THE BIOSYNTHETIC EFFECTS OF Euphorbia hirta
on Drosophila melanogaster
(FRUITFLIES)
An Investigatory project
TABLE OF CONTENTS
page
Title page
Abstract
Table of Content
Chapter 1
Introduction
ABSTRACT
A mutagen is any agent that cause heredity changes in DNA (genes). Almost everyone
is aware that x-rays and certain other radiation are mutagenic. Not everyone recognize
that phytochemicals can be mutagenic too.
The common fruit fly (Drosophila melanogaster) is an excellent organism for
genetic studies. It is easy to provide, easy to raise, completes its life cycle in a short time,
and is easy to observe.
The extract of Euphorbia hirta was used in the study as the basis to recognize that
lethal and mutagenic effects on fruit fly by Drosophila Technique and Mendelian Genetic
System. The fruit flies from the culture bottle were treated with plant extract. The Dihybrid
crossing of two heterozygous parents with the same trait (DdSs x DdSs) was also made.
Offspring from the first generation (F1) were culture again by pairing five males and five
females for the second generation (F2). Mortality rate was evaluated to determine the
lethal effects. Probability was computed to determine the genetic effects.
Based on the observation done, Euphorbia hirta extract has harmful effects on the
chromosomes that caused abnormalities of flies. Concentrated of plant extract exhibited
a lethal effect. Therefore, the study was able to scientifically prove that the4 plant extract
could increase the rate of gene mutation, and may be transmitted to future generation.
Chapter 1
INTRODUCTION
Background of the study:
Euphorbia hirta is erect and branched shrub up to a meter high, with cylindrical or
obscurely angled branches lined with shift, slender, divergent spines. Leaves are few,
alternate pale green, oblong- obviate, or short acuminate, up to 5 cm. long. The sap
contains latex which is toxic on ingestion and highly irritant externally, causing photo
sensitive skin reactions and severe inflammation, especially on contact with eyes or open
cuts. The toxicity can remain high even in dried plant material. Prolonged and regular
contact with the sap is inadvisable because of its carcinogenic nature. (www.
Stuartxchange.com/ corona De Espina, titul , 2008).
Not too many people know how carcinogenic photochemical are, and how they may also
cause mutation, according to the study of the World Health Organization (WHO) task
force against plants toxicity, (Philippine Daily Inquirer, 1991).
The National Center of Toxicological Research in Jefferson Arkansas is developing a test
to determine whether substances are mutagens. The problem increases all the time,
because new products are constantly being introduced by industries across the nation.
Many other functions in toxicological research are also carried out by the center in its role
as one of the bureaus of the Food and Drug Administration of the Department of Health,
Education and Welfare.
Typically, a substance being tested as a possible mutagen undergoes all the following
procedures; it is tested for its impurities or for its ingredients if it’s a mixture of substances.
Each ingredient or impurity, as well as the major compound or product itself, is reviewed
for its chemical relationship to any known mutagen. Tests are made using
microorganisms as indicators of mutagenic substances. The microorganisms are rapid
indicators of many mutagens Laboratory mammals, on the other hand, are more likely to
indicate whether a potential mutagen may affect human beings.
The proof that radiation causes mutations was presented by Dr. H.J Muller of the
University of Texas. He had worked with Dr. T.H Morgan and Dr. C.B Bridges during the
exciting years of Drosophila genetics. Later, Dr. Muller used Drosophila to plant breeding
experiments showed that high-energy radiation caused many other kinds of mutations on
all chromosomes of Drosophila (Mc Dougan, 1999).
Hence, the problems described here were undertaken to investigate the biosynthetic
effects of Euphorbia hirta extract on the mortality and heredity of Drosophila
melanogasler.
Statement of the Problem:
1. What are the distinguishing features of sex differences of male and female flies?
2. What is the mortality rate of Drosophila melanogasler in different plants per treatment?
3. What is the ratio of normal and dumpy wings, red and sepia eyes of Drosophila
melanogasler in different plants per treatment?
4. What is the probability of genetic events from first generation to second generation?
Statement of Hypothesis:
1.
The distinguishing feature of sex differences of male and female flies can be
determined.
2.
The mortality rates of Drosophila melanogaster in different plants per treatment
are all high value.
3.
The ratio of normal and dumpy wings, red and sepia eyes is high value.
4.
The probability of genetic events from first generation to second generation is high
value.
Significance of the Study:
The mutations become a part of the genetic variation of species both man and animals.
When substances cause mutations in the DNA, the mutations are likely to be passed
along to the next generation. Many genetic defects of certain agents are difficult to
measure because recessive mutations may not be expressed for many generations. It is
one of the ironies of scientific works that working with mutations prove that harmful
substances cause mutations.
The study will enable people to know if plant extracts have biosynthetic effect on heredity
and if these are mutagenic substances. The local government will also be able to pinpoint
which extract and their particular sources are carcinogenic, for prevention. This study with
efficient and effective information data would be helpful in the prevention and use in the
pest eradication.
This study approach is applicable for any suspected carcinogens in the other forms
of waste chemicals that may also pose great possibilities in their harmful effect on human
beings.
Scope and Limitation of the Study:
The study is limited to the use of only one species of Euphorbia hirta. However the source
of extracts comes from leaves and stems, separately.
It is also limited to particular species of fruity, Drosophila melanogaster, as specimen for
experimentation.
The study was conducted during the first week of September, and the
phytochemical analysis of the plant was conducted on DOST.
Due to time constraints, less treatment and observation periods were conducted.
Using the simple extraction of the plant was conducted. Further study and observation for
more periods of treatments and number of generation will be done.
Definition of Terms:
Alleles- Two chromosomes of a pair
Dyhybrid- a cross between two hybrids
Dominant gene- pure variety
Gametes- the offspring produced are four different types.
Genotypes- refer to the combination of genes that cause the trait.
Heterozygous- bearing the unlike genes
Homozygous- bearing like genes
Morgue- dead place
Review of Related Literatures
Euphorbia hirta (sometimes called asthma-plant) is a pantropical weed, possibly
native to India. It is a hairy herb that grows in open grasslands, roadsides and pathways.
It is used in traditional herbal medicine. This erect or prostrate annual herb can grow up
to 60 cm (24 in) long with a solid, hairy stem that produces an abundant white latex. There
are stipules present. The leaves are simple, elliptical, hairy (on both upper and lower
surfaces but particularly on the veins on the lower leaf surface), with a finely dentate
margin. Leaves occur in opposite pairs on the stem. The flowers are unisexual and found
in axillary cymes at each leaf node. They lack petals and are generally on a stalk. The
fruit is a capsules with three valves and produces tiny, oblong, four-sided red seeds. It
has a white or brown taproot. (Botanical Society of Britain and Ireland, 2015).
According to Sunil Kumar, et. Al, (2010), the oldest remedies known to mankind
are herbal medicines. India is known worldwide for its Ayurvedic treatment. Euphorbia
hirta is often used traditionally for female disorders, respiratory ailments (cough, coryza,
bronchitis, and asthma), worm infestations in children, dysentery, jaundice, pimples,
gonorrhea, digestive problems, and tumors. It is reported to contain alkanes, triterpenes,
phytosterols, tannins, polyphenols, and flavanoids. This review describes the medicinal
properties, chemical constituents, and other important aspects of Euphorbia hirta.
According to Fiskesjo (2000), chromosomal aberrations constitute a reliable and
effective bioassay for environmental pollutants. There are many organisms or plants that
can be used for the study of the toxic effects of substances in the environment. Among
the test systems is using Allium cepa chromosomes which have been found to be a good
method due to their high sensitivity to environmental changes. However, Drosophila
Technique is more efficient in terms of patterns of heredity because this method can
determine the effects until the next generation. It is, for this reason the researcher was
interested to use the Drosophila Technique due to its reliable response toward treatment.
From the book of Carale et. al, (1995), some agents can increase the rate of mutation.
These mutagens, as the agents can increase the rate of mutation. These mutagens, as
the agents are collective by called, include temperature, chemicals such as formaldehyde,
mustard gas, caffeine and nitrous acid, have also the potential ability to cause damage in
the chromosomes. These mutagens can even change nucleotides within DNA molecules.
Altering the bases chemically, such as mutagens can cause some changes in the code
sequence of the DNA. Most of these chemicals can be found in food additives and other
substances which contribute to air and water pollution.
Faith (2002) stated that many observations and experimentations now identify the genes
as nucleic acids, usually DNA. A gene is all or part of a DNA molecule that controls a
particular reaction in the cell. Together the genes control the life activities of a cell. There
are many kinds of genes in each of the millions of different spicies of organisms. How did
all these new kinds of genes arise? Before each cell division, every DNA molecule in the
cell duplicates itself. Because the nucleotides pair only in certain ways, according to the
Watson-crick DNA model, genes are able to make exact copies of themselves. This copy
mechanism almost always works, and genes can be duplicated millions of times without
a mistake. But occasionally a mutation does occur. For reasons that are not fully
understood, sometimes a different nucleotide is put into a DNA molecule, or a nucleotide
is omitted, or an extra nucleotide is added. This changes the nucleic acid molecule
slightly, so that it is no longer an exact copy of the original one. The genetic message,
written in the nucleotide alphabet, is also changed slightly, and may cause a slightly
different protein to be synthesized in the cell.
Mc Dougal (1999) cited that mutation may have only a minor effect in the cell. In fact, the
effect may be so small that it cannot be easily detected. On the other hand, if the mutation
affects the production of a very important enzyme or instructural protein, the change may
cause the death of the cell. This kind of mutation is called a lethal mutation because the
cell cannot survive. Most mutations that biologists study lie somewhere between these
extremes. These
mutations can cause observable effects, but they are not harmful enough to cause the
death of a cell. Genes may mutate in many ways. The normal eye color of Drosophila
is deep red, for example. But because of the mutation of various genes, the color of
the eyes may be white, pink, orange, purple, brown, or various shade of these colors.
But no one has not yet found a mutation blue or green eyes in the fruit fly, although
other species of these flies may have eyes of these colors. It seems then that the
genes or the raw materials to build blue or green pigments in the eye cells are not
present in the fruit fly (Bato Balani, 1989).
Biologists have observed that a particular gene may mutate to a number of
different forms, or alleles. Each form of these gene is still located at the place on
the chromosome. Chromosome breakage which occurs rarely, along with a few
other chromosomal accidents, can affect the gene’s location.
With the study of McLaren (1985), it has been found out that almost any aspect of
the life of an organism can be modified by genetic mutation. Mutations can affect
the size and the shape of the organism; its internal structure, the ability of the
nervous system to function, its behavior, or the efficiency of its cell activity.
PARADIGM OF THE STUDY
Independent Variables
Dependent variables

Distinguishing features of
sex differences

Mortality rate

Ratio of normal and dumpy
wings and red and sepia eyes

Probability of genetic events

Biosynthetic
effects
of
Euphorbia hirta on Drosophila
melanogaster Fruit Flies
Moderator Variable

Euphorbia hirta extract
-Leaves
-Stems

Fruit flies
Chapter II
METHODOLOGY
Preparing of Plant Extract:
After carefully gathering 500 grams of stems and leaves of Euphorbia hirta were prepared
50mL water for boiling in a clean container with the chopped leaves and stems. After 1
hour boiling, the decoction was separated through decantation and then was filtered.
Finally, the decoction was mixed with banana mashed inside the bottles. The
experimental bottles were covered with fish net in order to avoid insects. The set-ups
were exposed to fruit flies and observed for 3 days.
Drosophila Technique:
The collected fruit flies in different treatments were prepared to dampen the three (3)
cotton balls by several drops (about 5 drops) of ether alcohol. This was done to each of
the plants part in every treatment of three Drosophila vials (contact with ether alcohol may
possibility kill the flies), while one vial is not treated with extract, but with distilled water
and served as control. The bottoms of the glass vials were gently tabbed and the flies
should drop to the bottom. Quickly, the plug of the vials was removed and inserted the
cotton balls. The flies will overcome within a few seconds and they will fall to the bottom
of the bottles. The flies should not be treated with ether alcohol for more than a minute.
After the last fly stopped moving in the bottles, it was removed and replaced the plug,
then the flies were poured onto the index card for examination and the dead were placed
in the morgue. The flies can be injured easily; therefore, a bush or dissecting needles was
used to move them around. The flies were examined carefully using a stereoscopic
microscope or hand lens. Using a table to separate the male and female flies, the wild
type and the mutant parental strains, the cultures were stocked.
Mendelian Genetics
After 21 days, the stock cultures were etherized from different treatments which
previously used in the corresponding treatment. Offspring from a cross between the pure
wild and pure mutant flies were separated, with the male parents as the pure wild variety
and the females as the pure mutant variety. First generation was marked as F1. The flies
were classified one of each as sepia eyes to red eyes, and dumpy wings to normal wings.
Five (5) of the F1 males and five (5) of the F1 females were placed in the F2 bottle. The
bottles were laid on their side until the flies awakened. The cultured bottles were then
placed in a warm place, but away from windows, radiations and direct sunlight. The
temperatures should not change greatly, from night to day. The bottles were checked
each day
to see if the flies were in good condition. After 10 days, the F1 flies in the bottles were
etherized, and the dead were again placed in the morgue.
After another 10 days, all flies in the bottles are now F2 flies. Again, the flies were
etherized and each was classified as male or female, as mutant or wild type, sepia eyes
to red eyes, and dumpy wings to normal wings. All flies were put in the morgue.
F1 and F2 flies were counted. The ratio of the total number of flies with sepia, dumpy
wings, red and normal was computed with the Product Rule of Probability using the
formula;
Probability = No of change for particular event
Total no. of equality likely events
The genetic checker board was used to evaluate the expected genotypes and phenotypes
dominants.
Where: D- normal wings
d- dumpy wings
s- red eyes
s- sepia eyes
The cultured bottles, per treatment of plant extract, were labelled as “dumpy wings” and
“normal wings”. As the F2 flies appeared, they were sorted into “the” bottles according to
their type of wings. Another two bottles were labelled as “sepia eyes” and “red eyes”. As
“the” F2 flies appeared, they were also sorted into these bottles according to their eye
color. All cultured bottles were place in a warm place and left on their side till the flies
awakened.
After 20 days, they were crossed involving fire pairs of traits (dihybrid crosses). In the
original dihybrid cross made, Drosophila flies, with normal wings and red eyes:
Ds
Parents
DSd
DS
dS
ds
DDSs DDSS DdSS DdSs
DDss DDSs DdSs Ddss
DdSs dDSS ddSS ddSs
Ddss DdSs ddSs Ddss
Figure 1. Genetic Figure Board
Chapter III
RESULTS AND DISCUSSION
There are experiences related to incidents wherein people who accidentally touch the
thorn of Euphorbia hirta plant caused them severe pain, irritation and discomfort and
worst, skin infection. These stories are a few evidences that Euphorbia hirta contains
toxin which causes the skin irritation. The sap contains latex which is toxic on ingestion
and highly irritant externally, causing photo sensitive skin reaction and severe
inflammation, especially on contact with eyes or open cuts. The toxicity can remain high
even in dried plant material. Prolonged and regular contact with the sap is inadvisable
because of its carcinogenic nature.
Although DNA molecules usually duplicate themselves with great accuracy, occasional
errors result in mutations. Mutations are permanent change in the genetic message to the
cells. They may affect any aspect of an organism’s development or life. Some genes
mutate very rarely. When mutations of all the genes of an organism are considered,
however, it is clear that a large amount of genetic variation is introduced by mutation. The
genetic variation, however, is revealed slowly, because most mutations are recessive.
The effect will be covered up by the dominant allele from the other parent.
Table 1 presents the distinguishing features of Drosophila sex differences. In the
comparison of male to female, the male fly is relatively small in size while the female is
bigger. The kind of bonding on the abdomen is solid black on the tip of the abdomen for
the male, while it is brown for the female. The relative size of the abdomen for the male
is five small segments, while the female has six segments. The shape of the tip of the
abdomen for the male is rounded, while for the female, it is pointed. Sex combs are
present in the male, while absent in the female. The external genitalia have bristles in the
male, while the female’s genitalia are non-bristled.
TABEL 1
THE DISTINGUISHING FEATURES
OF SEX DIFFERENCES OF THE MALE AND
FEMALE (DROSOPHILA MELANAGASTER) FRUITFLIES
♂Distinguishing feature: Comparison:
Male♂
Female♀
1. Relative size
Small
Big
2. Kind of bonding
Tip of abdomen
Tip of abdomen
is solid black
is brown
on abdomen
3. relative size of abdomen Five small
Six small
4. Shape of tip of abdomen segments
segments
Rounded
Pointed
5. Sex combs
Present
Absent
6. External Genetalia
Black bristles
Non-bristled
around Genetalia
The mortality rate of Drosophila fruit flies of the first and second generation is on Table 2.
Flies in culture bottle 3 which were etherize and treated with Euphorbia leaves extract,
had the highest mortality rate of 10, followed by
Cultured
Bottle
TABLE 2
MORTALITY RATE OF
(DROSOPHILA MELANOGASTER) IN FIRST
(F1) AND SECOND (F2) GENERATIONS
Generation
Total Mean
(F1)
(F2)
Mortality
Rate
B1
(control)
0
0
0
0
0
3
5
8
4.0
4
(leaves)
9
11
20
10.0
10
Total
12
16
28
14.0
B2
(stems)
B3
12
10
F1
8
F2
6
4
2
0
B1
B2
B3
Cultured bottles
Graph 1 Mortality Rate of Drosophila melanogaster in First (F1) and Second (F2)
Generations
those in cultured bottle 2 which were etherize and treated with Euphorbia stems extract,
had a mortality rate of only 4, while from cultured bottle 1, serves as control and not
treated either stems or leaves, no fruit flies died, results to zero mortality. The different
bottles and treatments caused different mortality rates. But the mere fact that there were
recorded of mortality rates. It says that these plan’s extracts exhibited a lethal of fact on
fruit flies. The death of Drosophila fruit flies agrees with the statement contained in Health
Biology by McLaren (1985), that if the mutation affects the production of a very important
enzyme of structural protein, the change may cause the death of a cell, and thus called
lethal mutation.
TABLE 3
THE NUMBER OF GAMETES (PHENOTYPES)
IN FIRST GENERATION (F1) DTHYBRID CAUSE
Cultured Bottle
B1
(control)
B2
(stems)
B3
(leaves)
Total
Phenotypes
Total
Ratio
Probability
10
40
1:1
0
7
13
40
3.5:3.5
0.58
13
7
13
40
3.25:1.75
1.85
34
24
36
120
D
D
S
s
10
10
10
9
11
7
26
Table 3 presents the number of gametes (phenotypes) in first generation (F1)
dihybrid cross. Results showed that there are four kinds of phenotypes which were
inherited and combined from the parent’s genes. Both parents are heterozygous for two
traits (DdS x DdS), so with the mutations of all the gens of the flies, the genetic variation
was revealed slowly as seen in the calculated value of fraction/ratio in the following: for
132 dumpy wings and sepia eyes are dominant with ratio of 2:3, while in Bottle 3 dumpy
wings and sepia eyes are the same dominant with ratio of 3.25:1.75. However, in Bottle
1 (control), normal wings are equal to dumpy wings as well as eyes are equal to sepia
eyes, with ratio of 1:1. The probability value obtained was 0 for the control of equivalent
to 0% chances, compared to the Bottle 2 with probability of 0.66 at equivalent to 0.66%
chances, and Bottle 3 with probability of 1.85 at equivalent to 1.855 chances. In general,
the first generation (F1) has a total probability of 0.75 at equivalent to 0.75% chances.
The difference of each value are more or less equal and negligible, meaning the mutation
in the first generation (F1) is not so great. But it is said that the extracts have a mutagenic
impact on the fruit flies.
2
1,5
1
0,5
0
B1
B2
B3
Culture Bottles
Graph 3 The numbers of gametes (phenotypes) in first generation (f1) dihybrid cross
Cultured
Bottle:
B1
(control)
B2
(stems)
B3
(leaves)
Total
0.50
TABLE 4
THE NUMBER OF GAMETES (PHENOTYPES)
IN SECOND GENERATION (F1) DIHYBRID CROSS
Phenotypes
Ratio
Total
D
d
S
S
9.5
9
11
10
10
40
10.5
6:5:
7
13
6
14
40
13.5
3.5
4
16
3
17
40
16.5
20
40
19
41
120
1.77
0.47
1.02
Probability
0.9
2.07
4.71
Table 4 presents the number of gametes (phenotypes) in second generation (F2) dihybrid
cross. Results showed that Bottle 2 sepia eyes are dominant and dumpy wings are also
dominant, with ratio of 6.5:13.5 while Bottle 2 sepia eyes are dominant and dumpy wings
are also dominant, with ratio of 6.5:13.5, while Bottle 3, sepia eyes and dumpy wings are
also dominant with ratio of 3.5:16.5. However, in Bottle 1(control) the genetic variation
was revealed slowly as seen in the calculated value of ratio, dumpy wings are become
dominant with ratio of 9.5:10.5. The probability value obtained was 0.9 for the control at
equivalent to 0.9% chances, compared to the Bottle 2 with probability of 2.07 at equivalent
to 2.07% chances and Bottle 3 with highest probability of 4.71 at equivalent to 4.71%
chances. Results indicated that the mutation is increased as the generation increased.
Mutagenical impact on the fruit flies may be due to the increasing amount of extract that
they received or expose to. This agrees with the book of McLaren (1985) where it is
written that various chemicals synthetic and natural substances act to increase the rate
of gene mutation. Sometimes this rate increases very dramatically with continuous
exposure, and may even result to death. The probability value in general for the second
generation (F2) was 0.94 at equivalent to 0.95% chances. The extracts of Euphorbia plant
had significantly increased the phenotypes, which led to mutations of a dominant trait.
5
4,5
4
3,5
3
2,5
2
1,5
1
0,5
0
B1
B2
B3
CULTURED BOTTLES
Graph 3 The number of gametes (phenotypes) in second generation (F2) dihybrid cross.
Chapter IV
CONCLUSION AND RECOMMENDATION
Conclusions
The distinguishing features of sex differences of male and female flies, the male
fly is relatively small in size while the female is bigger. The kind of bonding on the
abdomen is solid black on the tip of the abdomen for the male, while it is brown for the
female. The relative size of the abdomen for the male is five small segments, while the
female has six segments. The shape of the tip of the abdomen for the male is rounded,
while for the female, it is pointed. Sex combs are present in the male, while absent in the
female. The external genitalia have bristles in the male, while the female’s genitalia are
non-bristled.
The mortality rate of Drosophila melanogasler in different plants per treatment is
10, It says that these plan’s extracts exhibited a lethal of fact on fruit flies. The ratio of
normal and dumpy wings, red and sepia eyes of Drosophila melanogasler in different
plants per treatment is 2:4, normal characteristics is lesser than the abnormal
characteristics. The probability of genetic events from first generation to second
generation is 50 percent chances.
Recommendations
References
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