Title: Cytochrome c plays an important functional role in the development of Aedes aegypti
1Cytochrome c plays an important functional role
in the development of Aedes aegypti
Liming Zhao, Julia W. Pridgeon, James J. Becnel,
Gary G. Clark and Kenneth J. Linthicum
Mosquito and Fly Research Unit CMAVE, ARS, USDA
2Research objective
Identify and investigate critical metabolic
pathways in mosquitoes and develop molecular
insecticides to disrupt these pathways
3Approach for the development of molecular
pesticides
Target essential pathways
Programmed cell death, mitochondria pathway
(Cyt C)
Molecular cloning (mRNA, cDNA, PCR)
(Aedes aegypti model)
Vector, carrier, formulation, microinjection
Molecular pesticide construction
Bioassays
(Aedes aegypti model)
4Why Cytochrome C (Cyt C)?
- At center of life in the oxygen-rich world-
crucial factor in programmed cell death and
critical for mitochondrial regulation of
apoptosis - Called a type of "moonlighting" protein which has
one function in one place and an entirely
different function in another place - Many lethal agents target the mitochondria and
cause release of Cyt C and other pro-apoptotic
proteins into the cytoplasm - Highly conserved protein- found in many
unicellular and multicellular organisms
5Studies of Cyt C expressionin Aedes aegypti
- Developmental regulation
- Environmental effects on gene expression
- Ultra violet radiation
- Temperature
- Pesticide (permethrin)
6Tools used in this study
- 1. Quantitative real-time polymerase chain
reaction (PCR) - 2. Western blot analysis (immunoblot)
-
7Developmental regulation of Cyt C
- Examined Cyt C gene expression during the
development of Aedes aegypti- stages from eggs to
adults were collected for RT-PCR analysis - Cyt C gene in Aedes aegypti AeaCytC
- CYTC protein in Aedes aegypti AeaCYTC
8AeaCytC gene expression level in different
developmental stages
Adults
Egg
1st Instar
2nd Instar
3rd Instar
4th Instar
Pupae
9Developmental protein expression of AeaCYTC
4th instar larvae
1st instar larvae
early pupae
early adult
late pupae
late adult
late egg
early egg
CYTC
12 KDa
ACTIN
42 KDa
10Environmental effects on AeaCytC gene expression
- Ultra-violet radiation light intensity
(irradiance) - 1.003 0.055 mW/cm2
- Exposure time 0, 15, 30, 180 and 240 min
- Temperatures 24ºC, 37ºC, 40ºC,
- relative humidity 56
- Exposure time 0, 15, 30, and 180 min
- Permethrin treatment 1.25 x 10-5?g per mosquito
using acetone as a carrier - Exposure time 0, 1, 3, 6, 12 hr
11AeaCytC gene expression in response to UV
treatments
12AeaCYTC protein expression in response to UV
treatments
3d Adults
10d Adults
30
0
180
30
0
180 min
CYTC
12 kDa
ACTIN
42 kDa
13Responses of AeaCytC gene expression to
temperature
14AeaCYTC protein expression in response to
temperature treatments
24ºC
37ºC
40ºC
30
0
180
0
30
180
0
30
180 min
CYTC
12 KDa
2-day adult
ACTIN
42 KDa
CYTC
12 KDa
9-day adult
ACTIN
42 KDa
15AeaCytC gene expression in response to permethrin
treatments
16AeaCYTC protein expression in response to
permethrin treatments
Acetone
Permethrin/acetone
1
0
3
6
12
0
1
3
6
12 hrs
CYTC
12 KDa
3-day adults
ACTIN
42 KDa
CYTC
12 KDa
10-day adults
ACTIN
42 KDa
17Microinjection for AeaCytC dsRNA
Primers used for the synthesis of dsRNA products
Primer Name
Sequence
TAATACGACTCACTATAGGGATGGGTATTCCAGCTGG
T7-AEACYTC-1F
T7-AEACYTC-327R
TAATACGACTCACTATAGGGTTAGGCGGTAGACGATTTC
T7-AEACYTC-157F
TAATACGACTCACTATAGGGTACA
CAGATGCCAACAAG
TAATACGACTCACTATAGGGGCCTTGTTGGCATCTGTGT
T7-AEACYTC-176R
All primers have a 5 T7 promoter sequence
18Microinjection for AeaCytC dsRNA
Table 1. Toxicities of dsRNA-AeaCytC products
against female adults of Aedes aegytpi using
microinjection
19Conclusions I.
- AeaCytC gene expression much higher in the
adult Ae. aegypti (especially in teneral adults)
than in egg, larval and pupal stages - AeaCYTC protein can only be detected in adult Ae.
aegypti - AeaCytC relative gene expression increased
(up-regulated) at 37ºC and 40ºC both in 2 day and
9 day-old adult after 15 min exposure
20Conclusions II.
- AeaCYTC protein is environmentally regulated by
the age of adults (3 day-old adults may have less
protection than 10 day-old adults under UV-light
treatment) - dsRNA AeaCytC microinjection of adult Ae. aegypti
resulting in higher mortality (gt 70) in full
Length (dsRNA CytC-F) of the gene -
21Acknowledgments
- Neil Sanscrainte
- Heather Furlong
- Lynn Jefferson
- Nathan Newlon
- Greg Allen
- Dr. Ulrich Bernier
- Dr. Brian Quinn
- Dr. Chris Geden
- Dr. Jerry Hogsette
- Dr. Ken Linthicum
- Dr. James Becnel
- Dr. Julia Pridgeon
- Dr. Gary Clark
- Dr. Sandra Allan
- Dr. Dan Kline
Mosquito and Fly Research Unit Center for
Medical, Agricultural, and Veterinary Entomology
ARS, USDA
Dr. Daniel Strickman
National Program Leader, Program 104, USDA-ARS
Deployed War Fighter Protection Research Program
22Thank you!