approach in the field, but also provides the first description of an ecological role for the C12 terpene, pregeijerene. To evaluate applied volatiles for the attraction of belowground natural enemies in the field, studies usually quantify mortality of a target pest by trapping adults emerging from soil [11]. This technique frequently results in low recovery and also gives no confirmation of the specific cause of mortality. In addition, it can be difficult to quantify populations of naturally occurring EPNs, which may be abundant in soil, but remain cryptic. We used real- time qPCR as an efficient method for describing EPN diversity and quantifying their abundance [32,44–46]. Moreover, we showed that pregeijerene was directly responsible for attracting five species of native EPNs in the soil so as to enhance pest mortality. Given the efficacy of this compound, there may be little need for exogenous application of non-native EPNs in systems with a rich fauna of native EPNs. In orchards with established EPN populations, large-scale introduction of non-native species may temporarily reduce native populations due to trophic cascades that increase predatory fungi and attenuate net efficacy of biological control [19,47]. Although it is known that artificially reared and commercially formulated EPNs can persist, it is possible that natives have advantages associated with habitat acclimation and response to HIPVs [9]; thus, further investigation of enhancing conservation biological control of belowground pests in concert with behavioral modification via HIPVs is warranted. The results of the experiment conducted in blueberries, an agricultural setting vastly different from citrus, demonstrate the potential broad applicability of pregeijerene on diverse species of EPNs. Timing application of pregeijerene to target the most susceptible instar of A. orientalis should optimize its efficacy (depending on EPN species, final (third)-instar A. orientalis may be less susceptible to EPN infection than earlier larval instars [48]). Our previous research suggests that volatile production in response to herbivore feeding differs between citrus species [8]. Thus, our current findings could have broad impacts not only for rootstock selection in commercial citriculture, but also for use of attractants in other agroecosystems as demonstrated in blueberry fields. Here, we identified an additional naturally occurring species of EPN responsive to pregeijerene that was not found in Florida. Pregeijerene may thus have extensive application for enhancing native biological control of root feeding insects, including those, which attack a wide range of crops. However, we recognize that plants should benefit from the proposed function of herbivore- induced responses. Our experiments were not designed to assess improved crop fitness as a result of attracting beneficial natural enemies by application of pregeijerene. However, there is strong evidence that plants benefit from the cascading effects caused by EPN-induced suppression of herbivores [49], and our future work will determine whether crop yield is affected by HIPV-mediated manipulation of EPN behavior. Where most aboveground studies have identified blends of volatiles as being responsible for the attraction of natural enemies [50], analogous belowground studies that identify an attractant in an agricultural system have demonstrated that a single compound can elicit natural enemy responses. This certainly makes applica- tion potentially less complex, but also points to an interesting potential property of belowground cues and natural enemy response. Future work should evaluate the complexity of belowground cues and the range of volatiles that cause below- ground natural enemies, like EPNs, to respond. Only recently have new methodologies been employed to investigate chemicals stimulating changes in EPN behavior [51] and much more progress is necessary to understand these relationships [52]. Subterranean Volatile Increases Biological Control Materials and Methods Insect Larvae D. abbreviatus larvae were obtained from a culture maintained at University of Florida’s Citrus Research and Education Center (CREC) in Lake Alfred, FL. This culture was periodically supplemented from a larger culture maintained at the Division of Plant Industry Sterile Fly Facility in Gainesville, FL. Larvae were reared on a commercially prepared diet (Bio-Serv, Inc., Frenchtown, NJ) using procedures described by Lapointe and Shapiro [53]. Larvae used in experiments were from third to sixth instars. Third-instar A. orientalis were collected from untreated turf areas at the Rutgers University Horticultural Research Farm (North Brunswick, NJ) in late April. The larvae were stored individually in the cells of 24-well plates in sandy loam at 10uC for two weeks and returned to room temperatures (21–24uC) for 24 h before use in experiments. Late instar G. mellonella larvae were obtained from Big Apple Herpetological (Hauppauge, NY). Plants ‘Swingle citrumelo’ (C. paradisi Macf. 6 P. trifoliata L. Raf.) rootstock is very prominent in commercial citrus production [54]. The extensive use of this rootstock in commercial citrus production justified its use in this investigation. All plants were grown and maintained at the CREC in Lake Alfred, FL in a greenhouse at 2663uC and 60–80% RH. Citrus seedlings used in the experiments were 25–35 cm long. R. graveolens was purchased as full grown plants 46–61 cm in height. The plants were immediately bare rooted and rinsed to remove as much soil material as possible; only roots were placed into vials containing dichloromethane for further extractions and purification. Nematodes Used for Laboratory qPCR and Bioassays The entomopathogenic nematodes, S. diaprepesi HK31, S. riobrave Btw1, Steinernema sp. LWD1, H. indica Ker1 and H. zealandica Btw1 were isolated from D. abbreviatus larvae buried in commercial citrus orchards in Florida. S. riobrave and S. carpocapsae isolates were descendants of commercial formulations intended for field application to manage D. abbreviatus. Other EPN species included in this study were S. scapterisci (provided by Dr. J.H. Frank, University of Florida, FL). All EPN species were cultured in last instar larvae of the greater wax moth, G. mellonella larvae, at approximately 25uC according to procedures described in Kaya and Stock [55]. Infective juveniles (IJs) that emerged from insect cadavers into emergence traps were stored in shallow water in tissue culture flasks at 15uC for up to two weeks prior to use. In situ Volatile Collection from Infested Roots in the Greenhouse Six ‘Swingle citrumelo’ plants were initially placed in glass root- zone chambers (300 ml volume capacity) (Analytical Research Systems, Gainesville, FL) filled with sand that had been autoclaved for one hour at 121uC and then adjusted to 10% moisture as described in Ali et al. [7,8] and El-Borai et al. [31]. All seedlings were given three days to adjust to their sand filled chambers. Three of the plants were subjected to feeding by weevil larvae for three days; the remaining three served as undamaged controls. During this period, each of the six root-zone chambers were connected to a vacuum pump (Analytical Research Systems, Gainesville, FL) with a suction flow of 80 ml/min [7]. Compounds emitted from chambers were collected on adsorbent traps filled with 50 mg Super-Q (800–1000 mesh, Alltech, Deerfield, IL) held in glass fittings between the chamber and vacuum pump [7]. PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e38146