Wolbachia is an obligate intracellular α-proteobacterium associated with arthropods and nematodes (O'Neill et al. 1997; Bazzocchi 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, Wolbachia can cause cytoplasmic incompatibility (Stouthamer et al. 1999), thelytoky (parthenogenesis) (Stouthamer et al. 1990), and alter aspects of fecundity (Grenier et al. 2002).

In Florida, colonies of the thelytokous endoparasitoid Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae) (Shafee, Alam and Agarwal) and the arrhenotokous ectoparasitoid Tamarixia radiata Waterston (Hymenoptera: Eulophidae) were imported from Taiwan and Vietnam, respectively, and released in a classical biological control program against the Asian citrus psyllid, Diaphorina 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 & Manjunath 2004).

Jeyaprakash and Hoy (2000) detected Wolbachia in the imported population of D. aligarhensis. We hypothesized that Wolbachia causes thelytoky in D. aligarhensis, and this was tested by attempting 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 Industries, St. Louis, MO), and placed in wooden-framed mesh cages (0.76 m × 0.91 m × 1.11 m) in a greenhouse at 20-32°C with a 16L:8D photoperiod. Adult female psyllids oviposited on the new growth (flush) produced by the trees. Adult D. aligarhensis were aspirated and released into the cages when immature D. citri reached the first or second instar. After emergence, adult D. aligarhensis were fed pure clover honey smeared on small strips of Kimwipes (Kimberly-Clark, Roswell, GA) and used to initiate the next generation. During a 4-year rearing period, all D. aligarhensis observed in this colony were females (J. Meyer, personal observation).

A preliminary toxicity test indicated that 10 mg/mL tetracycline + honey did not negatively influence 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 hydrochloride (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 parasitoids were released into a separate cage and maintained as described above. After the third generation, approximately 60 adult male D. aligarhensis 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 (Synoptics, Frederick, MD). Morphological differences were observed between female and male D. aligarhensis (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. 1B, E) and genitalia (Fig. 1C, F) of female and male D. aligarhensis were structurally distinguishable. The antennae of male D. aligarhensis in an arrhenotokous population from Asia (Shafee et al. 1975) were similar to those observed in male D. aligarhensis produced here.

Molecular analyses were used to determine if Wolbachia was eliminated from male D. aligarhensis. DNA was isolated from each of 3 individual female and male D. aligarhensis with PUREGENE reagents (Gentra Systems, Minneapolis, MN) according to the manufacturer's protocol. A 25-μL 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 (5'-TGATCAAATTTATAAT-3') and CI-N-2191 (5'-GGTAAAATTAAAATATAAACTTC-3') (Kambhampati & Smith 1995). PCR amplification products were analyzed, purified, cloned, and sequenced according to Hoy & Jeyaprakash (2005).

The wsp gene was PCR-amplified from all female D. aligarhensis (n = 3) but not from any males (n = 3) (Fig. 2). The mitochondrial COI gene was amplified from all samples indicating that the DNA was adequate for the PCR (Fig. 2). No amplification products were detected in the negative control for both the wsp and COI genes.

PCR products from the COI gene of both female and male D. aligarhensis were cloned and sequenced, and the resulting 552-bp sequences were 100% identical (GenBank accession EF431956). This indicated that the males were the same species as the female D. aligarhensis treated with tetracycline and that another parasitoid had not unexpectedly invaded the laboratory colony.

The morphological and molecular data support our hypothesis that Wolbachia causes thelytoky in our laboratory colony of D. aligarhensis, the first report of this phenomenon in this genus. Male production also has been documented following elimination of Wolbachia from other thelytokous parasitoids in the families Encyrtidae (Pijls et al. 1996), Trichogrammitidae (Stouthamer et al. 1990), Scelionidae (Arakaki et al. 2000), Eulophidae (Argov et al. 2000), and Aphelinidae (De Barro & Hart 2001). It is possible that the titer of Wolbachia in male D. aligarhensis was below the sensitivity of the high-fidelity PCR assay, which detects as few as 100 copies of the target template 100% of the time and as few as 10 copies 50% of the time (Hoy et al. 2001), but, if so, this titer reduction could still result in male production. No bacterial symbionts other than Wolbachia were detected in a molecular survey of the imported population of D. aligarhensis (Meyer 2007). Although unlikely, it cannot be excluded that unidentified microbial species in D. aligarhensis also influence thelytokous reproduction.

Male and female D. aligarhensis exhibited mating behavior when they were held together in a 50-mL centrifuge tube (Meyer 2007). First the male faced the female, then moved behind and climbed on the female, and finally attempted to copulate by bending its abdomen to contact the female abdomen. Males attempted to mate with multiple females. Further studies are needed to determine if males produce viable sperm that are transferred to the female during mating, and if a Wolbachia-free bisexual line of D. aligarhensis can be produced.

The authors thank Jennifer Zaspel for assistance with photography. This research was funded by the Davies, Fischer and Eckes Endowment in biological control to M. A. Hoy.

Summary

Wolbachia is associated with thelytokous reproduction in D. aligarhensis. Male D. aligarhensis were produced following antibiotic treatment of females in a thelytokous colony. The males lacked Wolbachia, were morphologically distinguishable from females, and exhibited mating behavior.