Evidence for Presence of Female Produced Pheromone Components in Male Scent Brush Extract of Castor Semi-Looper Moth Achaea Janata L
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Indian Journal of Experimental Biology Vol. 43, April 2005, pp. 335-341 Evidence for presence of female produced pheromone components in male scent brush extract of castor semi-looper moth Achaea janata L. K N Jyothi, A L Prasuna & A R Prasad Pheromone Laboratory, Organic Division I, Indian Institute of Chemical Technology, Tarnaka , Hyderabad 500 007, India. Received 4 October 2004; revised 3 January 2005 Hexane extract of male terminalia (along with scent brushes) of castor semi-looper moth, Achaea janata L, elicited significant olfactory responses in both male and female insects by electroantennogram recording technique. However, male extract in the wind tunnel evoked noticeable behaviour responses in the female insects only. Orientation response of the males to the male extract was not evident in wind tunnel experiments. Two electrophysiologically-active compounds were identified from the male extract. Based on GC retention times and mi'!ss spectrometry the two compounds were confirmed as (Z, Z)-9, 12- octadecadienal and (Z, Z, Z)-3, 6, 9-heneicosatriene. These two compounds are also constituents of female produced four component blend of A. janata. Keywords: Achaea janata, Electroantennogram, , Gas chromatography, (Z, Z, Z)-3, 6, 9-Heneicosatriene Male brush, (Z, Z)-9, 12-0ctadecadienal,Wind tunnel. Achaea janata L, Castor semi-looper (Lepidoptera: Electrophysiological studies of olfactory Noctuidae) moth is an important pest of castor, sensitivities to pheromones in insects are mostly Ricinus communis in India. Both caterpillars and adult concentrated on antennal receptor neurons of males, moths cause the damage. In severe infestation, larvae particularly on their sensitivity to female produced consume entire foliage leaving only the midribs and male attracting pheromones. Pheromones produced by stalks. Excessive loss of foliage causes reduction in males and their role in close range complex sexual 5 seed yield. Adults are fruit sucking moths and cause interplay have been relatively neglected . serious damage to citrus crop. Males of many species of Lepidoptera have scent Persoons et aI. l identified the sex pheromone organs located on the abdomen, thorax, legs and system in female semi-looper moth as a four wings, which vary from simple scales and hair tufts to component blend consisting of--{I) Heneicosane; (II) complex eversible structures. The noctuid male scent (Z, Z)-6, 9-Heneicosadiene; (III) (Z, Z, Z)-3, 6, 9- organs commonly referred to as hair pencils typically Heneicosatriene and (IV) (Z, Z)-9, 12-0ctadecadienal constitute a pair of brushes, scent glands and storage 6 in the ratio of l.0: l.0: 60 -70: l.0, respectively. pockets • The male scent brush apparatus of Ajanata Generally there is overwhelming behavioural and also possesses typical Noctuid morphology. The electrophysiological evidence that female moths are brushes extend and spread to a fan shape when a male non-responsive to the sex pheromones which they approaches a calling female. Sehgal et al. 7,8 have 2 produce . However, our electroantennogram studies described the morphological details of the external indicated that females are stimulated not only by the genitalia of Ajanata in detail. Although, volatile total pheromone blend, but also by each of the chemicals from males of some of the noctuid species individual pheromone components (Unpublished have been identified and biosynthetic pathways for observations, - Jyothi K N). Similar responsiveness of some of the compounds were known, the actual role 9 females to female produced pheromone has also been of most of the compounds is not clearly elucidated . demonstrated in Trichoplusia ni 2,3 and Choristonuera Aim of the present investigation was--{l) to 4 jumiferana . ascertain the electrophysiological and behavioural function of the hairbrush extract against both males and females of Ajanata; and (2) to isolate and Correspondent author: Tel. No. 27193136 E-maillO: [email protected] identify the chemical nature of secretions from male nCT communication No. 041219 abdominal tip extract (along with brushes) which may 336 INDIAN J EXP BIOL, APRIL 2005 possibly explain the reason for female-female capillary column HP-5 MS (0.25mm id; 30 m length attraction. at 0.25 f.Lm film thickness) was used. Ionization voltage was 70 e V and the temperature of the ion Materials and Methods source and the interface was 2300 and 2800 C, Insect culture--Culture of Ajanata was maintained respectively. Carrier gas was helium at a constant in the laboratory in an environmental chamber at flow rate of 1.2ml/min. The extract was analyzed 27°± 1°C, 70 % RH and 10: 14 dark/light under reverse using the same temperature program as described photoperiod conditions. The larvae were fed on fresh above for Gc. The identity of the various peaks was castor leaves until pupation. Pupae were segregated confirmed by comparing the unknowns with both the sex wise (based on the morphology of 8-10 abdominal standard spectra library and authentic samples (gift segments) and maintained in cubical wooden adult from TNO, The Netherlands). cages (36 x 36cm x 36cm). The moths after emergence were fed on 10 % honey solution soaked Chemicals on cotton pads in petri dishes. For identification and electrophysiological work, Male extracts (abdominal terminalia along with two compounds-(Z,Z)-9,12 octadecadienal and brushes) preparation-For extract preparation, 3-5 (Z,Z,Z)-3,6,9-heneicosatriene were obtained from day old male moths were utilized. Male terminalia Organic Division, nCT. Detailed synthesis procedures were everted by gently pressunzmg terminal are described elsewhere 1 I. abdominal segments during the period of mating activity 10 . With the help of micro scissors they were Electroantennogram recording technique (EAG) quickly excised into vials containing n-hexane. The The EAG technique was employed to study the hexane extracts were pooled and stored at -300 C sensitivity/selectivity of both male and female until further use. The extracts were filtered through a antennae of Ajanata to the crude male scent brush short silica column and were concentrated at room extract as well as the synthetic components. An EAG temperature with a stream of nitrogen for chemical profile indicated the senSItIvIty and relative analysis, electrophysiological and wind tunnel abundance of olfactory receptor neurons present on bioassay studies. the antenna for a particular compound tested. For electrophysiological recordings, Syntech EAG Analytical procedures (Syntech, Hilversum, The Netherlands) was Gas chromatography (GC)-Gas chromatography employed. The isolated antennae of different ages of of male extract and standard compounds were males and females Ajanata were individually performed on a Carlo Erba 5300 gas chromatograph mounted onto a metal electrode holder (stainless steel) fitted with a flame ionization detector and split with the help of an electrically conductive gel. The injector. Separations were performed on a 2m x 2.5 antenna was continuously flushed with charcoal mm id glass column packed with 3% CPSil-5 filtered and moistened air stream through a stainless chromo sorb phase (100-120 mesh). A temperature tube (8mm id) ending 2 cm before the antennal programme of 50°C (2min delay) increased to final preparation. The EAG signals were amplified and temperature of 260°C (5 min delay) at the rate of recorded with a data acquisition controller and 10°C/min was followed. Nitrogen was used as the software (Syntech, The Netherlands). carrier gas with a column head pressure of 100 kPa. The test compound (5 male equivalents of male Injector and detector temperatures were set at 275°C. extract/5-100 f.L g of synthetic compound) was spread The column effluent was split to send 1/6th of the on a filter paper. After complete evaporation of the extract to FlD detector and 5/6th to the glass capillary solvent, the filter paper strip was inserted into a embedded in dry ice for collection of volatiles ( a Pasteur pipette. Stimuli were provided by connecting custom made device of TNO, The Netherlands gifted the pipette for 300 ms into the air stream flushing to nCT). Two min fractions were collected in chilled over the antenna. An equal volume of solvent alone capillaries and subsequently bio-assayed by EAG (hexane) spread on the filter paper served as the technique. control. Control stimulation was made at the Gas chromatography-mass spectrometry (GC-MS) beginning and after every 2-3 EAG recordings. EAG -The extract was subjected to GC-MS instrument for responses were evaluated by measuring the maximum analysis. Agilent 6890 GC with 5973 NMSD with amplitude of depolarization triggered by stimuli. At JYOTHI et al.: PRESENCE OF FEMALE PHAROMORE COMPONENTS IN MALE 337 least 30 sec was allowed between two continuous Two kinds of stimuli were used in the wind tunnel. stimuli for recovery of the antenna. _EAG responses In the first test, the orientation behaviour of males and were recorded from five males and five females females separately towards the crude extract (five moths individually to the extract/synthetic compound. male equivalents) was evaluated. Filter paper loaded The EAG data in Figs. 2-4, represent corrected EAG with same volume of hexane served as control. In the figures (male extract/ synthetic compound minus second test, the behavioural response of compounds control). The EAG responses were subjected to identified from the male extract i.e., [Z, Z, Z)-3, 6, 9- analysis of variance (ANOV A). heneicosatriene and (Z, Z) - 9, 12-octadecadienal were evaluated. The test compound (100 I1g of each) Wind-tunnel bioassay dissolved in n-hexane was used as the stimulus. An indoor cylindrical wind tunnel (30 cm diam x 2 Numbers of males exhibiting different levels Of m) was used to test the biological activity of crude upwind flight behaviour in the tunnel were counted. extract/synthetic standards 12. The tunnel was housed All the experiments were performed in triplicate. in a room with temperature and relative humidity controls and an exhaust system from the tunnel to the Results outside of the room.