Biological Control 34 (2005) 215–221 www.elsevier.com/locate/ybcon

Targeted improvement of carpocapsae for control of the pecan , Curculio caryae (Horn) (Coleoptera: Curculionidae) through hybridization and bacterial transfer

David I. Shapiro-Ilan a,¤, Robin J. Stuart b, Clayton W. McCoy b

a USDA-ARS, Southeastern Fruit and Tree Nut Research Lab, Byron, GA 31008, USA b Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, FL 33850, USA

Received 4 April 2005; accepted 16 May 2005

Abstract

The entomopathogenic , Steinernema carpocapsae, has shown promise for control of adult pecan weevil, Curculio caryae, a key pest of pecan. In previous studies, the Italian strain of S. carpocapsae was found to be among the most virulent to C. caryae adults, but possessed poor heat and desiccation tolerance. In contrast, the DD-136 strain exhibited high levels of heat and des- iccation tolerance but low virulence to the weevil. Our objective was to determine the feasibility of developing improved strains of S. carpocapsae by transferring the nematode’s bacterial symbiont ( nematophila) from the Italian strain to the DD-136 strain, and through hybridization between the two wild-type . Three modiWed strains were created: one through bacterial transfer alone and two hybrids through controlled crosses. We hypothesized that the improvement approaches would result in strains possessing high levels of heat and desiccation tolerance similar to DD-136 and virulence similar to the Italian strain. The hypothesis was supported in two out of three modiWed strains. Heat and desiccation tolerance in all modiWed strains was more than 2.5-fold greater than the Italian strain and not diVerent from the DD-136 strain, except one hybrid had lower heat tolerance than DD-136. Mortality of adult C. caryae from the modiWed strains at 2 or 3 days post-treatment was greater than from DD-136 and similar to the Italian strain. Overall, the results indicate that bacterial transfer and hybridization could be a valuable tool in improv- ing the biocontrol eYcacy of S. carpocapsae. Published by Elsevier Inc.

Keywords: BeneWcial traits; Biological control; Entomopathogenic nematode; Genetic improvement; Steinernema Carpocapsae; Xenorhabdus

1. Introduction (Harris, 1985). Larvae develop within the nut and fourth instars drop to the ground where they burrow to a depth The pecan weevil, Curculio caryae (Horn), is a key of 8–25 cm, form a soil cell, and overwinter. The follow- pest of pecan throughout the Southeastern US as well as ing year, approximately 90% of the larvae pupate and in portions of Texas, Kansas, and Oklahoma (Payne and spend the next 9 months in the soil cell as adults (Harris, Dutcher, 1985). This insect has a 2- to 3-year life cycle 1985). The remaining 10% of the larval population spend (Harris, 1985). Adults emerge from soil in late July– an additional year in the soil as larvae and emerge as August at which time they feed on and oviposit in nuts adults in the third year (Harris, 1985). Control recommendations for C. caryae currently consist of foliar applications of chemical insecticides * Corresponding author. Fax: +1 478 956 2929. (e.g., carbaryl) to kill the adults (Harris, 1999). Due to E-mail address: [email protected] (D.I. Shapiro-Ilan). environmental and regulatory concerns, research toward

1049-9644/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.biocontrol.2005.05.006 216 D.I. Shapiro-Ilan et al. / Biological Control 34 (2005) 215–221 developing alternative control strategies is warranted. traits among heterorhabditid strains or species. Shap- Entomopathogenic nematodes, which occur naturally as iro et al. (1997) enhanced heat tolerance in Heteror- pathogens of C. caryae (Nyczepir et al., 1992), are one of habditis bacteriophora Poinar through hybridization. the potential alternatives (Shapiro-Ilan, 2003). The virulence (disease causing power) of H. megidis Entomopathogenic nematodes are biological con- Poinar, Jackson, and Klein strains to Tipula oleracea trol agents in the families and Het- (L.) was improved through novel combinations of nem- erorhabditidae (Adams and Nguyen, 2002). These atode and bacteria populations. To our knowledge, nematodes are parasites of insects that kill their hosts strain improvement using hybridization or bacterial with the aid of bacteria carried in the nematode’s ali- transfer approaches have not been investigated in mentary canal (steinernematids carry Xenorhabdus spp. steinernematid species. The objective of this study was whereas heterorhabditids carry Photorhabdus spp.) to determine the potential for strain improvement of (Adams and Nguyen, 2002; Poinar, 1990). The infective S. carpocapsae through hybridization and bacterial juvenile nematode (IJ), the only free-living stage, enters transfer. the host via natural openings, i.e., mouth, anus, spira- Among the important traits that entomopathogenic cles (Poinar, 1990), or occasionally through the insect nematodes must possess in order to achieve high levels cuticle (Bedding and Molyneux, 1982). The nematodes of pest suppression under Weld conditions are virulence then release their symbiotic bacteria, which take a to the target pest, and the ability to withstand prominent role in killing the host within 24–72 h environmental conditions (Kaya and Gaugler, 1993; (Dowds and Peters, 2002; Forst and Clarke, 2002). Shapiro-Ilan et al., 2002). In eight strains of S. carpocap- After the nematodes complete one to three generations sae, Shapiro-Ilan et al. (2003) characterized various ben- within the insect cadaver, IJs exit to Wnd new hosts eWcial traits deemed to be important for biocontrol of (Poinar, 1990). Entomopathogenic nematodes are C. caryae. The Italian strain was found to be among the capable of controlling a variety of economically impor- most virulent, but had poor environmental tolerance to tant insect pests including several weevil species (Klein, heat and desiccation. In contrast, the DD-136 strain was 1990; Shapiro-Ilan et al., 2002). found to possess relatively low virulence, but high levels Although only moderately susceptible in the larval of heat and desiccation tolerance. Thus, this strain stage, laboratory studies indicate that adult stage C. improvement study was targeted speciWcally at obtain- caryae are highly susceptible to entomopathogenic nem- ing an S. carpocapsae strain with heat and desiccation atodes, particularly to Steinernema carpocapsae (Weiser) tolerance similar or equal to that of DD-136, and viru- (Shapiro-Ilan, 2001a,b, 2003, Shapiro-Ilan et al., 2003). lence similar or equal to the Italian strain. We hypothe- S. carpocapsae could be applied to the soil surface in sized that this could be achieved by transferring the pecan orchards just prior to or during the time of adult bacteria from the more virulent Italian strain to the DD- weevil emergence, thereby creating a ‘nematode barrier’ 136 nematode and/or by hybridizing the two strains that the would have to pass through before through controlled crosses. reaching the tree (Shapiro-Ilan, 2003). Field trials with S. carpocapsae (All strain or Italian strain) using this approach only resulted in 60–80% suppression of the 2. Materials and methods adult weevils (Shapiro-Ilan et al., unpublished); higher levels of control will likely be required before nematodes 2.1. Nematodes, insects, and experimental conditions can be incorporated into a C. caryae management strat- egy (Shapiro-Ilan, 2003). One approach to achieving Nematodes, S. carpocapsae (Italian and DD-136 higher levels of Weld suppression is through strain strains), originally provided by K. Nguyen (University of improvement. Florida) were initially cultured in last instar Galleria Genetic methods that may be used for improvement mellonella (L.) (obtained from Webster’s Waxie Ranch, of entomopathogenic nematode strains include artiW- Webster, WI) according to Kaya and Stock (1997). cial selection, hybridization, and transgenic approaches Adult C. caryae were collected from Circle traps (Mul- (Burnell, 2002; Gaugler, 1987; Gaugler et al., 1989; der et al., 2003) on the USDA-ARS Research Station, Hashmi et al., 1995; Strauch et al., 2004; Tomalak, Byron, GA, and used in assays within 1 day of capture 1994; Shapiro-Ilan et al., 1997). Strain improvement (Shapiro-Ilan, 2001b). Prior to experimentation all treat- may also be achieved through novel combinations of ment and control nematode populations were passed compatible nematodes and bacterial symbionts (Ger- once through G. mellonella in parallel (minimum of Wve ritsen et al., 1998). Among these approaches, hybridiza- insects per population), and progeny nematodes were tion and bacterial transfer are perhaps the most rapid stored at 13 °C for up to 3 weeks until use. All experi- and simple, and are potentially quite eVective in trans- ments were conducted in the laboratory at approxi- ferring traits. Hybridization and bacterial transfer have mately 25 °C and were arranged as completely been shown to eVectively improve or transfer beneWcial randomized designs unless otherwise noted. D.I. Shapiro-Ilan et al. / Biological Control 34 (2005) 215–221 217

2.2. Bacterial transfer and hybridization (minimum of Wve insects per population), and progeny nematodes stored at 13 °C. Monoxenic in vitro culture and bacterial transfer were accomplished using procedures described by Lunau 2.3. Heat and desiccation tolerance et al. (1993) and Gerritsen et al. (1998) using nutrient agar (Becton Dickson, Sparks, MD, USA) as the growth Heat tolerance was measured using procedures medium. Infective juvenile S. carpocapsae, DD-136 described by Shapiro et al. (1996) and Shapiro-Ilan et al. strain, were surface sterilized in 0.1% Hyamine 10£ (2003). Approximately 2000 IJs in 0.2ml were pipetted (methylbenzethonium chloride, Sigma, St. Louis MO, into 5 ml of tap water in a 20-ml glass scintillation vial. USA) and placed on a lawn of Xenorhabdus nematophila The vial had already been equilibrated to 42°C prior to (Thomas and Poinar), which was isolated from the Ital- addition of nematodes. After incubation for 1.5h in a ian strain of S. carpocapsae. Gravid females were disinte- water bath shaker (70rpm) at 42 °C, 0.2ml of the suspen- grated in 0.4 M NaOH + 0.6% sodium hypochlorite. sion was transferred to a 60-mm petri dish containing 9ml After centrifugation the axenic eggs were pipetted onto a tap water. The dishes were incubated at 25 °C for 24h at fresh lawn of the Italian strain’s bacterial symbiont (on which time the percentage nematode mortality was deter- 100 mm petri dishes), and nematode development con- mined based on movement response when probed with a tinued at 25 °C. Thus, when the axenic nematode eggs dissecting needle. Treatments (wild-type, hybrid, and bac- hatched, they were exposed only to Italian strain bacte- terial transfer strains) were replicated four times and the ria. Four dishes in which bacteria were successfully experiment was repeated once (two trials). transferred from the Italian strain to the DD-136 strain Desiccation tolerance was compared among nema- were generated and collectively used to create a strain tode strains based on procedures described by Solomon hereafter referred to as the bacterial transfer strain. Once et al. (1999). Approximately 2000 IJs were pipetted onto IJs developed on the dishes, the nematodes were passed Wlter paper (55 mm, Whatman no. 1) in a 60-mm petri through G. mellonella (10 insects per dish), and progeny dish. Excess moisture was removed through vacuum IJs were combined and stored at 13 °C. Wltration. The Wlter paper containing nematodes was Hybridization procedures were based on those then placed in a plastic desiccator (23 cm maximum described by Shapiro et al. (1997) and Kaya and Stock diameter£ 24 cm height, Nalgene, Rochester, NY) that (1997). BrieXy, early juvenile (e.g., second instar) S. was set to 75% RH based on a saturated solution of carpocapsae Italian and DD-136 nematodes (grown on NaCl. Two trials were conducted; the nematodes were plates containing Italian strain’s bacterium) were trans- exposed to desiccating conditions for 48 h in the Wrst ferred individually to 60-mm petri dishes containing trial and 72 h in the second trial. After incubation at 75% nutrient agar and a fresh lawn of X. nematophila from RH, the Wlter paper was removed and placed in approxi- the Italian strain. The nematodes were transferred as mately 5 ml tap water for an additional 24 h at which early instars to ensure that mating had not yet occurred. time percentage nematode mortality was determined Once the nematodes reached adulthood, pairings were using procedures described above. Each treatment (the made on 60-mm dishes (with lawns of Italian strain’s same strains as in the heat tolerance assay) was repli- bacterium) using Wve DD-136 males to a single Italian cated four times in each trial. strain female; 10 replicate crosses of this nature were attempted. The crosses were also made reciprocally, i.e., 2.4. Virulence assays Wve male Italian strain nematodes to a single DD-136 female (with eight replicate crosses attempted). Two suc- Virulence assays were based on methods described by cessful matings were observed from the male DD-136/ Shapiro-Ilan (2001b) and Shapiro-Ilan et al. (2003). female Italian strain pairings, and none from the recipro- Experiments were conducted in plastic cups (Bioserv, cal crosses. The two successful crosses were kept sepa- Frenchtown, NJ) (3–4 cm in diameter, 3.5 cm deep) Wlled rate and the populations were labeled Hybrid 1 and with (oven-dried) soil from the USDA-ARS pecan Hybrid 2. When IJs developed on the plates, each hybrid orchard (Byron, GA). Each cup contained one adult population was exposed to two G. mellonella, and prog- C. caryae, and a slice of apple (ca. 1 § 0.4 g) provided as eny IJs were stored at 13 °C. a food and moisture source for the weevil (Shapiro-Ilan, To avoid any potential diVerential eVects of proce- 2001b). The soil was a loamy sand with the percentage dure (e.g., media eVects) between treatment and controls, sand:silt:clay D 84:10:6, pH 6.1, and organic monoxenic wild-type (i.e., control) populations of matter D 2.8% by weight. Approximately 100 IJs/cup (8/ S. carpocapsae Italian and DD136 strains were also cul- cm2) were pipetted onto the soil surface of each cup in tured in vitro by introducing nematode eggs to their 0.5 ml of water so that the Wnal moisture was standard- respective original bacteria using procedures described ized at Weld capacity (14%). Host mortality was deter- above. Once IJs developed on the in vitro culture plates, mined at 1, 2, and 3 days post-inoculation. The the nematodes were passed through G. mellonella experiment included an untreated control (only water 218 D.I. Shapiro-Ilan et al. / Biological Control 34 (2005) 215–221 added), the two wild-type strains, and the three modiWed the 2-day exposure, and F D 107.1; df D 4, 15; P D 0.0001 strains. There were four replicates of seven cups for each for the 3-day exposure) (Fig. 2). In both trials all three treatment, and the experiment was repeated once (two modiWed strains experienced lower mortality than the trials). Italian strain and did not diVer from the DD-136 strain (Fig. 2). The level of diVerence in mortality between 2.5. Data analysis modiWed strains and the Italian strain was at least 10- fold (Fig. 2). Treatment eVects in all experiments were analyzed Virulence assays detected diVerences in adult with ANOVA (P 6 0.05) (SAS, 2001). Data from identi- C. caryae mortality (F D 10.42; df D 5, 34; P D 0.0001, cally run trials (i.e., in heat tolerance and virulence F D 29.52; df D 5, 36; P D 0.0001, and F D 36.11; df D 5, assays) were combined if the trial¤treatment interaction 36; P D 0.0001 for 1, 2, and 3 days after treatment, was not signiWcant. Data from each trial in the desicca- respectively) (Fig. 3). Data from the two trials were com- tion assays were analyzed separately because the dura- bined because the trial ¤ treatment interaction was not tion of exposure diVered. If the ANOVA detected a signiWcant (P D 0.59, 0.48, and 0.94 for 1, 2, and 3, days signiWcant diVerence then treatment diVerences were elu- after treatment, respectively). Greater C. caryae mortal- cidated through LSD. Data were arcsine transformed ity was observed in all S. carpocapsae treatments relative (arcsine of square root) prior to analysis (Southwood, to the untreated control on all 3 days of evaluation. No 1978) (non-transformed means are presented in Wgures). diVerences in C. caryae mortality among nematode treatments were observed 1 day after treatment. Viru- lence of the Italian and Hybrid 2 strains were similar to 3. Results each other and greater than the DD-136 strain 2 days after treatment (at which time virulence of the other two Heat tolerance assays indicated signiWcant diVerences modiWed strains were not diVerent from either wild-type among treatments (F D 19.72; df D 4, 30; P D 0.0001) (Fig. strain) (Fig. 3). Three days after treatment, the virulence 1); data from the two trials were combined because the of all three modiWed strains was similar to the Italian trial ¤ treatment interaction was not signiWcant strain and higher than the DD-136 strain (Fig. 3). (P D 0.86). When exposed to heat, all three modiWed strains (the two hybrids and the bacterial-transfer strain) experienced more than 2.5 times lower mortality than the wild-type S. carpocapsae Italian strain (Fig. 1). DiVerences in heat tolerance were not detected between two of the modiWed strains (Hybrid 2 and the bacterial- transfer) and the DD-136 wild-type, but Hybrid 1 had lower heat tolerance (greater mortality) than DD-136 (Fig. 1). Desiccation tolerance assays indicated improvements in the modiWed strains relative to the wild-type S. carpo- capsae Italian strain (F D 78.94; df D 4, 15; P D 0.0001 for

Fig. 2. Mean percentage mortality (§SEM) in various S. carpocapsae Fig. 1. Mean percentage mortality (§SEM) in various S. carpocapsae strains following exposure to 75% RH for 2 or 3 days (2 DAT and 3 strains following exposure to 42 °C for 1.5 h. Nematodes included two DAT, respectively). Nematodes included two wild-type strains (Italian wild-type strains (Italian [Ital/wt] and DD-136 [136/wt]), two DD- [Ital/wt] and DD-136 [136/wt]), two DD-136 ¤ Italian strain hybrids 136 ¤ Italian strain hybrids (Hyb1 and Hyb 2), and the DD-136 nema- (Hyb1 and Hyb 2), and the DD-136 nematode containing Italian tode containing Italian strain’s bacterium (136 + bac).DiVerent letters strain’s bacterium (136 + bac). DiVerent letters above bars indicate sta- above bars indicate statistical diVerences (based on LSD tests). tistical diVerences (based on LSD tests). D.I. Shapiro-Ilan et al. / Biological Control 34 (2005) 215–221 219

widely separated in virulence as in heat and desiccation tolerance, which is a trend that was supported in prior studies (Shapiro-Ilan et al., 2003) and reduces the likeli- hood of observing diVerences when making comparisons with modiWed strains. Nonetheless, clear evidence of improvement in virulence was detected in the modiWed strains relative to DD-136. All modiWed strains caused C. caryae mortality similar to the Italian strain and greater than the DD-136 strain on at least 1 day of eval- uation, and mortality caused by DD-136 never exceeded any of the other strains. This strain improvement study was targeted speciW- cally at obtaining S. carpocapsae strains with heat and desiccation tolerance similar or equal to that of DD-136, and virulence similar or equal to the Italian strain. In this regard the goal was achieved in two of the three modiWed strains (the bacterial transfer strain and Hybrid 2). Further testing is required to determine if the advan- tages observed among modiWed strains in the laboratory translate into greater Weld eYcacy in suppression of C. caryae. EVects of bacterial transfer on virulence have been investigated in spp. (Gerritsen et al., 1998). Gerritsen et al. (1998) recombined the bacteria (Photorhabdus spp.) of four heterorhabditids with each other and compared the resulting population’s virulence to G. mellonella and T. oleracea. Out of the 12 novel combinations of nematodes and bacteria, most were either less virulent or about equal in virulence to the original populations. One of the novel combinations, Fig. 3. Mean percentage mortality (§SEM) of adult Curculio caryae however, produced greater mortality in T. oleracea than adults 1, 2 or 3 days after treatment (1, 2, or 3 DAT) with various S. either of the nematode strains with their own bacteria carpocapsae strains. Nematodes included two wild-type strains (Italian indicating a synergistic interaction between the symbi- [Ital/wt] and DD-136 [136/wt]), two DD-136 ¤ Italian strain hybrids (Hyb1 and Hyb 2), and the DD-136 nematode containing Italian onts (a phenomenon we did not observe in our combina- strain’s bacterium (136 + bac), Control D water only. DiVerent letters tion). Thus, the authors concluded that the virulence of above bars indicate statistical diVerences (based on LSD tests). the nematode–bacterium combination depends on the virulence of the nematode, the bacterium, as well as the interaction between the two. Successful bacterial transfer depends on compatibility between the nematode and bacteria strains being com- 4. Discussion bined. Incompatibility among strains of Photorhabdus spp. and heterorhabditids has led to unsuccessful combi- Steinernema carpocapsae strain manipulations involv- nations (Gerritsen and Smits, 1993; Gerritsen et al., ing bacterial transfer and hybridization resulted in nota- 1998; Han et al., 1990). For example, in the study by ble improvements relative to wild-type strains. All Gerritsen et al. (1998), one-fourth of the attempted novel modiWed strains possessed substantially superior heat Heterorhabditis–Photorhabdus combinations were and desiccation tolerance relative to the Italian strain, unsuccessful. Perhaps steinernematids are more amena- and except in one case (heat tolerance of Hybrid 1), their ble to bacterial transfer than heterorhabditids because heat and desiccation tolerance was not diVerent from the former might be considered less narrow in its symbi- DD-136. In terms of virulence, the diVerences detected otic association (and thus more capable of growing suc- between modiWed and wild-type strains were not as pro- cessfully on bacteria of other strains or species). In some nounced relative to diVerences detected in heat and des- cases, a single Xenorhabdus sp. (e.g., X. bovienii Akhurst) iccation tolerance (i.e., diVerences of up to 26% relative is naturally associated with several Steinernema spp. and, to diVerences greater than 2.5-fold). This result is not in contrast, the symbiotic relationship of a single Het- surprising because the diVerence in virulence between erorhabditis sp. (e.g., H. bacteriophora) is divided into wild-type strains (DD-136 and Italian) were also not as speciWc associations with several species or subspecies of 220 D.I. Shapiro-Ilan et al. / Biological Control 34 (2005) 215–221

Photorhabdus (Boemare, 2002). Additional evidence of a cases where nematode associated traits are already at a less fastidious nature in steinernematids is that they can desired level. For example, in our study, the preservation be reared axenically, whereas heterorhabditids cannot of heat and desiccation tolerance at levels akin to the (Lunau et al., 1993). Furthermore, we did not observe DD-136 strain could be expected to be most successful any incompatibility when transferring X. nematophila through bacterial transfer (using DD-136 as the host from the Italian strain of S. carpocapsae to the DD-136 nematode). Indeed this may explain why one of the strain; DD-136 nematodes grew readily on Italian strain hybrids did not exhibit heat tolerance equal to DD-136, bacteria on all plates where they were transferred. Addi- whereas the bacterial transfer strain did; heat tolerance tionally, we did not observe any incompatibility when in Hybrid 1 may have been diluted due to combination transferring the Agriotis strain of S. carpocapsae to Ital- with factors from the less heat tolerant Italian nematode ian strain bacteria (unpublished data). However, success- strain. Depending on the genetic makeup of the trait that ful combinations among just a few S. carpocapsae is targeted (e.g., dominance/recessive factors), it is always strains are certainly not suYcient to make overall gener- possible that some or all the hybrids will be inferior to alizations. Multiple combinations among various hete- the parent strain. Further investigation is required to rorhabditid and steinernematid species and strains and determine the level of variation in outcomes when their symbionts will be necessary before any solid con- employing bacterial transfer or hybridization for strain clusions can be drawn regarding whether one group is improvement. more amenable to bacterial transfer than the other. Per- Bacterial transfer and hybridization have some ceptions of speciWcity and compatibility among the advantages over other methods of entomopathogenic entomopathogenic nematodes and their bacteria rely on nematode strain improvement. Bacterial transfer and the accuracy of their ever-evolving , e.g., the hybridization are simpler than transgenic approaches, relatively recent division of the genus Photorhabdus into and probably more rapid than either transgenic or several species and subspecies (Fischer-Le Saux et al., selection methods. Furthermore, selection and trans- 1999) may explain some of the prior incompatibilities in genic manipulations generally focus on improvement transfers among heterorhabditids. of only one trait at a time, but we have demonstrated Other than the present study, the only other report of that improvement of several traits can be addressed at using hybridization to transfer beneWcial traits was in H. once through bacterial transfer and hybridization. bacteriophora (Shapiro et al., 1997). Heat tolerance was Selection also has the disadvantage of potential rever- transferred from the IS5 strain to a hybrid following sion back toward the trait’s original state once selec- controlled crosses with the (non-heat tolerant) HP88 tion pressure is removed, or selection for improvement strain (Shapiro et al., 1997). Hybridization among het- in a particular trait may inadvertently lead to selection erorhabditids can be challenging due to the diYculty of for an inferior level of another trait (Gaugler, 1987; discerning hermaphrodites from females (Adams and Gaugler et al., 1989, 1990). On the other hand, hybrid- Nguyen, 2002; Kaya and Stock, 1997; Koltai et al., ization and bacterial transfer are limited to improve- 1995). Shapiro et al. (1997) overcame this diYculty by ment increments within the genetic diversity or using marker mutations to verify that mating had interactions of the two strains being combined, whereas occurred. Hybridization of steinernematids is more selection can potentially maximize trait levels from the straightforward because only amphimictic forms exist in genetic diversity of numerous strains, and transgenic all species except one (Adams and Nguyen, 2002; Stock approaches can hypothetically utilize the genetic diver- et al., 2004). sity all organisms. When considering the two nematode improvement approaches, bacterial transfer and hybridization, we can expect that certain traits are more likely to be transmit- Acknowledgments ted or maintained through bacterial transfer rather than with hybridization, and vice versa. Certainly, bacterial We thank K. Halat, W. Evans, and G. Lathrop, for transfer is most likely to alter properties that are associ- technical assistance, and K. Nguyen for supplying nema- ated most closely with the nematode’s symbiont such as tode strains (Italian and DD-136). virulence. Virulence, however, may involve characteris- tics of the nematode as well, e.g., for penetration eYciency and immune suppression (Dowds and Peters, References 2002). Although bacteria quality may indirectly aVect some other traits in the nematode (e.g., through poor Adams, B.J., Nguyen, K.B., 2002. Taxonomy and systematics. In: Gau- nutrition), traits that are generally associated with the gler, R. (Ed.), Entomopathogenic Nematology. 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