Drosophila toxicological experiment

Protocol

To analyse the toxicity of the 3-pba, we use fruit fly to test it. We use the fruit fly to test its influence on reproductive capacity for it can affect the normal secreting of the sex hormne.

1.Material:

1.1 Animal:

Drosophila melanogaster(offered by college of animal science and technology Nanjing Agricultural University)

1.2 Instrument:

Light incubator GXZ-3001, Image measuring instrument, kitchen scale EK3820, Vertical pressure sterilizing pot LDZX-75KB, Vertical adjustable electric heating device, analytical balance, transfer liquid gun, flat bottom straight tube, milk bottle, injector, writing brush, tweezers, Carbon dioxide innovation

1.3 Reagent:

3-Phenoxybenzoic acid(AR), absolute ethyl alcohol[(C2O5)2O] (AR), propionic acid(CH3CH2COOH)(AR), Ager Poder(C12H18O9,M=3000-9000)(RT), dried yeast, corn flour, soft suga

2.Methods:

2.1 Confecting culture media

**Table 1 Formulations of basal media**
Corn Flour	Soft Sugar	Ager Powder	Dried Yeast	ddH₂O	Propionic Acid
56.7g	43.3g	3.3g	1.7g	500ml	2.7ml

**Table 2 confecting of media with different thickness* of 3-pba**
Thickness (µg/kg)	0	50	100	200	500
Amount of mother liquor (µl/50g)	0	125	250	500	1250

*Thickness of mother liquor: 20000mg/L

2.2 Steps for confecting medium(take 500ml of ddH2O for example):

Step one: Preparing a beaker(1000ml), adding 350ml of ddH2O , then adding soft suger. Stirring it until all of soft suger is melted, then adding corn flour.

Step two: Melting the yeast powder with 25ml of water and melting the agar powder with 25ml of water respectively.

Step three: Placing the beaker on the electric stove, heating, until getting gelatinized corn flour.

Step four: Adding 50ml of water to medium, then stirring well.

Step five: Adding propionic acid, then stirring well.

Step six: Adding dissolved agar powder, then stirring well.

Step seven: Cooling the medium down to about 37 centigrade degree.

Step eight: Adding dissolved yeast powder and the rest of water, then stirring well.

Step nine: Subpackaging medium into sterilized milk bottles and straight tubes immediately.

Step ten: Plugging these bottles and tubes with sterilized plugs. Store them for use.

Step eleven: Changing the medium every 7 days when feeding imagoes.

2.3 Steps for confecting medium with different concentrations of 3-pba:

Step one: Preparing the medium with the methods above, do not subpackage it into the pipes.

Step two: Weighing 50g of medium and adding the quantitative mother liquor of 3-pba according to the Table 2, then stirring the medium well. When doing this step, the medium should not be concretionary.

Step three: Subpackaging the medium into sterilized straight tubes immediately.

We set up 5 group with different concentration of 3-pba according to the Table 2. Every group has 5 repetitions. The group with no 3-pba is the negative control. The 3-pba will get into the fruit fly when the fruit fly is slurping the medium.

2.4 Steps for testing the reproductive capacity of the fruit fly:

Step one: Transferring all of the imagoes into the new bottles with new medium which has no 3-pba.(To collect the The newly emerged imagoes in old bottles )

Step two: Placing the bottles in the incubator.( temperature-25℃ relative humidity-65% natural light)

Step three: Collecting the new imagoes within 8 hours of eclosion in old bottles which haven`t mated. Hocusing these fruit flies with CO2, then distinguishing male and female.

Step four: Puttig 5 male fruit flies and 5 female fruit flies in each tubes which have medium with different concentration of 3-pba.

Step five:Removing parental flies in tubes on the 7th day.

Step six: On the 10th day, counting the new imagoes in each tubes, then removing these new imagoes.

Step seven:Repeating the step six in the next nine days.

Step eight: Analyzing all the data. We can see the data in Table 3.

2.5 Ending work

Step one: Collecting all of the medium which contains the 3-pba.

Step two: Collecting all of the flies which had eaten the medium with 3-pba.

Step three:Sending these things to the place where deal with toxic organic reagents specially.

Step four: Washing and sorting the apparatus that we have used.

2.6 Steps for Anesthesia and transfer of fruit flies:

Step one: Traversing bottle with fruit fly, pouring the CO2 into the bottle.

Step two:Sweep out the fruit flies with a brush.

Step three: Sweep the fruit flies into the bottle with new medium, the bottle can not be erective.

Step four: Stand up the bottle when the flies are awake.

Step five: Put the bottle into the incubator.

Thank Isotope Laboratory of college of science of Nanjing Agricultural University for offering us the materials , laboratory and the methods of testing.


Figure 3.1 The tubes that we have used in testing.	Figure 5 The Carbon dioxide innovation(used to hocus flies)

Figure 4 The incubator that we used during testing	Figure 3.2 The tubes that we have used in testing.

Figure 5.2 The Carbon dioxide device(used to hocus flies)

Thank Isotope Laboratory of college of science of Nanjing Agricultural University for offering us the materials , laboratory and the methods of testing.

Result

To analyse the toxicity of the 3-pba, we use fruit fly to test it. We use the fruit to test its influence on reproductive capacity for it can affect the normal secreting of the sex hormne.

We count the number of the new imagoes hatched by flies that were fed with medium with 3-pba before. The Concrete method is listed in protocles.

Table 1 numbers of the new imagoes
Numbers of repetition	The first day（7/25/2016）
Thickness(μg/kg)	0	50	100	200	500
1	53	14	5	11	0
2	54	22	16	10	0
3	49	47	24	13	0
4	51	51	9	8	0
5	59	42	29	18	0
Numbers of repetition	The second day
1	17	22	19	17	2
2	18	23	21	10	5
3	15	16	15	20	6
4	11	22	18	7	4
5	16	16	17	16	2
Numbers of repetition	The 3th day
1	21	2	4	23	15
2	20	8	17	20	15
3	21	11	12	21	14
4	22	16	11	10	11
5	23	10	15	22	8
Numbers of repetition	The 4th day
1	18	17	8	25	15
2	14	11	13	27	24
3	18	20	13	20	8
4	16	16	11	20	11
5	21	15	17	24	16
Numbers of repetition	The 5th day
1	11	9	6	16	11
2	9	4	11	11	12
3	8	1	9	9	9
4	11	10	6	6	12
5	8	1	8	8	15
Numbers of repetition	The 6th day
1	6	8	6	15	10
2	4	9	4	4	5
3	2	14	7	7	4
4	13	5	3	11	9
5	5	7	7	7	14
Numbers of repetition	The 7th day
1	5	1	2	10	8
2	8	12	3	8	13
3	11	6	2	5	6
4	6	4	4	2	12
5	2	2	6	10	5
Numbers of repetition	The 8th day
1	3	4	5	3	4
2	6	9	4	6	14
3	12	2	4	5	4
4	4	4	1	3	11
5	5	3	1	7	8
Numbers of repetition	The 9th day
1	7	16	10	5	15
2	11	1	10	11	10
3	5	3	9	8	8
4	14	6	4	17	13
5	8	4	9	13	13
Numbers of repetition	The 10th day
1	3	8	0	5	5
2	5	16	8	8	5
3	5	9	7	6	4
4	7	9	7	7	5
5	9	1	11	7	8

Figure 1.1 the changing in numbers of the new imagoes

FIG.1.1.The horizontal axis is the hatching days, the vertical axis is number of the new imagoes hatched each day.The line graph is drawed by using GraphPad Prism.

From the data we will find that the number of the new imagoes decrease with the increasing of the thickness of the 3-pba. Also the maximum day of hatching delays with the increasing of the thickness of the 3-pba.

On the 20th day, we found that some of the new imagoes become smaller than the normal imagoes. We think that these imagoes are the later periods of the new imagoes appearing from the first day to the 10th day for the fruit needs 12 to 15 days to growing from eggs to imagoes under 25℃. And on the 20th day, the eggs yielded from the parental flies should be hatched completely.


Figure 2 comparision of the normal imagoes and abnormal imagoes F1G.2.The left imagoe is the normal one, and the right imagoe is the abnormal one.

Figure 6 The tubes from different groups. Fig.6. The thickness of the medium in the tubes are 0 , 50 ,100, 200, 500(µg/kg), from left to the right.

Through the experiment, we found that the 3-pba can decrease the reproductive capacity of the fruit fly and delaying the growth of the fruit fly.

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Team:NAU-CHINA/Drosophila