Lab Report on Crosses of Fruit Flies and Observation of Phenotypes

This lab report discusses the crosses of fruit flies and observation of phenotypes in the progeny. The report includes the methodology, results, and discussion of the experiments conducted. The report also explains the significance of using Drosophila melanogaster as an experimental model for genetic research.

Running head: LAB REPORT
LAB REPORT
Name of the Student:
Name of the University:
Author Note:

1LAB REPORT
Abstract:
The aim of this lab work was to cross the diverse traits of fruit fly (Drosophila
melanogaster) and observe the corresponding phenotypes within the progeny. The lab work was
typically divided into two practical. The aim of the first practical was to make use of light
microscopy in order to set the crossing of fruit flies and record the effect of mutation to the
homeotic, Ultrabithorax gene (Ubx) within the F1 generation. A proportion of flies with the
homeotic mutation known as the Antennapedia (Antp) was also observed. This was achieved by
crossing the Ubx mutant/TM1 male with Def (Df(3r)P2) female. The progeny displayed the
mutant phenotypes of triple/quadruple, double and pbx1. Typically the mutations were marked by
body colour, eye markers, wing markers, bristle markers and other significant physically visible
characteristics. The striking Antennapedia (Antp) mutation was observed which was
characterized by the antenna to leg transformation, however in place of the antennae only a part
of the structure of leg was observed.
The aim of the second practical was to make use of light microscopy and score the
crosses of the previous practical. The F1 generation from the previous experiment was studied to
evaluate the effect of mutation to the homeotic gene (Ubx). Characteristic mutation in the form
of eye color, wing type and body type were identified and recorded.

2LAB REPORT
Introduction:
Fig: Drosophila melanogaster
(Source: Campos-Ortega and Hartenstein 2013)
The Drosophila melanogaster is an invertebrate model that has been used for more than a
century to understand the principles of Mendelian Genetics. The selection of fruit fly as an
experimental model can be explained as the economic feasibility and the abundant availability
within the environment. The experimental model of Drosophila became popular during the Neo-
Darwinian era. Thomas H. Morgan used the Drosophila experimental model to understand the
process of evolution and how organisms are naturally selected in the course of evolution. The
experiments conducted by T.H Morgan led to a number of important discoveries about the
characteristics of the fruit fly. In this regard, it should be mentioned that the discovery of the
white mutation in the mass breeding experiment helped in understanding its relation to the X-
chromosome. The discovery marked the wide use of the Drosophila model for genetic research
(Attrill et al. 2015). In this context, it is important to note that second half of the 20th century
served as the bridging period that marked the use of Drosophila experiments to study other
disciples of Biology including animal behavior, molecular biology and developmental biology
(Buchon et al. 2014). Further research significantly pointed that 75% of the disease genes in

3LAB REPORT
human are already present within the genome of fruit flies. Research studies indicate a number of
reasons that state why fruit flies are the best experimental models to perform Genetic
experiments (Attrill et al. 2015). The reasons primarily include, the convenient availability and
lower economic price to obtain the flies. This further explains the reason why the flies can be
stocked on laboratory trays hat provide ample opportunity to perform a number of experiments.
Further, the fruit fly generation is 10 days in length which enables researchers to evaluate and
monitor several generations (Wolfweb.unr.edu 2019). The genome in Drosophila is lower
redundant than other higher organisms (Gonzalez 2013). This means that only one or at the most
a handful of genes code for a similar class of proteins. Also, Drosophila helps in performing
unbiased screening for genes that direct a concerned biological process (Gonzalez 2013). This
also forms the basis of forward genetics and this allows researchers to design a number of
different screening strategies. In addition to this, research studies indicate that every gene present
in the fruit fly can be targeted and manipulated to study a unique trait. Also the FlyBase contains
a huge gamut of information about the genetic characteristics of Drosophila and these factors
collectively make fruit fly the most ideal animal model for studying genetic interaction between
two genes. Also, manipulation and observation of the cells as well as the tissues in fruit fly are
convenient to be carried out in-vivo. The flies have relatively simple organs and can be studied
using simple staining and fixation experimental protocols applied to the whole organism.
Life cycle of Drosophila:
Prior to proceeding with the discussion on the experimental study conducted for the lab
work, it is important to develop an understanding about the life cycle of Drosophila. The females
that are already fertilized store the sperm inside the receptaculum seminis and that is used for the
fertilization of hundreds of eggs which are laid in the consecutive days. The embryonic

4LAB REPORT
development occurs at 25ᵒ C and lasts for a period of approximately 21 hours. The 1st instar
larvae after hatching takes a period of 2 days to molt and advance into the 2nd and then to the 3rd
instar larvae. The 3rd instar larvae then continues feeding for one more day which is also referred
to the foraging stage. After this, the larvae stops feeding and then advances to the pupa stage.
The pupal stage marks the onset of degeneration of all the organs and begins the process of
metamorphosis. The metamorphosis stage marks the transformation to the adult stage. After 10
days of egg laying, the adult flies emerge out from the pupal case. It should be mentioned here
that after the process of enclosure the males need up to 8 hours to acquire sexual maturity which
can be used for fertilizing the female flies. In cases where the flies are raised at 18ᵒC, the flies
need double of the mentioned time to mature.
In order to introduce random mutations within the fruit flies a large number of flies are
needed which are treated chemically using Ethyl methanesulfonate which is also known as EMS
and has carcinogenic properties. In other cases the flies might be mutated through P-elemnt
mutagenesis or exposure to irradiation. Gene knockout systems such as screening with RNAi is
also used to over express a gene.
In this regard, it should be mentioned that the objectives of the two experiments
conducted intended to analyze the effect of the mutation on the homeotic Ultrabithorax and
Antennapedia gene and investigate the phenotypic appearance of the mutations in the F1
generation.
Methods:
Mutations introduced within the homeotic gene (Ubx) leads to a transformation in the
third thoracic region (T3 region) segment to the second thoracic (T2 region) segment. It should

5LAB REPORT
be noted here that null mutations within the Ubx lead to the production of embryonic lethal
phenotypes where the T3 region and A1 region are transformed to T2. However, mutations
within the control regions of the Ubx lead to limited transformations within T3 as well as A2 and
produce viable adult flies. In order to test the effect of the ambiguous regulation of the Ubx a
quadruple Ubx was crossed with a (Df(3R)P2) which is deficit of the Ubx. The mutants were
characteristically maintained in the heterozygosity to the TM1 which is the balancer
chromosome. The rationale for the use of TM1 can be explained as the property of suppressing
crossing over which is effective in following a particular mutation in a crossing scheme. The
methodology for the experiment included the following procedure:
 The Ubx mutant males were collected and crossed with the Ubx deficit females. The
males and virgin female flies were already provided in the vials.
 An empty vial was provided with fly food and was marked with the cross, date and group
name
 The cross was prepared using the male from a quadruple Ubx mutant cell line (line
101566, abx1, bx3, 61d, pbx1/TM1) and the female from the deficiency line (line 107058,
Df(3R)P2/TM3 Sb [1], Ser [1]
 Step 3 was done with the help of a plastic pasteur which was 1ml in length of Fly nap to
the cotton of the anaesthetizer that was used. The anaesthetizer was recomposed so that
the funnel was on top of the glass vial.
 The next step included emptying the vial with abx1, bx3, 61d, pbx1/TM1 males remaining
inside the anaesthetizer and the flies were observed to enter the slumber state. After a
brief span of 1-2 minutes, the anaesthetized flies were observed under the microscope
being kept in a petridish.

6LAB REPORT
 The observation in terms of eye colour, body structure, wing structure and sexes were
identified and documented
 The same drill was repeated with Df (3R) P2/TM3 Sb[1], Ser [1] females which was
crossed with abx1, bx3, 61d, pbx1/TM1 males
 A vial of quadruple Ubx mutant males crossed with Def females were obtained
Materials:
 2 flies vial were kept on the bench. One comprised of the Ubx mutants while the
other had female Def flies in it
 A dissecting microscope was provided with brushes to move the flies and note the
observation
 A flynap solution was provided to anaesthetize the flies
 A plastic Pasteur was provided
 A petri dish was provided
The material used for the second experiment comprised of the following:
 5 cm petri dishes with 100% ethanol, a marker pen, funnel and brush
 1 vial containing flies of the line 101566, abx1, bx3, 61d, pbx1/TM1
 I vial of flies from the cross previously set using (abx1, bx3, 61d, pbx1/TM1)
 Plastic Pasteur pipettes in order to place flies in separate petri dishes for
investigation
 A dissecting light microscope
 Twizzers
 A flynap solution for anaesthesia

7LAB REPORT
 A fly anaesthetiser
 Precautionary measure was observed while handling 100% ethanol as it is highly
flammable
The methodology for the second experiment included the following procedure:
 The thorax of the flies labeled line 101566, abx1,bx3,61d,pbx1/TM1 used in
the previous experiment were observed under the microscope
 Two 5cm petri-dishes were taken and 1 ml of ethanol was poured. In one
petridish a male fly was placed and in another a female fly was placed
 The tube containing the abx1,bx3,61d,pbx1/Def flies were then taken and
anesthetized and placed in another petri-dish
 On crossing 101566, abx1,bx3,61d,pbx1/TM1 with abx1,bx3,61d,pbx1/Def, 8
the F1 generation showed 8 different phenotypes which were recorded
 8 petri dishes of 5 cm were labeled according the observed phenotypes of Red
eyes mutation, Reddish eyes wing vein mutation, red eyes dark body and red
eyes;wing vein mutated with dark body.
Results:

8LAB REPORT
Fig: Antennapedia cross prepared in the experiment 1
Explanation: The chromosome of interest in the cross included the X chromosome, chromosome
II, chromosome III and Chromosome IV both in the male as well as the female fruit flies. The
chromosome X in the male contained the W1118/Y gene refers to the white mutant males. The
chromosome III comprises of the UbxMut/TM1 gene that regulates the structuring of the
thoracic segmentation. It should be mentioned in this regard that the rationale for the choice of
white mutant type trait and Ubx mutant trait included the stark significant phenotype related to
the mutation of these genes. A study conducted by Ferreiro etal. (2017) suggested that the white
mutant type was significantly linked to retinal degeneration. Also, white mutants were found to
lack climbing ability, experience a shortened life span and lack of conferring ability to cope with
environmental stressors. The choice of the Ubx gene could be explained as the significant
mutation observed within the thoracic segment of the mutated flies.
Fig: Simplified cross as Ch II and Ch IV are wild type
Since there was no mutation trait present within the chromosome II and IV (the positive sign
represents wild type), therefore, the cross can be simplified to X w1118/Y; III UbxMut/TM1 X
X w1118/w1118; III Df(3R)P2/TM3,Sb1,Ser1.

9LAB REPORT
Fig: Punnet Square of the Ubx cross
Explanation: On crossing the male gamete (X w1118/Y; III UbxMut/TM1) with the female
gamete ( X w1118/w1118; III Df(3R)P2/TM3,Sb1,Ser1) a total of 8 phenotypes were observed.
The gametes could be differentiated to w1118; Df(3R)P2, w1118;TM3;Sb1;Ser1 and w1118;
TM1, Y;UbxMut,Y;UbxMut and Y; TM1. The phenotype comprised of mutated females with red
eyes, females with red eyes and dark body, females with red eyes and mutated wing veins,
females with reddish eyes, dark body and mutated wing veins. In addition to this, the observed
phenotype also comprised of mutated males with red eyes, wild type males with red eyes and
dark body. Also, there were males with reddish eyes and mutated wing veins and males with red
eyes, mutates wing veins and dark body.

10LAB REPORT
Explanation: On the basis of the Ubx mutant cross explained above, the F1 generation included a
total of 24 mutated male flies with Red eyes. 23 of the male flies had Red eyes (ish) and had
mutated wing veins which were difficult to spot. 26 of the male flies had red eyes and dark body.
27 of the flies had Red eyes (ish) with wing vein mutation difficult to spot and distinct dark body
appearance.

11LAB REPORT
Explanation: On the basis of the Ubx mutant cross the F1 generation constituted a total of 22
female flies with Brick Red eyes that were mutated. 25 of the female flies had red eyes (ish) with
mutated wing veins. 28 of the female flies had red eyes and dark body. Whereas, 25 of the flies
had red eyes (ish) with mutated wing difficult to spot and dark body. Therefore, it can be said
that the phenotypic characteristics were determined by the interplay of the dominant mutant trait
in the flies.

12LAB REPORT
Discussion:
Fig: Hox genes in Drosophila
(Source: Baek, Enriquez and Mann 2013)
Hox genes in Drosophila are the key regulator genes that direct the development process
of the flies. Research studies suggest that when homeotic genes are inactivated or over-
stimulated by inducing mutations, structures of the body might start developing at a separate
place (Holland 2013). In this case it should be mentioned that the homeotic gene Antennapedia
when normally expressed becomes the second segment of the thorax of the fruit fly. However, on
inducing a mutation leg like structures emerges in place of antenna in the fly. Also the
Ultrabithorax gene which is responsible for the third thorax segmentation on mutation and direct

13LAB REPORT
the emerging of wings from the second segment by repressing the third segment (Rhee etal.
2014). On inactivation of the gene due to mutation, wings that come out from the third segment
are accompanied with a second set of wings at the second segment placed behind the third
segment (Wangler et al. 2015). Therefoe, it can be said that the fruit flies contain a set of 8 major
homeotic genes which on inducing mutation display different traits that help in developing an
understanding about the interplay of genes in the regulation of the development process.
Conclusion:
Therefore, to conclude it can be mentioned that Hox genes are regulated by the protein
products on the basis of gap genes and pair genes. The switching on and off mechanism of these
genes by inducing mutation help in the identifying a number of different phenotypic traits. The
study of the genetic traits helps in understanding the manner in which genes interact to dictate
the process of development. Also, these experiments typically help in understanding the
functionality of genetic interaction and the fundamentals of classical Genetics. The two
experiments typically helped in gaining an understanding about the homeotic genes in the fruit
flies and also helped in understanding the impact of mutation on the F1 generation obtained after
crossing for Ubx mutation.

14LAB REPORT
References:
Attrill, H., Falls, K., Goodman, J.L., Millburn, G.H., Antonazzo, G., Rey, A.J., Marygold, S.J.
and FlyBase Consortium, 2015. FlyBase: establishing a Gene Group resource for Drosophila
melanogaster. Nucleic acids research, 44(D1), pp.D786-D792.
Baek, M., Enriquez, J. and Mann, R.S., 2013. Dual role for Hox genes and Hox co-factors in
conferring leg motoneuron survival and identity in Drosophila. Development, 140(9), pp.2027-
2038.
Buchon, N., Silverman, N. and Cherry, S., 2014. Immunity in Drosophila melanogaster—from
microbial recognition to whole-organism physiology. Nature reviews immunology, 14(12),
p.796.
Campos-Ortega, J.A. and Hartenstein, V., 2013. The embryonic development of Drosophila
melanogaster. Springer Science & Business Media.
Ferreiro, M.J., Pérez, C., Marchesano, M., Ruiz, S., Caputi, A., Aguilera, P., Barrio, R. and
Cantera, R., 2018. Drosophila melanogaster white mutant w1118 undergo retinal
degeneration. Frontiers in Neuroscience, 11, p.732.
Gonzalez, C., 2013. Drosophila melanogaster: a model and a tool to investigate malignancy and
identify new therapeutics. Nature Reviews Cancer, 13(3), p.172.
Holland, P.W., 2013. Evolution of homeobox genes. Wiley Interdisciplinary Reviews:
Developmental Biology, 2(1), pp.31-45.

15LAB REPORT
Rhee, D.Y., Cho, D.Y., Zhai, B., Slattery, M., Ma, L., Mintseris, J., Wong, C.Y., White, K.P.,
Celniker, S.E., Przytycka, T.M. and Gygi, S.P., 2014. Transcription factor networks in
Drosophila melanogaster. Cell reports, 8(6), pp.2031-2043.
Wangler, M.F., Yamamoto, S. and Bellen, H.J., 2015. Fruit flies in biomedical
research. Genetics, 199(3), pp.639-653.
Wolfweb.unr.edu 2019. [online] Wolfweb.unr.edu. Available at:
https://wolfweb.unr.edu/homepage/pmiura/drosophila/Roote_Prokop_SupplMat1_v1.2.pdf
[Accessed 14 Mar. 2019].