Ostracod Eye
Evolution
Jules A Manfreda RET I
Oakley Lab
Mentor - Ajna Rivera
Funded by - National Science
Foundation (NSF)
Introduction
 This research project
focuses on evolutionary
eye development in
male vs. female
Ostracods (i.e.
Euphilomedes morini)
 Genes involved (opsin,
glass, toy, hh, and wnt)
are being isolated
METHODS
Ostracod Sampling
 Goleta beach pier
 Ostracods are not
always in the same
location
 Get samples from
different areas using
Eckman grab
 Separate the Ostracods
from other organisms
and debris
Ostracod Eye Dissection
 A tricky affair
 Performed under a
dissection scope
 Female eyes are red
(small)
 Male compound eyes
are black (large)
 Eyes are placed in
Trizol
 Begin RNA extraction
40x
3.0x
1.5x
.67x
PCR
 6/19 RT template
 Primers hhfw1 / hhfw2
and hhrev1 / hhrev2
used
 Fragments copied
(Amplified)
Methods (con’t)
 RNA Extraction
 RNA
DNA
 RT PCR
 Reverse Transcriptase
Polymerase Chain
Reaction
 Check DNA
 H3a PCR
 Run gels
 Degenerate PCR for eye
genes
 http://www.dnalc.org/d
dnalc/resources/pcr.htm
l
RESULTS
Gel
 Gel Electrophoresis
used (agrose)
 DNA fragments isolated
 hh fragments ≈ 200bp
 Strong presence @ 200
bp for hhfw1 / hhfw2
and hhrev1 / hhrev2
DNA
Ladder (bp)
500
400
300
200
100
Tri1 #3
hh2 #2
DISCUSSION / ANALYSIS
Cloning hh
 Isolated hh fragments
where cut out and
removed (gel)
 Attempted cloning using
bacteria cells (e coli)
 Bacteria did not grow
 Cloning process failed
 If successfully cloned,
genes would have then
been sequenced
Expression Profile
Template
 Female
ostracods
 PCR results
were
compared
for genes
present
(expressed)
H3a
28S
Opsin hh
Toy
Glass
C2 C # 1
     
Tri #3
 
RT 6/30

?
?
?
X

hh2
6/19
(Adult
female)
?
?
?

?
?
(Adult
female)
(Adult
female)
(Adult
female)
= positive
X
 
?
X = negative ? = unknown
Expression Profile (con’t)
 Results surprising
 Females low levels of the genes / no
proteins
 Females are similar to male juveniles
 Males should have much higher levels
Conclusion
My RET I summer program allowed me to
regain a respect for scientific research. I had
forgotten how much time, effort, and
fortitude research scientists display in the
continual evolution of science.
Some of my laboratory techniques were
strengthened (pipeting, dissection, and
sampling) while others had to be learned or
relearned (PCR, agrose gels, RNA extraction,
etc)
Overall this experience was wonderful. It
allowed my to be work in a high academic
research environment for 6 weeks stimulating
me as an educator
Special Thanks
 Ajna Rivera and Todd Oakley
 Frank, Martina, and MRL
 NSF