Genetic improvement of host-seeking ability in the entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora toward the Red Palm Weevil Rhynchophorus ferrugineus

Biological Control 100 (2016) 29–36

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Genetic improvement of host-seeking ability in the entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora toward the Red Palm Weevil Rhynchophorus ferrugineus Velayudhan Satheeja Santhi, Dana Ment, Liora Salame, Victoria Soroker, Itamar Glazer ⇑ Department of Entomology & Nematology, Institute of Plant Protection, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 S. carpocapsae and H. bacteriophora

host-seeking was enhanced by genetic selection.  Selection pressure was more effective in S. carpocapsae than H. bacteriophora.  Selection also improved infectivity and tolerance to desiccation stress.

a r t i c l e

i n f o

Article history: Received 11 January 2016 Revised 17 April 2016 Accepted 10 May 2016 Available online 11 May 2016 Keywords: Genetic improvement Steinernema carpocapsae Heterorhabditis bacteriophora Red palm weevil Attraction Infectivity

a b s t r a c t Rhynchophorus ferrugineus (red palm weevil) is highly susceptible to infection by the entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora. However, to reach and penetrate the insect in its habitat with good efficacy, the nematode infective juveniles (IJs) need to move through the tunnels bored by the feeding insect larvae in the tree trunk. We used a genetic-improvement approach to enhance host-seeking ability (HSA) of these nematode species’ IJs. The IJs were allowed to move through a 45-cm L-shaped tube toward red palm weevil larvae. The IJs which reached within 5 cm of the insect larvae were collected and reared. Selection cycles were repeated 15 times. The HSA of S. carpocapsae IJs was enhanced 11-fold (from 3.7 to 39.8% of all IJs reaching proximity of the larvae) and 8.5-fold (from 2.3 to 19.7%) for H. bacteriophora after 10 cycles of selection. Further selections (cycles 11–15) had no significant impact on improving HSA. HSA of the selected lines was highly specific to R. ferrugineus larvae. Selection for improved HSA also enhanced infectivity to R. ferrugineus, Galleria mellonella and Spodoptera littoralis. In addition, it enhanced host penetration 2.7-fold for S. carpocapsae and 1.5-fold for H. bacteriophora, and desiccation tolerance improved 1.2- and 1.7-fold, respectively. Heat tolerance and fecundity of the selected lines were somewhat reduced (MT50 by factors of 1.02 and 1.2 and fecundity by factors of 0.93 and 0.99, respectively) as compared to the foundation population. Improved HSA with substantial improvement of other traits may enhance field performance of these biocontrol agents. Ó 2016 Published by Elsevier Inc.

1. Introduction

⇑ Corresponding author E-mail address: [email protected] (I. Glazer). http://dx.doi.org/10.1016/j.biocontrol.2016.05.008 1049-9644/Ó 2016 Published by Elsevier Inc.

The red palm weevil (RPW), Rhynchophorus ferrugineus (Olivier) (Coleopetera: Curculionidae), is a devastating insect pest on different palm tree species worldwide. It originated in Southeast Asia,

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and later spread to the Mediterranean countries and the rest of the world, and has become a serious pest for date palms (Phoenix dactylifera L.) and Canary palms (Phoenix canariensis Chabaud). Damage caused by RPWs is mostly due to the larval stages that feed inside the tree’s trunk (Saleh and Alheji, 2003; Soroker et al., 2005). In date palm trees, the adult weevils lay hundreds of eggs in the lower part of the stem where the offshoots join the trunk, or in the wounds of detached offshoots. The neonate larvae start feeding extensively on the tissues, generating feeding tunnels within the trunk. Heavy infestation compromises stem stability and the palm eventually collapses, usually before any visible symptoms appear. The biology and concealed-feeding behavior of the RPW make it a most challenging pest to control (Blumberg, 2008). Management of crown infestation by chemical or biological means is possible, but delivering them to the infested trunk is difficult. One possible biological control agent is the entomopathogenic nematode (EPN), which detects, reaches, invades and kills its prospective host. The EPNs Steinernema carpocapsae and Heterorhabditis bacteriophora, belonging to the families Steinernematidae and Heterorhabditidae, respectively, are lethal obligatory parasites of different insect pests (Griffin, 2012). They play a major role as biocontrol agents in crop protection (Ehlers, 2001). The third-stage infective juvenile (IJ) is the non-feeding stage of the EPN. It lives freely in the environment, targets potential insect hosts via its hostseeking ability (HSA), invades the insect’s body and releases a symbiotic bacterium (Xenorhabdus for the Steinernematidae and Photorhabdus for the Heterorhabditidae). The released bacteria rapidly multiply and kill the insect by septicemia. The EPN feeds on the symbiotic bacteria as well as the tissues of the dead insect, develops, reproduces and releases numerous IJs (Forst and Clarke, 2002), which in turn search for further target insects. S. carpocapsae and H. bacteriophora are well documented for their infectivity against RPWs (Elawad et al., 2007; Atakan et al., 2012; Manachini et al., 2013). Santhi et al. (2015) recently showed that all larval, pupal and adult developmental stages of RPW are susceptible to nematode infection. To obtain effective control of target pests, it is necessary to ensure that the applied nematodes reach the target pest in sufficient quantities. Santhi et al. (2015) showed that under simulated natural conditions in tubes, exposure of RPW larvae to IJs of both nematode species increased the proportion of IJs found near the insect 2- to 2.3-fold compared to natural distribution without the presence of the insect. However, the proportion that reached the insect’s proximity was relatively low. Increasing the proportion of IJs that are attracted to pests in the tunnels bored into the trunk would likely enhance the efficacy of these biological control agents. One of the most successful approaches to enhancing biological control agent performance is genetic improvement (Hoy, 1990). In EPNs, genetic improvement has been used to enhance important traits, including virulence, stress tolerance, storage and hostseeking (Gaugler and Campbell, 1989, 1991; Shapiro-Ilan et al., 2012; Anbesse et al., 2013a,b; Nimkingrata et al., 2013; for review see Glazer, 2015). In the present study, we demonstrate the use of this system to select for and enhance the HSA of S. carpocapsae and H. bacteriophora for RPW. We also verified some fitness traits of the selected population.

2. Materials and methods 2.1. EPNs and insects: sources and culture The initial foundation strains (FSs) of S. carpocapsae and H. bacteriophora, which were subjected to selection, were obtained from e-nema GmbH (Schwentinental, Germany) and further reared in

the late-instar larvae of Galleria mellonella (Kaya and Stock, 1997). RPW larvae were obtained from cultures at the Department of Entomology, ARO and ‘‘Eden” Experimental Station in Bet Shean Valley, Israel. They were reared by capturing adult RPW by ‘‘Picosan” trap (Sansan, Náquera, Valencia, Spain). The larvae emerging from eggs laid by the captured females were kept in plastic containers and fed pieces of sugarcane. The insects were kept in the dark at 27 ± 2 °C. For selection purposes, 20–35-mm long insect larvae (big stage) were used. For the infectivity assay, we used larvae of G. mellonella and Spodoptera littoralis. Both were reared at the Department of Entomology, ARO, according to standard procedures (Woodring and Kaya, 1988; Ben-Aziz et al., 2006). 2.2. Genetic selection for HSA toward RPWs We used the L-shaped tube system described by Santhi et al. (2015), comprised of a 45-cm L-shaped plastic PVC tube with an inner diameter of 4.5 cm. The tube was filled with moist (10% w/ w) coconut pith. One big stage RPW larva (20–35-mm long) encased inside a perforated 5-cm Petri dish was placed on the upper end of the vertical side of the tube. Approximately 10,000 IJs were inoculated at the opposite, lower end of the horizontal side of the tube. Control treatment consisted of a similar tube without insect larvae. HSA was measured as the proportion (%) of IJs that reached the upper 5 cm of the tube in proximity of the insect larvae after 24 h (the incubation period was intensified to 16 h after the sixth selection cycle). The number of nematodes was quantified after recovering them by Baermann funnel technique (Salame et al., 2010; Santhi et al., 2015). The collected IJs were then exposed to the 10 last-instar larvae of G. mellonella in 5-cm diameter Petri dishes layered with moist filter paper. The IJs that emerged from the G. melonella cadaver were used immediately for the next round of selection in the L-shaped tubes. Fifteen selection cycles were performed before determining the fitness of the selected populations of both nematode species. In each selection cycle, the selected population was compared in all treatments to nonselected IJs (FSs) of S. carpocapsae and H. bacteriophora. Each selection cycle consisted of three replicates/tubes. 2.3. Fitness bioassays 2.3.1. Infectivity by dose-response assay The dose-response assay was conducted with G. mellonella lastinstar larvae, RPW (10–20-mm long medium stage) larvae and Sp. littoralis last-instar (larvae). The insects were exposed to various concentrations (0, 125, 250, 500 or 1000) of selected IJs (from the 15th selection cycle—Selected Population = SP15) or nonselected IJs in a Petri dish (5.0 cm diameter) lined with moist Whatman No. 1 filter paper for 96 h at 25 ± 1 °C. Each Petri dish contained five larvae of the different insect species for G. mellonella and Sp. littoralis and one medium-stage larva of RPW. After the incubation period, mortality of the insect larvae was recorded (Salame et al., 2010). For each insect species, the dose-response assay was performed in five replicates (Petri dishes) and repeated twice. 2.3.2. Invasion-rate assay An invasion-rate assay (Glazer and Lewis, 2000) was used as an additional measure of SP15 infectivity. Five last-instar G. mellonella larvae were exposed to 500 EPN IJs for 24 h at 24 ± 1 °C in a Petri dish containing moist filter paper. Then the larvae were washed to remove the nematodes on their surface and incubated on fresh Petri dishes for 48 h at 24 ± 1 °C. The larvae were then dissected in Ringer’s solution and the invading nematodes were counted under a stereo microscope (Glazer and Lewis, 2000). The

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experiment was repeated twice, each experiment with five replications (total 10 Petri dishes and each Petri dish has 5 last-instar G. mellonella larvae).

2.3.3. Attraction to other insects A coconut pith-filled 15-cm PVC tube (4.5 cm inner diameter) was used to attract SP15 S. carpocapsae and H. bacteriophora in the presence or absence of last-instar larvae of G. mellonella and Sp. littoralis. One end of the column was closed with the four insect larvae encased inside a perforated Petri dish and other end of the column was inoculated with 10,000 IJs. The column setup was incubated at 25 °C in a horizontal position for 48 h. Then the coconut pith substrate inside the column was divided into three segments: material near the IJ-inoculated end (A), central region of the tube (B), and the section near the insect host end (C), each segment comprised of 5.0 cm of filling material. The IJs dispersed across each of the segments were recovered using a Baermann funnel and quantified under a stereo microscope (Salame et al., 2010).

2.3.4. Fecundity/reproduction capacity Five G. mellonella last-instar larvae, with an average weight of 0.7 ± 0.06 g (each), were exposed to 500 IJs of the selected or non-selected populations in small Petri dishes (5-cm diameter) lined with moist Whatman No. 1 filter paper. After 72 h exposure at 25 ± 1 °C, the dead larvae were transferred to ‘White Traps’ (White, 1927) and incubated for 14 more days. The IJs emerging from the cadavers were collected daily from day 8 to day 14 and total IJs were counted. Fecundity capacity was determined as IJs produced per milligram live larva weight. The experiments were conducted twice and each experiment had five Petri dishes.

2.3.5. Desiccation tolerance Desiccation tolerance was determined according to the procedure described by Solomon et al. (2000). IJs (n = 10,000) were filtered on a Whatman No. 1 filter paper using air suction from a vacuum pump. The IJ-covered filter paper was air-dried at room temperature for 30 min to remove excess water and anhydrobiosis was induced at 97% relative humidity (RH) for 72 h by incubating in a sealed desiccator with 60 ml saturated K2SO4 salt solution at 24 ± 1 °C. Then 5000 batches of IJs were desiccated at 93% RH in desiccators with a saturated KNO3 solution and at 85% RH in desiccators with a saturated KCl solution at 24 ± 1 °C (Solomon et al., 2000). IJ survival was recorded after 24, 48, 72 and 96 h. The control treatment consisted of keeping IJs in water at 24 °C for the different times up to 96 h. Ten replicates were tested for each nematode population.

2.3.6. Heat tolerance Heat-stress tolerance of the selected populations was analyzed for the SP15. IJs (n = 10,000) suspended in 3 ml tap water were transferred to 20-ml disposable glass vials and exposed to 35 °C for 3 h for heat-stress induction. Heat-stress tolerance was determined by transferring a batch of 300 IJs to the vial in 5 ml tap water and exposing them to different temperature regimes of 37, 39 and 41 °C for 2 h. After the heat treatment, the IJs were transferred to Ringer’s solution for 24 h at 24 ± 1 °C and the surviving nematodes were counted under the microscope (Ehlers et al., 2005). The control consisted of nematodes kept at 24 °C in water. Control and treatments consisted of five replicates and the experiments were repeated twice. The heat tolerance was defined by the mean temperature tolerated by 50, 30 and 10% of the population (MT50, MT30 and MT10 respectively).

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2.4. Statistical analysis Statistical analysis of all of the data was performed using JMP10 (SAS Institute Inc., Cary, NC). Briefly, analysis of variance (ANOVA) (P 6 0.05) was performed to determine the difference between foundation and genetically selected lines. To delineate the means, one-way ANOVA with Student’s t-test or Tukey-Kramer HSD multiple-comparison test (P 6 0.05) was performed. 3. Results 3.1. Genetic selection of EPNs for host-seeking ability toward RPW The percentages of S. carpocapsae and H. bacteriophora found per cm3 in the upper 5-cm region of the L-shaped tube over the different generations are shown in Fig. 1. The percentage of nematodes attracted to the RPWs was always higher among selected populations and it was significantly different from the dispersal of IJs in the L-tube with no RPW at the end of upper 5-cm section (S. carpocapsae: F = 35.39, df = 2, P < 0.0001; H. bacteriophora: F = 57.75, df = 2, P < 0.0001). In the FS populations, 3.7 ± 0.23% of S. carpocapsae IJs and 2.3 ± 0.16% of H. bacteriophora IJs reached the proximity of the insects in the L-shaped tube system. After the first selection cycle, the proportion of IJs of S. carpocapsae and H. bacteriophora attracted to the insect increased by 2- to 3-fold (7.6 ± 1.3 and 7.06 ± 1.2%, respectively; Fig. 1A and B, respectively). After six selection cycles of S. carpocapsae IJs in the L-shaped tube, the percentage of nematodes, which had increased gradually in each generation, reached up to 7-fold (3.7 ± 0.23–25.9 ± 2.46%). After selection intensification (16 h incubation), a drastic reduction was recorded in the proportion of selected IJs reaching the target insect. This was followed by a further increase in attraction to 38 ± 1.35–40 ± 1.71% of the population in the subsequent generation. Despite the continuous selection, the level of attraction did not increase after the 10th selection cycle (Fig. 1A), reaching an 11-fold increase in attraction to the host compared to the original non-selected lines. The response of H. bacteriophora to selection differed from that of S. carpocapsae (Fig. 1B). Initially, a rapid increase in host-seeking was observed, reaching 17-fold (from 2.3 ± 0.16 to 39 ± 2.46%) that of the non-selected population after four selection cycles (Fig. 1B). In the fifth and sixth selection cycles, a decline in host attraction was recorded despite the selection pressure. Following the intensification, there was a gradual increase in attraction to the host, reaching 19 ± 1.03% at the 10th selection cycle. No further increase in host attraction was recorded among the H. bacteriophora IJs in the subsequent selection cycles (Fig. 1B). The rate of H. bacteriophora IJs’ HSA was lower than that of S. carpocapsae IJs (Fig. 1A and B). Even though H. bacteriophora exhibited higher attraction than S. carpocapsae under the 24-h selection pressure, its percentage increase in attraction was not homogeneous relative to S. carpocapsae. Overall, the selection pressure at 16 h improved HSA 11-fold (3.7 ± 0.23–39.8 ± 1.71%) in S. carpocapsae and 8.5-fold (2.3 ± 0.16–19.7 ± 1.03%) in H. bacteriophora. The improvements in HSA for these two nematodes differed significantly from each other (F = 106.65, df = 2, P = 0.0092). 3.2. Infectivity based on dose-response assay Infectivity based on a dose-response evaluation of the genetically selected populations also displayed improvement over the control. The infectivity of S. carpocapsae was more pronounced than that of H. bacteriophora. The treatment with 250, 500 and 1000 S. carpocapsae nematodes did not show a dose-dependent

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Fig. 1. Effect of repeated exposure of S. carpocapsae (A) and H. bacteriophora (B) infective juveniles (IJs) to larvae of the red palm weevil (RPW) R. ferrugineus in an L-shaped tube. Nematodes that were found near the larvae 24 h after exposure were collected, reared on G. mellonella larvae, and their progeny were re-exposed to the RPW in the tube. After six cycles of selection, the regime was intensified by reducing the exposure time to 16 h. Controls consisted of FS = non-selected foundation strains and Cont = tubes with no insects. Error bars depict SEM of the average percentage of IJs/cm3 in the top 5-cm section of the L-shaped tube. Levels with different letters are significantly different by ANOVA with Tukey-Kramer HSD multiple comparison test, P < 0.05, n = 3.

effect due to the high infectivity of the selected and non-selected populations of this EPN (Fig. 2). However, a significant variation in infectivity was observed with the lowest number of IJs (125 per dish) tested for SP15 against the non-selected S. carpocapsae (F = 36.0, df = 9, P < 0.0001). In the case of H. bacteriophora, the selected populations displayed enhanced infectivity relative to controls. The selected H.

bacteriophora at the lowest dose (125 IJs per dish) caused 98 ± 2.0% mortality of the G. melonella larvae, differing significantly from controls (F = 187.27, df = 9, P < 0.0001); 100% mortality of G. melonella larvae was achieved at the higher doses (250–1000 IJs per dish) and differed significantly from the FS lines (F = 6.0, df = 9, P = 0.0248). The selected populations of these two EPNs also displayed enhanced infectivity of RPW and Sp. littoralis larvae.

Fig. 2. Infectivity against G. mellonella based on dose-response assay. FS = non-selected foundation strains and SP15 = selected populations after 15 selection cycles. Infectivity assay was performed in five replicates (Petri dishes) and repeated twice. Each bar denotes mean percentage of larval mortality ± SEM; levels with different uppercase and lowercase letters are significantly different according to ANOVA with Tukey-Kramer HSD multiple comparisons, P < 0.05. IJ, infective juvenile.

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3.3. Attraction to G. mellonella and Sp. littoralis The selection pressure to enhance HSA toward RPWs did not have a similar impact on the EPNs’ attraction to other insects. The selected populations of S. carpocapsae and H. bacteriophora did not show any stronger attraction to G. mellonella or Sp. littoralis compared to their FSs (Fig. 3). After 48 h, only 4.31 ± 0.53% of the selected S. carpocapsae IJs were observed near G. mellonella, 15fold less than the number of FS IJs (60.48 ± 3.91%) attracted to this insect. Similarly, the percentage of selected S. carpocapsae observed near Sp. littoralis (8.05 ± 3.83%) was 5.9-fold less than that of the FSs (47.05 ± 0.31%). In addition, the average number of selected H. bacteriophora IJs observed near G. mellonella and Sp. littoralis (14.26 ± 2.42 and 18.02 ± 2.93%, respectively) was nearly 3-fold less than that of the FSs (46.38 ± 8.94 and 48.10 ± 9.79%, respectively). 3.4. Infectivity by invasion rate Selected populations of S. carpocapsae and H. bacteriophora IJs displayed improved infectivity, with the number of nematodes invading each larva increasing through the generations (Fig. 4). The average number of invading S. carpocapsae nematodes in the selected population was 42.2 ± 3.92, significantly different (F = 33.34, df = 9, P < 0.0001) from the number of invading nematodes observed in the non-selected population (15.7 ± 3.65). H. bacteriophora also displayed improved infectivity upon selection pressure. The average number of invading H. bacteriophora IJs of the selected line was 29.1 ± 2.92, significantly different from the number of invading IJs (19 ± 2.88) of the non-selected population (F = 6.19; df = 9; P = 0.0479). Enhancement of the infectivity trait was documented more often in S. carpocapsae than in H. bacteriophora. The overall increase in infectivity by means of invasion rate was 2.7-fold in S. carpocapsae and 1.5-fold in H. bacteriophora.

Fig. 4. Infectivity based on nematode invasion of G. mellonella. FS = non-selected foundation strains and SP15 = selected populations after 15 selection cycles. Assay was performed in five replicates (Petri dishes) and repeated twice. Levels with different letters are significantly different by ANOVA with Student’s t-test, P < 0.05.

3.5. Fecundity/reproduction capacity Reproduction capacity of the genetically selected S. carpocapsae population was significantly reduced (SP15: F = 8.23, df = 9, P = 0.0102) by a factor of 1.2 compared to the non-selected population. But the H. bacteriophora population selected for attraction to the RPW showed no significant (SP15: F = 0.07, df = 9, P = 0.7958) changes in fecundity relative to the non-selected population (Fig. 5). These results suggest that in the EPN species used

Fig. 5. Reproduction capacity of the nematodes genetically selected for attraction to red palm weevil. FS = non-selected foundation strains and SP15 = selected populations after 15 selection cycles. The assay was repeated twice and each experiment had five replicates (Petri dishes). Levels with different letters are significantly different based on ANOVA with Student’s t-test, P < 0.05. IJ, infective juvenile.

Fig. 3. Attraction of selected populations to G. mellonella and S. littoralis. FS = non-selected foundation strains and SP15 = selected populations after 15 selection cycles. Error bars depict SEM of the average percentage of nematodes observed in the IJ-inoculated end (A), central region (B), and insect host end (C) of the tube. Levels with different letters are significantly different by ANOVA with Tukey-Kramer HSD multiple comparison test; P < 0.05, n = 4.

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in this study, the selection process played a minor role in changing fecundity capacity.

3.6. Tolerance to heat stress The heat tolerance of genetically selected S. carpocapsae and H. bacteriophora populations deteriorated compared to their respective FSs (Fig. 6). Among the controls kept at 24 °C, there was no significant difference between the non-selected population and the SP15 of S. carpocapsae (F = 1.07, df = 9, P = 0.3151). Survival of the selected lines under induced heat stress (35 °C for 3 h) was 2.6fold less than that of the non-selected population. The latter attained MT50 at 35.5 °C. At 37 °C, MT10 was recorded for the genetically selected S. carpocapsae and a significant difference was observed for the survival of selected and non-selected S. carpocapsae (F = 140.05, df = 9, P 6 0.0001). The heat tolerance of the selected H. bacteriophora was also lower than that of their FSs. The non-selected H. bacteriophora showed MT10 and selected lines MT30 at 37 °C, a statistically significant difference (F = 164.39, df = 9, P > 0.0001). Overall the SP15 displayed reduced MT50, by a factor of 1.02 and 1.2 for S. carpocapsae (35.5–35.2 °C) and H. bacteriophora (37.5–35 °C), respectively.

3.7. Desiccation stress The survival of the selected and FS EPN populations exposed to evaporative desiccation stress is shown in Fig. 7. Desiccation tolerance was enhanced among the selected populations of S. carpocapsae and H. bacteriophora. Statistical analysis also revealed a significant difference in desiccation tolerance level at 85% RH (S. carpocapsae: F = 11.84, df = 9, P = 0.0029 and H. bacteriophora: F = 253.89, P < 0.0001) and 93% RH (S. carpocapsae: F = 49.80, df = 9, P < 0.0001 and H. bacteriophora: F = 151.78, df = 9, P < 0.0001) between the selected populations and FSs. Overall, among the selected lines, desiccation tolerance was enhanced 1.2- to 1.4-fold (47 ± 2.3–53 ± 2.0%) for S. carpocapsae and 1.4- to 1.7-fold (38 ± 1.9–61 ± 1.1%) for H. bacteriophora at 96 h.

4. Discussion Repetitive selection of both S. carpocapsae and H. bacteriophora dramatically increased (11-fold and 8.5-fold, respectively) their HSA toward RPW larvae. Similarly, 13 rounds of selection of Steinernema feltiae also resulted in a 20- to 27-fold increase in its HSA (Gaugler et al., 1990). Furthermore, genetic improvement of HSA in the heterogeneous populations of S. feltiae showed more than 75% of the nematodes closest to G. mellonella in a sand column after 25 selection cycles (Salame et al., 2010). All of these studies reported improvement of HSA by the selection process. The genetic architecture of HSA in EPNs is not well understood. In the present study, the pattern of enhancement over the selection cycles differed between the two nematode species. The differences in response to selection pressure may be attributed to their level of genetic heterogeneity as commercial strains which have been cultured in vitro for many generations. These differences may be affected by their reproductive biology. While S. carpocapsae develop into reproductive males and females (i.e. amphimictic reproduction), resulting in higher heterogeneity, H. bacteriophora reproduce hermaphroditically and may have a higher level of homogeneity. The genetic-selection process is associated with changes in behavioral and physiological traits (Gilbert, 2001). Similar to this report, the present study also adds HSA to this list. Both behavior and physiology of EPNs are pertinent to infectivity and adaptation to environmental stress conditions. The repeated selection pressure applied in each cycle pushes the nematodes to migrate quickly in the L-tube as they target their host, resulting in enhanced competitive ability to reach the host. The HSA of the selected nematode populations began to drift back (4.1% for S. carpocapsae and 4.3% for H. bacteriophora) to close to that of the FSs when the selection cycle was relaxed to three consecutive cycles of rearing in G. melonella and it was restored after imposing selection pressure for four to five generations (18% for S. carpocapsae and 14.5% for H. bacteriophora). This phenomenon is generally due to genetic homeostasis (Gaugler and Campbell, 1989) and the selected traits can be stabilized by inbreeding and further selection. Trait stability was not evaluated in the present study. Bai et al. (2005) suggested stabilizing beneficial traits in

Fig. 6. Tolerance to heat stress of the selected nematodes after 15 cycles of selection (SP15) compared to that of the non-selected foundation strains (FS). The assay was performed with five replicates and the experiments were repeated twice. Error bars depict SEM of the average percentage of surviving nematodes under each temperature regime and statistical significance was determined by ANOVA with Tukey-Kramer HSD multiple comparison test; P < 0.05.

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Fig. 7. Tolerance to desiccation stress of the genetically selected S. carpocapsae and H. bacteriophora vs. the foundation population. FS = non-selected foundation strains and SP15 = selected populations after 15 selection cycles. Levels with different letters are significantly different by ANOVA with Tukey-Kramer HSD multiple comparison test; P < 0.05, n = 10.

H. bacteriophora through the creation of genetically homozygous inbred lines that can deter beneficial trait decline. Other studies also suggested that selection relaxation results in a reduction in performance (Shapiro-Ilan et al., 1996; Anbesse et al., 2013a). Relaxation of selection pressure produced a decrease in hostfinding among the selected S. feltiae populations (Gaugler and Campbell, 1989) and repeated sub-culturing also reduced the HSA of H. bacteriophora populations (Bilgrami et al., 2006). Effective performance of the EPNs depends on enhancement of their HSA, reproductive potential and environmental stress tolerance (Shapiro and Glazer, 1996; Glazer, 2015). In the present study, the selected lines were improved for HSA along with a few other traits, such as host penetration and desiccation tolerance. The S. feltiae population selected for enhanced downward dispersal in a sand column displayed significantly higher infectivity than the foundation population (Salame et al., 2010). Similar to that report, selection pressure for attraction to the RPW also enhanced the infectivity trait, more so among the S. carpocapsae lines as compared to H. bacteriophora. Infectivity (dose response) was enhanced against a range of host insects; RPW, G. mellonella and Sp. littoralis. The studies of host-seeking improvement performed with S. feltiae reported a gain in host penetration and reproductive potential of the selected lines after 13 rounds of host-finding assays (Gaugler and Campbell, 1989; Gaugler et al., 1990). Similar to that report, the present study also showed a 1.5- to 2.7-fold gain in host penetration. In contrast, the selected populations displayed only minor changes in their reproduction capacity. The reason for the minor reduction (S. carpocapsae) or no significant change (H. bacteriophora) in reproduction capacity of the selected populations is not precisely known, but it may be speculated on: H. bacteriophora generally exhibits self-fertile hermaphroditic reproduction potential and development (Zioni et al., 1992) and S. carpocapsae shows a reproduction cycle that involves development into distinct, individual male and female nematodes (Griffin, 2012). Genetic selection for attraction to insects caused some reduction in heat tolerance. Similarly, nematode species in Bilgrami et al. (2006) also suffered from deterioration of heat tolerance in a series of in vivo culturing. However, in another study, a S. feltiae population subjected to selection for desiccation tolerance displayed enhanced heat tolerance relative to the foundation population (Salame et al., 2010). Hence, selection for certain traits also positively or negatively affects their associated traits, depending on the nature of the trait under investigation. Less tolerance to heat

stress might need to be taken into account for the control of insect pests under field conditions. Although it has not been tested specifically under field conditions, it restricts the use of selected EPN populations to favorable temperature regimes. Desiccation tolerance is the ability to survive dehydration stress via reversible cessation of metabolism (Phillips et al., 2002) and it is essential for survival and persistence under dry conditions (Glazer, 2015). The enhanced desiccation tolerance of the selected populations of S. carpocapsae (1.2- to 1.4-fold) and H. bacteriophora (1.4- to 1.7-fold) denotes their better fitness to withstand desiccation stress. Similarly, the selected populations of S. feltiae and liquid cultures of H. bacteriophora exposed to desiccation stress demonstrated better tolerance levels than their non-selected counterparts (Salame et al., 2010; Anbesse et al., 2013a). Overall, the selection process improved attraction, infectivity, and survival of desiccation stress with deterioration of heat tolerance. The selection pressure was more effective in improving the HSA of S. carpocapsae than that of H. bacteriophora. The role of genetic selection in the EPNs’ behavior in controlling insect pests and their performance in a natural environment remain to be investigated. Acknowledgments This work was supported by a grant from the Chief Scientist of the Ministry of Agriculture, Program No. 131-1686-11. The authors thank the ARO Postdoctoral Fellowship Program 2013–2015 for support of Dr. Satheeja Santhi. References Anbesse, S., Sumaya, N.H., Dörfler, A.V., Strauch, O., Ehlers, R.-U., 2013a. Selective breeding for desiccation tolerance in liquid culture provides genetically stable inbred lines of the entomopathogenic nematode Heterorhabditis bacteriophora. Appl. Microbiol. Biotechnol. 97, 731–739. Anbesse, S., Sumaya, N.H., Dörfler, A.V., Strauch, O., Ehlers, R.-U., 2013b. Stabilisation of heat tolerance traits in Heterorhabditis bacteriophora through selective breeding and creation of inbred lines in liquid culture. Biocontrol 58, 85–93. Atakan, E., Yüksel, O., Soroker, V., 2012. Current status of the red palm weevil in Canary Island date palms in Adana. Türk. Entomol. Bült. 2, 11–22. Bai, C., Shapiro-Ilan, D.I., Gaugler, R., Hopper, K.R., 2005. Stabilization of beneficial traits in Heterorhabditis bacteriophora through creation of inbred lines. Biol. Control 32, 220–227. Ben-Aziz, O., Zeltser, I., Bhargava, K., Davidovitch, M., Altstein, M., 2006. Backbone cyclic pheromone biosynthesis activating neuropeptide (PBAN) antagonists:

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