I. Introduction
Background of the Study
It was in 1865 that Gregor Mendel proposed Laws of Dominance, Segregation, and
Independent Assortment in his Experiments in Plant Hybridization. According to Mendel’s Law
of Segregation, two different alleles segregate from each other during the formation of gametes.
In addition, this segregation highly relies on chance. Staying in monastery for quite a long period
of time, Mendel tested this and two of his acclaimed laws with garden peas (Pisum sativum).
In 1906, however, Thomas Hunt Morgan tested Mendel’s Laws with one of the most
commonly used test organisms today – the fruit fly (Drosophila melanogaster). It is an insect
that thrives on the flesh of fruit, hence the name. It can usually be found in houses and stores. Its
larvae breed in decaying fruits and other organic materials.
Since 1909, D. melanogaster has been a subject of plenty experiments in genetics,
making it the among the most popular test subject in the world (Ross 1965). The fruit flies’ short
life cycle has made scientists and researchers create more tests and experiments on these species.
Moreover, it is easy to handle in the laboratory and it reproduces quickly. Through these species,
geneticists obtained opportunities to study the mechanism of genes in affecting the formation of
its body parts and to critically examine its life cycle from an egg to the adult fly (Snustad et. al
2010; Roberts 1986).
To prove the Law of Segregation, Morgan employed principles of sex-linked traits (See
Chapter II- Sex-linked traits). Drosophila eye is known to be a sex-linked trait. This study
contributed to the acceptance of the Chromosome Theory of Heredity.
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In this study, several generations of Drosophila will be cultured to observe the validity or
invalidity of Mendel’s Law of Segregation.
Statement of the Problem
The study was conducted to validate whether Mendel’s Law of Segregation is followed in
the mating of several generations of Drosophila melanogaster. Specifically, the study was
undertaken to answer the following inquiries:
. How is trait inheritance affected when the trait is a sex-linked one?
. What will be the effect of a sex-linked trait white eye to the number of male and female second
filial generation flies?
. How does the Law of Segregation followed in mating of flies?
. How does the actual number of male and female second filial generation flies compared to
expected number of flies?
Statement of the Null Hypothesis
There is no significant difference in the expected (in relation to population size) and
observed number of second filial generation Drosophila. The Law of Segregation states that two
different alleles segregate from each other during the formation of gametes. Because of this,
alleles of sex-linked traits, such as white-eye trait in flies, are crossed separately. Thus, sexspecific traits may be exhibited by the other trait in future generations. However, the affected
number of individuals may be minimal because of its sex-linked nature.
Half of male F2 individuals are hypothesized to be white-eyed, and the other half to be
red-eyed. Since, white-eye trait is said to be a sex-linked one, only one-fourth of female F2
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individuals are expected to exhibit white-eye trait while the rest are hypothesized to have red
eyes.
Significance of the Study
Mendel’s laws are being taken quite well for more than a decade now. The study is
deemed to be significant because the experiment will test the validity of a law observed since the
earlier times to the modern-day setting. Though a suave execution of the experiment, the
applicability of the concept to other organisms will be tested, as well.
Scope, Limitations, and Delimitations
The study extends to the expanse of one organism only, Drosophila. The study used only
five red-eyed female, five red-eyed male, five white-eyed female, and five red-eyed male as
starting organisms. The culture media used is a simple one which is composed only of gelatine
bar, propionic acid, yeast, oatmeal, and tap water.
The study is limited to the F2 generation of Drosophila. This batch of flies is the product
of mass-mating of F1 individuals. The count of organisms presented in the experiment is limited
to the flies present five days after the first sighting of mature fly. However, some F1 flies are not
used in mass-mating set-up because whether they died or the total number of flies is just too
many.
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II. Review of Related Literature and of Related Studies
Studies of Gregor Mendel
Selectively breeding of plants and animals to produce more useful hybrids had been done
many years ago by farmers and herders. However, there was no accuracy in results because they
did not know the concept regarding inheritance.
Information about genetic mechanisms was gathered through laboratory breeding
experiments done over the last century. Genetics research focused on understanding what really
happens in the passing of hereditary traits from parents to children. There were hypotheses
suggested to explain heredity. However, Gregor Mendel, a European monk, was able to get an
explanation. His ideas was published in 1866 but was not unrecognized until 1900. His early
adult life was spent doing basic genetics research.
Even though Mendel's research used plants, the basic principles behind heredity that he
discovered are also applicable to people and other animals. This is because the concepts of
heredity are the same for all complex life forms (Russell 1992).
Mendel worked with pea plants (P. sativum). He chose to study seven traits each
occurring in two forms. For example, one trait was pod color. Some pea plants have green pods
and others have yellow pods. Because pea plants can self fertilize, Mendel was able to produce
true-breeding plants. A true-breeding yellow-pod plant for instance would only produce yellowpod offspring. He started the experiment to find out the result of would cross-pollinated truebreeding yellow pod plant with a true-breeding green pod plant. He called to the two parental
plants as the parental generation or P generation and the resulting offspring were called the first
filial or F1 generation.
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When Mendel completed the cross-pollination between a true-breeding yellow pod plant
and a true-breeding green pod plant, he observed that all the F1 (first filial) generation offspring
were green. Then, he allowed all of the green F1 plants to self-pollinate. The offspring were then
called F2 (second filial) generation. Mendel noticed a 3:1 ratio in pod color. Three-fourths of the
F2 plants have green pods and one-fourth have yellow pods. Based on these Mendel ian
experiments, Law of Segregation was formulated (O’Neil 2009).
Mendel's Law of Segregation states that allele pairs segregate or separate during gamete
formation, then randomly fuse during fertilization. There are four main ideas involved in this
concept, namely:
1. There are different forms for genes. A gene can exist in more than one form.
2. Organisms inherit two forms of gene for each trait one from each parent, called alleles.
3. Allele pairs segregate when gametes are produced. Sex cells have partially the compliment of
genes.
4. If the two alleles of a pair vary, one is dominant and the other is recessive. One trait is
expressed and the other is hidden. If a pair of alleles for a trait is the same they are called
homozygous and heterozygous if different.
Mendel thought of what would happen if he studied plants with two different traits.
Through his experiments, Mendel developed the Law of Independent Assortment. Dihybrid
crosses were done in plants which were true-breeding for two traits. A plant with green pod color
and yellow seed color was cross-pollinated with a plant with yellow pod color and green seeds.
Green pod color (GG) and yellow seed color (YY) traits are dominant. Yellow pod color (gg)
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and green seed color (yy) are recessive. The output of F1 was all heterozygous for green pod
color and yellow seeds (GgYy) (Rothwell 1993).
After observing the results of the dihybrid cross, Mendel allowed all of the F1 plants to
self-pollinate and they produce F2 generation. A 9:3:3:1 ratio was observed. Nine of the F2
plants have green pods and yellow seeds, three have green pods and green seeds, three have
yellow pods and yellow seeds and only one have a yellow pod and green seeds.
Same experiments were done focusing on other traits like seed color and seed shape, pod
color and pod shape, and flower position and stem length. He observed similar ratios in each
case. Mendel's law of independent assortment says that allele pairs separate independently in the
stage of formation of gametes. Thus, traits are passed from parents to offspring independently
from one another (O’Neil 2009; Rothwell 1993).
Drosophila melanogaster
Fruit flies (Drosophila melanogaster) are abundant in tropical areas. Translated as “lover
of dew”, Drosophila has a dire need for moist environment. These species cannot endure cold
temperatures which inhibit the development of their offspring and normal body functioning, as
well. Majority of the species occupy plant material mainly on rotten plants and fruits. Eggs are
laid on unripened or ripened fruits, and when it starts to rot, the larva use the rotten fruit as their
source of nourishment (Miller 2000).
Drosophila melanogaster can easily be cultured at home or for scientific researches.
Some fruit flies cultured at home are used to feed on smaller reptiles and other insects. Culturing
fruit flies entails proper temperature, ventilation, maintenance and feeding. Cultures of these
species are kept in a glass bottle. A paper towel or cotton is attached to the top of the bottle to
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provide proper ventilation for the fruit flies. Food mixtures are used when cultures are done
regularly and they contain gelatin, oat meal, and water. A cardboard can be added to provide an
area for rest for third instar. Usually, at this stage the “fly” leaves the substrate and looks for a
dry place to further develop. Finally, temperatures from 21°C to 29°C ensure the proper
development of the cultured fruit flies (Brooks 2003).
Anatomy of Drosophila
The body of the fruit fly (D. melanogaster) is divided into three well-defined regions: the
head, the thorax, and the abdomen. The head carries the mouthparts and the organs of special
sense. The thorax has the locomotor organs, such as the legs and wings. The abdomen has the
structures concerned with reproduction and is the site of metabolic processes (Imms 1960).
The fruit fly (D. melanogaster) has three pairs of legs. It has expanded wings which are
either normal (called as wanting) or short-vestigial (called as lacking). These wings are equipped
with a complete system of veins termed venation. Modified wings, called halteres, can be found
in the thorax. They are movable and are capable of vibration. They have compound eyes and
bristles on the thorax (McGrath 1999).
Sexual Dimorphism
As with most organisms, fruit flies have two sexual orientations- male and female. These
sexual orientations are easily identified because of their recognizable physical features. Such
differences that allows easy identification of sex is termed as sexual dimorphism.
Female Drosophila has relatively longer body lengths, approximately 2.5 millimeters
long. On the other hand, male fruit flies have shorter body lengths (See Fig. 1). Sex combs are
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present on the tarsus of the first leg of the male species while these are absent in females
(McGrath 1999).
Figure 1. Sexual dimorphism of Drosophila.
The abdominal shape on the posterior side of females is pointed as males have rounded
posterior ends. A short rounded abdomen with six segments is possessed by the males while the
females have a long pointed abdomen that consists of eight segments. The abdominal color of the
species also greatly determines its gender. Males have relatively darker patches on their abdomen
than that of females. Moreover, the bands in the abdomen of female are more numerous and
uniformly distributed while males have fewer bands which are broadly and solidly pigmented
(Roberts 1986; McGrath 1999).
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Bristles on the ventral side of the species are mostly present in males. Long, dark,
radiating and spiky bristles are present in males. These are being used as anchor that attaches to
the female during mating. Females, however, lack these bristles on their ventral side.
The adjoining table (See Table 1.) summarizes the comparison of male and female
features of Drosophila melanogaster.
TABLE 1. Comparison of prominent physical distinctive features of
male and female fruit fly (Drosophila melanogaster).
MALE
FEMALE
Body Length
Shorter
Longer
Sex Combs on Forelegs
About 10 on first tarsal
---
Abdominal Shape (Posterior side)
Rounded
Pointed
Abdominal Color (patch)
Dark
Light
Abdominal Cross Bands
Fewer
More
Cross Bands (Posterior side)
Solidly pigmented
Uniformly distributed
Dark, Long Bristles (Ventral side)
Present (on penis)
---
Short Bristles (Ventral side)
Many (penile area)
Fewer (vaginal area)
Sexual Behavior of Drosophila
The different external features of D. melanogaster are essential in differentiating the male
and female fruit flies. Moreover, D. melanogaster base their mating rituals on these
characteristics. The more different their characteristics are the more excited they are for mating
(Spieth et al 1983; Demerec et al 1994). To put it in other way, flies are easily excited whenever
they see a species of the opposite sex.
The fruit fly (D. melanogaster) has their own courtship behavior. During this courtship,
the females will give an invitation signal to a male that she has chosen. The male fruit fly (D.
melanogaster) will then extend his proboscis to touch the female’s genitalia. If the female is
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aroused, then two will proceed with the mating (Spieth et al. 1983). The female will lay their
eggs between the fourth and seventh day after mating. During this period, the female lays eggs at
a rate of 50-70 eggs per day (McGrath 1999).
Life Cycle of Drosophila
Life cycle is a process by which an organism undergoes a series of stages towards a maturity and
full development. Each organism in the ecological sector passes through the growth, expansion,
maturity, saturation and decline stages (Demerec et al 1994).
From the hatching of the eggs to adulthood, the Drosophila undergoes metamorphosis
which is a period of growth and physical change. Metamorphosis is a feature of insects that
allows their hatched form to be morphologically different from its adult form (Imms 1960).
Drosophila belongs to order Diptera which, in turn, belongs to a group called Holometabola.
Every member of Holometabola undergoes a complex metamorphosis where the young are
larvae and the adult or imago is preceded by a pupal instar. This type of metamorphosis is also
referred to as indirect or complete metamorphosis (Imms 1960).
Some structures found in its early stages vanish as it reaches its adult form. The
organism’s tissues grow by either cell division or cell growth. Since its body wall cannot stretch,
it periodically shed its old skin and replaces it with a larger one in order to accommodate its
increase in size as it undergoes metamorphosis (Ross 1965).
The first stage in metamorphosis involves the egg which is oval-shaped, whitish and less
than half a millimeter in length. Its inner membrane is called the vitelline membrane and covered
by the chorion. This outer covering branches out to form two filaments which prevent the egg
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from sinking or drowning the embryo within it. A small opening in its anterior is called the
micropyle in which the sperm can enter and thus fertilize the egg (See Fig. 2).
Figure 2. Life cycle of Drosophila.
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The second stage involves the larva. It is whitish, worm-like, wingless and burrowing.
The larva increases in size over time as it is voracious. The larval stage is subdivided into three
substages as it sheds its skin to accommodate its growth. The form assumed by an insect after a
stadium, an interval between moltings, is called an instar (Imms 1960; McGrath 1999).
Drosophila has three instars.
The first instar evolves after the first molting after the hatch of the egg where the mouth
parts have also shed. They are about 1 mm in length. The second instar is twice as long as the
first instar (2 mm). It appears after the molting of the first instar. The increase in length is merely
due to the enlargement of cells. The third instar forms after the molting of the second instar. It is
twice the length of the second instar (4 mm). At this substage, the larva stops growing and stops
feeding. It proceeds to a dry place where the pupal stage begins (Powell 1997).
Next comes the pupal stage. The pupa is a non-feeding, quiescent stage where the
“structures of the adult are rebuilt from tissues of earlier stages” (Ross 1965). The pupa’s
epidermis excretes the cuticle which serves as the pupal covering. They are glued together by
proteins excreted by the salivary glands. It is in this stage that the tissues and organ systems of
the adult are formed and the larval structures are removed. At the emergence of the adult, the life
cycle of Drosophila ends and eventually the life cycle of baby Drosophila begin (Roberts 1986).
Life cycle is such a substantial part in the development of Drosophila. It holds true for
majority of animals (insects, at least). The logic behind this metabolic process is that they do
have live a miniscule amount of time as compared to human. They have to “grow fast” since they
can not survive for a long amount of time in a non-adult stage (Ross 1965; Powell 1997).
Temperature (as well as atmospheric conditions, pH level and nearby water salinity)
affects the life span and generation time of Drosophila. Females live about 39 days in warm
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weather or up to 70 days in cooler weather. Though males don’t seem to have the same weather
sensitivity, they generally and typically live a much shorter life (Demerec et al. 1986).
Sex-linked Traits
Traits expressed due to genes with loci in the X chromosome are called sex-linked or Xlinked traits. Such traits are known to be more prominent in male who have Y chromosome
instead. This results from the fact males have one X and one Y chromosome. Thus, males need
only to have one dominant allele and the Y chromosome to exhibit the trait.
If the trait is recessive, males possessing a recessive gene will definitely not exhibit the
trait. In females, a possession of recessive gene does not necessarily translate to devoid of trait.
A female needs at least one dominant allele to exhibit the trait (Snustad and Simmons 2010;
Roberts 1986).
Sex-linkage was discovered in 1906 in Abraxis moth by Doncaster and was also observed
when a group of researchers headed by Thomas Hunt Morgan crossed a normal female red-eyed
Drosophila with a mutated male white-eyed one (Roberts 1986). The first generation offspring
were all red-eyed while the second generation, progeny of crossed males and females from the
first generation, resulted to approximately 3:1 red-eyed: white-eyed ratio. While some males are
red-eyed, F2 white-eyed Drosophila flies are all males. Meanwhile, all females have red eyes
(Powell 1997; Russell 1992).
Sex-linkage also applies to body color, wing and eye shape in Drosophila. It also applies
to other animals including humans. These include red-green color blindness and hemophilia. In
red-green color blindness, what usually happens is that a recessive sex-linked gene affects the
pigment sensitive to green light found in cones in the eyes’ retina. This is caused by the protan
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gene. In another form of color-blindness that affects perception of red light, another gene called
deutan causes the defect. Each gene has three allelic forms which corresponds to normal,
defective and absence of pigments in the cones. The allele that codes for normal pigments is
dominant over the two other alleles (Russell 1992).
Meanwhile, the allele that codes for the absence of pigments can be masked by the
presence of the allele that expresses the defective pigments. Like in the eye color of Drosophila,
color-blindness is more common to human males. A human female must have two copies of the
recessive gene for color-blindness present in both of her X chromosomes for it to manifest. This
can happen if both her parents have color-blindness or only her father has it but her mother is
heterozygote and carries a recessive gene (Rothwell 1993).
Hemophilia is another sex-linked trait that is famously recorded up to today as it present
in most European royal families, especially those with ties with Queen Victoria of England. The
recessive h allele that codes for hemophilia is a mutation of the dominant form of the gene H.
Marrying between close kin made the inheritance of hemophilia more prevalent in most
European royal families. In hemophilia, defective genes cause missing cofactors and enzymes in
the blood that makes clotting difficult. Hemophilia A is caused by an anomaly in the gene that
codes for factor VIII that activates factor IX that, in turn, activates thrombin. When thrombin is
inhibited, fibrin clot cannot form. Factor IX production can also be inhibited by a mutation in the
gene that codes for this cofactor and causes hemophilia B. Both genes for factor VIII and IX are
X-linked. Hemophilia in females is rare and was once believed to be impossible (Rothwell 1993;
Russell 1992).
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Sex-Linked Traits: Eyes of Drosophila
In Morgan’s experiment, the X chromosomes play a major role in this phenomenon. Red
eyes are dominant, indicated by W, while white eyes are recessive, indicated by w. Morgan and
his team concluded that the characteristic observed (eye-color) was carried by a gene whose
locus can only be found in the X chromosome. Females have two X chromosomes while males
only have one. In the P generation, the red-eyed female was homozygous WW. The white-eyed
male, however, had wY, indicating that it is heterogametic since it has only one X chromosome
and the other chromosome is a Y. It is also hemizygous since it has only has one gene present for
eye color, found in the single X chromosome (Rothwell 1993; McGrath 1999).
The gametes of the first generation flies would be WW for the red-eyed female and WY
for the red-eyed male. This would result into three red-eyed flies, two of which are females and
one white eyed fly. The genotype and phenotype of the flies are as follows: WW red-eyed female,
Ww red-eyed female, WY red-eyed male and wY white-eyed male. A male with a w allele will
automatically become white-eyed because it only has one X chromosome since the gene for eyecolor is not found in the Y chromosome and there is no other allele to mask or enhance the effect
of the recessive allele. For a female to become white-eyed, it should carry two w alleles in its two
X chromosomes since white-eye is a recessive characteristic. This can happen when a
heterozygous red-eyed female is crossed with a white-eyed male producing: Ww red-eyed
female, ww white-eyed female, Wy red-eyed male and wy white-eyed male. Morgan estimated
this to be in an approximately 1:1:1:1 ratio (Rothwell 1993).
In a reciprocal cross of white-eyed females with red-eyed males, in relation to Morgan’s
original cross of red-eyed females with white-eyed males, the obtained results are different. The
P generation constitutes the male fly with WY genes and the female with ww genes. F1
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generation results to two red-eyed females and two white-eyed males. This illustrates a pattern of
inheritance called crisscross. This occurs when the phenotype of the female parent is expressed
in male offspring while the characteristic of the male parent is manifested in female descendants.
Meanwhile, the result of the cross between a red-eyed female with a white-eyed male from F1 is
the same as in the abovementioned example wherein it resulted to one Ww red-eyed female, one
ww white-eyed female, one Wy red-eyed male and one wy white-eyed male (Rothwell 1993;
Russell 1992).
The compound eye of the Drosophila fruit fly is composed of hundreds of minute units
that sense light. Seven hundred fifty to eight hundred of these structures, called ommatidia, are
organized in a hexagonal manner. This is composed of 19 cells; 8 photoreceptors, 4 cone cells
and 6 pigment cells; and a bristle that sense movement. It develops from the epithelial eye
imaginal disk bilayer. Differentiation of photoreceptors begins in the third larval instar and
continues up to the final larval instar after successive division of cells during the first and second
larval instar stages (Pappu et al. 2002).
Four major genes are responsible for the development of the eyes of Drosophila fruit flies
and constitute the retinal development (RD) network. Eyeless (ey) and twin of eyeless (toy),
genes under the Pax family, regulate the development of the retina. The roles of the
differentiated cells are finalized by the genes eyes absent (eya), sine oculis (so) and dachshund
(dac). Aside from these genes, two more genes are included in the RD network: the hedgehog
(hh) and decapentaplegic (dpp) genes. Genes in the RD network each code for proteins which
are needed in the development of the retina. Abnormal expression of any gene, or most
combination of these genes, is sufficient to initiate retinal development (Pappu et al. 2002).
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The genes toy and ey encode DNA transcription factors that are sequence-specific. The
eye could partially or completely be lost with the hypomorphic mutation, mutation in which the
expression of the gene is similar but is weaker than the expression the normal gene would have
expressed, of the ey gene. There are no reported mutations of toy gene yet. Development of the
eyes in the legs, wings and antennae, called ectopic expression where a body part will form in
areas other than the intended place, could happen if these genes are expressed in imaginal disks
other than the eye disk. Most abnormalities of the eyes of Drosophila are most likely resultant of
mutations of the toy and ey genes. Evidences point that the expression of the toy gene is not
affected by the expression or mutation of the ey gene (Pappu et al. 2002).
eya and so coordinates with the ey and toy and code for proteins necessary for the
development of the retina. Deletion mutation of eya and so regulatory sequences results in the
loss of the eye of the fruit fly. They are important in the differentiation of photoreceptors.
Abnormal, ectopic retinal development in major appendages can occur if eya is misexpressed,
which is lesser in extent than the incorrect expression of ey. ey works precedently over eya. In
turn, eya precedes the works of so. However, ey and eya regulates each other and concurrent
expression of both leads to ectopic development of the retina. Simultaneous expression of so and
eya also results to abnormal retinal development (Pappu et al. 2002).
In the second and third instar larval stages, dac is expressed in the posterior margin of the
eye disk. Loss of eyes will result from null mutation of dac. It is important in the organization of
the eyes’ ommatidia. The first three genes take precedence over dac and they do not need to wait
for dac to be activated for them to be expressed. However, if dac is expressed incorrectly, the
other genes will be expressed. Misexpression of dac follows the wrong expression primarily of
ey and/or eya and results to ectopic development.
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Aside from these genes, there are a few more that can cause ectopic development of the
eyes. These include teashirt (tsh), optix (optx), eyegone (eyg), homothorax (hth) and
extradenticle (exd). They are not part of the RD network and their effects on the RD network
have not been fully explained yet (Pappu et al. 2002).
The fate of the cells in development is regulated by three genes: hedgehog (hh),
decapentaplegic (dpp) and wingless (wg). hh codes for the secretion of a signaling molecule
important in the patterning of most tissues. The movement of the morphogenetic furrow is
inhibited if hh loses its function. This does not necessary impede the differentiation of
photoreceptors. dpp is involved mostly with the development of the furrow. dpp works with the
RD network genes in the development of photoreceptors. Like hh, wg is also associated with a
signaling molecule. It is important for the segmentation and development of the imaginal disks.
It helps in arranging the ommatidia of the eyes (Pappu et al. 2002).
Recent developments on Drosophila melanogaster
Though the mechanism is not thoroughly understood, it has been known that reduced
dietary intake can increase the life span of fruit flies. Recently, however, it has been found that
the smell of food can also shorten the life span of fruit flies. Researchers from Bolyer College of
Medicine in Houston, New Mexico State University in Las Cruces and the University of Houston
report that when fruit flies on a diet are allowed to smell food, their life spans are modulated and
the effect of their diet, which is longevity, is partially reversed. The reduction to their life span
ranges from 6 to 18 percent. The research also conducted studies with mutated flies to determine
whether the loss of olfactory function increases life spans. The tests performed confirmed this
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hypothesis. This study pointed out that life span may not just be a result of slower metabolism,
the fruit flies’ sense of smell can also play a part in it (EurekAlert- Researchers find 2007).
Scientists at the University of Bonn discovered a gene that regulates fat metabolism and
called it schlank, which is German for slim. Larvae lacking this gene lose fat reserves absolutely.
Since mammals have genes that are similar in structure, and probably in function as well, with
the schlank gene. It is hoped that medicines to fight off obesity can be developed with this
discovery. Schlank has been discovered to instruct ceramide synthase, ceramide being the raw
materials for the membranes that enclose the cells of the body. Shclank also promotes the
synthesis of lipids and at the same time inhibits the movement of fat from fat reserves
(ScienceDaily 2009).
Like humans, fruit flies can also get drunk when intoxicated with a significant amount of
alcohol. Researchers at North Carolina State University identified genes in fruit flies that are
linked to alcohol sensitivity. Moreover, 23 of these genes are orthologs or equivalent to those
genes found in humans. This suggests that these genes observed in fruit flies which are
apparently linked to alcohol sensitivity can be used in studying drinking behaviour or addiction
in humans. The researchers bred 25 generations to come up with two separate groups- one that is
sensitive to alcohol and another that is resistant or tolerant to alcohol. After performing wholegenome transcript expression analysis, 1000 genes that were expressed differently between the
two groups were identified and were suggested to be the genes determining alcohol sensitivity.
To verify whether these genes were indeed linked to alcohol sensitivity, mutated versions of the
genes were studied. 35 mutated genes were tested and 32 of them turned out to produce
sensitivity to alcohol. This study might further develop the genetic basis of the action of alcohol
in humans (EurekAlert- Bar Flies 2007).
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It is normal behaviour for male flies to avoid mating with non-virgin females. However,
when certain males do not have the gene called Gr32a, not only do they court the mated females,
they court males as well. Researchers at Duke University Medical Center identified the Gr32a
gene which is responsible for the production of a certain pheromone receptor. It turns out that
this gene is directly connected to the part of the fly’s brain that controls behaviour, indicating
that the signals from the outside of the fly do not “have to go through the processing stations in
the chemosensory system before being connected to the higher-order brain structures”. Male flies
without the Gr32a gene still try to mate with virgin females. However, when in competition with
male flies who possess the gene, the former do not only become outperformed, they court their
male competitors as well. Moreover, to verify their findings, the researchers covered virgin
female flies with male pheromones (normally it is these pheromones that tell the males that the
female has already mated since the female receives pheromones from her first mate). As
expected, the Gr32a-negative males still tried to mate with the females (ScienceDaily 2008;
Science Daily 2009).
Previously, it was believed that the determination of sex in D. melanogaster was the ratio
of X chromosomes to the non-sex chromosomes or autosomes. Recently however, through the
work of James Erickson and Jerome Quintero of Texas A&M University, it has been discovered
that it is the number of X chromosomes that are significant. Furthermore, two X chromosomes
are enough to initiate feminization of the embryo.(EurekAlert 2007- Researchers find).
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III. Methodology
Preparation of food media
One bar of white gelatin was torn into piece and mixed in 800 mL of water. Then, 40 mL
of oatmeal was added. The mixture was continuously heated until the components were cooked.
While boiling, 10 mL dried baker's yeast and 5 mL propionic acid, a mold inhibitor, were
added. After cooking, the media was distributed into Gatorade bottles and were then allowed to
solidify. Labels were made for each of the bottles, with each of them corresponding to specific
stages in the experiment.
Subculturing the flies
Flies were allowed to settle at the bottom of their culture bottles after which the cover of
the bottle was removed and taken over quickly by the new culture bottle. The positions of the
two bottles were switched such that the old culture bottle with all the flies was now at the top.
The flies were then directed into the new culture bottle, sometimes by shaking the flies into it.
The new culture bottle was replugged after the flies have migrated. All the culture bottles in this
experiment, including the stock culture bottles, were placed in a tray with some water on it to
make sure that ants would not tamper with the bottles (See Crossing the fruit flies).
Etherizing the flies
The base of the bottle was jarred with the palm of the hand so that the flies would gather on the
bottom of the bottle. Then, the cover of the culture bottle was removed and was replaced quickly
with etherizing bottles. The position of the bottles was reversed where the culture bottles were
21
placed on top. The bottles were placed near the source of light and were shook to direct the flies
towards the etherizer. When the flies were transferred, the bottles were replugged. Ten to twenty
seconds were allowed to immobilize the flies (See Crossing the fruit flies).
Crossing the fruit flies
Five white-eyed male and five white-eyed female flies were placed in one culture bottle
as starting material. Also, five red-eyed male and five red-eyed female flies wee placed in a
separate bottle. The flies were allowed to mate. When pupae appeared, both sets of adult flies
were removed from their previous bottles and were placed into separate stock culture bottles- one
stock for the white-eyed flies and another for red-eyed flies. These stock flies were subcultured
once every two weeks.
After setting aside the stock flies, their progeny which served as the parental flies in the
experiment were given some time to grow (See Fig. 3 in the next page). Once the new adult flies
emerged, they were etherized (See Etherizing the flies in the previous page). With the flies
immobilized, males of a certain phenotype (i.e., red-eyes) were identified and separated from the
females (also having red eyes) using a dissecting microscope.
Five female flies and five male flies were collected. The males and females were placed
into different culture bottles. The same was done with the flies having the opposite phenotype
(i.e., white eyes). The etherized flies were transferred by brushing them into the area near the
mouth of the new culture bottle which, in turn, was on its side. The culture bottle was kept this
way until the flies were awake and when this happens the bottle is set upright.
22
Figure 3. Flowchart of methodology.
23
Since it is of utmost importance that virgin flies were used, to verify that the females
obtained from the previous step were virgins, a four-day observation period was conducted to see
if pupae appeared on the culture bottles where the collected females were placed.
Five virgin females of a certain phenotype were crossed with five males of the opposite
phenotype (i.e, white-eyed females and red-eyed males). They were placed in a single culture
bottle. The reciprocal cross was then set up in a separate culture bottle (i.e., red-eyed females and
white-eyed males). While waiting for pupae to emerge, the two culture bottles of the two crosses
were placed in a pan with water to prevent ants from going into the bottles. The bottles were kept
in this arrangement for four days at room temperature. Once pupae emerged, the parent flies
were removed from the bottles.
On the seventh day after the new adult flies produced by the crosses appeared from the
pupae, the F1 progeny was scored. After collection of data, flies from the F1 progeny of a certain
cross were made to mate with one another in a fresh culture bottle to obtain the F2 progeny. The
same was done with the F1 flies of the opposite cross. The culture bottles containing the F1
crosses were then placed in a pan with water for four days at room temperature. Once pupae
appeared, the parent F1 flies were removed. On the seventh day after adult F2 flies appeared, the
F2 progeny was scored.
24
IV. Presentation and Analysis of Data
The law of segregation states the occurrence of the separation of two different alleles in a
heterozygote at some point in the formation of gametes. The principle discusses the transmission
of an allele even in the presence of a different allele to the next generation (Snustad et al. 2010).
The results show that the union of red-eyed males and white-eyed females as well as white-eyed
males and red-eyed females produced heterozygote offspring. Table 1C below shows that a cross
between a true-breeding red-eyed fruit fly and a true-breeding white-eyed fruit fly produced
organisms with both red and white alleles (i.e. all are heterozygous). Since red eye color is
dominant, the allele of white eye color is masked. Therefore, the F2 generation carried with them
two different alleles in which the other one is masked by the dominant allele. As the F2
generation mated, the alleles are segregated and the offsprings produced were both
phenotypically red-eyed and white-eyed. Therefore, as seen in Table 2 red-eyed fruit flies have a
3:1 ratio with white-eyed fruit flies.
Parents:
R – red-eyed
w – white-eyed
Table 1A Punnet square for Red-eyed P1 generation
R
R
R
RR
RR
R
RR
RR
Table 1B Punnet square for White-eyed P1 generation
25
w
w
w
ww
ww
w
ww
ww
Table 1C Punnet square for F1 generation
R
R
w
Rw
Rw
w
Rw
Rw
Table 1D Punnet square for F2 generation
R
w
R
RR
Rw
w
Rw
ww
Table 2 Summary of F2 generation
Phenotypes
Genotypes
Genotypic Ratio
Phenotypic Ratio
Red-eyed
RR
1
3
Rw
2
ww
1
White-eyed
1
26
To determine if the two monohybrid crosses performed in this experiment; red-eyed virgin
females crossed with white-eyed males, and its reciprocal cross, virgin white-eyed females
crossed with red-eyed males; conform to the Mendelian law of segregation, the chi-square test
was done.
The null hypothesis for this experiment is: If linkage does not occur and deviations can only be
attributed purely to chance, then the two F2 generation offsprings between red-eyed and whiteeyed fruit flies conform to the 3:1 red-eyed/white-eyed phenotypic ratio as stated by the law of
segregation.
The chi-square test works by getting the value of chi-square or
where
using the formula:
is the data observed in the experiment,
is the expected data for the experiment, and
Σ indicates that the values obtained per class were summed up.
The chi-square value determined using the data from the experiment was then used together
with the value indicated by the degree of freedom into which the experiment falls under. The
degree of freedom
experiment or
is simply one subtracted to the number of classes or categories used in the
. Chi-square obtained is then compared to the critical or p value
associated with degree of freedom of the experiment. If the chi-square value exceeds the critical
value, the null hypothesis is rejected. However, if it is well within or lower than the p value, then
we fail to reject the null hypothesis (Broker RJ 2005, Leland et al. 2008, Snustad et al. 2010).
For this experiment, the obtained chi-square value was 5. 429. We observed four classes; redeyed male, red-eyed female, white-eyed male and white-eyed female; therefore
. The
27
critical value associated with this is 7.815, five percent of the time and comparing it with
, the
latter is smaller. Therefore, we fail to reject the null hypothesis. This shows that the law of
segregation has been exhibited in the experiment.
28
V. Conclusions and Recommendations
Restatement of the Problem
The study was conducted to validate whether Mendel’s Law of Segregation is followed in
the mating of several generations of Drosophila melanogaster. Specifically, the study was
undertaken to answer the following inquiries:
. How is trait inheritance affected when the trait is a sex-linked one?
. What will be the effect of a sex-linked trait white eye to the number of male and female second
filial generation flies?
. How does the Law of Segregation followed in mating of flies?
. How does the actual number of male and female second filial generation flies compared to
expected number of flies?
Conclusions
After subjecting pure white-eyed and red-eyed fly cultures to regulated mating and then
to mass mating, one-half of male second filial (F2) generation are red-eyed and the other half is
white-eyed (1.154 red: 1.0 white). Since white-eye trait is sex-linked, male individuals need only
a copy of the recessive allele in partnership with the Y-chromosome to exhibit the trait.
However, it is harder to exhibit white-eyed trait in females because they are required to
possess two white-eyed alleles to demonstrate the trait. Thus, only one-fourth of the female are
white-eyed while the others are red-eyed (1.0 white: 3.759 red).
29
When computed for its degree of deviations between the expected and observed cases
through chi-square with 5% accuracy, the critical value (7.815) is higher than the computed chisquare (5.429). Performing the chi-square test ensures the accuracy of the result of the
experiment. With a small chi-square compared to the critical value associated with the
appropriate degree of freedom, we fail to reject the null hypothesis.
Thus, the F2 generation monohybrid crosses between red-eyed virgin females and whiteeyed males, and its reciprocal cross, virgin white-eyed females crossed with red-eyed males;
conform to the Mendelian law of segregation. Thus, for the Drosophila F2 generation, there is no
significant difference in the observed-experimental individuals and the natural distribution of
white-eyed and red-eyed traits for both sexes. Any deviations from the expected value are
attributable to chance.
Because of all of these, Mendel’s Law of Segregation is followed. Thus, two different
alleles segregate from each other during the formation of gametes. Heterozygous Ww and w+Y
chromosomes separate randomly before being distributed to the offspring.
Recommendations
With the consideration of the laid facts, the researchers strongly recommend that better
safe-keeping methods must be employed in the activity. In addition, variations in experimental
set-ups can still be more uniform by preparing the culture media for just a single time.
For further studies, Law of Segregation can still be explored by observing two traits- one
being they eye color, and the other wing size and venation. In this more advanced study,
researchers can explore on the interrelatedness of certain traits.
30
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