P6) Drosophila Breeding
Lab 6 Photographs
Lab 6 Photographs
Fly Types and Combinations
We were given the option of 4 types of Drosophila to breed:
1.
2.
3.
4.
Ebony vestigials
Wild type
Scarlet eyes
White eyes
After research, I found that ebony fruit flies produce an excess of melanin, and thus appear
darker in body than wild type flies. 'Vestigial' refers to shortened wings in mutated
individuals. Scarlet eyed flies produce only red drosopterin pigments in the eye (instead of
the drosopterins and brown xanthommatin pigments expressed in wild type flies) (Howells
and Ryall, 1975; Ewart and Howells, 1998). White eyed flies produce no pigment in the eye,
thus the lack of colour.
Certain eye colours, such as white eyes, are linked to the sex chromosomes. Drosophila sex
chromosomes are XX in females, and XY in males. These eye colours is carried on the X
chromosome. This means in this circumstance that we would expect male fruit fly offspring
to inherit their eye colour from their mother (as the Y was supplied by the father), and that a
male will display the sex-linked colour it inherited. However, if a male fruit fly inherited
white eyes on its X chromosome, but also inherited the autosomal scarlet gene, it would
display scarlet eyes, complicating findings.
We would therefore expect a white-eyed female (with two copies of the white-eye gene as it
is recessive) to produce only white-eyed male offspring, and only wild type female offspring
when bred to a red-eyed (wild type) male. All female offspring of this pairing would be
heterozygous for both the white and red-eyed genes though, so further breeding with a whiteeyed male from the same generation could reproduce either gender offspring with either
colour eyes.
For our breeding experiment, I needed to consider what I wanted to find. Ebony, vestigial
wings and scarlet eyes are caused by autosomal genes, so are not sex linked. These would
show mendelian inheritance, and as all are recessive, the offspring would all be
phenotypically wild type. This would confirm the recessive status of these genes. Breeding
different eye-colours of fly would confirm the sex linked nature of those genes. Breeding a
male white-eyed fly, for example, to an female ebony vestigial fly, would produce a first
generation of offspring that appear to be wild type, but that carry recessive genes.
Breeding sex linked genes seems to be the more interesting choice, as the interaction of these
is not simply medelian inheritance. I would probably breed female scarlet-eyed flies to male
white-eyed fruit flies to investigate the dominance relationship in those genes. It would be
interesting to see if the presence of a white-eyed gene reduces the vividity of scarlet eyes in
offspring that carry both genes. The second breeding experiment would then be to either
breed a male scarlet-eyed fly to a female white-eyed fly to see if eye-colour ratio of male
versus female offspring would reverse, or to breed a white eyed fly to a wild type eye-colour
fly (although this fly could be an ebony vestigial, as these traits don't affect eye-colour, and
simultaneously we could show that the ebony and vestigial genes were recessive and
autosomal) to observe the dominance pattern of abnormal types against wild type.
Fly Sexing
I decided to find out how to sex flies, as in the sex-linked breeding experiments listed above,
this would be very important to find significant results. I found that female Drosophila have
two extra segments in their abdomens, and so appear more banded than males. They also
have more pointed and larger abdomens when compared to males, which have smaller,
rounded, more bristled abdomens that appear black at the end. I found a fruit fly sexing game
that I have linked below, and tried to test out this knowledge. Sexing the game's flies was
quite easy, so I hope that it will be relatively easy for the experiment.
Fruit Fly Sexing Game
Fruit Fly Sexing Game
Drosophila Sexing
Last modified on 09 May, 2013 2:10 PM
Although I originally thought that sexing would be easy, the differences between sexes,
particularly between banding on sexes, is not always obvious. Males are generally smaller
than females but this is not always true, and I didn't have the microscope set at a high enough
magnification to look for sex combs on males' legs, which would have been more reliable.
The most reliable way, according to this website on sexing drosophila, is to look for the
genetalia of the flies. I believe that on repeating this experiment a further time (see below for
second set of breedings), I would use smaller, more sensitive tweezers for better positioning
of flies, and look at the genitals of the flies along with the banding and possible presence of
sex combs.
In experiment 7 we appeared to have two flies of the same gender, and no breeding had taken
place in this tube. More experience and looking at genetalia would prevent errors such as
this. Below are three photographs of white eyed flies. The first appears male, the second
female, and the last was too difficult to sex as it was newly hatched.
male white eyes
female white eyes
newly hatched white eye fly
After the Breeding Experiment
As competition for certain types of flies was high, and as some flies
appeared to be injured, different combinations of phenotypes were bred.
These were:
Vial 1: Scarlet-eyed female / 2 WT males
Vial 2: White-eyed female / Ebony vestigial (WT eyes) male
The resulting expected F1 generations will be shown below in a punnet
square. However, as these flies will be breeding for 5 weeks, F2
generations will be predominant by the time results are taken.
Vial 1 investigates dominance of eye colour. Wild type is usually
dominant, so I expect heterozygotes to be wild type-eyed phenotypically.
Vial 1 F1 and F2 Eye Colour Cross
Scarlet eyes against WT eyes is simple to plot.
WT WT
s WTs WTs
s WTs WTs
The F1 generation would be entirely made up of heterozygote flies that
were phenotypically wild type.
WT
s
WT WTWT WTs
s
WTs
ss
In the F2 generation, 75% of offspring would appear to be wild type, with
25% being homozygous. 25% would be scarlet-eyed.
If we had 20 offspring in this generation, 15 would be wild type, and 5
scarlet eyed. However, due to chance it is likely that the proportions may
differ.
Vial 2 investigates the inheritance of white eyes, ebony colour, and
vestigial wings. Any of these traits could been seen in individuals of the
F2 generation, perhaps seperately if the genes are not linked.
Vial 2 F1 and F2 Eye Colour Cross
XWT
Y
Xw XwXWT XwY
Xw XwXWT XwY
This shows that the F1 generation would have heterozygote WT females
that carry white eyes, and white eyed males. All offspring would carry
ebony and vestigial.
Xw,E,V
Xw,E,v
Xw,e,V
Xw,e,v
Y,E,V
Y,E,v
Y,e,V
Y,e,v
XWT, XWTXw, XWTXw, XWTXw, XWTXw, XWTY, XWTY, XWTY, XWTY,
E,V EE,VV
EE,Vv
Ee,VV
Ee,Vv
EE,VV EE,Vv Ee,VV Ee,Vv
XWT, XWTXw, XWTXw, XWTXw, XWTXw, XWTY, XWTY, XWTY, XWTY,
E,v
EE,Vv
EE,vv
Ee,Vv
Ee,vv
EE,Vv
EE,vv
Ee,Vv
Ee,vv
XWT, XWTXw, XWTXw, XWTXw, XWTXw, XWTY, XWTY, XWTY,e XWTY,
e,V
Ee,VV
Ee,Vv
ee,VV
ee,Vv
Ee,VV Ee,Vv
e,VV
ee,Vv
XWT, XWTXw, XWTXw, XWTXw, XWTXw, XWTY, XWTY, XWTY,e XWTY,
e,v
Ee,Vv
Ee,vv
ee,Vv
ee,vv
Ee,Vv
Ee,vv
e,Vv
ee,vv
Xw,E, XwXw,E XwXw,E XwXw,E XwXw,E XwY,EE XwY,EE XwY,Ee, XwY,Ee
V
E,VV
E,Vv
e,VV
e,Vv
,VV
,Vv
VV
,Vv
Xw,E, XwXw,E XwXw,E XwXw,E XwXw,E XwY,EE XwY,EE XwY,Ee, XwY,Ee
v
E,Vv
E,vv
e,Vv
e,vv
,Vv
,vv
Vv
,vv
Xw,e, XwXw,Ee XwXw,E XwXw,ee XwXw,ee XwY,Ee, XwY,Ee XwY,ee, XwY,ee,
V
,VV
e,Vv
,VV
,Vv
VV
,Vv
VV
Vv
Xw,e, XwXw,Ee XwXw,E XwXw,ee XwXw,ee XwY,Ee, XwY,Ee XwY,ee, XwY,ee,
v
,Vv
e,vv
,Vv
,vv
Vv
,vv
Vv
vv
The F2 generation was considerably more time consuming to plot out (see
the second version below). Assuming these genes are not linked, the
offspring would be:
 14.06%
WT eyed and bodied females, and 14.06% males of this
phenotype
 4.69% WT eyed females with ebony bodies, and 4.69% males of
this phenotype
 4.69% WT eyed females with vestigial wings, and 4.69% males of
this phenotype
 1.56% WT eyed females that are ebony vestigial, and 1.56% males
of this phenotype
 14.06% white eyed females with WT bodies, and 14.06% males of
this phenotype
 4.69% white eyed females with ebony bodies, and 4.69% males of
this phenotype
 4.69% white eyed females with vestigial wings, and 4.69% males of
this phenotype
 1.56 white eyed females that are ebony vestigial, and 1.56 males of
this phenotype
As this will be the generation present when we check the results, this will be useful. If there
were around 20 offspring, 5-6 would be totally wild type, and 5-6 would be white eyed with
WT bodies, around 2 could be WT with ebony bodies, another 2 white eyed with ebony
bodies, around 2 WT with vestigial wings, another 2 white eyes with vestigial wings, and
there could be a WT eyed ebony vestigial and/or a white eyed ebony vestigial. This is likely
not to be the case as above due to chance, and the possibility that wild type individuals could
be healthier.
I practiced punnet squares on the following website: Punnett Square
Calculator
Punnett square
A cross of flies from F1, both heterozygote for ebony and vestigial, with the male being white
eyed and the female WT eyed carrying white eyes.
X= Wild type eyes
x= White eyes
E= Non ebony
e= Ebony
V= Non Vestigial
v= Vestigial
White eyes had to be shortened to a lowercase x as the punnett square programme did not
allow multiple letters, ie. Xw. Made using the punnett square calculator.
Results
After 5 weeks we were able to see if our flies had bred. We had not given
any specific instructions, so we knew the flies we collected should have
been F2 flies.
Vial 1
In this vial, we seemed to have a mixture of scarlet and wild type
offspring as expected, although it is difficult to differentiate the two eye
colours, as they are both shades of red. It is possible that heterozygotes
may be somewhat inbetween wild type and scarlet expression. Our rough
count of individuals appeared to show that numbers of scarlet males and
females, and wild type males and females were about the same.
Vial 2
This vial was populated purely by white eyed flies, contrary to predictions.
Second Set of Results
Vial 2
This vial had the adult flies removed, and was left a further week. The flies that we counted
were likely all of, or mostly of, the F3 generation. However, all were white again. There were
14 males and 14 females, which is a perfect statistic for expected genders, although there was
one fly that had just hatched and so could not be sexed.
Vials 3 & 4
In these vials we crossed:
Vial 3: 2 White eyed females and WT male
Vial 4: White eyed female and 2 WT males
This would have just shown if numbers of offspring differed. However, neither breeding was
successful. In vial 3 all flies had died, while in vial 4, one had died and 2 males were left.
Discussion
Improvements that could be made
Our results were not as expected in vial 2, but as expected in vial 1. Vial 2 was a cross
between a white eyed female and a ebony vestigial male. However, the F2 and F3 generations
were all white eyed, so it seems that the female may have been pregnant before she was
placed with the male. Vial 1 did seem to be populated with both WT and scarlet eyed
individuals, but differentiating the two was difficult. Along with the problems with mortality
and possibly missexing in vials 3 and 4, there are many things that I would change if I
repeated this experiment again. One factor I improved between experiments was holding the
vial upside down as the flies were flynapped, as in the first experiment it was hard to remove
flies from the food substrate, and the substrate clung to flies, making sexing difficult and
trapping the flies as it dried. In the second breedings, I held the vials upside down as the flies
were flynapped, and the live flies dropped onto the sponge stopper instead, meaning that all
flies were easily removed. Sexing was easier this time, but I noticed that many flies are not
obviously male or female. The reflection above elaborates on this.
The next thing I would change is that I would have several vials for the same experiment, or
several flies with the same genetics. This reduces risk of failure via reduced fertility, or death.
However, if repeating this experiment I would also select newly hatched flies. This is a
benefit as females will be virgins and males at their most fertile. I would do this by removing
all adult flies from the sample vial, and collecting the flies that were to be bred in the next 8
hours. Any flies that will have hatched will be virgins, as it takes longer than this for the flies
to become ready to breed (Unknown, 2003), and they will be young, and therefore less likely
to die. The best time to take out the flies would be at night, as most flies hatch in the
morning. As we had already been collecting flies in the morning, this may have aided us in
the example of vial 1, for example, as the parent generation may have been younger.
I would also keep the flies at a stable temperature of 25 degrees celcius, as this is optimum
breeding and rearing temperature, although this may reduce generation times (Gerolt, 1957).
Finally, I would ensure only around 4 grains of yeast were included in the food substrate, and
would use small tweezers that clasped more gently, as this would reduce the risk of harming
flies while they are transported.
If I was repeating the experiment several times, it could also be useful to check whether other
methods of knocking out the flies were more or less effective (ie. cooling and CO2
administration).
Factors that affected results
The plastic vials we used were ideal, as they were of an appropriate size for the smaller
populations that were involved, and were sterile, so disease risk was minimised. Using
rehydrated food substrate is only a negative if rehydration does not reach all of the substrate
(Unknown, 2003). This would limit water availability to the flies and likely reduce success.
However, the substrate we used appeared to be well saturated.
Analysis of Results
Vial 1 results were that the numbers of scarlets to wild type were about the same. Following
mendelian inheritance, the proportions should have been about 3 wildtypes to every 1 scarlet
eye. This was not the case, however our results were not very reliable, and so some flies may
have been categorised incorrectly. High microscope settings would make both sexing and eye
colour discrimination easier, and the use of reference flies for each phenotype could be useful
in the latter. If it was assumed that our sorting had been correct, it could have been the low
numbers (around 20 offspring) that allowed by chance a higher proportion of scarlet eyes.
Vial 2 (F2 and F3) was unexpected. However, as our flies may not have been virgins, it
seems very likely that the female white eyed fly selected was pregnant when she was moved
to the vial. It is highly unlikely that by chance all of the F2 and F3 generation flies were white
eyed and not ebonies or vestigials. To improve this experiment we would use only virgin
flies. As we never saw the ebony vestigial male after his anaesthetisation he may have died,
or could have been so old that he could not effectively breed. As mentioned above, the
collection of virgins avoids this. This experiment would be the most valuable to repeat, due to
the lack of success and the many phenotypes that could result.
It is mentioned in the "Pleiotropy" tab that ebony vestigials may suffer some deleterious
effects. This may reduce breeding success.
Vial 3 and Vial 4 were both unsuccessful. The improvements stated above (using young
virgins, several parents in the first breedings, and more precise tweezers) would hopefully
increase chances of success in future experiments. It could be anticipated that vial 3 would
likely have produced more offspring in the F1 generation, as two females were involved
rather than one.
Conclusions
Not many conclusions can be drawn from this data. The 1:1 results for vial 1 are more
indicative of sex linked genes, rather than autosomal genes. However, as there are many
sources stating that scarlet eyes are autosomal, and as our sample size was small, it seems that
our results were likely not representative of what the normal ratio of offspring is.
The data from vial 2 does not appear to relate to the cross that was intended, but did allow
some sexing practice. Vials 3 and 4 were not successful, which could be avoided with the use
of several young virgin flies. There are many experiments that could be repeated to find more
conclusions.