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FROM GENES TO PHENOTYPE IN A FLY:
A DEFICIENCY SCREEN TO IDENTIFY GENE REGIONS AFFECTING
FEMALE FERTILITY IN DROSOPHILA MELANOGASTER
Whatever it may have been alleged to be, it [the scientific life] is in reality exciting,
rather passionate and – in terms of hours of work - a very demanding and sometimes
exhausting occupation.
-Peter B. Medawar, “Advice to a Young Scientist”
With all his amateurish fumbling, Martin had one characteristic without which there
can be no science: a wide-ranging, sniffing, snuffling, undignified, unself-dramatizing
curiosity and it drove him on.
-Sinclair Lewis, “Arrowsmith”
ABSTRACT
Many of us discovered the joy of science while conducting research. One way to provide opportunities
for our students to also discover the joys and challenges of experimental research while strengthening
their understanding of Developmental Biology is to implement original research projects into class-based
laboratories. This Learning Object describes a ~4 week long laboratory unit during which students use a
classic model organism, Drosophila melanogaster, to conduct a deficiency screen to identify chromosome
regions influencing components of female fertility (or another phenotype of their choice). By conducting
this series of activities, students improve both their understanding of and ability to communicate about: 1)
the strengths and limits associated with the use of model organisms/systems, 2) the genetic basis of
physiological processes such as female fertility, 3) one use of bioinformatics as a predictive instrument,
and 4) the scientific process – particularly hypothesis development and sources of experimental error.
Students develop a more sophisticated understanding of the integrated and data-rich fields of genetics and
development while they discover something new about Drosophila development. Students who get
‘hooked’ can satisfy their curiosity by extending their studies to test their hypotheses regarding individual
gene function or further characterizing the identified mutant phenotype.
INDEX
Instructor Notes
Student Exercises
pages
2-5
6-27
1
INSTRUCTOR NOTES
In this series of laboratory exercises, students engage in original scientific research. They use one forward
genetic approach, a deficiency screen, to identify Drosophila melanogaster genes with potential effects
on female fertility (or a phenotype of their choice).
RATIONALE
Developmental Biology is a dynamic field with a rapidly growing and increasingly technical knowledge
base. To become tomorrow’s developmental biologists, today’s biology students need a solid foundation
in scientific content, skills in experimental design and quantitative literacy, and skills in collaboration and
communication (pg 2, NRC 2002). Laboratory explorations on previously unexplored and engaging
topics are appropriate venues to develop these skills in an integrative, discovery-based approach that
fosters student development via frequent interactions with classmates and instructor. Such types of
inquiry-based learning are recommended as effective approaches to teach students scientific content,
process and skills (pg 16. NRC 2002).
Small-scale genetic screens are excellent opportunities to conduct discovery science in a traditional
laboratory setting. They can focus on biological processes of interest to the course instructor and thereby
serve as a valuable collaboration whereby students learn about the content and process of biology and
faculty advance their research program (Lengyel and Merriam 2001; Elgren and Hensel 2006). By
examining the genetic basis of developmental processes, students integrate an understanding across levels
of biological organization. Since screens often generate relatively large data sets and utilize organisms
with published genomes, screens can provide an accessible entry into the world of bioinformatics. There
is a rich history of genetic screens being used to understand development and laboratory work can be
coupled with readings that illustrate the role of screens in major biological developments (see selected
examples, below). Finally, screens provide a great deal of flexibility in terms of student responsibility for
selecting a phenotype of interest and developing an assay to detect it This type of experience can be
particularly valuable in terms of giving “students a sense of empowerment over a body of knowledge and
instills in them the confidence to succeed” (pg. 17, NRC 2002).
Drosophila melanogaster has become a workhorse in undergraduate education. Its history as a laboratory
research organism has had close ties with undergraduate students and instructors since its development as
a model organism in the early twentieth century (pp 33-37 Kohler 1994). While remaining a sturdy
teaching tool it has also become a model system par excellence for addressing biological questions from
chromosome structure to evolution. This usefulness stems from two characteristics of the animal. First, it
is relatively easy to rapidly raise large populations of flies in a laboratory setting. Second, the genetics of
this animal facilitates the examination of gene function in vivo (reviewed in O’Grady 2003).
SELECTED READINGS TO ACCOMPANY THE LABORATORY
 St Johnston D. 2002. The art and design of genetic screens: Drosophila melanogaster. Nature
Reviews, Genetics. 3: 176-188.
 Roman G. 2004. The genetics of Drosophila transgenics. BioEssays 26:1243-1253.
 Karim FD, Chang HC, Therrien M, Wasserman DA, Laverty T, Rubin GM. 1996. A screen for
genes that function downstream of Ras1 during Drosophila eye development. Genetics 143:315329.
 Kohler RE. 1994. Lords of the Fly. Chicago: Chicago University Press. pp. 321.
 Nusselein-Volhard C and Wieschaus E. 1980. Mutations affecting segment number and polarity
in Drosophila. Nature 287: 795-801.
 Weiner J. 1999. Time, Love, Memory. New York: Vintage Books. Pp. 300.
2
TIMELINE FOR LABORATORY SET-UP
Days before the
student lab
32
Preparatory Tasks
Quantities
student research groups
n=
Establish one large (bottle) culture of wildtype flies (++; such as Oregon R) per research
group.
Wild type cultures needed:
(1 x n) + 2 =
Start fresh small (vial) cultures of each
deficiency line to be tested. Establish extra as a
buffer.
Deficiency cultures needed:
(1 x n) + (0.25 x n) =
20-22
Collect virgin wild-type (++) females. Plan to
collect ~20 virgin females for each
experimental cross. Collect extra females to
compensate for deaths and losses due to
females who are not virgins.
Virgin ++ females needed:
(25 x n) + (6 x n) =
17
Set up one experimental cross for each student
research group. Set up one extra cross for
every four crosses to compensate for lines that
do not provide expected progeny phenotypes.
Experimental crosses needed:
(1 x n) + (0.25 x n) =
To do this, use 20 of the females collected 3-5
days prior. Check the vials carefully to be
certain there are no larvae in the vials
indicating that the females are not virgins.
Collect 12-20 males from each deficiency line
to cross with the virgin wild-type females. Be
certain that you are able to identify the
phenotype marker carried by the balancer.
Establish each cross in a bottle with media and
supplemented with yeast.
3-5
Establish one wild-type culture for each
experimental cross established.
Wild-type cultures needed:
(1 x n) + 2 =
Collect virgin F1 female progeny from each
experimental culture established ~12 – 14 days
prior. Collect 20 – 30 females of each
phenotype. While it is useful to check the
genotype of the deficiency stock because
exceptions occur, generally the females with
markers carry the balancer (bal) and females
with a wild-type phenotype carry the
deficiency (df). Group females of the same
genotype ~5 to a vial.
Virgin females needed:
Collect an equal number of wild-type males.
50-60 ++ males x n =
20-30 df/+ females x n =
20-30 bal/+ females x n =
3
MATERIALS
 Polystyrene vials (~2.3 cm x 9.5 cm) for maintaining stock cultures, storing virgins prior to mating &
screen progeny collection. You will need ~120 vials per line screened plus additional vials for culture
maintenance and temporarily holding virgin males and females.
Genesee Scientific (1-800-789-5550: www.geneseesci.com)
Drosophila vials, narrow, polystyrene bulk, 500 vials/unit. Catalogue number 32-116.
I order one or two boxes of vials in holders and the rest in large boxes – that way I get cardboard trays
that students use to keep their flies in order.
 Glass bottles for expanded wild-type lines and crosses to generate experimental females
Plan to use ~4 bottles per line you plan to screen (two bottles for wild-type cultures – you’ll be using one
and need to transfer flies into the other - and one for the experimental cross).
Half Pint Glass Stock Bottle, 48 Bottles/unit. Genesee Scientific, catalogue number 50-102.
Starbuck’s Frappachino bottles (empty and cleaned) also make suitable culture bottles.
 Cotton balls
Extra large for bottles: cotton balls, extra large, fits stock bottles, 2000 balls/unit. Genesee Scientific,
catalogue # 51-102B
Large for vials: Cotton balls, Large, fits narrow vials, 2000/unit. Genesee Scientific, catalogue # 51-101.
 Materials for collecting virgin flies
Dissecting stereoscope
Source of CO2 (compressed gas, dry ice + water, or antacids + water) to anesthetize flies.
Paintbrush for moving flies
Extra vials with reconstituted media
 Fly media: WARD’s instant Drosophila medium, blue, 4L. Catalogue # 38 V 0592.
 Additional Materials & Supplies
Sharpie marking pens
Data sheets (sample included in Appendix C).
Large freezer to kill and store progeny prior to counting.
Aspirators for transferring flies (to set up matings and remove males after mating)
Fisher Brand vinyl tubing ¼” inner diameter, 7/16” OD
(Fisher Scientific catalogue # 14-169-7D)
Cut to length of 27” (~68 cm)
Cut the distal 2cm (~3/4”) off of a 200ul pipette tip
Position a small piece (~ ¾” x ¾”) of mesh over the wide end of the pipette tip and insert
the end (covered with mesh) into one end of the tubing. Be certain that the mesh openings
are smaller than a fruit fly!
4
MODIFICATIONS TO DECREASE THE PREPARATION WORK INVOLVED
Modification 1: Smaller classes with advanced students can do much of the preparatory work themselves.
I have also conducted this lab as a semester-long research class in which students selected which
deficiency line they want to screen, collected virgins and set up experimental crosses.
Modification 2: Select another phenotype to screen. Phenotypes that do not involve reproduction do not
require the collection of virgin females, single pair matings, and transfers. An added benefit is that
students might feel additional ownership for the project if they select the phenotype to be examined. It is
still useful to cross the deficiency line with the wild type line to create a control.
LITERATURE CITED
Elgren T and Hensel N. 2006. Undergraduate Research Experiences: Synergies between Scholarship and
Teaching. peerReview. Winter:4-7.
Kohler RE. 1994. Lords of the Fly. Chicago: Chicago University Press. pp. 321
Lengyel JA and Merriam, JR. 2001. Translation of a new genetic screening method (ectopic expression)
from the research lab to a teaching lab. Drosophila Information Services. 84: 218-224.
Lewis S. 1925. Arrowsmith. New York: Signet Classics. Pp. 459.
Medawar PB. 1979. Advice to a Young Scientist. New York: Basic Books, Inc. pp. 109.
National Research Council. 2003. Bio2010: transforming undergraduate education for future research.
Washington, DC: The National Academies Press. pp. 191.
O’Grady PM. 2003. Drosophila melanogaster. In Resh, VH and Cardé RT (eds). Encyclopedia
of Insects. New York: Academic Press.
Weiner J. 1999. Time, Love, Memory. New York: Vintage Books. Pp. 300.
5
FROM GENES TO PHENOTYPE IN A FLY:
A DEFICIENCY SCREEN TO IDENTIFY GENE REGIONS AFFECTING
FEMALE FERTILITY IN DROSOPHILA MELANOGASTER
LABORATORY OVERVIEW
Understanding how organisms reproduce is a fundamental question in biology. This is because there is
great interest in managing (both limiting and augmenting) populations, to understand the evolution of
diverse reproductive phenotypes (behaviors, morphologies and physiologies), and to better understand
how two (or sometimes one) haploid cells give rise to a multicellular individual.
The fruit fly, Drosophila melanogaster provides a particularly tractable system for studying mechanisms
of physiological processes (such as fertility). This usefulness stems from two characteristics of the animal.
First, it is relatively easy to rapidly raise large populations of flies in a laboratory setting. Second, the
genetics of this animal facilitates the examination of gene function in vivo (elaborated in O’Grady 2003).
Over the following weeks, you will conduct a deficiency screen (one forward genetic approach) on a
portion of the fly’s third chromosome to identify gene regions affecting female fertility in a manner
consistent with a failure to properly store sperm.
LEARNING OBJECTIVES
 Describe advantages and limits of using Drosophila melanogaster as a model organism
 Explain how a study of genetics enriches our understanding of animal form & function
o Define and be able to explain the role of the following terms in Drosophila genetics:
genetic deficiency, balancer, dominant marker, hemizygous, and haploinsufficient
o Explain how genetic screens are used to elucidate the genetic basis of phenotypes
 Explore proximate and ultimate explanations for high female fertility
o Define and relate the following terms: fertility, fecundity, sperm storage, and larval
viability
o Describe fitness consequences of sperm storage variants
 Utilize a bioinformatics approach to develop testable hypotheses regarding the role of specific
genes on female sperm storage.
o Use FlyBase to identify genes within the deficiency region
o Describe predicted and determined biological roles for these genes
o Given a candidate gene within a deficiency region, develop a model describing how it
might affect female sperm storage
 Further develop an understanding of the process of scientific investigation
o Identify and control/account for sources of variation in results
o Present results in written form
o Suggest new avenues of research within this field.
I. INTRODUCTION
A. THE RESEARCH SUBJECT: FEMALE SPERM STORAGE IN DROSOPHILA MELANOGASTER
Female insects have a phenomenal ability to produce large numbers of offspring. Their high fertility is
possible because females produce and fertilize many eggs. Studying insect fertility helps us elucidate
basic mechanisms involved in sexual reproduction – a fundamental developmental process. Furthermore,
understanding female fertility has direct applications for managing insect populations. The physiological
events occurring within the female reproductive tract between sperm transfer and fertilization remain
poorly understood. Several observations make understanding these intervening events interesting.
6
 Among many animals species, females retain sperm within specific regions of their
reproductive tract (often chambers called storage organs) for various periods of time between the
start of sperm transfer and when sperm leave the reproductive tract (Bloch Qazi et al., 2003). This
phenomenon is called female sperm storage.
 Females frequently mate repeatedly over the course of their adult life (Ridley, 1988). Often remating before sperm from the previous mating are depleted thus producing a situation in which
sperm from more than one male can potentially exist within the female’s reproductive tract.
 In most animals with internal fertilization, males transfer many more sperm than the female can
possibly use to fertilize her eggs (Ridley, 1988). Sperm that do not fertilize an egg have one of
several fates: death, digestion, or expulsion from the female reproductive tract (Bloch Qazi et al.,
2003; Snook and Hosken, 2004). Since this sperm ‘gauntlet’ occurs within the female’s
reproductive tract, females likely play a critical role determining sperm fate (Eberhard, 1996).
Although female sperm storage is a common phenomenon in the animal world, we understand relatively
little about the female’s role determining sperm fate.
You are using the well-established animal model Drosophila melanogaster to study mechanisms of
female sperm storage. There are many reasons why D. melanogaster is appropriate for experimental
work. First, they are easy to maintain within a laboratory. They are small, develop rapidly, and have
simple dietary requirements. Second, there are literally thousands of different genetic stocks available
serving as 'tools' to examine physiological processes. Furthermore, D. melanogaster's genome has been
sequenced facilitating the identification of genes involved in physiological processes such as female
sperm storage. Third, the behavior and ecology of these flies have been examined. Thus, this animal
serves as a tractable system to understand the genetic basis of physiological mechanisms, and then to
relate that understanding to the whole animal. Fourth, genetic sequence similarities between D.
melanogaster and humans makes flies excellent models to study human diseases and conditions such as
cancer, neurodegenerative diseases, senescence and immune function. To explore this for yourself, try the
website Homophilia (http://superfly.ucsd.edu/homophila/) to identify Drosophila sequence homologues to
human genes associated with disease (such as BRCA1 which is associated with some breast and ovarian
cancers). In summary, we have many excellent reasons to use these hardy little insects to learn about
ourselves and the larger biological world!
The Drosophila melanogaster lifecycle
Drosophila melanogaster is a true fly (Order Diptera). Like all flies, Drosophila are holometabolous
meaning that they undergo a radical change in body form from the juvenile (larval or maggot) to the
adult (fly) stage.
Adult females lay eggs directly onto the surface of food. Recently mated females can lay >50 eggs per
day. A first instar larva emerges from the eggshell within 24 hours and feeds upon the media as well
as surface bacteria and fungi. The juvenile undergoes three larval molts. At the end of the third larval
instar, the larva leaves the food and crawls to a dry, undisturbed place. A pupa emerges after the third
instar molt. Within the puparium the larval body is dismantled and an adult body is assembled. Under
standard room conditions, a fly takes 10 – 12 days to develop. The emerging adult is teneral; it is
lightly pigmented and very soft. However, within minutes of emerging, the wings expand and the body
hardens and darkens. Males are able to mate within 6 – 8 hours after emergence. Both males and
females are fully mature within 3 days of eclosion. Adults live for approximately one month.
7
B. RESEARCH OBJECTIVES
The ultimate goal of this research is to elucidate mechanisms of female sperm storage in Drosophila
melanogaster by identifying genes affecting this process. The particular experimental approach includes
screening the fly genome to identify small chromosomal regions whose gene(s) influence female fertility,
then further characterizing the fertility mutants to identify sperm storage mutants. The experimental
question is: do females from particular chromosome deficiency lines show abnormal fertility in a pattern
consistent with failure to store sperm normally?
This research is considered basic research since it addresses questions about fundamental biological
processes. Specifically, the mechanisms by which female animals in general, and insects in particular,
determine the fate of the sperm they receive during mating. The ultimate goal of this research is to explain
how animals control the process of fertilization in order to better understand why female sperm storage is
such a common phenomenon. These types of questions are important in the study of animal development,
behavior, and evolution. This research also has more direct applications related to insect conservation and
pest control.
C. PROJECT DESCRIPTION
Over the next several weeks, you will be contributing to a specific type of genetic screen called a
deficiency screen. A deficiency is a type of genetic rearrangement resulting in the deletion of a number
of adjacent genes along a chromosome. Normally animals have two copies of every gene. One copy is
derived from each parent. Flies possessing a deficiency have only one copy of some genes (called
hemizygous) and two copies of all other genes (figure 1).
Chromosome containing
balancers
Chromosome with deficiency regions
(shown slightly shorter in length)
Figure 1: Cartoon of two homologous chromosomes. The one on the top, derived from one
parent, contains the full complement of genes. The light gray bars represent a region containing
genes present on one chromosome, but not the other (homologous) chromosome. The
chromosome on the bottom, derived from the other parent, contains a deficiency and is therefore
missing a number (usually <100) of genes normally present at this location on the chromosome.
Deficiency screens are used to identify genes that, when present in only a single copy, are insufficient to
direct normal characteristic expression (St. Johnston 2002). Animals showing this condition are called
haploinsufficient. For example, if normal sperm storage organ development requires two copies of gene
sso, then a female from the deficiency strain missing one copy of gene sso is predicted to have malformed
sperm storage organs and store a different quantity of sperm than the controls. Each deficiency line (aka a
stock) contains a deficiency in a distinct location within their genome. By screening all available
deficiency stocks, one screens most of the genome.
You might be wondering what protects the deficiency: what keeps the genome from repairing this missing
region and the flies possessing it viable? The answer is that another type of genetic rearrangement, called
a balancer, exists on the homologous chromosome. The most frequently used balancers have three
properties making them a useful tool for Drosophila geneticists (Greenspan 1997):
1. They are associated with a marker, an allele for a dominant phenotypic trait. That way, you
always know whether or not the balancer is present.
2. Balancers contain alleles that are lethal recessives – sometimes the very markers identified in #1.
Flies homozygous for the balancer die early in their development. Larvae or adults carrying the
marker must therefore also carry the genetic anomaly on the corresponding chromosome.
8
3. They prevent genetic exchange between the homologous chromosomes along the length of the
deficiency region. This maintains the integrity of the deficiency region. Thus, by selecting the
appropriate balancer, one can protect a specific anomaly on the corresponding chromosome.
What this looks like in terms of what you will be doing in this experiment is described below.
II. EXPERIMENTAL METHODS
In order to examine whether females with a particular deficiency have abnormal fertility, it is important to
define what constitutes normal fertility. Thus, it is critical to define an appropriate control. Comparing
deficiency lines to each other or to the wild-type, Oregon-R, strain is not a very accurate control.
Deficiency lines composing the third chromosome ‘kit’ were created from different fly lines in many
different labs (via different methods…). Therefore, these lines differ genetically in ways unrelated to the
deficiencies themselves. We say that they have different genetic backgrounds. Therefore, we might
expect different fly lines to have slightly different physiologies and behaviors since they do not share the
same alleles. In order to decrease genetic heterogeneity among the different deficiency lines and create an
internal positive control for the experiment, males from each deficiency line are crossed to females from
the wild-type line (P). The female progeny from this cross (F1s) will are the females whose fertility you
will examine and compare, called experimental females. You will be conducting single pair matings
between experimental females (deficiency and control) and wild-type males, then collecting and
quantifying the progeny from each mated female.
To generate experimental flies, ~20 Oregon-R (symbolized ++) females were crossed with ~20 males
containing a specific deficiency on the third chromosome ‘over’ a balancer on the homologous
chromosome. F1 progeny of this cross either possess two copies of every gene (positive control) or are
hemizygous for a small number of genes (deficiency). These two progeny genotypes can be distinguished
phenotypically because the flies with the balancer express a mutant phenotype. This is represented
schematically below (figure 2). You will use specific phenotypic markers to identify which flies are wildtype, which contain the balancer and which contain the deficiency.
Generation
Female
Parental (P)
+/+
Male
x
Deficiency/Balancer
The balancer contains either stubble or
serrate as a dominant marker
F1
Deficiency)/+
Balancer/+
hemizygous for a number of genes
(deficiency group)
two copies of every gene
(control group)
Figure 2: The method of generating experimental deficiency and control females by crossing
males from the deficiency line with wild-type females.
A. AN EXERCISE TO PRACTICE DISTINGUISHING FLY GENOTYPES BY THEIR PHENOTYPES
You have been provided with a sample of the F1 progeny (cross described above) that will be used in your
experiment. You will be told which dominant allele is carried by the balancer. Carefully observe some of
the flies. In the space below describe the mutant phenotype and compare it to the wild type phenotype.
Sort your flies into two groups: those that would compose the deficiency treatment and those that would
compose the control treatment. To do this, think about how the phenotype reflects the fly genotype.
# flies in the deficiency treatment:
# flies in the control treatment:
9
B. AN EXERCISE TO PRACTICE TRANSFERRING FLIES
This research project requires the selection and transfer of individual flies. This activity is intended to
help you develop and practice skills transferring individual flies and establishing single pair matings.
Individual flies are collected and moved most easily using an aspirator (figure 3). You will make your
own aspirator by wrapping a small piece of fine nylon mesh over the large end of a clipped 200 µl
micropipette tip. Insert this end into one end of the provided plastic tubing. Into the other end of the
tubing insert a clipped 1 ml pipette tip. Label the tube clearly so you do not inadvertently “share” tubes
with each other. By gently inhaling through the pipette tip, you can collect an individual fly and retain it
within the 200 µl tip. To keep the fly in the aspirator, you will need to inhale continuously or put your
finger over the open end of the pipette tip. Talking, laughing and sneezing result in flies being shot out of
the tube at a high speed and are generally impossible to recover.
Figure 3: Aspirator ‘anatomy’. From left to right, the 200 µl micropipette tip with a piece of
mesh at its largest end is inserted into a length of plastic tubing. Into the other end of the tube is
inserted a 1 ml tip. By gently inhaling through the larger tip, you can collect a single fly to
transfer elsewhere.
To collect a single fly from a culture containing several flies, tip the vial so the bottom containing the
media is towards the ceiling. Flies are negatively gravitropic and will move toward the ceiling (‘bottom’
of the upside-down vial). Loosen the cotton plug, but do not remove it completely and slide the aspirator
tip into the vial. Pick a fly and inhaling gently. Carefully re-stopper the vial. The fly can be released into
its new vial by gently breathing out through the tube.
* Transfer a single male to each sample vial.
* Transfer a female into each of vial containing a single male. Observe how long it takes until
they are all mating. It is also interesting to watch them court (description below).
Drosophila males perform a series of
stereotyped courtship behaviors before
copulation occurs (figure 4). The male
approaches the female and orients towards
her, tapping her with his foreleg. He
follows her, vibrating his wing to produce a
courtship song. Then, he goes toward her
posterior and licks her genitalia. Finally, he
attempts to copulate by mounting her.
Normally, unmated females mate readily,
but previously mated females are generally
unreceptive to courting males for some time
after mating. Females that have previously
mated will actively reject male courtship by
fluttering their wings, depressing or
elevating their abdomen, running away,
falling off the side of the vial, kicking and
extruding their ovipositor (figure 4).
Figure 4. The sequence of D. melanogaster courtship
behaviors. Arrows represent sensory information exchanged
(+ stimulatory; - inhibitory signals). From Greenspan &
Ferveur 2000.
10
C. FERTILITY ASSAY
Once you feel comfortable transferring flies, you are ready to start the matings!
1. Make up 60 vials with instant media following the directions in Appendix A.
2. Transfer a wild type, Oregon-R male to each vial.
3. Transfer a female to each vial. Immediately label the vial so you know what genotype the female is
(e.g. Df or Bal) and lay the vial on its side where it will not be disturbed. At this point it becomes critical
that you do not bump the table or otherwise disturb the flies.
4. Continue to set up vials containing a single male and a single female fly. Set up a total of 30 vials with
females from the deficiency treatment and another 30 with females from the control (balancer) treatment.
5. Observe each vial at least every 10 minutes and note when you see the flies mating. When a pair
mates, label the vial with a unique number corresponding to the order in which the mating occurred
(beginning with 1, 2, 3, and so on) and record the appropriate information on the data sheet provided
(Appendix C) for your experimental line.
6. Use your aspirator to remove the male from the vial within 1 h of the end of mating and dispose of him.
There should now be a single female in a vial of fresh media. Make certain that each vial is securely
plugged with the cotton ball. Flies are capable of squeezing through remarkably small spaces!
7 DAYS LATER:
(input date)
7. At the next laboratory period, transfer each female to a new vial. Label the vial so you know the female
identity (genotype and ID number) and that it is the second collection week (e.g. Df14.2, Bal3.2). Record
any dead females on your data sheet.
14 DAYS LATER:
(input date)
8. Remove and dispose the female.
17 DAYS LATER:
(input date)
9. Freeze the vials containing progeny from the first week’s collection – and only those vials!
24 DAYS LATER:
(input date)
10. Freeze the vials containing progeny from the second week’s collection.
11
III. INTERPRETING PATTERNS IN FEMALE FERTILITY
A. BACKGROUND
The ultimate objective of this screen is to identify genes that are associated with a female’s ability to
manage sperm received during mating. Directly observing the quantity of viable sperm in the storage
organs over time would take a tremendous amount of time and resources (i.e. many students for many
hours for many days). To increase the throughput of the screen, you will be examining sperm storage by
observing patterns in offspring production over time.
The developmental path from the inception of an oogonium to the formation of a reproductively capable
adult (or even a pupa) consists of multiple, interdependent processes. Each of which is required in order
for the next one to occur. Biologists define these processes and then study them to better understand
organismal development and life history. On the female side, it begins with the initial division of a
number of stem cells to produce oocyte precursor cells. These cells further develop during oogenesis to
eventually produce oocytes, or eggs. The rate of gamete production (oogenesis) is dependent on the
female genotype and environmental factors such as diet quality and quantity. After copulating with a
male, the female stores sperm in her reproductive tract where they undergo changes necessary to
transition from ‘movers’ – cells capable of translocating – to ‘fertilizers’ – cells prepared to fertilize an
oocyte. The union of oocyte and sperm is coordinated and results in fertilization (i.e. syngamy) when the
sperm enters the oocyte, the oocyte completes meiosis and a diploid zygote is formed. Females must
deposit the embryo in a suitable habitat and embryogenesis must occur normally for the first instar larva
to emerge successfully from egg’s chorion. This larva must then proceed through the developmental
stages normally (grow, molt, and metamorphose), compete successfully with other larvae for space and
resources, and avoid infection by microbes in its environment. Clearly, development is a complex
process requiring multiple physiological events to ‘go right’ in order for development to proceed
normally.
Given all of these complex interactions between cells within the organism and between the organism and
its environment, there is a lot that can go wrong. It is not the case that every stem cell division within the
female’s ovariole results in a breeding adult. In order to identify the developmental processes that have
the greatest impact on the probability of surviving to become a breeding adult, it is helpful to define a
series of episodes encompassing a collection of processes during which selection acts. The episodes listed
below define portions of the life-cycle (Endler, 1986):
 Sexual: includes competition for mates, preference among potential mates and mating success
 Gametic: meiotic drive, sperm competition and sperm preference
 Fecundity: the rate of gamete production and the rate of zygote production (also referred to as
fertility)
 Viability: survival of embryos, survival of other life stages (larval, pupal and adult),
developmental rate and age at first reproduction
B. QUESTIONS FOR DISCUSSION
o Into which stage should female sperm storage be categorized?
o When you are counting pupae to assess the number and quality of sperm stored within the
female, what other stages are confounded with the one including female sperm storage?
o What changes in the number of offspring (measured as pupal cases) over time would be
consistent with a defect in female sperm storage? In other words, what patterns in
progeny production would reflect a mother’s failure to store sperm normally?
12
IV. SOURCES OF EXPERIMENTAL VARIATION
A. SOURCES OF VARIATION
When scientists record data, variation seems to be the rule. Sometimes this variation reflects differences
in the subject(s) or object(s) being measured (called subject error1). This variation can be of critical
biological importance – differences among individuals in a population are the fodder upon which natural
selection acts. Differences between treatments or relationships between variables can only be detected if
the measured units differ (i.e. vary) from each other. Other times, the variation is due not to the entity
being measured, but to error introduced by the experimenter or random fluctuations in the subject being
measured (called random error). Identifying the general source of measurement variation is significant:
variation attributable to the measured subject is of potential biological importance while variation due to
the way the subject is measured or chance fluctuations (i.e. random variation) is unlikely to be
biologically meaningful.
Example: A scientist is interested in exploring the effect of diet quality on female fly
fecundity. She hypothesizes that, since female insects are often protein limited and that
protein is a large component of eggs, diets supplemented with protein-rich yeast will
result in higher average female fecundity than diets lacking yeast. To test this
hypothesis, the scientist sets up two treatments: recently mated females are randomly
assigned to vials containing media supplemented with yeast (experimental) or vials
containing normal media (control). She then counts the daily number of eggs each
female fly lays over a two-week time span. Not surprisingly, the total number of eggs
produced by each fly is different. At this point, the scientist doesn’t know the cause(s)
of these fecundity differences.
A consequence arising from failure to distinguish between subject and random error is that the recorded
variation can appear to be greater than it actually is. Experimenter-introduced variation can even be larger
in magnitude than the biological variation resulting in an inability to see the biological variation through
the introduced variation. In other words, one cannot see the signal because of the noise. When it comes
time to analyze your results statistically, you might fail to detect a hypothesized difference or relationship
(i.e. reject your null hypothesis) not because the difference or relationship does not exist, but because you
are unable to detect the effect. This failure to reject a null hypothesis when, in fact, it truly should be
rejected is called a Type II statistical error. While this sounds ominous, no one will cart you away for
committing this error, but the tragedy lies in the consequence: you worked extremely hard to conduct the
experiment, collect the data and analyze the results and you are left unable to say with statistical
confidence that your experimental hypothesis received any support!
We can, to a large extent, take matters into our own hands – literally. While random error is due to
variation introduced by both the experimenter and unexplained fluctuations, removing your contribution
(i.e. experimenter) to the variation can have significant (both biological and statistical) results. Before we
even begin our experiment we can think carefully to identify uncontrolled, and therefore potentially
confounding, variables. This requires some knowledge of the system being studied. You must understand
what you are doing well enough to predict what could influence the outcome. It is important to list these
variables, evaluate their potential effect on the variable(s) you plan to measure, and then plan how you
will control for their effect. Take some time to think about variables that might affect your project and
what actions you can take to control for their effects.
Example: Our scientist thinks about her experiment before she ever mated a single
female. She knows that females are not fully sexually mature until approximately 72 h
post-eclosion and that as females age they tend to produce fewer eggs over time.
Therefore, our researcher made the astute decision to use females that are at least three
1
In this context, error means variation and not that something is incorrect.
13
days post-eclosion, but not more than 5 days post-eclosion. Furthermore, to the extent
that it is possible, all of the females are within 48 h of age from each other and females
of each age are represented in each experimental treatment group. Our scientist also
knows that eggs are small and that females like to lay eggs in crevices in with media.
Therefore, she practices counting eggs before she counts for her experiment and she
uses a microscope to be certain that she can see every egg that is deposited. Finally,
females lay more eggs in the morning than in the evening and eggs hatch
approximately 24 h after being laid, so our researcher plans out the experiment in
advance so she is certain to be able to count eggs at the same time each day.
B. MEASUREMENT ACCURACY & PRECISION
We can further improve our chances of distinguishing the biological variation from the random variation
(thereby increasing our confidence that observed differences reflect real differences in the entity being
measured) by deliberately maximizing the accuracy and precision of our measurements. Accuracy
describes how close a measurement is to the actual characteristic being observed. In conceptual terms; if
you shot an arrow at a target, accuracy would describe how close the arrow was to the bull’s eye. In
biological terms; if you measure the mass of several flies to the nearest milligram (mg), accuracy would
describe how close the measurements you made (2 mg, 2 mg, 3 mg, etc.) were to the true mass of the flies
(which might be 1.98 mg, 2.30 mg, and 2.61 mg in reality). Precision describes the consistency of
repeated measurements. Again, in conceptual terms, if several arrows are repeatedly shot at the target,
precision measures how close (clustered) the arrows are to each other. In biological terms, if you
measure the mass of a single fly repeatedly, precision describes the consistency of your repeated
measurements (for example, 2 mg the first time you measure, 3 mg the second time, and 2 mg the third
time).
Example: Our scientist does a couple of things to improve her accuracy: she uses the
microscope to have a clear, magnified view of the media surface. She is careful to not add
too much yeast and uses vials with smooth surfaces to increase the chances of seeing
every egg that is laid. She divides the media surface into quadrants and she counts each
one thereby decreasing the chances that she will accidentally miss or double-count an
egg-containing area. In order to improve her precision, she counts each quadrant three
times noting the number of eggs counted each time. She feels confident that her counts
are reasonably precise if she arrives at the same number two out of three times.
While high precision in our measurements gives us confidence that our measurements are accurate
(although we never know with absolute certainty…), how do we know that our measurements are precise?
Precision can be quantified and our measurements accepted as sufficiently precise when variation in our
repeated measurements is at or below some predetermined quantity. Precision can be quantified in a
number of ways including measuring the correlation between pairs of measurements, comparing the
variation between repeated measurements of the same subject and measurements of distinct but similar
subjects (called repeatability), and quantifying the variation among repeated measurements standardized
by the mean measurement value (coefficient of variation). It is this last measurement that we will use to
have some confidence that your measurements reflect a biological event rather than the effects of changes
in the room temperature (or your state of awareness!) on a given day. The coefficient of variation is
calculated as:
Coefficient of variation (CV or V) =
standard deviation (s)  100
sample mean
It is generally expressed as a percent. The higher the CV, the greater are the differences between
measurements. In order to calculate a meaningful coefficient of variation, you will need to make three
measurements of the same entity. Additional measurements can increase the precision of your
measurement, which is reflected in a lower the coefficient of variation. So, what will you measure?
14
C. AN EXERCISE TO MEASURE THE PRECISION OF OFFSPRING COUNTS
In your project, you are indirectly observing female sperm storage by counting the
number of pupal cases observed in vials housing the female for each of two
successive weeks. The pupa is a small (~3 mm) long tan tube with tapered ends
(figure 5). Generally, third instar larvae creep up the sides of the vial to pupate,
but sometimes they pupate right in the media itself where it is difficult to see
them. It is useful to hold the vial at an angle to see the pupae without glare from
overhead lights and then make a small mark with a Sharpie pen onto the plastic
over the pupal case. That way, you can decease the chances of missing an area
~3 mm
containing pupae or counting some pupae twice. Only count pupae, late third
instar larvae are excluded (although their presence should be noted on your Figure 5. A Drosophila
datasheet).
pupa. Image from NASA.
Before you begin, list all of the variables you can think of that might influence how many pupal cases you
count in the space provided below. Then, with a check () categorize each variable as one of the
following: controllable, uncontrollable but accountable, and uncontrollable & unknown.
controllable
uncontrollable &
accountable
uncontrollable &
unaccountable
Try to conduct your counts in such as way as to prevent the variables in the first category from
influencing your results and to carefully keep track of the variables in the second category. The variables
in the third category will not be addressed since they are unknown.
How can you be precise when you are counting pupae? You can methodically and meticulously search the
vial for each visible pupal case. You can also keep track of each pupa you count, to ensure that all are
accounted for only once. To gauge your precision, you will make repeated measurements of the vial and
calculate the coefficient of variation and then figure out how many re-counts are necessary to get a good
estimate of progeny production. To begin, make five counts of the same vial, then calculate the
coefficient of variation for the first three counts and compare that value to the value for all five counts.
CVthree counts =
CVfive counts =
Does the coefficient of variation improve (i.e. get smaller)? When you are done, we will talk as a group to
decide what coefficient of variation is considered acceptable and how many counts will be needed to meet
that standard. How could you improve the accuracy of your measurements? How could you improve the
precision of your measurements?
15
V. DATA MANAGEMENT & ANALYSIS
A. STATISTICS
Statistics, the numerical analysis of observations, are a critical component of biological research. They
enable scientists to characterize a collection of observations and to make inferences from their
observations to larger biological phenomena. Statistics are categorized into two major groups:
Descriptive statistics summarize a collection of observations and describe qualities of the collection such
as the central tendency of the data (mean, median, mode) and the amount of dispersion about the central
tendency (variance, interquartile range, standard error, coefficient of variation), and the shape of the
distribution (skew and kurtosis).
Inferential statistics are used to make inferences about a population. These statistical tests are used to
determine the likelihood an observed relationship or difference occurred by chance.
B. ANALYZING YOUR DATA
You have counted the number of progeny produced over two, week-long collection periods by females
hemizygous for a number of genes and control females. You will now use both descriptive statistics and
inferential statistics in order to describe, in general, how these two groups of females are similar or differ
in terms of progeny production (descriptive statistics) and whether any observed differences are likely to
reflect biological differences between the two female groups (inferential statistics).
While you are welcome to work with classmates on this section, each student is to complete the analysis
on his/her own. Partners may not submit a duplicated analysis.
Analysis proceeds in a series of steps:
Entering your data into a spreadsheet for statistical analysis
Open SigmaStat 3.5
Double click on column headings to add variable names
Row 1: FID (Deficiency female ID)
Row 2: Line (corresponds to data set column #2)
Row 3: Prog1Def (Number of pupal cases from week 1 deficiency females)
Row 4: Prog2Def (Number of pupal cases from week 2 deficiency females)
Row 5: FID (Control female identity)
Row 6: Prog1Con (Number of pupal cases from week 1 control females)
Row 7: Prog2Con (Number of pupal cases from week 2 control females)
Enter data into the spreadsheet (see example below)
Save the file as the deficiency stock number (e.g. 1145)
16
To calculate the change in progeny production over the two week test period, select ‘transforms’
from the toolbar, then select ‘quick transforms’ and ‘subtract’. Under ‘data for input’ select
‘Prog2Def’, then ‘Prog1Def’. Under ‘output’ select ‘first empty’. Rename this column ‘DiffDef’.
Repeat for the control females naming the new column (#9) ‘DiffCon’.
Exclude data for a female if she died or escaped during any portion of the two week period.
Would there be any other reasons to exclude data from females?
Inferential statistical analysis
You will conduct two Mann-Whitney Rank Sum tests. This test is a non-parametric alternative to
a t-test. Essentially, you are asking two statistical questions:
1) Do deficiency and control females differ in their change in progeny production over two
weeks?
2) Do deficiency and control females differ in the number of offspring produced the first
week after mating?
The null hypotheses (hypothesis of no difference or no effect) are, respectively, that:
1) No difference exists between the two groups of females in progeny production over time,
and
2) No difference exists between the two groups of females in offspring production the first
week after mating
The alternative hypothesis in each case is that there is a difference in progeny production between
deficiency females and control females. You will need to repeat the analysis twice, once for each
hypothesis. The steps are essentially the same. To conduct each analysis;
Select ‘statistics’ from the toolbar
Select ‘two groups’
Select ‘rank sum test’
Data format is ‘raw’
For ‘selected columns’ choose ‘DiffDef’ and ‘DiffCon’ and finish.
You will receive a report (example shown on the next page).
17
The resulting output states the probability that the observed difference is due to chance.
The probability value of interest is labeled “P(exact)”. If it is <0.05, then we reject our
null hypothesis of no difference and accept the alternative hypothesis that there is a real
(i.e. biological) difference between the two groups. In short, we say that the two groups
are significantly different from each other. If you get this, congratulations, you have
identified a candidate line! Repeat the analysis for the second question.
Save your output! Save the spreadsheet by the line number and specify ‘data’ (e.g.
1145data). Save the analysis by line number and specify ‘anal’ (e.g. 1145anal).
Create a figure representing the analyzed data: Making a box plot
Select ‘Graph’ and ‘Create a graph’ from the toolbar.
Select ‘Boxplot’ and ‘Next’
Select ‘Many Y’
Select each of the four columns: “ProgDef1”, “ProgDef2”, “ProgCon1”, and
“ProgCon2”, then ‘Finish’.
If all goes well, a figure will appear!
Now modify the image to add useful details such as: samples sizes, informative axis and
tick labels (both X and Y axes), and a figure caption that includes that statistical results
(see comments in section C below).
Print a hard copy of the spreadsheet, the analysis and the figure.
18
C. DESCRIBING THE RESULTS QUANTITATIVELY AND QUALITATIVELY
It is useful to describe the observed differences between the two female groups. Using information of the
median numbers of progeny produced in each of the four groups, write a brief description under the figure
you have printed. This description should address both the median difference in progeny production over
time between the two female groups as well as the median difference in week 1 progeny production
between the two female groups.
D. INTERPRET YOUR RESULTS IN A BIOLOGICAL CONTEXT
Do you have evidence that a gene contained within the deficiency region likely to have an effect on
female sperm storage? Why or why not?
19
VI. BIOINFORMATICS:
USING FLYBASE TO MAKE INFERENCES ABOUT GENE IDENTITY & FUNCTION
FlyBase is a web-based resource for many topics related to Drosophila genetics. FlyBase catalogues the
lines carrying particular alleles and published descriptions of their origin/discovery and characterization.
This week you will use the site to list the identified genes/loci within a deleted region for one of the third
chromosome deficiency candidate lines. Candidate lines are those that produce fewer progeny over a two
week time period when compared to their genetic controls. It will then be possible for you to review
publications describing the expression pattern(s) and function(s) of these genes. Finally, you will
summarize what is known and propose a molecular or physiological mechanism by which the hemizygous
condition resulted in aberrant progeny production in a particular deficiency line.
A. DEFINING DEFICIENCY BOUNDARIES
To identify the genes located within the deficiency region (and therefore in the hemizygous state), you
will need to identify the deficiency breakpoints. These breakpoints are identified cytologically or by
sequencing. Once you have identified the locations of the breakpoints, you can “crawl” along a map of
the chromosome identifying the genes present. To do this:
1. Go to FlyBase (http://flybase.org/)
2. Under ‘Data class’ select ‘stocks’
3. Enter your stock number (i.e. 1145) and ‘Search’
4. Select the hyperlink of the genotype associated with the stock (e.g. Dp(1;Y)B[S];
Df(2R)en30/SM5).
5. By the ‘FlyBase Genotype’, select the deficiency region only (e.g. Df(2R)en30).
6. The line ‘Deleted Segment’ refers to the cytological location of the deficiency break points.
Note both breakpoints (e.g. 48A3--48C8).
Your breakpoints:
7. Return to the site homepage and select the box ‘GBrowse’ from the top of the screen.
Accessed 25/11/2008
20
8. In the empty box to the right of ‘Advanced search: cytolocation’, input one of the breakpoint
positions. Under ‘Scoll/Zoom’ select ‘1 kb’ to limit the range that you are shown. Select ‘start’.
The window will update and you will be given the location, in numbered bases, of your
breakpoint (e.g. 2R:7490029). The 2R means that the breakpoint is on the right side of the second
chromosome. Which chromosome are you screening? Record the smaller number corresponding
to your breakpoint below and repeat the process for the other break point to define the region, in
basepairs, that is deleted in the deficiency.
Left (lower numbered) breakpoint:
Right (higher numbered) breakpoint:
9. Now, to increase the window you are screening, change the ‘Scroll/Zoom’ setting to 100 kb.
This means that you will be searching the genome in units of 100,000 bases at a time. Check that
you are still in the region of the first breakpoint, and you are ready to start listing genes!
B. IDENTIFYING GENES/LOCI WITHIN THE DEFICIENCY REGION
10. The overview box shows you your general location along the chromosome you ore screening.
Below this box, in the ‘Details’ box, are listed genes (blue bars) located in this region. In the
example provided below, note that genes toutatis (tou), Enigma (Egm) and CG9005 are located
within this region. If you scroll the mouse over this bar, additional information will be provided
including the full gene name, its identification number (called an accession number), and
information about its hypothesized function and/or biological role(s). See the example provided
below:
21
C. SUMMARIZING WHAT IS KNOWN ABOUT THE IDENTIFIED GENE
11. In the data sheet provided, record the following information:
 Gene name
 Accession number
 Cytological location (be certain it is within the deficiency region!)
 Molecular functions
 Biological processes
 Phenotypes
For tou, it would look like this:
Line
1145
Gene name
Accession #
Cyto Loc
Mol Fun
Biol. Proc.
Phenotypes
toutatis
CG10897
48A2-48A3
Binding:
Chromatin,
DNA,
Protein,
Phosphopantet
heine,
Zinc ion;
H+
transmembran
e transporter
activity
Chromatin
remodeling,
nervous
system
development
Scutellar
bristle,
Dorsocentral
bristle, wing,
wing vein L5.
Enigma
12. Repeat for all of the genes within the deficiency region. You can scroll along the genome by
selecting the
button to the right of the ‘Scroll/Zoom’ box. Genes may overlap, so be certain to
check the window all the way down to the ‘mRNA’ region (see example below). You do not need
to list sno:RNAs. For deficiency line 1145, this includes 36 different genes!
13. If you wish to learn more about any of the genes, double click on the corresponding blue bar
and you will be redirected to a window that lists publications describing that gene’s identification
and characterization. Explore!
22
FINAL ASSIGNMENT
The report of this project is worth 30 points. It will consist of the following:
 Completed data sheet including raw data of progeny counts
 A hard copy of the data table used for analysis and the statistical analysis
 A figure with caption presenting the progeny counts each weekly interval for each group of
experimental females
 A table containing the following information: fly line tested and a line for each gene within the
deficiency region. For each gene provide: its name, accession number, cytolocation, molecular
functions, the biological processes in which it may be involved, and any described phenotypes. A
model of the information needed in provided in section VI C 11 of this handout
 A carefully composed paragraph in which you provide the following information for one gene of
interest:
o What type of protein is coded for by this gene? Does this protein remain within the cell or
is it secreted? If it is intracellular, does it appear to be a transcription factor, an enzyme, a
structural protein or something else? If it is secreted, what is it predicted to do?
o
What role(s) does this gene appear to have in Drosophila? What phenotype(s) do
individuals missing or with altered form(s) of the gene have? Is there information
whether the gene is dominant or recessive?
o
Using the above information, generate a hypothesis explaining how this gene might affect
female Drosophila fertility.
LITERATURE CITED
Bloch Qazi, MC., Heifetz Y. and Wolfner MF. 2003. The developments between gametogenesis & fertilization:
ovulation and female sperm storage in Drosophila melanogaster. Developmental Biology 256, 195-211.
Eberhard, WG. 1996. Female Control: Sexual Selection by Cryptic Female Choice. Princeton University Press,
Princeton, NJ.
Endler, JA. 1986. Natural Selection in the Wild. Monographs in Population Biology 21. Princeton: Princeton
University Press.
Greenspan, R. J. 1997. Fly Pushing: The Theory and Practice of Drosophila Genetics. Woodbury, NY: Cold Spring
Harbor Laboratory Press.
Greenspan, R. J. and J.-F. Ferveur. 2000. Courtship in Drosophila. Annual Review of Genetics 34: 205-232.
O’Grady, PM. 2003. Drosophila melanogaster. In Resh, VH and Cardé RT (eds). Encyclopedia of Insects. New
York: Academic Press.
Ridley, M. 1988. Mating frequency and fecundity in insects. Biological Reviews 63, 509-549.
Snook, RR. and Hosken, DJ. 2004. Sperm death and dumping in Drosophila. Nature 428, 939 - 941.
St. Johnston, D. 2002. The art and design of genetic screens: Drosophila melanogaster. Nature Reviews 3: 176-188.
ACKNOWELDGEMENTS
Students of 2005 Directed Research class (BIO 396), 2008 Entomology class (BIO376) and research students at
Gustavus Adolphus College provided thoughtful and constructive feedback. Dr. Eric Cole suggested that Starbuck’s
bottles made good fly culturing bottles. Dr. Frank Avila and two anonymous reviewers provided feedback on earlier
versions of this exercise.
23
APPENDIX A: PREPARING INSTANT MEDIA
This is what you will use for all of your fly needs. Make it up immediately prior to use. It will not be good
more than one day after it has been made up. Measure 1 teaspoon of dry instant media into a single plastic
vial. Use a funnel to minimize spillage. Add 5 ml of DI water to the dry media being careful to wet the
entire surface. Sprinkle a few (<10) grains of dried Brewers’ yeast onto the surface and immediately
stopper with a cotton plug. Be extremely careful to exclude unwanted flies from the vials!
APPENDIX B: STATISTICAL DIRECTIONS FOR USING SPSS FOR STATISTICAL
ANALYSES
Open SPSS 15.0 for Windows (licensed to GAC)
Select ‘type in data”
Designate variable names in Variable View window
Row 1: FID (Female ID
Row 2: Line (corresponds to data set column #2)
Row 3: Genotype
Row 4: Prog1 (Number of pupal cases from week 1)
Row 5: Prog2 (Number of pupal cases from week 1)
Row 6: Diff (Prog 2- Prog1)
Save the file with the stock number (e.g. 1145)
Return to the Data View window
Enter data into the spread sheet.
To add data to column 6, Diff, calculate the difference in progeny production between
week 1 and week 2. Select ‘transform’ from the toolbar, then select ‘compute’. The target
variable is the title for column 6, “Diff”. For ‘numeric expression’ type “Prog2-Prog1”.
Select ‘ok’.
Exclude data for a female if she died or escaped during any portion of the two week
period. Would there be any other reasons to exclude data from females?
Run inferential statistical analysis
You will conduct two Mann-Whitney U tests. This test is a non-parametric alternative to a t-test.
Essentially, you are asking two statistical questions:
3) Do deficiency and control females differ in the number of offspring produced the first
week after mating?
4) Do deficiency and control females differ in their change in progeny production over two
weeks?
The null hypotheses (hypothesis of no difference or no effect) are, respectively, that:
3) there is no difference between the two groups of females in offspring production the first
week after mating, and
4) there is no difference between the two groups of females in progeny production over time
The alternative hypothesis in each case is that there is a difference in progeny production between
deficiency females and control females. You will need to repeat the analysis twice, once for each
hypothesis. The steps are essentially the same, only the dependent variable selected will differ. To
conduct each analysis;
Select ‘analyze’ from the toolbar
Select ‘nonparametric tests’
24
Select ‘2-independent samples’
Test variable: select the dependent variable: either ‘Diff’
or “Prog1”.
Grouping variable: select the independent variable (‘Genotype’).
Define the groups by typing in a ‘1’ (hemizygous) and ‘2’ (control)
Run
Sample Output:
Ranks
Diff
Genotype
1
2
Total
N
33
32
65
Mean Rank
39.47
26.33
Test Statisticsa
Sum of Ranks
1302.50
842.50
Mann-Whitney U
Wilcoxon W
Z
As ymp. Sig. (2-tailed)
Diff
314.500
842.500
-2.802
.005
a. Grouping Variable: Genotype
The resulting output will give you a probability value that the observed difference is due to
chance. The probability value of interest is labeled “Asymp. Sig. (2-tailed)” If it is <0.05,
then the chance that the difference observed between hemizygous and control female groups
is sufficiently unlikely (< 5% chance) that we reject our null hypothesis of no difference and
accept the alternative hypothesis that there is a real (i.e. biological) difference between the
two groups. In short, we say that the two groups are statistically significantly different from
each other. If you get this, congratulations, you have identified a candidate line!
Save your output! Save the spreadsheet by the line number and specify ‘data’ (e.g.
1145data). Save the analysis by line number and specify ‘anal’ (e.g. 1145anal).
3. Create a figure representing the analyzed data: Make a box plot.
Select Graphs from the toolbar, then ‘Interactive’, and finally ‘Boxplot’
Copy and paste your data to create four columns corresponding to the following
information: df – week #1, df – week #2, bal – week #1, and bal – week #2.
Select ‘Graph’, then ‘Create graph’
Select ‘Box Plot’, then ‘next’
Select ‘Vertical Box Plot’, then ‘next’
Select ‘Many Y’, then ‘next’
Select df-week #1 (column 1) for “Data for Box 1”. Continue until all four columns of
data have been selected.
Select ‘finish’
An image should appear!
Now modify the image to add useful details. Add data to reflect the sample size of each
bar. Label the axes with variable names and clearly label treatment groups below each
bar.
25
APPENDIX C: DEFICIENCY SCREEN DATA SHEET
Fly line:
Genotype:
Males collected:
n=
date:
Oregon-R females collected:
n=
date:
n=
date:
n=
date:
n=
date:
n=
date:
Date of cross:
Progeny collected:
Experimental matings:Df/wt ♀ x wt ♂
Date experimental matings conducted:
Number pairs established:
# mating:
ID
# progeny
week 1
# progeny
week 2
Notes
.1
.2
.3
.4
.5
.6
.7
.8
.9
.10
.11
.12
.13
.14
.15
.16
.17
.18
.19
.20
26
ID
#P1
#P2
Notes
.21
.22
.23
.24
.25
.26
.27
.28
.29
.30
.31
.32
.33
.34
.35
.36
.37
.38
.39
.40
.41
.42
.43
.44
.45
.46
.47
.48
.49
.50
.51
.52
.53
.54
.55
.56
.57
.58
.59
.60
.61
.62
.63
.64
27