Biological Control 54 (2010) 75–82
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Biological Control
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Compatibility of Heterorhabditis indica (Rhabditida: Heterorhabditidae) and Habrobracon hebetor (Hymenoptera: Braconidae) for biological control of Plodia interpunctella (Lepidoptera: Pyralidae)
George N. Mbata a,*, David I. Shapiro-Ilan b a Department of Biology, Fort Valley State University, 1005 University Drive, Fort Valley, GA 31030, USA b USDA, Agricultural Research Service, Southeastern Fruit and Tree Nut Research Laboratory, 21 Dunbar Road, Byron, GA 31008, USA article info abstract
Article history: The potential for integrating the application of Heterorhabditis indica Poinar, Karunakar, and David (Homl Received 12 August 2009 strain) and release of Habrobracon hebetor (Say) in the management of the Indianmeal moth, Plodia inter- Accepted 22 April 2010 punctella (Hübner), was investigated in the laboratory. A combination of the nematode and the parasitoid Available online 27 April 2010 was observed to increase the mortality of P. interpunctella. The interaction between the nematodes and parasitoids was not antagonistic but could possibly be additive or synergistic. Release of parasitoids or Keyword: application of nematodes alone generated between 62.25% and 71.25% mortality of the P. interpunctella Biological control larvae whereas combination of the two resulted in 98.0–99.25% mortality. The nematode was found to Entomopathogenic nematodes be virulent to the larvae of the parasitoid but not to the pupae and the adults. Adult female parasitoids Heterorhabditis indica Plodia interpunctella that were exposed to both uninfected and nematode-infected P. interpunctella larvae in a free-choice Habrobracon hebetor arena were unable to distinguish between the two. In contrast, infective juvenile nematodes preferen- Stored-products tially infected parasitized host larvae compared with healthy non-parasitized host larvae. Nematode reproduction was not significantly different in parasitized and non-parasitized host larvae. The combined application of H. indica and H. hebetor for the control of P. interpunctella may be beneficial if the detrimen- tal effects of the nematode on the parasitoid can be minimized through optimum timing. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction breakfast cereals and candies made with nuts and chocolate (Platt et al., 1998). Among all stored-product moth pests the Indianmeal moth, Plo- Pest management in post-harvest systems in the USA, from raw dia interpunctella (Hübner) (Lepidoptera: Pyralidae), is especially grain storage to food processing to value-added retail products, is important in the US and worldwide. P. interpunctella is a pest in facing a critical situation. Chemical insecticides have been used milling machinery and other food processing plants, warehouses, historically at all facets of the post-harvest handling of commodi- bakeries, and it contributes significantly to losses caused by insects ties. Some insecticides are effective and needed in some cases; oth- of stored commodities, particularly stored peanuts, stored cereals ers are probably ineffective and only add potentially harmful and processed food (Mbata and Osuji, 1983; Mbata, 1985, 1986). residues to food, which is cause for public concern (Foschi, P. interpunctella larvae spin silken threads, and mat infested prod- 1989). The Food Quality Protection Act of 1996 (FQPA, Anonymous ucts together using their silken webs. Female moths lay large num- (1996)) called for a re-evaluation of all pesticide labels and is tar- bers of eggs in food and on the walls and roofs of storage buildings geting organophosphate insecticides among which are some resid- (Mbata and Osuji, 1983; Mbata, 1985, 1986). The moth larvae hide ual insecticides (chlorpyrifos methyl, chlorpyrifos ethyl and in crevices and build up residual populations, which are responsi- malathion) commonly used by the food industry. Methyl bromide, ble for infestation of uninfested fresh commodities. Female moths an important fumigant in the management of stored-product in- lay eggs on or near food packages in response to food odors and sects, is an ozone-depleting substance that falls under an interna- also prior larval contamination (Phillips and Strand, 1994). Storage tional ban along with other organo-halides as mandated by the moths such as the P. interpunctella are considered among the most Montreal Protocol (Bell, 1996). In addition to the environmental is- common and visually obvious invaders of pet food, baking mixes, sues surrounding chemical insecticides are the facts that many populations of stored-product insects are resistant to commonly used chemicals (Georgiou and Saito, 1983; Subramanyam and Hag-
* Corresponding author. Fax: +1 (478) 825 6104. strum, 1995) and consumers continue to demand high quality food E-mail addresses: [email protected], [email protected] (G.N. Mbata). that is also pest and residue free. Since an efficient replacement for
1049-9644/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2010.04.009 76 G.N. Mbata, D.I. Shapiro-Ilan / Biological Control 54 (2010) 75–82 methyl bromide has yet to be found, it is reasoned that integration including Steinernema riobrave Cabanillas, Poinar, and Raulston, of IPM (integrated pest management) tools may be a plausible ap- which had previously been reported to possess the highest viru- proach. To overcome the challenges faced by the stored-product lence among several steinernematids (Ramos-Rodríguez et al., industry, alternatives to pesticides are proposed for the manage- 2006). ment of storage insects. The use of natural enemies such as parasit- Deployment of entomopathogenic nematodes with parasitoids oids, Habrobracon hebetor (Say) (Hymenoptera: Habrobraconidae), could be a pest management strategy against residual populations and entomopathogenic nematodes such as Heterorhabditis indica of P. interpunctella in warehouses or in other enclosed post-harvest Poinar, Karunakar, and David (Homl strain) (Rhabditida: Heteror- storage systems. Interactions between some hymenopteran para- habditidae), have the potential to become alternative pest manage- sitoids and entomopathogenic nematodes have been investigated, ment tools in post-harvest storage systems (Cline and Press, 1990; and the ensuing results were mixed with respect to virulence of Brower and Press, 1990; Mbata and Shapiro-Ilan, 2005; Shapiro- the nematodes to the beneficial insects (Shannag and Capinera, Ilan et al., 2009). 2000; Sher et al., 2000; Head et al., 2003; Lacey et al., 2003; Dillon Habrobracon hebetor is a gregarious, ectoparasitoid that attacks et al., 2008). Sher et al. (2000) found that eulophid parasitoid wasp the wandering stage of larvae of most stored-products moths Diglyphus begini Ashmead was not susceptible to infection by the (Akinkurolere et al., 2009; Benson, 1974; Eliopoulos and Stathas, entomopathogenic nematode Steinernema carpocapsae (Weiser). 2008). The life cycle of the parasitoid is characterized by a short Dillon et al. (2008) found that the entomopathogenic nematodes, larval stage and a long pupal stage (Payne, 1933). The pupa is ade- Heterorhabditis downesi Stock Burnell and Griffin, and S. carpocap- cticous and exarate and pupation takes place in a cocoon. When sae, did not affect natural populations of Bracon hylobii developing reared on the pyralid moth, Ephestia kühniella Zeller, at tempera- on Hylobius abietis. However, other studies observed antagonistic tures between 20 and 27 °C, eggs of H. hebetor hatched between relationship between parasitoids and entomopathogenic nema- 2 and 4 days, larval developmental period was between 5 and todes. Shannag and Capinera (2000) observed that S. carpocapsae 8 days, while pupae enclosed in 6–8 days (Payne, 1933). However, infected newly emerged and cocoon-spinning larvae of Cardichiles when reared on P. interpunctella at 25 °C and 70% RH, both larval diaphaniae Marsh, a solitary internal wasp parasitoid of the melon- and pupal developmental periods were slightly extended to about worm, Diaphania hyalinata (L.), and pickleworm D. nititalis (Stoll). 7–9 days and 8–10 days for larvae and pupae, respectively (Mbata, Lacey et al. (2003) found that S. carpocapsae caused 70.7–85.5% unpublished observation). H. hebetor has been investigated for its mortality in larvae of two ectoparasitic ichneumonid species of potential as a biological control agent (Ullyett, 1945; Press et al., the codling moth, Cydia pomonella (L.). 1974, 1982; Cline, 1989; Cline et al., 1984; Cline and Press, 1990; Our objective was to determine how P. interpunctella’s idiobiont Brower and Press, 1990). In laboratory tests, H. hebetor produced parasitoid, H. hebetor, might interact with the heterorhabditid a 97% reduction of residual population of Ephestia cautella (Walk- nematode, H. indica, in a concomitant use of the two for control er), a species related to P. interpunctella, in food debris (Reinert of residual moth populations. In laboratory experiments, we inves- and King, 1971; Press et al., 1982). Cline et al. (1984) and Cline tigated the effects of combined versus single applications of the and Press (1990) observed that H. hebetor caused a significant two biocontrol agents on P. interpunctella mortality, the impact of reduction in the level of E. cautella infestation in small experimen- combined application on nematode and parasitoid yield, direct vir- tal packages of corn meal and raisins by H. hebetor. Benson (1974) ulence of the nematode to the parasitoid (outside of the P. inter- observed that mortality of E. cautella was caused by the interaction punctella host), and choice tests to determine the ability of the with H. hebetor in a delayed-density dependent manner. H. hebetor parasitoid or nematode to distinguish between parasitized/in- has also been shown to cause significant suppression of P. inter- fected hosts. punctella (Brower and Press, 1990; Grieshop et al., 2006). Brower and Press (1990) with Grieshop et al. (2006) reported 71% suppres- 2. Materials and methods sion of P. interpunctella in ground cornmeal. Entomopathogenic nematodes in the genera Heterorhabditis and 2.1. Rearing of P. interpunctella Steinernema are potential alternatives to chemical insecticides (Grewal et al., 2005). In nature these nematodes are obligate para- The culture of P. interpunctella was originally obtained from sites that kill insects with the aid of mutualistic bacteria (Photor- USDA-ARS, Grain Marketing and Research Laboratory, Manhattan, habdus spp. for heterorhabditids and Xenorhabdus for Kansas, in 2001 and had been reared for nine generations at the steinernematids) that inhabit the intestine of the nematode (Poin- Department of Biology, Fort Valley State University, prior to the ar, 1990; Boemare, 2002). The free-living infective juveniles (IJs) commencement of this study. The culture was reared on a diet of enter their host through natural openings such as the mouth, anus cornmeal, chick laying mash, chick starter mash, oats and glycerin and spiracles, or occasionally through the cuticle, and then release (volumetric mixture at 4:2:2:2:1) and in an environmental cham- their bacteria that kill the host within 48 h (Poinar, 1990; Dowds ber set at 28 ± 1.5 °C, 70 ± 5% RH and a photoperiod of 16:8 (L:D). and Peters, 2002). The nematodes generally complete two to three generations before leaving the host-insect. These nematodes, which are harmful to insects but innocuous to mammals including 2.2. Rearing of H. hebetor humans, are important biological control agents in the manage- ment of several crop insect pests (Kaya and Gaugler, 1993; Shap- H. hebetor used in this study was obtained from a colony main- iro-Ilan et al., 2002; Grewal et al., 2005). tained at the Department of Entomology and Plant Pathology, Several species and strains of entomopathogenic nematodes Oklahoma State University, Stillwater, OK, USA. Adults of H. hebetor were investigated for virulence to P. interpunctella (Mbata and were transferred to a 1 L rearing jars containing about 50 last instar Shapiro-Ilan, 2005; Ramos-Rodríguez et al., 2006; Shapiro-Ilan of P. interpunctella at the wandering stage. The jars containing both et al., 2009). Of seven different heterorhabditid strains and species the moth larvae and the wasps had their tops screened with filter investigated, H. indica (HOM1) was found to be the most virulent to paper and placed in environmental chamber maintained at larval and adult stages of P. interpunctella (Mbata and Shapiro-Ilan, 28 ± 1.5 °C, 70 ± 5% RH and a photoperiod of 16:8 (L:D). Except 2005). Additionally, when targeting P. interpunctella larvae, Shap- where otherwise stated, H. hebetor used in experiments were 3- iro-Ilan et al. (2009) reported higher virulence in H. indica com- d-old adult males and females that were provided with 3–5 host pared with five other entomopathogenic nematode species larvae on emergence. Apart from the host larvae the wasps were G.N. Mbata, D.I. Shapiro-Ilan / Biological Control 54 (2010) 75–82 77 not provisioned with any source of drinking water or sucrose 2.6. Direct virulence of H. indica to H. hebetor solution. Parasitoid larvae were exposed to IJs that had been applied to 2.3. Nematode culture 90 mm filter paper (Whatman grade 40) in 1 ml of deionized water at a concentration of 8000 IJs/ml. The moist filter paper containing H. indica was reared at 25 °C in last instar greater wax moth, the nematodes was placed on the base of four 1 L storage jars. Galleria mellonella (L.), according to procedures described by Twenty 9-day old parasitoid larvae (from egg deposition) of H. heb- Woodring and Kaya (1988). The larvae of G. mellonella were ob- etor were placed on the moist filter papers in each of the four jars. tained from Webster’s Waxie Ranch (Webster, WI). Nematodes Four P. interpunctella larvae paralyzed by adult H. hebetor and were stored at 13 °C for 15 d or less before being used for experi- rinsed in distilled water to remove any parasitoid eggs were placed ments. The culture of H. indica used in experiments was originally in each jar to provide food for the developing parasitoid larvae. An- from Integrated BioControl Systems (Lawrenceburg, IN). other set of four jars containing parasitoid larvae along with para- lyzed P. interpunctella as a food source but no nematodes was set 2.4. Combined versus single applications of the two biocontrol agents up as control. The above procedure was duplicated for the pupae of H. hebetor with the exception that P. interpunctella larvae were The interaction between H. hebetor and H. indica was evaluated not provided to the pupae. The jars were placed in a chamber at two P. interpunctella larval densities (20 and 40) in 1 L storage maintained at 28 ± 1.5 °C and 70 ± 5% RH until the parasitoid larvae jars (7.4 cm id 16.8 cm h). For each replicate, a set of four jars and pupae developed into adults. The experiment continued until was set up at each density (20 or 40) of the host larvae. The jars no adults emerged for seven consecutive days. This experiment were distributed for the following four treatments: In treatment was run three times and data generated were combined. one, nematodes only were dispensed onto 9 cm filter papers (Whatman grade 40) with 2 ml of water containing 8000 IJs 2.7. Effect of parasitism on nematode yield in P. interpunctella larvae (=200 and 400 IJs per host-insect). In treatment two, parasitoids only were distributed six adults (three males and three females) Plastic Petri dishes (60 mm diam.) lined with filter papers were per jar. For treatment 3, both control agents were dispensed at inoculated with IJs at a concentration 4000 IJs in 0.35 ml of water the exposure rates and numbers indicated above, and the control per dish. Three P. interpunctella larvae previously killed through (treatment 4), contained filter paper with distilled water (2 ml). parasitism by H. hebetor were added to one dish inoculated with The tops of all jars were screened off with filter papers to prevent nematodes, and a second set of three healthy non-parasitized P. insects from escaping. The jars were placed in a rearing chamber interpunctella larvae was added to a second dish with nematodes. maintained at about 28 ± 1.5 °C, 70 ± 5% RH and LD 16:8 h for The dishes were held in a control chamber maintained at 25 °C 3 days after which the jars were checked for mortality of P. inter- and allowed to incubate for about 44 h. Dead P. interpunctella lar- punctella larvae and the parent parasitoids. The jars containing P. vae from each replicate were transferred to White traps and the interpunctella larvae exposed to parasitoids or to both IJs and par- number of IJs produced was counted 28 d post-inoculation (Wood- asitoids were returned to the rearing chambers until the emer- ring and Kaya, 1988). The total number of IJs per White trap was gence of F1 parasitoids. There were four replicates of each divided by the number of P. interpunctella larvae to obtain the yield treatment and the entire experiment was run three times (thus per larva. There were four replicates of each treatment. The exper- comprising three generations of P. interpunctella and H. hebetor). iment was repeated with three replicates of one larva per White trap. 2.5. Effect of prior exposure of P. interpunctella larvae to IJs on survival and progeny production of H. hebetor 2.8. Ability of H. hebetor to distinguish between nematode-infected and non-infected P. interpunctella larvae in a free-choice arena Sets of twenty and forty P. interpunctella larvae were transferred to 1 L storage jars with moist filter paper that had received either A 4 L beaker (height 25 cm; id 15 cm) was partitioned with a 4000 or 8000 IJs in 2 ml water. The tops of the jars were screened cardboard of 10 cm height across the diameter. The experiment off with filter papers. The jars were placed in a rearing chamber was carried out on a table with an incandescent lamp above. The and maintained at 28 ± 1.5 °C and 70 ± 5% RH for 3 days. Thereaf- illumination the lamp provided on the table was 720 l . One ter, six adults (three males and three females) of H. hebetor were fourth-instar P. interpunctella that was infected with H. indica (fol- placed in each of the jars. All P. interpunctella larvae exposed to lowing 2-day exposure of ten moth larvae to a total of 8000 in IJs at the rate of 400/larva were dead while some of the moth lar- 2 ml) was placed in one of the sectors of the beaker. The infected vae exposed at the rate of 200 IJs/P. interpunctella larva survived larva was dead and infection was evident by change of color of after a 3-day exposure period. However, all the P. interpunctella lar- the larva. A live 4th instar P. interpunctella unexposed to the nem- vae exposed to nematodes were used in setting up experiment atodes was placed in the second sector of the beaker. The top of the without regard to whether the larvae were infected or not by the beaker was screened off with muslin exposing only a 2-cm opening nematodes. Seven jars were set up at each nematode exposure rate. for introduction of two females of H. hebetor that had been kept A group of seven jars containing 20 healthy and unexposed P. inter- with males for 3 days, and were presumed to have been mated. punctella larvae was set up as the control with six adults (three The parasitoids were naïve with respect to exposure to P. inter- males and three females) of H. hebetor. The jars were placed in a punctella larvae as they were removed from culture jars as pupae. growth chamber with temperature and relative humidity as spec- Following the introduction of the female parasitoids into the bea- ified above but in addition, the chamber was illuminated (LD ker, the top of the beaker was completely taped off. The parasitoids 16:8 h). The adult parasitoids were checked for survival and re- were allowed 15 min to settle prior to the first observation. Subse- moved after six days. The jars were inspected for the emergence quently, the number of visits that the female parasitoids made to of parasitoid progeny starting on the 14th day after the experiment the P. interpunctella larvae was recorded. The larvae were observed was initiated. The number of emerged parasitoids was recorded. every 10 min for 2 h. If a parasitoid was noticed inspecting (walk- Inspection of jars for parasitoids continued until no parasitoids ing or landing on larva), stinging, or resting on one of the P. inter- emerged for six consecutive days. This experiment was run three punctella larvae, this was recorded as visit. Observation was times and resulting data were pooled and analyzed. discontinued and data discarded for any female parasitoid that ap- 78 G.N. Mbata, D.I. Shapiro-Ilan / Biological Control 54 (2010) 75–82 peared docile or did not walk or fly for two consecutive minutes. Seven replicate trials were carried out. Prior to each trial, the bea- ker was rotated through 180° to counter the effect of uneven illu- mination. Data generated for the seven trials were pooled and analyzed.
2.9. Ability of H. indica to distinguish between parasitized and non- parasitized P. interpunctella larvae in a free-choice arena
This experiment compared the attractiveness of parasitized P. interpunctella larvae versus non-parasitized larvae to the nema- todes. The parasitized P. interpunctella larvae were paralyzed and never developed further but died and provided nutrient for larval parasitoids. One parasitized and one non-parasitized P. interpunc- tella larvae were placed on base of a 60 mm Petri dish lined with filter paper (Whatman No. 1). Approximately 4000 IJs were added to the dish in 0.35 ml tap water. After 46 h, the P. interpunctella lar- vae were removed, rinsed in distilled water to remove any IJs adhering to the surface and dissected; the number of IJs invading each P. interpunctella larva was determined (Kaya and Stock, 1997). There were 10 replicate dishes in the experiment, and the experiment was repeated once in time.
2.10. Statistical analyses
Treatment effects were determined through analysis of variance (ANOVA; Proc GLM). Statistically significant treatment differences were further elucidated using the Student-Newman-Keul’s test (/ = 0.05) (SAS, 2001; Steel and Torrie, 1980). Prior to analysis, all data in percentages were arcsine of square root transformed
(Steel and Torrie, 1980). In experiments investigating combined Fig. 1. Mean (±SEM) percentage mortality of Plodia interpunctella larvae exposed to versus single application of the two biocontrol agents on P. inter- the nematode Heterorhabditis indica, the parasitoid Habrobracon hebetor, or their punctella mortality, the nature of the interaction (synergy, additiv- combinations. D.W. = distilled water (control), IJs = infective juvenile nematodes, ity, or antagonism) was determined through a comparison of IMM = Indianmeal moth (P. interpunctella). Means followed by the same letter are expected and observed mortality (Koppenhöfer and Kaya, 1997; not significantly different (P < 0.05; N = 40; 1a = 200 IJs/P. interpunctella larva; 1b = 400 IJs/P. interpunctella larva). Shapiro-Ilan et al., 2004). The expected additive proportional mor- tality ME for nematode–parasitoid combinations was calculated by ME = MN + MP (1 MN), where MN and MP are proportional mortal- ically stated whether the interaction between the parasitoid and ities caused by nematodes and parasitoids alone, respectively. Chi the nematode is either synergistic or additive because mortality 2 2 square (v ) value was calculated as (MNP – ME) /ME, where MNP is of P. interpunctella larvae was high at both rates of exposure to the observed mortality for the combination of nematodes and par- nematodes and parasitoids. asitoid. If the calculated value exceeded 3.84 (as specified for 1 df), Percentage H. hebetor parent adults (F0) used in nematode-par- a non-additive effect of the two control agents was indicated (Fin- asitoid experiment above that survived for up to two weeks ranged ney, 1964). If the difference MNP ME had a positive or negative between 70.0% for treatments that had parasitoids and nematodes value, a significant synergistic or antagonistic interaction was ad- at exposure rate of 400 IJs/P. interpunctella larva and 80.0% treat- duced, respectively. ments with parasitoids only (control, Table 1). The percentage sur- viving parent parasitoids were not significantly different (F = 2.08; 3. Results df = 3, 35; P = 0.1222). However, the mean numbers of H. hebetor progeny (F1 adults) that completed development under the various 3.1. Combined versus single applications of the two biocontrol agents treatments were significantly different (Table 1; F = 6.40; df = 2 and 35; P = 0.001). Fewer H. hebetor progeny were produced in the The effects of H. indica IJs, H hebetor adults, or a combination of both on the mortality of P. interpunctella larvae are given in Fig. 1. Table 1 At the two IJ exposure rates investigated, 200 and 400 IJs per P. Mean survival of parent Habrobracon hebetor (out of 6) and parasitoid progeny (F1 interpunctella larva (4000 and 8000 IJs/2 ml), the mortality of P. adults) on Plodia interpunctella larvae with or without exposure to Heterorhabditis interpunctella was highest when exposed to a combination of both indica.a nematodes and the parasitoid. Analysis of variance showed that % Surviving F0 ± SE Mean no. progeny ± SE mortality values resulting from the treatments were significantly Nematode 78.0 ± 6.96A 19.80 ± 4.43B different (for 200 IJs/insect: F = 276.23; df = 3, 36; P = 0.0001; for (200 IJs) + parasitoid 2 400 IJs/insect: F = 110.02; df = 3, 36; P = 0.0001). Result of the v Nematode 70.0 ± 7.56A 13.25 ± 3.17B indicated that the effects of the nematode and the parasitoid on (400 IJs) + parasitoid the mortality of P. interpunctella larvae were not antagonistic but Parasitoids alone 80.0 ± 9.43A 41.20 ± 6.44A could possibly be additive or synergistic at both nematode expo- a Nematodes (H. indica) were applied at 200 or 400 infective juveniles (IJs) per P. sure rates (v2 = 1.81 and 2.26; Me = 0.87 and 0.84; P < 0.05; at interpunctella larva. Means with each row followed by the same letter are not sig- 200 IJs/larva and 400 IJ/larva, respectively). It could not be categor- nificantly different (P < 0.05). G.N. Mbata, D.I. Shapiro-Ilan / Biological Control 54 (2010) 75–82 79 combination treatment (mean = 19.8 ± 4.42 at 200 Ijs/larva; and 100 13.25 ± 3.17 at 400 IJs/larvae) than in the parasitoid-alone treat- 90 ment (mean = 41.2 ± 6.44). 80 Control Parasitoids A 3.2. Effect prior exposure of P. interpunctella larvae to IJs on survival 70 Parasitoids exposed to nematodes A A and progeny production of H. hebetor 60 50 Exposure of parent adult parasitoids to nematode IJs had no sig- 40 nificant effect on the survival of adult parasitoids (Fig. 2; F=0.40; df = 2, 25; P = 0.6731). However, production of progeny by parasit- 30 B oid was significantly affected by the exposure of parasitoids to 20 nematode-infected P. interpunctella larvae (Fig. 3; F = 278.33; Adults of F0 Survivial Percentage 10 df = 2, 25; P < 0.0001). H. hebetor produced more progeny when of- 0 fered uninfected P. interpunctella (44.71 ± 2.50) compared to when Larvae Pupae Larvae Pupae offered nematode-infected P. interpunctella (infected at the 400 IJs per insect). The mean numbers of progeny produced at exposure Fig. 4. Mean (±SEM) percentages of Habrobracon hebetor parasitoid larvae and rates of 400 IJs/larva and 200 IJs/larva of P. interpunctella were 0, pupae exposed to Heterorhabditis indica nematodes that completed development. Means followed by the same letter are not significantly different and larvae are and 42.1 ± 2.95. The production of progeny by H. hebetor at compared only with larvae and pupae only with pupae (P < 0.05; N = 20). 200 IJs/ larva of P. interpunctella was not significantly different from the number produced from uninfected P. interpunctella larvae. man–Keul’s test showed that the mean number of parasitoid larvae 3.3. Direct virulence of H. indica to H. hebetor exposed to distilled water that completed development (50.8) was significantly higher than that exposed to H. indica IJs (20.4) Exposure of larval H. hebetor to H. indica IJs significantly affected (P < 0.0001). However, no difference was detected in mean number the number parasitoids that completed development (Fig. 4; of parasitoid pupae completing development. F = 19.08; df = 1, 18; P < 0.0005). Analysis with the Student–New- 3.4. Effect of parasitism on nematode yield in P. interpunctella larvae
100 The yield of nematode IJs in parasitized and non-parasitized P. 90 A A A interpunctella larvae is given in Fig. 5. No difference was detected 80 in nematode yields in non-parasitized versus parasitized hosts 70 (F = 1.44; df = 1, 12; P = 0.2782). 60 50 3.5. Ability of H. hebetor to distinguish between infected and non- 40 infected P. interpunctella larvae 30 The number of visits by H. hebetor was not significantly differ- 20 ent for nematode-infected and non-infected P. interpunctella larvae 10 (Fig 6; F = 0.36; df = 1, 12; P = 0.5686).
Mean % (± SE) F0 Parasitoid survival Mean % (± SE) F0 Parasitoid 0 400 IJs/IMM Larva 200 IJs/IMM Larva 0 IJs/IMM Larva 3.6. Ability of H. indica to distinguish between parasitized and non- Fig. 2. Mean (±SEM) percentage survival of the parasitoid, Habrobracon hebetor parasitized P. interpunctella larvae in a free-choice arena adults provided to Plodia interpunctella larvae previously exposed to the nematode Heterorhabditis indica at two exposure rates. IJs = infective juvenile nematodes, IMM = Indianmeal moth (P. interpunctella). Means followed by the same letter are The mean number of H. indica that chose parasitized P. inter- not significantly different (P < 0.05; N = 36). punctella larvae was significantly higher than those that chose non-parasitized larvae (Fig. 7; F=5.47; df = 1, 37; P < 0.0012).
50 A A 45 16000 A 40 14000 35 12000 30 A 25 10000 20 8000 15 10 6000 5 B 4000 Mean No. of Nematodes
Mean (± SE)Number of F1 Parasitoids 0 400 IJs/IMM Larva 200 IJs/IMM Larva 0 IJs/IMM Larva 2000
Fig. 3. Mean (±SEM) number of progeny of the parasitoid, Habrobracon hebetor 0 adults provided to Plodia interpunctella larvae previously exposed to the nematode Parasitized IMM Larvae Non-parasitzed IMM Larvae Heterorhabditis indica at two exposure rates. IJs = infective juvenile nematodes, IMM = Indianmeal moth (P. interpunctella). Means followed by the same letter are Fig. 5. Mean (±SEM) number of Heterorhabditis indica nematode yields in parasit- not significantly different (P < 0.05; N = 36). ized and non-Plodia interpunctella larvae (IMM; N = 14). 80 G.N. Mbata, D.I. Shapiro-Ilan / Biological Control 54 (2010) 75–82
5.0 In contrast to the lack of effects on adult parasitoids, production 4.5 of parasitoids had an inverse relationship to the exposure rate to A A nematode IJs. Reduction in the number of parasitoid progeny in 4.0 our study suggests that the nematode is virulent to the develop- 3.5 mental stages of H. hebetor. Indeed, the parasitoid larvae experi- 3.0 enced high mortality when exposed directly to the nematodes,
2.5 though the pupal stage was not susceptible. The results of some other studies on nematode virulence to parasitoids are analogous 2.0 to ours, whereas the results of other studies differ. For instance, 1.5 similar to our results with the adult parasitoid, Sher et al. (2000) 1.0 reported that adult D. begini were not susceptible to the nematode Mean No. Of Visits/ 2h period Visits/ Mean No. Of S. carpocapsae. Lacey et al. (2003) observed that combined applica- 0.5 tion of S. carpocapsae and developing larvae of two ectoparasitic 0.0 parasitoids, Mastrus ridibundus and Liotryphon caudatus, of the cod- Nematode Infected Larvae Non-Infected larvae ling moth, Cydia pomonella (L.), resulted in high mortality of the Fig. 6. Mean (±SEM) number of visits (inspections, landing, stinging) per 2-h period ectoparasitoids but diapausing larvae within cocoons were pro- from female Habrobracon hebetor parasitoids made to Heterorhabditis indica tected from nematode infection. Shannag and Capinera (2000) nematode-infected or non-infected Plodia interpunctella larvae in a free-choice determined from the study of infectivity of S. carpocapsae to C. arena. Means followed by the same letter are not significantly different (N = 14; diaphaniae Marsh, a solitary parasitoid of the melonworm and pic- P < 0.05). kleworm, that cocoon-spinning wasp larvae are less susceptible to infection than those without cocoons, and wasp pupae nearly unaf- 900 fected. Everard et al. (2009) observed that eclosion rate was not re- A duced when nematode H. downesi was applied to fully formed 800 cocoon (pupae) of the Braconidae B. hylobii. However, Everard 700 et al. (2009) observed that all the emerging adult parasitoids died but in the present study, the eclosed adults from pupae of H. heb- 600 etor exposed to nematodes survived. These studies imply that var- 500 ious developmental stages of parasitoids have varying B 400 susceptibilities to entomopathogenic nematodes and, thus confirm an earlier observation that older parasitoid stages are less affected 300 than young parasitoid stages by entomopathogenic nematodes Mean No. of Nematodes 200 (Kaya, 1978). It is probable that the older parasitoid stages may have developed an effective immunity against infection by the 100 nematode. In addition, the pupal cuticular surface and cocoon 0 may have provided a barrier that reduced infection. Since different Parasitized IMM Larvae Non-parasitzed IMM Larvae stages of parasitoids may exhibit various levels of susceptibility to infective juvenile of nematodes, combined use of the two natural Fig. 7. Mean (±SEM) numbers of Heterorhabditis indica nematodes choosing Plodia interpunctella larvae (IMM) that were either parasitized or non-parasitized by agents would be reliant on precise timing of parasitoid releases Habrobracon hebetor. Means followed by the same letter are not significantly (Sher et al., 2000). In addition, since the nematode IJs had little different (N = 40; P < 0.05). or no effect on the survival of adult female parasitoids, which are responsible for both killing P. interpunctella larvae and for progeny production, the combined use of the two natural enemies may call 4. Discussion for periodic augmentation release of adult parasitoids. When provided a paired choice of nematode-infected and non- Our data indicate that the parasitoid, H. hebetor, might be inte- nematode-infected host larvae, adult female wasps did not dis- grated with the application of the entomopathogenic nematode, H. criminate between the two. The inability of female parasitoid indica (HOM1 strain) for control of P. interpunctella, but the benefits wasps to distinguish between nematode-infected and non-infected and negative aspects of the combination must be evaluated care- host larvae has been observed in some nematode-parasitoid com- fully before adoption. On the positive side, our result showed no plexes. For instance, Head et al. (2003) reported that adult Digly- antagonistic interaction between the parasitoid and the nematode phus isaea did not discriminate between nematode-infected and but an interaction that could possibly be additive or synergistic. non-infected leafminer larvae. Sher et al. (2000) observed that The combined application resulted in enhanced mortality of the adult female wasps of D. begini were unable to discriminate be- P. interpunctella larvae compared to the use of either parasitoid tween nematode-infected and non-infected leafminer larvae. How- or nematode alone. Enhanced host-insect mortality has been previ- ever, Lacey et al. (2003) observed that M. ridibundus and L. caudatus ously observed when entomopathogenic nematodes were com- females were able to detect and avoid ovipositing on nematode-in- bined with parasitoids. For example, Dillon et al. (2008) observed fected cocooned codling moth. The inability of H. hebetor to distin- that interaction between the nematodes H. downesi or S. carpocap- guish between nematode-infected and non-infected host larvae sae and the parasitoid B. hylobii enhanced the mortality of the host- decreases the compatibility of the two agents and reduces their po- insect, Hylobius abietis. In contrast, Sher et al. (2000) observed sim- tential for concurrent use. However, we only tested recognition ilar mortality in larvae of the leafminer, Liriomyza trifolii when the shortly after infection; it may well be that as the infection process parasitoid wasp D. begini was applied alone compared with com- progresses in the host, the inability of H. hebetor to distinguish be- bined application of the parasitoid and S. carpocapsae. The en- tween infected and non-infected hosts may change over time (La- hanced effect of the nematodes and the parasitoids on the cey et al., 2003; Everard et al., 2009). mortality of the P. interpunctella larvae might have resulted from Interestingly, when provided a choice between parasitized and the fact that the nematode IJs had no significant impact on the sur- non-parasitized P. interpunctella larvae, the nematodes preferen- vival of adult parasitoids. tially invaded the parasitized host larvae. In a previous study, the G.N. Mbata, D.I. Shapiro-Ilan / Biological Control 54 (2010) 75–82 81 nematode, S. carpocapsae showed equal orientation between para- Everard, A., Griffin, C.T., Dillon, A.B., 2009. Competition and intraguild predation sitoid-parasitized and non-parasitized larvae (Sher et al., 2000). In between the braconid parasitoid Bracon hylobii and entomopathogenic nematode Heterorhabditis downesi, natural enemies of the large pine weevil, fact, to our knowledge this is the first report of an entomopatho- Hylobius abietis. Bullettin Entomological Research 99, 151–161. genic nematode’s preferential infection in a host that was parasit- Foschi, S., 1989. Pesticide residues in food. Difesa delle Piate 12, 41–64. ized by an insect. We contend that our findings support the Georgiou, G.P., Saito, T., 1983. Pest Resistance to Pesticides. Plenum Press, New York. Grewal, P.S., Ehlers, R.U., Shapiro-Ilan, D.I. (Eds.), 2005. Nematodes as Biocontrol hypothesis of Fushing et al. (2008), which predicts that entomo- Agents. 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