Pheromone extracts act as boosters for entomopathogenic nematodes efficacy

https://doi.org/10.1016/j.jip.2019.04.008Get rights and content

Highlights

  • Pheromone treated EPNs exhibited enhanced dispersal in soil columns.

  • Pecan weevils, and black soldier flies had lowest survival with pheromone treated EPNs.

  • Pheromones increase EPN dispersal and host mortality.

Abstract

Inconsistency in entomopathogenic nematode (EPN) efficacy is still one of the biggest challenges for the wider adoption of EPNs as biocontrol agents. Previous studies demonstrated that extracts from EPN-infected hosts enhance dispersal and efficacy, two key factors in success of EPNs. Some active components in the insect host cadavers responsible for dispersal, ascarosides, have been identified as nematode pheromones. We hypothesized that pheromone extracts increase dispersal of EPN infective juveniles (IJs) leading to increased efficacy. First, we determined whether pheromone extracts improved IJ movement/dispersal in soil columns baited with Tenebrio molitor larvae. We found that pheromone extracts induced higher numbers of Steinernema carpocapsae and Steinernema feltiae IJs to move towards T. molitor larvae in the bottom of the column compared to IJs treated with infected cadaver macerate and water, positive and negative controls, respectively. Furthermore, the number of S. carpocapsae IJs that invaded T. molitor larvae was higher for the pheromone extract treatment than the controls. S. feltiae IJs that were pretreated with pheromone extracts and macerate (positive control) infected T. molitor at the same rate but invasion was superior to IJs that were treated with water. Consistent with the soil column tests, both S. carpocapsae and S. feltiae IJs treated with pheromone extracts performed better in killing larvae of two economically important insect larvae, pecan weevil, Curculio caryae, and black soldier fly, Hermetia illucens, in greenhouse tests compared to IJs treated with water. We demonstrated pheromone-mediated behavioral manipulation of a biological control agent to enhance pest control potential. Conceivably, nematodes can be exposed to efficacy-enhancing pheromones prior to field application.

Introduction

Entomopathogenic nematodes (EPNs) alone or as part of integrated insect pest management programs can offer sustainable pest control in greenhouse or field settings (Shapiro-Ilan et al., 2017). Species belonging to the families Heterorhabditidae and Steinernematidae are commercially available and can be applied via standard farming equipment (Shapiro-Ilan and Dolinski, 2015). However, inconsistencies in efficacy remain an important challenge impeding the adoption of EPNs in pest management programs (Georgis et al., 2006). Traditional efforts to improve EPN efficacy have largely focused on strain improvement, refining formulation and application methods (Shapiro-Ilan and Dolinski, 2015). More recently, plant volatiles and other aspects of EPN chemical ecology have also been explored as factors impacting nematode efficacy (Ali et al., 2010, Hiltpold et al., 2010, Kaplan et al., 2012, Turlings et al., 2012, Willett et al., 2018).

Some insect species or developmental stages of target insects are naturally more resistant to EPN infection than others. For example, a test of 15 EPN strains showed that nematode-induced mortality in pecan weevil larvae, Curculio caryae (Horn) (Coleoptera: Curculionidae) was moderate at best (<40%) (Shapiro-Ilan, 2001a), while pecan weevil adults were highly susceptible to the same strains (Shapiro-Ilan, 2001b). Pecan weevil is a major insect pest of pecan production in Southern United States (Lacey and Shapiro-Ilan, 2008). Adults emerge from the soil from July through August and are the primary stage targeted for control via aboveground insecticide applications (Lacey and Shapiro-Ilan, 2008, Shapiro-Ilan et al., 2011). Larvae develop within the pecan nut and fourth instars drop to the soil where they burrow up to 25 cm depth and spend 9 months to 2 years in an earthen cell before pupation and adult emergence (Lacey and Shapiro-Ilan, 2008). Pecan weevil biology makes larval control in the soil difficult to achieve; developing technologies to manage pecan weevil larvae could provide an extra option for the control of this pest. Likewise, the larvae of black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae) are poor hosts for EPNs, most likely due to their thick cuticle (Tourtois et al., 2017). Soldier fly eggs are oviposited in or near food sources (Booth and Sheppard, 1984) and pass through six instars during a relatively short developmental period of approximately 3 weeks (Tomberlin et al., 2002, Tomberlin et al., 2009). In this study, black soldier flies were selected because of their relatively low level of susceptibility to EPN (Tourtois et al., 2017) and their commercial availability.

Efficacy of entomopathogenic nematodes is based on host finding and infectivity. After nutrients in the insect host have been depleted, new infective juveniles (IJs) emerge and leave the insect cadaver in search of new hosts. IJs are the only EPN stage that survive in the soil in the absence of hosts. IJs find new hosts and navigate in the environment using several environmental cues such as plant volatiles (Rasmann et al., 2005), electromagnetic fields (Ilan et al., 2013), substrate vibration (Torr et al., 2004), temperature (Burman and Pye, 1980); and host recognition factors including CO2 (Ramos-Rodríguez et al., 2007); feces (Grewal et al., 1993) and odorants (Dillman et al., 2012). IJs enter hosts through natural openings or direct penetration of cuticle and, once inside the hemocoel, release symbiotic bacteria, Photorhabdus spp. (associated with heterorhabditids) or Xenorhabdus spp. (associated with steinernematids) that infect and kill the insect host within 24–72 h. IJs feed on degraded host tissues and complete one to three generations before leaving the host cadaver (Kaya and Gaugler, 1993).

The most common EPN application method is spraying IJs in aqueous suspension (Shapiro-Ilan and Dolinski, 2015), however, approaches to improve efficacy by enhancing EPN dispersal have been explored. For example, applying EPNs with phoretic hosts led to increased insect control (Shapiro-Ilan and Brown, 2013). In an alternative “cadaver approach” the EPNs are applied directly to the target site in their infected hosts; pest suppression is subsequently achieved by the IJs that emerge from the infected hosts (Shapiro-Ilan et al., 2003a). EPNs applied directly in insect host cadavers exhibit greater dispersal and infectivity compared to IJ application in aqueous suspension (Shapiro and Glazer, 1996, Shapiro and Lewis, 1999, Shapiro-Ilan et al., 2003a). Moreover, Heterorhabditis bacteriophora Poinar (Hb strain) (Rhabdita: Heterorhabditidae) applied in aqueous suspension amended with infected cadaver extract increased Galleria mellonella (L.) (Lepidoptera: Pyralidae) infection rates relative to IJ emergence directly from cadavers into sand (Shapiro and Lewis, 1999). Similarly, EPN-infected host macerate also enhanced dispersal of Steinernema carpocapsae (Weiser) (All strain), Steinernema feltiae (Filipjev) (SN strain) (Rhabdita: Steinernematidae) and H. bacteriophora within a soil profile (in soil columns) (Wu et al., 2018).

A synthetic mixture of ascarosides obtained from Caenorhabditis elegans (strain N2) Maupas (Rhabdita: Rhabditidae) induced the dispersal of S. feltiae IJs in petri dish arenas (Kaplan et al., 2012). Moreover, the ascaroside ascr#9 was found in both Steinernema spp. and Heterorhabditis spp. infected cadavers suggesting that ascarosides are produced by a broad range of EPN species (Kaplan et al., 2012). Even though pheromone extracts can disperse IJs on agar, we do not know whether they can disperse them in soil or whether agar plate dispersal will translate into increased insect mortality or improved IJ efficacy for pest suppression. In this study, we tested whether exposing S. feltiae and S. carpocapsae IJs to pheromone extracts derived from infected hosts enhances EPN efficacy in soil. The objectives of this study were to (i) test the impact of pheromone extracts on the dispersal of IJs in sand columns baited with T. molitor and on host infection; (ii) assess the efficacy of pheromone extract-treated IJs against larvae of pecan weevils and black soldier flies in greenhouse tests.

Section snippets

Insect sources and nematode treatments

Galleria mellonella and Tenebrio molitor L. (Coleoptera: Tenebrionidae) used in this experiment were obtained from Vanderhorst Wholesale, Inc. (St. Mary’s, Ohio) and Southeastern Insectaries (Perry, GA), respectively. Fourth (final) instar pecan weevil larvae were obtained from pecans collected at the USDA- ARS Southeastern Fruit and Tree Nut Research Unit, Byron, GA (Shapiro-Ilan, 2001a). Black soldier flies were obtained from Fluker’s Cricket Farm (Port Allen, LA).

Infective juveniles of S.

Soil column studies

A significant treatment effect was found in all analyses (P < 0.05). The average number of S. carpocapsae IJs recovered from the bottom column together with IJs recovered from dissections was highest in pheromone extract treated nematodes (1114 ± 108), followed by the macerate treatment (832 ± 81) then control nematodes (367 ± 36) (F2,15 = 34.81, Pr < 0.0001) (Fig. 1). The same pattern was true for S. carpocapsae excised from T. molitor dissections only; 60 (±14), 20 (±5), 4 (±1) for pheromone

Discussion

Our results show that pre-treatments of pheromone extracts can improve EPN efficacy by increasing IJ dispersal leading to higher insect encounter and mortality. This is consistent with previous findings that indicated the importance of host factors in IJ dispersal and infectivity (Shapiro and Glazer, 1996, Shapiro and Lewis, 1999, Kaplan et al., 2012, Wu et al., 2018). Two of the components identified in the host cadavers were ascaroside pheromones (Kaplan et al., 2012). In the current study, a

Acknowledgments

We thank Stacy Byrd, Nick Morris, Chloe Gallegos and Benjamin Sloniker for technical assistance. We also thank Mr. Karl Cameron Schiller for his assistance in the preparation of pheromone extracts. We also acknowledge the USDA-National Institute of Food and Agriculture (NIFA) award 2017-00120 (Small Business Innovation Research, SBIR) and USDA-NIFA award 2018-67013-28064 (Agriculture and Food Research Initiative, AFRI) for funding a portion of the research. Mention of a proprietary product name

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