kept in 70% EtOH.
3. Samples are placed on glass slides and then photographed
under a camera-equipped stereomicroscope. All photos are
transferred to ImageJ software to carry out the measurements.
The measurement of pupae and eggs is carried out from the
most proximal to the most distal point of them. The length of
a larva, viewed laterally, is measured between the most distal
parts of the head and the eighth abdominal segment. The
width of a larva, viewed laterally, is measured between the ven-
tral and dorsal surfaces at the junction of the fifth and sixth
abdominal segments. Dorsal views of adult images are taken to
measure the length of the adult.
4. All statistical comparisons are performed using origin 9.0 soft-
ware. The significance of treatments in groups is examined by
two-way ANOVA test with the least significances of P < 0.05.
To test the molecular mechanism of teratogenic effect, one of the
most widely used techniques is to determine protein expression
and localization in fixed samples using specific antibodies.
1. Embryos treated with teratogens are collected and dechorion-
ated as described in the above section.
2. The dechorionated embryos are fixed with 3.7% paraformalde-
hyde in PBS for 20 min and devitellinized by vigorous shaking
with methanol (see Note 9).
3. After fixation, rinse embryos twice with methanol. Discard the
methanol and add PBST. Let embryos settle and rinse three
times for 5 min with PBST.
4. Block the embryos in blocking buffer for 30 min at room tem-
perature (see Note 10).
5. Dilute the primary antibody to desired concentration in
blocking buffer and incubate 90 min at room temperature
(see Note 11).
 Study of Teratogenicity in Drosophila melanogaster 239

Fig. 2 Irradiation induced cell death in Drosophila embryo. Wild-type embryos (stage 4) are irradiated with
10 Gy by a Precision X-Ray X-RAD 320 iX and labeled with anti-cleaved caspase-3 antibody

6. Rinse embryos three times for 10 min with PBST.

7. Prepare the secondary antibody mix in blocking buffer and
incubate for 60 min at room temperature (see Note 12).
8. Rinse embryos three times for 10 min with PBST.
9. After rinsing in PBST the stained embryos are then transferred
to a drop of mounting medium onto a glass slide using a cutoff
yellow pipet tip. Mix the embryos carefully with the mounting
medium and place a coverslip onto the sample.
10. Seal the edges of the coverslip with nail polish. To prevent
movement of the coverslip, first add nail polish to the four
corners of the coverslip and let it harden. Then add nail polish
to the edges.
11. Imaging immune-labeled Drosophila embryos by confocal
microscopy (see Note 13). An example of this methodology is
present in Fig. 2.

4  Notes

1. Instead of Tween 20, Triton X-100 can be used, which is a

stronger detergent and thus promotes permeability of the tis-
sue by extraction of membranes.
2. A collection of excellent monoclonal antibodies for various
Drosophila antigens is available through the Developmental
Studies Hybridoma Bank (DSHB) in Iowa City (http://www.
uiowa.edu/~dshbwww/).
3. Commercial mounting medium is also available for Vectashield
Mounting Medium for fluorescence (Vector, Burlingame, CA).
4. Yeast can be easily prepared by mixing dry baker’s yeast with a
bit of water in a plastic beaker and mold it with a metal spatula
into a thick paste. Do not make the paste too watery, o ­ therwise
flies will stick to it. Keep the yeast paste in the fridge in a large
glass beaker covered with aluminum foil.
240 Dong Li and Xiaolin Bi

5. Flies die if they are not fed for 2 days. Fly cages need to be
cleaned thoroughly between the fly generations.
6. Wear a lab coat and gloves when handling sodium hypochlorite
to avoid bleaching of your clothes.
7. Avoid foaming during homogenization.
8. Some teratogens are heat-labile enterotoxin and inactivated at
high temperatures, so to avoid disruption of the compound,
cool the food to an appropriate temperature before adding the
drugs.
9. The methanol treatment affects both the antigenicity and the
localization of proteins. To fix these problems, the vitelline
envelope can also be removed manually after fixation using a
27-gauge syringe needle under a dissecting scope.
10. The blocking step can be extended up to 2 days (over week-
end) on a rocking plate at 4 °C without any noticeable loss of
staining quality.
11. Alternatively incubate overnight on a rocking plate at 4 °C. This
step can also be extended for up to 2 days. However, in our
experience long incubation times often increase background
staining.
12. The incubation is carried out in the dark to prevent fluores-
cence bleaching.
13. The images are collected as single optical sections or as Z-series.

Acknowledgment

This work was supported by grants from National Natural Science

Foundation of China (Grant No. 31771437, 31501165, and
31271480), and Hundred Talent program of Chinese Academy of
Sciences, Pandeng scholarship of Liaoning province and Qizhen
Gongcheng from Dalian Medical University to X.B.

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