1. Tyler Eaton, Dan Horton, Ann Amis, Ted Cottrell, Tracie Jenkins
DNA Markers for Plum curculio (Conotrachelus nenuphar) (Herbst) collected from peaches in Byron, Georgia
Tyler Eaton, Dan Horton, Ann Amis, Ted Cottrell, Tracie Jenkins

2. Background
Plum curculio is a pest of stone fruit in eastern North America
There are two strains: northern univoltine and southern multivoltine
The southern multivoltine strain is a source of concern because second generation larvae may be present in the fruit at harvest

3. Purpose
To expand on the work of D. Pfeiffer by focusing on samples collected in Georgia
To determine if DNA markers can distinguish samples collected in Georgia from others
To determine how many DNA markers can distinguish between samples

4. Purpose mtDNA vs. Wolbachia wsp gene Sequencing vs. RFLP
Power of mtDNA cytochrome oxidase I gene (COI) to differentiate geographically-specific collections
Zhang et al Ann. Entomol. Soc. Am 101:

5. Power of COI sequence to differentiate PC: Haplotype (State)
A (MA) E (MA)
B (MA) F (NY)
C (NY) G (NY, MA)
D (NY)
100
Curculio hilgendorfi
Northern
R (NJ)
S (VA)
T (NJ, VA, WV)
U (NJ)
V & W (VA)
Mid-Southern/ Mid-Atlantic
X (GA, Byron 2011)
H (MA)
I (FL)
J ( FL)
K (GA)
L (VA, FL, GA, WV)
M (WV)
N (VA)
O, P, Q (WV)
Southern

6. Mid-Southern (Mid-Atlantic)
Wolbachia Strains in PC Across Eastern US
Geographic Populations
Strains (w1- w3, GenBank GU GU013552)
Northern
w1, w2, w1 + w2
Mid-Southern (Mid-Atlantic)
w2, w1 + w2, few w3
Southern
w3 only in GA (all samples tested to date), w1 + w2 and w3 in FL

7. Male X Female Mating Examples*
Wolbachia: Inter-strain Effects on Fecundity
Male X Female Mating Examples*
Fecundity
w1 X w1
High
w1 X w2
Reduced
w1 X w3
w2 X w3
*FL females have all strains and reciprocal crosses (females X males) compatible across the range

8. What is the affect of Wolbachia on intracolony fecundity in Byron,GA?
Date Collected (2012)
Period Covered
Females
Males
2/21
2/18-21 38 33
2/23
2/22-23 35
2/24 19 21
2/27
2/25-27 28
2/29
2/28-29 27 25
TOTALS
150
152
2 = , df = 1, P > 0.9
~ 1:1 ratio with no apparent reduction in fecundity because males and females both carry the same Wolbachia strain

9. PCR-generated 590 bp wsp gene cut with restriction enzyme HindIII
No cut site w2
461 bp cut w3
253 bp cut
DNA Marker w w w3
600 bp
500 bp
400 bp
300 bp
200 bp
100 bp

10. Observations
(1) mtDNA can distinguish Byron samples from other samples
(2) There are three Wolbachia strains (w1, w2, w3) along the Eastern U.S.
(3) Only one strain, w3, has been identified to date in GA
(4) RFLP can be used to determine which Wolbachia strain is causing infection

11. Conclusions Total sequencing expense is more than RFLP expense.
Sequencing cost is $8 - $12 more per sample than RFLP.
PCR-generated RFLP fingerprints provide promise as a low cost alternative to sequencing, but need further investigation.

12. Thank You