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Wael Osman

BIOL 223 Lab – Section B6

Ms. Rouba Hilal

October 22nd 2020

Fly Mitochondrial DNA Extraction

A. Pre-Laboratory Project Report

I. Obtaining the Fly

I was planning to catch my fly around AUB campus (upper campus behind the Lee

Observatory) since the campus is rich in biodiversity which raises the chances of catching a rare

fly. However, I could not make it to AUB due to the restrictions which impede entering campus.

Thus, I limited my search to the living room’s balcony (coordinates determined by Google Map:

33.892668, 35.483730). It took several attempts to find a fly; however, I finally found a fly

which was on the balcony flowers. The fly then landed on the balcony’s table, and I carefully

and swiftly covered it with a glass jar, then covered the lid before it could fly away. I caught the

fly on Wednesday October 21st at 4:16 PM. The outside temperature was 28ºC and it was sunny

with little humidity.

II. Identification of the Fly

The fly exhibited the following phenotype: orange and black patterned abdomen, black eyes, and

hairlike extensions. I referred to Cedar Creek’s website to identify my fly which turned out to be

Musca autumnalis (“Family MUSCIDAE”, 2000). It is commonly known as “face fly” or

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“autumn housefly”, is usually about 7-8 mm long and has characteristics such as red/dark red

eyes, a tan body, and black back, leg, and body bristles (“Musca autumnalis”, 2019). It is worth

noting that during daylight hours these flies feed on plant sugars and juices which further

validates my identification (Bennet, 2003). Moreover, it is worth mentioning that since the

balcony has glass curtains and they were half open, this may indicate too that the fly was trying

to get indoors, as these flies enter buildings only during autumn to dispause (Bennet,2003).

This fly’s scientific classification is:

Kingdom: Animalia

Phylum: Arthropoda

Class: Insecta

Order: Diptera

Superfamily: Muscoidea

Family: Muscidae

Subfamily: Muscinae

Genus: Musca

Species: autumnalis

According to the Diptera ND1 sequences catalog on Moodle (2020), Musca autumnalis is closely

related to both Haematobia irritans irritans (horn fly) and Stomoxys calcitrans (stable fly) since

they belong to the same family, namely Muscidae. For the purpose of this lab, I will rely on the

mtND1 gene sequence for the Haematobia irritans irritans (horn fly).
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Figures 3 and 4: Picture of Musca

Figures 1 and 2: Picture of Musca
autumnalis from Wildlife Natural
autumnalis which I caught

III. Predicted True9 Digestion of Horn Fly

The mtND1 gene sequence from the Diptera mt nd1 Sequence (2020) on moodle is:

5’ATCATATCGAAATCGAGGTAAAGTTCCACGAACTCAAATAAATATAAAAGAAAC

AAAAGTTAATTTAATATAAAATATAAAAGAAAATACATCTCTTCCTAAAAATATAAC

ACAAAATAATATTCTTATAAATAAAATACTTGCATATTCAGCTAAAAAAATTAAAGC

AAATCCTCCTCTTCTATATTCTACATTAAATCCAGATACTAATTCAGATTCCCCTTCA

GCAAAATCAAAAGGAGTTCGATTAGTTTCAGCTAAAGAAATTCTAAATCAAACTAA

TGCTATAGGAAATATAATAAATAAAAATCAAATAAATATTTGATATTTATAAAATAT

TAATATATTATAACCTCCAATTAAAAAAATAAAACTTAATAAAACCAAAGCTAATCT

AACTTCATAAGAAATAGTTTGAGCAACAGCTCGTAATCCACCTAATAATGCATAATT

TGAATTAGAAGATCATCCCGCAATTATAACAGTATAAACACCTAAACTTGTACAACA
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TAAAAAAAATAATAACCCTAAATTAAAAGAAAATAATTTAATAAATATAGGTATAC

ATATTCAAACTAATAAAGACAAAAATAAAGAGAAAATAGGAGAAAAATAATAAGA

AATATAATTAGATAATAAAGGATAAGTTTGTTCCTTAGTAAATAATTTAATTGCATC

ACAAAAAGGTTG 3’

The digestion by Tru9 I (isoschizomer of MseI, Tru 1I), for the closest relative’s sequence (horn

fly), occurs at 10 restriction sites and results in 11 restriction fragments. Tru9 I cuts between the

two thymine nucleotides in each restriction site (T*TAA) (Hajjar et al., 2020).

The predicted fragments are as follows:

1. 5’ATCATATCGAAATCGAGGTAAAGTTCCACGAACTCAAATAAATATAAAAG

AAACAAAAGT 3’ (Size: 60 bp)

2. 5’TAATT 3’ (Size: 5 bp)

3. 5’TAATATAAAATATAAAAGAAAATACATCTCTTCCTAAAAATATAACACAAA

ATAATATTCTTATAAATAAAATACTTGCATATTCAGCTAAAAAAAT 3’ (Size:

97bp)

4. 5’TAAAGCAAATCCTCCTCTTCTATATTCTACAT3’ (Size: 32 bp)

5. 5’TAAATCCAGATACTAATTCAGATTCCCCTTCAGCAAAATCAAAAGGAGTTC

GATTAGTTTCAGCTAAAGAAATTCTAAATCAAACTAATGCTATAGGAAATAT

AATAAATAAAAATCAAATAAATATTTGATATTTATAAAATAT 3’ (Size: 145 bp)

6. 5’TAATATATTATAACCTCCAAT 3’ (Size: 21 bp)

7. 5’TAAAAAAATAAAACT 3’ (Size: 15 bp)

8. 5’TAATAAAACCAAAGCTAATCTAACTTCATAAGAAATAGTTTGAGCAACAGC

TCGTAATCCACCTAATAATGCATAATTTGAATTAGAAGATCATCCCGCAATTA
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TAACAGTATAAACACCTAAACTTGTACAACATAAAAAAAATAATAACCCTAA

AT 3’ (Size: 158 bp)

9. 5’TAAAAGAAAATAATT 3’ (Size: 15 bp)

10. 5’TAATAAATATAGGTATACATATTCAAACTAATAAAGACAAAAATAAAGAG

AAAATAGGAGAAAAATAATAAGAAATATAATTAGATAATAAAGGATAAGTT

TGTTCCTTAGTAAATAATT 3’ (Size: 120 bp)

11. 5’TAATTGCATCACAAAAAGGTTG 3’ (Size: 22 bp)

IV. Predicted True9 Digestion for Drosophila melanogaster

The mtND1 gene sequence from the laboratory manual on Moodle (Hajjar et al, 2020):

Drosophila melanogaster GenBank NC_001709 bp 11819 to 12508.

5’ATCATAACGAAATCGAGGTAAAGTTCCTCGAACTCAAATAAAAACAAAAGAAAT

AAAAGTTAATTTTATATAAATTAATAAATTAAACACATCACAAGGTAAAAAAATAA

CGCAAAATAATATTCTTATAAATAAAATTCTCGCATATTCAGCTATAAAAATTAAAG

CCAAACCCCCTCTTCTATATTCTACATTAAATCCTGAAACTAATTCTGATTCTCCTTC

AGCAAAATCAAAAGGATTCCGATTAGTTTCAGCTAATGAAATAGATACTCAAACTA

AAGCTATAGGAAATAAAATAATTAAAAATCATATATAAACTTGATAAAAAAAAAAA

TAAATTATATTATAACTTCCAATTAAAAAAATAAAAGATAATAAAATTAAAGCTAA

ACTAACTTCATAAGAAATAGTCTGAGCCACAGCTCGCAAACCTCCTAATAAAGCATA

ATTAGAATTAGACGACCAACCAGCTACTATAACAGTATAAACCCCCAATCTAGTACA

ACATAAAAAAAATAAACCACCCAAATTAAAAGAATATAATTTTACAAAAAAAGGCA

TACATATTCAAACAAATAATGATAAAAATAAAGAAAAAATTGGAGAAATATAATAT
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CTTAAATAATTAGATAATAAAGGATAAGTTTGTTCTTTTGTAAATAATTTAATCGCA

TCACAAAAAGGTTG 3’

Upon digestion by Tru9 I at 11 restriction sites (T*TAA), we obtain 12 restriction fragments,

which are:

1. 5’ATCATAACGAAATCGAGGTAAAGTTCCTCGAACTCAAATAAAAACAAAA

GAAATAAAAGT 3’ (Size: 60 bp)

2. 5’ TAATTTTATATAAAT 3’ (Size: 15 bp)

3. 5’ TAATAAAT 3’ (Size: 8 bp)

4. 5’TAAACACATCACAAGGTAAAAAAATAACGCAAAATAATATTCTTATAAATA

AAATTCTCGCATATTCAGCTATAAAAAT 3’ (Size: 79 bp)

5. 5’ TAAAGCCAAACCCCCTCTTCTATATTCTACAT 3’ (Size: 32 bp)

6. 5’TAAATCCTGAAACTAATTCTGATTCTCCTTCAGCAAAATCAAAAGGATTCC

GATTAGTTTCAGCTAATGAAATAGATACTCAAACTAAAGCTATAGGAAATAA

AATAAT 3’ (Size: 109 bp)

7. 5’TAAAAATCATATATAAACTTGATAAAAAAAAAAATAAATTATATTATAACT

TCCAAT 3’ (Size: 57 bp)

8. 5’ TAAAAAAATAAAAGATAATAAAAT 3’ (Size: 24 bp)

9. 5’TAAAGCTAAACTAACTTCATAAGAAATAGTCTGAGCCACAGCTCGCAAACC

TCCTAATAAAGCATAATTAGAATTAGACGACCAACCAGCTACTATAACAGTA

TAAACCCCCAATCTAGTACAACATAAAAAAAATAAACCACCCAAAT 3’ (Size:

149 bp)

10. 5’TAAAAGAATATAATTTTACAAAAAAAGGCATACATATTCAAACAAATAATG

ATAAAAATAAAGAAAAAATTGGAGAAATATAATATCT 3’ (Size: 88 bp)

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11. 5’ TAAATAATTAGATAATAAAGGATAAGTTTGTTCTTTTGTAAATAATT 3’

(Size: 47 bp)

12. 5’ TAATCGCATCACAAAAAGGTTG 3’ (Size: 22 bp)

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Works Cited

“Diptera mt nd1 Sequence.” BIOL223 Genetics (L. Hajjar),2020,

https://lms.aub.edu.lb/course/view.php?id=9860.Accessed 21 October 2020.

“Family MUSCIDAE.” Cedar Creek Ecosystem Science Reserve, 13 Feb. 2020,

https://www.cedarcreek.umn.edu/insects/029100n.html. Accessed 21 October 2020.

Accessed 21 October 2020.

Hajjar, Layane, et al. Laboratory manual, 2020, https://lms.aub.edu.lb/course/view.php?id=9860.

Accessed 21 October 2020.

“Musca autumnalis.” Wildlife Natural, 2019, https://www.wildlifenatural.com/Musca-

autumnalis/i-mLbBcTW. Accessed 21 October 2020.

Bennet, Stuart. “Musca autumnalis.” Pied Piper,2003,

https://www.the-piedpiper.co.uk/th6f.htm. Accessed 21 October 2020.