Behavioral and electrophysiological responses of mango hopper, Idioscopus nitidulus (Walker) (Hemiptera: Cicadellidae) to host cues

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1 Behavioral and electrophysiological responses of mango hopper, Idioscopus nitidulus (Walker) (Hemiptera: Cicadellidae) to host cues GUNDAPPA 1, KAMALA JAYANTHI, P. D 2., RAVINDRA M AURADE 2, VIVEK KEMPRAJ 2, RAVINDRA, K. V 3., BHAKTAVATSALAM, N 3. and ABRAHAM VERGHESE 3 1 ICAR-Central Institute for Sub-tropical Horticulture, Lucknow , India 2 ICAR-Indian Institute of Horticultural Research, Bangalore , India 3 ICAR-National Bureau of Agriculturally Important Insects, Bangalore , India gundu6100@gmail.com ABSTRACT : Mango hopper, Idioscopus nitidulus (Walker), is a perilous pest monophagous to mango. Through co-evolution, hoppers have been tuned to detect mango inflorescence, which is their sole feeding and oviposition forte. Here, we studied the behavior of the mango hopper, I. nitidulus, towards headspace volatiles of mango (cv. Totapuri) inflorescence through olfactometer bioassays and GC-EAD. The chemical composition of inflorescence volatiles were analyzed by GC-MS/MS. The time spent by adult I. nitidulus in the treatment and control was found significantly different. Electrophysiological responses of hoppers to volatiles of mango flowers revealed fifteen volatile compounds. This study provides evidence that chemical cues in mango flower volatiles may act as probable attractants. Keywords: Hopper, mango, semiochemicals, volatile INTRODUCTION Flowers volatiles from the higher plants serve as potential attractants for herbivores (Visser, 1986). Hostproduced volatile chemical cues (kairomones) play an important role in enabling insects to recognize their host plants at a distance. Usually, herbivores may avoid certain non-host plant volatiles (Dicke, 1986; Pallini et al. 1997), but attracted to their host plant volatiles (Loughrin et al. 1995; Bolter et al, 1997; Pallini et al. 1997; Dicke & Vet 1999). In many cases the volatiles are specific for herbivory, or even for the herbivore species that inflicts the damage. This is all the more so in case of monophagous insects like mango hoppers, Idioscopus nitidulus (Walker) (Homoptera: Cicadellidae) that exclusively feeds on mango inflorescence. Such host specialization tunes the monophagous species to volatiles that are specific to their host plants. Our thorough understanding of insect plant interactions could strengthen our current pest management programmes as discovery of potent chemical cues (=host plant derived kairomones) is helpful for designing sustainable, semiochemical based IPM programs (Kamala Jayanthi et al. 2015). Mango hopper, Idioscopus nitidulus (Walker) (Hemiptera: Cicadellidae) is the most serious pest and widespread throughout the country. Enormous numbers of nymphs and adults are found clustering on the inflorescence during spring, sucking the sap. The infested flowers shrivel, turn brown and ultimately fall off causing non-setting of flowers and dropping of immature set fruits, thereby reducing the yield. On attaining maturity the hoppers leave the blossoms and move on to the leaves. Swarms of adults are commonly seen hovering in mango groves and sitting on all plant parts. The hoppers excrete honeydew that covers the inflorescence, leaves and fruits encouraging black sooty mold, which affects photosynthetic activity of leaves and market quality of fruits (Verghese & Kamala Jayanthi 2001). Since the mango hopper is a monophagous pest confining to mango only, it is assumed that it may respond the mango inflorescence chemical cues (kairomones) that may have potential for use in mango hopper pest management programs in future. The present investigation was undertaken to identify the potent host volatile cues from mango inflorescence to mango hopper I. nitidulus. MATERIALS AND METHODS The study was carried out at Indian Institute of Horticultural Research (IIHR), Bangalore, India (12 o 58 N; 77 o 35 E). The mango hoppers, I. nitidulus and the mango inflorescence used for the study were collected from the experimental mango orchards of IIHR. 118

2 Response of Mango hoppers to host cues Insects : The experiments were carried out with adult mango hoppers that were collected from the unsprayed infested mango trees cv. Totapuri using polythene covers. The polythene covers (45 cm length x 30.5 cm breadth) were draped on to the hopper infested mango inflorescences and the inflorescences were tapped to dislodge the adult hoppers present on them in to the polythene covers. After collection of hoppers, the polythene covers were removed from the inflorescence, tied with rubber bands to prevent the escape of hoppers and brought to the laboratory. Each individual hopper in the polythene covers was separated and placed in glass vials covered with muslin cloth and starved for 1 hr before experimentation. Air entrainment : Headspace samples of volatiles from mango inflorescences (cv. Totapuri) were collected by air entrainment (Agelopoulos et al. 1999). The mango inflorescences were collected from the field during the early hours of the day (between 9-10 am). The whole inflorescence on the tree was cut with secateurs and the stem end was kept immediately in conical flask (50 ml) containing 50 ml distilled water to prevent the desiccation. Further, the conical flask was covered with polythene cover and brought to the laboratory to collect the volatiles. Before volatile collection, glassware and aluminium plates were washed with aqueous Teepol detergent, rinsed with distilled water and acetone, and then dried in a hot air oven at 120 C for 2 h. The Porapak Q tubes used for collection of volatiles were eluted with redistilled diethyl ether and heated at 120 C for 2 h in hot air oven to remove contaminants. The field collected cut inflorescences along with conical flasks were placed individually inside a glass vessel (200 mm high x 150 mm wide), closed with an aluminium plate at the bottom and fitted with a collection port at the top serving as outlet and a second port at the side wall of the bottom serving as an inlet. The aluminium plates were clipped at the open end of the glass vessel using flange to make air tight. Air purified by passage through an activated charcoal filter, was pumped into the glass vessel at 600 ml/min through the inlet port. Air was drawn out at 800 ml/min through Porapak Q (50 mg, 60/80 mesh; Supelco, Sigma Aldrich, India) in a 5 mm diameter glass tube. All connections were made with polytetrafluoro ethylene (PTFE) tubing with brass ferrules and fittings. The volatiles from mango inflorescence were entrained for 2 hrs and the Porapak Q filters were eluted with 750 µl of redistilled diethyl ether, providing a solution that contained the isolated volatile compounds that served as test sample. Sample was stored in glass vial in a freezer (-20 C) until use. Olfactometer Bioassays: A Perspex four-arm olfactometer (Pettersson, 1970) was used to determine the behavioural responses of adult I. nitidulus to headspace samples of volatiles. Prior to each experiment, all glassware were washed with Teepol, rinsed with acetone and distilled water, and baked in an oven overnight at 160 C. Perspex components were washed with Teepol solution, rinsed with 80% ethanol solution and distilled water, and left to air dry. Experiments were conducted in a isolated room (25 ± 2 o C, 60% RH) to avoid contaminant odours. The olfactometer had four glass side arms feeding into a central arena which was divided into four odour fields. The central area was fitted with a filter-paper base (Whatman No. 1, 12 cm diameter) to provide traction for the walking insect. The olfactometer was illuminated from above by uniform lighting from white fluorescent light bulb (10 watts) covered with opaque dome to make it diffuse and was surrounded by a black wall cage to remove any external visual stimuli. The field collected and starved individual adult I. nitidulus was introduced through a hole in the top of the olfactometer. Each hopper was given 2 min to acclimatize in the olfactometer after which the experiment was run for 15 min for each replicate. The olfactometer was rotated 90 degrees every 2 min to eliminate any directional bias in the room. Air was drawn through the central hole at the rate of 900 ml min -1. The central arena of the olfactometer was divided into four discrete odour fields corresponding to each of four glass inlet arms. Of four glass arms, one contained the treatment and the other three arms served as controls. Test samples (10µl) were pipetted onto the filter paper strips and the solvent was allowed to evaporate prior to their placement in the treatment arm. The filter paper strips with solvent (diethyl ether) served as controls in the remaining three arms. In the bioassays, a response of adult I. nitidulus to the host cues collected from mango inflorescences was studied. Time spent and number of entries into each olfactometer arm was recorded with Olfa software (F. Nazzi, Udine, Italy). Nine replicates were carried out. The mean time spent in treated/ control regions and number of entries in treated/ control arms were compared using a paired t-test (SPSS version 16). Electrophysiology Coupled Gas Chromatography - Electroantennography (GC-EAD) : Mango hopper, I. nitidulus antennal responses to extracts of poropak 119

3 Gundappa et al. volatiles from mango Cv. Totapuri were studied by gas chromatography coupled electroantennographic detector (GC EAD) GC model Agilent 7890A (Singapore) coupled with Syntech EAG Model IDAC 2 (Intelligent Data Acquisition Controller) system with GCEAD interface temperature controller, TC 02 and with stimulus controller, CS-55. For separation of volatiles, the GC column having Agilent 19091J-413 HP-5 5% Phenyl Methyl Siloxane capillary column (30 m x 320 ìm x 0.25 ìm) with maximum temperature of 325 o C and Helium (99.999% purity) as the carrier gas (at a constant flow 2.0 ml/min) was used. The oven temperature maintained initially at 70 o C (2 min hold) to 260 o Cwith with a ramp of 10 C per minute. The Transfer line (Syntech Laboratories, Germany) connects between the GC and EAD temperature were maintained at 2 C above the maximum temperature in order to avoid condensation. The electronic Splitter with makeup gas was used to split the injected sample (1µl) in equal proportion to both EAD and FID. The EAG preparations were obtained using the indifferent electrode being placed within the head capsule and the recording electrode was slipped over the one of the antennae and the electrodes are filled with 25 mm Nacl (Ringer solution). The Purified fraction were eluted from the transfer line were carried out by a flow of humidified air with flow rate of 80cm/s. The antennal response eluted against the GC peaks were recorded by Syntech GCEAD software version Peaks eluting from the GC column were judged to be active if they elicited EAG activity in two or more of the coupled runs. GC-MS Analysis : Coupled Gas Chromatography- Mass Spectrometry (GCMS) Chemical composition of Porapak Q elutes were analyzed by GC-MS/MS using Varian 3800 apparatus equipped with coupled MS/MS (Saturn 4000). A capillary column (DB-5ms) of 30 m length and 0.25 mm ID and 0.25 mm film thickness was used to examine samples. Oven temperature was programmed at C with ramping at 3 C min -1 for 60 min. Helium was used as carrier gas at a flow rate of 1 ml -1. MS was in full scan mode (70 ev) and AMU ranged from 50 to 350. Two micro liter sample were injected in split mode (1:20) with injection temperature at 270 C. Compounds were identified by GC retention time, mass spectrum and KOVATS index using NIST 2007 and Wiley library as reference. RESULTS In four arm olfactometer, choice was given between the volatiles from mango flowers as a treatment and solvent diethyl ether as control to adult I. nitidulus to study behavioral responses. The time spent by adult I. nitidulus in the treatment and control was significantly different (t = 2.05; P < 0.04; N = 9) (fig 1). The crude extract of mango flowers (cv. Totapuri) was subjected for GS-MS/MS analysis. Wherein 24 compounds were identified based on the retention time viz., a-pinene, b- Phellandrene, b-pinene, Trans- Ocimene, Limonene, cis - Ocimene, Allo -Ocimene, Ethyl benzoate, Methyl salicylate, Thymol, a -Copaene, a - Gurjunene, b- Caryophyllene, a- Guaiene, a-humulene, a-muurolene, (+)-Aromandendrene, Chemigrene, d Guaiene, a - Elemene, Methyl hexadecanoate, 2- Methylhexadecan-1- ol and Hydrocarbons. Among them most abundant compound was trans-ocimene, whereas less abundant was á-muurolene. Flower volatiles, classified according to their chemical structure, are found in seven major classes of compounds: aliphatics, benzenoids/ phenyl propanoids, C5-branched compounds, terpenoids, nitrogen-containing compounds, sulphur-containing compounds, and a class of various cyclic compounds (Knudsen et al. 2006). In the current study majority of identified mango flower volatiles belongs to the group benzenoids/ phenyl propanoids. Time (min) t = 2.05; p<0.037 Fig 1. Time spent by I. nitidulus in treatment (mango flower volatile) and control (Diethyl ether) in four-arm Olfactometer bioassay (t =2.05; P=0.04; N=9) Headspace collections from mango flowers were analyzed by GC EAD (Fig. 2). Fifteen volatile compounds in the headspace collections consistently elicited significant EAD responses from the antennae of adult I. nitidulus. Among them, a -Pinene, b-phellandrene, Trans-Ocimene, Allo- Ocimene, Ethyl benzoate, Thymol, a-copaene, a -Gurjunene, b-caryophyllene, were the most potent attractant cues for mango hopper, I. nitidulus and were present in abundance. However, 120

4 Response of Mango hoppers to host cues (Coleoptera: Chrysomelidae) to Solanum tuberosum L. (Solanaceae) plants, Citrus weevil Cratosomus flavofasciatus Guerin Coleoptera: Curculionidae) to Cordia curassavica. Further, Bucephalogonia xanthophis (Hemiptera:Cicadellidae) is an important vector of the citrus variegated chlorosis that reported to be attracted to volatiles of Vernonia condensata (Asteraceae) (Dickens, 2002; Arab & Bento, 2006; Bento et al. 2008). Some herbivorous arthropods can detect their hosts using volatile compounds released by healthy plants, which can also stimulate oviposition and courtship behaviors in these organisms (Rojas et al. 2003). Fig 2. Antennal responses of adult mango hopper, I. nitidulus as EAD and FID peaks of gas chromatogram in the headspace volatiles of mango flowers (Cv. Totapuri). Compounds that elicited consistent response are a- Pinene (1); b- Phellandrene (2); Trans-Ocimene (3); Allo- Ocimene (4); Ethyl benzoate (5);Methyl salicylate (6);Thymol (7); a-copaene (8); a-gurjunene (9); b-caryophyllene (10); a-guaiene (11); a-humulene (12); a-muurolene (13); Chemigrene (14) and á-elemene (15) antennae of adult I. nitidulus also responded to compounds that were relatively less abundant in these extracts, such as Methyl salicylate, a -Guaiene, a - Humulene, a-muurolene Chemigrene and a-elemene. DISCUSSION Locating a host plant is crucial for a phytophagous (herbivorous) insect to fulfill its nutritional requirements and to find suitable oviposition sites. Insects can locate their hosts even though the host plants are often hidden among an array of other plants. Plant volatiles play an important role in this host-location process. The recognition of a host plant by these olfactory signals could occur by using either species-specific compounds or specific ratios of ubiquitous compounds (Bruce et al. 2005). In the current study behavior of the I. nitidulus was studied in the olfactometer assays where mango flower volatiles could attract the hoppers. This attraction can be attributed to the compounds in the volatiles. This was further supported by GC-EAD analysis, where antennal response was elicited for the 15 compounds. Using a combination of electrophysiological and behavioral experiments, few studies have identified plant volatile substances that enhance the ability of insects to locate host (Ngi-Song et al. 2000). The herbivorous insects which are also attracted for the host volatiles are Colorado potato beetle Leptinotarsa decemlineata Say The host chemical cues were shown to play role in host selection of female pear psylla Cacopsylla bidens, a specialized homopteran (Soroker et al. 2004). Similarly, the present study revealed for first time that the mango hopper, I nitidulus responds positively to mango flower volatiles. Considering the fact that host plants provide a plethora of specific chemical cues of variable volatility (Soroker et al. 2004, Bernays & Chapman 1994, Schoonhoven et al. 1998), it is expected that volatile cues would play a significant role in host location of as observed in the present study. Further, previous studies reported that appearance of inflorescence on the tree is the critical event for the migration of hibernating hoppers, I. nitidulus from main trunk to inflorescence (Gundappa et al. 2014). Current management programs for hopper are dominated by insecticidal applications with future research efforts to explore the role of the identified chemical cues for hopper monitoring and management programs will be envisaged. ACKNOWLEDGEMENTS We are grateful to Director, Indian Institute of Horticultural Research, Hessaraghatta, Banagalore for providing facility for this study. Senior author is grateful to Director, Central Institute for Subtropical Horticulture, Rehamakhera, Lucknow for permitting to undergo professional attachment training. REFERENCES Agelopoulos, N. G., Hooper, A. M., Maniar, S. P., Pickett J. A. and Wadhams L. J A novel approach for isolation of volatile chemicals released by individual leaves of a plant in situ. Journal of Chemical Ecology, 25: Arab, A. and Bento, J. M Volatiles: new perspectives for research in Brazil. Neotropical Entomology, 35:

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