A weevil sex pheromone serves as an attractant for its entomopathogenic nematode predators

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1 Chemoecology (2017) 27: DOI /s CHEMOECOLOGY ORIGINAL ARTICLE A weevil sex pheromone serves as an attractant for its entomopathogenic nematode predators Monique J. Rivera 1 Xavier Martini 2 Ashot Khrimian 3 Lukasz Stelinski 1 Received: 28 June 2017 / Accepted: 23 August 2017 / Published online: 30 August 2017 Ó Springer International Publishing AG 2017 Abstract Diaprepes abbreviatus is an invasive pest of citrus in the USA from the Caribbean. Entomopathogenic nematodes (EPNs) are used as biological control agents in citrus agroecosystems against D. abbreviatus. EPNs respond to herbivore-induced volatiles from citrus roots to assist in the location of insect hosts. Here, we investigated EPN response to the male-produced sex pheromone of D. abbreviatus. In the laboratory, we used two-choice tests to investigate the behavioral response of Steinernema diaprepesi, Heterorhabditis indica, Steinernema riobrave, and the plant parasitic nematode, Tylenchulus semipenetrans, to the synthetic sex pheromone of D. abbreviatus, as well as its natural source, beetle frass. Nematodes were not attracted by volatiles of citrus plant origin or carvacrol, a non-pheromone volatile associated with beetle frass. S. diaprepesi and H. indica were attracted to the frass and the pheromone of D. abbreviatus. The response of S. diaprepesi to the pheromone was greater than that of H. indica at all doses tested; the greatest response from both species occurred at 0.12 and 1.2 ng of pheromone/ll of solvent. Deploying the pheromone in a citrus grove increased the Handling Editor: Liliane Ruess. & Monique J. Rivera monique.rivera@ufl.edu Entomology and Nematology Department, Citrus Research and Education Center, University of Florida, Lake Alfred, FL, USA Entomology and Nematology Department, North Florida Research and Education Center, University of Florida, 155 Experiment Road, Quincy, FL 32351, USA Invasive Insect Biocontrol and Behavior Laboratory, USDA- ARS, NEA, Bldg. 007, Rm. 301, Baltimore Avenue, Beltsville, MD 20705, USA mortality of caged Diaprepes larvae as compared to control larvae deployed with solvent alone. Also, more EPNs were found in the soil surrounding the pheromone-baited larvae than the surrounding controls. Keywords Entomopathogenic nematodes Nematode chemotaxis Pheromone Diaprepes abbreviatus (E)-3-(2-Hydroxyethyl)-4-methyl-2-pentenoate Introduction Diaprepes abbreviatus (Linnaeus) (Coleoptera: Curculionidae) is an invasive pest of citrus in the USA. It attacks approximately 293 different plant species including citrus, sugarcane, vegetables, potatoes, strawberries, woody ornamentals, sweet potatoes, papaya, guava, mahogany, containerized ornamentals, and non-cultivated wild plants (Simpson et al. 1996). Since its arrival in the USA from the Caribbean, it has significantly contributed to the spread of disease and damage to citrus, ornamental plants, and other crops (Weissling et al. 2002). D. abbreviatus is distributed across a large area of central and southern Florida where it causes approximately $70 million damage annually (Weissling et al. 2002). Larval root feeding can girdle the taproot and prevent the plant from taking up water and nutrients, which results in plant death (Schroeder 1992). Young plant hosts can be killed by a single larva, while several larvae can cause serious decline of older, established hosts (Weissling et al. 2002). This damage also exposes the plant roots to higher incidence of secondary infections by Phytophthora species (Graham et al. 1996, 2002).

2 200 M. J. Rivera et al. The most effective method for controlling larvae found on roots is with entomopathogenic nematodes (EPNs). Release of mass-produced EPNs has been implemented for over two decades to reduce larval populations of D. abbreviatus (Downing et al. 1991; Bullock et al. 1999; Duncan et al. 1999). EPNs are soil-dwelling roundworms from the genera Heterorhabditis or Steinernema. They are obligate parasites that kill their host with the aid of symbiotic bacterium (Poinar 1990). Native and introduced EPNs are infectious to all larval stages of D. abbreviatus and possibly to adults (Adair 1994; Schroeder 1990). Host finding behavior of EPNs is regulated by signals and cues from conspecific nematodes and their host plant/ environment. Vertebrate parasitic nematodes exhibit both thermo- and chemotactic host finding behavior responding to warmth and using CO 2 and/or host-specific chemicals (Granzer and Hass 1991; Castelletto et al. 2014). EPNs also respond to nonvolatile cues exuded by the host such as sodium chloride gradients used by the human parasite, Strongyloides stercoralis (Forbes et al. 2003). Similarly, S. ratti is attracted by serum proteins (Koga and Tada 2000). In addition, vertebrate parasitic nematodes are attracted to host-specific chemicals such as urocanic acid from skin and human-specific 7-octenoic acid (Castelletto et al. 2014; Safer et al. 2007). Environmental cues also direct host finding behavior. For example, the ruminant parasite, Haemonchus contortus, responds to grass odors (Castelletto et al. 2014). Likewise, EPNs use CO 2, host, and environmental cues to locate insect larvae (Dillman et al. 2012; Lewis et al. 1992). The role of environmental cues, particularly herbivoreinduced plant volatiles (HIPVs) released by plants in response to herbivory by the insect host, is well established for EPNs in multiple systems. Heterorhabditis megidis responds to HIPVs released by the white cedar Thuja occidentalis (van Tol et al. 2001) and (E)-b-caryophyllene released by maize (Rasmann et al. 2005). Finally, plant parasitic nematodes are also attracted by CO 2 that is emitted by roots, as well as root exudates (Robinson 1995; Perry 1996; Xu et al. 2015). In citrus, multiple EPN species use HIPVs as foraging cues for locating D. abbreviatus larvae. Both Steinernema and Heterorhabditis species are specifically attracted to pregeijerene, released by citrus upon root damage by D. abbreviatus feeding (Ali et al. 2010). To date, pregeijerene has been the most consistently identified cue attracting EPNs of root herbivores in citrus and has also been shown to attract EPNs in the blueberry agroecosystem (Ali et al. 2012). Adult D. abbreviatus feed on foliage and lay eggs between leaves (Schroeder 1992). Upon hatching, the larvae fall to the soil and crawl to the roots of plants where later instars feed and develop (Schroeder 1992; Weissling et al. 2002). While adults feed, they excrete frass (feces), which contains a male-produced pheromone [(E)-3-(2-hydroxyethyl)-4-methyl-2-pentenoate] that attracts females for mating (Lapointe et al. 2012). Diaprepes abbreviatus adults congregate in large numbers on single trees (referred to as party trees, Wollcott 1936). This likely occurs because of pheromone-induced arrestment behavior in areas where conspecifics are present for mating (Lapointe and Hall 2009). This is caused, in part, by the beetle response to male-produced pheromone (Lapointe et al. 2012) found specifically in beetle frass (Jones and Schroeder 1984). The frass falls to the ground along with first instar larvae as they hatch and burrow into the soil. Thus, we explored the hypothesis that this pheromone may be exploited by subterranean EPNs as another infochemical for host location. Our results indicate that two species of EPNs from two genera that natively co-occur with D. abbreviatus are specifically attracted to the adult beetle-produced pheromone, while a recently introduced EPN species, as well as a plant parasitic species, are not. Furthermore, we show that the adult beetle-produced pheromone increases feral EPN response and associated mortality of larval beetles in cultivated citrus in the field. Methods and materials Insects Diaprepes abbreviatus larvae were obtained from a laboratory colony maintained at the University of Florida s Citrus Research and Education Center (CREC) in Lake Alfred, FL, USA. This culture was initiated from a larger colony maintained at the Division of Plant Industry Sterile Fly Facility in Gainesville, FL, and periodically supplemented with beetles collected from citrus orchards in central Florida. Larvae were reared on a commercially prepared diet (Bio-Serv, Inc., Frenchtown, NJ, USA) using procedures described by Lapointe and Shapiro (1999). Larvae used in experiments were from third to sixth instars. Nematodes The EPNs, S. diaprepesi and H. indica, were isolated from D. abbreviatus larvae buried in commercial citrus orchards in Florida. S. riobrave were descendants of commercial formulations intended for field application to manage D. abbreviatus. All EPN species were cultured in last-instar larvae of the greater wax moth, Galleria mellonella, at 25 C according to procedures described in Kaya and Stock (1997) and were continuously reared for 6 9 months prior to the initiation of the investigation. IJs that emerged from insect cadavers into White traps (White 1927) were stored in shallow water in tissue flasks at 15 C for up to

3 A weevil sex pheromone serves as an attractant for its entomopathogenic weeks prior to use. Tylenchulus semipenetrans were obtained from infected field-grown citrus approximately 6 months prior to initiation of experiments. Infected roots and surrounding soil were soaked and IJ nematodes were subsequently extracted via sieving and centrifugation flotation (Southey 1986). Chemicals Carvacrol, (±)-linalool [50 (R): 50 (S)], and geraniol [blend of its (Z, E)-isomers referred to as nerol and geraniol, respectively] were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were [97% pure. (E)-3-(2- Hydroxyethyl)-4-methyl-2-pentenoate (E/Z/4-isopropyl- 5,6-dihydro-2H-pyran-2-one, 83:11:6) was synthesized and verified for identity and purity following a previously detailed procedure (Lapointe et al. 2012). Laboratory behavioral assays The behavioral responses of nematodes to collected beetle frass samples and synthetic chemicals were quantified in a two-choice, sand-filled olfactometer described thoroughly in Ali et al. (2010). Briefly, the olfactometer consists of three detachable sections: two opposing 16-ml glass jars which contained treatments and a central connecting tube 3 cm in length with an apical hole into which nematodes were applied (Ali et al. 2010). Initially, we tested the hypothesis that EPNs respond to a chemical found in beetle frass. Frass was collected from a chamber containing groups of 20 male and 20 female weevils feeding on Swingle citrumelo (Citrus paradisi Macf. 9 Poncirus trifoliata L. Raf.) rootstock. An insect frass sample consisted of 1 g of the feces macerated onto 2-cm diameter disk of filter paper (Whatman no. 1). This amount was based on preliminary assays to determine a feasible amount of frass to collect and replicate using the olfactometer described below. Negative controls consisted of clean filter paper. Thereafter, filter papers were placed on the bottom of each glass jar, which were subsequently filled with 10% saturated (dry wt. sand: water volume; w/v) sterilized sand (Ali et al. 2010). The central chamber connecting the two arms of the olfactometer was also filled with sterilized and moistened sand. Nematodes (c IJs) were applied into the central orifice of the connecting tube and given 48 h to respond. Following the incubation period, the column was disassembled and the nematodes from the two collection jars were extracted using Baermann funnels. The experiment was replicated 20 times for each nematode species and volatile treatment combination. The control treatment for each nematode species consisted of solvent blanks placed in each arm of the olfactometer. This double blank treatment produced identical results for each nematode species (no directional response, see Results ). Following assays with D. abbreviatus adult frass showing that certain EPN species exhibit attraction, we postulated that the pheromone of D. abbreviatus, present in and released from the Diaprepes weevil s frass (Lapointe et al. 2012), caused the behavioral effect. Subsequent assays tested this hypothesis and compared EPN response to the synthetic pheromone. We simultaneously tested EPN response to carvacrol, geraniol, and (±)-linalool. Carvacrol was investigated because it is the only other behaviorally active volatile identified from the frass of male D. abbreviatus that attracts female D. abbreviatus adults when blended with the host plant volatiles linalool and (Z)-3- hexen-1-ol (Otálora-Luna et al. 2009). Geraniol and linalool were chosen as additional negative controls, given that these are common citrus leaf volatiles and not known to specifically affect behavior of the EPN species belowground. The choice treatments compared for each nematode species in the comprehensive experiment were: (1) blank versus blank; (2) blank versus beetle frass; (3) blank versus pheromone; (4) blank versus linalool; (5) blank versus geraniol; (6) blank versus carvacrol; (7) blank versus 1:1:1:1 blend of linalool:geraniol:citral:pheromone. All chemicals (except D. abbreviatus pheromone) were diluted in hexane and 300 ng of chemical in 100 ll ofsolventwaspipettedontofilter paper. The pheromone was diluted in 10:1 hexane:ethyl acetate and 12 ng was applied onto filter paper in 100 ll of solvent. The solvent was allowed to evaporate for 60 s prior to insertion into olfactometers. Negative controls consisted of either relevant solvent only applied to filter paper. Two-choice bioassay to determine optimal dosage to attract EPNs Bioassays were conducted in olfactometers as described above for S. diaprepesi and H. indica. In the olfactometer, the EPNs were given a choice between a solvent blank and one of five logarithmically diluted doses of pheromone dissolved in hexane:ethyl acetate. Volatiles were released from filter paper as described above. EPNs were collected by disassembling olfactometers and assessing the choice after 48 h when they had moved into either arm of the olfactometer containing treatments. Response of feral EPNs in field citrus A field experiment was conducted in sandy soil (97:2:1, sand:silt:clay; ph 7.1; 0.1% OM) citrus orchard in Lake Alfred, FL ( N, W). The experiment was placed within a section of mature orange trees spaced (without beds) 4.5 m within and 8.1 m between rows that was irrigated with microsprinklers. A randomized complete block

4 202 M. J. Rivera et al. design was used to place treatments between trees in eight adjacent rows. Cylindrical wire mesh cages containing autoclaved sandy soil (10% moisture) and a single D. abbreviatus larva (reared on artificial diet for 3 5 weeks) were buried 20 cm deep in the soil beneath the tree canopies. The location was chosen, in part, because it has been investigated previously and was known to contain populations of nematode species relevant to our laboratory experiments (Ali et al. 2012). Cages were made of 225-mesh stainless steel cylinders (7 length 9 3 cm diameter) secured at each end with polypropylene snap-on caps. A replicate consisted of two cages placed equidistantly from one another in a circle pattern (48 cm diam) for each treatment (see figure S5 in Ali et al. 2012). All cages were baited with one of two treatments per replicate: (1) D. abbreviatus pheromone or (2) blank solvent control. There were 20 replicates of two cages per treatment. Treatments were applied by pipette as 100 ll aliquots(120 ng of pheromone) to 3 cm diameter filter paper discs (Whatman #1). The solvent was allowed to evaporate for 30 s prior to insertion of filter papers at the base of each cage. The cages were left buried for 72 h. Eight soil core samples (2.5 cm diameter 9 30 cm deep) were taken from the soil surrounding the treatment arena before the cages were removed to measure the number of EPNs attracted to the surrounding treatment arena. Nematodes from the soil surrounding each cage were recovered from the soil using Baermann extractors; extracted nematodes were collected and counted with a dissection scope. Recovered larvae were rinsed and placed on moistened filter paper within individual Petri dishes to confirm EPN infection by subsequent infective juvenile emergence from cadavers. The mortality of the larvae caused by EPNs was recorded from 0 to 72 h after removal from soil. Statistical analysis Nematode two-choice experiments and nematode subsample count associated with Diaprepes larvae in the field were analyzed using paired, two-tailed T tests at a = Data from in-field deployment of sentinel Diaprepes larvae were analyzed using a two-tailed McNemar s test with continuity correction. Dose response data recorded for S. diaprepesi and H. indica were analyzed using a two-way analysis of variance by treatment dosage and species. Results Nematode two-choice experiments More S. diaprepesi were attracted to the pheromone (t = 11.13, df = 19, P B ), frass (t = 8.175, df = 19, P B ) and the blend of volatiles (t = 7.763, df = 19, P B ) than by the solvent control (Fig. 1). S. diaprepesi showed no directional bias when exposed to the solvent blank in both arms of the olfactometer (t = 1.343, df = 19, P = ) and were not attracted to linalool (t = , df = 19, P = ), geraniol (t = 0.169, df = 19, P = ), or carvacrol (t = , df = 19, P = ) as compared to the solvent control. Similarly, more Heterorhabditis indica were attracted to the pheromone (t = 8.756, df = 19, P B ), frass (t = 5.314, df = 19, P B ), and the synthetic blend of volatiles (t = 6.837, df = 19, P B ) than by the control (Fig. 2). H. indica exhibited no directional preference when presented with a choice between two solvent blanks (t = 0.444, df = 19, P = ) and was not attracted to linalool (t = , df = 19, P = ), geraniol (t = , df = 19, P = ), or carvacrol (t = , df = 19, P = ) as compared to the control. Steinernema riobrave was not attracted to any treatment compared the solvent blank: vs. blank (t = 1.059, df = 19, P = ), linalool (t = , df = 19, P = ), geraniol (t = , df = 19, P = ), carvacrol (t = , df = 19, P = ), pheromone (t = , df = 19, P = ), frass (t = , df = 18, P = ), or the synthetic blend (t = , df = 19, P = ). The plant parasitic nematode, Tylenchulus semipenetrans, was also not attracted to any of the treatments compared with the blank: vs. blank (t = 1.058, df = 19, P = ), linalool (t = , df = 19, P = ), geraniol (t = 1.61, df = 19, P = 0.9), carvacrol (t = , df = 19, P = ), pheromone (t = 1.247, df = 19, P = ), frass (t = , df = 19, P = ), or the synthetic blend (t = 1.008, df = 19, P = ). Nematode response to pheromone over dosage range in laboratory two-choice assay Steinernema diaprepesi and Heterorhabditis indica were exposed to increasing dosages of the pheromone versus a solvent blank. Attraction to the pheromone varied by dose (F (6) = 43.67, P B 0.001) and nematode species (F (2) = , P B 0.001) (Fig. 3). Response of S. diaprepesi to the pheromone was greater than that of H. indica at all dosages and most nematodes of each species were attracted at the 0.12 and 1.2 lg dosages. Response of feral EPNs in field citrus Diaprepes larvae were more likely to be infected with EPN when deployed in the field with D. abbreviatus pheromone as compared to the solvent control (v 2 = 5.818; df = 1, P = ). More nematodes were counted in samples taken surrounding the area where the pheromone was

5 A weevil sex pheromone serves as an attractant for its entomopathogenic 203 Fig. 1 Mean (±SE) number of Steinernema diaprepesi responding to: a hexane solvent blank vs. solvent blank, b linalool vs. solvent blank, c geraniol vs. solvent blank, d carvacrol vs. solvent blank, e Diaprepes abbreviatus pheromone vs. solvent blank, f D. deployed as compared to the solvent control (t = 3.382, df = 19, P = ). Discussion Our results indicate that two EPN species from two genera that natively co-occur with D. abbreviatus were specifically attracted to the adult beetle-produced sex pheromone. However, a recently introduced EPN species, abbreviatus frass vs. solvent blank and g the synthetic 1:1:1:1 blend of linalool:geraniol:citral:pheromone vs. solvent blank in a twochoice olfactometer (N = 20). Means without an asterisk did not differ significantly (a = 0.05) from their associated blank control as well as a plant parasitic nematode species, was not attracted to the beetle pheromone. No other known chemical components of the beetle frass were attractive to EPNs (Fig. 1); however, the diffusion of frass components into the soil requires further investigation (Som et al. 2017). Deploying the pheromone in cultivated citrus increased feral EPN response and associated mortality of larval beetles. This result confirms increasing evidence of local adaptation of EPNs to host signals (Willett et al. 2015). Furthermore, this may be the first example of

6 204 M. J. Rivera et al. Fig. 2 Mean (±SE) number of Heterorhabditis indica responding to: a hexane solvent blank vs. solvent blank, b linalool vs. solvent blank, c geraniol vs. solvent blank, d carvacrol vs. solvent blank, e Diaprepes abbreviatus pheromone vs. solvent blank, f D. abbreviatus frass vs. EPNs exploiting an insect pheromone as a host-seeking cue. The ability of EPNs to detect its host s pheromone likely represents an important step in the evolutionary development of host association. Our results are congruent with investigations of necromenic Pristionchus nematodes, which also orient to their host insect s pheromone (Hong et al. 2008). These nematodes exist on the surface of larvae of soil-dwelling beetles, primarily Scarabaeidae, and wait solvent blank and g the synthetic 1:1:1:1 blend of linalool:geraniol:citral:pheromone vs. solvent blank in a two-choice olfactometer (N = 20). Means without an asterisk did not differ significantly (a = 0.05) from their associated blank control until the larva dies to colonize the carcass. This genus of nematodes, similar to other free-living nematodes, can compete with EPNs for resources by concurrently feeding on hosts killed by EPNs (Blanco-Pérez et al. 2017). Pristioncus nematodes can exist in the soil, but also exist on all beetle life stages moving to the soil in search of other hosts at various points in the insect s life cycle (Hong et al. 2008). Beetle larvae exist in the soil, but generally do not release pheromones; therefore, these nematodes likely

7 A weevil sex pheromone serves as an attractant for its entomopathogenic 205 Fig. 3 Mean (±SE) number of Steinernema diaprepesi and Heterorhabditis indica responding to Diaprepes abbreviatus pheromone at increasing dosages in a two-choice olfactometer (N = 20). Infective juvenile EPNs were allowed to choose between pheromone at various dosages versus a blank solvent control. Means differ significantly (a = 0.05) by letter and *** indicates a statistical significance between species at P B come into contact with the pheromone following oviposition by adults or from adult beetle frass. In the case of D. abbreviatus in citrus, local populations of EPN likely come in contact with the pheromone through frass. Adult weevils feed on and lay their eggs on citrus foliage. Frass drops to the ground from the feeding adult weevils and eventually larvae drop to the ground to feed on the citrus tree roots (McCoy et al. 2000). Leaves with deposited pheromone or frass containing the pheromone falling to the soil could serve as sources of pheromone release detected by nearby EPNs. Our data suggest that these cues likely serve to improve host location by EPNs, but the extent to which these cues may increase the efficiency of predation may depend on both the nematodes predominant foraging strategy and vertical distribution of the EPN species in the soil column. The pheromone-responsive EPNs, S. diaprepesi and H. indica, exhibit cruising behavior; they actively move toward their hosts. These two species are considered intermediate cruisers along a continuum as compared to other EPN species investigated here (Ali et al. 2011). H. indica moves rapidly and is considered a cruiser (Manimaran et al. 2012), while S. riobrave displays jumping behavior during movement (Dillman et al. 2012). However, our sampling of nematode species in the current investigation with regard to predominant foraging strategy and movement behavior was insufficient to discern patterns of response based on this specific trait. Steinernema diaprepesi exhibited greater attraction to the beetle pheromone than H. indica (Fig. 3), which suggests the former species may be more closely associated with D. abbreviatus. Despite this difference, the known dispersal capabilities and cruising behavior of both S. diaprepesi and H. indica to host-related chemical cues may explain why these two species exhibited such consistent chemotactic responsiveness to an attractive cue (their host s pheromone) in the current investigation. It is, however, possible that these species are vertically stratified in the soil in a manner that gives certain species occurring in the upper soil column greater advantage in exploiting a chemical cue found near the soil surface as compared with species that occur lower in the soil column. The vertical distribution of EPNs and how it may impact their chemotactic behavior with respect to host finding requires further investigation. The EPN species, S. diaprepesi and H. indica, are known to exploit herbivore-induced volatiles released by citrus roots upon damage by D. abbreviatus larvae to locate their hosts (Ali et al. 2011). Our current results indicate that these same EPN species are also attracted to the maleproduced pheromone of adult weevils. Therefore, EPNs appear to use multiple, redundant cues specific to their hosts during foraging by chemotaxis. It remains to be determined whether these distinct cues (plant source versus insect source) act additively or synergistically to attract EPNs and their role in host location at various distances from the source. In other words, do multiple cues provide more information to nematodes at the same distance away from the host or are these two different cues detected and used sequentially by EPNs at different distances away from the source? Does one cue indicate general presence of the host within a location (insect pheromone), while another more precisely indicate an active living host larva (HIPV) and can nematodes discriminate the difference and use this information for making directed behavioral adjustments? Moreover, the underlying peripheral physiology of nematode chemotaxis and integration of this complex information prior to exhibiting behavior deserves investigation. Specific questions include: (1) Are there multiple separate peripheral receptors for detecting these very different chemical cues? (2) How are these odors coded and integrated downstream of the peripheral nervous system? Acknowledgements We thank Ian Jackson and Daniel Diaz for technical assistance. We are grateful to Drs. F. El-Borai and L. Duncan for assistance with nematode culture establishment and for providing guidance regarding their maintenance. This work was partially supported by a USDA-CSREES special Grant and University of Florida Research Foundation Professorship Grant to L. L. S. References Adair RC (1994) A four-year field trial of entomopathogenic nematodes for control of Diaprepes abbreviatus in Flatwoods citrus grove. Proc Fla State Hortic Soc 107:63 68

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