Wolbachia-Associated Thelytoky in Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae), A Parasitoid of the Asian Citrus Psyllid

Wolbachia is an obligate intracellular ct-pro teobacterium associated with arthropods and nematodes (O'Neill et al. 1997; Bazzochi et al. 2000). Wolbachia is transovarially transmitted by females to their progeny, and infections often are associated with reproductive anomalies in their host (O'Neill et al. 1997). In parasitoids, Wol bachia can cause cytoplasmic incompatibility (Stouthamer et al. 1999), thelytoky (parthenogen esis) (Stouthamer et al. 1990), and alter aspects of fecundity (Grenier et al. 2002). In Florida, colonies of the thelytokous endo parasitoid Diaphorencyrtus aligarhensis (Hymen optera: Encyrtidae) (Shafee, Alam and Agarwal) and the arrhenotokous ectoparasitoid Tamarixia radiata Waterston (Hymenoptera: Eulophidae) were imported from Taiwan and Vietnam, respec tively, and released in a classical biological control program against the Asian citrus psyllid, Diapho rina citri Kuwayama (Hemiptera: Psyllidae) (Hoy & Nguyen 1998, 2000; Hoy et al. 1999; Skelley & Hoy 2004). Worldwide, these parasitoids have a significant impact on reducing populations of D. citri, which is the most economically important citrus pest in regions where it vectors citrus greening disease (Chien 1995; Halbert & Manju nath 2004). Jeyaprakash and Hoy (2000) detected Wolba chia in the imported population of D. aligarhen sis. We hypothesized that Wolbachia causes thely toky in D. aligarhensis, and this was tested by at tempting to eliminate Wolbachia with antibiotics following the previous work of Stouthamer et al. (1990). This research is important because D. aligarhensis populations are low in Florida, and this could be due to its low reproductive rate (Skelley & Hoy 2004) which may be influenced by Wolbachia. A laboratory colony of D. aligarhensis was maintained as follows. Ten small citrus trees (20 50 cm tall) grown in 15.2-cm diameter pots were pruned each week, fertilized with Peter's 20-20-20 (N-P-K) water-soluble fertilizer (United Indus tries, St. Louis, MO), and placed in wooden framed mesh cages (0.76 m x 0.91 m x 1.11 m) in a greenhouse at 20-32?C with a 16L:8D photope riod. Adult female psyllids oviposited on the new growth (flush) produced by the trees. Adult D. ali garhensis were aspirated and released into the cages when immature D. citri reached the first or second instar. After emergence, adult D. aligar hensis were fed pure clover honey smeared on small strips of Kimwipes (Kimberly-Clark, Roswell, GA) and used to initiate the next gener ation. During a 4-year rearing period, all D. ali garhensis observed in this colony were females (J. Meyer, personal observation). A preliminary toxicity test indicated that 10 mg/mL tetracycline + honey did not negatively in fluence longevity in adult female D. aligarhensis, so this dosage was adopted for this experiment. For 3 consecutive generations, 50 newly-emerged female D. aligarhensis were administered pure clover honey + 10 mg/mL tetracycline hydro chloride (Sigma Chemical Co., St. Louis, MO) (Stouthamer et al. 1990) for 24 h at 70-75% RH, 24-25?C with a 16L:8D photoperiod. Treated par asitoids were released into a separate cage and maintained as described above. After the third generation, approximately 60 adult male D. ali garhensis were observed and collected. Female and male D. aligarhensis were placed on a glass slide and submerged in 95% EtOH or Euparal mounting medium (BioQuip, Rancho Domingez, CA) for photography with the Auto Montage Pro system with software ver. 5.02 (Syn optics, Frederick, MD). Morphological differences were observed between female and male D. ali garhensis (Fig. 1). The male abdomen was small and all black, but the female abdomen was larger and was yellowish and black (Fig. 1A, D). Both the geniculate antennae (Fig. IB, E) and genitalia (Fig. 1C, F) of female and male D. aligarhensis were structurally distinguishable. The antennae of male D. aligarhensis in an arrhenotokous pop ulation from Asia (Shafee et al. 1975) were simi lar to those observed in male D. aligarhensis pro duced here. Molecular analyses were used to determine if Wolbachia was eliminated from male D. aligarhen sis. DNA was isolated from each of 3 individual fe male and male D. aligarhensis with PUREGENE reagents (Gentra Systems, Minneapolis, MN) ac cording to the manufacturer's protocol. A 25-uL high-fidelity polymerase chain reaction (PCR) was conducted according to Hoy et al. (2001) to detect the wsp gene of Wolbachia with the primers wsp 81F (5'-TGGTCCAATAAGTGATGAAGAAAC-3') and wsp 691R (5'-AAAAATTAAACGCTACTCCA 3') (Braig et al. 1998). For a DNA template control, the mitochondrial cytochrome c oxidase I gene (COI) was amplified with the primers CI-J1632