Moorea, Problematic or Not?
Jack Meghreblian, Evgenia Glukhov, and William H. Gerwick
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, La Jolla, California
92093, United States
Brine Shrimp Assay
G:\20160722_2251G
0.00
5.92 9.15
uAU
0
100
0.00
25.66
20.62
27.23
27.55
28.39
24.43
50
17.95
12.84
7.68 10.69
2.05
0
100
20.59
17.55 20.27
50
0.67 3.73
0
0
9.40
5
27.14
28.02
28.24
26.42
12.87 14.30
10
1.77
100000
5.92 9.15
0
100
NL: 1.01E7
Base Peak F: + c ESI Full ms
[190.00-2000.00] M S
20160722_2251G
20
25
0
100
7.68 10.69
20.59
17.55 20.27
50
30
17.95
12.84
2.05
0.67 3.73
0
15
27.23
27.55
28.39
24.43
50
NL: 2.45E7
Base Peak F: + c ESI Full ms
[190.00-2000.00] M S
20160722_2251h
25.66
20.62
0
9.40
5
26.42
12.87 14.30
10
15
Time (min)
20
27.14
28.02
28.24
25
NL: 1.01E7
Base Peak F: + c ESI Full ms
[190.00-2000.00] M S
20160722_2251G
NL: 2.45E7
Base Peak F: + c ESI Full ms
[190.00-2000.00] M S
20160722_2251h
30
Time (min)
20160722_2251G #740 RT: 20.41 AV: 1 NL: 1.51E6
F: + c ESI Full ms [190.00-2000.00]
808.28
20160722_2251h #758 RT: 20.59 AV: 1 NL: 2.45E7
F: + c ESI Full ms [190.00-2000.00]
808.32
830.49
1400000
20000000
1200000
830.51
1000000
100
The ten fractions including the crude had little affect on
the brine shrimp’s health. There was little death found
in each of the wells showing that the compounds
toxicity level were small at 30 ug/mL . Despite having a
small amount of death, there were large percent errors
found. The leading cause of the large percent error is
most likely because of the population of brine shrimps
was larger than others.
809.20
800000
831.51
810.24
600000
15000000
10000000
809.32
832.59
400000
831.47
5000000
200000
846.36
851.69
821.66
793.33
871.34
800
820
840
860
810.31
832.50
887.31
811.31 822.97
807.42
0
880
800
810
820
840.20
830
846.48
840
m/z
850
859.30 865.23
860
870
m/z
20160722_2251G #662 RT: 18.31 AV: 1 NL: 2.50E5
F: + c ESI d Full ms2 [email protected] [210.00-820.00]
444.04
20160722_2251h #668 RT: 18.28 AV: 1 NL: 8.78E5
F: + c ESI d Full ms2 [email protected] [210.00-820.00]
444.10
800000
200000
700000
600000
30 ug/mL
150000
100000
416.14
245.61
605.96
468.08
659.32
780.18
0
80
400000
100000
245.72
379.07
561.11 613.75 691.18 743.28 790.55
450.22
0
300
400
500
600
700
800
300
m/z
60
543.10
330.96
200000
50000
100
500000
300000
543.17
331.07
120
Survival, %
1.77
100000
NL: 2.37E5
Total Scan PDA
20160722_2251G
200000
Intensity
0.00
200000
100
100
57
84
75
70
38
100
100
RT: 0.00 - 32.00
1.41
NL: 2.37E5
Total Scan PDA
20160722_2251G
Intensity
0.00
crude
7/22/2016 4:24:18 PM
RT: 0.00 - 32.00
1.41
Intensity
Moorea sp. is a species of cyanobacteria that grow in tropical oceans.
The species of cyanobacteria increases everyday due to overfishing
and pollution. They thrive from nitrogen and human waste that flow to
the ocean due to rain and ocean runoff, increasing their population
and decreasing the amount of oxygen in the water. Cyanobacteria are
now being considered as toxic to the environment. To answer this
question, this research used cyanobacteria from American Samoa on
brine shrimp and cancer cells to help give us a better understanding
of how toxic cyanobacteria is on living organisms.
Brine shrimp viewed
under a microscope
Avgerag
M2
e death Error
M1
30
30
30
30
ug/mL 30 ug/mL ug/mL ug/mL ug/mL
MeOH
0
0
0.00
0.00
A
0
0
0.00
0.00
B
0.2 0.67
0.44
0.33
D
0.13 0.20
0.16
0.05
E
0.50 0.00
0.25
0.35
F
0.00 0.40
0.20
0.28
G
0.25 0.50
0.38
0.18
H
0.00 0.00
0.00
0.00
I
0.00 0.00
0.00
0.00
Avgerage
survival M1,
%
Intensity
The purpose of this project was to extract compounds from cyanobacteria Moorea
sp. from American Samoa and test their toxicity on brine shrimp and cancer cells.
Cyanobacteria are microorganisms that contain chlorophyll and are capable of
conducting photosynthesis. Nine fractions were produced from the cyanobacteria
and used to test their toxicity on brine shrimp and cancer cells. The compounds
were found nontoxic and created little to no harm on the brine shrimp and cancer
cells. Fractions G and H were later used to see what compounds could be found in
the cyanobacteria. Six compounds were discovered that can potentially be found in
the cyanobacteria used in this research.
Introduction
Example LC-MS Results
uAU
Abstract
400
500
600
700
800
m/z
30 ug/mL
40
20
0
Negative
control
A
B
D
E
F
G
H
I
crude
2251 ASZ24JUL14-2
Cytatoxicity Assay
Ten fractions including the crude had little affect on cancer cells. Instead of using sea water and a
microscope, human blood was used along with a plate reader to enhance the living condition for
a cancer cells. 10 ug/mL and 1ug/mL of each fraction were used. Each of these concentrations of
the fractions were unable to kill any cancer cells.
Methods
Discussion and Conclusions
All the fractions were ineffective towards all organisms used in this research. Despite how
the fractions had little effect on the organisms, this unsuccessful testing hopefully
provides other researchers on what to do to succeed. I gained a lot from taking part in
lab work from rotovaping to determining what compounds exist in molecular networks.
This learning experience has not only been a great opportunity, but has also further
motivated me to do research in marine biology.
Molecular Network
References
1- Engene N. et al Moorea producens gen. nov., sp. nov. and Moorea bouillonii comb. nov., tropical marine cyanobacteria rich in
bioactive secondary metabolites National Institute of Health Institute 2012 May; 62(Pt 5): 1171–1178.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542135/
Acknowledgments
There were multiple compounds that were found from the fractions. The molecules
that contained a mass of 872.793 and 873.57 are pheophytin, a chemical compound
that is commonly found in cyanobacteria. The other molecule was unable to be
identified directly but is most likely pitipeptolide A because not only is it commonly
found in cyanobacteria, but also can be found close to American Samoa.
Sincerest thanks to the Academic Connections program and Professor Komives
for directing the Research Scholars program, to Professor Glukhov for helping
me with my research.