From: Paola Colmenares
Date: 09 May 2003
Time: 16:39:01 -0400
Remote Name: 10.4.6.136
In Mendelian genetics, a variety of organisms can be used to demonstrate the significance of Mendelian laws (Drosophila Guide, 2003).Scientists have used varies types of animals and insects such as Drosophila’s’ and maize in order to fully understand the genetic outcome (Drosophila Guide, 2003). Therefore, for this in class experiment we used Drosophila melanogaster, as our insect for the genetic experiment (Drosophila Guide, 2003). We chose these insects because they are especially suited to experimental crosses in the laboratory (Drosophila Guide, 2003). This very small fly passes through a complete metamorphosis in ten to fourteen days at room temperature (Drosophila Guide, 2003). Because of its short life span, these insects hold an abundance of genetic variability, higher prolific and a convenient and inexpensive organism to study (Drosophila Guide, 2003). In this experiment we sought to discover the basic procedures for culturing and experimenting with Drosophila melanogaster (Drosophila Guide, 2003). We also sought to interpret Drosophila and maize F2 data and be able to understand Mendel’s law of segregation (Drosophila Guide, 2003). To begin, in the nineteenth century Gregor Mendel discovered the principles of inheritance through a series of careful experiments with peas (The Biology Place.com). We know now that physical characteristics are transmitted from generation to generation according to general patterns (The Biology Place.com). By have sufficient information about the parents; we can predict the occurrence of traits in offspring (The Biology Place.com). By analyzing the offspring, we can discern the mode of transmission. The mode of transmission relates back to being monohybrid or dihybrid, sex linked or autosomal (The Biology Place.com). Drosophila has been cultured in numerous medium (The Biology Place.com). However, for this experiment we used “instant medium” (The Biology Place.com). We add water to the medium and it would automatically become usable (Drosophila Guide, 2003). The culture medium is very important because the Drosophila tends to lay her eggs on or near the surface of the culture medium (Drosophila Guide, 2003). After one day of being laid on the surface of the medium, these little eggs hatch into very tiny, white larvae insects (Drosophila Guide, 2003). The white larvae dig deep into the culture medium and feed on the yeast cells that were added (Drosophila Guide, 2003). After seven days, the larvae attain full size and they can climb onto the side of the bottle and pupate (Drosophila Guide, 2003). Pupate is the generic term for the pupal stage of insects that can last for two days (Drosophila Guide, 2003). Once they have developed out of the pupal stage, these insects have now emerged into mature flies (Drosophila Guide, 2003). At room temperature, the entire life cycle of Drosophila melanogaster has been completed within ten days (Drosophila Guide, 2003). Secondly, the life cycle of male and female Drosophila melanogaster, are the same but their sexual dimorphism is very distinguishable (Drosophila Guide, 2003). Male Drosophila’ is much smaller than female Drosophila’ (Drosophila Guide, 2003). The male displays a more rounded abdomen and the abdomen is black-tipped rather than striped (Drosophila Guide, 2003). Black pigment is more extensive on the abdominal part of the male than on the female fruit fly (Drosophila Guide, 2003). The black markings of the male extend completely around the abdomen which joins the ventral side (Drosophila Guide, 2003).The female displays a more pointy abdomen and it is striped colored (Drosophila Guide, 2003). On the female, the pigment occurs only in the dorsal side of her abdomen (Drosophila Guide, 2003). The male has an enlarge foreleg that shows special bristles which is called the sex comb (Drosophila Guide, 2003). This ex comb is small tuft black bristles that are found on the anterior margin at the basal tarsal joint of each front leg (Drosophila Guide, 2003).However, the female lacks the sex comb because it is strictly a male genital part (Drosophila Guide, 2003). Like previously mentioned, it s life cycle is the same they start of as an egg which converts into larvae, which leads to pupa and finally becomes a mature adult fly(Drosophila Guide, 2003). Crosses of flies and other animals were the main point of discovery of the classical Mendel laws. To begin a cross between two varieties of flies, we secured a virgin female fly (Drosophila Guide, 2003). A virgin female fly is categorized as being pure of insemination from a male fly and laying eggs (Drosophila Guide, 2003). After obtaining a virgin fly, researchers artificial inseminate the fly which retains viable sperm for several days (Drosophila Guide, 2003). By using a virgin female fly, we can insure a controlled mating between different genetic stocks (Drosophila Guide, 2003). The most common virgin flies derive from the pupa case (Drosophila Guide, 2003). These virgin flies do not mate for eight hours after coming out of the pupa (Drosophila Guide, 2003). In order to produce a significant number of crosses by using varies different types of Drosophila, we must consider only those characters in which the parents distinguish from (Drosophila Guide, 2003). For instance, in crossing flies with vestigial body color and white eyes, we must only focus on the body and eye color of the flies (Drosophila Guide, 2003). After the crosses we must compare and contrast by using Mendel’s law of segregation. As the law of segregation states, each allele of the pair sorts to a different gamete (GHS, IB Genetics.com). This law would lead us then to the different type of genotype and phenotype ratios of the cross breeding between the distinct fruit flies (GHS, IB Genetics.com).Thus, this experiment will enable us to recognize the morphological characteristics from the male and female Drosophila melanogaster and two interpret the crosses done by between various Drosophila’s and linking it with Mendel’s law of segregation.
Materials and Methods I.Creating the medium of the fruit flies. 1.Obtain three bottles labeled Drosophila wild type (+), drosophila ebony, drosophila white and drosophila vestigial. 2.Obtain three more empty bottles and labeled them as mentioned above. 3.Within the three empty bottles, measure out a small cup of white medium and pour. 4.After the white medium has been added, measure out approximately three grams of yeast. This is used as a diet for the white larvae once the eggs have been hatched within the bottles. 5.Measure out one cup of water and pour into the three bottles with the white medium and yeast supplement. Once the water is added the medium will turn a moist bright blue color. Shake the bottles when the water has been added. 6.Place in the mesh into the bottles. This will help keep the flies from getting stuck to the medium. II.Etherizing and examining of the flies 1.Place a few drops of ether on the absorbent material of the etherizer 2. Hit the base of the culture bottle lightly on the palm of your hand so that the flies will drop to the bottom of the container. 3. Remove the culture bottle plug (blue cap) quickly and replace it with the mouth of the etherizer. Invert the bottle over the etherizer and shake the flies to the etherizer.4. Leave the etherizer for thirty seconds or after the flies have stopped moving. If the etherizer has been left for too much time the flies will die. This process just allows the flies to remain in a sleep state. III. Viewing the Flies and their morphological characteristics 1. Transfer the etherizer flies to a clean sheet of paper. 2. Using a dissecting microscope at 10times to 25times magnification, examine the etherized flies and their body composition. 3.If the flies revive before you are done viewing them, just add a few drops of ether and cover the flies with the white paper on the stage of the microscope for a few seconds. 4.If flies are not needed after observation, they may be discarded. IV. Crosses between varies types of Drosophila melanogaster 1.First we mated- White eye, vestigial wing Female (virgin) mated with wild Male. This was a dihybrid cross. 2. Secondly, we mated- Wild female (virgin) mated with white eye, vestigial wing Male. This was a reciprocal cross.
RESULTS Dihybrid Cross: a) White eye, vestigial wing Female (virgin) mated with wild Male. F2 results (using virgin female) F2 Female: wild (382); vestigial (119); white (365); white, vestigial (136) F2 Male: wild (371); vestigial (127); white (383); white, vestigial (122) ____________________________________ b) Reciprocal Cross Wild female (virgin) mated with white eye, vestigial wing Male. F2 results (using virgin female) F2 Female: wild (741); vestigial (255); white (0); white, vestigial (0) F2 Male: wild (377); vestigial (123); white (361); white, vestigial (129)
Phenotype Observed No. (o) Expected No. (e) (o - e) (o - e)2
White Eyes Vestigial wing (F) Virgin 1002 1000 2 4 0.004 Wild Type (+) (M) 1003 1000 3 9 0.009
Fig. 1.0- Dihybrid Cross: a) White eye, vestigial wing Female (virgin) mated with wild Male. Chi square- x2 – 0.013
b) Reciprocal Cross Wild female (virgin) mated with white eye, vestigial wing Male. Phenotype Observed No. (o) Expected No. (e) (o - e) (o - e)2
Wild Virgin (+) (F) 996 1000 4 16 0.016 White eyed, vestigial (M) 1003 1000 3 9 0.009
Chi square- x2 – 0.025
Fig. 1.3 – Description table of morphological traits between Drosophila melanogaster
Trait Wild type (+) Ebony White Vestigial Body color Light brown Dark brown Dark brown Brown Eye color Reddish Reddish White Red with black dot Eye shape Oval shape Oval shape Round shape Oval Wing shape and size Round and long wings Long wings and round Round long wings (+) Short wings Antenna shape and size Short and string like wings Short string like wings Short string like wings Short string like wings
Discussion Based on the results, that was obtained one can deduce the following for this experiment The chi square shows that the standard deviation (0.013) compared to p<0.05, is relatively low. Based on this result one can conclude that the observed and expected results run very close to each other which allows for the hypothesis to be stated as a correct guess. Based on the chi square fig. 1.0, we can conclude that the traits have a degree of freedom that ranges as three. This means that four traits were being compared and deduced that the expected and observed results are highly close together and could account or a justifiable the given hypothesis. Therefore the probability due to chance is considered a possibility because it falls under the p< 0.05 rule for probability and standard deviation. In Fig. 1.2, one can also deduce the above statement because of the expected and observed results running close together. The chi square table shows how the deviation of traits within this specie can have a high deviancy and the probability of happening due to chance alone can also be concluded as a correct hypothesis. This is so because of the p<0.05 rule and it does fall under this category which allows for the hypothesis to stand corrected. The di- hybrid cross displayed that all the females were wild type for white eyes and vestigial wings traits. The males were appeared as white eyed and vestigial wing like the wild type. When the second cross was performed (F2) the wild females (w+/_ vg+/_) (3/8), the vestigial females (w+/_ vg/vg) (1/8), the white eyed (w/w vg+/_) (3/8), white vestigial (w/w vg/vg). this was the considered hypotheis because of its low deviation it can be considred as the coreect hypotheisis for both experiment. Lastly, the trait table displayed the different morphological characteristics of the fruit fly. Most of the fruit flies remain with their characteristics that followed the wild type fly. For example, the eye shape of the wild type was oval and so was it for ebony and vestigial flies. The antenna shape and size for the wild type was short and string like. These characteristics were inherited throughout the ebony, white and vestigial flies. In conclusion, the fly lab was one of the most interesting labs of all. I really learned from this lab how easily these types of flies can convert and duplicate into distinguishable types of flies. The chi square also helped in producing a deviation of traits and changes to each different type of fly. Thus, Drosophila melanogaster, is a very easy and useful species to adhere to when it comes to genetics and could provide us with rather fascinating conclusions and discoveries for future genetic experiments.
REFERENCES Carniege Institute, “Drosophila Guide”,Washington DC. Carnegie Institute. 2003, pg. 1-9. Http:/www.The Biology Place.com. Drosophila melanogaster experiment. 2003 Http:/www.GHS, IB Genetics.com Drosophila genome project. 2003