Tyler Eaton, Dan Horton, Ann Amis, Ted Cottrell, Tracie Jenkins

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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

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

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

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. 2008. Ann. Entomol. Soc. Am 101: 824-832

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

Mid-Southern (Mid-Atlantic) Wolbachia Strains in PC Across Eastern US Geographic Populations Strains (w1- w3, GenBank GU013550-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

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

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 = 0.0134, df = 1, P > 0.9 ~ 1:1 ratio with no apparent reduction in fecundity because males and females both carry the same Wolbachia strain

PCR-generated 590 bp wsp gene cut with restriction enzyme HindIII No cut site w2 461 bp cut w3 253 bp cut DNA Marker w1 w2 w3 600 bp 500 bp 400 bp 300 bp 200 bp 100 bp

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

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.

Thank You