Journal of Pest Science (2018) 91:799–813 https://doi.org/10.1007/s10340-017-0942-0

ORIGINAL PAPER

Increasing entomopathogenic biodiversity reduces efcacy against the Caribbean fruit fy Anastrepha suspensa: interaction with the parasitoid Diachasmimorpha longicaudata

William K. Heve1 · Fahiem E. El‑Borai1,2 · Daniel Carrillo3 · Larry W. Duncan1

Received: 17 August 2017 / Revised: 22 November 2017 / Accepted: 1 December 2017 / Published online: 8 December 2017 © Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract Entomopathogenic nematode (EPN) species richness merits studies towards making rational decisions for efective manage- ment of Caribfy, Anastrepha suspensa (Loew) in southern Florida. Competition for Caribfy and efcacies of EPN biodi- versity were examined under laboratory conditions. Similar EPN species treatments to Caribfy-infested fruits, periodically obtained from the ground in groves which were also infested by the parasitoid Diachasmimorpha longicaudata Ashmead (Braconidae), were studied in a series of feld trials. Treatments with individual EPN species and their mixtures caused similar mortalities of Caribfy larvae, though the various EPN species competed for larvae in multiple-species treatments. Laboratory trials showed that mortalities of EPN-treated Caribfy pupae were mostly inversely related to EPN diversity. In the feld, population densities of emerging adult Caribfy increased with increasing number of EPN species combined in treatments. Thus, single-EPN species treatments proved to be more efective for the management of fruit-to-soil stages of Caribfy. Relative to controls, the proportions of surviving adult Caribfy observed in EPN treatments with Heterorhabditis bacteriophora (exotic in Florida), Steinernema feltiae (exotic EPN) and (the endemic species) in feld plots were 22.5 ± 6, 45 ± 13 and 47 ± 13%, respectively. Number of emerging D. longicaudata in each of EPN species treat- ments was similar to that observed in control, suggesting that none of the EPN species signifcantly afected the emergence of D. longicaudata, a parasitoid of Caribfy. Heterorhabditis bacteriophora will be more promising, with insignifcant side efects on D. longicaudata in Caribfy-integrated pest management.

Keywords Entomopathogenic nematode species richness · Heterorhabditis bacteriophora · Steinernema feltiae · Heterorhabditis indica · Antagonistic efects · Psidium guajava

Key messages

• The use of entomopathogenic nematode (EPN) species richness to manage Caribfy in south Florida, where the parasitoid Diachasmimorpha longicaudata is endemic, Communicated by M. Traugott. has not been investigated. * Larry W. Duncan • In both laboratory and feld trials, single-EPN species [email protected] treatments were more efcacious than their multiple-

1 species treatments. Citrus Research and Education Centre, Institute of Food • In the feld, no EPN treatments signifcantly reduced den- and Agricultural Sciences, University of Florida, 700 D. longicaudata, Experiment Station Road, Lake Alfred, FL 33850, USA sities of a parasitoid of Caribfy. 2 • Heterorhabditis bacteriophora was more efective than Department of Plant Protection, Faculty of Agriculture, Heterorhabditis indica Steinernema feltiae Zagazig University, Zagazig, Egypt or in the feld and will be more useful in Caribfy IPM. 3 Tropical Research and Education Centre, Institute of Food and Agricultural Sciences, University of Florida, 18905 SW 280th Street, Homestead, FL 33031, USA

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Introduction in pest control (Choo et al. 1996; Neumann and Shields 2008; Jabbour et al. 2011; O’Callaghan et al. 2014; Demir The majority of fruit fies are pests because they deposit et al. 2015). The possible efects following augmentation eggs into fruits, resulting in blemishes and quality dam- of multiple EPN species to manage Caribfy in the felds age to infested fruits (Weems et al. 2014; Qin et al. 2015; can be negative or positive. Moreover, the two species S. Suckling et al. 2016; Villalobos et al. 2017). Several man- feltiae and H. bacteriophora are not native to south Florida agement strategies such as bagging fruits in trees, fruit where A. suspensa has been causing problems in guava fy baiting (or male-annihilation) methods, the use of groves for several decades (Weems et al. 2014; Heve et al. insecticides (e.g. organophosphate products and spinosad 2017). from the bacterium Saccharopolyspora spinosa), trapping The parasitoid, D. longicaudata Ashmead (Hymenoptera: adult fruit fies on sticky materials and the release of teph- Braconidae), has established in regions infested by Anastre- ritid parasitoids, among others, have been used to manage pha fruit fies following deliberate introductions in biologi- fruit fies in orchards (Weems et al. 2014; Suckling et al. cal control programs in south Florida, the Caribbean, Pacifc 2016). However, numerous challenges are associated with Islands and Central and South American countries, among tephritid pest control approaches (Suckling et al. 2016). others (Vargas et al. 2012; Meirelles et al. 2013; Thompson Methyl eugenol, Beta-caryophyllene, Trimedlure and 2014; Weems et al. 2014; Schliserman et al. 2016). Dia- CeraTrap­ ® are fruit fy-specifc baits for monitoring the chasmimorpha longicaudata oviposits eggs into fruit fy oriental fruit fy (Bactrocera dorsalis Hendel), the Asian larvae (when in fruits), develops feeding internally on fruit guava fruit fy (B. correcta Bezzi), the Mediterranean fy larvae and emerges from fruit fy pupae (when in soils) fruit fy (Ceratitis capitata Wiedemann) and Anastrepha (Bézier et al. 2009; Hoy 2013; Thompson 2014; Simmonds fruit fies (A. ludens and A. obliqua), respectively (Weems et al. 2016). In the process of augmenting EPNs to manage et al. 2014; Qin et al. 2015; Suckling et al. 2016; Vil- Caribfy in fruit on the ground in orchards, Caribfy larvae lalobos et al. 2017; Wee et al. 2017). The sterile parasitized by D. longicaudata will also receive EPN treat- techniques have not been successfully used to manage ments. Studies are needed to examine whether release of some economically important fruit fies (e.g. A. suspensa EPN species over fruits on soil surface will afect emergence Loew) (Hoy 2013; Qin et al. 2015; Suckling et al. 2016). of the Caribfy and the benefcial parasitoids in a similar Moreover, feld sanitation whereby infested fruits on the manner. ground are regularly collected and destroyed to kill fruit We hypothesized that (1) multiple EPN species will be fies before they migrate from decaying fruits into soils, more efective than single EPN species to manage Caribfy where majority of the larvae pupate, is not often observed and (2) EPN species treatments to fruits on the soil surface in large commercial groves due to the amount of labour will negatively afect emergence of D. longicaudata. Our this requires. Based on several reports, we considered the main objectives were to (1) assess competition between use of entomopathogenic (EPNs) to target fruit- multiple EPN species for resources (or Caribfy), (2) com- to-soil stages of fruit fies in infested fruits dropped to the pare the performance of exotic EPN species S. feltiae and ground in groves (Dolinski et al. 2012; Dolinski 2015; H. bacteriophora to that of the endemic nematode species Minas et al. 2016; Heve et al. 2017; Kapranas et al. 2017). H. indica in feld conditions using the multiple-species The practice of using EPNs may also be integrated with approach and (3) establish population dynamics (or den- the use of organic matter inputs in the rocky calcareous sity patterns) of Caribfy and D. longicaudata, using the soils of south Florida by letting the EPN-treated fruits rot infested-fruit approach, for the purposes of monitoring, fore- on the ground in groves (Dolinski 2015; Li 2015). casting and making rationale decisions in Caribfy-integrated In recent studies, Steinernema feltiae, H. bacteriophora pest management programmes. and Heterorhabditis indica signifcantly reduced popula- tion densities of adult Caribfy in laboratory assays (Heve et al. 2017). Perhaps, combinations of these virulent EPN Materials and methods species in the feld may be useful for biocontrol of Caribfy, because collective activities of diverse natural enemies can Laboratory trial I: competition between EPN species improve the management of some pests on crops (Hooper for Caribfy larvae in autoclaved soils et al. 2005; Cardinale et al. 2006; Neumann and Shields 2008; Grifn et al. 2009; Ramirez and Snyder 2009; Finke Samples of Rockdale soils from south Florida (Fig. 1) and Snyder 2010; Jabbour et al. 2011). However, previous were autoclaved to kill all naturally occurring native studies have shown that the efects of combining diferent EPNs (Tables 1, 2) and then moistened to obtain initial −1 EPN species can be antagonistic, synergistic or additive gravimetric moisture content of 7% (mass ­mass or w/w). Twenty-five millilitres of the moistened soils was

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Fig. 1 A map of Florida show- ing the distribution of selected orchards around the Tropical Research and Education Centre of University of Florida, Insti- tute of Food and Agricultural Sciences (TREC-UF/IFAS) in Homestead FL within Miami- Dade County, USA

added to each Petri dish (diameter = 50 mm). The infec- mortalities of the inoculated larvae were assessed in each tive juveniles (IJs) of H. indica (native isolate in Florida, treatment, and the experiment was repeated once. USA), S. feltiae (exotic nematode) and H. bacteriophora (exotic species) were used in EPN species treatments. Laboratory trial II: competition between EPN Ten third instar Caribfly larvae from reared colonies species for Caribfy larvae in air‑dried soils were inoculated with 1 ml of 200 IJs ­−1 in the soil microcosms in Petri dishes using a pipette, thereby adjust- Previous procedures were used with the following changes ing the final soil moisture in Petri dishes to 12% (w/w) (Heve et al. 2017): air-dried calcareous soils (Table 1) in all single EPN species treatments (Heve et al. 2017). were moistened to initial moisture content of 7% (w/w). The 1 mL of 200 IJs ­larva−1 in Petri dish was maintained EPN inoculation rate was reduced from 200 to 20 IJs in ratios 1:1 and 1:1:1 for two- and three-EPN species larva­ −1. For single-EPN species treatments, ten actively treatments, respectively (Jabbour et al. 2011). Controls moving third instar Caribfy larvae were treated with 1 mL were 1 mL of tap water. Five replicates were made for of 20 IJs larva−1 in soil microcosm [25-mL of the moist each EPN treatment, and Parafilm was used to wrap Petri soils in a Petri dish (diameter = 5 cm)], using a pipette dishes and then incubated at 27 °C. (Heve et al. 2017). The 1 mL of 20 IJs larva−1 was main- On the fourth day following date of inoculation, tained in ratios 1:1 and 1:1:1 for two- and three-EPN spe- dead larvae and pupae retrieved from soils were placed cies treatments, respectively (Jabbour et al. 2011). Ten on a 710-µm sieve and pressurized tap water was gen- replicates were made for each EPN treatment, with con- tly sprayed over them to remove all soil particles and trols (or 1 mL of tap water). Petri dishes were sealed using any nematodes on their cuticle. Clean cadavers were Paraflm and incubated at 27 °C. Five days after inocula- placed in White traps (one per trap), and emerging IJs tion, all dead larvae observed in controls were washed were examined under a microscope. Based on their mor- and opened in drops of distilled water on glass slide and phological features, suspected coinfecting two or three then examined under a microscope for any baited native nematode species in cadavers in mixed EPN species treat- EPNs in non-autoclaved soils. Following eclosion of pupae ments were confirmed using the molecular qPCR analysis (assessed and recorded in each replicate between 2 and (Table 2; Shapiro-Ilan and Gaugler 2002; Stock and Hunt 3 weeks, according to Heve et al. (2017)), fnal mortalities 2005; Campos-Herrera et al. 2011a, b). Proportions of in replicates numbered 1–10 were calculated by subtract- infected larvae for each EPN species as well as the final ing proportions of emerging adult Caribfy in replicates of EPN treatments from the proportions of emerging adults

1 3 802 Journal of Pest Science (2018) 91:799–813 TC SCL SCL CL SCL SCL CL CL CL CL SCL SCL SCL SCL SL SL SCL SCL SCL 8.4 8.8 22.8 12.8 22.4 22.0 22.4 26.4 24.4 24.4 24.8 16.4 12.8 18.8 20.8 Silt 20.8 24.4 22.4 26.8 32.4 30.0 22.4 26.4 28.4 32.4 30.4 28.4 22.4 22.4 22.4 22.4 Clay 12.4 14.4 18.4 20.8 26.4 50.4 54.8 47.6 55.6 51.2 45.2 43.2 45.2 46.8 61.2 64.8 58.8 Particle size distribu - tion (%) 56.8 Sand 79.2 76.8 60.8 54.8 51.2 ) −1 30 31 30 34 30 32 32 31 31 29 29 30 31 CEC (cmol ­ kg 28 29 29 31 30 5.52 7.52 3.92 7.04 3.75 7.42 5.41 OC (%) 5.78 5.72 3.26 3.91 6.58 6.58 2.75 2.59 2.98 4.95 4.93 8.01 6.40 5.33 8.24 5.40 8.76 7.62 H 7.63 7.81 5.51 6.84 8.02 7.75 5.65 6.79 5.58 7.75 5.31 a 86.96 73.7 89.06 78.23 88.89 79.57 84.62 84.84 85.01 90.71 89.12 85.62 85.62 Ca 89.39 88.44 91.18 87.76 89.82 4.04 5.05 8.73 4.91 7.62 5.23 5.14 5.07 3.07 3.28 5.16 5.38 4.28 4.14 2.71 3.71 3.82 17.6 Mg 0.98 2.3 0.54 4.80 0.79 4.05 2.53 Base saturation (%) Base saturation K 2.37 2.09 0.69 0.74 1.18 1.25 0.65 0.61 0.51 0.76 1.03 clay loam; SL sandy loam CL clay loam; class; SCL sandy clay textural c 0.13 0.59 0.40 0.74 0.44 0.80 0.69 Total N (%) Total 0.59 0.65 0.42 0.46 0.67 0.71 0.36 0.35 0.43 0.56 0.53 ) −1 84 27 20 83 28 81 79 80 21 29 44 41 99 33 56 22 121 101 Total P Total (mg kg 7.5 7.7 7.7 7.5 7.6 7.2 pH 7.5 7.3 7.4 7.7 7.6 7.4 7.6 7.6 7.4 7.8 7.6 7.7 Native EPNs Native detected H. indica No EPNs No No EPNs No H. indica No EPNs No H. indica H. indica H. indica No EPNs No H. indica H. indica H. indica H. indica H. indica H. indica No EPNs No H. indica H. indica 0–15 0–25 0–5 0–25 0–5 0–25 Depth (cm) 0–5 0–5 0–5 0–25 0–5 0–25 0–5 0–25 0–5 0–25 0–5 0–5 b W W W W W W W W W W W W W W W W ′ 19.44 ″ W N, 80°32 ′ 43.50 ″ N, N, 80°32 ′ 36.73 ″ N, N, 80°32 ′ 36.41 ″ N, N, 80°32 ′ 37.13 ″ N, N, 80°32 ′ 35.52 ″ N, N, 80°32 ′ 38.31 ″ N, N, 80°32 ′ 44.28 ″ N, N, 80°30 ′ 26.57 ″ N, N, 80°30 ′ 16.56 ″ N, N, 80°30 ′ 33.78 ″ N, N, 80°30 ′ 35.27 ″ N, N, 80°29 ′ 00.19 ″ N, N, 80°28 ′ 59.65 ″ N, N, 80°30 ′ 54.98 ″ N, N, 80°30 ′ 58.58 ″ N, N, 80°25 ′ 19.09 ″ N, N, 80°25 N, only, detected by using both by detected H. indica only, native for soils in southern positive properties Florida, were and chemical of Rockdale the where majority orchards from of soil samples Physical in base saturation ≈ 0% + Field trials were conducted at this location, because the grove had never been irrigated and was not infested by Caribfy by infested been irrigated not and was had never conducted at this trialsField location, because the grove were A non-commercial avocado orchard that received little N-fertilizer inputs. The procedures for EPN-baiting and quantitative polymerase chain reaction analysis have been described in reports of have analysis reaction chain polymerase EPN-baiting and quantitative for The procedures inputs. little N-fertilizer that received orchard avocado A non-commercial

Na 2 in Table presented details are Campos-Herrera ( 2011a , b 2013 2016 ). Additional et al. Coordinate of grove within distri- of grove Coordinate bution of A. suspensa 1 Table a b c microscopy and quantitative polymerase chain reaction analysis following EPN-baiting method following analysis reaction chain polymerase and quantitative microscopy TC capacity; carbon; CEC cation exchange OC organic nematodes; EPNs entomopathogenic N, 80° 30.179 ′ W) (25° 30.654 ′ N, 25°29 ′ 12.39 ″ 25°29 ′ 12.53 ″ 25°29 ′ 06.85 ″ 25°29 ′ 21.65 ″ 25°29 ′ 18.63 ″ 25°29 ′ 18.61 ″ 25°29 ′ 21.43 ″ 25°35 ′ 30.39 ″ 25°35 ′ 27.40 ″ 25°35 ′ 27.50 ″ 25°35 ′ 26.38 ″ 25°34 ′ 31.96 ″ 25°34 ′ 29.10 ″ 25°34 ′ 43.82 ″ 25°34 ′ 42.02 ″ 25°33 ′ 29.88 ″ 25°33 ′ 28.67 ″

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Table 2 Correct amounts of ingredients to which 1 µL of DNA in each PCR well was added for the detection of specifc EPN species against standard DNAs of known EPNs, using the qPCR technique at specifc temperature Source of standard ­DNAsa The amount of ingredients in µL needed in a qPCR well Required temperature Primer Primer Probe BSAe Mastermix RNAse- Total excluding b c ® d ® (°C) (forward) (backward) ­(Taqman ) ­(Taqman ) free ­H2O 1 µL DNA

Hi, Hb 0.25 0.25 1.00 0.80 10.00 6.70 19.00 59 Sf 0.40 0.40 1.00 0.80 10.00 6.40 19.00 59 Hz 0.60 0.60 1.00 0.80 10.00 6.00 19.00 59 Sd, Sx, Sc, Sg 0.40 0.40 1.00 0.80 10.00 6.40 19.00 59 Hf 0.60 0.60 1.00 0.80 10.00 6.00 19.00 57 a Hi: H. indica; Hb: H. bacteriophora; Sf: S. feltiae; Hz: H. zealandica; Sd: S. diaprepesi; Sx: S. glaseri-group; Sc: S. carpocapsae; Sg: S. gla- seri; Hf: H. foridensis b (5′–3′): Hi: CAT​CGA​TAA​CTG​GTG​GCT​GA; Hb: AAC​GAG​AGT​TCG​GTG​ATA​CTG​ACA​AC; Sf: CGT​TTT​TCT​TGC​CGA​CTG​ATT​GGT​ ACA; Hz: CCG​CCT​TGC​GAT​ATT​TGA​T; Sd: CTT​TCG​CTA​ACG​CTT​CTG​CT; Sx: ACG​ACT​ATT​CGT​TGT​GTC​CGC; Sc: CTA​ACG​GCT​TTG​ AAA​GGT​TTC​TAC; Sg: TTT​TTC​GAC​GAG​CTA​TGT​ACG​ c (5′–3′): Hi: CCA​TCG​CTA​AGG​CTA​TTT​TCC; Hb: GCC​GTG​TAT​ACG​ACA​CGT​CA; Sf: TGC​GCA​ATT​CCT​CGA​CAA​ACGA; Hz: CGC​TGA​ AGC​TAA​TAC​ATT​GTGC; Sd: AGT​TCC​AGT​TGC​GTT​CGT​CA; Sx: GCG​CCG​AAC​CGT​AAAG; Sc: TGC​ATC​AAC​GAG​TGA​CAC​AAT​TAT​ CGA; Sg: TGA​TAG​CGG​ACA​CGA​CAA​AA d (5′–3′): Hi: FAM-CAA​CCG​CCA​CTA​TCG​GTA​AT-IB®FQ; Hb: FAM-CGT​TCA​AGT​ATC​TTT​ATG​GGG​CAA​C-MGB; Sf: FAM-ATT​TTT​ CAG​AAT​TTT​TCA​GAG​GCC​CTTAC-MGB; Hz: VIC-TCG​TTA​TCT​TGT​CTC​TTG​GAT​ACG​-MGB; Sd: FAM-TTG​CCA​GTT​GAC​TTG​TAC​ GC-IB®FQ; Sx: FAM-CTG​ACT​TGT​ACG​CGA​TTC​-IB®FQ; Sc: FAM-GTA​CAT​TGT​TAT​CTA​AGC​GTT​TCG​TC-MGB; Sg: VIC-TGT​ACC​ TCG​TTC​GGT​GTG​AA-MGB e BSA = Acetylated bovine serum albumin (Promega­ ®)

observed in the corresponding replicates of controls (Heve et al. 2017). This experiment was repeated once.

Laboratory trial III: competition between EPN species for Caribfy pupae in air‑dried soils

Ten normal-looking 4–7 days old Caribfly pupae were buried in grooves (at depth 2–5 mm; each per pupa) in a 25-mL soil microcosm of air-dried soils (initial moisture = 11% (w/w); Table 1) in Petri dishes (diam- eter = 5 cm) (Heve et al. 2017). The IJ stock suspension of each EPN species was concentrated and adjusted to 500 IJs per 100 μL and then immediately applied. For all single-EPN species treatments, the ten pupae per Petri dish were inoculated with 400 μL of 2000 IJs, using pipette: The high IJ dose was applied to pupae because of resistance of fruit flies at pupal stage to EPN infections (Toledo et al. 2006; Minas et al. 2016; Heve et al. 2017). Similar IJ dose [the 400 μL of 2000 IJs] per ten pupae Fig. 2 in each Petri dish was maintained in ratios 1:1 and 1:1:1 Experimental plot with screen cage covering Caribfy-infested guavas and yellow sticky trap, placed beneath an avocado tree. Cages for two- and three-EPN species treatments, respectively were frmly supported on the ground using pieces of water hose flled (Jabbour et al. 2011). Ten replicates for each EPN treat- with sand to maintain the soil structure in plots. The properties of ment including controls (400 μL of tap water only) were calcareous soils in the avocado (P. americana) orchard located at the ′ ′ made, and all Petri dishes were sealed using Parafilm and coordinate (25°30.654 N, 80°30.179 W) are in Table 1. (Color fgure online) then incubated at 27 °C until surviving adults emerged between 2 and 3 weeks for assessment of final mortalities

1 3 804 Journal of Pest Science (2018) 91:799–813 in EPN species treatments (Heve et al. 2017). This experi- of water in each treatment to 120 mL. Similar volume for ment was repeated two times. the 352 IJs cm­ −2 was maintained in ratios 1:1 and 1:1:1 for two- and three-EPN species treatments to fve guavas ­cage−1, Field trials: EPN species richness in treatments respectively (Jabbour et al. 2011). Controls were 120 mL of to guavas (Psidium guajava L.) infested by Caribfy tap water used to prepare inocula. Similar EPN treatments with D. longicaudata were applied to 5 guavas plot­ −1 in subsequent months [see ‘Result’ section for the dates in image 4 or 5]. Weather data An avocado (Persea americana Mill) grove [not infested were recorded over entire period (3–6 weeks) between the by Caribfy to avoid variability in data between treatments; date infested fruits were treated and the date emergence of not irrigated so that resilience of each exotic EPN species adult fies and parasitoids ended (https://fawn.ifas.uf.edu). to climatic variability could be accounted] was used for feld trials (Table 1). Forty conical screen cages (diameter Data analysis of the base = 80–100 cm; height = 50 cm) were fxed on the ground near avocado trees (Fig. 2): intrarow distances The R software [R.3.3.2 & R.Studio.v.1.0.44; R core group between successive cages were 22.8 ± 1.3 m, whereas the in Vienna, Austria] was used for all statistical tests. Mean interrow distances were 16.5 ± 0.5 m. Five replicates (or values (± standard errors) of variables for each EPN treat- screen cages) were labelled for each of the EPN species ment repeated once in experiments were compared using treatments and then arranged in completely randomised paired t-tests, whereas the Tukey’s HSD at P ≤ 0.05 was block design form within fve rows of the non-host avocado used to separate mean values among several EPN treatments. plants. Field observations in June 2016 were not included in the On 25 June 2016, at least 200 guava fruits dropped to the statistical analyses because the infested fruits (in cages) were ground in guava groves (which were infested by Caribfy not treated with EPNs in the frst month. However, the graph with D. longicaudata) were transferred to the cages in the for population dynamics of the emerging adult fruit fies avocado grove. But, because ca. 0.56 guava fruits ­tree−1 ha−1 (A. suspensa) included those data. The spread of variables were observed on the ground in guava groves, fve guava (weather data and number of days for complete emergence fruits were added to plot under each cage with a sticky trap of both adult Caribfy and parasitoids) relative to their mean (Fig. 2). The decaying guavas were maintained in 40 labelled values was ascertained using the coefcient of variability. cages until both adult Caribfy and D. longicaudata emerged Regression analysis was used to determine the linear rela- between 2 and 4 weeks following 25 June 2016 and were tionship between each weather element (the predictor) and trapped by sticky traps. Having observed that population the number of days (the dependent variable) required for densities of both emerging Caribfy and D. longicaudata emergence of adults (Caribfy and D. longicaudata) from from the fve guava fruits ­cage−1 were not signifcantly dif- soils. Two-way ANOVA (analysis of variance) was used to ferent between cages (Tukey’s HSD at P < 0.05), fve guava partition variance among the treatment factors with their fruits dropped to the ground were periodically transferred interactions observed in the feld trials, having assessed the from Caribfy-infested groves to each cage for EPN species normal distributions of the data of emerging Caribfy as well treatments. as emerging parasitoids from infested guavas in EPN treat- Freshly produced active IJs of H. bacteriophora, H. ments using the quantile-quantile plots (Gotelli and Ellison indica (the native) and S. feltiae in 3.78 L gallons, contain- 2013). ing ample tap water to avoid overcrowding IJs, were stored at 6–8 °C in dark conditions for 5 days before concentrating and adjusting IJ stock suspension of each EPN species to Results 2500 IJs mL−1 (Laznik et al. 2010; Shapiro-Ilan et al. 2015; Heve et al. 2017): nematodes were immediately applied on Efects of competition between EPN species 29 July 2016, as single-, two- and three-EPN species treat- following treatments to Caribfy larvae and pupae ments to fve Caribfy-infested guavas (in circular plot of 710 cm2 soil surface) in each screen cage (Jabbour et al. In experiments 1 and 2, proportion of infected larvae in sin- 2011). Based on previous observations, each single-EPN gle-EPN species treatments was similar to that observed in species treatment received 100 mL of 2500 IJs mL−1, which the majority of the multiple-EPN species treatments (Fig. 3). was evenly spread over the fve guavas ­plot−1 using a 100- The lowest proportion of infected larvae was observed in mL measuring cylinder, thereby making the equivalent rate the treatment [H. bacteriophora + H. indica] (Fig. 3A, B). to be 352 IJs cm−2 (Toledo et al. 2006; Minas et al. 2016). In each of the multiple-EPN species treatments, the propor- Twenty millilitres of tap water was used to rinse the cylinder tion of infected Caribfy larvae observed for the individual and then added to treatment, thus adjusting fnal volume EPN species was signifcantly diferent between S. feltiae,

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Fig. 3 Proportion of larvae infected by the individual EPN species following inoculation of third instar Caribfy larvae with 1 mL of 200 IJs larva­ −1 in experiments A 1 and B 2. The proportion of infected Caribfy larvae observed for the individual EPN species in each of the multiple-EPN species treatments was signifcantly diferent (Tukey’s HSD tests at P ≤ 0.05). Diferent letters on top of bars indicate that the total numbers of infected larvae are signifcantly diferent between treatments (Tukey’s HSD tests at P ≤ 0.05)

H. bacteriophora and H. indica (Fig. 3). Numerous IJs of mortalities of inoculated third instar Caribfy larvae were S. feltiae were observed in a large proportion (48–76%) not signifcantly diferent between one-, two- and three- of infected larvae, whereas few IJs of H. bacteriophora EPN species treatments in either experiment 1 or 2 (Table 3; or H. indica were observed in a few dead larvae, follow- Fig. 4A). However, mortalities of EPN-treated Caribfy ing EPN species treatments with [S. feltiae + H. bacterio- pupae decreased with increasing number of EPN species in phora], [S. feltiae + H. indica] and [S. feltiae + H. bacterio- treatments in experiments 1 and 2, whereas mean values of phora + H. indica] (Fig. 3A, B). In the treatment with [H. observed mortalities of pupae in experiment 3 were more bacteriophora + H. indica], the proportion of infected larvae directly related linearly to EPN biodiversity (coefcient of recorded for H. bacteriophora was similar to the proportion correlation, R = 0.69; P value = 0.515) (Fig. 4B). of infected larvae observed for H. indica in experiment 1 (Fig. 3A). In experiment 2, H. bacteriophora was observed Efcacies of EPN species treatments to infested in 20% of infected larvae, whereas H. indica was observed in fruits in feld trials 50% of the infected larvae retrieved from the treatment, [H. bacteriophora + H. indica] (Fig. 3B). Multiple EPN species Changing climatic elements (especially soil temperatures, were observed in a few EPN-killed Caribfy larvae in the evapotranspiration and, to some extent, solar radiation) multiple-EPN species treatments (Fig. 3A, B). with time were inversely related to the number of days In some replicates of EPN species treatments, either no required for emergence of adult Caribfy from infested gua- nematodes were observed in a few cadavers or uninfected vas on the ground in cages (Table 4). In efect, population larvae pupated and developed to adult fies (results are not densities of emerging Caribfy varied signifcantly between shown). Consequently, the means (± standard errors) of fnal the dates infested guavas were added to plots (Fig. 5A;

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Table 3 Observed fnal EPN species treatments, with control Final mortalities (Mean ± SE) % P-value mortalities (uncorrected) [paired following inoculation of third Experiment 1 Experiment 2 t-test] instar Caribfy larvae with 1 mL −1 of 200 IJs ­larva in autoclaved Control 14 ± 4.96 a 10 ± 4.29 a 0.569 Rockdale soils H. bacteriophora (Hb) only 96 ± 2.8 b 86 ± 4.96 b 0.024* S. feltiae (Sf) only 100 ± 0 b 88 ± 4.64 b 0.013* H. indica (Hi) only 100 ± 0 b 84 ± 5.24 b 0.004** Sf + Hb 90 ± 4.29 b 92 ± 3.88 b 0.743 Sf + Hi 97.96 ± 2.04 b 97.96 ± 2.04 b 1 c Hb + Hi 92 ± 3.88 b 85.71 ± 5.05 b 0.371 Sf + Hb + Hi 97.96 ± 2.04 b 96 ± 2.8 b 0.569

The 200 IJs ­larva−1 was proportionally maintained in single-, two- and three-EPN species treatments with controls Tukey’s HSD tests at P ≤ 0.05: Excluding results in controls, the lowercase letter ‘b’ against mean (± SE) values in the same column indicates no signifcant diferences in fnal mortalities between single- and mul- tiple-EPN species treatments in experiment 1 or 2 Paired t-tests: * and ** indicate signifcant diference at P ≤ 0.05 and P ≤ 0.01, respectively, between fnal mortalities in experiments 1 and 2. c: Exact equality was observed in the treatment [Sf + Hi]

Fig. 4 Proportions of fnal mortalities, corrected according to Heve et al. (2017), follow- ing EPN species treatments in air-dried calcareous soil microcosms. A Treatments at 20 IJs ­larva−1: No native EPNs were baited by few dead larvae in controls. Paired t-tests: ‘ns’ means no signifcant difer- ence at P ≤ 0.05, but * and ** indicate signifcant diference at P ≤ 0.05 and P ≤ 0.01, respectively, between fnal mor- talities in experiments 1 and 2. According to Tukey’s HSD tests at P ≤ 0.05, similar bars (± SE) have the same letter ‘a’ or ‘b’. B Treatments at 200 IJs pupa­ −1: proportions of fnal mortalities were inversely related to the number of EPN species (in treatments) in experiment 1 or 2, whereas the mean values of pupal mortalities in experiment 3 were more directly related (or proportional) to EPN biodi- versity

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Table 4 Regression analysis for the linear relationship between length of period (or number of days required for emergence of Caribfy and para- sitoids in treatments) and each weather element in south Florida. Coefcients of variation for mean values of variables are included Details Length of Weather ­elementsb,d period (in days)a Average evapo- Total precipitation Average solar Average soil tem- Average relative transpiration (mm) observed in a radiation perature (°C, humidity (%) (mm) period ­(Wm−2) depth = 10 cm)c

Mean ± standard devia- 29 ± 6 3.28 ± 1.04 95.3 ± 73.5 192.8 ± 38.1 27.08 ± 3.3 75.45 ± 14.9 tion Coefcient of variation 20.25 31.84 77.13 19.75 12.04 19.71 (CV) % Regression analysis: P value for analysis of 0.001 0.191 0.071 <0.0001 0.647 covariance Constant ± standard 44.7 ± 3.47*** 32.41 ± 2.85*** 45.9 ± 8.36*** 76.19 ± 4.42**** 33.74 ± 10.01** error Slope ± standard error − 4.8 ± 1.01*** − 0.035 ± 0.02 − 0.087 ± 0.04 − 1.74 ± 0.16**** − 0.062 ± 0.13 Coefcient of determi- 0.71 0.19 0.32 0.93 0.024 nation (R2) a Emerging D. longicaudata and emerging Caribfy in cages were both observed in similar period of time, though the majority of the parasitoids emerged in 1–3 days before emergence of Caribfy b Each weather element was considered as the predictor for the length of period required (the response) c Soil temperatures (at depth ≈ 10 cm) were assessed because Caribfy pupae were observed at depths ≤ ca. 7 cm in Rockdale calcareous soils in south Florida d P = Signifcance level; **, *** and **** indicate signifcance at P ≤ 0.01, P ≤ 0.001 and P ≤ 0.0001, respectively

Table 5). In controls, high population densities of emerg- densities of emerging D. longicaudata were similar in all ing Caribfy were observed in November and in the long EPN species treatments with control, but varied between period from March to August, whereas very few fies were the dates infested fruits were added to plots (Fig. 6A, B; obtained in the remaining months (or parts) of the year Table 5). (Fig. 5A). Intensive pruning of guava trees observed in Jan- uary–March 2017 reduced densities of fruits from ca. 1.05 to 0.03 guava fruits tree­ −1 ha−1 on the ground in the groves, Discussion but the population densities of emerging Caribfy observed in controls increased with increasing soil temperatures in An equivalent number of IJs in EPN species treatments, the subsequent months (Fig. 5A). No other tephritid pests comprising either single or multiple species, produced than adult Caribfy emerged from infested guavas. similar mortalities of Caribfy larvae in all trials, whereas Other significant sources of variation in densities of single-species treatments to pupae in the laboratory or emerging adult Caribfy included the EPN biodiversity and Caribfy-infested fruits in the feld outperformed the spe- the interactions between EPN biodiversity and dates infested cies mixtures. Based on the results in the current study fruits were added to plots (Fig. 5A; Table 5). Similar to compared to the observations in the previous studies, the observations in experiments 1 and 2 (Fig. 4B), accumulated efect of EPN biodiversity on biocontrol efcacy appears to densities of emerging Caribfy increased with increasing depend in part on the EPN species combined in treatments, EPN biodiversity (Fig. 5B). The lowest population densi- the insect hosts and the developmental stages of the hosts ties of emerging adult Caribfy were observed in treatment (Choo et al. 1996; Neumann and Shields 2008; Jabbour to fruits, with the exotic species H. bacteriophora (Fig. 5B). et al. 2011; Demir et al. 2015). Diferent foraging (cruis- ing, ambushing, herding and the intermediate between cruising and ambushing) behaviours of IJs belonging to Emergence of the parasitoid D. longicaudata diferent EPN genera and species may account for the vari- following EPN treatments to Caribfy‑infested guava ations in efcacies of multiple virulent EPN species to fruits hosts (or insect pests) in treatments (Demir et al. 2015; Lortkipanidze et al. 2016). In both laboratory and green- Only D. longicaudata was observed from Caribfly- house trials, Choo et al. (1996) observed less control of infested guavas in southern Florida. The observed the spotted cucumber Diabrotica undecimpunctata

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Fig. 5 Curves show A fuctua- tions in population densities of emerging adult Caribfy in treatments as soil temperatures (at soil depth = 10 cm) varied between 17 and 36 °C, with time or dates; B cumulative densities of emerging Caribfy in treatments, excluding densi- ties of adults observed in the frst month, June 2016, when no EPNs were applied over guava fruits. Same letter(s) at the tips of curves indicate(s) that the total densities of adult Caribfy are not signifcantly diferent between treatments, according to Tukey’s HSD at P ≤ 0.05. The dates with the symbol ‘[→]’ indicate that EPNs were applied on the third–ffth day following these dates. Intensive pruning of guava trees was observed in the months with symbol ‘♣’

(Mannerheim) by mixed species rather than single-EPN signals emanating from the highly active Caribfy larvae species treatments of H. bacteriophora, Steinernema sp., may have attracted and induced social movement of single S. riobravis and S. carpocapsae. Some combinations of and even multiple EPN species towards them (Torr et al. S. glaseri, H. bacteriophora and S. weiseri were antago- 2004; Willett et al. 2015). The immobile, less-active and nistic, whereas others were additive or synergistic in their more resistant fruit fy pupae, which produce resistance- efects on larvae of the elephas (Gyllen- inducing tyrosine hydroxylase (Chen et al. 2017), may be hal) and Polyphylla fullo (L.) (Demir et al. 2015). Con- less attractive to IJs which may have the efect of causing sistently, synergistic and additive efects of multiple EPN greater EPN dispersal in search of host signals (Kaplan species against the beetle pests Otiorhynchus ligustici et al. 2012; Heve et al. 2017). There is increasing evidence (L.) and Leptinotarsa decemlineata (Say) have also been that EPNs generally exhibit herding or group movement reported (Neumann and Shields 2008; Jabbour et al. 2011). during foraging, which presumably results in higher infec- For the Caribfy under the conditions of our studies, the tion rates and greater potential to overcome host physical rapid development from short susceptible larval stage to and immunological defences (El-Borai et al. 2011; Shap- long resistant pupal stage may be a key factor restrict- iro-Ilan et al. 2014; Willett et al. 2015). Although the herd- ing any value in the use of multiple EPN species (Toledo ing signals are conserved across EPN genera (Willett et al. et al. 2006; Minas et al. 2016). Chemical and vibrational 2015), further work on the interspecifc communication

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Table 5 Two-way analysis of variance for the sources of variation in from July 2016 to May 2017, excluding data obtained in June 2016 population densities of surviving adult Caribfy and D. longicaudata when no EPNs were applied to guavas observed following EPN treatments to infested fruits in screen cages

Sources of variation df Surviving adults observed following EPN species treatments to infested ­guavasb,c Caribfy (A. suspensa) The parasitoid, D. longicaudata MS F P MS F P

Dates infested fruits were added to plots (Months)a 9 1879.33 14.3196 < 2.2 × 10−16 3.3667 4.1337 4.401 × 10−5 Number of EPN species in treatments with control 3 2411.19 18.3721 4.112 × 10−11 1.6589 2.0368 0.10837 (EPN biodiversity) Interaction between ‘Months’ and ‘EPN biodiversity’ 27 305.72 2.3295 0.0002686 1.1583 1.4222 0.08217 Residuals 360 131.24 0.8144 a From Table 4, it could be deduced that climatic variability markedly varied population densities of both Caribfy and parasitoids between the dates infested guavas were added to treatments or plots b df Degree of freedom; MS mean sum of squares; F F-statistic; P probability statistic for signifcance level c In bold, all values of P ≤ 0.05 indicate that values of their corresponding F are signifcant between EPN species used here is warranted. Possibly, suppress other nematode species (Koppenhofer et al. 1995; the proportionately reduced densities of IJs of individual Duncan et al. 2003; Puža and Mrácek 2009; Hearne et al. EPN species in the mixed species treatments compared 2017). With time over the long Caribfy pupal stage, all to the whole IJ density of EPN species in single-species these reported interactions or factors may likely cause a treatments could reduce the ftness of the mixed species more rapid decline in density of active nematodes in mixed cohort if intraspecifc communication in single-EPN spe- species treatments than there may be active nematodes in cies treatments was more efective than interspecifc inter- single-EPN species treatments (Koppenhofer et al. 1995; action between EPN species in mixtures. Duncan et al. 2003; Sicard et al. 2005; Puža and Mrácek When the EPN species competed for Caribfy larvae, S. 2009; O’Callaghan et al. 2014; Grifn 2015; Hearne et al. feltiae dominated both heterorhabditid species in terms of 2017; Lu et al. 2017). reproductive success. Although there may have been higher The native H. indica is the only commonly encountered levels of coinfection than we detected, some nematode spe- EPN in south Florida, compared to the species of diverse cies compete vastly better for food resources than do others EPN communities in central Florida where soils are more (Duncan et al. 2003; Puža and Mrácek 2009; O’Callaghan acidic (Campos-Herrera et al. 2013, 2016; El-Borai et al. et al. 2014; Hearne et al. 2017). For example, free-living 2016). Perhaps, the abundance of H. indica in the alkaline nematodes suppressed reproduction by S. riobrave more soils of south Florida is due in part to its heat tolerance (Ma than that by S. diaprepesi in EPN-killed larvae et al. 2013). Despite its dominance in this region, naturally (Diaprepes abbreviatus L.) (Duncan et al. 2003), and repro- occurring H. indica in calcareous soils did not appear to duction by S. afne was much higher than that by S. kraus- interfere with the performance of the more virulent exotic sei following mixed infections of (L.) H. bacteriophora. Indeed, H. bacteriophora occurs widely (Puža and Mrácek 2009). Numerous mechanisms in reports throughout subtropical, tropical and Mediterranean areas may possibly interfere with EPNs in multiple-species treat- and is thus well equipped to compete in similar climatic ments (Grifn 2015). The symbiotic bacteria of one EPN conditions of south Florida (Hatting et al. 2009). species can inhibit the development or activities of another Virulence of EPNs to Caribfy in the feld trials was inde- species (Sicard et al. 2005). Probably, toxic secretions from pendent of the emergence of D. longicaudata, because our individual EPN species similar to the lethal venoms found results showed that the mean values of accumulated popula- in S. carpocapsae may more rapidly antagonize, kill or para- tion densities of emerging D. longicaudata in all the EPN lyse other EPN species in mixed species treatments than in species treatments were not signifcantly less than those of single-species treatments (Lu et al. 2017). Male steinerne- control. Possibly, the EPNs and D. longicaudata may be matids have recently been observed killing large numbers compatible to efectively manage tephritid pests because of males and females of other species, when EPN species both are known for killing a wide range of fruit fies (Vargas were combined in treatments (O’Callaghan et al. 2014; et al. 2012; Meirelles et al. 2013; Thompson 2014; Weems Zenner et al. 2014; Grifn 2015). In mixed infections, some et al. 2014; Schliserman et al. 2016; Heve et al. 2017). Prob- nematode species signifcantly develop faster and tend to ably, the endosymbiotic rhabdoviruses associated with D.

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Fig. 6 Emerging parasitoid D. longicaudata from fve infested fruits ­plot−1 or ­cage−1 observed in zero-, one-, two- and three- EPN species treatments to infested fruits at 352 IJs ­cm−2. A Population dynamics of D. longicaudata from the time (or date) EPN treatments started; B Cumulative population densities of D. longicaudata. Tukey’s HSD tests at P ≤ 0.05: The let- ter ‘a’ at the ends of curves indi- cates no signifcant diferences between accumulated densities of the wasps in treatments. Dates with the symbols ‘[→]’ and ‘♣’ are described in Fig. 5

longicaudata in hosts may deter EPN species from attacking ≤ 9% of Caribfy-infested guava fruits), suggesting that aug- developing D. longicaudata in hosts, similar to protection mentation of these important hymenopterans is required in of EPNs (in EPN-killed ) aforded by the reported south Florida. scavenger deterrent factors from symbiotic bacteria of EPNs Seasonal variations in weather conditions throughout or, perhaps, D. longicaudata is naturally resistant to EPN the year produced no signifcant negative impacts on the infections (Bézier et al. 2009; Gulcu et al. 2012; Hoy 2013; insecticidal efcacy of the exotic EPN species, H. bacte- Simmonds et al. 2016). Moreover, the rapid pupation of riophora and S. feltiae, when compared to performance third instar Caribfy larvae to resistant pupae may quickly of the endemic species H. indica in the feld. The south shield the koinobiont parasitoid D. longicaudata, similar Florida climate seems to be well suited for optimum activ- to the protection against EPNs aforded to the parasitoid ity by the subtropical and tropical EPN isolates used here Microplitis rufventris (Kokujev) in the late larval instar of (Grewal et al. 2005; Hatting et al. 2009; Shakeela and Hus- the cotton leafworm Spodoptera littoralis (Boisd.) (Atwa saini 2009; Ma et al. 2013; Shapiro-Ilan et al. 2014). But et al. 2013). However, the population densities of D. lon- our Caribfy emergence data suggest that the long period gicaudata observed in this study were extremely low (i.e. (March–August) and the short period (October–Novem- ber) are the most critical time for EPN augmentation in

1 3 Journal of Pest Science (2018) 91:799–813 811 conjunction with other IPM tactics. Fruit fies have gen- Campos-Herrera R, Johnson EG, EL-Borai FE, Stuart RJ, Graham JH, erally required high EPN application rates for efective Duncan LW (2011b) Long-term stability of entomopathogenic nematode spatial patterns in soil as measured by sentinel insects control (Toledo et al. 2006; Minas et al. 2016). Ongoing and real-time PCR assays. Ann Appl Biol 158:55–68. https://doi. EPN dose response trials will assess the more virulent H. org/10.1111/j.1744-7348.2010.00433.x bacteriophora in the feld towards developing economic Campos-Herrera R, Pathak E, El-Borai FE, Gutiérrez C, Rodríguez- EPN-augmentation approaches that will optimize Caribfy Martín JA, Stuart RJ, Graham JH, Duncan LW (2013) Geospa- tial patterns of soil properties and the biological control poten- control in south Florida. tial of entomopathogenic nematodes in Florida citrus groves. Soil Biol Biochem 66:163–174. https://doi.org/10.1016/j. soilbio.2013.07.011 Authors’ contributions Campos-Herrera R, El-Borai FE, Rodríguez MJA, Duncan LW (2016) Entomopathogenic nematode food web assemblages in Flor- ida natural areas. Soil Biol Biochem 93:105–114. https://doi. The frst author (WKH) conducted the experiments with full org/10.1016/j.soilbio.2015.10.022 support of FEE, DC and LWD and analysed the data and Cardinale BJ, Srivastava DS, Dufy JE, Wright JP, Downing AL, then drafted the manuscript. All authors contributed to the Sankaran M, Jouseau C (2006) Efects of biodiversity on the func- tioning of trophic groups and ecosystems. Nature 443:989–992 revision of the manuscript. Chen EH, Hou QL, Wei DD, Dou W, Liu Z, Yang PJ, Smagghe G, Wang JJ (2017) Tyrosine hydroxylase coordinates larval-pupal Acknowledgements The frst author thanks the authorities as well as tanning and immunity in oriental fruit fy (Bactrocera dorsalis). individuals of both CREC-UF/IFAS (in Lake Alfred FL, USA) and Pest Manag Sci. https://doi.org/10.1002/ps.4738 TREC-UF/IFAS (in Homestead FL, USA) for providing him accommo- Choo HY, Koppenhofer AM, Kaya HK (1996) Combination of two dations and research facilities during his PhD studies at these research entomopathogenic nematode species for suppression of an insect centres. We thank Joshua Q. Fluty of CREC-UF/IFAS in Lake Alfred, pest. J Econ Entomol 89(1):97–103 Florida, Rita Duncan of TREC-UF/IFAS in Homestead, Florida, Demir S, Karagoz M, Hazir S, Kaya HK (2015) Evaluation of Suzanne Frazer of the FDACS in Gainesville, Florida, and stafs of entomopathogenic nematodes and their combined application the USDA [Dr. Nancy Epsky and Mr. G. Micah in Miami, Florida] against Curculio elephas and Polyphylla fullo larvae. J Pest Sci for their support. We appreciate Tropical Fruit Producers for allowing 88(1):163–170. https://doi.org/10.1007/s10340-014-0571-9 access to their orchards. Dolinski C (2015) Entomopathogenic nematodes against the main guava insect pests. Biocontrol 61(3):325–335. https://doi. Funding This research was funded by the University of Florida for org/10.1007/s10526-015-9695-y education and training of a PhD candidate (i.e. the frst author) in the Dolinski C, Choo HY, Duncan LW (2012) Grower acceptance of Department of Entomology and Nematology. entomopathogenic nematodes: case studies on three continents. J Nematol 44(2):226–235 Compliance with ethical standards Duncan LW, Dunn DC, Bague G, Nguyen K (2003) Competition between entomopathogenic and free-living bactivorous nema- todes in larvae of the weevil Diaprepes abbreviatus. 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