Electrophysiological and Behavioral Responses of Theocolax Elegans (Westwood)(Hymenoptera: Pteromalidae) to Cereal Grain Volatiles

Hindawi Publishing Corporation BioMed Research International Volume 2016, Article ID 5460819, 8 pages http://dx.doi.org/10.1155/2016/5460819 Research Article Electrophysiological and Behavioral Responses of Theocolax elegans (Westwood) (Hymenoptera: Pteromalidae) to Cereal Grain Volatiles Giacinto Salvatore Germinara,1 Antonio De Cristofaro,2 and Giuseppe Rotundo2 1 Department of the Sciences of Agriculture, Food and Environment, University of Foggia, Via Napoli 25, 71122 Foggia, Italy 2Department of Agricultural, Environmental and Food Sciences, University of Molise, Via De Sanctis, 86100 Campobasso, Italy Correspondence should be addressed to Giacinto Salvatore Germinara; [email protected] Received 2 October 2015; Revised 23 December 2015; Accepted 5 January 2016 Academic Editor: Johannes Stokl¨ Copyright © 2016 Giacinto Salvatore Germinara et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Volatiles emitted by the host’s food would be the first signals used by parasitoids in the host location process and are thought to play an important role in host habitat location. In this study, the olfactory responses of Theocolax elegans (Westwood), a Pteromalid wasp that parasitizes immature stages of stored-product insect pests developing inside cereal or leguminous grains, to volatiles emitted by healthy wheat grains, their hexane extracts, and different doses of three individual compounds previously identified in cereal grain odors were investigated in Y-tube olfactometer and Petri dish arena behavioral bioassays and electroantennogram recordings. In Y-tube olfactometer bioassays, odors from healthy wheat grains and their hexane extracts were attractive to both sexes of T. elegans. Moreover, hexane extracts elicited arresting effects in Petri dish arena. The three synthetic compounds valeraldehyde, maltol, and vanillin elicited dose-dependent responses in both male and female adult wasps confirming the capability of the peripheral olfactory systems to perceive cereal volatiles. In behavioral bioassays, different doses of vanillin were significantly attractive to both sexes. 1. Introduction parasitic wasps [10–14], including some Pteromalidae species parasitizing stored-grain insect pests [15–17]. These volatiles, The use of natural enemies is considered to be an important generally acting as long distance cues, would be the first component of integrated pest management of stored-grain signals used by parasitoids in the host location process and are insect pests [1], particularly in storage areas. Because the adult thought to play an important role in host habitat location [18– beneficial insects are external to the grain, they can easily be 20]. The identification of such synomones has a great practical removed using normal grain-cleaning procedures [2]. interest since they could be used as parasitoid behavior mod- In their search for hosts, food and mates parasitoids are ifying compounds to enhance their biological performances. guided by multisensory information including visual, vibra- Theocolax elegans (Westwood) is a solitary ectoparasi- tional, and olfactory signals [3–5] which are frequently used toid of immature stages of economically important stored- in an interactive manner [6–8]. product insect pests which develop as internal feeders of The success of parasitic wasps in suppressing pest pop- cerealandlegumegrainsincludingRhyzopertha dominica ulation depends on their ability to locate hosts; therefore, (Fabricius), Sitophilus spp., Sitotroga cerealella (Olivier), and understanding the host location process is critical for the suc- Callosobruchus spp. [21]. T. elegans has been extensively stud- cessful implementation of biological control programs. Host- ied as a biological control agent of some cereal pests. In large- seeking behavior in parasitoids has been divided into four scale experiments, the wasp was effective in reducing Sitophi- phases, habitat location, host location, host recognition, and lus zeamais Motschulsky populations by up to 50% [22] and host acceptance [3, 9]. Orientation to host plant or host food those of R. dominica by 50–99% depending on the temper- volatiles in the absence of any host, host damage, and host ature [23–25]. Moreover, augmentative release of T. elegans derived materials have been demonstrated for a number of to control R. dominica resulted in a 61–92% reduction of the 2 BioMed Research International total number of insect fragments in wheat flour [2] indicating fluorescent lamps providing uniform lighting (2500 lux) a positive impact on the quality of stored cereal products. insidethetube.Apurified(activatedcharcoal)andhumid- Preliminary behavioral bioassays suggested a preferential ified airflow maintained at 6 mL/min by a flowmeter was orientation of T. elegans adults to odors of healthy wheat pumped through each arm. grains [26] and the sensitivity of the antennal olfactory sys- In order to test the attractiveness of volatiles emitted tems of both sexes towards a wide range of volatiles identified by healthy wheat grains and their hexane extracts to T. from various cereal grains [27–29] was shown by electroan- elegans males and females two dual-choice experiments were tennogram (EAG) recordings [30]. performed: (1) wheat grains (25 g) versus clean air and (2) In the present study, the behavioral responses of T. elegans wheat grain hexane extracts versus control hexane. In this 2 males and females to odors of uninfested wheat grains and latter, the odor chamber contained a filter paper disk (1.0 cm ) their hexane extracts were carefully investigated using Y-tube loaded with 50 Lofa10mggrainequivalent/L hexane olfactometer and Petri dish arena bioassays. Moreover, the extract, while the control chamber contained a filter paper biological activity of three individual volatiles (valeraldehyde, disk loaded with 50 L of hexane. Both disks were suspended maltol, and vanillin) previously identified in the aroma of in the center of the cross section. In third set of experiments fresh cereal grains [27] and attractive to the two T. elegans the behavioral responses of the adult wasps to different doses hosts Sitophilus oryzae [31] and S. granarius [32] was investi- of vanillin were evaluated. The test stimulus was a filter paper gated in a range of doses by electroantennographic and behav- disk loaded with 10 Lof5g/L vanillin hexane solution, ioural bioassays. whileasimilarfilterpaperdiskloadedwith10L of hexane was used as control. 2. Material and Methods Individual T. elegans adults were released at the open end of the stem. Each experiment lasted for 5 min. A choice was 2.1. Insects. AcolonyofR. dominica parasitized by T. elegans recorded when the insect moved 3 cm up an arm of the Y- was collected from a cereal storehouse and set up in glass jars × tube crossing the decision line (marked on both arms) and (15 cm diameter 25 cm) half filled with uninfested wheat remained beyond that line for more than 30 sec. Insects that kernels and closed with fine metallic net (0.2 mm mesh). ∘ did not make a decision within 5 min were considered as no Colonies were maintained at 28 ± 2 C, 60 ± 5%relative response and discarded. humidity, and 12L : 12D photoperiod. During the following After 5 individuals had been tested, the olfactometer was four months, wheat kernel samples (5 g) from the rearing jars ∘ were periodically transferred to transparent plastic containers cleaned with acetone and dried (200 Cfor30min)andtreat- (6 cm diameter × 8 cm) and held to check for the emergence ments between arms were switched to avoid position bias. For each test stimulus at least 20 male and female wasps were of adult wasps. Emerging insects were collected daily and kept 2 individually in glass vials (1.5 cm diameter × 5cm)without tested. A test was employed to determine the significance foodsupplyandintheabsenceofcerealodorsforatleast of differences between the number of wasps choosing the 12 h before the EAG and behavioral experiments. The sex of treatment or control arm of the olfactometer. the insects tested was determined by observing their genitalia with a stereomicroscope. 2.5. Electroantennography (EAG). The electrophysiological response of male and female T. elegans antennae to increas- 2.2. Extract Preparation. Solvent extracts of wheat kernels ing concentrations of synthetic valeraldehyde, maltol, and (Triticum durum var. Simeto) were prepared by immersing vanillin was measured by the EAG technique used in previous wheat grains (40 g) in hexane (20 mL) for 36 hours at room studies [30, 34]. Antennae were excised from 1- to 2-day- temperature. Extracts from 3 grain samples were combined old insects. The base of the antenna was put into a glass and concentrated to 10 mg grain equivalent/Lusingagentle ∘ pipette filled with Kaissling saline [35] which served as the stream of nitrogen and stored at −20 C until being needed. neutralelectrode.Thetipoftheantennawasputincontact with the end of a similar pipette (0.1–0.2 mm diameter) which 2.3. Chemicals. Valeraldehyde (1-pentanal), maltol (3-meth- provided the active electrode. oxy-2-methyl-4-pyrone), vanillin (3-methoxy-4-hydroxyben- AgCl-coated silver wires were used to maintain the elec- zaldehyde), and hexanal used as stimuli and hexane, mineral trical continuity between the antennal preparation and an oil, acetone, and chloroform used as solvents were purchased AC/DC UN-6

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