Olfactometer studies on preference of Rhynocoris marginatus Fabricius to three lepidopteran preys

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1 Olfactometer studies on preference of Rhynocoris marginatus Fabricius to three lepidopteran preys K. PRAVALIKA 1, T. UMAMAHESWARI 1 and CHITRA SHANKER 2 * 1 Professor Jayashankar Telangana Agricultural University, Hyderabad , India 2 ICAR - Indian Institute of Rice Research, Hyderabad , India * chitrashanker@gmail.com ABSTRACT: The assassin bug, Rhynocoris marginatus is a generalist predator and can be deployed across agro ecosystems. In a study to assess its interactions with three polyphagous lepidopteran pests, the predator showed significantly higher preference to Spodoptera litura (F.) (50 %) over Mythimna separata (25.3 %) and Sesamia inferens (24.7%). The prey predator interaction of three prey species and R. marginatus was assessed in a Y-arm olfactometer and a six-arm olfactometer. Though R. marginatus responded to all the hexane extracts of tested insect pests, it showed maximum preference to S. litura (85.7 %) followed by M. separata (57.1%) and S. inferens (42.8 %) in the Y arm olfactometer compared to control. The approaching time and handling time were more for S. litura i.e., (10.9±1.99 min) and (18.3 ± 2.62 min) when compared to M. separata (10.2 ± 2.49 min), (17.7 ± 2.92 min) and S. inferens (7.33 ± 2.51 min) (17.6 ± 3.51 min) respectively. Similarly in a multi choice test, R. marginatus highly preferred the hexane extract of S. litura (42.8%) followed by M. seperata (28.5%) and least preference was to S. inferens (21.4 %) which was on par with hexane extract alone. Keywords: Host prey preference, Mythimna seperata, Sesamia inferens, Spodoptera litura INTRODUCTION The family Reduviidae includes important generalist predators associated with a wide array of pests in diverse agroecosystems(ambrose, 2003). Among reduviids, Rhynocoris marginatus Fabricius was reported as a key predator on various insect pests belonging to Lepidoptera. Although R. marginatus is polyphagous, it certainly exhibited certain degree of hostspecificity and also preferred a particular stage of prey. Hence, knowledge on prey preference of this polyphagous predator is imperative before employing it in biological warfare (Nordlund and Morrison, 1990; Hattingh and Samways, 1992). Moreover, such an understanding will facilitate the augmentation of the predator for biological control programmes. Be havioura l chemic als tha t provide cues for orientation of predators in the prey finding sequence includes secondary plant metabolites and chemicals directly associated with the host prey. Antennectomy studies by Ambrose et al. (1994) and Ambrose and Mayamuthu (1994) on the predatory behaviour of reduviids revealed that, these predators use the prey chemical cues to locate their prey. Thus, it seemed likely that the reduviids utilize chemicals released by their prey as kairomone in prey location. Hence the present study aims at investigating the behaviour of R. marginatus to the chemical cues of Spodoptera litura, M. separata and Se samia infe re ns a nd ide ntifying the compounds that elicit the host-seeking response of R. marginatus. MATERIALS AND METHODS Host prey preference Preference of adult R. marginatus to thre e lepidopteran preys, S. litura, S. inferens and M. separata were studied in plastic containers (16 x 7 cm). The predator and the larval prey were released into the container at the rate of one predator to two of each larvae in each container where 24 hrs pre starved predator could be used for the study. As the predator chooses the prey of interest prey preference was assessed by choice experiments i.e. in terms of percentage of each prey species preferred (consumed) by the predator in 24 hrs. Five replicates were maintained and observations were recorded for five days. Olfactometer studies The body extracts of the three insect pests were prepared following the methodology of Yasuda (1997). Fifteen fifth instar larvae of S. litura, S. inferens and M. separata were kept in reagent bottles having 1:2 mixtures of hexane and acetone for 30 minutes at room 28

2 Oflactometer studies on Rhynocoris marginatus temperatures, separately and subsequently stored in a freezer overnight. The following day the solvent extracts were then filtered through a Whatman No. 1 filter paper. Thereafter, the filtered solvent extracts were evaporated in a vacuum desiccator under room temperature and the residues were dissolved in 100 ml of ether separately. The ether was washed off with 50 ml of distilled water thrice. Then the ether-soluble layer was dried over Sodium sulfate. The solvent was removed by using vacuum desiccator under room temperature and the residue was dissolved in hexane. The resultant extracts were stored at below -20 C until further use. Bioassay for orientation/approaching behaviour A Y-shaped olfactometer made up of glass with a main stem 20 cm length, two arms 15 cm length and 5 cm diameter, each and 90 angle between them, was used for the bioassay studies. The olfactometer has two arms that are connected to 6.5 cm diameter glass chambers (odour cells), in which the prey solvent extracts (odour sources) could be placed. Before starting the experiment, the Y-shaped olfactometer with odour cells were cleaned with 70% alcohol followed by continuous blowing of air by an aerator for 15 min to remove the unwanted odour from the odour cells. The air was blown into the two arms of the olfactometer using a small T tube and the air was allowed to pass outside through the exit tubes of odour cells. A small piece of sterile cotton impregnated with the body extract of insect larvae (100 ul of a sample) was used as test and cotton impregnated with only hexane was used as control (100 ul of hexane). Pre dators sta rved for 24 h we re introduced through the main stem and their predatory behaviour was observed for 30 min continuously for 7 observations. The predatory behaviour was observed in terms of approaching and sucking time. From these the handling time was calculated by summing up both (Cohen, 2000). The predators choose either the test chamber with body extract or the control chamber with hexane or neither. If the predator chose the test chamber or control chamber,it was considered as positive choice or negative choice, respectively. If the predator chooses neither of the chambers, then it was considered as no choice. The experiment was replicated five times with 24 h starved and inexperienced predators on each body extract of insect pests, separa tely. T he data was subjected to ANOVA. The approaching time taken by the predators in the olfactometer to different body extracts of different larvae was converted into an index called Excess Proportion Index (EPI) (Sakuma and Fukami, 1985) using the following formula: NS - NC EPI = NS + NC where, NS is number of predators choosing the sample cell NC is number of predators choosing the control cells EPI values from +1 to -1 These terms simply express the polarity of the directional choice. Positive values indicate a positive approach response. The assay for contact chemicals consists of counting antennation and probing frequencies towards e ac h te st s ample, a t give n pe riod of time.observations were recorded on percent predator preference for each extract against control (hexane extract). Six-arm olfactometer The six arm olfactometer was used to find out the orientation of the predator towards the body extracts of different pest larvae. The six arm olfactometer consisted of central chamber of 6 cm diameter and 3 cm length from which six glass tubes of 20 cm length and 2.5 cm diameterwere projected outwards at equidistance with 60 angle between two tubes. The middle chamber hada opening of 2.5 cm diameter at the top. At a time, three cells with prey extract (100 ul each) impregnated in sterile cotton and another three cells as control with hexane alone (100 ul each) impregnated in sterile cotton was used. RESULTS AND DISCUSSION When percentage prey preference was calculated for individual days over five days, R. marginatus significantly preferre d S. litur a (50%) while pre fe re nc e for M. separata (25.3%) and S. inferens (24.7%) was lesser than S. litura but on par with each other (Table 1). The offered prey was ranked according to the first second and third preferred etc. until all prey offered at a time was ranked. The unfed prey was given the same rank following the rank of the last fed prey. The rank data was subjected to the Kurskal Wallis non parametric analysis for ascertaining individual prey preferences of the predators tested. The results showed significance for prey preference. The overall H-statistic indicated a high level of significance for prey preference opposing the null hypothesis that all prey will be equally preferred 29

3 Pravalika et al. Table 1. Preference of predator, Rhynocoris marginatus to different species of the prey Day of experimentation Predatory preference (%) Spodoptera litura* Mythimna separata* Sesamia inferens* (45.00) 25.0(30.00) 25.0(30.00) (45.00) 16.7(24.12) 33.3(35.24) (50.77) 20.0(26.57) 20.0(26.57) (39.23) 40.0(39.23) 20.0(26.57) (45.00) 25.0(30.00) 25.0(30.00) AVG 50.0 a (45.00) 25.3 b (21.20) 24.7 b (21.42) CD (0.01) 7.68** *Average of fifteen larvae ** Significant (Shapiro Wilk statistic of normalcy 0.93 at P-value 0.000; Kruskal Wallis statistic at P-value <0.001 assuming a chi-square distribution with 2 df). The means separated by Fisher s LSD showed that preference was highest for S. litura followed by M. separata and then S. inferens. The median preference rank of individual predators for S. litura was 2.5 which included the larvae that were not fed upon while it was 4.5 for both M. separata and S. inferens (Figure 1). Prey preference varied among generalist predators. Some are random while others are Fig 1. Rank preference of Rhynocoris marginatus to three different species of the prey/host Kurskal Wallis analysis of the average ranking of prey preference of Rhynocoris marginatus (n = 25). The box represents the interquartile range (25th to 75th percentile) of the data, and the line in the centre of the box is the median value of prey preference. The dots are outliers, and the whiskers are the outer range of data. (Shapiro Wilk statistic of normalcy 0.93 at P-value 0.000; Kruskal Wallis s tatistic at P-value < ass uming a chi-square distribution with 2 d.f.). cue based. In our studies through multiple-choice test, S. litura larvae were found to be the most preferred. Reduviid pre dators generally prefer lepidopte ran caterpillars due to their soft cuticle and slow movement (Ables, 1978). Harpactorine reduviid predators, viz., R. kumarii, Rhynocoris fuscipes (F.), Rhynocoris marginatus (F.) and Sycanusversi color Dohrn uniformly preferred soft bodied lepidopteran larvae followed by termites, bugs and beetles (Ambrose, 1999). Among the lepidopteran prey it preferred S. litura. McMahan (1983) noted that reduviids preference for one prey to another, in a choice test might be also influenced by smell or unpleasant taste of the prey. Attacked caterpillars rolled themselves and emitted a fluid. But it may or may not deter a predator as was found in the case of R. kumarii (Ambrose, 1999). The low preference exhibited by R. marginatus to S. inferens could be due to their chemical cues or speed of movement. Hatting and Samways (1992) reported that the ability of predatory insects to choose between prey types has an important bearing on the outcome of a biocontrol programmes. In this perspective it becomes imperative that we study prey preferences to ascertain the biocontrol utility of R. marginatus. Orientation/approaching behaviour The bioassay experime nts with the Y-sha ped olfac tomete r reve ale d that when the preda tor R. marginatus was released into the main chamber of the Y-shaped olfactometer, it oriented towards the odour source present in the sterile cotton with antennae directing towards the odour source (Table 2). After getting perfect orientation the reduviid palpated its 30

4 Oflactometer studies on Rhynocoris marginatus Table 2. Response of Rhynocoris marginatus in a Y arm olfactometer Parameter Spodoptera litura Mythimna seperata Sesamia inferens Prey preferred (%) Control preferred (%) Approaching time (min) 10.9± ± ±2.51 Handling time (min) 18.3± ± ±3.51 EPI (Excess Proportion Index) antennae, followed by rubbing their legs, rostral cleaning and extended rostrum towards the odour source. Once the predator entered the sample cell it exhibited quick walking and approaching with antennae, wings and legs cleaning and rostral protrusion.the hexane fractions of all the insect pests elicited a positive approaching response in R. marginatus. It showe d the highest response to the hexane extract of S. litura (10.9± 1.99 min) followed by M. separata (10.2± 2.49 min), and S. inferens (7.33± 2.51min). The preference to S. litura and M. separata body extracts was greater than that of S. inferens (Table 2). The predator R. marginatus exhibited the highest handling time in S. litura extract (18.3±2.62 min) followed by M. separata (17.7±2.92 min) and S. inferens (17.6± 3.51 min). The EPI values indicated positive response of R. marginatus in the order S. litura>m. separata>s.inferens. Highest preference of hexane extract of S. litura was also observed in a related predator species, R. fuscipes (Nagarajan and Ambrose, 2013) when tested in a Y shaped olfactometer. The cues stimulate receptors generating sensory inputs and finally behavioural responses. The predatory behavioural pattern of R. marginatus is arousal-approachrostral probing behaviour-injecting toxic saliva-paralyzingsucking and post predatory behaviour. R. marginatus oriented towards the prey with facing antenna, after getting perfect orientation, the predator palpated its antennae, then aroused and subsequently showed the other behavioral responses (Ambrose, 1999) as observed in this reduviid predator. The handling time of R marginatus to the infochemicals from the three pests studied conformed to results obtained by Maran (1999) with the reduviids R. kumarii Ambrose and Livingstone and R. marginatus (Fabricius) and by those of Yasuda and Wakamura (1996). They postulated that the chemical cues or kairomones of the prey stimulate the predators to respond towards them. It was very clear and from observations that R. marginatus exhibited approaching, rostrum protrusion and handling the body extracts on the cotton swabs. Similar observations were made by Yasuda and Wakamura (1996) and Yasuda (1997) with the predatory bug, Eocanthe cona f urce llata (Wolff) (Heteroptera: Pentatomidae) towards larval extracts of S. litura. A number of sa turated hydrocarbons were identified in the scales as well as whole body wash of many lepidopteran insects and their kairomonal activity has also been demonstrated (Jones et al., 1973; Madhu et al., 1997). Kairomones involved in the foraging beha viour of organisms have been published and reviewed during the past decades (Ruther et al, 2002). It includes chemical cue s exploited by preda tors (Hendrichs et al, 1994; Mendel et al, 1995; Dejean and Beugnon, 1996; Kielty et al, 1996) as well as parasitoids (Vinson, 1976; Godfray, 1994; Leprmce et al., 1994; Carroll et al., 1995; Carroll et al., 1998; Dougherty et al., 1999) during their search for food or oviposition sites or both. The involvement of antennae in the perception of volatile chemicals was proved by Crocker (1977). In his experiment, the detection of Trichoplusia ni (Hubner) eggs by the big eyed bug Geocoris punctipes (Say) was reduced, if the distal half of the antennae was removed. It was further proved by the antennectomy studies by Ambrose et al. (1991) in Acanthas pissiva (Distant); Ambrose et al. (1994) in Euagoras plagiatus (Bermeister) and Ambrose and Mayamuthu (1994) in R. marginatus (Fabricius), in which the antennectomized predator exhibited delayed arousal, approach capturing, paralyzing and sucking suggesting the role of antennae in predation. Response to chemical cues through Six-arm olfactometer C he mica lly me diated hos t preference of R. marginatus to different insect pest extracts were assessed in terms of percentage preference (Table 3). When the predator was released into the central camber, the predator exhibited behavioural responses, due to chemical 31

5 Pravalika et al. Table 3. Response of Rhynocoris marginatus to chemical cues in a six arm olfactometer Test insect Predator orientation (%) Approaching time (min) Handling time (min) Spodoptera litura ± ±1.45 Mythimna seperata ± ±0.97 Sesamia inferens ± ±2.39 Control ± ±3.48 cues elicited from the cotton impregnated with body extracts of pests viz., S. litura, M. separata and S. inferens. Due to the chemical cues from the samples the predator moved towards the respective odour cell. In this experiment, R. marginatus highly preferred the he xane extrac t of S. litura (42.8%) followed by M. separata (28.5%) and least preference to S. inferens which was on par with hexane extract alone. The hexane fractions of all the insect pests elicited a positive approaching response in R. marginatus. It showed the highest responses to the hexane extract of S. litura (12.6±1.20 min) followed by M. separata (10.2±1.88 min), and then to S. inferens (8.8±1.33min). The approaching time for control was 7.6±2.02 min. The preference to S. litura and M. separata body extracts was greater than to S. inferens (Table 3). The predator R. marginatus exhibited the highest handling time to S. Litur a extrac t (21. 6±1. 45 min) followed by M. separata (19.2±0.97 min), S. inferens (15.7±2.39 min) and only hexane (12.6±3.48). The cues stimulate receptors generating sensory input and after being processed and integrated by the central nervous system, interpreted as a positive or negative signal and the decision made as to whether to make a certain behaviour response (Heard, 2001). In this present investigation, R. marginatus responded to the hexane extracts of S. litura, M. separata and S.inferens, in that order which was inferred by the approaching response of R. marginatus to the sample cell. Moreover the predators extended their antennae and rostrum towards the sample loaded cotton and rubbed it, suggesting that volatile chemicals attracted these predators and stimulated them to extent their rostrum. At first the inactive predators orients toward the odour source with antennae (Ra ma chandran a nd Norris, 1991; Rani, 1998). Harpactorine reduviids oriented towards the prey with exte nded a nd palpating ante nnae at a rous al a nd subsequently approach the prey (Ambrose, 1999) as observed in this harpactorine reduviid. Reduviid predators generally prefer lepidopteran larvae (Edward 1962; Ables 1978; Ambrose 1999, 2000a, 2003). McMahan (1983) noted that in reduviids, preference for one prey over another in choice test might be influenced by the noxious smell or unpleasant taste of the pre y. Among the lepidopterans it preferred mostly S. litura than S. inferens and M. separata because the S. litura. This may possibly be due to the fact that S. litura is exposed on leaves where as S. inferens and M. separata are hidden feeders and it is more difficult for the predator to find its prey and hence predators have instinctively learnt to recognise the chemical cues of such prey. Capture success generally depends on prey mobility and access to a refuge (Eubanks and Denno, 2000) and may influence the preference of one prey over another. REFERENCES Ables, J. R Feeding behaviour of assassin bug, Zelusrenardii. Annals of the Entomological Society of America, 71(4): Ambrose, D. P Assassin Bugs. Science Publishers, New Hampshire, USA and Oxford & IBH Publishing Company Private Limited, New Delhi, India, pp Ambrose, D.P. 2000a. Assassin Bugs (Reduviidae excluding Triatominae), In: Heteroptera of Economic Importance, (eds.) Schaefer, C.W. and Panizzi, A.R., CRC Press, Florida, U.S.A. pp Ambrose, D.P Biocontrol potential of assassin bugs (Hemiptera: Reduviidae). Journal of Experimental Zoology India,6(1): Ambrose, D.P. and Mayamuthu, T Impact of sex, starvation, antennectomy, eye blinding and tibial comb coating on the predatory behaviour of Rhinocoris fuscipes Fabricius (Insecta: Heteroptera: Reduviidae). Journal of Advanced Zoology, 15(2): Ambrose, D.P., Kulandaisamy, A.M.J. and Sahayaraj, K Impact of starvation, eye blinding and antennectomy on the predatory behaviour and efficiency of Euagora splagiatus Burmeister (Insecta: Heteroptera: Reduviidae). Bulletin of Entomology, 35(1-2): Ambrose, D.P., Samuel, M and Sahayaraj, K Impact of antennectomy, eye blinding and fossulaspongiosa 32

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