Fruit fly lab report

?Genetic Fruit Fly lab report. Includes?Abstract (approx. 250 word), Introduction (approx 2 pages), Materials and method, results, discussion, references and appendix.***What needs to be included in Abstract, Introduction, Materials and method, results, discussion, references and appendix are included in Document 1 (attached below)? ***A checklist is included in Document 2 (attached below)?*** Procedure and other information can be found in Lab 8 attached as Document 3?***Document 4 (lab 10) shows how the chi Square and other results should be analyzed?***Document 5 (Class Data)
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Bio103 Laboratory – Student’s Guide
John Jay College, C.U.N.Y
Multi-week Laboratory:
Genetic Crosses with Drosophila
Introduction
Drosophila melanogaster, the common fruit fly, is a two-winged insect that belongs to the
order Diptera. Drosophila has been one of the most widely used model organisms for the study
of genetics, molecular biology, evolution, and developmental biology since the early 1900s and
many consider it the single most studied organism on earth. Although a few scientists had used
Drosophila before him, it was the work of Thomas Hunt Morgan with Drosophila that
demonstrated the enormous advantages of fruit flies and led to their becoming a common model
system for scientific research.
In genetics, it is important to know the genotype and phenotype of your parentals and
offspring. The genotype describes the genes an individual has while the phenotype describes
what those genes look like or how those genes are expressed. In describing a phenotype, you
may describe an individual as exhibiting the dominant or recessive trait. For example, lets say
gene “L” that controlled leg length in flies. The dominant condition causes a fly to have long
legs, while the recessive condition leads to short legs. Having long or short legs would describe
the phenotype of an individual. If we were to look at genotype possibilities, you can describe an
individual as being homozygous or heterozygous. Homozygous refers to those individuals that
have two similar alleles. This is also described as “true-breeding”. This can be homozygous
dominant (or LL in our example) or homozygous recessive (ll). Individuals would be described
as heterozygous if they have different alleles, one dominant and one recessive (or Ll).
Hypotheses Development
An important step in developing an experiment involves outlining what your hypotheses
will be. These are carefully constructed statements that are used to test your results against.
This typically involves a two-step process. First you determine what your null hypothesis will be.
The null hypothesis typically involves considering the neutral situation, or the result if there was
no effect. This is termed Ho. Secondly, you determine what your alternative hypothesis will be.
This typically involves considering the situation if there was an effect.
For example, in insects temperature can be an important factor that can have an effect
development. In investigating this, a scientist may have a null hypothesis (Ho) that temperature
does not have an effect on development.
The alternative hypothesis (Ha) would be that
temperature does have an effect on development. Once the experiment was conducted and the
data was collected, the scientist would run statistical analysis on the results. The statistical
analysis is tested against the null hypothesis. In this case, if there was no significant effect
detected (or no significant difference in the data), then the null hypothesis can’t be rejected, and
it is therefore accepted to be true. The scientist would accept Ho and state that temperature did
not have effect on insect development. If there was a significant effect detected, then the null
hypothesis is rejected, the alternative hypothesis is accepted and the scientist would state that
temperature did have an effect on insect development.
Lab #8 – Page 1
Bio103 Laboratory – Student’s Guide
John Jay College, C.U.N.Y
Basic Biology of the Fruit Fly
The following are the major taxonomic groups of the scientific classification of Drosophila
melanogaster, with some common properties of the taxa in parentheses:
Kingdom
Animalia
ingestive heterotrophs
Phylum
Arthropoda
jointed limbs, exoskeletons
Class
Insecta
three body segments, six legs
Order
Diptera
two-winged flies
Family
Drosophilidae
small, fruit-loving flies
Genus
Drosophila
means “dew loving”
Species
D. melanogaster
means “black belly”
Wild-type Drosophila (genetically “normal,” as would be found in the wild) have dark red
eyes and yellow-brown bodies with black rings around the abdomen segment. They are
sexually dimorphic (two distinct gender forms – male and female) and about 2.5mm in length.
The average lifespan is 30 days but varies widely, based on the ambient temperature, as does
the rate of growth and development. Drosophila have four pairs of chromosomes: a pair of sex
chromosomes (X and Y) and three pairs of autosomes, named 2, 3, and 4. (The fourth
chromosome is so tiny that it is often ignored.)
At 25oC, a well-fed mating pair can give rise to 800 offspring over their lifetime. Following
an elaborate courtship and copulation, taking about 30 minutes, a female lays an average of five
eggs shortly thereafter, directly into the food source. The eggs hatch 12-15 hours later and the
initial hatchlings emerge looking like small white meal worms, barely visual to the naked eye.
The larval stage lasts 5-8 days, and the larvae molt twice during this period. Next is the
metamorphosis stage during which the larvae are wrapped in a pupa (similar to a cocoon) for 46 days. Thus, the total time required to go from egg to mature fly is 9-14 days, and mature flies
typically live for ~3 weeks.
Usefulness as a Model Organism
D. melanogaster is an ideal model system for the study of genetics, molecular biology,
and development biology because of the following distinct advantages:
1. They are small and thus easy and cheap to grow in the laboratory.
2. They have a short generation time (~2 weeks) and high rate of reproduction.
3. The mature larvae have giant many-times-re-replicated chromosomes in the salivary
glands called polytene chromosomes containing visible “puffs” that indicate regions of
gene activity.
4. Drosophila have only four pairs of chromosomes: three autosome pairs, and one sex
chromosome pair.
5. Males do not show meiotic recombination, simplifying genetic studies.
6. Genetic transformation techniques are easy and have been available since 1987.
7. Its compact genome (much less noncoding DNA than humans) is fully sequenced and
well-annotated.
Lab #8 – Page 2
Bio103 Laboratory – Student’s Guide
John Jay College, C.U.N.Y
Mating Fruit Flies
In this week’s laboratory experiment, which will continue for three sessions, we will need
to be able to cross different strains of Drosophila to observe and count the resulting offspring. In
order to set up crosses successfully, we need to first be confident that we can isolate males of
one strain and females of another. This is made significantly easier because Drosophila have
distinctive sex-specific markings. The diagrams below show the most conspicuous differences
between males and females.
?
?
?
?
Homework Exercise #1:
HQ-1. Closely examine the pictures above and make a list of four noticeable differences
between males and females that make good features for determining the sex of a fly. Complete
the following chart.
Physical Characteristic
Male (?)
Lab #8 – Page 3
vs.
Female (?)
Bio103 Laboratory – Student’s Guide
John Jay College, C.U.N.Y
Another important consideration when mating flies is to know that the females have not
already copulated, because male sperm can survive in the female reproductive tract for some
time. Thus, one must collect “virgin” females. Because females are not receptive to copulation
for 8-12 hours after they first emerge from the pupa, there is a short “window” of time after an
adult female fly emerges from her pupal case that one can be reasonably sure that she has not
yet copulated. This is essential when performing genetic crosses; to be sure that the cross
really is what it was intended to be.
Mutant Phenotypes in Drosophila
In fruit flies, the nomenclature for genes is a bit backward from what one might think. The
name of a gene is usually given based on the phenotype that a mutant form of the gene causes.
For example, there is a gene in flies that is necessary for the normal red pigment to develop in
the eyes. One might think that “red eyes” would be a suitable name for this gene, but, by
convention, that is not how it is done with Drosophila. Instead, the name white is given to this
gene, abbreviated w, because this gene was first discovered as a mutant nonfunctional allele
and the flies that carry two of these alleles have white eyes. In the same way, an imaginary
gene that helps a fly maintain its normal weight might be called obese if the flies that suffer
mutations in this gene cannot maintain a normal body weight. In the fly crosses that we are
going to set up for this laboratory, we are concerned with the following three genes:
Lab #8 – Page 4
Bio103 Laboratory – Student’s Guide
John Jay College, C.U.N.Y
white (w) As described above, the white gene is responsible for normal pigment production in
Drosophila. The normal or “wild type” allele of this gene is written w+, which codes for the
normal red eye color. The mutant allele w causes a failure in pigment production leading to
eyes that lack the red pigment and thus appear white.
w+
w
sepia (se) is another gene involved in eye pigment production. The product of this gene is an
enzyme involved in the final stages of pigment processing. Thus, the eyes in mutants of this
genes are still pigmented, but the pigment is much darker than normal, a color called sepia.
se+
Lab #8 – Page 5
se
Bio103 Laboratory – Student’s Guide
John Jay College, C.U.N.Y
vestigial (vg) is a gene involved in normal wing development in flies. The wild type allele of
this gene is written vg+ and codes for normal-sized wings. The mutant allele vg causes a
drastic failure in wing development in which flies with the vestigial phenotype harbor tiny,
shriveled, and totally nonfunctional wings.
vg+
vg
vg
Lab #8 – Page 6
vg
Bio103 Laboratory – Student’s Guide
John Jay College, C.U.N.Y
Laboratory Procedures
Your instructor will guide you with the necessary background and demonstrations. For the
entire fruit fly experiment, everyone will work as individuals – not lab pairs. Each student will
have their own cross to set up and analyze.
Exercise One: Designing the Experiment
When performing these crosses, to save time, you are already working with F1 flies. In one set
of experiments (sex-linked vs. autosomal), you will be examining one trait. In this cross, you are
crossing a recessive (mutant) white-eyed female with a dominant wild type, red-eyed male. All
adults are considered true-breeding.
Answer the following questions:
What are the genotypes of these parents if these genes are autosomal?
Male (?)
X
Female (?)
In this cross, what would the F1 generation genotypes be?
What are the genotypes of these parents if the genes are sex-linked and carried on the X
chromosome?
Male (?)
X
Female (?)
In this cross, what would the F1 generation genotypes be (be sure to indicate males and
females)?
i. Consider the phenotype of white eyes in fruit flies. Design a cross that would reveal if the
white gene is on the X chromosome or an autosome. Give the expected results for these
two possibilities. You are starting with the F1 generation (from above exercise).
Autosome
F1 Parent Genotypes:
?
X?
F2 Genotype Ratio:
————————————————————————————————————————–X-chromosome F1 Parent Genotypes:
?
X
?
NOTE: for this cross, you are using white-eyed males and red-eyed females from the F1 generation
F2 Genotype Ratio:
————————————————————————————————————————–Lab #8 – Page 7
Bio103 Laboratory – Student’s Guide
John Jay College, C.U.N.Y
In another set of experiments (unlinked genes vs. linked genes), you will be examining two
traits. In this cross, the original parentals can be described as follows. The females are
described as homozygous recessive for vestigial wings but homozygous for wild type, red eyes.
The males are described as being homozygous for wild type wings but homozygous for mutant
(or sepia) eyes. These traits are autosomal.
What are the genotypes of these parents?
Male (?)
X
Female (?)
In this cross, what would the F1 generation genotypes be?
ii. Working with the F1 flies (from the above cross), you want design a cross to determine if
these genes are linked or not. When you cross your dihybrid F1 flies with and what would
your expected outcomes be if the genes were unlinked versus linked?
X
Genotypes: Parent 1
Parent 2
EXPECTED OUTCOMES:
If the genes were unlinked:
F2 Genotype Ratio:
————————————————————————————————————————–If the genes were linked:
F2 Genotype Ratio:
—————————————————————————————————————————
Exercise Two: Setting up the fly crosses
Preparation of vials for flies (Week 1):
1. Your instructor will demonstrate this, but the general directions are below.
2. Gather a vial, plug, and cap for each student, including those absent, and 2-4 extra
vials per table, if there are enough flies. (Students that finished first will be asked to
make a couple extra vials.)
3. If your lab partner is absent, you must make a vial (and a cross) for them also.
4. Label each vial with last name, section, and cross.
Lab #8 – Page 8
Bio103 Laboratory – Student’s Guide
John Jay College, C.U.N.Y
5. Take an equal amount of water and food (measure them separately in different small
beakers) and then mix together thoroughly. This can be done as a large batch for
several students to share.
6. Place mixture in vials to a height of 2-3cm.
7. Press down to remove air pockets and smooth the top of mixture.
8. Clean the sides of vials.
9. Only now: add six grains (pieces) of yeast onto top of mixture. Don’t add more than 6
or the yeast will proliferate so fast that they consume the oxygen faster than it can
confuse through the cotton plug.
10. Place the vials on their side – for the next several hours, the vials must be on their
sides, or else the sleeping flies will fall into the fresh food and get stuck.
11. Clean up.
Anesthetization of flies (Weeks 1, 2, and 3):
1. Remove blue cap.
2. Do not remove white plug.
3. Place vial on side (leave on side until flies removed).
4. For Week 1 when working with F1 flies: netting should be on sides and not on top
and bottom (this will allow the flies to fall in between the netting and not get caught in
the netting when they fall asleep).
5. Place applicator in Fly Nap for 3 seconds.
6. Cap the bottle of Fly Nap.
7. Place wet applicator into the vial: mid-way in and on top (follow the instructors
demonstration carefully and do not remove the white plug).
8. Leave applicator in for 3-5 minutes (one full minute after last fly stops moving).
9. Remove plug and applicator and gently tap out flies onto prepared paper.
10. Plug and cap the vial from which you took the flies.
Working with anesthetized flies:
Week 1:
1. Separate F1 flies by sex.
2. Divide flies up as equally as possible.
3. If some flies start moving while you are doing this: carefully bring wet Fly Nap
applicator up to the fly for 2-3 seconds while not touching it (otherwise you will
probably kill the fly).
4. Minimum needed is 4 female and 4 male flies per vial (preferably, 6 female and 6
male).
5. Gently introduce flies to vials with brush (vials always on sides).
6. Plug and cap vials (vials always on their sides)
7. Place vials in box (vials on their sides for several hours, until flies wake up.)
Week 2:
1. Put the F1 flies to sleep and tap them out on paper as per instructions above.
2. Plug and cap the vials.
3. If the cap has any brown stains (Fly Nap), get a new cap – the residual Fly Nap will
kill the flies.
4. Place these flies in the fly morgue (a beaker of water on your table) – do not let any
escape or they will end up in the Cafeteria!
Lab #8 – Page 9
Bio103 Laboratory – Student’s Guide
John Jay College, C.U.N.Y
Week 3:
1. Prepare paper to receive flies as per instructor’s direction: remember, you will have 4
separate types of flies.
2. Put the F2 flies to sleep and tap them out on paper as per instructions above.
3. Separate flies into the 4 types.
4. After you have finished your identification, separation, and counting: place these flies
into the fly morgue.
5. Keep your results and give them to your instructor as well.
6. The class will now proceed to a Chi Square analysis.
Lab #8 – Page 10
Bio103 Laboratory – Student’s Guide
John Jay College, C.U.N.Y
Post-Laboratory Homework Questions
1. Consider the crosses you did today. One investigated whether the genes were
autosomal and sex-linked and one investigated whether two genes were link or unlinked.
You started with F1 generation flies. Clearly state the possible outcomes of the two
crosses that you set up today. Include a diagram of the crosses, as well as the parent
genotypes and offspring ratios. (NOTE: you should have four crosses, one for autosomal,
one for sex chromosomes, one for linked genes and one for unlinked genes).
2. Consider your cross that examines whether or not a trait is autosomal or sex-linked.
What is the null hypothesis of your cross (Ho)?
Ho:
What is your experimental hypothesis (Ha)?
Ha:
If your null hypothesis is accepted, what can you conclude?
If your null hypothesis rejected, what can you conclude?
Lab #8 – Page 11
Bio103 Laboratory – Student’s Guide
John Jay College, C.U.N.Y
3. Now consider the cross that examines whether or not two traits are linked (on the same
chromosome) or unlinked (located on different chromosomes).
What is the null hypothesis of your cross (Ho)?
Ho:
What is your experimental hypothesis (Ha)?
Ha:
If your null hypothesis is accepted, what can you conclude?
If your null hypothesis rejected, what can you conclude?
4. The flies in the vial that you made actually eat mostly yeast as their principle food source.
So what is the purpose of the blue “fly food?”
Lab #8 – Page 12
The Chi-squared Statistical Test
Your required reading/activity for Lab 10 is about the Chi-squared statistical test.
A chi-square test can be used to analyze categorical data, which is data that is
qualitative or can be classified based on a characteristic. For instance, you can classify
the population according to sex, or race. If you wanted to analyze this, you could use a
chi-squared test to determine if there is a relationship or trend in the population. Let’s
say you surveyed students at John Jay and wanted to determine if males preferred
steak for dinner, or if females preferred vegan food instead. You would need to design a
survey, hand it out to 50 females and 50 males and record what they would choose for
dinner: steak or a vegan meal.
Now, let’s say that you want to see if there was a trend in the data. In order to do this,
you would first need to consider what the data would look like if there weren’t a
preference. If males and females didn’t care if they received a steak or vegan dinner,
then you would expect to see an equal number of males and females choosing steak
and vegan dinner options. If we surveyed 100 people (50 male, 50 females), the data
may resemble the following:
“Expected Values”
Category
If there wasn’t a preference
Males that want steak
25
Males that want vegan
25
Females that want steak
25
Females that want vegan
25
However, once you perform the survey, you may find the actual/observed data looks
like this:
“Observed Values”
Category
Observed Values
Males that want steak
30
Males that want vegan
20
Females that want steak
22
Females that want vegan
28
You may want to ask if your observed data matches what you originally expected – that
there were no preferences between males and females in their dinner choice. To do
this, we could use a Chi-squared goodness-of-f
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